Mammalian Neuraminidases in Immune-Mediated Diseases: Mucins and Beyond

Abstract

Mammalian neuraminidases (NEUs), also known as sialidases, are enzymes that cleave off the terminal neuraminic, or sialic, acid resides from the carbohydrate moieties of glycolipids and glycoproteins. A rapidly growing body of literature indicates that in addition to their metabolic functions, NEUs also regulate the activity of their glycoprotein targets. The simple post-translational modification of NEU protein targets—removal of the highly electronegative sialic acid—affects protein folding, alters protein interactions with their ligands, and exposes or covers proteolytic sites. Through such effects, NEUs regulate the downstream processes in which their glycoprotein targets participate. A major target of desialylation by NEUs are mucins (MUCs), and such post-translational modification contributes to regulation of disease processes. In this review, we focus on the regulatory roles of NEU-modified MUCs as coordinators of disease pathogenesis in fibrotic, inflammatory, infectious, and autoimmune diseases. Special attention is placed on the most abundant and best studied NEU1, and its recently discovered important target, mucin-1 (MUC1). The role of the NEU1 - MUC1 axis in disease pathogenesis is discussed, along with regulatory contributions from other MUCs and other pathophysiologically important NEU targets.

Full text

Mammalian Neuraminidases
in Immune-Mediated Diseases:
Mucins and Beyond
Erik P. Lillehoj
1
*, Irina G. Luzina
2,3
and Sergei P. Atamas
2
1
Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD, United States,
2
Department of
Medicine, University of Maryland School of Medicine, Baltimore, MD, United States,
3
Research Service, Baltimore Veterans
Affairs (VA) Medical Center, Baltimore, MD, United States
Mammalian neuraminidases (NEUs), also known as sialidases, are enzymes that cleave off
the terminal neuraminic, or sialic, acid resides from the carbohydrate moieties of
glycolipids and glycoproteins. A rapidly growing body of literature indicates that in
addition to their metabolic functions, NEUs also regulate the activity of their
glycoprotein targets. The simple post-translational modification of NEU protein
targets—removal of the highly electronegative sialic acid—affects protein folding, alters
protein interactions with their ligands, and exposes or covers proteolytic sites. Through
such effects, NEUs regulate the downstream processes in which their glycoprotein targets
participate. A major target of desialylation by NEUs are mucins (MUCs), and such post-
translational modification contributes to regulation of disease processes. In this review, we
focus on the regulatory roles of NEU-modified MUCs as coordinators of disease
pathogenesis in fibrotic, inflammatory, infectious, and autoimmune diseases. Special
attention is placed on the most abundant and best studied NEU1, and its recently
discovered important target, mucin-1 (MUC1). The role of the NEU1 - MUC1 axis in
disease pathogenesis is discussed, along with regulatory contributions from other MUCs
and other pathophysiologically important NEU targets.
Keywords: post-translational modification, sialidase, sialic acid, fibrosis, inflammation, infection, autoimmunity
INTRODUCTION
Protein glycosylation is a biologically ubiquitous, enzymatic post-translational modification
involving the attachment of one or more carbohydrate moieties to a protein molecule, typically
at the nitrogen atom of the side chain amide group of an Asn residue (N-linked glycosylation) or the
oxygen atom of the side chain hydroxyl group of a Ser or Thr residue (O-linked glycosylation).
Glycosyltransferases catalyze the transfer of the glycan to the protein acceptor, while glycosidases
catalyze the reverse reaction. Sialyltransferases and neuramindases (NEUs), also known as
sialidases, are subclasses of glycosyltransferases and glycosidases, respectively, that specifically
add or remove sialic acids to/from the terminal position of glycan chains (Figure 1). Sialic acids
comprise a family of more than 50 closely related, but structurally diverse, nine-carbon
monosaccharides (1–3). N-acetylneuraminic acid (Neu5Ac or NANA) is the predominant sialic
acid in most mammalian cells and is often used interchangeably with sialic acid. Most commonly,
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 8830791
Edited by:
Gaby Palmer,
Universite
´de Genève,
Switzerland
Reviewed by:
Tamara Nowling,
Medical University of South Carolina,
United States
Eugenio Monti,
University of Brescia, Italy
*Correspondence:
Erik P. Lillehoj
elillehoj@som.umaryland.edu
Specialty section:
This article was submitted to
Autoimmune and
Autoinflammatory Disorders,
a section of the journal
Frontiers in Immunology
Received: 24 February 2022
Accepted: 21 March 2022
Published: 11 April 2022
Citation:
Lillehoj EP, Luzina IG and Atamas SP
(2022) Mammalian Neuraminidases
in Immune-Mediated Diseases:
Mucins and Beyond.
Front. Immunol. 13:883079.
doi: 10.3389/fimmu.2022.883079
SYSTEMATIC REVIEW
published: 11 April 2022
doi: 10.3389/fimmu.2022.883079

the C-2 carbon atom of the a-anomer of Neu5Ac forms a
covalent bond with the C-3 hydroxyl group of galactose (Gal),
or the C-6 hydroxyl group of Gal or N-acetylgalactosamine
(GalNAc), to form a(2,3)- and a(2,6)-linked structures. In a
small number of proteins, polysialic acid has been identified as a
unique, a(2,8)-linked homopolymer of up to 150 sialic acid
residues that confers an exceptionally high electronegative
charge (4). With their terminal location and negative charge at
physiological pH, sialic acids are strategically positioned to
influence intermolecular and intercellular interactions through
steric hindrance and/or electronic effects. Consequently, sialic
acids, and the enzymes that add or remove them from glycan
chains, influence protein tertiary conformation, bioactivity,
and proteolysis.
NEUs –GENERAL FEATURES
As early as 1960, mammalian sialidase activity was detected in
fractioned human serum (5). Later, crude sialidase activity was
reported in a variety of eukaryotic cell and tissue extracts (6–11).
Prior to isolation and characterization of mammalian NEU
genes, the biochemical characterization and enrichment/
purification of these various sialidases demonstrated the
presence of enzymes with different molecular weights,
subcellular localization, substrate specificities, and pH
optimums in vitro (12). Klotho, a protein originally associated
with senescence and aging, also has been reported to exhibit
sialidase activity, although it lacks conserved structural domains
characteristic of canonical NEU proteins (13).
NEUs are found in organisms of diverse evolutionary lineages,
from viruses to mammals, and functional consequences of their
enzymatic activity across and within taxa are remarkably broad and
complex. There are four mammalian NEUs, NEU1 –NEU4, and
they are all exosialidases, meaning that they remove the terminal
sialic residues from the glycan moieties of glycoproteins and
glycolipids. Mammalian NEUs are differentially expressed across
tissues and differentially distributed at the subcellular level. Their
relative expression levels are usually different, with NEU1 being the
predominant isoform, often closely followed by the relatively
abundant NEU3, whereas NEU2 and NEU4 are commonly
expressed minimally if at all; this generalization is not always
supported as it may be hindered by specifics of a particular
disease process, tissue involved, or differentiation state of the cells
engaged. Based on their catalytic function, NEUs are primarily
metabolic enzymes; mutations of the NEU1 gene lead to a metabolic
FIGURE 1 | Opposing catalytic activities of sialyltransferases and sialidases. In the reaction catalyzed by sialyltransferases, sialic acid is transferred from its activated
nucleotide sugar donor, cytidine 5’-monophosphate (CMP)-sialic acid, to the penultimate galactose (illustrated here) or N-acetylgalactosamine residue of a glycan
chain, thereby releasing free CMP. The resulting sialic acid-galactose disaccharide is illustrated in an a(2-3) linkage. In the sialidase-catalyzed reaction, the sialic acid-
galactose/N-acetylgalactosamine covalent bond is hydrolyzed to released release free sialic acid.
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 8830792

disorder, sialidosis (14). The research of the past two decades has
revealed that NEUs also regulate important biological functions and
are involved in disease pathogenesis (12,15–17). This is not
surprising, considering that many homeostatic mediators involved
in metabolic regulation, development, reproduction, and immune
tolerance, as well as pathophysiological regulators involved in
immune and inflammatory diseases, fibrosis, atherosclerosis,
cancer, and other diseases are sialylated glycoproteins. NEU
targets and binding partners include multiple glycoproteins
known to play important roles in the mechanisms of immunity,
inflammation and fibrosis (Table 1).
At the mechanistic level, the downstream effects of
desialylation are substantially defined by the properties of sialic
acid, which is a highly electronegative sugar. Desialylation affects
the charge of the target molecules and even the often much
sialylated cell surface. At the molecular level, desialylation can
directly unmask or mask glycoproteins’bindings sites for their
molecular partners. Desialylation can also affect intramolecular
interactions and, subsequently, protein folding, again revealing
or sequestering binding sites for low molecular weight ligands or
high molecular weight interactors, thus affecting the target
glycoproteins’functions as enzymes, receptors, or signal
transducers. More specifically, several mechanistic pathways
can be envisioned and have been experimentally validated (12,
15–17) through which neuraminidases control their glycoprotein
targets and their downstream functioning. Desialylation also
affects the target glycoproteins’folding, thus additionally
regulating protein-protein interactions. Desialylation of the
entire cell surface has far-going consequences for cell-cell
interactions, thus affecting pathophysiological processes on the
large scale. Such changes in folding may induce transitions
between functionally active and inactive states of enzymes,
attenuation or augmentation of ligand binding by receptors,
and masking or unmasking of proteolytic sites. On a more
direct level, siglecs bind to sialic acid of glycoproteins, whereas
galectins bind to Gal residues which become exposed following
desialylation, as Gal is typically the subterminal carbohydrate to
which the terminal sialic acid is attached in the glycan portions of
glycoproteins and glycolipids. Thus, NEU-mediated desialylation
diminishes siglec binding and allows for galectin binding to
glycoproteins, affecting such interactions that are known to
regulate immunity and inflammation.
Originally, the four mammalian NEUs were characterized by
their characteristic subcellular localization, including lysosomal
NEU1, cytosolic NEU2, plasma membrane NEU3, and lysosomal,
mitochondrial, and endoplasmic reticular NEU4 (12). In the
lysosome, NEU1 is associated with its chaperone/transport
protein, protective protein/cathepsin A (PPCA), and b-
galactosidase (60). It became quickly recognized that in addition
to such predominant localization, each NEU may be distributed
more broadly. For example, in addition to the predominantly
membrane-targeted NEU3, the other NEUs can be found on the
cell surface, including NEU1 (20,41,44–47,53,55,56,60–75),
NEU2 (76–78), and NEU4 (79,80). NEU1 on the plasma
membrane has been reported either in the presence (62,64–66,
72,75)orabsence(68,71) of PPCA. Membrane localization allows
NEUs to act as structural and functional modulators of extracellular
soluble and membrane-bound molecules (17). Further information
TABLE 1 | Selected confirmed and putative glycoprotein substrates of NEUs and NEU interactors with regulatory contributions to immune, inflammatory, and fibrotic
processes.
Glycoprotein NEU Isozyme References
ApoB100 NEU1, NEU3 (18)
ATG5 NEU2 (19)
CD5 NEU1 (20)
CD18 (ITGB2) NEU1, NEU3 (21–24)
CD31 (PECAM1) NEU1 (22,25)
CD36 NEU1 (22)
CD42b (GPIba) NEU1, NEU3 (26,27)
CD44 NEU1 (28–30)
CD54 (ICAM1) NEU1 (23)
CD64 (FCgR) NEU1 (31)
CD104 (ITGB4) NEU1 (32)
CD107a/b (LAMP-1, LAMP-2) NEU1 (33,34)
CD140 (PDGFR) NEU1 (35)
CD220 (insulin receptor) NEU1 (36–39)
CD221 (IGF-1R) NEU1 (35,36)
EGFR NEU1, NEU3 (40–42)
HGFR/Met NEU1 (43)
MMP9 NEU1 (22,41,44–47)
MUC1 NEU1 (40,48–51)
TGF-b/LAP NEU3 (52)
TLR2 NEU1 (22,53)
TLR3 NEU1 (53)
TLR4 NEU1 (45,47,53–58)
TLR7 NEU1 (44)
TLR9 NEU1 (44)
TrkA NEU1 (46,59)
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 8830793

on the subcellular, cellular, and tissue distribution, substrate
specificity, catalytic properties, and amino acid homologies of the
four mammalian NEUs can be found in prior review articles (12,15,
81–87).
MUCs –GENERAL FEATURES
Mucus is a complex mixture of ions, salts, peptides, proteins,
glycoconjugates, and water covering the surface of mucosal tissues.
The primary protein component of mucus are mucins, high
molecular weight, extensively glycosylated proteins containing
variable numbers of tandem repeats (VNTRs) in which Ser, Thr,
and Pro amino acids are highly enriched. Mucin glycosylation
primarily occurs through O-glycosidic linkages between the first
GalNAc residue of the oligosaccharide side chains and Ser andThr
amino acids of the polypeptide backbone. More than 20 mucin
glycoproteins have been identified and are broadly subdivided into
secreted and cell membrane-tethered molecules (88,89). Secreted
mucins are further subdivided into gel-forming and non-gel-
forming mucins. Examples of membrane-bound mucins include
MUC1, MUC3A, MUC3B, MUC4, MUC13, and MUC16, while
secreted mucins include MUC2, MUC5AC, MUC5B, MUC6,
MUC7, MUC19, and MUC20.
MUC1 is the prototype membrane-tethered mucin. The MUC1
glycoprotein consists of a large extracellular ectodomain (MUC1-
ED), a single-pass transmembrane domain, and an intracellular
cytoplasmic domain (MUC1-CD) (90–92). The MUC1-ED is
primarily composed of varying numbers (25–125)ofhighlyO-
glycosylated, 20-amino acid VNTRs, with the number of repeats
being a polymorphic genetic trait. The MUC1-CD contains multiple
Ser, Thr, and Tyr residues as potential phosphorylation sites. MUC1
is initially synthesized as a single polypeptide chain, but
autoproteolytic cleavage at a Gly-Ser peptide bond in the
endoplasmic reticulum generates two subunits that are together
transported to the cell surface. The larger subunit (> 250 kDa) is
derived from most of the MUC1-ED and the smaller subunit (20-30
kDa) contains a juxtamembrane region of the MUC1-ED, the
membrane-spanning domain, and the MUC1-CD (93). MUC1
autoproteolysis occurs within its SEA (sea urchin sperm protein,
enterokinase, agrin) domain, a 120-amino acid region highly
conserved in glycosylated, mucin-like proteins (94,95). While the
two autoproteolytic cleavage fragments constitute separate
polypeptide chains, they remain tightly associated on the cell
surface, although they are not linked through disulfide bonds, and
are only separated under extreme dissociating conditions, for
example in the presence of sodium dodecyl sulfate (96). However,
the MUC1-ED can be untethered from the cell surface under
physiologic conditions by additional proteolytic cleavage by
proteasessuchasneutrophilelastase(97,98), TNF-aconverting
enzyme (TACE) (99), matrix metalloprotease-14 (MMP-14) (100,
101), and g-secretase (102). Finally, an extracellular form of the
MUC1-ED, MUC1-SEC, can be produced through alternative
splicing of the MUC1 mRNA to introduce a translation stop
codon prior to its transmembrane region (103).
The 72-amino acid MUC1-CD contains 9 Ser, 6 Thr, and 7
Tyr residues as potential phosphorylation sites. Many of these
residues are located within consensus sequence binding motifs
for signaling kinases and adapter proteins. Among the most well-
characterized of these are phosphatidylinositol 3-kinase (PI3K),
Shc, phospholipase Cg(PLCg), protein kinase Cd(PKCd),
glycogen synthase kinase-3b(GSK3b), c-Src, epidermal growth
factor receptor (EGFR), b-catenin, and Grb-2 (104–107). The
pattern of MUC1-CD Tyr phosphorylation is similar to that of
some cytokine and growth factor receptors, but unlike cytokine/
growth factor receptors, the MUC1-CD is not capable
of autophosphorylation.
Carbohydrate analysis of MUC1 glycans reveals a high
content of sialic acid (108,109). Because MUC1 is the major
membrane-bound mucin expressed in the airways and NEU1 is
the major sialidase expressed in the respiratory tract (40), studies
were performed to determine the molecular relationship
between NEU1 and MUC1 in airway epithelial cells. By
immunohistochemical staining, airway expression of NEU1
matches with that seen for MUC1 in these same tissues, i.e. at
the superficial surface of airway epithelia, including the brush
border of the trachea and bronchus, but not in subepithelial
tissues (40,110,111). Overexpression of NEU1 in cultured
human airway epithelial cells increased its association with
MUC1 and decreased the sialic acid content of the MUC1-ED
as assessed by lectin blotting with the sialic acid-reactive lectin,
Maakia amurensis lectin (40). In contrast, NEU1 silencing in
these cells had the opposite effects. Thus, the MUC1-ED is a
substrate for NEU1. However, it is unknown whether NEU1
desialylates MUCs other than MUC1, and whether NEUs other
than NEU1 desialylate MUC1.
NEU1 desialylates not only MUC1, but also EGFR (40).
Because MUC1 forms a molecular complex with EGFR (112–
114), and NEU1 plays a role in programmed cell death (115,
116), studies were performed to examine the relationship
between NEU1, MUC1, and EGFR in autophagy (117).
Immunohistochemically, human triple-negative breast cancer
cells expressed high levels of EGFR, but low levels of MUC1
and NEU1. The levels of two autophagy pathway proteins, PI3K
and beclin-1, were positively correlated with both NEU1 and
MUC1-ED levels, while only PI3K correlated with the MUC1-
CD. These results led to the authors to speculate that a NEU1 –
MUC1 –EGFR axis is dysregulated in breast cancer cells
exhibiting reduced autophagy.
KL-6
In 1988, a mouse monoclonal antibody, Krebs von den Lungen-6
(KL-6) (from the German “cancer from the lungs”)was
described that was raised against human pulmonary
adenocarcinoma cells (118). In addition to the immunizing
cells, the KL-6 antibody reacted with normal human type 2
alveolar pneumocytes and bronchiolar epithelial cells, and with
an unidentified high molecular weight mucin-like serum
glycoprotein whose levels were increased in patients with lung
adenocarcinoma. The epitope on the KL-6 antigen recognized by
the KL-6 antibody was initially defined as a sialylated glycan
becausetreatmentofthepurified antigen with sialidase
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 8830794

diminished its reactivity with the cognate antibody (118).
Carbohydrate analysis identified Gal, GalNAc, and Neu5Ac
(sialic acid) in the KL-6 epitope (119). Subsequently, Ohyabu
et al. (120) reported that the minimal antigenic structure of the
KL-6 epitope consisted of the glycan, Neu5Aca2,3Galb
1,3GalNAca, attached to the Thr residue in the peptide, Pro-
Asp-Thr-Arg-Pro-Ala-Pro (Figure 2). This carbohydrate
structure also is referred to as the 2,3-sialyl T antigen of the
core 1-type O-glycan. More recent analysis suggests that the KL-
6 antibody also recognizes a 6′-sulfo-Gal/GalNAc glycan epitope
(121). Interestingly, the KL-6 epitope only is expressed in
humans and apes and not other mammals (122).
On the basis of flow cytometric and immunohistochemical
studies,KL-6wasinitiallycategorizedasaCluster9/MUC1
antigen in the lung tumor and differentiation antigen
classification system (123). Biochemical analysis of purified
KL-6 established its identity with MUC1 (119). Amino acid
analysis indicated a relatively high content of Pro (19.1 mol
%), Thr (13.7%), and Ser (12.3%), whilecarbohydrateanalysis
revealed enrichment of Neu5Ac (30.2% per weight), Gal
(18.9%), and GalNAc (11.9%). Most convincingly, in
Western blot analysis the purified KL-6 glycoprotein reacted
with the established MUC1 antibody, DF3. Because the KL-6
antibody reacts with the surface of type II pneumocytes and
bronchiolar epithelial cells, and KL-6 is present in
bronchoalveolar lavage fluid (BALF) and serum, the
mechanism through which KL-6 is transported between the
cell –BALF –blood compartments has been speculated (124,
125). While it is unclear how KL-6 is released from the cell
surface into the alveolar fluid that is collected as BALF, the
identification of two established MUC1-ED sheddases, MMP-
14 and g-secretase, in airway epithelial cells (51)suggestsa
proteolytic mechanism of KL-6 shedding.
NEUs AND MUCs IN PULMONARY
FIBROSIS
Exaggerated, often irreversible, and sometimes relentless
scarring, known as fibrosis, can affect any organ or tissue.
Excessive activation of fibroblasts, manifesting as their
transdifferentiation into collagen-depositing, apoptosis-
resistant, myofibroblasts underlies fibrosis. The ensuing
replacement of the functional parenchyma with the scar tissue
can become functionally and cosmetically debilitating in the skin
or outright deadly when fibrosis develops in the lung, heart,
kidney, or liver. There is no cure for fibrosis, and existing
therapies do not consistently prevent fibrosis progression. The
mechanistic involvement of NEUs in fibrosis has been suggested
relatively recently, mostly in the lungs, where NEU activation
promotes fibrosis (48,52,126–130); but also heart, with reports
suggesting a profibrotic role for NEU1 (131) and an antifibrotic
role for NEU3 (132). Mucins have also been suggested as
mechanistic contributors to pulmonary fibrosis and,
furthermore, NEUs and MUCs interplay mechanistically, as
reviewed in detail below. Therefore, the following discussion
focuses mostly on pulmonary fibrosis. The mechanisms of
fibrosis are highly diverse, complex, redundant, and organ-
specific, yet there are numerous unifying features across organ-
and tissue-specificfibroses. In relevance to pulmonary fibrosis,
these mechanisms include, but are not limited to, activation of
transforming growth factor-b(TGF-b)(133,134), changes in the
levels of other cytokines regulating both inflammation and
fibrosis (135–142), oxidative stress (143,144), coagulation
disturbances (145,146), changes in biomechanical forces (147,
148), cellular senescence (149–155), defective autophagy (156,
157), and dysregulated epithelial cell –fibroblast crosstalk
(158–161).
Pulmonary fibrosis is usually considered in the context of
interstitial lung disease (ILD), an umbrella entity that comprises
a large and diverse group of human disorders characterized by
variously proportioned pulmonary inflammation and scarring.
Idiopathic pulmonary fibrosis (IPF) represents the most severe,
debilitating, and poorly understood ILD (162,163). Connective
tissue disease related ILD, especially in patients with scleroderma
and rheumatoid arthritis, also poses a serious biomedical
problem (164–166). Although the roles for NEU contributions
to ILD have been suggested recently, early indications were
observed in the past. Lambreet al. (167)first reported sialidase
activity in the BALF from patients with IPF, while BALF from
healthy subjects and serum from both IPF patients and controls
had no such activity. Later, NEU1 mRNA and protein levels were
shown to be increased in lung epithelial and endothelial cells, and
fibroblasts, of IPF patients compared with healthy controls (128).
In the same study, NEU1 overexpression in cultured lung
fibroblasts increased the levels of collagen types I and III and
MMP-14 degradation products, while NEU1 overexpression in
mice increased lung collagen and TGF-blevels and promoted
lung leukostasis of CD8
+
, and to a lesser extent CD4
+
, T cells. A
later report from the same group demonstrated that selective
pharmacological inhibition of NEU1 using C9-butyl-amide-2-
FIGURE 2 | Proposed minimal antigenic structure of the KL-6 epitope based
on Ohyabu et al. (120).
Lillehoj et al. NEUs and MUCs in Disease
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deoxy-2,3-dehydro-N-acetylneuraminic acid (C9-BA-DANA),
attenuated collagen accumulation in the lungs and pulmonary
lymphocytosis in the acute and chronic bleomycin models of
lung fibrosis (48). Taken together, these results indicated that
NEU1 plays a central role in the pathogenesis of pulmonary
fibrosis and NEU1-selective inhibition may offer a potential
therapeutic intervention for pulmonary fibrotic diseases. Other
studies have implicated a role for NEU3 in IPF pathogenesis (52,
126,127,129,130).
NEU1 –MUC1 Axis in ILD
Kohno and colleagues (168) reported that serum levels of the KL-
6 antigen positively correlated with disease activity in patients
with interstitial pneumonitis, including IPF, hypersensitivity
pneumonitis, and sarcoidosis. Elevated levels of KL-6 also were
observed in the BALF of patients with ILD and there was a
positive correlation between KL-6 serum and BALF levels (125,
169). Serum KL-6 levels were demonstrated to be proportionally
higher in interstitial pneumonitis patients compared with other
biomarkers of disease activity, such as type III procollagen N-
terminal peptide (PIIIP) and type IV collagen 7S (7S collagen),
while the latter two were more sensitive markers of alveolar
pneumonia (170). In subsequent investigations, elevated serum
levels of KL-6 were shown to be present in a greater proportion
of patients with ILD compared with other reported disease
biomarkers, including sialyl-Lewis a (sLe
a
), sialyl-Lewis x
(sLe
x
), surfactant protein-A, surfactant protein-D, monocyte
chemoattractant protein-1 (MCP-1), and C-C motif chemokine
ligand 18 (CCL18) (171–174). In addition to its value as a
diagnostic biomarker of ILD, KL-6 serum levels have been
reported to be of prognostic value for predicting disease
severity, acute exacerbations, and patient survival (125,175,
176). Further, the value of KL-6 as a biomarker for ILD
extends to diverse ethnic populations throughout the world,
despite the fact that polymorphisms in the MUC1 gene affect
serum levels of KL-6 (177–179). Based on these collective studies,
KL-6 has been approved by the Japanese Health Insurance
Program as a clinical diagnostic biomarker for ILD (124).
Several relevant review articles can be consulted for further
information on the diagnostic and prognostic value of KL-6 in
IPF/ILD (125,180–183).
KL-6 has been implicated in the pathogenesis of IPF. Purified
KL-6 increased the chemotaxis of human lung and skin
fibroblasts in vitro, which was augmented by fibronectin (119).
KL-6 also promoted lung fibroblast proliferation and migration,
and inhibited apoptosis, which were synergized by TGF-b(184).
In a mouse model of bleomycin-induced pulmonary fibrosis,
administration of anti-KL-6 antibody following bleomycin
administration reduced the number of BALF inflammatory
leukocytes, diminished the content of hydroxyproline in lung
tissues, reduced the expression of collagen type I, TGF-b, and
KL-6, increased hepatocyte growth factor (HGF) expression, and
inhibited airway epithelial cell apoptosis (185). Bleomycin-
induced lung fibrosis was decreased in MUC1-deficient mice,
and in vitro silencing of MUC1 expression diminished fibroblast
proliferation (186). In vitro cultures of lung fibroblasts and
epithelial cells treated with the antifibrotic drug, pirfenidone,
had reduced TGF-b-induced MUC1-CD phosphorylation at
Thr-42 and Tyr-46, and decreased nuclear translocation of the
MUC1-CD/SMAD3/b-catenin complex (187). MUC1-ED levels
were increased in the BALF of bleomycin-challenged mice, and
the NEU1-selective inhibitor, C9-BA-DANA, dose-dependently
inhibited bleomycin-induced increases in MUC1-ED levels (48).
Further, MUC1-ED in BALF of bleomycin-challenged mice was
desialylated, and C9-BA-DANA reduced bleomycin-provoked
MUC1-ED desialylation. Transgenic mice expressing human
MUC1 had increased serum KL-6 levels following bleomycin
challenge (122). Serum KL-6 levels were positively correlated
with BALF albumin levels indicating that increases in serum KL-
6 after bleomycin administration were associated with disruption
of the alveolar-capillary barrier. In an experimental model of
silica-induced pulmonary fibrosis using mice expressing the
human MUC1 transgene, BALF and serum levels of MUC1
were increased compared with saline controls (188).
Unexpectedly, however, MUC1 knockout mice had increased
lung collagen deposition and pulmonary inflammation
compared with wild type littermates. Collectively, these studies
have led us to propose a NEU1 –MUC1 axis as a central
component of the pathogenesis of pulmonary fibrotic diseases.
Several unanswered questions remaining concerning the roles
of KL-6/MUC1 and NEU1 in the pathogenesis of IPF/ILD. 1) Do
all secreted/extracellular forms of MUC1 that arise from different
proteolytic cleavages (elastase, TACE, MMP-14, g-secretase)
and/or alternative mRNA splicing (MUC1/SEC) contribute to
the appearance of KL-6? 2) Do genetic polymorphisms that
dictate the number of MUC1-ED tandem repeats influence the
development of IPF? 3) Are KL-6/MUC1 molecules in the serum
and BALF of IPF patients desialylated to a similar extent? 4) Do
pharmacologic inhibitors that target protease-mediated MUC1-
ED shedding inhibit IPF development? 5) And ultimately, what
is the molecular mechanism through which NEU1-induced, KL-
6/MUC1 desialylation contributes to the profibrotic,
proinflammatory phenotype that characterizes IPF?
Increased KL-6 levels in BALF of ILD patients likely reflects
the presence of high glycoprotein levels on the surface of
regenerating type II pneumocytes (168,189). Upregulation of
MUC1-ED sheddases in these cells during disease manifestation
remains an alternative possibility, and elevated levels of MMP-14
have been described in pulmonary fibrosis (190,191). High KL-6
levels in serum also may be causally related to increased
regenerating pneumocytes and/or greater alveolar capillary
permeability to proteins following disruption of the alveolar-
capillary barrier (124).
Mucins as Substrates for NEU2, NEU3,
and NEU4
NEU2 mRNA and sialidase activity were detected in the acellular
mucin-enriched capsule of the equine pre-implantation
conceptus (192). Recombinant human NEU2, expressed in
Escherichia coli and purified to homogeneity, exhibited
sialidase activity against bovine submaxillary gland mucin
(BSM) (193). Although the particular mucin expressed by the
bovine submaxillary gland has yet to be fully characterized,
cDNA sequence analysis shows it to be highly similar to
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MUC19 (194–196). A human NEU4 cDNA, expressed in
monkey COS-7 cells, desialylated BSM to a greater extent
compared with other NEU substrates, including 4-MU-NANA,
sialyllactose, and mixed bovine gangliosides (197). By contrast,
while recombinant NEU3 desialylated BSM to a greater extent
than 4-MU-NANA, BSM was an inferior NEU3 substrate
compared with sialyllactose and bovine gangliosides.
Recombinant cDNAs of both the short and long isoforms of
human NEU4 (NEU4-S and NEU4-L), expressed in COS-7 cells,
also exhibited in vitro sialidase activity against BSM (198).
NEU4-S expressed in human embryonic kidney 293T
(HEK293T) cells, but not NEU1, NEU2, or NEU3, desialylated
the sLe
a
mucin-expressing antigen (79). NEU4-S also exhibited
enzymatic activity against the sLe
x
antigen, while NEU3 was
moderately active and NEU1 and NEU2 were unreactive against
sLe
x
. Compared with untreated controls, treatment of human
THP-1 macrophages with the phytochemical, thymoquinone,
stimulated an increase in endogenous NEU4 activity that
exhibited sialidase activity against BSM (80,199). Therefore, in
addition to the established role of NEU1 in desialylating MUC1,
as discussed previously, other NEUs are capable of desialylating
other MUCs. Several such mucins—potential desialylation
targets of mammalian NEUs—have been suggested or
demonstrated to play a role in ILD. A specific single nucleotide
polymorphic (SNP) MUC2 gene variant was significantly
associated with IPF (200), and the expression levels of MUC2
protein were decreased in patients with IPF (201). Similarly, a
specific SNP variant of the MUC5AC gene was associated with
IPF (202). However, the expression of MUC5AC was reported to
be either increased (203) or decreased (201,204) in patients with
pulmonary fibrosis. In a mouse model, overexpression of
MUC5AC protected against inflammation and fibrosis through
several mechanisms (205). These apparent discrepant results
might be explained, in part, by differences in disease etiologies,
patient demographics, and/or lung sampling techniques between
the various studies (201,203,204). The expression levels of
MUC4 and MUC16 were elevated in patients with IPF, with
evidence for each MUC4 and MUC16 promoting the fibrotic
process in collaboration with TGF-b(206–209). Of particular
prominence is a strong association of a gain-of-function MUC5B
gene promoter variant (rs35705950) with pulmonary fibrosis
(210–215). To date, the MUC5B rs35705950 SNP remains the
strongest and most reproducible risk factor for development of
IPF. It should be expected that specific details of the interplay
between NEU isozymes and these MUCs might transpire in
the future.
NEUs AND MUCs IN INFECTIOUS
DISEASES
NEU1 and MUC1 in Pseudomonas
aeruginosa Infection
P. aeruginosa is a Gram-negative, motile, opportunistic pathogen
responsible for a wide range of human infections (216). In the
respiratory tract, P. aeruginosa contributes to the morbidity and
mortality of cystic fibrosis, chronic obstructive pulmonary
disease, and bronchiectasis. P. aeruginosa bacteria isolated
from the airway secretions of cystic fibrosis patients are tightly
bound to mucins (217–219). In in vitro cell adhesion assays, the
MUC1-ED bound to P. aeruginosa through bacterial flagellin,
the major structural protein of the flagellar filament (220,221).
Flagellin binding to the MUC1-ED stimulates two distinct
molecular events, Tyr phosphorylation of the MUC1-CD
leading to activation of the extracellular signal-regulated kinase
1/2 (ERK1/2) signal transduction pathway and NEU1-catalyzed
MUC1-ED shedding from the cell surface as a soluble decoy
receptor (40,49,51,222). MUC1-CD diminishes the host
inflammatory response to P. aeruginosa lung infection through
a mechanism involving EGFR-catalyzed Tyr phosphorylation of
the MUC1-CD and inhibition of TLR signaling (104,105,107,
112,223–229). As discussed above, EGFR and TLRs are
established NEU1 substrates, but it is unclear to what extent
NEU1-mediated desialylation of EGFR and/or TLRs impacts P.
aeruginosa-initiated, MUC1-CD-inhibited airway inflammation.
Overexpression of NEU1 in cultured human airway epithelial
cells increased P. aeruginosa adhesion to MUC1-expressing airway
epithelia,andpromotedMUC1-EDdesialylationandflagellin-
stimulated, MUC1-dependent ERK1/2 phosphorylation, while
NEU1 silencing had the opposite effects (40). To extend these
results to a physiologically relevant context, additional studies were
performed to assess whether P. aeruginosa flagellin, the MUC1
ligand, might regulate NEU1-mediated MUC1-ED desialylation
and/or P. aeruginosa adhesion to airway epithelial cells (51).
NEU1 overexpression in cultured human airway epithelial cells
increased MUC1-dependent adhesion of flagellin-expressing P.
aeruginosa to the cells, but not the adhesion of a flagellin-
deficient P. aeruginosa isogenic mutant (fliC¯). Treatment of these
cells with purified P. aeruginosa flagellin dose-dependently
increased bacterial adhesion, which was abolished by NEU1
silencing. In coimmunoprecipitation assays, P. aeruginosa flagellin
increased MUC1 association with both NEU1 and the NEU1
chaperone, PPCA, in a dose-dependent manner. Airway epithelial
cells treated with P. aeruginosa flagellin also dose-dependently
increased MUC1-ED desialylation, which was abrogated by NEU1
knockdown. Because sialic acid residues can mask protease
recognition sites (230), additional studies were conducted to
assess the role of NEU1 and P. aeruginosa flagellin in MUC1-ED
shedding from the cell surface (51). NEU1 overexpression and
flagellin stimulation dose-dependently increased shedding of
desialylated MUC1-ED into the supernatants of cultured airway
epithelial cells. Moreover, the desialylated MUC1-ED that was shed
in response to flagellin stimulation decreased P. aeruginosa
adhesion to airway epithelial cell-associated MUC1-ED. Thus, the
MUC1 ligand, P. aeruginosa flagellin, increased MUC1 association
with NEU1, leading to desialylation and shedding of the MUC1-ED
from the airway epithelial cellsurface,andtheshedMUC1-ED
competitively blocked P. aeruginosa adhesion to MUC1-ED
residing on the cell surface.
Using an intact, physiologically relevant, mouse model of P.
aeruginosa pneumonia, further studies were performed to assess
whether P. aeruginosa-derived flagellin might stimulate NEU1-
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dependent MUC1-ED desialylation and shedding (49). Mice
administered intranasally with viable bacteria or purified
flagellin exhibited both dose- and time-dependent increases in
shed MUC1-ED in their BALF. No increases in BALF levels of
MUC1-ED were evident in mice administered with the flagellin-
deficient P. aeruginosa fliC¯ mutant strain. P. aeruginosa lung
infection of mice also increased NEU1-MUC1 and PPCA-MUC1
association and MUC1-ED desialylation, and inhibition of NEU1
activity in vivo with the NEU1-selective inhibitor, C9-BA-DANA
(231), abrogated both of these effects. Shed, desialylated MUC1-
ED purified from the BALF of P. aeruginosa-infected mice
diminished both flagellin and bacterial binding to cultured
airway epithelial cells, and a similar effect was observed using a
nonglycosylated recombinant MUC1-ED protein expressed in E.
coli. Co-administration of the recombinant MUC1-ED protein
with P. aeruginosa to mice also decreased bacterial lung burden,
diminished BALF levels of keratinocyte-derived chemokine (KC)
and tumor necrosis factor-a(TNF-a), inhibited pulmonary
leukostasis, and increased 5-day mouse survival from 0% to 75%.
To extend these results to an in vivo human setting, studies
were conducted to determine whether shed MUC1-ED levels
might be elevated in BALF of patients with P. aeruginosa lung
infection, and whether the recombinant MUC1-ED protein might
influence in vitro parameters of the host proinflammatory
response to bacterial infection (232). BALF from patients with
P. aeruginosa pneumonia contained higher levels of MUC1-ED,
compared with either BALF from noninfected patients or patients
infected with microorganisms other than P. aeruginosa,andshed
MUC1-ED in the BALF of P. aeruginosa-infected patients was
desialylated, presumably from the action of NEU1. P. aeruginosa-
derived flagellin also was detected in the BALF of P. aeruginosa-
infected patients, and a positive correlation existed between BALF
levels of MUC1-ED and flagellin. Finally, E. coli-expressed
recombinant MUC1-ED dose-dependently decreased P.
aeruginosa motility and biofilm formation, diminished P.
aeruginosa-stimulated interleukin-8 (IL-8) production by airway
epithelial cells, and promoted neutrophil-mediated bacterial
phagocytosis. Taken together, these results indicate that not only
do BALF levels of shed MUC1-ED provide a diagnostic biomarker
of P. aeruginosa lung infection, but that NEU1-mediated
desialylation of the MUC1-ED generates a flagellin-targeting
releasable decoy that provides a protective component of the
proinflammatory response to P. aeruginosa at the human airway
epithelial surface (Figure 3).
In subsequent studies, coimmunoprecipitation and glutathione-
S-transferase (GST) pull down assays were performed to identify the
biochemical mechanism through which NEU1 associates with
MUC1 (233). Unexpectedly, NEU1 associated with the MUC1-
CD, but not with the sialic acid-containing MUC1-ED. Inhibition of
NEU1 enzymatic activity using C9-BA-DANA did not affect
NEU1-MUC1-CD association. The NEU1 binding site was
localized to the membrane-proximal 36 amino acids of the
MUC1-CD, while the MUC1-CD binding site was mapped to
NEU1 amino acids 1-139. NEU1-MUC1-CD association was
direct and did not require the NEU1 chaperone protein, PPCA.
NEU1-mediated MUC1-ED desialylation in response to
P. aeruginosa flagellin was equivalent in cells either expressing or
not expressing the MUC1-CD, indicating that NEU1-MUC1-CD
interaction was not required for MUC1-ED desialylation. Finally,
overexpression of NEU1 in airway epithelial cells reduced MUC1-
CD association with PI3K and diminished downstream Akt
phosphorylation. These results indicate that NEU1 associates with
the juxtamembrane region of the MUC1-CD to inhibit PI3K-
Akt signaling.
Several unanswered questions remain concerning the NEU1 –
MUC1 axis. 1) What is the molecular mechanism through which
engagement of P. aeruginosa flagellin by the MUC1-ED leads to
recruitment of NEU1? 2) Do other flagellated respiratory
pathogens other than P. aeruginosa activate the flagellin –
MUC1 –NEU1 axis? 3) Can the E. coli-expressed recombinant
MUC1-ED offer a clinically useful therapeutic intervention to
treat P. aeruginosa lung infections either alone or in combination
with anti-pseudomonal antibiotics? 4) Can measurement of
MUC1-ED and/or flagellin levels in BALF be developed into a
dependable and rapid diagnostic assay to identify patients with P.
aeruginosa lung infections without the need for bacterial culture
or genotyping techniques?
NEUs and MUCs in Other
Bacterial Infections
In mouse models of E. coli-induced sepsis or S. enterica serovar
Typhimurium-induced colitis, or in mice administered
intraperitoneally with E. coli lipopolysaccharide (LPS),
circulating levels of both NEU1 and NEU3 were increased
compared with saline controls (234). No increases in NEU1 or
NEU3 were observed in TLR4 knockout mice. Streptococcus
pneumoniae-induced murine sepsis also increased serum NEU1
and NEU3 levels, but these increases were similar in both wild type
and TLR4-deficient mice, suggesting an effect of the bacterial-
encoded neuraminidase, NanA. Mechanistic studies revealed that
increased NEU1/NEU3 levels promoted desialylation and
clearance of alkaline phosphatase by the Ashwell-Morell
receptor, thereby decreasing LPS dephosphorylation and
increasing TLR4-dependent host inflammatory responses. In
other studies, repeated gastric administration of S. enterica
Typhimurium or LPS increased intestinal sialidase activity and
NEU3 mRNA and protein levels in the gut in a TLR4-dependent
manner with a corresponding increase in alkaline phosphatase
desialylation and decrease phosphatase activity (235). Oral
administration of zanamivir (Relenza), an influenza virus NEU
inhibitor, blocked the bacterial-induced increased intestinal NEU
activity, maintained alkaline phosphatase sialylation, and
prevented disease. No increases in intestinal sialidase activity,
NEU expression, or alkaline phosphatase desialylation were seen
following gastric administration of S. enterica Typhimurium to
NEU3 knockout mice (236). NEU3-deficient, S. enterica
Typhimurium-infected mice also demonstrated decreases in LPS
phosphorylation, intestinal levels of proinflammatory cytokines,
and gut infiltration of CD3
+
Tcells,Gr1
+
neutrophils, and F4/80
+
monocyte/macrophages, and protected against the development of
bacterial colitis. The NEU inhibitor oseltamivir (Tamiflu),
originally developed against influenza virus NEU, but also active
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against NEU1 (41,237–239), reduced murine lung neutrophil
infiltration, IL-17 and TNF-alevels in BALF and serum, and
mortality in both experimental E. coli sepsis and the cecal ligation
puncture model of sepsis (240).
MUC1 serves as a cell surface receptor for Helicobacter pylori in
gastric epithelia (100,241–246). Here, the MUC1-ED is released as a
soluble decoy receptor to limit H. pylori infection (100,247,248),
but the role of NEU1 in H. pylori-stimulated MUC1-ED shedding is
unclear. MUC1 expression downregulates H. pylori-driven gastric
inflammation (244,249–256). MUC1 expression also influences a
variety of other human bacterial infections, including those by E.
coli,S. enterica Typhimurium, S. pneumoniae,Staphylococcus
aureus,Bacillus subtilis,Campylobacter jejuni,andHaemophilus
influenzae (257–267). Based on these studies and the documented
role of the NEU1 –MUC1 axis in regulating airway inflammation
in response to P. aeruginosa (40,49,51,232), it is likely that this
same pathway impacts the interaction of a broad array of
pathogenic microorganisms with the human host. In summary,
these collective studies suggest that NEUs and MUCs play
important roles in regulating bacterial infections.
NEUs and MUCs in Viral Infections
DNA methylation analysis of primary nasal epithelial cells from
children with or without asthma implicated NEU1 in the host
immune response to human rhinovirus-16 infection (268).
Compared with non-asthmatic controls, NEU1 mRNA levels
were decreased in asthma subjects with rhinovirus-16 infection,
and a positive correlation between NEU1 gene methylation and
NEU1 transcript levels was observed. The hepatitis B virus core
protein (HBc) increased NEU1 gene promoter activity through
NF-kB binding sites, leading to activation of ERK1/2 and NF-kB
signaling pathways, and increased HBc-mediated migration and
proliferation of hepatoma cells (269). Knockdown of NEU2
expression in monkey Vero or human HEp-2 cells increased
FIGURE 3 | Hypothetical model for Pseudomonas aeruginosa flagellin-induced, NEU1-mediated MUC1-ED desialylation and shedding. Step 1. P. aeruginosa
flagellin engages cell-associated MUC1-ED in the airway epithelium. Step 2. NEU1 is recruited to the MUC1-CD. Step 3. NEU1 desialylates the MUC1-ED. Step 4.
Desialylated, cell-associated MUC1-ED binds to P. aeruginosa through its flagella. Step 5. The P. aeruginosa-MUC1-ED complex is proteolytically released from the
cell surface. Step 6. The P. aeruginosa-MUC1-ED complex is removed from the lungs by the combined actions of neutrophil phagocytosis and mucociliary
clearance. Image of the pseudostratified columnar epithelium from Anatomy and Physiology, Chapter 4: The Tissue Level of Organization, Section 4.2: Epithelial
Tissue by OpenStax College, Rice University (https://openstax.org/details/books/anatomy-and-physiology) and used under Creative Commons Attribution 4.0
International (CC BY) license/Modified from the original.
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hepatitis A virus replication by up to 200-fold, however the
mechanism was not elucidated (270). Overexpression of mouse
NEU3 in COS-7 cells diminished Newcastle Disease Virus
(NDV) infection and propagation through cell-cell fusion
(271). Because reduced cell surface levels of GD1a also were
decreased upon NEU3 overexpression, this ganglioside was
suggested to constitute an NDV receptor on host cells. The
expression of NEU1, but not NEU3 or NEU4, was increased in
upper and lower respiratory tract cells, and in lung infiltrating
neutrophils, of SARS-CoV-2-infected patients (240).
Interestingly, two recent reports have documented increased
MUC1 protein levels in the airways of SARS-Cov-2-infected
patients compared with healthy controls (272,273). MUC1 also
has been shown to play a critical role in several other virus
infections, including those by respiratory syncytial virus,
adenovirus, influenza virus, and human immunodeficiency
virus (274–280). Future studies are needed to elucidate the role
of NEUs, if any, in regulating the MUC1-dependent responses to
virus infections. In conclusion, these investigations reveal that a
variety of human and animal virus infections are impacted by the
expression and activity of NEU1, NEU2, NEU3, and MUC1.
NEU1 in Parasite Infections
In vitro infection of mouse J774.A1 macrophage cells with the
obligate intracellular protozoan parasite, Leishmania donovani,
reduced cell surface expression of NEU1 compared with
uninfected controls (55). Parasite infection enhanced TLR4
sialylation and reduced TLR4-NEU1 and TLR4-MyD88
association. NEU1 overexpression in L. donovani-infected
murine macrophages increased TLR4 desialylation, TLR4-
NEU1 and TLR4-MyD88 association, activation of the JNK,
ERK1/2, p38, and NF-kB pathways, and expression of IL-12,
interferon-g(IFN-g), and inducible nitric oxide synthase (NOS)
proinflammatory mediators, while decreasing expression of IL-4,
IL-10, and TGF-band reducing parasite burden, all
compared with mock-transfected cells. In other studies, NEU1
overexpression in L. donovani-infected mouse macrophages
reduced TLR4 ubiquitination and TLR4-siglec-E and siglec-E-
SHP1 association, but increased IL-1b, IL-6, and TNF-alevels
compared with nontransfected cells (281).
NEUS IN INNATE AND ADAPTIVE
IMMUNITY
NEUs and TLRs
Toll-like receptors (TLRs) are cell surface sialoglycoproteins that
recognize broadly-distributed pathogen-encoded molecules to
initiate MyD88- and TRIF-dependent intracellular signaling
pathways that activate host innate immune responses (282). It
is now established that sialidases, principally NEU1, regulate
TLR-dependent innate immunity (Table 2).
TLR ligands, including LPS, poly(I:C), Mycobacterium
butyricum, and zymosan, induced sialidase activity in mouse
BMC-2 and primary bone marrow macrophages that was
associated with NF-kB activation and the production of nitric
oxide, IL-6, and TNF-a(53). In these same studies, NEU1, but
not NEU2, NEU3, or NEU4, colocalized with TLR2, TLR3, and
TLR4 on the cell surface of unstimulated mouse macrophages,
which increased following stimulation with zymosan, poly(I:C),
or LPS, respectively. NEU1 knockout mice had reduced
granulocyte-colony stimulating factor (G-CSF), IL-1Ra, IL-6,
KC, and macrophage inflammatory protein-2 (MIP-2)
production in response to LPS administration compared with
NEU1-expressing mice. In other studies, NEU1- and NEU4-
deficient macrophages had reduced LPS-induced TLR4-MyD88
complex formation and NF-kB activation compared with wild
type littermates (57,80,199). Following LPS stimulation of
mouse dendritic cells (DCs), NEU1 translocated from the
lysosome to the cell surface where it associated with and
desialylated TLR4, and NEU1 silencing diminished LPS-
stimulated TNF-aproduction (70).Inthesesamestudies,
NEU1 deficiency in murine hematopoietic cells or in vivo
treatment with the sialidase pharmacologic inhibitor, 2-deoxy-
2,3-dehydro-N-glycolylneuraminic acid (Neu5Gc2en), protected
mice against lethal LPS challenge. NEU1 silencing in mouse 3T3-
L1 adipocytes increased TLR4 sialylation and decreased LPS-
stimulated NF-kB nuclear translocation and IL-6 and monocyte
chemoattractant protein-1 (MCP-1) production (54). NEU1
pharmacologic inhibition with oseltamivir decreased LPS-
stimulated IL-6, granulocyte-macrophage colony-stimulating
factor (GM-CSF), and MIP-1aproduction in mouse primary
renal mesangial cells through a TLR4 !p38/ERK pathway
(283). Treatment of mouse RAW264.7 macrophages with LPS
decreased NEU1 levels on the cell surface, and NEU1 silencing
promoted LPS-induced tolerance, while NEU1 overexpression
had the opposite effect (73). Finally, treatment of RAW264.7 cells
with TLR7 or TLR9 agonists promoted TLR7-NEU1 and TLR9-
NEU1 association, NF-kB activation, and TNF-aand MCP-1
production, all of which were reversed by NEU1 silencing or
pharmacologic inhibition with oseltamivir (44).
In human studies, NEU4 was identified as the strongest locus
associated with TLR4 stimulationinageneticanalysiscomparing
LPS-treated primary monocytes with untreated cells (299).
Overexpression of NEU1, but not NEU3, in HEK293T cells dose-
dependently increased LPS-stimulated, TLR4-mediated NF-kB
activation and treatment with the broad spectrum sialidase
inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid
(Neu5Ac2en or 2-deoxy-NANA) reduced NF-kB activation (58).
Mechanistically, it was proposed that NEU1-mediated desialylation
of TLR4 unmasked TLR dimerization sites to promote receptor
activation and initiation of the host proinflammatory response (53,
57). Additional studies revealed that NEU1, MMP-9, and G protein-
coupled receptors (GPCRs) formed a multiprotein complex on the
cell surface wherein GPCR agonists activated GPCR signaling to
stimulate MMP-9-dependent NEU1 activation through removal of
the elastin-binding protein, thereby allowing for TLR desialylation
and initiation of host inflammation (44,45,47). Further
investigations revealed NEU1 –MMP-9 cross-talk in combination
withtheGPCRagonist,neuromedinB,asnecessaryforEGFR
activation and downstream intracellular signaling (41). LPS
activation of mouse microglial BV-2 cells and rat primary
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microglia increased cell surface sialidase activity and desialylation of
the cell surface (300,301), which was abolished by NEU1
knockdown (56). NEU1 knockdown in microglia also reduced
LPS-stimulated IL-6 and MCP-1 expression, while NEU1
overexpression had the opposite effects (56). Using a cell surface
biotinylation assay, NEU1 and TLR4 were localized in close
proximity (within 100 nm) on the cell surface of LPS-activated
BV-2 cells, and LPS treatment of the cells increased TLR4
desialylation. Collectively, studies in both human and mouse
experimental systems indicate that NEU1, and to a lesser extent
NEU4, regulate TLR-driven intracellular signaling and host
innate immunity.
NEUs and PMNs
Polymorphonuclear leukocytes (PMNs) are blood-borne leukocytes
that play a critical role in the host innate immune response to
infectious pathogens through microbial phagocytosis, release of
soluble anti-microbial compounds, and generation of neutrophil
extracellular traps (NETs) (302). Sialidase activity was identified in
the primary and secondary granules of human PMNs as well as on
TABLE 2 | NEU-associated cells, signaling pathways, and cytokine responses for selected immune/inflammatory processes.
Immune/Inflammatory
Process
NEU
Isozyme
Cells Signaling Pathways Cytokine Responses References
TLR2, TLR3 activation NEU1 Macrophages NF-kBND(53)
TLR4 activation NEU1 Macrophages NF-kB↑IL-6, TNF-a(53,57)
TLR4 activation NEU1 Adipocytes NF-kB↑IL-6, MCP-1 (54)
TLR4 activation NEU1 Kidney mesangial cells ERK, p38 ↑IL-6, GM-CSF, MIP-1a(283)
TLR4 activation NEU1 HEK293T cells NF-kBND(58)
TLR4 activation NEU1 Microglia ND ↑IL-6, MCP-1 (56)
TLR4 activation NEU3 Dendritic cells ND ↑IL-6, IL-12, TNF-a(66)
TLR4 activation NEU4 Macrophages NF-kB↑IL-1b, IL-6, TNF-a, IFN-g(80,199)
TLR7, TLR9 activation NEU1 Macrophages NF-kB↑TNF-a, MCP-1 (44)
PMA activation NEU1 Macrophages ND ↑IL-1b, IL-6, TNF-a(64)
Staph. aureus,E. coli infection NEU1 Macrophages FcgR, Syk kinase ND (31)
Leishmania donovani infection NEU1 Macrophages JNK, ERK, p38, NF-kB↑IFN-g, IL-12 ↓IL-4, IL-10,
TGF-b
(55)
Salmonella enterica Typhimurium
infection
NEU3 T cells, Neutrophils,
Macrophages
ND ↑IL-1b, TNF-a, IFN-g, IL-10,
TGF-b
(236)
Pseudomonas aeruginosa
infection
NEU1 Airway epithelial cells PI3K, Akt ND (233)
Hepatitis B virus infection NEU1 Hepatoma cells ERK, NF-kBND (269)
Concanavalin A activation NEU1 T cells ND ND (284,285)
Anti-CD3 antibody activation NEU1 CD4
+
T cells ND ↑IL-2, IL-4 (286)
Anti-CD3/CD28 antibody
activation
NEU1,
NEU3
CD4
+
T cells ND ND (287)
Anti-CD3/CD28 antibody
activation
NEU1 CD4
+
T cells ND ↑IL-2, IL-4, IL-13, IFN-g(288)
Anti-CD3/CD28 antibody
activation
NEU3 CD4
+
T cells ND ↑IL-2, IL-13, IFN-g(288)
Anti-CD3/CD28 antibody
activation
NEU1 T cells ND ↑IFN-g(65)
Dermatophagoides farinae
activation
NEU1 CD4
+
T cells ND ↑IL-4, IL-5, IL-13 (30)
Ovalbumin activation NEU3 FoxP3
+
T cells ND ND (289)
Obesity-induced insulin
resistance
NEU1 FoxP3
+
, Th17
+
T cells ND ND (290)
Systemic lupus erythematosus NEU1 Kidney mesangial cells ND ↑IL-6, MCP-1 (291)
Rheumatoid arthritis NEU2,
NEU3
CD19
+
B cells, CD138
+
plasma cells
ND ND (292)
Type 1 diabetes NEU1 Liver, Muscle cells Insulin receptor kinase, Akt ND (39)
Type 1 diabetes NEU1 Liver cells p38, Akt ND (38)
Type 1 diabetes NEU1 Hepatoma cells Insulin receptor, Insulin receptor
substrate-1
ND (293)
Type 1 diabetes NEU3 Adipocytes, Muscle cells Insulin receptor, Insulin receptor
substrate-1, Grb-2
ND (294)
Type 1 diabetes NEU3 Liver cells Insulin receptor substrate-1, PPARgND (295)
Type 1 diabetes NEU3 Adipocytes Akt ND (296)
Diabetic cardiomyopathy NEU1 Cardiomyocytes AMPK, SIRT3 ND (131)
Inflammatory bowel disease NEU3 Intestinal mucosa ND ND (297)
Inflammatory bowel disease NEU1 Peripheral blood
mononuclear cells
ND ND (298)
ND, Not determined.
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the plasma membrane, and PMN activation induced translocation
of the enzymatically-active sialidase from the granules to the cell
surface (303). In addition, PMN homotypic aggregation and
adhesion to nylon or plastic surfaces was inhibited by 2-deoxy-
NANA. In other studies, a rabbit antibody prepared against the
Clostridium perfringens NEU cross-reacted with a sialidase
expressed on the surface of IL-8-stimulated human PMNs, but
not on unstimulated cells, and with sialidase proteins in PMN
lysates and granule preparations (304). This same antibody
inhibited human PMN sialidase activity in vitro and diminished
both pulmonary leukostasis in mice administered with cobra venom
factor and intrapulmonary transendothelial migration of PMNs into
the bronchoalveolar compartment of IL8-administered mice. In
subsequent experiments, the sialidase antibody was shown to
recognize recombinant human NEU3, but not NEU1, and
inhibited NEU3 sialidase activity against the synthetic fluorogenic
substrate, 4-MU-NANA (305).Inotherstudies,NEU1,NEU2,and
NEU3 mRNAs were detected in unstimulated murine PMNs and
IL-1bstimulation of human PMNs increased NEU1 and NEU2, but
not NEU3, expression (288). LPS-stimulated human PMNs had
decreased amounts of sialic acid on their surface that was inhibited
by oseltamivir and zanamivir (240). Both NEU1 inhibitors also
reduced PMN-mediated phagocytosis and killing of E. coli,LPS-
driven PMN activation, phorbol 12-myristate 13-acetate (PMA)-
stimulated reactive oxygen species production, and LPS-mediated
NET formation.
Utilizing the NEU3 cross-reactive antibody, Sakarya et al.
(306) reported reduced, antibody-dependent PMN adhesion to
pulmonary vascular endothelial cell monolayers in vitro, which
was associated with PMN activation. Preincubation of
endothelial cell monolayers with activated PMNs rendered the
cells hyperadhesive for freshly added PMNs via PMN sialidase-
dependent autodesialylation. Desialylation of both the CD11b
and CD18 protein subunits of b2-integrin, through mobilization
of an endogenous PMN sialidase, unmasked activation epitopes
on both proteins and increased binding to intracellular adhesion
molecule-1 (ICAM-1) (21). NEU1 was expressed on the surface
of PMNs and colocalized with CD18, the latter being increased
following PMN activation. Desialylation of immobilized ICAM-1
increased leukocyte arrest in vivo and treatment with 2-deoxy-
NANA diminished LPS-stimulated leukocyte adhesion to
ICAM-1. Taken together, these results indicate that NEU1,
NEU2, and NEU3 regulate the activities of PMNs.
NEUs and Monocytes, Macrophages, and
Dendritic Cells
Macrophages isolated from NEU1 knockout mice exhibited
reduced phagocytosis of Gram positive (S. aureus) and Gram
negative (E. coli) bacteria and phosphorylation of FcgR and Syk
kinase, and treatment of cultured NEU1-deficient macrophages
with purified mouse NEU1 restored their phagocytic activity (31).
NEU1-knockout macrophages displayed increased lysosomal
exocytosis as a consequence of reduced NEU1-mediated
desialylation of lysosomal-associated membrane protein-1
(LAMP-1) (34). High levels of NEU1 mRNA were detected in
unstimulated human monocytes, while NEU2 and NEU3 were
expressed at much lower levels (288). In vitro stimulation of
human monocytes with GM-CSF to promote differentiation into
macrophages temporally increased sialidase activity for the 4-MU-
NANA substrate up to 14-fold after 7 days in culture (307).
Correspondingly, NEU1 and NEU3, but not NEU4, mRNAs
and proteins were increased following monocyte-to-macrophage
differentiation. Following PMA-stimulated differentiation of
human primary monocytes and the THP-1 monocyte cell line to
macrophages, NEU1, but not NEU3, protein levels increased and
NEU1 translocated from the lysosomes to the plasma membrane
(64). NEU1 silencing reduced E. coli phagocytosis by PMA-
induced macrophages and diminished their production of IL-1b,
IL-6, and TNF-a. Differentiation of human monocytes into DCs
following in vitro culture in the presence of GM-CSF and IL-4
increased sialidase activity and elevated the mRNA and protein
levels of NEU1 and NEU3, but not NEU4 (66). Further,
monocyte-to-DC differentiation in the presence of zanamivir or
2-deoxy-NANA to inhibit NEU1 and NEU3 activities
decreased LPS-stimulated IL-6, IL-12, and TNF-aproduction.
Overexpression of NEU1 in THP-1 macrophages increased IL-1b
and TNF-aproduction while NEU1 silencing had the opposite
effect (308). In these same studies, NEU1 silencing in human
primary M1 macrophages suppressed IL-1band TNF-a
production. PMA-induced differentiation of THP-1 cells to M0
macrophages increased intracellular sialidase activity and
diminished sialoglycoconjugate levels on the cell surface (308).
However, further polarization of M0 macrophages into M1 or M2
macrophages did not affect the amount of sialic acid on the
cell surface.
NEU1 expression was increased in human circulating
monocytes isolated from patients with myocardial infarction
compared with healthy controls, and high NEU1 levels were
observed in macrophages isolated from atherosclerotic plaques
(308). In an animal model of atherosclerosis, NEU1-deficient
mice exhibited reduced monocyte and lymphocyte infiltration
into atherosclerotic lesions compared with NEU1-sufficient mice
(309). Reductions in serum and liver nonhigh-density
lipoprotein (nonHDL) cholesterol levels, atherosclerotic lesion
size, and lesion macrophage numbers were observed in
apolipoprotein E (ApoE) knockout mice expressing
hypomorphic levels of NEU1 (NEU
hypo
)comparedwith
ApoE
-/-
littermates (310). NEU1
hypo
ApoE knockout mice also
had reduced circulating PMNs, but increased numbers of M2
macrophages, compared with ApoE knockout mice expressing
normal NEU1 levels. Finally, treatment of ApoE
-/-
mice with the
general sialidase inhibitor, 2-deoxy-NANA, reduced liver
nonHDL cholesterol levels and atherosclerotic lesion size,
compared with saline controls. In another study, recombinant
human NEU1 and NEU3 desialylated apolipoprotein B 100 in
human low-density lipoprotein (LDL) in vitro, and ApoE-NEU1
and ApoE-NEU3 double knockout mice, or ApoE knockout mice
treated with pharmacologic inhibitors of NEU1 or NEU3,
exhibited reduced atherosclerosis compared with ApoE single
knockout mice or ApoE null mice treated with vehicle control
(18). In a mouse model of ischemia/reperfusion (I/R) cardiac
injury, elevated sialidase activity, and NEU1, PPCA, and b-
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galactosidase mRNA and protein levels, were seen in the heart
compared with sham controls (311). These authors also reported
that NEU
hypo
mice subjected to I/R injury had diminished
proinflammatory and increased anti-inflammatory macrophage
numbers compared with controls. Further, cardiomyocyte-
specific NEU1 overexpression in vivo was associated with
cardiac hypertrophy, but no changes in cardiac inflammation,
following I/R injury compared with wild type littermates.
Binding of pro-atherogenic elastin-derived peptides (EDP) to
the elastin receptor complex (ERC), a heterotrimeric association
of NEU1, PPCA, and the elastin binding protein, a splice variant
of b-galactosidase (312), led to NEU1-mediated desialylation of
the CD36 scavenger receptor and augmented uptake of oxidized
LDL by human macrophages in vitro (22). In a subsequent
report, EDP binding to the ERC also promoted NEU1-b2-
integrin association and NEU1-driven b2-integrin desialylation
in human monocytes, as well as NEU1-ICAM-1 association and
NEU1-mediated ICAM-1 desialylation in human umbilical vein
endothelial cells (23). As a result, EDP binding to the ERC
promoted monocyte adhesion to and migration across
endothelial cell monolayers through NEU1. In conclusion,
these studies strongly suggest that NEU1 regulates monocyte,
macrophage, and DC differentiation and function (Table 2).
NEUs and Lymphocytes
Landolfiand coworkers (284) reported that mouse T
lymphocytes possess an endogenous sialidase activity that
increased following in vitro stimulation with the T cell
mitogen, concanavalin A, and sialidase activity was controlled
by the NEU1 locus in the murine major histocompatibility
complex. Mouse strains carrying the H-2
v
haplotype (SM/J,
B10.SM) exhibited reduced sialidase activity compared with
other haplotypes. Previously, the SM/J mouse strain had been
reported to possess deficient sialidase activity (313). Subsequent
analysis revealed that a point mutation in the NEU1 gene,
resulting in a Leu-to-Ile substitution, was responsible for the
deficient sialidase activity in the SM/J stain (314). Increased
sialidase activity following T cell activation was associated with
desialylation of cell surface glycoproteins (285). Mouse T cell
activation through a mixed lymphocyte reaction (MLR) with
allogeneic B cells also resulted in increased sialidase activity and
desialylation of T cell surface glycoproteins (315). Wipfler et al.
(316) reported that total sialidase activity was greatest in
unstimulated human peripheral blood lymphocytes (PBLs),
intermediate in thymic T cells, and lowest in tonsillar T and B
cells. At the mRNA level, NEU1 was expressed at approximately
2.5-fold greater levels in PBLs compared with thymocytes and
tonsillar T and B cells, while NEU3 was expressed at up to 125-
fold greater levels in thymic T cells compared with PBLs and
tonsillar T and B cells. In a different study, NEU1 and NEU3
transcripts were identified in murine thymic T cells (20). Crude
membrane fractions of mouse thymocytes had sialidase activity
that was inhibited by 2-deoxy-NANA and the NEU1-specific
inhibitor, C9-BA-DANA. CD5 was identified as a substrate for
thymocyte cell surface NEU1 by peanut agglutinin (PNA) lectin
blotting and anti-CD5 antibody immunoprecipitation studies.
A subpopulation of murine thymic lymphocytes was identified
that co-expressed surface IgG, b2-integrin, and NEU1 (Neu-
medullocytes) (317,318). Real-time PCR studies revealed that
NEU2 was the major sialidase expressed in Neu-medullocytes
with NEU1 detected at low levels (319). Of five NEU2 mRNAs
arising as a result of alternative mRNA splicing (variants A, B, C,
D, N), only variant B was identified in murine thymocytes (320).
Up to 40% of NEU2 variant B sialidase activity was found in the
membrane fraction of transfected COS-7 cells. An analysis of
mouse thymic lysates prepared with different detergents
suggested that NEU2 was a peripheral membrane protein,
rather than a transmembrane or GPI‐anchored protein, with
enzymatic activity at pH 7.0 but not pH 4.5 (78). It should be
noted that other studies have established NEU2 as a cytosolic
enzyme with a pH optimum of 6.0-6.5 and an amino acid
sequence lacking protein domains typical of transmembrane or
GPI-anchored proteins (12,84,85).
The levels of IL-2 and a T helper 2 (Th2) cytokine, IL-4, were
increased following in vitro anti-CD3 antibody stimulation of
murine spleen cells, and reduced levels of IL-4, but not IL-2 or
IFN-g, were seen upon anti-CD3 antibody activation of SM/J
splenocytes (286). Addition of exogenous IL-4 during CD4
+
Tcell
activation enhanced NEU1 sialidase activity and cell surface levels of
asialoGM1 ganglioside, while activation of T cells in the presence of
2-deoxy-NANA diminished IL-4 production. In other studies,
treatment of IL-4-primed CD4
+
T cells with the GM3 ganglioside
diminished IL-4, IL-5, and IL-13 production, but had no effect on
IL-2 or IFN-g, through a mechanism involving mobilization of
intracellular calcium (321). Further, anti-CD3 antibody stimulation
of NEU1-deficient B10.SM strain CD4
+
T cells reduced intracellular
calcium levels compared with unstimulated cells. A COOH-
terminal tetrapeptide, Tyr-Gly-Thr-Leu, was suggested to
constitute an internalization signal in the NEU1 protein that
targets it to the lysosome in human lymphocytes, fibroblasts, and
COS-7 cells (62). Upon Tyr phosphorylation of the tetrapeptide in
activated lymphocytes, NEU1 sialidase activity was unchanged, but
the sialidase was redistributed from the lysosome to the cell surface.
NEU1 and NEU3 enzyme activities and mRNA levels were
increased in human CD4
+
T cells activated with anti-CD3 and
anti-CD28 antibodies (288). NEU3 was the major sialidase
expressed in the activated T cells. Overexpression of NEU1 in
human Jurkat T cells increased IL-2, IL-4, IL-13, and IFN-g
expression following CD3/CD28 costimulation, while these same
cytokines, with the exception of IL-4, were produced in NEU3-
overexpressing cells. CD3/CD28 costimulation of human primary T
cells increased IFN-gproduction, which was inhibited by zanamivir
and 2-deoxy-NANA (65). CD3/CD28 costimulation also increased
NEU1 mRNA levels and sialidase activity with minimal increase in
NEU3 activity, heightened NEU1 and PPCA cell surface expression,
and increased the levels of desialylated cell surface glycoconjugates,
all compared with unstimulated cells. While these studies suggested
that NEU1, and possibly NEU3, might be linked to Th2 immunity,
they also highlight the apparent contradiction to the studies
reviewed above implicating NEU1 in the Th1 response (65,66,308).
Altered expression of surface sialoglycoconjugates is a
characteristic feature of apoptosis (287). Treatment of Jurkat
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T cells with the proapoptotic drug, etoposide, increased sialidase
activity and desialylated cell surface glycoconjugates during the
early phase of apoptosis, which was reversed by 2-deoxy-NANA
(322). In subsequent studies, etoposide-induced apoptosis was
associated with increased cell surface GM3, reduced GD3, and
increased NEU3, but not NEU1, mRNA levels (323). Changes in
GM3, GD3, and NEU3 levels were blocked by caspase inhibition.
Phytohemagglutinin/IL-2-induced human T cell blasts
undergoing UV- or staurosporine-induced apoptosis exhibited
decreased surface staining for terminal a2,3- and a2,6-linked
sialic acids (324). Desialylation of surface glycoconjugates also
was observed on microparticles derived from apoptotic
lymphoblast membranes. Plasma membrane-derived apoptotic
microparticles had increased sialidase activity on their surface
(325). Treatment of viable Jurkat cell lysates with caspase-3
increased sialidase activity to the same degree as that of
apoptotic cell lysates, consistent with a predicted caspase-3
proteolytic cleavage site at Asp-135 in the NEU1 protein.
Further, the caspase inhibitor, zVAD, decreased sialidase
activity in etoposide-induced apoptotic Jurkat cells. Not only
was cell surface NEU1 responsible for desialylating Jurkat T cell
surface glycoconjugates in a cis-acting manner, but also in trans
on the surface of co-incubated human erythrocytes (67).
In a mite allergen model of allergic asthma, in vitro exposure
of Dermatophagoides farinae (Derf)-sensitized CD4
+
T cells
to the antigen increased desialylation of cell surface
glycoconjugates, augmented binding of hyaluronic acid (HA)
to its cognate receptor, CD44, and heightened NEU1, but not
NEU2 or NEU3, transcript levels (30). In NEU1-deficient cells
from SM/J mice, CD44 receptor activity for HA was reduced, as
were reductions in BALF eosinophil and Th2 cell numbers, Th2
cytokine levels, and airway hyperresponsiveness following Derf
challenge, compared with cells expressing normal NEU1 levels.
Subsequent studies using in vitro-differentiated ovalbumin-
specific CD4
+
T cells revealed that NEU1 and NEU3 mRNA
levels were greater in Th2, compared with Th1, cells (326).
Collectively, these studies indicated that NEU1 is required for
HA-CD44 interaction and development of acute asthmatic
inflammation, and suggested that NEU1-mediated desialylation
of CD44 on CD4
+
T cells alters the ability of CD44 to interact
with HA (28). In subsequent studies, analysis of mouse CD4
+
T
cell subsets revealed that while NEU1 mRNA was expressed at
approximately equal amounts in Th1, Th2, Th17, and induced
regulatory T (iTreg) cells, NEU3 transcript levels were greater in
iTreg cells compared with the other cell subsets (289). CD4
+
activated Treg (FoxP3
+
CD62L
−
) spleen cells expressed higher
levels of NEU3 transcripts compared with resting Treg
(FoxP3
+
CD62L
+
)andFoxP3
−
cells, and overexpression of
NEU3 in naïve CD4
+
T cells upregulated FoxP3 expression. In
a mouse model of obesity-induced insulin resistance, the
percentages of CD4
+
FoxP3
+
Treg cells and NEU1 levels were
decreased, while the percentages of CD4
+
Th17
+
cells and the
levels of the NEU1-targeting microRNA, miR-23b-3p, were
increased, all compared with nonobese controls (290).
Administration of the anti-obesity phytochemical, acacetin,
reversed these effects, while administration miR-23b-3p
exacerbated the outcomes. Co-administration of acacetin and
miR-23b-3p restored the obesity-related downregulated levels of
NEU1, while NEU1 overexpression offset the effects of miR-23b-
3p on the Treg/Th17 cell ratio. The authors concluded that
acacetin reverses insulin resistance in obese model mice by
regulating the Treg/Th17 cell balance through a miR-23b-3p –
NEU1axis.Finally,inhumanstudies,differentialgene
expression analysis identified 7 genes related to the lysosomal
pathway, including NEU1, that were downregulated in the
airways of asthma patients compared with healthy controls,
suggesting that lymphocyte lysosomal dysfunction might be
involved in the pathogenesis of allergic asthma (327).
Vitamin D is a potent immunomodulator (328).Vitamin D-
binding protein (DBP), also referred to as the group-specific
component (Gc) protein in humans, is the main transporter of
vitamin D in the circulation. Part of the immune-modulating
effects of vitamin D are mediated through the conversion of Gc/
DBP to macrophage activating factor (MAF) (329). In humans,
the Gc protein contains a mucin-type O-linked trisaccharide
composed of Thr-linked GalNAc with dibranched sialic acid and
Gal termini, while MAF contains only the Thr-linked
monosaccharide GalNAc (330). A two-step sequential action of
b-galactosidase on the surface of B cells followed by surface-
expressed NEU1 on T cells was identified as being responsible for
the conversion of Gc to MAF (331–333). In mice, DBP is not
sialylated and only the action of b-galactosidase on the Gal-
GalNAc disaccharide was required to deglycosylate DBP to
MAF. Taken together, these results indicate that NEU1, and to
a lesser extent NEU3, play in important role in regulation of
adaptive immunity through their effects on T and B
lymphocytes (Table 2).
NEUs AND MUCs IN AUTOIMMUNE
DISEASES
Autoimmune diseases comprise a diverse collection of
pathological conditions originating from a dysregulated
immune response to a self tissue or organ. As discussed above,
NEUs play a critical role in the activity of Treg cells (289,290)
suggesting the possible involvement of NEUs in the pathogenesis
of autoimmunity. Further, MUC1 has been suggested as a
checkpoint inhibitor on T cells (334), implicating MUC1, and
possibly other membrane-bound mucins, in the development of
autoimmune diseases. This section reviews the role of NEUs and
MUCs in the context of four selected autoimmune diseases,
systemic lupus erythematosus (SLE), rheumatoid arthritis
(RA), type 1 diabetes (T1D), and inflammatory bowel
disease (IBD).
Systemic Lupus Erythematosus
SLE is an autoimmune disease typically characterized by the
presence of anti-nuclear antibodies (ANA) and anti-extractable
nuclear antigen (anti-ENA) antibodies with immune complex
deposition in multiple organs, particularly the kidneys (335). A
growing body of evidence suggests a role for NEU1 in the
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pathogenesis of lupus nephritis (283,336–338). More
specifically, NEU1 expression and catalytic activity were
increased in the kidneys of MRL/lpr lupus mice (339), and
kidney mesangial cells from NEU1-haplodeficent mice had
reduced cytokine expression and secretion in response to LPS
or lupus serum (LS) compared with NEU1-expressing mice
(340). In other studies, overexpression of NEU1 in cultured
mouse MES13 kidney mesangial cells dose-dependently
increased spontaneous IL-6 and MCP-1 production and
treatment of primary mesangial cells from MRL/lpr lupus mice
with heat-aggregated IgG (HA-IgG), a mimic of immune
complex deposition, increased NEU1 expression (291).
Moreover, pharmacologic inhibition of NEU1 activity with
oseltamivir reduced HA-IgG- or LS-stimulated IL-6 and
MCP-1 production. In human studies, proteomic analysis of
renal tissues comparing patients with proliferative lupus
nephritis vs. healthy controls revealed NEU1 as the greatest
up-regulated protein of 48 identified proteins (341). NEU1
immunohistochemical staining and urinary excretion were
significantly elevated in lupus nephritis patients compared with
controls. Finally, flow cytometric analysis of human B cells from
SLE patients revealed a positive correlation between the cell
surface ratio of sialyltransferase ST3Gal-1 and NEU3 with
disease severity (342).
SLE patients with serositis had greater MUC16 levels and
disease duration compared with SLE patients without serositis
(343). Pseudo–pseudo Meigs’syndrome is a rare manifestation
in SLE patients presenting with ascites, pleural effusion, and
elevated serum MUC16 levels unrelated to malignancy (344–
346). MUC20 transcript levels were increased with the
progression of lupus in the kidneys of MRL/lpr lupus-prone
mice (347). Interestingly, mouse NEU1 (348) and mouse and
human MUC20 (347) are highly expressed in kidneys compared
with other organs, but it is unknown whether MUC20 might be a
NEU1 substrate.
Rheumatoid Arthritis
RA is an autoimmune disease of unknown etiology characterized
by synovitis and bone erosion (349). A positive correlation
between B cell surface ST3Gal-1/NEU3 ratio and RA disease
severity was seen in patients with mild or high disease activity,
but not patients in non-remission (342). In a follow-up study, B
cell surface NEU3 levels were found to positively correlate with
RA disease activity (350). NEU1 catalytic activity was increased
in the blood and liver of rats with Freund’s adjuvant-induced RA
(351). In a mouse model of bovine collagen type II-induced RA,
pharmacologic inhibition of NEU2 and NEU3 activity with
zanamivir dose-dependently diminished CD19
+
B cells and
CD138
+
/TACI
+
plasma cells numbers, anti-collagen type II
autoantibody levels, disease activity, and total sialidase activity
in arthritic joints (292). Corresponding, increased sialylation of
circulating IgG and a2,3-linked surface sialylation of CD138
+
/
TACI
+
plasma cells was noted.
MUC1 protein levels were elevated on T cells isolated from
the joint fluid of a patient with RA (352). The combination of
lung ultrasound and serum MUC1 levels, in addition to current
markers, was proposed for screening and following patients with
ILD associated with RA (353). A greater number of RA patients
expressed MUC3 protein in synovial lining cells and synovial
macrophages compared with normal controls (354). The
MUC5B gene promoter variant rs5705950 was linked to an
increased susceptibility to pulmonary fibrosis in RA patients
(213,215,355). Increased salivary MUC7 sulfation was detected
in RA patients compared with healthy controls (356). Synovial
fluid cells from RA patients contain a distinct population of
MUC18-positive T cells (357), and these cells were identified as a
biomarker for synovial membrane angiogenesis in RA
patients (358).
Type 1 Diabetes
T1D is an autoreactive disease arising from immune destruction of
insulin-producing pancreatic bcells (359). Both NEU1 and NEU3
have been implicated in the pathogenesis of T1D. NEU1 transcript
levels and catalytic activity were diminished in two strains of
diabetes-prone mice compared with nondiabetic littermates (360).
Treatment of cultured rat L6 myoblasts with purified mouse
NEU1 promoted desialylation of both the insulin receptor (IR)
and insulin-like growth factor-1 receptor (IGF-1R) compared with
untreated controls (36). Mice deficient in NEU1 activity and fed a
high fat diet (HFD) developed glucose intolerance and insulin
resistance compared with mice expressing normal levels of NEU1,
and HEK293 cells overexpressing both NEU1 and the insulin
receptor kinase (IRK) exhibited increased IRK-NEU1 association
and NEU1-mediated IRK desialylation (39). Liver and muscle cells
from NEU1-deficient, HFD-fed mice had decreased IR
phosphorylation and downstream Akt activation, and treatment
of HEK293 cells overexpressing IR and NEU1 promoted IR-NEU1
association and NEU1-mediated IR desialylation. Administration
of EDPs to normal mice expressing the NEU1-containing ERC
increased IR-NEU1 association and NEU1-mediated IR
desialylation in muscle tissues, both of which were reversed by
2-deoxy-NANA (37). Murine hepatocytes treated with EDPs in
the context of nonalcoholic steatohepatitis linked to insulin
resistance had decreased hepatic growth factor receptor (HGFR)
phosphorylation as a consequence of NEU1-mediated HGFR
desialylation (43). Overexpression of NEU1 in HEK293 cells
increased IR desialylation and dimerization, and NEU1
overexpression in human HepG2 liver cells reversed palmitate-
induced insulin resistance (38).Inthesesamestudies,
pharmacologic upregulation of NEU1 expression and activity
with ambroxol in mice led to desialylation of the IR, increased
IR Tyr phosphorylation and Akt signaling, and improved glucose
tolerance and insulin resistance in mice exposed to a HFD. NEU1
expression was increased in the spleen, liver, and kidneys of
streptozotocin (STZ)-treated diabetic rats (361). Similarly, NEU1
protein levels were increased in cardiomyocytes in the STZ mouse
model of diabetic cardiomyopathy, and NEU1 silencing protected
against STZ-induced cardiomyopathy and myocardial fibrosis,
inflammation, and apoptosis through an AMPK-SIRT3 pathway
(131). Inhibition of NEU1 activity with anti-NEU1 antibody or
oseltamivir reduced insulin-stimulated IGF-1R and insulin
receptor substrate-1 (IRS-1) phosphorylation in human
fibroblasts, and promoted IR-NEU1 association in rat HTC
hepatoma cells (69). Further, pharmacologic inhibition of
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MMP-9 in HTC cells dose-dependently reduced insulin-
stimulated NEU1 sialidase activity, and treatment of NEU1-
deficient human fibroblasts with olanzapine, an antipsychotic
drug associated with insulin resistance, increased NEU3 sialidase
activity, both compared with vehicle controls. Finally, a variety of
different G protein-coupled receptor agonists dose-dependently
increased NEU1 sialidase activity, IR activation, and IR and IRS-1
phosphorylation in human IR-overexpressing HTC cells (293).
NEU3-overexpressing mouse 3T3-L1 adipocytes and L6
myocytes, and muscle tissue from human NEU3 transgenic mice,
had decreased insulin-stimulated IR signaling, and administration
of insulin to NEU3 transgenic mice induced NEU3 Tyr
phosphorylation and NEU3-Grb-2 association (294). In vivo
overexpression of NEU3 in the livers of insulin-sensitive or
insulin-resistant mice increased hepatic levels of glycogen,
triglycerides, peroxisome proliferator-activated receptor g
(PPARg), ganglioside GM1, and IRS-1 phosphorylation,
compared with control mice, suggesting that NEU3 regulates
hepatic insulin sensitivity and glucose tolerance (295). Obese
strain rats fed a HFD had reduced NEU3 expression in adipose
tissue through an intracellular palmitate-driven, Akt-dependent
pathway (296). Glucose-stimulated insulin release by rat INS-1D
pancreatic bcells was increased by 2-deoxy-NANA, and blood
insulin levels were greater in NEU3-deficient mice compared with
wild type controls (362). The relationship between insulin resistance
and intestinal hypoxia was explained, in part, by the activation of
hypoxia-inducible factor-2a(HIF-2a) in hypoxic, HFD-fed mice
leading to NEU3 activation (363).
MUC1 downregulation is necessary for human and mouse
fetal-uterine implantation, and diabetic mice expressed increased
MUC1 mRNA and protein levels at fetal-uterine implantation
sites compared with nondiabetic controls (364). STZ-induced
diabetic mice administered with sodium hyaluronate eye drops
for treatment of diabetic ocular surface disease exhibited
increased MUC5AC expression in the conjunctival epithelium
compared with saline controls (365). STZ-induced diabetic
rats had decreased MUC10 protein levels in salivary
submandibular glands compared with nondiabetic controls
(366). Transcriptome analysis of nucleolar protein p120
(NOP2)-deficient vs. NOP2-expressing human colon cancer
cells identified the MUC19 gene as significantly upregulated,
and T1D as a gene pathway most significantly affected, by NOP2
silencing (367). Future studies are needed to determine whether
NEUs regulate MUC-associated diabetes.
Inflammatory Bowel Disease
IBD comprises a collection of idiopathic, intestinal autoimmune
diseases, the most common being Crohn’s disease (CD) and
ulcerative colitis (UC) (368). CD typically affects the small and
large intestines, while UC primarily involves the colon and
rectum. Elevated expression of intestinal NEU3 (297)was
correlated with diminished levels of intestinal alkaline
phosphatase (IAP) in patients with colitis (369), and human
genetic deficiency of IAP is associated with IBD (370), suggesting
thatNEU3mayplayaroleinthepathogenesisofIBD.
Compared with controls, a 2.0-fold increase in GM3
ganglioside levels and an 8.3-fold increase in NEU3 protein
levels were reported in the intestine of IBD patients (297).
Because NEU3 is responsible for the conversion of disialylated
GD3 to monosialylated GM3, the authors speculated that
increasing dietary GD3 intake may be beneficial for treatment
of IBD patients. A SNP, rs4947331, encoding a minor T allele
located in the 3’untranslated region of the NEU1 gene was
significantly associated with the presence of CD in North Indian
and Dutch populations (298). It was suggested that the NEU1
pharmacologic inhibitor, oseltamivir, might be applicable to treat
CD patients. Serum sialic acid levels were increased, presumably
as a consequence of increased NEU catalytic activity, and
positively correlated with established markers of disease
activity, in CD patients (371,372). Meta-analysis of six
genome-wide association studies in CD patients identified the
MUC1 gene as a candidate for disease susceptibility (373).
Elevated expression of multiple mucin gene products,
including MUC1, MUC4, and MUC13, were reported in IBD
patients compared with controls (374–376). By contrast, another
study reported decreased expression of these MUCs in IBD
patients (377). MUC1 knockout mice had more severe forms
of Th1- and Th2-induced colitis and increased Th17 cell-
mediated responses in the colon, compared with MUC1-
expressing mice (378). Studies in both human and animal
model systems indicated that MUC1 regulates the progression
of IBD to colon cancer (379–384). Alterations in MUC2
intestinal expression and glycosylation were reported in IBD
patients (385,386). MUC2 knockout mice had greater intestinal
inflammation and reduced body weight gain compared with
MUC2-expressing mice, despite equal food intake, and exhibited
intestinal microbiome, short chain fatty acid, and inflammatory
cytokine profiles similar to that of IBD patients (387,388). The
Winnie mouse strain possessing a MUC2 gene point mutation
encoding a Cys-to-Tyr missense mutation develops spontaneous
colitis with many of the same pathologic features seen in UC
patients (389). Other MUCs associated with CD and UC disease
include MUC3, MUC5AC, MUC5B, MUC6, MUC16, MUC19,
and MUC20 (390–394). Additional studies are needed to
establish a causal link between the expression and catalytic
activity of NEUs with MUCs in IBD. In summary, the
development and progression of SLE, RA, T1D, and IBD are
influenced by both NEUs (Table 2) and MUCs.
DISCUSSION
In this review, we have attempted to summarize historical and
recent research on the roles of mammalian NEUs and MUCs,
both individually and together, in the context of fibrotic and
immune-mediated human diseases. First, general features of the
20-member MUC family and 4-member NEU family were
summarized, including an updated tabulation of selected
known and putative NEU1 glycoprotein substrates relevant to
fibrotic and inflammatory processes. Particular attention was
paid to the KL-6 glycoprotein, a soluble form of the membrane-
bound MUC1-ED. The concept of a NEU1 –MUC1 axis in IPF/
ILD was presented. Evidence supporting the NEU1 –MUC1 axis
included publications demonstrating that NEU1 regulates
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Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 88307916

pulmonary collagen deposition, lymphocytosis, and fibrosis in
human and mouse models of IPF (128), KL-6 serves as a
diagnostic and prognostic biomarker for, and mediator of,
IPF/ILD (125,180–183), and most importantly, NEU1
pharmacologic inhibition reverses bleomycin-induced increases
in MUC1-ED desialylation and shedding from the cell surface
(48). Finally, the influence of NEU1 on human infections by
bacterial, viral, and parasitic pathogens, and the impact of NEUs
and MUCs on four selected human autoimmune diseases
was discussed.
A limitation of our analysis was that the expanse and
complexity of this field prevented us from an in-depth
exploration of the broader NEU/MUC biology. We utilized a
reductionist approach, which on the one hand, allowed for a
focused analysis, but on the other hand, limited the scope by
excluding related topics. Only mammalian NEUs and MUCs
were considered. Bacterial or viral exosialidases, endosialidases,
or trans-sialidases were not discussed despite their direct
relevance to disease processes. We did not consider catalytic
mechanisms of NEU activity but instead emphasized the
functional consequences relevant to health and disease. Further
reducing the complexity and sharpening the focus of this review,
only the immune-, inflammation-, and fibrosis-related functions
of mammalian NEUs and MUCs were discussed; the effects on
other processes, e.g., atherosclerosis, diabetes, or cancer, were
mentioned as far as they related to the immune, inflammatory, or
connective tissue aspects of those processes. Moreover, the
complexity of the mechanisms controlling NEU1 elevation or
decline in the diseases were not considered. We only reviewed
the functional consequences of glycoprotein desialylation, with
substantially less attention devoted to desialylation of another
major group of NEU substrates, glycolipids, and its functional
sequelae. We did not reflect on the balancing action of
sialyltransferases, which covalently attach sialic acid to glycans
in contrast to NEUs, which remove it. The biological effects of
free sialic acid were not considered either. An important topic
that was not discussed in depth relates to recent developments in
discovery and characterization of mammalian NEU isozyme-
specific inhibitors; these have been considered here only to the
extent that helps in reviewing NEU function. We refer the reader
interested in those aspects of NEUs excluded here to numerous
review articles that illuminate these topics (12,15,17,81,395).
Even with such reductions, the information summarized in this
review is rather voluminous and complicated, yet we hope that it
is sufficiently focused thematically to be useful.
In summary, human NEUs and MUCs, particularly NEU1
and MUC1, contribute alone, and in symphony, to the
development and progression of a variety of fibrotic,
inflammatory, and fibro-inflammatory human pathologies.
Future studies focusing on the details of the NEU1 –MUC1
relationship are expected provide new insights into the
mechanisms of selected disease processes characterized by
dysregulated fibrosis and inflammation that may someday lead
to novel bedside therapeutic interventions for the benefit
of humankind.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/supplementary material. Further inquiries can be
directed to the corresponding author.
AUTHOR CONTRIBUTIONS
All authors conceived and designed the review outline,
contributed to the writing and critical review of the
manuscript, and approved the final version of the manuscript.
FUNDING
This work was supported by grants from the National Institutes
of Allergy and Infectious Diseases (AI-144497) and the U.S.
Department of Defense (W81XWH1910056) to EL and the U.S.
Department of Defense (PR202031) to IL.
REFERENCES
1. Varki A. Sialic Acids in Human Health and Disease. Trends Mol Med (2008) 14
(8):351–60. doi: 10.1016/j.molmed.2008.06.002
2. Schauer R. Sialic Acids as Regulators of Molecular and Cellular Interactions.
Curr Opin Struct Biol (2009) 19(5):507–14. doi: 10.1016/j.sbi.2009.06.003
3. Varki A, Schnaar RL, Schauer R, Cummings RD, Esko JD, Stanley P, et al. Sialic
Acids and Other Nonulosonic Acids. In: A Varki, RD Cummings, JD Esko, P
Stanley and GW Hart, editors. Essentials of Glycobiology. NY: Cold Spring
Harbor Laboratory Press (2015). p. 179–95.
4. Finne J. Occurrence of Unique Polysialosyl Carbohydrate Units in
Glycoproteins of Developing Brain. J Biol Chem (1982) 257(20):11966–70.
doi: 10.1016/S0021-9258(18)33661-5
5. Warren L, Spearing CW. Mammalian Sialidase (Neuraminidase). Biochem
Biophys Res Commun (1960) 3:489–92. doi: 10.1016/0006-291x(60)90161-3
6. Corfield AP, Veh RW, Wember M, Michalski JC, Schauer R. The Release of N-
Acetyl- and N-Glycolloyl-Neuraminic Acid From Soluble Complex
Carbohydrates and Erythrocytes by Bacterial, Viral and Mammalian
Sialidases. Biochem J (1981) 197(2):293–9. doi: 10.1042/bj1970293
7. Kishore CS, Tulsiani DR, Bhavanandan VP, Carubelli R. Membrane-Bound
Neuraminidases of Rat Liver. Neuraminidase Activity in Golgi Apparatus.
J Biol Chem (1975) 250(7):2655–9.
8. Schengrund CL, Rosenberg A. Intracellular Location and Properties of Bovine
Brain Sialidase. J Biol Chem (1970) 245(22):6196–200. doi: 10.1016/S0021-9258
(18)62678-X
9. Mahadevan S, Nduguba JC, Tappel AL. Sialidase of Rat Liver and Kidney. J Biol
Chem (1967) 242(19):4409–13. doi: 10.1016/S0021-9258(18)99554-2
10. Sandhoff K, Jatkewitz H. A Particle-Bound Sialyl Lactosidoceramide Splitting
Mammalian Sialidase. Biochim Biophys Acta (1967) 141(2):442–4.
doi: 10.1016/0304-4165(67)90124-9
11. Carubelli R, Trucco RE, Caputto R. Neuraminidase Activity in Mammalian Organs.
Biochim Biophys Acta (1962) 60:196–7. doi: 10.1016/0006-3002(62)90392-x
12. Miyagi T, Yamaguchi K. Mammalian Sialidases: Physiological and
Pathological Roles in Cellular Functions. Glycobiology (2012) 22(7):880–96.
doi: 10.1093/glycob/cws057
13. Dalton GD, Xie J, An SW, Huang CL. New Insights Into the Mechanism of
Action of Soluble Klotho. Front Endocrinol (Lausanne) (2017) 8:323.
doi: 10.3389/fendo.2017.00323
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 88307917

14. Khan A, Sergi C. Sialidosis: A Review of Morphology and Molecular Biology
of a Rare Pediatric Disorder. Diagnostics (Basel) (2018) 8(2):29. doi: 10.3390/
diagnostics8020029
15. Glanz VY, Myasoedova VA, Grechko AV, Orekhov AN. Sialidase Activity in
Human Pathologies. Eur J Pharmacol (2019) 842:345–50. doi: 10.1016/
j.ejphar.2018.11.014
16. Bennasroune A, Romier-Crouzet B, Blaise S, Laffargue M, Efremov RG,
Martiny L, et al. Elastic Fibers and Elastin Receptor Complex:
Neuraminidase-1 Takes the Center Stage. Matrix Biol (2019) 84:57–67.
doi: 10.1016/j.matbio.2019.06.007
17. Pshezhetsky AV, Ashmarina LI. Desialylation of Surface Receptors as a New
Dimension in Cell Signaling. Biochem (Mosc) (2013) 78(7):736–45.
doi: 10.1134/S0006297913070067
18. Demina EP, Smutova V, Pan X, Fougerat A, Guo T, Zou C, et al.
Neuraminidases 1 and 3 Trigger Atherosclerosis by Desialylating Low-
Density Lipoproteins and Increasing Their Uptake by Macrophages. JAm
Heart Assoc (2021) 10(4):e018756. doi: 10.1161/JAHA.120.018756
19. Satyavarapu EM, Nath S, Mandal C. Desialylation of Atg5 by Sialidase (Neu2)
Enhances Autophagosome Formation to Induce Anchorage-Dependent Cell
Death in Ovarian Cancer Cells. Cell Death Discov (2021) 7(1):26. doi: 10.1038/
s41420-020-00391-y
20. Kijimoto-Ochiai S, Matsumoto-Mizuno T, Kamimura D, Murakami M,
Kobayashi M, Matsuoka I, et al. Existence of Neu1 Sialidase on Mouse
Thymocytes Whose Natural Substrate Is Cd5. Glycobiology (2018) 28
(5):306–17. doi: 10.1093/glycob/cwy009
21. Feng C, Zhang L, Almulki L, Faez S, Whitford M, Hafezi-Moghadam A, et al.
Endogenous Pmn Sialidase Activity Exposes Activation Epitope on Cd11b/
Cd18 Which Enhances Its Binding Interaction With Icam-1. J Leukoc Biol
(2011) 90(2):313–21. doi: 10.1189/jlb.1210708
22. Kawecki C, Bocquet O, Schmelzer CEH, Heinz A, Ihling C, Wahart A, et al.
Identification of Cd36 as a New Interaction Partner of Membrane Neu1:
Potential Implication in the Pro-Atherogenic Effects of the Elastin Receptor
Complex. Cell Mol Life Sci (2019) 76(4):791–807. doi: 10.1007/s00018-018-
2978-6
23. Bocquet O, Tembely D, Rioult D, Terryn C, Romier B, Bennasroune A, et al.
Characterization of Novel Interactions With Membrane Neu1 Highlights New
Regulatory Functions for the Elastin Receptor Complex in Monocyte
Interaction With Endothelial Cells. Cell Biosci (2021) 11(1):206.
doi: 10.1186/s13578-021-00718-x
24. Howlader MA, Li C, Zou C, Chakraberty R, Ebesoh N, Cairo CW.
Neuraminidase-3 Is a Negative Regulator of Lfa-1 Adhesion. Front Chem
(2019) 7:791. doi: 10.3389/fchem.2019.00791
25. Lee C, Liu A, Miranda-Ribera A, Hyun SW, Lillehoj EP, Cross AS, et al. Neu1
Sialidase Regulates the Sialylation State of Cd31 and Disrupts Cd31-Driven
Capillary-Like Tube Formation in Human Lung Microvascular Endothelia.
J Biol Chem (2014) 289(13):9121–35. doi: 10.1074/jbc.M114.555888
26. Li J, van der Wal DE, Zhu G, Xu M, Yougbare I, Ma L, et al. Desialylation Is a
Mechanism of Fc-Independent Platelet Clearance and a Therapeutic Target in
Immune Thrombocytopenia. Nat Commun (2015) 6:7737. doi: 10.1038/
ncomms8737
27. Jansen AJ, Josefsson EC, Rumjantseva V, Liu QP, Falet H, Bergmeier W, et al.
Desialylation Accelerates Platelet Clearance After Refrigeration and Initiates
Gpibalpha Metalloproteinase-Mediated Cleavage in Mice. Blood (2012) 119
(5):1263–73. doi: 10.1182/blood-2011-05-355628
28. Katoh S. Critical Involvement of Cd44 in T Helper Type 2 Cell-Mediated
Eosinophilic Airway Inflammation in a Mouse Model of Acute Asthma. Front
Immunol (2021) 12:811600. doi: 10.3389/fimmu.2021.811600
29. Faller CE, Guvench O. Terminal Sialic Acids on Cd44 N-Glycans Can Block
Hyaluronan Binding by Forming Competing Intramolecular Contacts With
Arginine Sidechains. Proteins (2014) 82(11):3079–89. doi: 10.1002/prot.24668
30. Katoh S, Maeda S, Fukuoka H, Wada T, Moriya S, Mori A, et al. A Crucial
Role of Sialidase Neu1 in Hyaluronan Receptor Function of Cd44 in T Helper
Type 2-Mediated Airway Inflammation of Murine Acute Asthmatic Model.
Clin Exp Immunol (2010) 161(2):233–41. doi: 10.1111/j.1365-2249.
2010.04165.x
31. Seyrantepe V, Iannello A, Liang F, Kanshin E, Jayanth P, Samarani S, et al.
Regulation of Phagocytosis in Macrophages by Neuraminidase 1. J Biol Chem
(2010) 285(1):206–15. doi: 10.1074/jbc.M109.055475
32. Uemura T, Shiozaki K, Yamaguchi K, Miyazaki S, Satomi S, Kato K, et al.
Contribution of Sialidase Neu1 to Suppression of Metastasis of Human Colon
Cancer Cells Through Desialylation of Integrin Beta4. Oncogene (2009) 28
(9):1218–29. doi: 10.1038/onc.2008.471
33. Zanoteli E, van de Vlekkert D, Bonten EJ, Hu H, Mann L, Gomero EM, et al.
Muscle Degeneration in Neuraminidase 1-Deficient Mice Results From
Infiltration of the Muscle Fibers by Expanded Connective Tissue. Biochim
Biophys Acta (2010) 1802(7-8):659–72. doi: 10.1016/j.bbadis.2010.04.002
34. Yogalingam G, Bonten EJ, van de Vlekkert D, Hu H, Moshiach S, Connell SA,
et al. Neuraminidase 1 Is a Negative Regulator of Lysosomal Exocytosis. Dev
Cell (2008) 15(1):74–86. doi: 10.1016/j.devcel.2008.05.005
35. Hinek A, Bodnaruk TD, Bunda S, Wang Y, Liu K. Neuraminidase-1, a Subunit
of the Cell Surface Elastin Receptor, Desialylates and Functionally Inactivates
Adjacent Receptors Interacting With the Mitogenic Growth Factors Pdgf-Bb
and Igf-2. Am J Pathol (2008) 173(4):1042–56. doi: 10.2353/
ajpath.2008.071081
36. Arabkhari M, Bunda S, Wang Y, Wang A, Pshezhetsky AV, Hinek A.
Desialylation of Insulin Receptors and Igf-1 Receptors by Neuraminidase-1
Controls the Net Proliferative Response of L6 Myoblasts to Insulin.
Glycobiology (2010) 20(5):603–16. doi: 10.1093/glycob/cwq010
37. Blaise S, Romier B, Kawecki C, Ghirardi M, Rabenoelina F, Baud S, et al.
Elastin-Derived Peptides Are New Regulators of Insulin Resistance
Development in Mice. Diabetes (2013) 62(11):3807–16. doi: 10.2337/db13-
0508
38. Fougerat A, Pan X, Smutova V, Heveker N, Cairo CW, Issad T, et al.
Neuraminidase 1 Activates Insulin Receptor and Reverses Insulin
Resistance in Obese Mice. Mol Metab (2018) 12:76–88. doi: 10.1016/
j.molmet.2018.03.017
39. Dridi L, Seyrantepe V, Fougerat A, Pan X, Bonneil E, Thibault P, et al. Positive
Regulation of Insulin Signaling by Neuraminidase 1. Diabetes (2013) 62
(7):2338–46. doi: 10.2337/db12-1825
40. Lillehoj EP, Hyun SW, Feng C, Zhang L, Liu A, Guang W, et al. Neu1 Sialidase
Expressed in Human Airway Epithelia Regulates Epidermal Growth Factor
Receptor (Egfr) and Muc1 Protein Signaling. JBiolChem(2012) 287
(11):8214–31. doi: 10.1074/jbc.M111.292888
41. Gilmour AM, Abdulkhalek S, Cheng TS, Alghamdi F, Jayanth P, O’Shea LK,
et al. A Novel Epidermal Growth Factor Receptor-Signaling Platform and Its
Targeted Translation in Pancreatic Cancer. Cell Signal (2013) 25(12):2587–
603. doi: 10.1016/j.cellsig.2013.08.008
42. Mozzi A, Forcella M, Riva A, Difrancesco C, Molinari F, Martin V, et al. Neu3
Activity Enhances Egfr Activation Without Affecting Egfr Expression and
Acts on Its Sialylation Levels. Glycobiology (2015) 25(8):855–68. doi: 10.1093/
glycob/cwv026
43. Romier B, Ivaldi C, Sartelet H, Heinz A, Schmelzer CEH, Garnotel R, et al.
Production of Elastin-Derived Peptides Contributes to the Development of
Nonalcoholic Steatohepatitis. Diabetes (2018) 67(8):1604–15. doi: 10.2337/
db17-0490
44. Abdulkhalek S, Szewczuk MR. Neu1 Sialidase and Matrix Metalloproteinase-9
Cross-Talk Regulates Nucleic Acid-Induced Endosomal Toll-Like Receptor-7
and -9 Activation, Cellular Signaling and Pro-Inflammatory Responses. Cell
Signal (2013) 25(11):2093–105. doi: 10.1016/j.cellsig.2013.06.010
45. Abdulkhalek S, Amith SR, Franchuk SL, Jayanth P, Guo M, Finlay T, et al.
Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-Talk Is Essential for
Toll-Like Receptor Activation and Cellular Signaling. J Biol Chem (2011) 286
(42):36532–49. doi: 10.1074/jbc.M111.237578
46. Jayanth P, Amith SR, Gee K, Szewczuk MR. Neu1 Sialidase and Matrix
Metalloproteinase-9 Cross-Talk Is Essential for Neurotrophin Activation of
Trk Receptors and Cellular Signaling. Cell Signal (2010) 22(8):1193–205.
doi: 10.1016/j.cellsig.2010.03.011
47. Abdulkhalek S, Guo M, Amith SR, Jayanth P, Szewczuk MR. G-Protein
Coupled Receptor Agonists Mediate Neu1 Sialidase and Matrix
Metalloproteinase-9 Cross-Talk to Induce Transactivation of Toll-Like
Receptors and Cellular Signaling. Cell Signal (2012) 24(11):2035–42.
doi: 10.1016/j.cellsig.2012.06.016
48. Luzina IG, Lillehoj EP, Lockatell V, Hyun SW, Lugkey KN, Imamura A, et al.
Therapeutic Effect of Neuraminidase-1-Selective Inhibition in Mouse Models
of Bleomycin-Induced Pulmonary Inflammation and Fibrosis. J Pharmacol
Exp Ther (2021) 376(1):136–46. doi: 10.1124/jpet.120.000223
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 88307918

49. Lillehoj EP, Guang W, Hyun SW, Liu A, Hegerle N, Simon R, et al. Neuraminidase
1-Mediated Desialylation of the Mucin 1 Ectodomain Releases a Decoy Receptor
That Protects Against Pseudomonas Aeruginosa Lung Infection. JBiolChem
(2019) 294(2):662–78. doi: 10.1074/jbc.RA118.006022
50. Hyun SW, Liu A, Liu Z, Cross AS, Verceles AC, Magesh S, et al. The Neu1-
Selective Sialidase Inhibitor, C9-Butyl-Amide-Dana, Blocks Sialidase Activity
and Neu1-Mediated Bioactivities in Human Lung In Vitro and Murine Lung
In Vivo.Glycobiology (2016) 26(8):834–49. doi: 10.1093/glycob/cww060
51. Lillehoj EP, Hyun SW, Liu A, Guang W, Verceles AC, Luzina IG, et al. Neu1
Sialidase Regulates Membrane-Tethered Mucin (Muc1) Ectodomain
Adhesiveness for Pseudomonas Aeruginosa and Decoy Receptor Release.
J Biol Chem (2015) 290(30):18316–31. doi: 10.1074/jbc.M115.657114
52. Karhadkar TR, Meek TD, Gomer RH. Inhibiting Sialidase-Induced Tgf-Beta1
Activation Attenuates Pulmonary Fibrosis in Mice. J Pharmacol Exp Ther
(2021) 376(1):106–17. doi: 10.1124/jpet.120.000258
53. Amith SR, Jayanth P, Franchuk S, Siddiqui S, Seyrantepe V, Gee K, et al.
Dependence of Pathogen Molecule-Induced Toll-Like Receptor Activation
and Cell Function on Neu1 Sialidase. Glycoconj J (2009) 26(9):1197–212.
doi: 10.1007/s10719-009-9239-8
54. Natori Y, Nasui M, Edo K, Sato S, Sakurai T, Kizaki T, et al. Neu1 Sialidase
Controls Gene Expression and Secretion of Il-6 and Mcp-1 Through Nf-
Kappab Pathway in 3t3-L1 Adipocytes. J Biochem (2017) 162(2):137–43.
doi: 10.1093/jb/mvx006
55. Karmakar J, Roy S, Mandal C. Modulation of Tlr4 Sialylation Mediated by a
Sialidase Neu1 and Impairment of Its Signaling in Leishmania Donovani
Infected Macrophages. Front Immunol (2019) 10:2360. doi: 10.3389/
fimmu.2019.02360
56. Allendorf DH, Franssen EH, Brown GC. Lipopolysaccharide Activates
Microglia Via Neuraminidase 1 Desialylation of Toll-Like Receptor 4. J
Neurochem (2020) 155(4):403–16. doi: 10.1111/jnc.15024
57. Amith SR, Jayanth P, Franchuk S, Finlay T, Seyrantepe V, Beyaert R, et al.
Neu1 Desialylation of Sialyl Alpha-2,3-Linked Beta-Galactosyl Residues of
Toll-Like Receptor 4 Is Essential for Receptor Activation and Cellular
Signaling. Cell Signal (2010) 22(2):314–24. doi: 10.1016/j.cellsig.2009.09.038
58. Feng C, Stamatos NM, Dragan AI, Medvedev A, Whitford M, Zhang L, et al.
Sialyl Residues Modulate Lps-Mediated Signaling Through the Toll-Like
Receptor 4 Complex. PLoS One (2012) 7(4):e32359. doi: 10.1371/
journal.pone.0032359
59. Woronowicz A, Amith SR, De Vusser K, Laroy W, Contreras R, Basta S, et al.
Dependence of Neurotrophic Factor Activation of Trk Tyrosine Kinase
Receptors on Cellular Sialidase. Glycobiology (2007) 17(1):10–24.
doi: 10.1093/glycob/cwl049
60. Bonten EJ, Annunziata I, d’Azzo A. Lysosomal Multienzyme Complex: Pros
and Cons of Working Together. Cell Mol Life Sci (2014) 71(11):2017–32.
doi: 10.1007/s00018-013-1538-3
61. Pshezhetsky AV, Hinek A. Where Catabolism Meets Signalling:
Neuraminidase 1 as a Modulator of Cell Receptors. Glycoconj J (2011) 28
(7):441–52. doi: 10.1007/s10719-011-9350-5
62. Lukong KE, Seyrantepe V, Landry K, Trudel S, Ahmad A, Gahl WA, et al.
Intracellular Distribution of Lysosomal Sialidase Is Controlled by the
Internalization Signal in Its Cytopla smic Tail. JBiolChem(2001) 276
(49):46172–81. doi: 10.1074/jbc.M104547200
63. Hinek A, Pshezhetsky AV, von Itzstein M, Starcher B. Lysosomal Sialidase
(Neuraminidase-1) Is Targeted to the Cell Surface in a Multiprotein Complex
That Facilitates Elastic Fiber Assembly. J Biol Chem (2006) 281(6):3698–710.
doi: 10.1074/jbc.M508736200
64. Liang F, Seyrantepe V, Landry K, Ahmad R, Ahmad A, Stamatos NM, et al.
Monocyte Differentiation Up-Regulates the Expression of the Lysosomal
Sialidase, Neu1, and Triggers Its Targeting to the Plasma Membrane Via
Major Histocompatibility Complex Class Ii-Positive Compartments. J Biol
Chem (2006) 281(37):27526–38. doi: 10.1074/jbc.M605633200
65. Nan X, Carubelli I, Stamatos NM. Sialidase Expression in Activated Human T
Lymphocytes Influences Production of Ifn-Gamma. J Leukoc Biol (2007) 81
(1):284–96. doi: 10.1189/jlb.1105692
66. Stamatos NM, Carubelli I, van de Vlekkert D, Bonten EJ, Papini N, Feng C,
et al. Lps-Induced Cytokine Production in Human Dendritic Cells Is
Regulated by Sialidase Activity. J Leukoc Biol (2010) 88(6):1227–39.
doi: 10.1189/jlb.1209776
67. Shkandina T, Herrmann M, Bilyy R. Sweet Kiss of Dying Cell: Sialidase
Activity on Apoptotic Cell Is Able to Act Toward Its Neighbors.
Autoimmunity (2012) 45(8):574–8. doi: 10.3109/08916934.2012.719951
68. D’Avila F, Tringali C, Papini N, Anastasia L, Croci G, Massaccesi L, et al.
Identification of Lysosomal Sialidase Neu1 and Plasma Membrane Sialidase
Neu3 in Human Erythrocytes. J Cell Biochem (2013) 114(1):204–11.
doi: 10.1002/jcb.24355
69. Alghamdi F, Guo M, Abdulkhalek S, Crawford N, Amith SR, Szewczuk MR. A
Novel Insulin Receptor-Signaling Platform and Its Link to Insulin Resistance
and Type 2 Diabetes. Cell Signal (2014) 26(6):1355–68. doi: 10.1016/
j.cellsig.2014.02.015
70. Chen GY, Brown NK, Wu W, Khedri Z, Yu H, Chen X, et al. Broad and Direct
Interaction Between Tlr and Siglec Families of Pattern Recognition Receptors
and Its Regulation by Neu1. Elife (2014) 3:e04066. doi: 10.7554/eLife.04066
71. Maurice P, Baud S, Bocharova OV, Bocharov EV, Kuznetsov AS, Kawecki C,
et al. New Insights Into Molecular Organization of Human Neuraminidase-1:
Transmembrane Topology and Dimerization Ability. Sci Rep (2016) 6:38363.
doi: 10.1038/srep38363
72. Scandolera A, Odoul L, Salesse S, Guillot A, Blaise S, Kawecki C, et al. The
Elastin Receptor Complex: A Unique Matricellular Receptor With High Anti-
Tumoral Potential. Front Pharmacol (2016) 7:32. doi: 10.3389/fphar.
2016.00032
73. Wu Y, Lan C, Ren D, Chen GY. Induction of Siglec-1 by Endotoxin Tolerance
Suppresses the Innate Immune Response by Promoting Tgf-Beta1
Production. JBiolChem(2016) 291(23):12370–82. doi: 10.1074/jbc.
M116.721258
74. Sasaki N, Itakura Y, Toyoda M. Sialylation Regulates Myofibroblast
Differentiation of Human Skin Fibroblasts. Stem Cell Res Ther (2017) 8
(1):81. doi: 10.1186/s13287-017-0534-1
75. Wahart A, Hocine T, Albrecht C, Henry A, Sarazin T, Martiny L, et al. Role of
Elastin Peptides and Elastin Receptor Complex in Metabolic and
Cardiovascular Diseases. FEBS J (2019) 286(15):2980–93. doi: 10.1111/
febs.14836
76. van der Wal DE, Davis AM, Mach M, Marks DC. The Role of Neuraminidase
1 and 2 in Glycoprotein Ibalpha-Mediated Integrin Alphaiibbeta3 Activation.
Haematologica (2020) 105(4):1081–94. doi: 10.3324/haematol.2019.215830
77. Nath S, Mandal C, Chatterjee U, Mandal C. Association of Cytosolic Sialidase
Neu2 With Plasma Membrane Enhances Fas-Mediated Apoptosis by
Impairing Pi3k-Akt/Mtor-Mediated Pathway in Pancreatic Cancer Cells.
Cell Death Dis (2018) 9(2):210. doi: 10.1038/s41419-017-0191-4
78. Kijimoto-Ochiai S, Doi N, Fujii M, Go S, Kabayama K, Moriya S, et al.Possible
Association of Neu2 With Plasma Membrane Fraction From Mouse Thymus
Exhibited Sialidase Activity With Fetuin at Ph 7.0 But Not at Ph 4.5. Microbiol
Immunol (2013) 57(8):569–82. doi: 10.1111/1348-0421.12076
79. Shiozaki K, Yamaguchi K, Takahashi K, Moriya S, Miyagi T. Regulation of
Sialyl Lewis Antigen Expression in Colon Cancer Cells by Sialidase Neu4.
J Biol Chem (2011) 286(24):21052–61. doi: 10.1074/jbc.M111.231191
80. Finlay TM, Jayanth P, Amith SR, Gilmour A, Guzzo C, Gee K, et al.
Thymoquinone From Nutraceutical Black Cumin Oil Activates Neu4
Sialidase in Live Macrophage, Dendritic, and Normal and Type I Sialidosis
Human Fibroblast Cells Via Gpcr Galphai Proteins and Matrix
Metalloproteinase-9. Glycoconj J (2010) 27(3):329–48. doi: 10.1007/s10719-
010-9281-6
81. Achyuthan KE, Achyuthan AM. Comparative Enzymology, Biochemistry and
Pathophysiology of Human Exo-Alpha-Sialidases (Neuraminidases). Comp
Biochem Physiol B Biochem Mol Biol (2001) 129(1):29–64. doi: 10.1016/s1096-
4959(01)00372-4
82. Monti E, Preti A, Venerando B, Borsani G. Recent Development in
Mammalian Sialidase Molecular Biology. Neurochem Res (2002) 27(7-
8):649–63. doi: 10.1023/A:1020276000901
83. Miyagi T. Aberrant Expression of Sialidase and Cancer Progression. Proc Jpn
Acad Ser B Phys Biol Sci (2008) 84(10):407–18. doi: 10.2183/pjab.84.407
84. Monti E, Bonten E, D’Azzo A, Bresciani R, Venerando B, Borsani G, et al.
Sialidases in Vertebrates: A Family of Enzymes Tailored for Several Cell
Functions. Adv Carbohydr Chem Biochem (2010) 64:403–79. doi: 10.1016/
S0065-2318(10)64007-3
85. Monti E, Miyagi T. Structure and Function of Mammalian Sialidases. Top
Curr Chem (2015) 366:183–208. doi: 10.1007/128_2012_328
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 88307919

86. Miyagi T, Takahashi K, Yamamoto K, Shiozaki K, Yamaguchi K. Biological
and Pathological Roles of Ganglioside Sialidases. Prog Mol Biol Transl Sci
(2018) 156:121–50. doi: 10.1016/bs.pmbts.2017.12.005
87. Bourguet E, Figurska S, Fra Czek MM. Human Neuraminidases: Structures
and Stereoselective Inhibitors. J Med Chem (2022) 65(4):3002–25.
doi: 10.1021/acs.jmedchem.1c01612
88. Ma J, Rubin BK, Voynow JA. Mucins, Mucus, and Goblet Cells. Chest (2018)
154(1):169–76. doi: 10.1016/j.chest.2017.11.008
89. Rose MC, Voynow JA. Respiratory Tract Mucin Genes and Mucin
Glycoproteins in Health and Disease. Physiol Rev (2006) 86(1):245–78.
doi: 10.1152/physrev.00010.2005
90. Gendler SJ, Lancaster CA, Taylor-Papadimitriou J, Duhig T, Peat N, Burchell
J, et al. Molecular Cloning and Expression of Human Tumor-Associated
Polymorphic Epithelial Mucin. J Biol Chem (1990) 265(25):15286–93. doi:
10.1016/S0021-9258(18)77254-2
91. Lan MS, Batra SK, Qi WN, Metzgar RS, Hollingsworth MA. Cloning and
Sequencing of a Human Pancreatic Tumor Mucin Cdna. J Biol Chem (1990)
265(25):15294–9. doi: 10.1016/S0021-9258(18)77255-4
92. Ligtenberg MJ, Vos HL, Gennissen AM, Hilkens J. Episialin, a Carcinoma-
Associated Mucin, Is Generated by a Polymorphic Gene Encoding Splice
Variants With Alternative Amino Termini. J Biol Chem (1990) 265(10):5573–
8. doi: 10.1016/S0021-9258(19)39399-8
93. Ligtenberg MJ, Kruijshaar L, Buijs F, van Meijer M, Litvinov SV, Hilkens J.
Cell-Associated Episialin Is a Complex Containing Two Proteins Derived
From a Common Precursor. J Biol Chem (1992) 267(9):6171–7. doi: 10.1016/
S0021-9258(18)42677-4
94. Levitin F, Stern O, Weiss M, Gil-Henn C, Ziv R, Prokocimer Z, et al. The
Muc1 Sea Module Is a Self-Cleaving Domain. JBiolChem(2005) 280
(39):33374–86. doi: 10.1074/jbc.M506047200
95. Macao B, Johansson DG, Hansson GC, Hard T. Autoproteolysis Coupled to
Protein Folding in the Sea Domain of the Membrane-Bound Muc1 Mucin.
Nat Struct Mol Biol (2006) 13(1):71–6. doi: 10.1038/nsmb1035
96. Julian J, Carson DD. Formation of Muc1 Metabolic Complex Is Conserved in
Tumor-Derived and Normal Epithelial Cells. Biochem Biophys Res Commun
(2002) 293(4):1183–90. doi: 10.1016/S0006-291X(02)00352-2
97. Blalock TD, Spurr-Michaud SJ, Tisdale AS, Gipson IK. Release of Membrane-
Associated Mucins From Ocular Surface Epithelia. Invest Ophthalmol Vis Sci
(2008) 49(5):1864–71. doi: 10.1167/iovs.07-1081
98. Kim KC, Wasano K, Niles RM, Schuster JE, Stone PJ, Brody JS. Human
Neutrophil Elastase Releases Cell Surface Mucins From Primary Cultures of
Hamster Tracheal Epithelial Cells. Proc Natl Acad Sci USA (1987) 84
(24):9304–8. doi: 10.1073/pnas.84.24.9304
99. Thathiah A, Blobel CP, Carson DD. Tumor Necrosis Factor-Alpha
Converting Enzyme/Adam 17 Mediates Muc1 Shedding. JBiolChem
(2003) 278(5):3386–94. doi: 10.1074/jbc.M208326200
100. Linden SK, Sheng YH, Every AL, Miles KM, Skoog EC, Florin TH, et al.
Muc1 Limits Helicobacter Pylori Infection Both by Steric Hindrance and by
Acting as a Releasable Decoy. PLoS Pathog (2009) 5(10):e1000617.
doi: 10.1371/journal.ppat.1000617
101. Thathiah A, Carson DD. Mt1-Mmp Mediates Muc1 Shedding Independent
of Tace/Adam17. Biochem J (2004) 382(Pt 1):363–73. doi: 10.1042/
BJ20040513
102. Julian J, Dharmaraj N, Carson DD. Muc1 Is a Substrate for Gamma-
Secretase. J Cell Biochem (2009) 108(4):802–15. doi: 10.1002/jcb.22292
103. Smorodinsky N, Weiss M, Hartmann ML, Baruch A, Harness E, Yaakobovitz
M, et al. Detection of a Secreted Muc1/Sec Protein by Muc1 Isoform Specific
Monoclonal Antibodies. Biochem Biophys Res Commun (1996) 228(1):115–
21. doi: 10.1006/bbrc.1996.1625
104. Kim KC, Lillehoj EP. Muc1 Mucin: A Peacemaker in the Lung. Am J Respir
Cell Mol Biol (2008) 39(6):644–7. doi: 10.1165/rcmb.2008-0169TR
105. Lillehoj EP, Kato K, Lu W, Kim KC. Cellular and Molecular Biology of
Airway Mucins. Int Rev Cell Mol Biol (2013) 303:139–202. doi: 10.1016/
B978-0-12-407697-6.00004-0
106. Lillehoj ER, Kim KC. Airway Mucus: Its Components and Function. Arch
Pharm Res (2002) 25(6):770–80. doi: 10.1007/BF02976990
107. Kato K, Lillehoj EP, Lu W, Kim KC. Muc1: The First Respiratory Mucin
With an Anti-Inflammatory Function. JClinMed(2017) 6(12):110.
doi: 10.3390/jcm6120110
108. Argueso P, Herreras JM, Calonge M, Citores L, Pastor JC, Girbes T. Analysis
of Human Ocular Mucus: Effects of Neuraminidase and Chitinase Enzymes.
Cornea (1998) 17(2):200–7. doi: 10.1097/00003226-199803000-00015
109. Patton S, Gendler SJ, Spicer AP. The Epithelial Mucin, Muc1, of Milk,
Mammary Gland and Other Tissues. Biochim Biophys Acta (1995) 1241
(3):407–23. doi: 10.1016/0304-4157(95)00014-3
110. Button B, Cai LH, Ehre C, Kesimer M, Hill DB, Sheehan JK, et al. A
Periciliary Brush Promotes the Lung Health by Separating the Mucus Layer
From Airway Epithelia. Science (2012) 337(6097):937–41. doi: 10.1126/
science.1223012
111. Stonebraker JR, Wagner D, Lefensty RW, Burns K, Gendler SJ, Bergelson JM,
et al. Glycocalyx Restricts Adenoviral Vector Access to Apical Receptors
Expressed on Respiratory Epithelium In Vitro and In Vivo: Role for Tethered
Mucins as Barriers to Lumenal Infection. J Virol (2004) 78(24):13755–68.
doi: 10.1128/JVI.78.24.13755-13768.2004
112. Kato K, Lillehoj EP, Kim KC. Pseudomonas Aeruginosa Stimulates Tyrosine
Phosphorylation of and Tlr5 Association With the Muc1 Cytoplasmic Tail
Through Egfr Activation. Inflammation Res (2016) 65(3):225–33.
doi: 10.1007/s00011-015-0908-8
113. Li Y, Ren J, Yu W, Li Q, Kuwahara H, Yin L, et al. The Epidermal Growth
Factor Receptor Regulates Interaction of the Human Df3/Muc1 Carcinoma
Antigen With C-Src and Beta-Catenin. J Biol Chem (2001) 276(38):35239–
42. doi: 10.1074/jbc.C100359200
114. Schroeder JA, Thompson MC, Gardner MM, Gendler SJ. Transgenic Muc1
Interacts With Epidermal Growth Factor Receptor and Correlates With
Mitogen-Activated Protein Kinase Activation in the Mouse Mammary
Gland. J Biol Chem (2001) 276(16):13057–64. doi: 10.1074/jbc.M011248200
115. Chen S, Li M, Jiang W, Zheng H, Qi LW, Jiang S. The Role of Neu1 in the
Protective Effect of Dipsacoside B on Acetaminophen-Induced Liver Injury.
Ann Transl Med (2020) 8(13):823. doi: 10.21037/atm-19-3850
116. Wang W, Liu L, Zhou Y, Ye Q, Yang X, Jiang J, et al. Hydroxychloroquine
Enhances the Antitumor Effects of Bc001 in Gastric Cancer. Int J Oncol
(2019) 55(2):405–14. doi: 10.3892/ijo.2019.4824
117. Garbar C, Mascaux C, Giustiniani J, Salesse S, Debelle L, Antonicelli F, et al.
Autophagy Is Decreased in Triple-Negative Breast Carcinoma Involving
Likely the Muc1-Egfr-Neu1 Signalling Pathway. Int J Clin Exp Pathol (2015)
8(5):4344–55.
118. Kohno N, Akiyama M, Kyoizumi S, Hakoda M, Kobuke K, Yamakido M.
Detection of Soluble Tumor-Associated Antigens in Sera and Effusions
Using Novel Monoclonal Antibodies, Kl-3 and Kl-6, Against Lung
Adenocarcinoma. Jpn J Clin Oncol (1988) 18(3):203–16.
119. Hirasawa Y, Kohno N, Yokoyama A, Inoue Y, Abe M, Hiwada K. Kl-6, a
Human Muc1 Mucin, Is Chemotactic for Human Fibroblasts. Am J Respir
Cell Mol Biol (1997) 17(4):501–7. doi: 10.1165/ajrcmb.17.4.2253
120. Ohyabu N, Hinou H, Matsushita T, Izumi R, Shimizu H, Kawamoto K, et al.
An Essential Epitope of Anti-Muc1 Monoclonal Antibody Kl-6 Revealed by
Focused Glycopeptide Library. J Am Chem Soc (2009) 131(47):17102–9.
doi: 10.1021/ja903361f
121. Seko A, Ohkura T, Ideo H, Yamashita K. Novel O-Linked Glycans
Containing 6’-Sulfo-Gal/Galnac of Muc1 Secreted From Human Breast
Cancer Ymb-S Cells: Possible Carbohydrate Epitopes of Kl-6(Muc1)
Monoclonal Antibody. Glycobiology (2012) 22(2):181–95. doi: 10.1093/
glycob/cwr118
122. Sakai M, Kubota T, Ohnishi H, Yokoyama A. A Novel Lung Injury Animal
Model Using Kl-6-Measurable Human Muc1-Expressing Mice. Biochem
Biophys Res Commun (2013) 432(3):460–5. doi: 10.1016/j.bbrc.2013.01.123
123. Stahel RA, Gilks WR, Lehmann HP, Schenker T. Third International
Workshop on Lung Tumor and Differentiation Antigens: Overview of the
Results of the Central Data Analysis. Int J Cancer Suppl (1994) 8:6–26.
doi: 10.1002/ijc.2910570704
124. Ishikawa N, Hattori N, Yokoyama A, Kohno N. Utility of Kl-6/Muc1 in the
Clinical Management of Interstitial Lung Diseases. Respir Investig (2012) 50
(1):3–13. doi: 10.1016/j.resinv.2012.02.001
125. Kohno N. Serum Marker Kl-6/Muc1 for the Diagnosis and Management of
Interstitial Pneumonitis. J Med Invest (1999) 46(3-4):151–8.
126. Karhadkar TR, Chen W, Gomer RH. Attenuated Pulmonary Fibrosis in
Sialidase-3 Knockout (Neu3(-/-)) Mice. Am J Physiol Lung Cell Mol Physiol
(2020) 318(1):L165–L79. doi: 10.1152/ajplung.00275.2019
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 88307920

127. Karhadkar TR, Pilling D, Cox N, Gomer RH. Sialidase Inhibitors Attenuate
Pulmonary Fibrosis in a Mouse Model. Sci Rep (2017) 7(1):15069.
doi: 10.1038/s41598-017-15198-8
128. Luzina IG, Lockatell V, Hyun SW, Kopach P, Kang PH, Noor Z, et al.
Elevated Expression of Neu1 Sialidase in Idiopathic Pulmonary Fibrosis
Provokes Pulmonary Collagen Deposition, Lymphocytosis, and Fibrosis. Am
J Physiol Lung Cell Mol Physiol (2016) 310(10):L940–54. doi: 10.1152/
ajplung.00346.2015
129. Pilling D, Sahlberg K, Karhadkar TR, Chen W, Gomer RH. The Sialidase
Neu3 Promotes Pulmonary Fibrosis in Mice. bioRxiv (2022)
2022:02.12.480197. doi: 10.1101/2022.02.12.480197
130. Chen W, Karhadkar TR, Ryu C, Herzog EL, Gomer RH. Reduced Sialylation
and Bioactivity of the Antifibrotic Protein Serum Amyloid P in the Sera of
Patients With Idiopathic Pulmonary Fibrosis. Immunohorizons (2020) 4
(6):352–62. doi: 10.4049/immunohorizons.2000043
131. Guo Z, Tuo H, Tang N, Liu FY, Ma SQ, An P, et al. Neuraminidase 1
Deficiency Attenuates Cardiac Dysfunction, Oxidative Stress, Fibrosis,
Inflammatory Via Ampk-Sirt3 Pathway in Diabetic Cardiomyopathy Mice.
Int J Biol Sci (2022) 18(2):826–40. doi: 10.7150/ijbs.65938
132. Ghiroldi A, Piccoli M, Creo P, Cirillo F, Rota P, D’Imperio S, et al. Role of
Sialidase Neu3 and Ganglioside Gm3 in Cardiac Fibroblasts Activation.
Biochem J (2020) 477(17):3401–15. doi: 10.1042/BCJ20200360
133. Inui N, Sakai S, Kitagawa M. Molecular Pathogenesis of Pulmonary Fibrosis,
With Focus on Pathways Related to Tgf-Beta and the Ubiquitin-Proteasome
Pathway. Int J Mol Sci (2021) 22(11):6107. doi: 10.3390/ijms22116107
134. Ye Z, Hu Y. Tgfbeta1: Gentlemanly Orchestrator in Idiopathic Pulmonary
Fibrosis (Review). Int J Mol Med (2021) 48(1):132. doi: 10.3892/ijmm.2021.4965
135. Huaux F. Interpreting Immunoregulation in Lung Fibrosis: A New Branch of
the Immune Model. Front Immunol (2021) 12:690375. doi: 10.3389/
fimmu.2021.690375
136. van Geffen C, Deissler A, Quante M, Renz H, Hartl D, Kolahian S. Regulatory
Immune Cells in Idiopathic Pulmonary Fibrosis: Friends or Foes? Front
Immunol (2021) 12:663203. doi: 10.3389/fimmu.2021.663203
137. Shenderov K, Collins SL, Powell JD, Horton MR. Immune Dysregulation as a
Driver of Idiopathic Pulmonary Fibrosis. J Clin Invest (2021) 131(2):e143226.
doi: 10.1172/JCI143226
138. Desai O, Winkler J, Minasyan M, Herzog EL. The Role of Immune and
Inflammatory Cells in Idiopathic Pulmonary Fibrosis. Front Med (Lausanne)
(2018) 5:43. doi: 10.3389/fmed.2018.00043
139. Kolahian S, Fernandez IE, Eickelberg O, Hartl D. Immune Mechanisms in
Pulmonary Fibrosis. Am J Respir Cell Mol Biol (2016) 55(3):309–22.
doi: 10.1165/rcmb.2016-0121TR
140. Luzina IG, Todd NW, Iacono AT, Atamas SP. Roles of T Lymphocytes in
Pulmonary Fibrosis. J Leukoc Biol (2008) 83(2):237–44. doi: 10.1189/
jlb.0707504
141. Luzina IG, Todd NW, Sundararajan S, Atamas SP. The Cytokines of
Pulmonary Fibrosis: Much Learned, Much More to Learn. Cytokine (2015)
74(1):88–100. doi: 10.1016/j.cyto.2014.11.008
142. Todd NW, Luzina IG, Atamas SP. Molecular and Cellular Mechanisms of
Pulmonary Fibrosis. Fibrogenesis Tissue Repair (2012) 5(1):11. doi: 10.1186/
1755-1536-5-11
143. Estornut C, Milara J, Bayarri MA, Belhadj N, Cortijo J. Targeting Oxidative
Stress as a Therapeutic Approach for Idiopathic Pulmonary Fibrosis. Front
Pharmacol (2021) 12:794997. doi: 10.3389/fphar.2021.794997
144. Otoupalova E, Smith S, Cheng G, Thannickal VJ. Oxidative Stress in Pulmonary
Fibrosis. Compr Physiol (2020) 10(2):509–47. doi: 10.1002/cphy.c190017
145. Lin C, Borensztajn K, Spek CA. Targeting Coagulation Factor Receptors -
Protease-Activated Receptors in Idiopathic Pulmonary Fibrosis. J Thromb
Haemost (2017) 15(4):597–607. doi: 10.1111/jth.13623
146. Crooks MG, Hart SP. Coagulation and Anticoagulation in Idiopathic
Pulmonary Fibrosis. Eur Respir Rev (2015) 24(137):392–9. doi: 10.1183/
16000617.00008414
147. Nho RS, Ballinger MN, Rojas MM, Ghadiali SN, Horowitz JC. Biomechanical
Force and Cellular Stiffness in Lung Fibrosis. Am J Pathol (2022) S0002-944
(22):00050–5. doi: 10.1016/j.ajpath.2022.02.001
148. Wu H, Yu Y, Huang H, Hu Y, Fu S, Wang Z, et al. Progressive Pulmonary
Fibrosis Is Caused by Elevated Mechanical Tension on Alveolar Stem Cells.
Cell (2020) 180(1):107–21 e17. doi: 10.1016/j.cell.2019.11.027
149. Kellogg DL, Kellogg DL Jr, Musi N, Nambiar AM. Cellular Senescence in
Idiopathic Pulmonary Fibrosis. CurrMolBiolRep(2021) 7(3):31–40.
doi: 10.1007/s40610-021-00145-4
150. Hernandez-Gonzalez F, Faner R, Rojas M, Agusti A, Serrano M, Sellares J.
Cellular Senescence in Lung Fibrosis. Int J Mol Sci (2021) 22(13):7012.
doi: 10.3390/ijms22137012
151. Pardo A, Selman M. The Interplay of the Genetic Architecture, Aging, and
Environmental Factors in the Pathogenesis of Idiopathic Pulmonary
Fibrosis. Am J Respir Cell Mol Biol (2021) 64(2):163–72. doi: 10.1165/
rcmb.2020-0373PS
152. Merkt W, Bueno M, Mora AL, Lagares D. Senotherapeutics: Targeting
Senescence in Idiopathic Pulmonary Fibrosis. Semin Cell Dev Biol (2020)
101:104–10. doi: 10.1016/j.semcdb.2019.12.008
153. Gulati S, Thannickal VJ. The Aging Lung and Idiopathic Pulmonary Fibrosis.
Am J Med Sci (2019) 357(5):384–9. doi: 10.1016/j.amjms.2019.02.008
154. Waters DW, Blokland KEC, Pathinayake PS, Burgess JK, Mutsaers SE, Prele
CM, et al. Fibroblast Senescence in the Pathology of Idiopathic Pulmonary
Fibrosis. Am J Physiol Lung Cell Mol Physiol (2018) 315(2):L162–L72.
doi: 10.1152/ajplung.00037.2018
155. Zank DC, Bueno M, Mora AL, Rojas M. Idiopathic Pulmonary Fibrosis:
Aging, Mitochondrial Dysfunction, and Cellular Bioenergetics. Front Med
(Lausanne) (2018) 5:10. doi: 10.3389/fmed.2018.00010
156. Zhao H, Wang Y, Qiu T, Liu W, Yao P. Autophagy, an Important
Therapeutic Target for Pulmonary Fibrosis Diseases. Clin Chim Acta
(2020) 502:139–47. doi: 10.1016/j.cca.2019.12.016
157. O’Dwyer DN, Ashley SL, Moore BB. Influences of Innate Immunity,
Autophagy, and Fibroblast ActivationinthePathogenesisofLung
Fibrosis. Am J Physiol Lung Cell Mol Physiol (2016) 311(3):L590–601.
doi: 10.1152/ajplung.00221.2016
158. Katzen J, Beers MF. Contributions of Alveolar Epithelial Cell Quality Control
to Pulmonary Fibrosis. J Clin Invest (2020) 130(10):5088–99. doi: 10.1172/
JCI139519
159. Selman M, Pardo A. The Leading Role of Epithelial Cells in the Pathogenesis
of Idiopathic Pulmonary Fibrosis. Cell Signal (2020) 66:109482. doi: 10.1016/
j.cellsig.2019.109482
160. Hewlett JC, Kropski JA, Blackwell TS. Idiopathic Pulmonary Fibrosis:
Epithelial-Mesenchymal Interactions and Emerging Therapeutic Targets.
Matrix Biol (2018) 71-72:112–27. doi: 10.1016/j.matbio.2018.03.021
161. Kulkarni T, de Andrade J, Zhou Y, Luckhardt T, Thannickal VJ. Alveolar
Epithelial Disintegrity in Pulmonary Fibrosis. Am J Physiol Lung Cell Mol
Physiol (2016) 311(2):L185–91. doi: 10.1152/ajplung.00115.2016
162. Lederer DJ, Martinez FJ. Idiopathic Pulmonary Fibrosis. N Engl J Med (2018)
378(19):1811–23. doi: 10.1056/NEJMra1705751
163. Moss BJ, Ryter SW, Rosas IO. Pathogenic Mechanisms Underlying
Idiopathic Pulmonary Fibrosis. Annu Rev Pathol (2022) 17:515–46.
doi: 10.1146/annurev-pathol-042320-030240
164. Akiyama M, Kaneko Y. Pathogenesis, Clinical Features, and Treatment
Strategy for Rheumatoid Arthritis-Associated Interstitial Lung Disease.
Autoimmun Rev (2022) 21(5):103056. doi: 10.1016/j.autrev.2022.103056
165. Shao T, Shi X, Yang S, Zhang W, Li X, Shu J, et al. Interstitial Lung Disease in
Connective Tissue Disease: A Common Lesion With Heterogeneous
Mechanisms and Treatment Considerations. Front Immunol (2021)
12:684699. doi: 10.3389/fimmu.2021.684699
166. Perelas A, Silver RM, Arrossi AV, Highland KB. Systemic Sclerosis-
Associated Interstitial Lung Disease. Lancet Respir Med (2020) 8(3):304–
20. doi: 10.1016/S2213-2600(19)30480-1
167. Lambre CR, Pilatte Y, Le Maho S, Greffard A, De Cremoux H, Bignon J.
Sialidase Activity and Antibodies to Sialidase-Treated Autologous
Erythrocytes in Bronchoalveolar Lavages From Patients With Idiopathic
Pulmonary Fibrosis or Sarcoidosis. Clin Exp Immunol (1988) 73(2):230–5.
168. Kohno N, Kyoizumi S, Awaya Y, Fukuhara H, Yamakido M, Akiyama M.
New Serum Indicator of Interstitial Pneumonitis Activity. Sialylated
Carbohydrate Antigen Kl-6. Chest (1989) 96(1):68–73. doi: 10.1378/
chest.96.1.68
169. Kohno N, Awaya Y, Oyama T, Yamakido M, Akiyama M, Inoue Y, et al. Kl-
6, a Mucin-Like Glycoprotein, in Bronchoalveolar Lavage Fluid From
Patients With Interstitial Lung Disease. Am Rev Respir Dis (1993) 148
(3):637–42. doi: 10.1164/ajrccm/148.3.637
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 88307921

170. Kohno N, Yokoyama A, Hirasawa Y, Kondo K, Fujino S, Abe M, et al.
Comparative Studies of Circulating Kl-6, Type Iii Procollagen N-Terminal
Peptide and Type Iv Collagen 7s in Patients With Interstitial Pneumonitis
and Alveolar Pneumonia. Respir Med (1997) 91(9):558–61. doi: 10.1016/
s0954-6111(97)90090-1
171. Hamai K, Iwamoto H, Ishikawa N, Horimasu Y, Masuda T, Miyamoto S,
et al. Comparative Study of Circulating Mmp-7, Ccl18, Kl-6, Sp-A, and Sp-D
as Disease Markers of Idiopathic Pulmonary Fibrosis. Dis Markers (2016)
2016:4759040. doi: 10.1155/2016/4759040
172. Ohnishi H, Yokoyama A, Kondo K, Hamada H, Abe M, Nishimura K, et al.
Comparative Study of Kl-6, Surfactant Protein-A, Surfactant Protein-D, and
Monocyte Chemoattractant Protein-1 as Serum Markers for Interstitial Lung
Diseases. Am J Respir Crit Care Med (2002) 165(3):378–81. doi: 10.1164/
ajrccm.165.3.2107134
173. Ohshimo S, Ishikawa N, Horimasu Y, Hattori N, Hirohashi N, Tanigawa K,
et al. Baseline Kl-6 Predicts Increased Risk for Acute Exacerbation of
Idiopathic Pulmonary Fibrosis. Respir Med (2014) 108(7):1031–9.
doi: 10.1016/j.rmed.2014.04.009
174. Yokoyama A, Kohno N, Kondo K, Ueda S, Hirasawa Y, Watanabe K, et al.
Comparative Evaluation of Sialylated Carbohydrate Antigens, Kl-6, Ca19-9
and Slx as Serum Markers for Interstitial Pneumonia. Respirology (1998) 3
(3):199–202. doi: 10.1111/j.1440-1843.1998.tb00121.x
175. Aloisio E, Braga F, Puricelli C, Panteghini M. Prognostic Role of Krebs Von
Den Lungen-6 (Kl-6) Measurement in Idiopathic Pulmonary Fibrosis: A
Systematic Review and Meta-Analysis. Clin Chem Lab Med (2021) 59
(8):1400–8. doi: 10.1515/cclm-2021-0199
176. Zhang H, Chen L, Wu L, Huang J, Li H, Wang X, et al. Diagnostic and
Prognostic Predictive Values of Circulating Kl-6 for Interstitial Lung Disease:
A Prisma-Compliant Systematic Review and Meta-Analysis. Med
(Baltimore) (2020) 99(16):e19493. doi: 10.1097/MD.0000000000019493
177. Bonella F, Long X, Ohshimo S, Horimasu Y, Griese M, Guzman J, et al. Muc1
Gene Polymorphisms Are Associated With Serum Kl-6 Levels and
Pulmonary Dysfunction in Pulmonary Alveolar Proteinosis. Orphanet J
Rare Dis (2016) 11:48. doi: 10.1186/s13023-016-0430-2
178. Horimasu Y, Hattori N, Ishikawa N, Kawase S, Tanaka S, Yoshioka K, et al.
Different Muc1 Gene Polymorphisms in German and Japanese Ethnicities
Affect Serum Kl-6 Levels. Respir Med (2012) 106(12):1756–64. doi: 10.1016/
j.rmed.2012.09.001
179. Janssen R, Kruit A, Grutters JC, Ruven HJ, Gerritsen WB, van den Bosch JM.
The Mucin-1 568 Adenosine to Guanine Polymorphism Influences Serum
Krebs Von Den Lungen-6 Levels. Am J Respir Cell Mol Biol (2006) 34
(4):496–9. doi: 10.1165/rcmb.2005-0151OC
180. van den Blink B, Wijsenbeek MS, Hoogsteden HC. Serum Biomarkers in
Idiopathic Pulmonary Fibrosis. Pulm Pharmacol Ther (2010) 23(6):515–20.
doi: 10.1016/j.pupt.2010.08.001
181. d’Alessandro M, Bergantini L, Cameli P, Vietri L, Lanzarone N, Alonzi V,
et al. Krebs Von Den Lungen-6 as a Biomarker for Disease Severity
Assessment in Interstitial Lung Disease: A Comprehensive Review.
Biomark Med (2020) 14(8):665–74. doi: 10.2217/bmm-2019-0545
182. Zhang Y, Kaminski N. Biomarkers in Idiopathic Pulmonary Fibrosis. Curr
Opin Pulm Med (2012) 18(5):441–6. doi: 10.1097/MCP.0b013e328356d03c
183. Vij R, Noth I. Peripheral Blood Biomarkers in Idiopathic Pulmonary
Fibrosis. Transl Res (2012) 159(4):218–27. doi: 10.1016/j.trsl.2012.01.012
184. Ohshimo S, Yokoyama A, Hattori N, Ishikawa N, Hirasawa Y, Kohno N. Kl-
6, a Human Muc1 Mucin, Promotes Proliferation and Survival of Lung
Fibroblasts. Biochem Biophys Res Commun (2005) 338(4):1845–52.
doi: 10.1016/j.bbrc.2005.10.144
185. Xu L, Yang D, Zhu S, Gu J, Ding F, Bian W, et al. Bleomycin-Induced
Pulmonary Fibrosis Is Attenuated by an Antibody Against Kl-6. Exp Lung
Res (2013) 39(6):241–8. doi: 10.3109/01902148.2013.798056
186. Milara J, Ballester B, Montero P, Escriva J, Artigues E, Alos M, et al. Muc1
Intracellular Bioactivation Mediates Lung Fibrosis. Thorax (2020) 75(2):132–
42. doi: 10.1136/thoraxjnl-2018-212735
187. Ballester B, Milara J, Cortijo J. Pirfenidone Anti-Fibrotic Effects Are Partially
Mediated by the Inhibition of Muc1 Bioactivation. Oncotarget (2020) 11
(15):1306–20. doi: 10.18632/oncotarget.27526
188. Kato K, Zemskova MA, Hanss AD, Kim MM, Summer R, Kim KC. Muc1
Deficiency Exacerbates Pulmonary Fibrosis in a Mouse Model of Silicosis.
Biochem Biophys Res Commun (2017) 493(3):1230–5. doi: 10.1016/
j.bbrc.2017.09.047
189. Ohtsuki Y, Fujita J, Hachi suka Y, Uomo to M, Okada Y, Yoshinouchi T,
et al. Immunohistochemical and Immunoelectron Microscopic Studies
of the Localization of Kl-6 and Epithelial Membrane Antigen (Ema) in
Presumably Normal Pulmonary Tissue and in Interstitial Pneumonia.
Med Mol Morphol (2007) 40(4):198–202. doi: 10.1007/s00795-007-
0382-7
190. Ballester B, Milara J, Cortijo J. The Role of Mucin 1 in Respiratory Diseases.
Eur Respir Rev (2021) 30(159):200149. doi: 10.1183/16000617.0149-2020
191. Pardo A, Cabrera S, Maldonado M, Selman M. Role of Matrix
Metalloproteinases in the Pathogenesis of Idiopathic Pulmonary Fibrosis.
Respir Res (2016) 17:23. doi: 10.1186/s12931-016-0343-6
192. Klein C, Troedsson M. Equine Pre-Implantation Conceptuses Express
Neuraminidase 2–A Potential Mechanism for Desialylation of the Equine
Capsule. Reprod Domest Anim (2012) 47(3):449–54. doi: 10.1111/j.1439-
0531.2011.01901.x
193. Tringali C, Papini N, Fusi P, Croci G, Borsani G, Preti A, et al. Properties of
Recombinant Human Cytosolic Sialidase Hsneu2. The Enzyme Hydrolyzes
Monomerically Dispersed Gm1 Ganglioside Molecules. J Biol Chem (2004)
279(5):3169–79. doi: 10.1074/jbc.M308381200
194. Bhargava AK, Woitach JT, Davidson EA, Bhavanandan VP. Cloning and
Cdna Sequence of a Bovine Submaxillary Gland Mucin-Like Protein
Containing Two Distinct Domains. Proc Natl Acad Sci USA (1990) 87
(17):6798–802. doi: 10.1073/pnas.87.17.6798
195. Eckhardt AE, Timpte CS, DeLuca AW, Hill RL. The Complete Cdna
Sequence and Structural Polymorphism of the Polypeptide Chain of
Porcine Submaxillary Mucin. JBiolChem(1997) 272(52):33204–10.
doi: 10.1074/jbc.272.52.33204
196. Jiang W, Woitach JT, Gupta D, Bhavanandan VP. Sequence of a Second
Gene Encoding Bovine Submaxillary Mucin: Implication for Mucin
Heterogeneity and Cloning. Biochem Biophys Res Commun (1998) 251
(2):550–6. doi: 10.1006/bbrc.1998.9515
197. Seyrantepe V, Landry K, Trudel S, Hassan JA, Morales CR, Pshezhetsky AV.
Neu4, a Novel Human Lysosomal Lumen Sialidase, Confers Normal
Phenotype to Sialidosis and Galactosialidosis Cells. J Biol Chem (2004) 279
(35):37021–9. doi: 10.1074/jbc.M404531200
198. Yamaguchi K, Hata K, Koseki K, Shiozaki K, Akita H, Wada T, et al.
Evidence for Mitochondrial Localization of a Novel Human Sialidase (Neu4).
Biochem J (2005) 390(Pt 1):85–93. doi: 10.1042/BJ20050017
199. Finlay TM, Abdulkhalek S, Gilmour A, Guzzo C, Jayanth P, Amith SR, et al.
Thymoquinone-Induced Neu4 Sialidase Activates Nfkappab in Macrophage
Cells and Pro-Inflammatory Cytokines In Vivo.Glycoconj J (2010) 27
(6):583–600. doi: 10.1007/s10719-010-9302-5
200. Kishore A, Zizkova V, Kocourkova L, Petrkova J, Bouros E, Nunes H, et al.
Association Study for 26 Candidate Loci in Idiopathic Pulmonary Fibrosis
Patients From Four European Populations. Front Immunol (2016) 7:274.
doi: 10.3389/fimmu.2016.00274
201. Plantier L, Crestani B, Wert SE, Dehoux M, Zweytick B, Guenther A, et al.
Ectopic Respiratory Epithelial Cell Differentiation in Bronchiolised Distal
Airspaces in Idiopathic Pulmonary Fibrosis. Thorax (2011) 66(8):651–7.
doi: 10.1136/thx.2010.151555
202. Lorenzo-Salazar JM, Ma SF, Jou J, Hou PC, Guillen-Guio B, Allen RJ, et al.
Novel Idiopathic Pulmonary Fibrosis Susceptibility Variants Revealed by
Deep Sequencing. ERJ Open Res (2019) 5(2):00071–2019. doi: 10.1183/
23120541.00071-2019
203. Seibold MA, Smith RW, Urbanek C, Groshong SD, Cosgrove GP, Brown KK,
et al. The Idiopathic Pulmonary Fibrosis Honeycomb Cyst Contains a
Mucocilary Pseudostratified Epithelium. PLoS One (2013) 8(3):e58658.
doi: 10.1371/journal.pone.0058658
204. Pang J, Luo Y, Wei D, Cao Z, Qi X, Song M, et al. Comparative
Transcriptome Analyses Reveal a Transcriptional Landscape of Human
Silicosis Lungs and Provide Potential Strategies for Silicosis Treatment.
Front Genet (2021) 12:652901. doi: 10.3389/fgene.2021.652901
205. He L, Feng QQ, Zhang Q, Zhang B, Wu SS, Gong JH. Protective Role of
Overexpressed Muc5ac Against Fibrosis in Mhv-68-Induced Combined
Pulmonary Fibrosis and Emphysema Mouse Model. J Med Virol (2020) 92
(12):3726–35. doi: 10.1002/jmv.26094
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 88307922

206. Hoffmann J, Wilhelm J, Marsh LM, Ghanim B, Klepetko W, Kovacs G, et al.
Distinct Differences in Gene Expression Patterns in Pulmonary Arteries of
Patients With Chronic Obstructive Pulmonary Disease and Idiopathic
Pulmonary Fibrosis With Pulmonary Hypertension. Am J Respir Crit Care
Med (2014) 190(1):98–111. doi: 10.1164/rccm.201401-0037OC
207. Maher TM, Oballa E, Simpson JK, Porte J, Habgood A, Fahy WA, et al. An
Epithelial Biomarker Signature for Idiopathic Pulmonary Fibrosis: An
Analysis From the Multicentre Profile Cohort Study. Lancet Respir Med
(2017) 5(12):946–55. doi: 10.1016/S2213-2600(17)30430-7
208. Milara J, Ballester B, Safont MJ, Artigues E, Escriva J, Morcillo E, et al. Muc4
Is Overexpressed in Idiopathic Pulmonary Fibrosis and Collaborates With
Transforming Growth Factor Beta Inducing Fibrotic Responses. Mucosal
Immunol (2021) 14(2):377–88. doi: 10.1038/s41385-020-00343-w
209. Ballester B, Milara J, Montero P, Cortijo J. Muc16 Is Overexpressed in
Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by
Transforming Growth Factor-Beta1 Canonical Pathway. Int J Mol Sci (2021)
22(12):6502. doi: 10.3390/ijms22126502
210. Seibold MA, Wise AL, Speer MC, Steele MP, Brown KK, Loyd JE, et al. A
Common Muc5b Promoter Polymorphism and Pulmonary Fibrosis. N Engl J
Med (2011) 364(16):1503–12. doi: 10.1056/NEJMoa1013660
211. Evans CM, Fingerlin TE, Schwarz MI, Lynch D, Kurche J, Warg L, et al.
Idiopathic Pulmonary Fibrosis: A Genetic Disease That Involves Mucociliary
Dysfunction of the Peripheral Airways. Physiol Rev (2016) 96(4):1567–91.
doi: 10.1152/physrev.00004.2016
212. Schwartz DA. Idiopathic Pulmonary Fibrosis Is a Genetic Disease Involving
Mucus and the Peripheral Airways. Ann Am Thorac Soc (2018) 15(Suppl 3):
S192–S7. doi: 10.1513/AnnalsATS.201802-144AW
213. Juge PA, Lee JS, Ebstein E, Furukawa H, Dobrinskikh E, Gazal S, et al. Muc5b
Promoter Variant and Rheumatoid Arthritis With Interstitial Lung Disease.
N Engl J Med (2018) 379(23):2209–19. doi: 10.1056/NEJMoa1801562
214. Hancock LA, Hennessy CE, Solomon GM, Dobrinskikh E, Estrella A, Hara
N, et al. Muc5b Overexpression Causes Mucociliary Dysfunction and
Enhances Lung Fibrosis in Mice. Nat Commun (2018) 9(1):5363.
doi: 10.1038/s41467-018-07768-9
215. Juge PA, Solomon JJ, van Moorsel CHM, Garofoli R, Lee JS, Louis-Sydney F,
et al. Muc5b Promoter Variant Rs35705950 and Rheumatoid Arthritis
Associated Interstitial Lung Disease Survival and Progression. Semin
Arthritis Rheum (2021) 51(5):996–1004. doi: 10.1016/j.semarthrit.
2021.07.002
216. Pier GB, Ramphal R. Pseudomonas Aeruginosa. In: GL Mandell, JE Bennett
and R Dolin, editors. Mandell, Douglas, and Bennett’s Primciples and Practice
Infectious Diseases,2. 7th ed. Philadelphia, PA: Churchill Livingstone
Elsevier (2010). p. 2835–60.
217. Ramphal R, Guay C, Pier GB. Pseudomonas Aeruginosa Adhesins for
Tracheobronchial Mucin. Infect Immun (1987) 55(3):600–3. doi: 10.1128/
iai.55.3.600-603.1987
218. Reddy MS. Human Tracheobronchial Mucin: Purification and Binding to
Pseudomonas Aeruginosa. Infect Immun (1992) 60(4):1530–5. doi: 10.1128/
iai.60.4.1530-1535.1992
219. Sajjan U, Reisman J, Doig P, Irvin RT, Forstner G, Forstner J. Binding of
Nonmucoid Pseudomonas Aeruginosa to Normal Human Intestinal Mucin
and Respiratory Mucin From Patients With Cystic Fibrosis. J Clin Invest
(1992) 89(2):657–65. doi: 10.1172/JCI115632
220. Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, et al. Muc1
Mucins on the Cell Surface Are Adhesion Sites for Pseudomonas Aeruginosa.
Am J Physiol Lung Cell Mol Physiol (2001) 280(1):L181–7. doi: 10.1152/
ajplung.2001.280.1.L181
221. Kato K, Lillehoj EP, Kai H, Kim KC. Muc1 Expression by Human Airway
Epithelial Cells Mediates Pseudomonas Aeruginosa Adhesion. Front Biosci
(Elite Ed) (2010) 2:68–77. doi: 10.2741/e67
222. Lillehoj EP, Kim H, Chun EY, Kim KC. Pseudomonas Aeruginosa Stimulates
Phosphorylation of the Airway Epithelial Membrane Glycoprotein Muc1
and Activates Map Kinase. Am J Physiol Lung Cell Mol Physiol (2004) 287(4):
L809–15. doi: 10.1152/ajplung.00385.2003
223. Lu W, Hisatsune A, Koga T, Kato K, Kuwahara I, Lillehoj EP, et al. Cutting
Edge: Enhanced Pulmonary Clearance of Pseudomonas Aeruginosa by Muc1
Knockout Mice. JImmunol(2006) 176(7):3890–4. doi: 10.4049/
jimmunol.176.7.3890
224. Ueno K, Koga T, Kato K, Golenbock DT, Gendler SJ, Kai H, et al. Muc1
Mucin Is a Negative Regulator of Toll-Like Receptor Signaling. Am J Respir
Cell Mol Biol (2008) 38(3):263–8. doi: 10.1165/rcmb.2007-0336RC
225. Williams MA, Bauer S, Lu W, Guo J, Walter S, Bushnell TP, et al. Deletion of
the Mucin-Like Molecule Muc1 Enhances Dendritic Cell Activation in
Response to Toll-Like Receptor Ligands. J Innate Immun (2010) 2(2):123–
43. doi: 10.1159/000254790
226. Choi S, Park YS, Koga T, Treloar A, Kim KC. Tnf-Alpha Is a Key Regulator
of Muc1, an Anti-Inflammatory Molecule, During Airway Pseudomonas
Aeruginosa Infection. Am J Respir Cell Mol Biol (2011) 44(2):255–60.
doi: 10.1165/rcmb.2009-0323OC
227. Kato K, Lillehoj EP, Park YS, Umehara T, Hoffman NE, Madesh M, et al.
Membrane-Tethered Muc1 Mucin Is Phosphorylated by Epidermal Growth
Factor Receptor in Airway Epithelial Cells and Associates With Tlr5 to
Inhibit Recruitment of Myd88. J Immunol (2012) 188(4):2014–22.
doi: 10.4049/jimmunol.1102405
228. Umehara T, Kato K, Park YS, Lillehoj EP, Kawauchi H, Kim KC. Prevention
of Lung Injury by Muc1 Mucin in a Mouse Model of Repetitive
Pseudomonas Aeruginosa Infection. Inflammation Res (2012) 61(9):1013–
20. doi: 10.1007/s00011-012-0494-y
229. Milara J, Peiro T, Armengot M, Frias S, Morell A, Serrano A, et al. Mucin 1
Downregulation Associates With Corticosteroid Resistance in Chronic
Rhinosinusitis With Nasal Polyps. J Allergy Clin Immunol (2015) 135
(2):470–6. doi: 10.1016/j.jaci.2014.07.011
230. Bachert C, Linstedt AD. A Sensor of Protein O-Glycosylation Based on
Sequential Processing in the Golgi Apparatus. Traffic(2013) 14(1):47–56.
doi: 10.1111/tra.12019
231. Magesh S, Moriya S, Suzuki T, Miyagi T, Ishida H, Kiso M. Design, Synthesis,
and Biological Evaluation of Human Sialidase Inhibitors. Part 1: Selective
Inhibitors of Lysosomal Sialidase (Neu1). Bioorg Med Chem Lett (2008) 18
(2):532–7. doi: 10.1016/j.bmcl.2007.11.084
232. Verceles AC, Bhat P, Nagaria Z, Martin D, Patel H, Ntem-Mensah A, et al.
Muc1 Ectodomain Is a Flagellin-Targeting Decoy Receptor and Biomarker
Operative During Pseudomonas Aeruginosa Lung Infection. Sci Rep (2021)
11(1):22725. doi: 10.1038/s41598-021-02242-x
233. Hyun SW, Imamura A, Ishida H, Piepenbrink KH, Goldblum SE, Lillehoj
EP. The Sialidase Neu1 Directly Interacts With the Juxtamembranous
Segment of the Cytoplasmic Domain of Mucin-1 to Inhibit Downstream
Pi3k-Akt Signaling. JBiolChem(2021) 297(5):101337. doi: 10.1016/
j.jbc.2021.101337
234. Yang WH, Heithoff DM, Aziz PV, Haslund-Gourley B, Westman JS,
Narisawa S, et al. Accelerated Aging and Clearance of Host Anti-
Inflammatory Enzymes by Discrete Pathogens Fuels Sepsis. Cell Host
Microbe (2018) 24(4):500–13 e5. doi: 10.1016/j.chom.2018.09.011
235. Yang WH, Heithoff DM, Aziz PV, Sperandio M, Nizet V, Mahan MJ, et al.
Recurrent Infection Progressively Disables Host Protection Against
Intestinal Inflammation. Science (2017) 358(6370):eaao5610. doi: 10.1126/
science.aao5610
236. Yang WH, Westman JS, Heithoff DM, Sperandio M, Cho JW, Mahan MJ,
et al. Neu3 Neuraminidase Induction Triggers Intestinal Inflammation and
Colitis in a Model of Recurrent Human Food-Poisoning. Proc Natl Acad Sci
USA (2021) 118(29):e2100937118. doi: 10.1073/pnas.2100937118
237. Chen QQ, Ma G, Liu JF, Cai YY, Zhang JY, Wei TT, et al. Neuraminidase 1 Is
a Driver of Experimental Cardiac Hypertrophy. Eur Heart J (2021) 42
(36):3770–82. doi: 10.1093/eurheartj/ehab347
238. Thulasiraman P, Kerr K, McAlister K, Hardisty S, Wistner A, McCullough I.
Neuraminidase 1 Regulates Proliferation, Apoptosis and the Expression of
Cadherins in Mammary Carcinoma Cells. Mol Cell Biochem (2019) 462(1-
2):207–15. doi: 10.1007/s11010-019-03623-7
239. Kapishon V, Allison S, Whitney RA, Cunningham MF, Szewczuk MR,
Neufeld RJ. Oseltamivir-Conjugated Polymeric Micelles Prepared by Raft
Living Radical Polymerization as a New Active Tumor Targeting Drug
Delivery Platform. Biomater Sci (2016) 4(3):511–21. doi: 10.1039/
c5bm00519a
240. Formiga RO, Amaral FC, Souza CF, Mendes D, Wanderley CWS, Lorenzini
CB, et al. Neuraminidase Inhibitors Rewire Neutrophil Function In Vivo in
Murine Sepsis and Ex Vivo in Covid-19. bioRxiv (2021) 2020:11.12.379115.
doi: 10.1101/2020.11.12.379115
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 88307923

241. Teixeira A, David L, Reis CA, Costa J, Sobrinho-Simoes M. Expressionof Mucins
(Muc1, Muc2, Muc5ac, and Muc6) and Type 1 Lewis Antigens in Cases With and
Without Helicobacter Pylori ColonizationinMetaplasticGlandsoftheHuman
Stomach. JPathol(2002) 197(1):37–43. doi: 10.1002/path.1083
242. Linden S, Nordman H, Hedenbro J, Hurtig M, Boren T, Carlstedt I. Strain-
and Blood Group-Dependent Binding of Helicobacter Pylori to Human
Gastric Muc5ac Glycoforms. Gastroenterology (2002) 123(6):1923–30.
doi: 10.1053/gast.2002.37076
243. Linden S, Mahdavi J, Hedenbro J, Boren T, Carlstedt I. Effects of Ph on
Helicobacter Pylori Binding to Human Gastric Mucins: Identification of
Binding to Non-Muc5ac Mucins. Biochem J (2004) 384(Pt 2):263–70.
doi: 10.1042/BJ20040402
244. McGuckin MA, Every AL, Skene CD, Linden SK, Chionh YT, Swierczak A,
et al. Muc1 Mucin Limits Both Helicobacter Pylori Colonization of the
Murine Gastric Mucosa and Associated Gastritis. Gastroenterology (2007)
133(4):1210–8. doi: 10.1053/j.gastro.2007.07.003
245. Costa NR, Mendes N, Marcos NT, Reis CA, Caffrey T, Hollingsworth MA,
et al. Relevance of Muc1 Mucin Variable Number of Tandem Repeats
Polymorphism in H Pylori Adhesion to Gastric Epithelial Cells. World J
Gastroenterol (2008) 14(9):1411–4. doi: 10.3748/wjg.14.1411
246. Radziejewska I, Borzym-Kluczyk M, Leszczynska K. Are Lewis B and HType
1 on Helicobacter Pylori Involved in Binding of Bacteria to Muc1 Mucin?
Adv Clin Exp Med (2013) 22(3):347–53.
247. Koning N, Kessen SF, van der Voorn JP, Appelmelk BJ, Jeurink PV, Knippels
LM, et al. Human Milk Blocks Dc-Sign-Pathogen Interaction Via Muc1.
Front Immunol (2015) 6:112. doi: 10.3389/fimmu.2015.00112
248. Radziejewska I, Borzym-Kluczyk M, Leszczynska K. Luteolin Alters Muc1
Extracellular Domain, St Antigen, Adam-17, Il-8, Il-10 and Nf-Kappab
Expression in Helicobacter Pylori-Infected Gastric Cancer Crl-1739 Cells:
A Preliminary Study. BioMed Rep (2021) 14(2):19. doi: 10.3892/br.2020.1395
249. Guang W, Ding H, Czinn SJ, Kim KC, Blanchard TG, Lillehoj EP. Muc1 Cell
Surface Mucin Attenuates Epithelial Inflammation in Response to a
Common Mucosal Pathogen. JBiolChem(2010) 285(27):20547–57.
doi: 10.1074/jbc.M110.121319
250. Guang W, Twaddell WS, Lillehoj EP. Molecular Interactions Between Muc1
Epithelial Mucin, Beta-Catenin, and Caga Proteins. Front Immunol (2012)
3:105. doi: 10.3389/fimmu.2012.00105
251. Lillehoj EP, Guang W, Ding H, Czinn SJ, Blanchard TG. Helicobacter Pylori
and Gastric Inflammation: Role of Muc1 Mucin. J Pediatr Biochem (2012) 2
(3):125–32. doi: 10.3233/JPB-2012-00058
252. Park YS, Guang W, Blanchard TG, Kim KC, Lillehoj EP. Suppression of Il-8
Production in Gastric Epithelial Cells by Muc1 Mucin and Peroxisome
Proliferator-Associated Receptor-Gamma. Am J Physiol Gastrointest Liver
Physiol (2012) 303(6):G765–74. doi: 10.1152/ajpgi.00023.2012
253. Sheng YH, Triyana S, Wang R, Das I, Gerloff K, Florin TH, et al. Muc1 and
Muc13 Differentially Regulate Epithelial Inflammation in Response to
Inflammatory and Infectious Stimuli. Mucosal Immunol (2013) 6(3):557–
68. doi: 10.1038/mi.2012.98
254. Guang W, Czinn SJ, Blanchard TG, Kim KC, Lillehoj EP. Genetic Regulation
of Muc1 Expression by Helicobacter Pylori in Gastric Cancer Cells. Biochem
Biophys Res Commun (2014) 445(1):145–50. doi: 10.1016/j.bbrc.2014.01.142
255. Ng GZ, Menheniott TR, Every AL, Stent A, Judd LM, Chionh YT, et al. The
Muc1 Mucin Protects Against Helicobacter Pylori Pathogenesis in Mice by
Regulation of the Nlrp3 Inflammasome. Gut (2016) 65(7):1087–99.
doi: 10.1136/gutjnl-2014-307175
256. Sheng YH, Ng GZ, Summers KM, Every AL, Price G, Hasnain SZ, et al.
InfluenceoftheMuc1CellSurfaceMucin on Gastric Mucosal Gene
Expression Profiles in Response to Helicobacter Pylori Infection in Mice.
Front Cell Infect Microbiol (2020) 10:343. doi: 10.3389/fcimb.2020.00343
257. Kardon R, Price RE, Julian J, Lagow E, Tseng SC, Gendler SJ, et al. Bacterial
Conjunctivitis in Muc1 Null Mice. Invest Ophthalmol Vis Sci (1999) 40
(7):1328–35.
258. DeSouza MM, Surveyor GA, Price RE, Julian J, Kardon R, Zhou X, et al. Muc1/
Episialin: A Critical Barrier in the Female Reproductive Tract. J Reprod Immunol
(1999) 45(2):127–58. doi: 10.1016/s0165-0378(99)00046-7
259. McAuley JL, Linden SK, Png CW, King RM, Pennington HL, Gendler SJ,
et al. Muc1 Cell Surface Mucin Is a Critical Element of the Mucosal Barrier to
Infection. J Clin Invest (2007) 117(8):2313–24. doi: 10.1172/JCI26705
260. Sando L, Pearson R, Gray C, Parker P, Hawken R, Thomson PC, et al. Bovine
Muc1 Is a Highly Polymorphic Gene Encoding an Extensively Glycosylated
Mucin That Binds Bacteria. J Dairy Sci (2009) 92(10):5276–91. doi: 10.3168/
jds.2009-2216
261. Parker P, Sando L, Pearson R, Kongsuwan K, Tellam RL, Smith S. Bovine
Muc1 Inhibits Binding of Enteric Bacteria to Caco-2 Cells. Glycoconj J (2010)
27(1):89–97. doi: 10.1007/s10719-009-9269-2
262. Kyo Y, Kato K, Park YS, Gajghate S, Umehara T, Lillehoj EP, et al.
Antiinflammatory Role of Muc1 Mucin During Infection With
Nontypeable Haemophilus Influenzae. Am J Respir Cell Mol Biol (2012) 46
(2):149–56. doi: 10.1165/rcmb.2011-0142OC
263. Ng GZ, Sutton P. The Muc1 Mucin Specifically Inhibits Activation of the
Nlrp3 Inflammasome. Genes Immun (2016) 17(3):203–6. doi: 10.1038/
gene.2016.10
264. Boll EJ, Ayala-Lujan J, Szabady RL, Louissaint C, Smith RZ, Krogfelt KA,
et al. Enteroaggregative Escherichia Coli Adherence Fimbriae Drive
Inflammatory Cell Recruitment Via Interactions With Epithelial Muc1.
mBio (2017) 8(3):e00717–17. doi: 10.1128/mBio.00717-17
265. Dhar P, Ng GZ, Dunne EM, Sutton P. Mucin 1 Protects Against Severe
Streptococcus Pneumoniae Infection. Virulence (2017) 8(8):1631–42.
doi: 10.1080/21505594.2017.1341021
266. Li X, Bleumink-Pluym NMC, Luijkx Y, Wubbolts RW, van Putten JPM,
Strijbis K. Muc1 Is a Receptor for the Salmonella Siie Adhesin That Enables
Apical Invasion Into Enterocytes. PLoS Pathog (2019) 15(2):e1007566.
doi: 10.1371/journal.ppat.1007566
267. Li X, Wubbolts RW, Bleumink-Pluym NMC, van Putten JPM, Strijbis K. The
Transmembrane Mucin Muc1 Facilitates Beta1-Integrin-Mediated Bacterial
Invasion. mBio (2021) 12(2):e03491–20. doi: 10.1128/mBio.03491-20
268. Pech M, Weckmann M, Konig IR, Franke A, Heinsen FA, Oliver B, et al.
Rhinovirus Infections Change DNA Methylation and Mrna Expression in
Children With Asthma. PLoS One (2018) 13(11):e0205275. doi: 10.1371/
journal.pone.0205275
269. Kong F, Li N, Tu T, Tao Y, Bi Y, Yuan D, et al. Hepatitis B Virus Core Protein
Promotes the Expression of Neuraminidase 1 to Facilitate
Hepatocarcinogenesis. Lab Invest (2020) 100(12):1602–17. doi: 10.1038/
s41374-020-0465-9
270. Murray J, Todd KV, Bakre A, Orr-Burks N, Jones L, Wu W, et al. A Universal
Mammalian Vaccine Cell Line Substrate. PLoS One (2017) 12(11):e0188333.
doi: 10.1371/journal.pone.0188333
271. Anastasia L, Holguera J, Bianchi A, D’Avila F, Papini N, Tringali C, et al.
Over-Expression of Mammalian Sialidase Neu3 Reduces Newcastle Disease
Virus Entry and Propagation in Cos7 Cells. Biochim Biophys Acta (2008)
1780(3):504–12. doi: 10.1016/j.bbagen.2007.11.011
272. Lu W, Liu X, Wang T, Liu F, Zhu A, Lin Y, et al. Elevated Muc1 and Muc5ac
Mucin Protein Levels in Airway Mucus of Critical Ill Covid-19 Patients.
J Med Virol (2021) 93(2):582–4. doi: 10.1002/jmv.26406
273. Kim D, Maharjan S, Kim J, Park S, Park JA, Park BK, et al. Muc1-C
Influences Cell Survival in Lung Adenocarcinoma Calu-3 Cells After Sars-
Cov-2 Infection. BMB Rep (2021) 54(8):425–30. doi: 10.5483/
BMBRep.2021.54.8.018
274. Habte HH, de Beer C, Lotz ZE, Tyler MG, Kahn D, Mall AS. Inhibition of
Human Immunodeficiency Virus Type 1 Activity by Purified Human Breast
Milk Mucin (Muc1) in an Inhibition Assay. Neonatology (2008) 93(3):162–
70. doi: 10.1159/000108414
275. Saeland E, de Jong MA, Nabatov AA, Kalay H, Geijtenbeek TB, van Kooyk Y.
Muc1 in Human Milk Blocks Transmission of Human Immunodeficiency
Virus From Dendritic Cells to T Cells. Mol Immunol (2009) 46(11-12):2309–
16. doi: 10.1016/j.molimm.2009.03.025
276. Li Y, Dinwiddie DL, Harrod KS, Jiang Y, Kim KC. Anti-Inflammatory Effect
of Muc1 During Respiratory Syncytial Virus Infection of Lung Epithelial
Cells In Vitro.Am J Physiol Lung Cell Mol Physiol (2010) 298(4):L558–63.
doi: 10.1152/ajplung.00225.2009
277. Nguyen Y, Procario MC, Ashley SL, O’Neal WK, Pickles RJ, Weinberg JB.
Limited Effects of Muc1 Deficiency on Mouse Adenovirus Type 1
Respiratory Infection. Virus Res (2011) 160(1-2):351–9. doi: 10.1016/
j.virusres.2011.07.012
278. Mthembu Y, Lotz Z, Tyler M, de Beer C, Rodrigues J, Schoeman L, et al.
Purified Human Breast Milk Muc1 and Muc4 Inhibit Human
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 88307924

Immunodeficiency Virus. Neonatology (2014) 105(3):211–7. doi: 10.1159/
000357201
279. McAuley JL, Corcilius L, Tan HX, Payne RJ, McGuckin MA, Brown LE. The
Cell Surface Mucin Muc1 Limits the Severity of Influenza a Virus Infection.
Mucosal Immunol (2017) 10(6):1581–93. doi: 10.1038/mi.2017.16
280. Li K, Cao P, McCaw JM. Modelling the Effect of Muc1 on Influenza Virus
Infection Kinetics and Macrophage Dynamics. Viruses (2021) 13(5):850.
doi: 10.3390/v13050850
281. Karmakar J, Mandal C. Interplay Between Sialic Acids, Siglec-E, and Neu1
Regulates Myd88- and Trif-Dependent Pathways for Tlr4-Activation During
Leishmania Donovani Infection. Front Immunol (2021) 12:626110.
doi: 10.3389/fimmu.2021.626110
282. Takeda K, Akira S. Toll-Like Receptors in Innate Immunity. Int Immunol
(2005) 17(1):1–14. doi: 10.1093/intimm/dxh186
283. Sundararaj K, Rodgers J, Angel P, Wolf B, Nowling TK. The Role of
Neuraminidase in Tlr4-Mapk Signalling and the Release of Cytokines by
Lupus Serum-Stimulated Mesangial Cells. Immunology (2021) 162(4):418–
33. doi: 10.1111/imm.13294
284. LandolfiNF, Leone J, Womack JE, Cook RG. Activation of T Lymphocytes
Results in an Increase in H-2-Encoded Neuraminidase. Immunogenetics
(1985) 22(2):159–67. doi: 10.1007/BF00563513
285. LandolfiNF, Cook RG. Activated T-Lymphocytes Express Class I Molecules
Which Are Hyposialylated Compared to Other Lymphocyte Populations.
Mol Immunol (1986) 23(3):297–309. doi: 10.1016/0161-5890(86)90057-x
286. Chen XP, Enioutina EY, Daynes RA. The Control of Il-4 Gene Expression in
Activated Murine T Lymphocytes: A Novel Role for Neu-1 Sialidase.
J Immunol (1997) 158(7):3070–80.
287. Bilyy R, Stoika R. Search for Novel Cell Surface Markers of Apoptotic Cells.
Autoimmunity (2007) 40(4):249–53. doi: 10.1080/08916930701358867
288. Wang P, Zhang J, Bian H, Wu P, Kuvelkar R, Kung TT, et al. Induction of
Lysosomal and Plasma Membrane-Bound Sialidases in Human T-Cells Via
T-Cell Receptor. Biochem J (2004) 380(Pt 2):425–33. doi: 10.1042/
BJ20031896
289. Kaminuma O, Katoh S, Miyagi T, Watanabe N, Kitamura N, Nishimura T,
et al. Contribution of Neuraminidase 3 to the Differentiation of Induced
Regulatory T Cells. Genes Cells (2018) 23(2):112–6. doi: 10.1111/gtc.12553
290. Wei Y, Jing J, Peng Z, Liu X, Wang X. Acacetin Ameliorates Insulin
Resistance in Obesity Mice Through Regulating Treg/Th17 Balance Via
Mir-23b-3p/Neu1 Axis. BMC Endocr Disord (2021) 21(1):57. doi: 10.1186/
s12902-021-00688-8
291. Sundararaj K, Rodgers JI, Marimuthu S, Siskind LJ, Bruner E, Nowling TK.
Neuraminidase Activity Mediates Il-6 Production by Activated Lupus-Prone
Mesangial Cells. Am J Physiol Renal Physiol (2018) 314(4):F630–F42.
doi: 10.1152/ajprenal.00421.2017
292. Sehnert B, Mietz J, Rzepka R, Buchholz S, Maul-Pavicic A, Schaffer S, et al.
Neuraminidase Inhibitor Zanamivir Ameliorates Collagen-Induced
Arthritis. Int J Mol Sci (2021) 22(3):1428. doi: 10.3390/ijms22031428
293. Haxho F, Haq S, Szewczuk MR. Biased G Protein-Coupled Receptor
Agonism Mediates Neu1 Sialidase and Matrix Metalloproteinase-9
Crosstalk to Induce Transactivation of Insulin Receptor Signaling. Cell
Signal (2018) 43:71–84. doi: 10.1016/j.cellsig.2017.12.006
294. Sasaki A, Hata K, Suzuki S, Sawada M, Wada T, Yamaguchi K, et al.
Overexpression of Plasma Membrane-Associated Sialidase Attenuates
Insulin Signaling in Transgenic Mice. J Biol Chem (2003) 278(30):27896–
902. doi: 10.1074/jbc.M212200200
295. Yoshizumi S, Suzuki S, Hirai M, Hinokio Y, Yamada T, Yamada T, et al.
Increased Hepatic Expression of Ganglioside-SpecificSialidase,Neu3,
Improves Insulin Sensitivity and Glucose Tolerance in Mice. Metabolism
(2007) 56(3):420–9. doi: 10.1016/j.metabol.2006.10.027
296. Lipina C, Nardi F, Grace H, Hundal HS. Neu3 Sialidase as a Marker of
Insulin Sensitivity: Regulation by Fatty Acids. Cell Signal (2015) 27(9):1742–
50. doi: 10.1016/j.cellsig.2015.05.010
297. Miklavcic JJ, Hart TD, Lees GM, Shoemaker GK, Schnabl KL, Larsen BM,
et al. Increased Catabolism and Decreased Unsaturation of Ganglioside in
Patients With Inflammatory Bowel Disease. World J Gastroenterol (2015) 21
(35):10080–90. doi: 10.3748/wjg.v21.i35.10080
298. Bhagavatula S, Banerjee P, Sood A, Midha V, Thelma BK, Senapati S.
Multiple Allelic Associations From Genes Involved in Energy Metabolism
Were Identified in Celiac Disease. J Biosci (2021) 46:61. doi: 10.1007/s12038-
021-00184-0
299. Kim S, Becker J, Bechheim M, Kaiser V, Noursadeghi M, Fricker N, et al.
Characterizing the Genetic Basis of Innate Immune Response in Tlr4-
Activated Human Monocytes. Nat Commun (2014) 5:5236. doi: 10.1038/
ncomms6236
300. Allendorf DH, Puigdellivol M, Brown GC. Activated Microglia Desialylate
Their Surface, Stimulating Complement Receptor 3-Mediated Phagocytosis
of Neurons. Glia (2020) 68(5):989–98. doi: 10.1002/glia.23757
301. Nomura K, Vilalta A, Allendorf DH, Hornik TC, Brown GC. Activated
Microglia Desialylate and Phagocytose Cells Via Neuraminidase, Galectin-3,
and Mer Tyrosine Kinase. J Immunol (2017) 198(12):4792–801. doi: 10.4049/
jimmunol.1502532
302. Hickey MJ, Kubes P. Intravascular Immunity: The Host-Pathogen Encounter
in Blood Vessels. Nat Rev Immunol (2009) 9(5):364–75. doi: 10.1038/nri2532
303. Cross AS, Wright DG. Mobilization of Sialidase From Intracellular Stores to
the Surface of Human Neutrophils and Its Role in Stimulated Adhesion
Responses of These Cells. J Clin Invest (1991) 88(6):2067–76. doi: 10.1172/
JCI115536
304. Cross AS, Sakarya S, Rifat S, Held TK, Drysdale BE, Grange PA, et al.
Recruitment of Murine Neutrophils In Vivo Through Endogenous Sialidase
Activity. J Biol Chem (2003) 278(6):4112–20. doi: 10.1074/jbc.M207591200
305. Feng C, Li J, Snyder G, Huang W, Goldblum SE, Chen WH, et al. Antibody
Against Microbial Neuraminidases Recognizes Human Sialidase 3 (Neu3):
The Neuraminidase/Sialidase Superfamily Revisited. mBio (2017) 8(3):
e00078–17. doi: 10.1128/mBio.00078-17
306. Sakarya S, Rifat S, Zhou J, Bannerman DD, Stamatos NM, Cross AS, et al.
Mobilization of Neutrophil Sialidase Activity Desialylates the Pulmonary
Vascular Endothelial Surface and Increases Resting Neutrophil Adhesion to
and Migration Across the Endothelium. Glycobiology (2004) 14(6):481–94.
doi: 10.1093/glycob/cwh065
307. Stamatos NM, Liang F, Nan X, Landry K, Cross AS, Wang LX, et al.
Differential Expression of Endogenous Sialidases of Human Monocytes
During Cellular Differentiation Into Macrophages. FEBS J (2005) 272
(10):2545–56. doi: 10.1111/j.1742-4658.2005.04679.x
308. Sieve I, Ricke-Hoch M, Kasten M, Battmer K, Stapel B, Falk CS, et al. A
Positive Feedback Loop Between Il-1beta, Lps and Neu1 May Promote
Atherosclerosis by Enhancing a Pro-Inflammatory State in Monocytes and
Macrophages. Vascul Pharmacol (2018) 103-105:16–28. doi: 10.1016/
j.vph.2018.01.005
309. Gayral S, Garnotel R, Castaing-Berthou A, Blaise S, Fougerat A, Berge E, et al.
Elastin-Derived Peptides Potentiate Atherosclerosis Through the Immune
Neu1-Pi3kgamma Pathway. Cardiovasc Res (2014) 102(1):118–27.
doi: 10.1093/cvr/cvt336
310. White EJ,Gyulay G, LhotakS, Szewczyk MM, Chong T, Fuller MT, et al.Sialidase
Down-Regulation Reduces Non-Hdl Cholesterol, Inhibits Leukocyte
Transmigration, and Attenuates Atherosclerosis in Apoe Knockout Mice. JBiol
Chem (2018) 293(38):14689–706. doi: 10.1074/jbc.RA118.004589
311. Heimerl M, Sieve I, Ricke-Hoch M, Erschow S, Battmer K, Scherr M, et al.
Neuraminidase-1 Promotes Heart Failure After Ischemia/Reperfusion Injury
by Affecting Cardiomyocytes and Invading Monocytes/Macrophages. Basic
Res Cardiol (2020) 115(6):62. doi: 10.1007/s00395-020-00821-z
312. Tembely D, Henry A, Vanalderwiert L, Toussaint K, Bennasroune A, Blaise
S, et al. The Elastin Receptor Complex: An Emerging Therapeutic Target
Against Age-Related Vascular Diseases. Front Endocrinol (2022) 13:815356.
doi: 10.3389/fendo.2022.815356
313. Potier M, Lu Shun Yan D, Womack JE. Neuraminidase Deficiency in the
Mouse. FEBS Lett (1979) 108(2):345–8. doi: 10.1016/0014-5793(79)80560-8
314. Rottier RJ, Bonten E, d’Azzo A. A Point Mutation in the Neu-1 Locus Causes
the Neuraminidase Defect in the Sm/J Mouse. Hum Mol Genet (1998) 7
(2):313–21. doi: 10.1093/hmg/7.2.313
315. Taira S, Nariuchi H. Possible Role of Neuraminidase in Activated T Cells in
the Recognition of Allogeneic Ia. J Immunol (1988) 141(2):440–6.
316. Wipfler D, Srinivasan GV, Sadick H, Kniep B, Arming S, Willhauck-
Fleckenstein M, et al. Differentially Regulated Expression of 9-O-Acetyl
Gd3 (Cd60b) and 7-O-Acetyl-Gd3 (Cd60c) During Differentiation and
Maturation of Human T and B Lymphocytes. Glycobiology (2011) 21
(9):1161–72. doi: 10.1093/glycob/cwr050
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 88307925

317. Kijimoto-Ochiai S, Koda T, Suwama T, Matsukawa H, Fujii M, Tomobe K,
et al. Low Expression of Neu2 Sialidase in the Thymus of Sm/J Mice-
Existence of Neuraminidase Positive Cells “Neu-Medullocyte”in the Murine
Thymus. Glycoconj J (2008) 25(8):787–96. doi: 10.1007/s10719-008-9126-8
318. Kijimoto-Ochiai S, Doi N, Matsukawa H, Fujii M, Tomobe K. Localization of
Sialidase-Positive Cells Expressing Mac-1 and Immunoglobulin in the
Mouse Thymus. Glycoconj J (2004) 20(6):375–84. doi: 10.1023/B:
GLYC.0000033994.99464.ce
319. Kijimoto-Ochiai S, Kamimura K, Koda T. Neu-Medullocytes, Sialidase-
Positive B Cells in the Thymus, Express Autoimmune Regulator (Aire). Sci
Rep (2019) 9(1):858. doi: 10.1038/s41598-018-37225-y
320. Koda T, Kijimoto-Ochiai S, Uemura S, Inokuchi J. Specific Expression of
Neu2 Type B in Mouse Thymus and the Existence of a Membrane-Bound
Form in Cos Cells. Biochem Biophys Res Commun (2009) 387(4):729–35.
doi: 10.1016/j.bbrc.2009.07.100
321. Chen XP, Ding X, Daynes RA. Ganglioside Control Over Il-4 Priming and
Cytokine Production in Activated T Cells. Cytokine (2000) 12(7):972–85.
doi: 10.1006/cyto.1999.0596
322. Azuma Y, Taniguchi A, Matsumoto K. Decrease in Cell Surface Sialic Acid in
Etoposide-Treated Jurkat Cells and the Role of Cell Surface Sialidase.
Glycoconj J (2000) 17(5):301–6. doi: 10.1023/a:1007165403771
323. Azuma Y, Sato H, Higai K, Matsumoto K. Enhanced Expression of
Membrane-Associated Sialidase Neu3 Decreases Gd3 and Increases Gm3
on the Surface of Jurkat Cells During Etoposide-Induced Apoptosis. Biol
Pharm Bull (2007) 30(9):1680–4. doi: 10.1248/bpb.30.1680
324. Meesmann HM, Fehr EM, Kierschke S, Herrmann M, Bilyy R, Heyder P,
et al. Decrease of Sialic Acid Residues as an Eat-Me Signal on the Surface of
Apoptotic Lymphocytes. J Cell Sci (2010) 123(Pt 19):3347–56. doi: 10.1242/
jcs.066696
325. Bilyy RO, Shkandina T, Tomin A, Munoz LE, Franz S, Antonyuk V, et al.
Macrophages Discriminate Glycosylation Patterns of Apoptotic Cell-Derived
Microparticles. JBiolChem(2012) 287(1):496–503. doi: 10.1074/jbc.M111.273144
326. Katoh S, Kaminuma O, Hiroi T, Mori A, Ohtomo T, Maeda S, et al. Cd44 Is
Critical for Airway Accumulation of Antigen-Specific Th2, But Not Th1,
Cells Induced by Antigen Challenge in Mice. Eur J Immunol (2011) 41
(11):3198–207. doi: 10.1002/eji.201141521
327. Lu LQ, Liao W. Screening and Functional Pathway Analysis of Genes
Associated With Pediatric Allergic Asthma Using a DNA Microarray. Mol
Med Rep (2015) 11(6):4197–203. doi: 10.3892/mmr.2015.3277
328. Hewison M. Vitamin D. And Innate and Adaptive Immunity. Vitam Horm
(2011) 86:23–62. doi: 10.1016/B978-0-12-386960-9.00002-2
329. Yamamoto N, Homma S, Millman I. Identification of the Serum Factor Required
for In Vitro Activation of Macrophages. Role of Vitamin D3-Binding Protein
(Group Specific Component, Gc) in Lysophospholipid Activation of Mouse
Peritoneal Macrophages. J Immunol (1991) 147(1):273–80.
330. Yamamoto N, Naraparaju VR. Role of Vitamin D3-Binding Protein in
Activation of Mouse Macrophages. J Immunol (1996) 157(4):1744–9.
331. Yamamoto N, Naraparaju VR. Vitamin D3-Binding Protein as a Precursor
for Macrophage Activating Factor in the Inflammation-Primed Macrophage
Activation Cascade in Rats. Cell Immunol (1996) 170(2):161–7. doi: 10.1006/
cimm.1996.0148
332. Naraparaju VR, Yamamoto N. Roles of Beta-Galactosidase of B
Lymphocytes and Sialidase of T Lymphocytes in Inflammation-Primed
Activation of Macrophages. Immunol Lett (1994) 43(3):143–8.
doi: 10.1016/0165-2478(94)90214-3
333. Yamamoto N, Kumashiro R. Conversion of Vitamin D3 Binding Protein
(Group-Specific Component) to a Macrophage Activating Factor by the
Stepwise Action of Beta-Galactosidase of B Cells and Sialidase of T Cells.
J Immunol (1993) 151(5):2794–802.
334. Agrawal B, Gupta N, Konowalchuk JD. Muc1 Mucin: A Putative Regulatory
(Checkpoint) Molecule of T Cells. Front Immunol (2018) 9:2391.
doi: 10.3389/fimmu.2018.02391
335. Nowling TK, Gilkeson GS. Mechanisms of Tissue Injury in Lupus Nephritis.
Arthritis Res Ther (2011) 13(6):250. doi: 10.1186/ar3528
336. Nowling TK, Rodgers J, Thiyagarajan T, Wolf B, Bruner E, Sundararaj K,
et al. Targeting Glycosphingolipid Metabolism as a Potential Therapeutic
Approach for Treating Disease in Female Mrl/Lpr Lupus Mice. PLoS One
(2020) 15(3):e0230499. doi: 10.1371/journal.pone.0230499
337. Sundararaj KP, Thiyagarajan T, Molano I, Basher F, Powers TW, Drake RR,
et al. Fli1 Levels Impact Cxcr3 Expression and Renal Infiltration of T Cells
and Renal Glycosphingolipid Metabolism in the Mrl/Lpr Lupus Mouse
Strain. J Immunol (2015) 195(12):5551–60. doi: 10.4049/jimmunol.1500961
338. Richard EM, Thiyagarajan T, Bunni MA, Basher F, Roddy PO, Siskind LJ,
et al. Reducing Fli1 Levels in the Mrl/Lpr Lupus Mouse Model Impacts T Cell
Function by Modulating Glycosphingolipid Metabolism. PLoS One (2013) 8
(9):e75175. doi: 10.1371/journal.pone.0075175
339. Nowling TK, Mather AR, Thiyagarajan T, Hernandez-Corbacho MJ, Powers
TW, Jones EE, et al. Renal Glycosphingolipid Metabolism Is Dysfunctional in
Lupus Nephritis. J Am Soc Nephrol (2015) 26(6):1402–13. doi: 10.1681/
ASN.2014050508
340. Rodgers J, Sundararaj K, Bruner E, Wolf B, Nowling TK. The Role of
Neuraminidase 1 (Neu1) in Cytokine Release by Primary Mouse Mesangial
Cells and Disease Outcomes in Murine Lupus Nephritis. Autoimmunity
(2021) 54(3):163–75. doi: 10.1080/08916934.2021.1897978
341. Mao Z, Tan Y, Yu F, Zhao M. Discovery of Neu1 as a Candidatedone. Renal
Biomarker for Proliferative Lupus Nephritis Chronicity. Lupus Sci Med
(2021) 8(1):e000569. doi: 10.1136/lupus-2021-000569
342. Liou LB, Huang CC. Sialyltransferase and Neuraminidase Levels/Ratios and
Sialic Acid Levels in Peripheral Blood B Cells Correlate With Measures of
Disease Activity in Patients With Systemic Lupus Erythematosus and
Rheumatoid Arthritis: A Pilot Study. PLoS One (2016) 11(3):e0151669.
doi: 10.1371/journal.pone.0151669
343. Yang Z, Liang Y, Li C, Zhong R. Serum Ca125 Elevation Is Independently
Associated With Serositis in Sle Patients. Clin Exp Rheumatol (2012) 30(1):93–8.
344. Meena DS, Kumar B, Gopalakrishnan M, Kachhwaha A, Kumar S, Sureka B,
et al. Pseudo-Pseudo Meigs’Syndrome (Ppms) in Chronic Lupus Peritonitis:
A Case Report With Review of Literature. Mod Rheumatol Case Rep (2021) 5
(2):300–5. doi: 10.1080/24725625.2021.1916160
345. Quintero-Munoz E, Gomez Pineda MA, Araque Parra C, Vallejo Castillo
CA, Ortega Marrugo V, Bonilla Jassir J, et al. Is There Any Relationship
Between Massive Ascites and Elevated Ca-125 in Systemic Lupus
Erythematosus? Case Report and Review of the Literature. Mod Rheumatol
Case Rep (2021) 5(2):292–9. doi: 10.1080/24725625.2021.1909213
346. Tansir G, Kumar P, Pius A, Sunny SK, Soneja M. Pseudo-Pseudo Meigs’
Syndrome: A Rare Presentation of Systemic Lupus Erythematosus.
Reumatismo (2019) 71(2):108–12. doi: 10.4081/reumatismo.2019.1140
347. Higuchi T, Orita T, Nakanishi S, Katsuya K, Watanabe H, Yamasaki Y, et al.
Molecular Cloning, Genomic Structure, and Expression Analysis of Muc20, a
Novel Mucin Protein, Up-Regulated in Injured Kidney. J Biol Chem (2004)
279(3):1968–79. doi: 10.1074/jbc.M304558200
348. de Geest N, Bonten E, Mann L, de Sousa-Hitzler J, Hahn C, d’Azzo A.
Systemic and Neurologic Abnormalities Distinguish the Lysosomal
Disorders Sialidosis and Galactosialidosis in Mice. Hum Mol Genet (2002)
11(12):1455–64. doi: 10.1093/hmg/11.12.1455
349. Radu AF, Bungau SG. Management of Rheumatoid Arthritis: An Overview.
Cells (2021) 10(11):2857. doi: 10.3390/cells10112857
350. Liou LB, Jang SS. Alpha-2,3-Sialyltransferase 1 and Neuraminidase-3 From
Monocytes in Patients With Rheumatoid Arthritis Correlate With Disease
Activity Measures: A Pilot Study. J Chin Med Assoc (2019) 82(3):179–85.
doi: 10.1097/JCMA.0000000000000027
351. Marchand NW, Kishore GS, Carubelli R. Neuraminidase Activity in the
Blood and Liver of Arthritic Rats. Exp Mol Pathol (1978) 29(3):273–80.
doi: 10.1016/0014-4800(78)90070-9
352. Correa I, Plunkett T, Vlad A, Mungul A, Candelora-Kettel J, Burchell JM,
et al. Form and Pattern of Muc1 Expression on T Cells Activated In Vivo or
In Vitro Suggests a Function in T-Cell Migration. Immunology (2003) 108
(1):32–41. doi: 10.1046/j.1365-2567.2003.01562.x
353. Wang Y, Chen S, Zheng S, Lin J, Hu S, Zhuang J, et al. The Role of Lung
Ultrasound B-Lines and Serum Kl-6 in the Screening and Follow-Up of
Rheumatoid Arthritis Patients for an Identification of Interstitial Lung
Disease: Review of the Literature, Proposal for a Preliminary Algorithm,
and Clinical Application to Cases. Arthritis Res Ther (2021) 23(1):212.
doi: 10.1186/s13075-021-02586-9
354. Volin MV, Shahrara S, Haines GK3rd, Woods JM, Koch AE. Expression of
Mucin 3 and Mucin 5ac in Arthritic Synovial Tissue. Arthritis Rheum (2008)
58(1):46–52. doi: 10.1002/art.23174
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 88307926

355. Morisset J, Lee JS. New Trajectories in the Treatment of Interstitial Lung
Disease: Treat the Disease or Treat the Underlying Pattern? Curr Opin Pulm
Med (2019) 25(5):442–9. doi: 10.1097/MCP.0000000000000600
356. Flowers SA, Ali L, Lane CS, Olin M, Karlsson NG. Selected Reaction Monitoring
to Differentiate and Relatively Quantitate Isomers of Sulfated and Unsulfated
Core 1 O-Glycans From SalivaryMuc7ProteininRheumatoidArthritis.Mol Cell
Proteomics (2013) 12(4):921–31. doi: 10.1074/mcp.M113.028878
357. Pickl WF, Majdic O, Fischer GF, Petzelbauer P, Fae I, Waclavicek M, et al.
Muc18/Mcam (Cd146), an Activation Antigen of Human T Lymphocytes.
J Immunol (1997) 158(5):2107–15.
358. Neidhart M, Wehrli R, Bruhlmann P, Michel BA, Gay RE, Gay S. Synovial
Fluid Cd146 (Muc18), a Marker for Synovial Membrane Angiogenesis in
Rheumatoid Arthritis. Arthritis Rheum (1999) 42(4):622–30. doi: 10.1002/
1529-0131(199904)42:4<622::AID-ANR4>3.0.CO;2-Y
359. Richardson SJ, Pugliese A. 100 Years of Insulin: Pancreas Pathology in Type
1 Diabetes: An Evolving Story. J Endocrinol (2021) 252(2):R41–57.
doi: 10.1530/JOE-21-0358
360. Natori Y, Ohkura N, Nasui M, Atsumi G, Kihara-Negishi F. Acidic Sialidase
Activity Is Aberrant in Obese and Diabetic Mice. Biol Pharm Bull (2013) 36
(6):1027–31. doi: 10.1248/bpb.b12-00995
361. Inim MD, Ibrahim MA, Isah MB, Onyike E. Variations in the Mrna
Expression Level of Udp-Glcnac Epimerase/Mannac Kinase and
Neuraminidase 1 Genes in Organs of Type 2 Diabetic Animals. Glycoconj
J(2021) 38(1):129–34. doi: 10.1007/s10719-021-09979-7
362. Minami A, Fujita Y, Shimba S, Shiratori M, Kaneko YK, Sawatani T, et al.
The Sialidase Inhibitor 2,3-Dehydro-2-Deoxy-N-Acetylneuraminic Acid Is a
Glucose-Dependent Potentiator of Insulin Secretion. Sci Rep (2020) 10
(1):5198. doi: 10.1038/s41598-020-62203-8
363. Xia QS, Lu FE, Wu F, Huang ZY, Dong H, Xu LJ, et al. New Role for
Ceramide in Hypoxia and Insulin Resistance. World J Gastroenterol (2020)
26(18):2177–86. doi: 10.3748/wjg.v26.i18.2177
364. Albaghdadi AJ, Kan FW. Endometrial Receptivity Defects and Impaired
Implantation in Diabetic Nod Mice. Biol Reprod (2012) 87(2):30.
doi: 10.1095/biolreprod.112.100016
365. Di G, Qi X, Zhao X, Zhang S, Zhou Q. Efficacy of Sodium Hyaluronate in
Murine Diabetic Ocular Surface Diseases. Cornea (2017) 36(9):1133–8.
doi: 10.1097/ICO.0000000000001271
366. Mednieks MI, Szczepanski A, Clark B, Hand AR. Protein Expression in
Salivary Glands of Rats With Streptozotocin Diabetes. Int J Exp Pathol (2009)
90(4):412–22. doi: 10.1111/j.1365-2613.2009.00662.x
367. Bi J, Huang Y, Liu Y. Effect of Nop2 Knockdown on Colon Cancer Cell
Proliferation, Migration, and Invasion. Transl Cancer Res (2019) 8(6):2274–
83. doi: 10.21037/tcr.2019.09.46
368. Baumgart DC, Carding SR. Inflammatory Bowel Disease: Cause and
Immunobiology. Lancet (2007) 369(9573):1627–40. doi: 10.1016/S0140-
6736(07)60750-8
369. Tuin A, Poelstra K, de Jager-Krikken A, Bok L, Raaben W, Velders MP, et al.
Role of Alkaline Phosphatase in Colitis in Man and Rats. Gut (2009) 58
(3):379–87. doi: 10.1136/gut.2007.128868
370. Parlato M, Charbit-Henrion F, Pan J, Romano C, Duclaux-Loras R, Le Du
MH, et al. Human Alpi Deficiency Causes Inflammatory Bowel Disease and
Highlights a Key Mechanism of Gut Homeostasis. EMBO Mol Med (2018) 10
(4):e8483. doi: 10.15252/emmm.201708483
371. Chen Y, He Y, Zhan X, Chen D, Feng P, Yan Y, et al. Sialic Acid as a Suitable
Marker of Clinical Disease Activity in Patients With Crohn’s Disease. Lab
Med (2022) lmac006. doi: 10.1093/labmed/lmac006
372. Baba R, Yashiro K, Nagasako K, Obata H. Significance of Serum Sialic Acid
in Patients With Crohn’s Disease. Gastroenterol Jpn (1992) 27(5):604–10.
doi: 10.1007/BF02774974
373. Franke A, McGovern DP, Barrett JC, Wang K, Radford-Smith GL, Ahmad T,
et al. Genome-Wide Meta-Analysis Increases to 71 the Number of
Confirmed Crohn’s Disease Susceptibility Loci. Nat Genet (2010) 42
(12):1118–25. doi: 10.1038/ng.717
374. Furr AE, Ranganathan S, Finn OJ. Aberrant Expression of Muc1 Mucin in
Pediatric Inflammatory Bowel Disease. Pediatr Dev Pathol (2010) 13(1):24–
31. doi: 10.2350/08-06-0479.1
375. Begue B, Verdier J, Rieux-Laucat F, Goulet O, Morali A, Canioni D, et al.
Defective Il10 Signaling Defining a Subgroup of Patients With Inflammatory
Bowel Disease. Am J Gastroenterol (2011) 106(8):1544–55. doi: 10.1038/
ajg.2011.112
376. Miro L, Amat C, Rosell-Cardona C, Campbell JM, Polo J, Perez-Bosque A,
et al. Dietary Supplementation With Spray-Dried Porcine Plasma Attenuates
Colon Inflammation in a Genetic Mouse Model of Inflammatory Bowel
Disease. Int J Mol Sci (2020) 21(18):6760. doi: 10.3390/ijms21186760
377. Moehle C, Ackermann N, Langmann T, Aslanidis C, Kel A, Kel-Margoulis
O, et al. Aberrant Intestinal Expression and Allelic Variants of Mucin Genes
Associated With Inflammatory Bowel Disease. J Mol Med (Berl) (2006) 84
(12):1055–66. doi: 10.1007/s00109-006-0100-2
378. Nishida A, Lau CW, Zhang M, Andoh A, Shi HN, Mizoguchi E, et al. The
Membrane-Bound Mucin Muc1 Regulates T Helper 17-Cell Responses and
Colitis in Mice. Gastroenterology (2012) 142(4):865–74 e2. doi: 10.1053/
j.gastro.2011.12.036
379. Beatty PL, Plevy SE, Sepulveda AR, Finn OJ. Cutting Edge: Transgenic
Expression of Human Muc1 in Il-10-/- Mice Accelerates Inflammatory
Bowel Disease and Progression to Colon Cancer. J Immunol (2007) 179
(2):735–9. doi: 10.4049/jimmunol.179.2.735
380. Beatty P, Ranganathan S, Finn OJ. Prevention of Colitis-Associated Colon
Cancer Using a Vaccine to Target Abnormal Expression of the Muc1 Tumor
Antigen. Oncoimmunology (2012) 1(3):263–70. doi: 10.4161/onci.18950
381. Beatty PL, Narayanan S, Gariepy J, Ranganathan S, Finn OJ. Vaccine Against
Muc1 Antigen Expressed in Inflammatory Bowel Disease and Cancer
Lessens Colonic Inflammation and Prevents Progression to Colitis-
Associated Colon Cancer. Cancer Prev Res (Phila) (2010) 3(4):438–46.
doi: 10.1158/1940-6207.CAPR-09-0194
382. Poh TW, Madsen CS, Gorman JE, Marler RJ, Leighton JA, Cohen PA, et al.
Downregulation of Hematopoietic Muc1 During Experimental Colitis
Increases Tumor-Promoting Myeloid-Derived Suppressor Cells. Clin
Cancer Res (2013) 19(18):5039–52. doi: 10.1158/1078-0432.CCR-13-0278
383. Khemiri M, Doghri R, Mrad K, Friedrich K, Oueslati R. Mucin-1 Expression
and Localization in Epithelial Cells Shows Characteristic and Distinct
Patterns in Inflammatory Bowel Diseases and Colorectal Cancer. Int J Clin
Exp Pathol (2019) 12(5):1731–7.
384. Li W, Zhang N, Jin C, Long MD, Rajabi H, Yasumizu Y, et al. Muc1-C Drives
Stemness in Progression of Colitis to Colorectal Cancer. JCI Insight (2020) 5
(12):e137112. doi: 10.1172/jci.insight.137112
385. Liu Y, Yu X, Zhao J, Zhang H, Zhai Q, Chen W. The Role of Muc2 Mucin in
Intestinal Homeostasis and the Impact of Dietary Components on Muc2
Expression. Int J Biol Macromol (2020) 164:884–91. doi: 10.1016/
j.ijbiomac.2020.07.191
386. Engler DB, Leonardi I, Hartung ML, Kyburz A, Spath S, Becher B, et al.
Helicobacter Pylori-Specific Protection Against Inflammatory Bowel Disease
Requires the Nlrp3 Inflammasome and Il-18. Inflammation Bowel Dis (2015)
21(4):854–61. doi: 10.1097/MIB.0000000000000318
387. Van der Sluis M, De Koning BA, De Bruijn AC, Velcich A, Meijerink JP, Van
Goudoever JB, et al. Muc2-Deficient Mice Spontaneously Develop Colitis,
Indicating That Muc2 Is Critical for Colonic Protection. Gastroenterology
(2006) 131(1):117–29. doi: 10.1053/j.gastro.2006.04.020
388. Estaki M, Morck DW, Ghosh S, Quin C, Pither J, Barnett JA, et al. Physical
Activity Shapes the Intestinal Microbiome and Immunity of Healthy Mice
But Has No Protective Effects Against Colitis in Muc2(-/-) Mice. mSystems
(2020) 5(5):e00515–20. doi: 10.1128/mSystems.00515-20
389. Heazlewood CK, Cook MC, Eri R, Price GR, Tauro SB, Taupin D, et al.
Aberrant Mucin Assembly in Mice Causes Endoplasmic Reticulum Stress
and Spontaneous Inflammation Resembling Ulcerative Colitis. PLoS Med
(2008) 5(3):e54. doi: 10.1371/journal.pmed.0050054
390. Kyo K, Muto T, Nagawa H, Lathrop GM, Nakamura Y. Associations of Distinct
Variants of the Intestinal Mucin Gene Muc3a With Ulcerative Colitis and
Crohn’sDisease.J Hum Genet (2001) 46(1):5–20. doi: 10.1007/s100380170118
391. Kyo K, Parkes M, Takei Y, Nishimori H, Vyas P, Satsangi J, et al. Association
of Ulcerative Colitis With Rare Vntr Alleles of the Human Intestinal Mucin
Gene, Muc3. Hum Mol Genet (1999) 8(2):307–11. doi: 10.1093/hmg/8.2.307
392. Buisine MP, Desreumaux P, Leteurtre E, Copin MC, Colombel JF, Porchet N,
et al. Mucin Gene Expression in Intestinal Epithelial Cells in Crohn’s
Disease. Gut (2001) 49(4):544–51. doi: 10.1136/gut.49.4.544
393. Yamamoto-Furusho JK, Ascano-Gutierrez I, Furuzawa-Carballeda J,
Fonseca-Camarillo G. Differential Expression of Muc12, Muc16, and
Lillehoj et al. NEUs and MUCs in Disease
Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 88307927

Muc20 in Patients With Active and Remission Ulcerative Colitis. Mediators
Inflamm (2015) 2015:659018. doi: 10.1155/2015/659018
394. Takagawa T, Kitani A, Fuss I, Levine B, Brant SR, Peter I, et al. An Increase in
Lrrk2 Suppresses Autophagy and Enhances Dectin-1-Induced Immunity in a
Mouse Model of Colitis. Sci Transl Med (2018) 10(444):eaan8162.
doi: 10.1126/scitranslmed.aan8162
395. Lipnicanova S, Chmelova D, Ondrejovic M, Frecer V, Miertus S. Diversity of
Sialidases Found in the Human Body - A Review. Int J Biol Macromol (2020)
148:857–68. doi: 10.1016/j.ijbiomac.2020.01.123
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