Macrophages in xenotransplantation

Abstract

Xenotransplantation refers to organ transplantation across species. Immune rejection of xenografts is stronger and faster than that of allografts because of significant molecular differences between species. Recent studies have revealed the involvement of macrophages in xenograft and allograft rejections. Macrophages have been shown to play a critical role in inflammation, coagulation, and phagocytosis in xenograft rejection. This review presents a recent understanding of the role of macrophages in xenograft rejection and possible strategies to control macrophage-mediated xenograft rejection.

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ⓒ 2019 The Korean Society for Transplantation
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Macrophages in xenotransplantation
Jae Young Kim
Department of Life Science, Gachon University, Seongnam, Korea
Xenotransplantation refers to organ transplantation across species. Immune rejection of xenografts is stronger and faster
than that of allografts because of significant molecular differences between species. Recent studies have revealed the involve-
ment of macrophages in xenograft and allograft rejections. Macrophages have been shown to play a critical role in inflammation,
coagulation, and phagocytosis in xenograft rejection. This re vi ew presents a recent understanding of the role of macrophages
in xenograft rejection and possible strategies to control macrophage-mediated xenograft rejection.
Keywords: Xenotransplantation; Macrophages; Inflammation; Coagulation
Received December 9, 2019
Revised December 19, 2019
Accepted December 19, 2019
Correspondence to: Jae Young Kim
Department of Life Science, Gachon University, 1342 Seongnam-
daero, Sujeong-gu, Seongnam 13120, Korea
Tel: +82-31-750-4762
E-mail: jykim85@gachon.ac.kr
Review Article
INTRODUCTION
Xenotransplantation is the transplantation of organs, tis-
sues, or cells across species. Pigs are considered to be
an ideal organ source for xenotransplantation due to their
physiological similarity to humans and the feasibility of
pig breeding. However, immune rejection of xenografts
is believed to be stronger and faster than that of
allografts. This can be attributed to the significant mo-
lecular differences between species. Xenograft rejection
can be temporally divided into four interlinked immune
rejection types: hyperacute rejection (HAR), acute vas-
cular and cellular rejections, and chronic rejection [1].
HAR is mediated by human antibodies against carbohy-
drate moieties, such as galactose 1,3-galactose (-gal)
and N-glycolylneuraminic acid, present on the surfaces
of pig endothelial cells [2]. Binding of pre-existing anti-
bodies to these antigens and subsequent complement ac-
tivation destroy pig endothelial cells, resulting in xeno-
graft rejection within a few minutes [3]. During the last
two decades, outcomes of pig-to-non-human primate
organ transplantation have been markedly improved ow-
ing to development of genetically-engineered pigs;
transplants from such pigs help avoid HAR [4].
However, many aspects of immune responses to xeno-
grafts still pose a critical problem for successful
transplantation.
Macrophages are phagocytic innate immune cells that
play a crucial role in host defense. Recent studies have
revealed the involvement of macrophages in immune re-
jection of organ transplants. In animal allotransplant
models, macrophages recognize allogeneic antigens, in-
duce immune responses, and thus contribute to graft re-
jection [5,6]. In addition, they are able to kill allogeneic
cells by phagocytosis [7]. Clinical studies exhibit a pos-
itive correlation between macrophage infiltration and
graft rejection [8-11]. The recently arising role of mac-
rophages in allograft rejection must be one of the inter-
esting topics in the field of transplant immunology.
Therefore, the purpose of this paper is to review the re-
cent understanding of the role of macrophages in xeno-
graft rejection and possible strategies to control macro-
phage-mediated xenograft rejection.
Korean J Transplant 2019;33:74-82
https://doi.org/10.4285/jkstn.2019.33.4.74

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Kim JY. Macrophages in xenotransplantation
HIGHLIGHTS
∙Damage-associated molecular pattern (DAMP) re-
lease during ischemia reperfusion injury is one of the
main causes of activation of macrophages, which play
a critical role in inflammation and coagulation in xeno -
graft rejection.
∙Cross-talk between macrophages, hepatocytes, and
vascular endothelial cells by producing immune medi-
ators, such as monocyte chemoattractant protein 1
(MCP-1), interl eu ki n ( IL)-6, and creac ti ve protein
(CRP) may play a critical role in inflammatory re-
sponses and coagulation in pig-to-baboon organ
transplantation.
∙Early generation of MCP-1, IL-6, and CRP as well
as DAMPs needs to be controlled to avoid inflammation
and coagulation.
ROLE OF MACROPHAGES IN
XENOGRAFT REJECTION
Inflammation and Coagulation
Inflammation, triggered by innate immune cells as a de-
fense mechanism against infectious agents or tissue dam-
age, is a major problem in organ transplantation.
Damaged host cells release or secrete various dam-
age-associated molecular patterns (DAMPs) [12]. In or-
gan transplantation, DAMP release from injured tissues
is inevitable during ischemia reperfusion injury (IRI) and
levels of the released DAMPs increase following IRI.
DAMPs are recognized by cellular receptors of various
cell types including, the Toll-like receptors (TLRs) of
macrophages, and the inflammasomes, which trigger in-
tracellular signaling that leads to inflammatory responses
and subsequent initiation of rejection of the transplanted
organ [5,6]. As shown in Table 1, IRI-induced release
of various DAMPs, such as high-mobility group box 1
(HMGB1) [13-16], nuclear DNAs or mitochondrial DNAs
[17-19], heat shock proteins (HSPs) [20-23], and his-
tones [24], was detected in different types of solid or-
gans after transplantation.
Unlike other DAMPs, adenosine triphosphate (ATP) is
a relatively small molecule and is recognized by specific
cell surface receptors, such as P2X and P2Y. Binding of
extracellular ATP to these receptors induces in-
flammatory responses of macrophages [25]. A study us-
ing a murine liver allotransplantation model has suggested
extracellular ATP involvement in increased graft dys-
function and the involvement of the reduction of regu-
latory T cell frequency in overall graft survival [26].
The role of the coagulation pathway in IRI and its
crosstalk with the inflammatory pathway have been re-
cently proposed [27]. Tissue factor (TF), which is the
primary initiator of coagulation and is expressed on both,
monocytes/macrophages and endothelial cells, is a central
player in providing a bridge between these pathways
[28]. Macrophages play a critical role in coagulation as
well as inflammation in xenograft rejection (Fig. 1).
During IRI, DAMPs activate monocytes/macrophages,
which then, produce proinflammatory cytokines. In re-
sponse to DAMPs and the pro-inflammatory cytokines,
TF is rapidly induced by these cells in the graft recipi-
ents, and becomes exposed to blood [29,30]. Cell surface
TF can complex with factor VIIa, and thereby trigger co-
agulation by activating factor X and subsequent coagulat-
ing factors. TF can also induce protease-activated re-
ceptor-mediated signaling, which leads to the production
of more inflammatory cytokines, upregulation of adhesion
molecules, and suppression of thrombomodulin (Fig. 1A)
[31].
One of the reasons for the stronger immune rejection
of xenografts than that of allografts is associated with
vascular endothelial cells. In the early phase of organ
xenotransplantation, xenoantigen-specific innate anti-
bodies can bind to and activate the endothelial cells,
causing the release of proinflammatory immune media-
tors, as well as the activation of the complement medi-
ated destruction of the endothelial layer. Monokines,
such as interleukin (IL)-1, IL-6, and tumor necrosis
factor-, secreted by monocytes/macrophages, can also
activate vascular endothelial cells across pig and human
species [32]. Activated endothelial cells release CD62,
von Willebrand Factor, platelet factor 4, and CD40 ligand
(Fig. 1A) in addition to proinflammatory cytokines. All
these mediators eventually lead to the recruitment of
more inflammatory cells and activation of coagulation
[31]. The interplay between the inflammatory responses

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Korean J TransplantㆍDecember 2019ㆍVolume 33ㆍIssue 4
Table 1. DAMPs identified in inflammatory diseases and upon clinical organ transplantation [39,40]
Origin DAMP Receptor Organa)
Extracellular matrix Biglycan
Decorin
Heparan sulfate
Hyaluronan
Fibrinogen
Fibronectin
Tenascin C
Versican
TLR2, 4, NLRP3
TLR2, 4
TLR4
TLR2, 4, NLRP3
TLR4
TLR4
TLR4
TLR2, 6, CD14
K
H, Lu
H, K
H, K, Lu
Intracellular compartment
cytosol ATP
-amyloid
Cyclophilin A
F-actin
HSP
S100 proteins
Uric acid
P2X7, P2Y2
TLR2, NLRP1, 3, CD36, RAGE CD147
DNGR-1
TLR2, 4, CD91, LOX-1
TLR2, 4, RAGE
NLRP3, P2X7
H, Li
H, K, Li, Lu
Nuclei DNA
Histones
HMGB1
HMGN1
IL-1
IL-33
RNA
SAP130
TLR9, AIM2
TLR2, 4
TLR2, 4, RAGE
TLR4
IL-1R
ST2
TLR3, 7, 8, RIG-I, MDA5, Mincle
H, K, Li, Lu
H, K, Li, Lu
Mitochondria Formyl peptide
mtDNA
mtROS
mtTFA
FPR1
TLR9
NLRP3
RAGE
H, Li, Lu
Granule Cathelicidin
Defensins
EDN
Granulysin
P2X7, FPR2
TLR4
TLR2
TLR4
Plasma membrane Glypicans
Syndecans
TLR4
TLR4
Endoplasmic reticulum Calreticulin CD91
DAMP, damage-associated molecular pattern; K, kidney; H, heart; Lu, lun g ; ATP , a deno s i n e tri p h osph a t e; Li , l i v er; HS P , hea t s h ock
protein;
IL, interleukin; mtDNA, mitochondrial DNA; mtROS, mitochondrial reactive oxygen species; mtTFA, mitochondrial transcription
factor
A; EDN, eosinophil-derived neurotoxin.
a)Designated organs where indicated DAMPs have been identified and studied in clinical solid organ transplantation.
and the coagulation system plays a significant role in
xenograft rejection [33-36].
Phagocytosis
The mechanism by which macrophages distinguish be-
tween self and allogeneic non-self organs, tissues, cells,
or antigens and promote organ rejection has been re-
cently clarified [37]. Mice that lack T, B, and natural
killer cells could distinguish allogeneic antigens from
those of self-tissues and induce an innate response. This
innate allo-activation is triggered by mismatch between
donor and recipient signal regulatory protein  (SIRP),
which is a cell surface molecule interacting with CD47.
Similarly, macrophages are able to recognize and destroy
xenografts through their cell surface interactions between
CD47 and SIRP 1 (Fig. 1B). Due to their molecular in-
compatibility, an impaired interaction between pig CD47
and human SIRP 1 can result in the phagocytic killing
of pig endothelial cells by human macrophages [38].
However, pig hematopoietic cells expressing human

77
Kim JY. Macrophages in xenotransplantation
Fig. 1. (A-C) Central role of activ-
ated macrophages in
inflammation,
coagulation, phagocytosis, and ant-
igen presentation [31]. TNF,
tumor
necros; IL, interleukin; MCP-1, mo
-
nocyte chemoattractant protein
1;
DAMP, damage-associated
molecular
pattern; TLR, Toll-like receptor;
TF,
tissue factor; PLT, platelet;
VWF,
von Willebrand factor; PF4,
platelet
factor 4; TM, thrombomodulin; PAR
,
protease-activated receptor; ICA-
M-1, intercellular adhes io n mole-
cule-1; VCAM-1, vascular cell adhe-
sion molecule-1; SIRP, signal regul-
atory protein .
Fig. 2. Possible cross-talk between macrophages, hepatocytes,
and
vascular endothelial cells through the production of
interleukin
(IL)-6, monocyte chemoattractant protein 1 (MCP-1), and
C-
react i ve p r o t e i n ( C RP) in inf l am m a t o r y r e spons e s a n d
coagulation
in pig-to-baboon organ transplantation. TF, tissue factor.
CD47 could be protected from phagocytic killing by hu-
man macrophages in hematopoietic cell engraftment ex-
periments [41].
Chemotaxis and Acute Phase Responses
Macrophages are the major cells infiltrating into an allog-
raft during severe rejection [42]. Similarly, macrophage
infiltration occurs just after IRI of a xenograft and per-
sists until graft rejection [43,44]. The infiltration level
of macrophages was significantly higher in -gal knock-
out xenogeneic islets than in allogeneic islets [45]. The
mechanism of monocyte accumulation within a xenograft
is thought to be associated with the production of chemo-
kines, such as monocyte chemoattractant protein 1
(MCP-1), in the graft [35,46].
In pig-to-baboon heart and kidney transplantation, it
was observed that early elevated serum levels of MCP-1,
IL-6, and C-reactive protein (CRP), which is an acute
phase protein synthesized by hepatocytes in response to
proinflammatory cytokines [47], precede consumptive
coagulopathy [35]. In addition, increased numbers of
monocytes were associated with enhanced expression of
TF [35]. These results, taken together with those of
previous reports indicate that IL-6 provokes liver cells
to produce CRP [48], which stimulates endothelial cells
to produce MCP-1 [49], and both IL-6 [50] and CRP
[51] promote TF expression, suggesting the occurrence
of cross-talk between macrophages, hepatocytes, and
vascular endothelial cells through the production of these
mediators. MCP-1, produced by the activated vascular
endothelial cells, recruit monocytes/macrophages, which
are eventually activated to produce IL-6, which stim-
ulates liver cells to release CRP, which in turn activates
vascular endothelial cells. This positive feedback loop
may play a critical role in inflammatory responses and
coagulation in pig-to-baboon organ transplantation (Fig.
2). Therefore, early upregulation of CRP, IL-6, and
MCP-1 levels needs to be suppressed to avoid in-
flammation-induced coagulopathy.

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Korean J TransplantㆍDecember 2019ㆍVolume 33ㆍIssue 4
STRATEGIES TO CONTROL
MACROPHAGE-MEDIATED
XENOGRAFT REJECTION
A recent study has suggested that there is increasing
evidence for sustained inflammatory response in
pig-to-baboon xenograft recipients, and this systemic
inflammation is a critical hurdle for successful xeno-
transplantation [52]. Therefore, therapeutic prevention
of inflammation is necessary to achieve successful pig or-
gan xenotransplantation.
Anti-inflammatory Drugs
A commonly used immunosuppressive regimen in recent
studies of pig-to-non-human primate xenotrans-
plantation includes antithymocyte globulin, cobra venom
factor (CVF), corticosteroids, and an immunophilin in-
hibitor, such as cyclosporine A, tacrolimus, or rap-
amycin. In addition to this regimen, several other se-
lective biological drugs aimed at suppressing immune re-
sponses after xenotransplantation have been tested in
separate studies, such as anti-CD154 monoclonal anti-
body (mAb), CTLA-4 fusion proteins, anti-CD40 mAb,
anti-CD20 mAb, anti-CD25 mAb, anti-IL-6 receptor
mAb, and anti-LFA1 mAb [45,53]. Among them, mAbs
for blocking the interaction between CD40 and CD154,
and CTLA-4 fusion protein appear to be essential for
achieving long term xenograft survival. CD40–CD154 in-
teractions stimulate the inflammatory response. CD40
expression is induced in activated monocytes/macro-
phages while CD154 is also expressed on monocytes/
macrophages during inflammation [54]. Blockade of the
IL-6 receptor with the anti-IL-6 receptor mAb, tocili-
zumab, resulted in a reduction in the levels of CRP [36]
and serum histones [55] upon pig-to-non-human pri-
mate xenotransplantation.
Other inflammatory drugs were also tested. Nuclear
factor kappa B inhibitor, parthenolide, significantly sup-
pressed histone-induced pig endothelial cell death in in
vitro study [55]. CVF, which had been originally used
to deplete complements causing HAR following xeno-
transplantation, was found to reverse the increased IL-6
and MCP-1 levels in pig-to-baboon heart and artery
patch transplantation [56].
Since the effective treatment of an established in-
flammatory response to DAMPs is relatively difficult, se-
lective and rapid blocking or scavenging of released
DAMPs would be a more promising therapeutic strategy.
Indeed, anti-histone therapy was found to prevent his-
tone-induced inflammation in xenotransplantation [55].
Mice treated with HMGB1 antibody were protected
against pulmonary dysfunction and had improved lung al-
lograft outcomes [16]. Blockade of HMGB1 secretion by
small molecule inhibitor was found to be beneficial to pre-
vent the loss of islet grafts and to reverse diabetes in
murine syngeneic islet transplantation [57]. Administr-
ation of ATP antagonist to a recipient mouse for 2 weeks
led to prolonged survival of the transplanted allogeneic
heart [58].
Laboratory studies have suggested possible manipu-
lation of the inflammatory response by using a DAMP an-
tigen rather than using an antibody or antagonist.
Ischemic preconditioning with HMGB1 protected grafts
from IRI through TLR4 signaling in renal and hepatic al-
lotransplantation [59,60]. In addition, genetic over-
expression of HSP27 could reduce IRI-induced apoptosis
of graft cells and delay the onset of acute rejection in
murine heart allotransplantation [61].
Targeting Macrophages
Deletion or inhibition of macrophages can attenuate graft
injury and prolong graft survival [62]. In recent animal
and clinical studies, some macrophage subsets have been
reported to act as regulatory cells, and the adoptive
transfer of these macrophages significantly prolonged
graft survival. A subset of macrophages was found to
suppress allogeneic T cell proliferation and inhibit den-
dritic cell maturation [63,64]. Furthermore, adoptive
transfer of these macrophages promotes graft survival
and minimizes immunosuppression [64,65].
Although immunological memory has long been thought
to be driven exclusively by adaptive immunity, new evi-
dence suggests that various tissue-derived factors can
induce epigenetic changes, leading to the formation of in-
nate memory of macrophages [66]. Further under-
standing of the mechanisms of innate memory could allow

79
Kim JY. Macrophages in xenotransplantation
us to dampen the response to DAMPs induced during the
xenograft rejection.
CONCLUSIONS
Although T cells are major players of organ transplant
rejection, early inflammation after transplantation is
critical for T-cell mediated immune rejection. Thus,
prevention of inflammation following transplantation
might help avoid acute graft rejection. Monocytes/mac-
rophages are critical immune mediators of inflammation,
and thus are important targets for immune modulation.
Owing to phenotypical and functional heterogeneity of
macrophages, the identification and/or isolation of dis-
tinct subsets of macrophages involved in graft rejection
or tolerance is essential to develop macrophage-based
therapeutic strategies. Compared to allotransplantation,
xenotransplantation has some advantages, including fea-
sibility of genetic modification and preconditioning for
transplantation. Thus, understanding precise mecha-
nisms of macrophage-mediated immune responses in or-
gan xenotransplantation will enable establishment of
strategies to modulate macrophage function, which can
improve the outcomes of xenotransplantation in future
clinical practice.
ACKNOWLEDGMENTS
Conflict of Interest
No potential conflict of interest relevant to this article
was reported.
Funding/Support
This study was supported by research grant from the Korean
Society for Transplantation (2019-04-03001-002).
ORCID
Jae Young Kim https://orcid.org/0000-0001-9323-3580
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