Innate Immunity and Pathogenesis of Biliary Atresia

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published: 25 February 2020
doi: 10.3389/fimmu.2020.00329
Frontiers in Immunology | www.frontiersin.org 1February 2020 | Volume 11 | Article 329
Edited by:
Martin Herrmann,
University Hospital Erlangen, Germany
Reviewed by:
Kushagra Bansal,
Harvard Medical School,
United States
Paramananda Saikia,
Cleveland Clinic, United States
*Correspondence:
Sujit Kumar Mohanty
sujit.mohanty@cchmc.org
Specialty section:
This article was submitted to
Molecular Innate Immunity,
a section of the journal
Frontiers in Immunology
Received: 24 October 2019
Accepted: 10 February 2020
Published: 25 February 2020
Citation:
Ortiz-Perez A, Donnelly B, Temple H,
Tiao G, Bansal R and Mohanty SK
(2020) Innate Immunity and
Pathogenesis of Biliary Atresia.
Front. Immunol. 11:329.
doi: 10.3389/fimmu.2020.00329
Innate Immunity and Pathogenesis of
Biliary Atresia
Ana Ortiz-Perez 1, Bryan Donnelly 2, Haley Temple2, Greg Tiao 2, Ruchi Bansal 1and
Sujit Kumar Mohanty 2
*
1Department of Biomaterials Science and Technology, Technical Medical Centre, Faculty of Science and Technology,
University of Twente, Enschede, Netherlands, 2Department of Pediatric and Thoracic Surgery, Cincinnati Children’s Hospital
Medical Center, Cincinnati, OH, United States
Biliary atresia (BA) is a devastating fibro-inflammatory disease characterized by the
obstruction of extrahepatic and intrahepatic bile ducts in infants that can have fatal
consequences, when not treated in a timely manner. It is the most common indication
of pediatric liver transplantation worldwide and the development of new therapies, to
alleviate the need of surgical intervention, has been hindered due to its complexity
and lack of understanding of the disease pathogenesis. For that reason, significant
efforts have been made toward the development of experimental models and strategies
to understand the etiology and disease mechanisms and to identify novel therapeutic
targets. The only characterized model of BA, using a Rhesus Rotavirus Type A infection
of newborn BALB/c mice, has enabled the identification of key cellular and molecular
targets involved in epithelial injury and duct obstruction. However, the establishment
of an unleashed chronic inflammation followed by a progressive pathological wound
healing process remains poorly understood. Like T cells, macrophages can adopt
different functional programs [pro-inflammatory (M1) and resolutive (M2) macrophages]
and influence the surrounding cytokine environment and the cell response to injury. In
this review, we provide an overview of the immunopathogenesis of BA, discuss the
implication of innate immunity in the disease pathogenesis and highlight their suitability
as therapeutic targets.
Keywords: biliary atresia, liver fibrosis, rotavirus, innate immunity, macrophages
INTRODUCTION
Biliary atresia (BA) is a devastating obliterative cholangiopathy that affects exclusively infants and is
characterized by a progressive fibro-inflammatory obstruction of the extrahepatic and intrahepatic
bile ducts that can lead to cirrhosis and liver failure (1–4). BA occurs in 1 out of 15,000 births
in the US (5), affecting all ethnic groups, (6) and with a higher frequency in girls (7). Despite its
low incidence, BA is the most common cause of neonatal cholestasis (3), end-stage liver disease in
children and the number one indication of pediatric liver transplant worldwide (8,9). The first
disease symptoms include jaundice, alcoholic stools, dark urines (3), and high levels of serum
bilirubin (10). A conclusive diagnosis of BA is based on an exploratory surgery where obstruction
of the extrahepatic biliary tree can be observed and confirmed by a histological analysis of liver
or biliary tissue biopsy (3). At the time of diagnosis, about 60 days of life on average (4), the
obstructed extrahepatic remnants are removed and hepatoportoenterostomy (HPE, called Kasai) is
performed to restore the bile flow (11). However, even if the Kasai procedure is performed during

Ortiz-Perez et al. Immunopathogenesis of Biliary Atresia
the first month of life and the cholestasis is resolved, bile duct
proliferation, and fibrosis persist (9) resulting in the development
of variable degrees of liver fibrosis, cirrhosis, portal hypertension,
or other severe hepatic complications (12). Notably, the long-
term survival of BA patients has extraordinarily improved in the
last decades—from 70% in the 1990s to 80–90% in 2009 (13)—but
the treatment still relies on surgery (HPE, transplantation), which
is palliative, thereby highlighting the necessity of developing
novel targeted therapies to prevent or reverse liver injury.
CLASSIFICATION AND MOLECULAR
SIGNATURES
Traditionally, BA patients were divided into
“embryonic/developmental” BA (<20%) and
“perinatal/acquired” BA (>80%) depending on their onset
(14–16). The former is believed to originate during the first
trimester of pregnancy and the accompanying clinical features
suggest a developmental origin (4), the latter is thought to appear
shortly after birth when the first symptoms become recognizable
(10). The presence of splenic malformations—polysplenia but
also asplenia—is characteristic of the Biliary Atresia Splenic
Malformation (BASM) syndrome, the most representative form
of embryonic BA (about 10%). The infants within this group were
found to have a worse prognosis than infants with isolated BA
(17). The remaining sub-group comprises patients with at least
one non-splenic malformation. This group is also often included
in the category of non-syndromic BA, since the presence of
the underlying defects does not necessarily worsen the disease
or implicates different mechanisms of pathogenesis (11,18).
Notably, BASM patients may also have another concomitant
defect, such as cardiovascular and laterality defects (17).
In 2012, Davenport proposed the latest reference classification
incorporating the cytomegalovirus (CMV)-associated and cystic
BA variants to the aforementioned non-syndromic BA and
BASM groups (19). CMV-associated BA refers to a subgroup of
infants whose liver biopsies stained positive for immunoglobulin
M (IgM) antibodies against CMV. The presence of these
antibodies has been linked to the poorest HPE outcome and
highest mortality, and the tissue biopsies revealed an exacerbated
pro-inflammatory response (20): the predominant cellular profile
observed in most of the BA patients (16). By contrast, cystic
BA, an anatomic variant in which a cyst is formed close to the
site of obstruction and a Th2-response is primed, was associated
with an improved drainage after HPE and a better long-term
outcome (21).
ETIOLOGY
The etiology of BA is heterogeneous and has not been fully
elucidated yet. Diverse theories regarding the causes of
the disease have been formulated, including embryonic or
developmental abnormalities (17,21), exposure to exogenous
triggers such as viruses or toxins (16,22), immune immaturity
(11,23), immune dysregulation (24,25), and autoimmunity
(26–29). Furthermore, numerous susceptibility factors—
such as genetic predisposition (30), maternal diabetes (17),
or microchimerism (31)—have also been implicated in the
pathogenesis of the disease. This complex cocktail of variables
and factors supports the claim that biliary atresia is not a disease
with a single etiology but a combination of different phenotypes
that share certain clinical features, such as the obliteration of the
biliary tree early in life (32).
Animal Models and Etiological Agents
The characteristic lesions of BA such as the obstruction of the
extrahepatic biliary tree and cholestasis, have been successfully
reproduced and investigated in several animal models—such
as lamb, calf, zebrafish, and mouse. The first three forms of
experimental BA in lamb, calf and zebrafish are induced through
toxins, while the murine models are achieved upon viral infection
(5,33,34).
One of the first observations of BA-like pathologies in animals
was reported in the Australian outbreak in 1964, 1988, and
2007 when lambs were born with cholestasis after pregnant
livestock was exposed to unidentified toxic environmental factors
in extreme drought conditions (1,22,35), which arose the
suspicion that the toxic effect could come from the grass. A
group of scientists from the university of Pennsylvania imported
a plant species characteristic of that area and used zebrafish
bioassays to identify the substance responsible: an isoflavonoid
that they named biliatresone (22). This toxic compound, capable
of inducing biliary atresia phenotype, is the basis of the theory
that implicates hepatotoxins as etiological agents.
The other leading theory about the origin of the disease
points toward a viral insult (16,36). The first implication of
an hepatotropic virus as causative factor in BA was suggested
by Benjamin Landing (37). Despite the initial contradictory
findings regarding the presence and role of reovirus in BA (38–
41), numerous viruses have been implicated in the pathology
of the disease and evidence of preceding viral infection—MxA
proteins (Myxovirus resistance protein 1)—could be found even
in the absence of viral material (42–44). Whether the virus is
the primary causative factor or an accidental secondary event
remains unclear (44,45).
Rhesus Rotavirus-Induced Murine Model
Among all viruses, rhesus rotavirus type A (RRV) is the gold
standard to model BA in mice. The use of this murine model
has facilitated the study of different aspects of the disease, such
as the underlying mechanisms of the pathogenesis (26–28,46–
50) or the identification of novel therapeutic targets (51). This
experimental form of BA uses BALB/c newborn mice that,
when challenged with RRV within the first hours of life (12–
48 h), can recapitulate many aspects of human BA (52) such
as time-restricted susceptibility to the viral infection, portal
tract infiltration of inflammatory cells and obstruction of both
extrahepatic and intrahepatic biliary tree (5,34). This in vivo
model allows for the comprehensive study of the early events
of the disease that cannot be explored directly in humans, since
they happen before the time of diagnosis. However, the RRV
model is not yet suitable to study the progression of the disease
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Ortiz-Perez et al. Immunopathogenesis of Biliary Atresia
after duct obstruction, due to the high mortality rate of the mice
before the development of liver fibrosis and related long-term
complications (5). Previous studies have examined the fibrogenic
response in RRV model and observed insufficient fibrosis (Ishak
score 1–2) when determined at 2 weeks’ time (Figure 1A) (53,
54). These limitations (e.g., high mortality and poor fibrogenic
responses), however, could be tackled by optimizing the model
induction using reassortant viruses. Recently, a novel RRV-
TUCH rotavirus reassortant (TUCH for Tulane University and
Cincinnati Children’s Hospital) could recapitulate an obstructive
jaundice phenotype with lower mortality rates when injected into
newborn mice (54). This new model recapitulates the late events
of the disease such as liver fibrosis (Ishak score 3–5) and showed
a unique resemblance to the human BA, significantly different
from CCl4and bile duct ligation models (54) (Figure 1B). This
model, therefore, not only improves our current understanding
about BA disease pathogenesis but will also contribute toward the
identification of new therapeutic targets.
Other Virus Induced Models
Cytomegalovirus (CMV) has also been used to recapitulate
BA in animal models (55). For instance, a regulatory T
cell (Treg)-depleted neonatal mouse, when infected with
low-dose CMV (LD-CMV) to study BA, induced extensive
inflammation, atresia of intrahepatic bile ducts and partial
obstruction of the extrahepatic bile ducts. Liver mononuclear
cells showed increased percentages of CD3/CD8 T cells and
serum autoantibodies (α-enolase) reactive to bile duct epithelial
proteins, suggesting the involvement of cellular and humoral
autoimmune responses in LD-CMV BA mouse model. There was
also an increased hepatic expression of Th1-related genes (tumor
necrosis factor α, TNF-α), interferon γ(IFN-γ)-activated genes
(signal transducer and activator of transcription 1, STAT-1) and
Th1 cytokines/chemokines (lymphotactin, interleukins IL-12p40
and macrophage inflammatory protein 1-alpha, MIP-1α).
Evidence of Viruses as a Causative Agent
of BA
As mentioned earlier, viruses have been proposed as etiological
agents in BA. These viruses activate pathways that might
predispose certain individuals to develop the disease. In the
animal model, the RRV Viral Protein 4 (VP4) gene has been
demonstrated to be the major determining factor required for
the pathogenesis of BA (49). Rotavirus strains with 87% or more
homology to RRV’s VP4 were capable of infecting murine bile
ducts and inducing the disease as well as activating mononuclear
cells, independent of viral titers (56). Further research led to
the identification of a key amino acid sequence “SRL” in VP4, a
sequence specific to those rotavirus strains that cause obstructive
cholangiopathy (57). This tripeptide “SRL” on RRV VP4 was
found to bind specifically to the cholangiocyte membrane
protein heat shock cognate 70 (Hsc70), defining a novel binding
site governing VP4 attachment (57). To gain insight into the
mechanisms involved upon VP4-mediated infection, a reverse
genetics system was developed to create a mutant of RRV with
a single amino acid change in the VP4 protein and compared
to that of wild-type RRV (where the arginine “R” in “SRL”
region was replaced with glycine “G”) (58). The mutant virus,
when injected to mice, demonstrated reduced symptoms and
lower mortality in neonatal mice, resulting in an attenuated
form of biliary atresia indicating the importance of “SRL”
region (57). This “SRL” peptide was also found either on the
capsid or the attachment protein of other viruses including
reovirus, cytomegalovirus, human papillomavirus, Epstein-Barr
virus, bluetongue virus, polyomavirus, coronavirus, respiratory
syncytial virus, adenovirus, rodent paramyxovirus, and herpes
FIGURE 1 | Time line of events in the murine model of BA upon RRV challenging, depicting (A) the standard RRV model in comparison with (B) the modified model
using a novel viral reassortant [TR(VP2,VP4 )]; this virus reassortant was engineered by replacing the VP2 and VP4 gene of TUCH for the corresponding RRV’s VP2 and
VP4.
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Ortiz-Perez et al. Immunopathogenesis of Biliary Atresia
simplex virus 1. Several of these (cytomegalovirus, Epstein-
Barr virus, human papillomavirus, and reovirus) have been
detected in explanted livers of infants with BA (59–63). Thus,
this sequence in the above-mentioned viruses might be involved
in cholangiocyte binding in a similar fashion to the RRV “SRL”
peptide. Binding of these viruses to Hsc70 might activate the
innate immune system through different pathways. The role
of Hsc70 binding in human BA induction as a function of
these proteins and their influence in oxidative stress and cell
metabolism remain largely unexplored.
IMMUNOPATHOGENESIS OF BILIARY
ATRESIA
Cholangiocyte Immunobiology
Biliary epithelial cells (cholangiocytes) are not only a physical
barrier that drains the bile into the duodenum but they are also
immunocompetent cells involved in tissue homeostasis, capable
of recognizing microbial conserved motifs known as Pathogen
Associated Molecular Patterns (PAMPs) through pattern-
recognition receptors (PRRs) and initiating an inflammatory
response (64–67). Four main families of PRRs have been
described, including toll-like receptors (TLRs), retinoic acid
inducible gene 1 (RIG-I)-like receptors (RLRs), nucleotide-
binding oligomerization domain (NOD)-like receptors (NLRs),
and C-type lectin receptors (CLRs) (68).
From the ten types of TLRs that have been identified
in mammals, at least 5 of them have been described in
mice and human cholangiocytes (64). Among them, TLR-
4 is responsible for sensing lipopolysaccharides (LPS) and
TLR-3, 7, 8, and 9 are involved in recognition of viral and
bacterial RNA or DNA. Activation of these receptors triggers
an inflammatory response via Mitogen-activated protein kinases
(MAPK), interferon regulatory factor 3 (IRF3) and/or nuclear
factor κB (NF-κB) characterized by the production of type I
interferons (IFNs) and/or pro-inflammatory cytokines. MAPK
signaling is a multifunctional pathway that is pivotal in the
innate immune response and viral infection. Among the three
central members of the MAPK pathway, extracellular signal-
regulated kinase (ERK) 1/2 and p38 activation play the most
important roles in RRV infection of cholangiocytes as they seem
to be involved in both viral replication and epithelial injury (69).
Further studies revealed that ERK phosphorylation and calcium
influx appear to be essential to RRV infection, and RRV’s viral
protein 6 (VP6) drives ERK phosphorylation (70).
TLRs depend on adaptor molecules– myeloid differentiation
primary response 88 (MyD88) or toll/interleukin-1 receptor
domain-containing adaptor protein (TRIF)—to effectively
initiate and transduce the downstream signal to the nuclei,
differentiating them into two main TLR signaling pathways
(Figure 2A) (68). In the MyD88-dependent pathway (associated
to TLR 1–5, except for TLR-3), the Interleukin-1 receptor-
associated kinase (IRAK)-1, −2 and −4 upregulate the
production of Type I IFNs and pro-inflammatory cytokines
(IL-1β, IL-6, and TNF-α) via MAPK, IRF3, and NF-κB pathways
(65,67,68). It has been demonstrated that the pathogenesis of
murine BA is independent of the MyD88 signaling pathway (71).
In MyD88/IRAK-M independent pathway, the activation of
TLR-3, 7/8 or 9, associated with the TRIF-dependent signaling,
results in the activation of NF-κB and IRF3 signaling cascades
(65,68). This different level of regulation could explain why
“endotoxin tolerance” to enteric bacteria can be induced in
cultured cholangiocytes by treating them with TLR-4 ligands
(like LPS) (72) but “viral tolerance” could not be achieved using
the same approach (73).
The RLR family (74) is comprised of cytosolic sensors,
including RIG-1 and melanoma differentiation-associated
protein 5 (MDA-5) that are capable of binding to dsRNA
(75–77). This interaction triggers a conformational change
that exposes the two caspase activation and recruitment
domains (CARDs) at their N-terminus, which are responsible
to recruit the complementary protein mitochondrial antiviral-
signaling protein (MAVS) and transduce the signal to the
nuclei to produce type I interferons and pro-inflammatory
cytokines (Figure 2B) (75,78). NLRs (e.g., NLRP3), are also
cytosolic innate immune receptors that are activated upon
recognition of viral dsRNA. Rather than contributing to the
initial events of the acute inflammatory response, they amplify
the immune response, release late mediators (IL-1β, IL-18 and
high mobility group box 1, HMGB-1) and regulate pyroptosis
(pro-inflammatory programmed cell death) through the
formation of inflammasomes (Figure 2C) (79).
The last group of PRRs described are the large family of CLRs.
They are transmembrane receptors, with an immunoreceptor
tyrosine-based activation motif (ITAM) or an immunoreceptor
tyrosine-based inhibition motif (ITIM), that are able to induce
a pro-inflammatory response or modulate it through a crosstalk
with other PRRs such as TLRs. CLRs play a crucial role in
maintaining immune homeostasis against pathogens and in
mounting a pro-inflammatory and/or antiviral response (80–
82). Alterations of CLRs have been implicated in different
pathological conditions, including gastrointestinal cancers,
autoimmune disorders, or allergies (82). It is known that
cells from myeloid lineage such as dendritic cells (DCs) and
macrophages, as well as some endothelial and epithelial cells,
express CLRs; however, it has not been reported in biliary
epithelium yet.
Although cholangiocytes play a central role in initiating an
immune response upon exposure to the exogenous substances,
they are however not capable of mounting an inflammation that
is sufficient to induce chemotaxis and recapitulate the obstructing
phenotype of BA without the involvement of macrophages and
DCs (83–86).
Mechanisms of Epithelial Injury and Duct
Obstruction
Upon viral infection, cholangiocytes, macrophages, and DCs
(RRV cellular targets) trigger the anti-viral response through
type I interferons in an autocrine and paracrine manner in
both infected and surrounding cells to prevent the virus from
spreading (5). In infected cells, type I IFNs promote biliary
apoptosis by upregulation of tumor necrosis factor related
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Ortiz-Perez et al. Immunopathogenesis of Biliary Atresia
FIGURE 2 | Innate immune receptors present in cholangiocytes. (A) Toll-like receptors (TLRs) and schematic representation of the two main signaling pathways: the
MyD88 dependent pathway (characteristic of all toll-like receptors except TLR 3) and MYD88 independent pathway (characteristic of TLR3). (B) Cytosolic viral sensing
of Retinoic-acid-inducible gene I (RIG-I)-like receptors, capable of triggering a pro-inflammatory and antiviral response, and (C) nucleotide-binding oligomerization
domain (NOD)-like receptors that have the ability to perpetuate the immune response through the formation of inflammasomes, induction of cell death and release of
late mediators.
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Ortiz-Perez et al. Immunopathogenesis of Biliary Atresia
FIGURE 3 | Mechanism of obstruction in biliary atresia. (A) RRV infection and activation of the anti-inflammatory and anti-viral response. (B) Innate immune cell
recruitment & tissue specific attack to epithelia. (C) Activation of adaptive immunity (D) Th1-primed polarization and alternatively (E) Th2 polarization.
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Ortiz-Perez et al. Immunopathogenesis of Biliary Atresia
apoptosis ligand (TRAIL) (TNF receptor p55) and CD95
(Fas/Apo1 ligand) (87). In surrounding tissue, IFNs trigger the
production of antiviral proteins (Mx) that provide protection
against viral infection (Figure 3A) (88). The production of
pro-inflammatory cytokines and chemokines by cholangiocytes,
macrophages and DCs creates the favorable microenvironment
to recruit and activate inflammatory cells, and to promote an
immune effector tissue-specific attack (Figure 3B) (84,85,89).
Among the chemokines produced, the most relevant are IL-
8 and IL-15. IL-8, mostly produced by macrophages but also
by cholangiocytes (90), recruits and modulates the action of
neutrophils (85), basophils, monocytes, and T cells (64,67,90);
while IL-15, secreted primarily by DCs, attracts and regulates
the activity of natural killer (NK), natural killer T (NKT), and
gamma-delta cells (89). The recruited inflammatory effector
cells are engaged to target specifically the biliary epithelium
in a contact dependent manner (91), through IFN-γ-related
cytokines (48) and/or cytotoxic agents (perforins, granzymes)
(92). Recruited neutrophils produce reactive oxygen species
(ROS), leukotrienes, and neutrophil defensins (90). NK cells,
activated by DCs via IL-15 (89), induce cholangiocyte death
in a contact-dependent manner through Natural killer group
2d (Nkg2d) ligand that interacts with ribonucleic acid export
1 (RAE1) receptors, expressed in infected cells (91) and via
the secretion of IFN-γ, perforins, and granzymes (92). In
a similar fashion, the cytotoxic power of neonatal CD8+T
cells is exerted through cytotoxic agents (perforin, granzymes,
IFN-γ) (92) and in a contact-dependent manner by invading
the epithelium (27). Mechanistical studies using the RRV-
infected BALB/c murine model showed that depletion of NK
cells, blockage of the receptor Nkg2d or depletion of CD8+
T cells (with impairment of IFN-γmechanisms) reduced
cholangiocyte death, evaded rupture of the epithelium and
ultimately prevented the obstruction of the extrahepatic biliary
tree (27,91). Likewise, epithelial integrity was preserved by
depleting plasmacytoid DCs or blocking the IL-15 signaling,
responsible for NK cell activation (86,89). These results
highlight the specific role of DCs, NK, and CD8+T cells in
the model.
As the inflammation progresses without being resolved,
DCs and macrophages interact chiefly with helper CD4+
T cells (Th0) to promote their activation, oligoclonal
expansion (93) and differentiation into a specialized phenotype
depending on the predominant cytokine microenvironment
at the time (Figure 3C). In most of BA patients, this
microenvironment is pro-inflammatory (Th1), characterized
by IFN-γproduction and the activation of effector cells
(macrophages, CD8+T cells and B cells) to perpetuate the
tissue damage (Figure 3D) (11,16). In some cases, the infants
are not capable of mounting a Th1 response, therefore,
the polarization primed is Th2, with IL-13 [produced by
type 2 innate lymphoid cells (ILC2)] as a predominant
cytokine, responsible for the tissue damage mediated by
ductal proliferation and activation of hepatic stellate cell
(HSCs) and portal fibroblasts. This is typically the case for
the aforementioned cystic variant of BA (94), as depicted
in Figure 3E.
Humoral Immunity
In contrast to T-cell polarization, very little is known about the
implication of humoral immunity in the pathogenesis of BA.
In the early stage of the disease, humoral-related genes (i.e.,
immunoglobulins) are transiently suppressed (95). However, B
lymphocytes seem to play a role as antigen presenting cells
for effector T cell activation as also shown in Figure 3C. An
evidence for the role of B lymphocytes has been proposed in
a study where the depletion of B-cells in experimental BA was
associated with impaired effector T-cell activation and protection
against biliary injury (96). Furthermore, humoral duct-specific
autoimmunity has been demonstrated in experimental BA (26)
but the role of B lymphocytes remains unclear in human BA.
Human-based studies regarding humoral activity in BA include
the description of immunoglobulins IgM and IgG deposits in the
biliary epithelium basement membrane (97) and the detection
of autoantibodies (28,29). Lu et al. (28) detected autoantibodies
against α-enolase in the RRV induced mouse model of BA and
in serum samples from patients, indicating a role of humoral
auto-immunity in disease pathogenesis. The cross-reactivity
between an anti-enolase antibody and RRV proteins indicates
that molecular mimicry might activate humoral autoimmunity in
BA patients. However, further investigation is needed to provide
more insight into the implication of humoral immunity in BA.
Immune Dysregulation
A subset of helper CD4+T cells known as regulatory T cells
(Tregs)—that expresses CD25 and forkhead box P3 (FOXP3)—
has a pivotal role in immunoregulation and induction of
peripheral tolerance. Neonatal Tregs (98,99) prevent the
activation of autoreactive T cells and inhibit the action of
several immunocompetent cells (B and T cells, macrophages,
dendritic cells, and natural killer cells) (50,98,100,101). In
neonatal mice, Tregs populate the spleen from day 3 of life
(102) which corresponds the susceptibility time window in the
RRV model (100,103). Moreover, adoptive transfer of Tregs
to pups before RRV infection prevented the obstruction of
the extrahepatic bile ducts (50,100,101). In infants with BA,
gene expression of regulatory cytokines (IL-10, transforming
growth factor β, TGF-β] and transcription factors (FOXP3) are
upregulated in the liver (100), but there is a deficit in number
of circulating Tregs in peripheral blood and their regulatory
function seems to be impaired (25,104). Even though the exact
underlying mechanisms of Treg malfunctioning and immune
dysregulation are not fully understood, epigenetic changes might
play a major role. For instance, hypomethylation of FOXP3
promoter was associated with improper functioning of Tregs
(25), while hypermethylation of DNA in lymphocytes elicited
them to promote an exacerbated inflammatory response (24).
MECHANISMS OF POST-OBSTRUCTION:
CHRONIC INFLAMMATION, DUCT
PROLIFERATION, AND FIBROSIS
After obstruction, regardless of the restoration of the bile
flow, the immune-mediated biliary damage persists (9) and the
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Ortiz-Perez et al. Immunopathogenesis of Biliary Atresia
initial Th1-predominant milieu shifts toward a Th2 with the
simultaneous emergence of the Th17 subset (Figure 4).
On one hand, apoptotic and necrotic cells release endogenous
molecules known as damage-associated molecular patterns
(DAMPs)—recognizable by PRRs—as excessive damage or
“danger signals” (68). One of these DAMPs is the interleukin
IL-33 that, when released by cholangiocytes and hepatocytes,
accumulates in the extracellular matrix (ECM) and promotes
inflammation and fibrosis. High levels of IL-33 has been
detected in serum and tissue biopsies in both patients and
experimental BA (105). In this context, IL-33 in the liver is
believed to engage with liver-resident innate helper cells (ILC2)
that express IL-33 receptor (ST2 or IL-1R4) to produce pro-
fibrotic Th2-related cytokines (IL-4, IL-5, IL-9, and IL-13)
(106). Among them, IL-13 upregulates the expression of TGF-β
and matrix metalloproteinase 9 (MMP9); activates HSCs via
IL-4Ra and STAT6, promoting fibrosis in a TGF-β1/SMAD-
independent mechanism (107); and stimulates collagen synthesis
by myofibroblasts (activated HSCs and portal fibroblasts).
Simultaneously, IL-33 was shown to drive duct proliferation in
both intra- and extra-hepatic ducts (105). This IL-33-ILC2-IL13
axis is depicted in Figure 4A.
On the other hand, damaged cholangiocytes are shown to
produce IL-1β, IL-6, and IL-23 (65). IL-1β, IL-6 are required for
Th17 commitment, and IL-23 is needed for the maintenance of
this phenotype (108). IL-17A is the representative cytokine of this
panel, which induces the production of several pro-inflammatory
cytokines and chemokines. Lages et al. identified Th17 cells as
the main source of IL-17A after the obstruction of the biliary
tree in experimental BA. In this study, a model of biliary
injury perpetuation was proposed in which IL-17A stimulated
cholangiocytes to produce C-C motif chemokine ligand 2 (CCL2)
that recruited inflammatory macrophages expressing IL-17AR
to target the epithelium (51), as shown in Figure 4B. In this
model, depletion of Th17 cells or blockage of CCL2 prevented
bile duct paucity and the number of Th17 cells correlated with
the concentration of gamma glutamyl transpeptidase (GGT), a
biochemical marker of bile duct injury (51). In BA patients, the
presence of Th17 in the biliary tree and peripheral blood has
been confirmed, as well as Th17-related markers in liver tissue
FIGURE 4 | Disease progression mechanisms after bile duct obstruction. (A) IL-33-ILC2-IL-13 axis, implicated in fibrosis and duct proliferation and (B)
Th17-Macrophage axis, as a mechanism of chronic inflammation and damage perpetuation.
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Ortiz-Perez et al. Immunopathogenesis of Biliary Atresia
[IL-17A and retinoic acid-related orphan receptor (ROR)-γt] and
serum IL-23. In addition, a high ratio between Th17 and Tregs
has been characterized in peripheral blood (109), a trend that
has also been observed in chronic liver diseases such as primary
biliary cirrhosis (108).
In addition, damaged or pro-apoptotic as well as
inflammatory cells (especially Kupffer cells and macrophages)
can express or produce hedgehog (Hh) ligands under
pathological conditions (110). Cholangiocytes stimulated
with Hh ligands (in an autocrine or paracrine manner) produce
a wide assortment of cytokines—including IL-6 and TGF-β
(111)—and chemokines that attract different populations of
inflammatory cells, including neutrophils, monocytes, and
lymphocytes (112). Inflammatory cells stimulated by Hh
ligands sustain inflammation, while activated HSCs continue
to proliferate in response to this stimulus (113). Abnormal
over-activation of the Hedgehog pathway has been observed in
the context of chronic inflammation-related fibrosis (114,115),
human cholangiopathies (116), and biliary atresia (117). A
characteristic Hh ligand in BA is osteopontin (OPN) that has
been correlated with severity of the disease (118).
MACROPHAGES, MICROENVIRONMENT,
AND AGE-RAGE
Like T cells, macrophages can adopt different polarization states
depending on the surrounding tissue microenvironment (119).
Characterization of these functional programs is important since
they seem to have vast implications in the outcome of several
chronic auto-inflammatory and degenerative diseases (120).
Conventionally, they are divided into classically activated M1
(pro-inflammatory) and alternatively activated M2 (restorative)
macrophages (119). Polarization into M1 macrophages is driven
by activation of TLR signaling through LPS and IFN-γchallenge;
while stimulation with regulatory cytokines (IL-4, IL-10) primes
a M2 polarization. Several reports have pointed that, in many
contexts, the dichotomy M1/M2 may not be sufficient to
describe a relevant macrophage population because of its
heterogeneity, the complexity of the activation stimuli, and
surrounding tissue microenvironment (121–123). However, in
the context of fibrosis, two distinct macrophage population
have been described for its role in modulating the body
response to chronic injury: pro-fibroinflammatory and resolutive
macrophages, often associated with M1 and M2 features,
respectively. These polarizations have the ability to influence the
tissue microenvironment and with it, the net cellular response
and outcome of the disease. For instance, pro-inflammatory
macrophages, displaying high levels of inflammatory marker
lymphocyte antigen 6 complex, locus C (Ly6C), are characterized
by a high production of chemokines (such as CCL2) that
attract inflammatory cells to the site of injury, pro-inflammatory
cytokines (such as TNF-αand IL-1β) that perpetuate hepatic
damage and TGF-βthat activates HSCs into ECM-producing
myofibroblasts. On the contrary, restorative macrophages,
displaying low levels of Ly6C, seem to be responsible for inducing
HSCs apoptosis (through TRAIL and MMP9), digesting the
excess of ECM and promoting clearance of the profibrotic
stimuli, thereby facilitating tissue regeneration (122–125).
Both tissue-resident and monocyte-derived macrophages can
acquire these functional programs. However, the latter is the
predominant population during tissue injury (122), highlighting
the relevance of infiltration of inflammatory cells in the course of
the disease.
Pro-fibroinflammatory macrophages exhibit a wide
assortment of mechanisms that allow them to activate and
perpetuate inflammation and fibrosis in both TGFβ-dependent
and independent circuits. One way to modulate the surrounding
cellular response is by influencing the tissue microenvironment.
An important component of this microenvironment is the
level of oxidative stress, intimately linked to the Advanced
Glycation End-Products (AGE)-Receptor of AGEs (RAGE)
pathway (120,122). AGEs refer to a heterogeneous group of
toxic by-products that are a result of irreversible non-enzymatic
reactions between sugars and proteins as consequence of
elevated intra-cellular oxidative species. In normal physiological
conditions, AGEs are produced in small amounts, released into
the extracellular space, and cleared by specialized phagocytic
cells: principally macrophages through scavenger receptors
(Figure 5A). However, during chronic injury, under continuous
oxidative stress, the production of AGEs is higher than their
clearance and this leads to their accumulation in the extracellular
space, affecting surrounding cells. Interaction of AGEs (or/and
other RAGE ligands, such as S100 proteins and HMGB1) with
their receptor triggers a signal transduction cascade through
different pathways, resulting in numerous cellular responses
such as inflammation, fibrosis, or apoptosis (120,126,127), as
depicted in Figure 5B.
In the murine model, RRV has the ability to infect the
macrophages, resulting in their activation (85). Activated pro-
inflammatory macrophages are one of the main sources of
AGEs but damaged cholangiocytes and hepatocytes have also
been shown to produce several RAGE ligands in response
to injury. In patients with BA, the serum levels of soluble
RAGE has been correlated with the severity of the disease
(128). A recent network analysis study involving the three main
human cholangiopathies (including BA), identified a common
connectome in which AGE-RAGE pathways occupy central
nodes (129). Remarkably, we have observed an induction of
oxidative species and production of AGE-RAGE ligands in RRV-
infected cholangiocytes (unpublished work), which suggests
an involvement of oxidative stress circuits from the onset of
the disease.
THERAPEUTICS AND CLINICAL TRIALS
The routine treatments of BA patients after HPE are
ursodeoxycholic acid, antibiotics, and fat-soluble vitamin
formulations that have not substantially improved the outcomes
of the disease. In a double-blind, placebo-controlled study
(START trial) corticosteroid administration within 3 days
of the HPE did not change the outcome of the BA cohort
while increased the risk of serious adverse effects as compared
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Ortiz-Perez et al. Immunopathogenesis of Biliary Atresia
FIGURE 5 | Schematic representation of macrophages and tissue micro-environment: (A) Clearance of receptor of advanced glycation end products (RAGE) ligands
and Damage-Associated Molecular Patterns (DAMPs) under physiological conditions and (B) accumulation of RAGE ligands and DAMPs and consequent
perpetuation of damage through the induction of oxidative stress, inflammation, and fibrosis.
to placebo controls (130). Although corticosteroids in BA
infants younger than 2 weeks of age did appear to improve
biliary drainage, with pending data on native liver survival
(131) suggesting a possibility of corticosteroids use on these
subsets of infants. In the future, the agents which are currently
being tested in cholestatic and fibrotic liver diseases in adults
(132) can also be investigated in BA, such as the farnesoid X
receptor (FXR) agonist, obeticholic acid, and the modified bile
acid norursodeoxycholic acid, which are also currently used
in primary biliary cholangitis (PBC) and primary sclerosing
cholangitis (PSC) patients (133,134). Other agent such as apical
sodium-dependent bile acid transporter (ASBT) inhibitor may
reduce bile acid burden in the liver. The two other agents
that are currently used in clinics for pediatric liver diseases—
bile acid sequestrants (cholestyramine or colesevelam) and
ursodeoxycholic acid—are yet to be thoroughly tested in clinical
trials in BA (135).
CONCLUSION AND FUTURE
PROSPECTIVE
Due to the establishment of experimental models of BA,
especially the RRV murine model, some of the driving
mechanisms of epithelial injury and duct obstruction have been
elucidated, and the corresponding key cellular and molecular
targets have been identified. However, the real applicability
of these targets for therapy is hindered due to the lack of
early diagnosis and screening tools, and that many questions
regarding the etiology of the disease remain unanswered.
The molecular and cellular mechanisms in which the disease
progresses are still under investigation. Increasing evidence
suggests a deeper implication of intricated mechanisms of
the innate immunity from the onset of the disease: namely,
oxidative stress, altered metabolism, and induction of long-
term/abnormal epigenetic changes. Among them, AGE-RAGE
Frontiers in Immunology | www.frontiersin.org 10 February 2020 | Volume 11 | Article 329

Ortiz-Perez et al. Immunopathogenesis of Biliary Atresia
pathway has attracted most of the attention since it encompasses
key circuits involved in the pathogenesis of several chronic
inflammatory and degenerative diseases, including biliary atresia.
Further investigation is needed to determine the extent of
implication of the AGE-RAGE pathway and its crosstalk with
other fibro-inflammatory circuits. Because macrophages are
one of the main drivers of AGE-RAGE and their functional
polarizations seem to occupy a central role in the modulation
of the tissue response and outcome in chronic conditions,
future research should interrogate these cell populations in
the context of biliary atresia. Imperatively, there is a need to
develop new or improve existing experimental platforms to
perform mechanistical studies of later events of the disease and
facilitate the identification and implication of cell populations
and pathways. In addition, deeper understanding of the model
induction through other viruses and/or toxins could shed some
light into the etiology of the disease and aid the development
of new therapies to manage BA patients without the need
of surgery.
AUTHOR CONTRIBUTIONS
AO-P drafted the manuscript. BD, HT, RB, GT, and SM
supported the writing of the manuscript, implemented it, and
ensured scientific quality. AO-P, RB, and SM designed the figures.
AO-P, RB, GT, and SM made the final corrections. All authors
corrected and approved the manuscript.
FUNDING
This work was supported in part by National Institutes of Health
Grants R01 DK-091566 (to GT and SM).
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
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