Discussion
Our initial goal was to clarify the association between LILRA3
polymorphism and IBD susceptibility. Although no significant association
with IBD development was observed between rs103294, rs410852 and the
6.7-kb deletion genotypes, we demonstrated that homozygous 6.7-kb
deletion results in undetectable LILRA3 levels, which is consistent with
previous studies[18,19]. In addition, we detected
a much higher deletion ratio among Han population of Central China
compared with CHB. Unexpectedly, we found LILRA3 expression in IBD
patients was significantly higher than in healthy controls. LILRB1 and
LILRB2 are two members of LILRB family. LILRB1 was reported to block the
combination of CD8 molecular with HLA-I hence to regulate
CD8+ T cells. LILRB2 promoted hematopoietic stem cell
(HSC) proliferation. Considering that LILRA3 is homologous to LILRB1 and
LILRB2, additionally, LILRA3 alleles were in strong linkage
disequilibrium with LILRB2 alleles. Previous study reported that LILRA3
might affect functions of LILRB2 and LILRB1. Based on these findings, we
speculate that LILRA3 might play certain roles in immune system.
LILRA3, a member of the highly homologous family of receptors primarily
co-expressed on mono-myeloid leukocytes, is increasingly recognized as
regulating innate immune responses[24,35]. In
vitro, recombinant LILRA3 shows strong and specific binding to the
surface of primary peripheral blood mononuclear cells (PBMCs), U937
monocyte cells and B cells[23]. Additionally,
recombinant LILRA3 could dramatically abrogate lipopolysaccharide
(LPS)-induced TNF-α secretion by monocytes, and increases in LILRA3 have
been detected in MS and SLE patients. All these data indicate a critical
role for LILRA3 in inflammatory diseases. The intestinal LP contains a
diverse array of mononuclear phagocyte (MNP) subsets, including
conventional dendritic cells, monocytes and tissue-resident macrophages
that collectively play an essential role in mucosal homeostasis,
infection and inflammation. In our study, we also found that LILRA3 is
mainly expressed on macrophages located in the LP of intestinal and CD
patients possessed more CD68+LILRA3+cells. Intestinal macrophages are mainly derived from monocytes in
circulation, and LILRA3 was reported to mainly expressed on monocytes.
Since IBD is characterized by immunologic disorder and monocytes are
critical regulators in immune responses and played important roles in
IBD pathogenesis. We speculate that LILRA3 might exert certain function
on monocytes. To deduce the effect of LILRA3 on immune responses, in our
study, human U937 monocytes were employed to establish
LILRA3-overexpressing cells, and we observed that LILRA3 could markedly
decrease IFN-γ, TNF-α and IL-6 secretion and increase IL-10 secretion.
This finding further confirms an anti-inflammatory effect of LILRA3 in
immune responses.
Monocytes account for 2–10% of all leukocytes in human body. These
cells circulate through the blood and lymphatic system and are recruited
to damaged tissue or infected sites to differentiate into Mø and DCs,
thus triggering adaptive immune responses[36].
Therefore, stimulation of monocyte migration can help to boost
inflammatory responses. In vivo, migration is largely mediated by
interaction between chemokines and chemokine receptors. We found that
the migration ability of U937 cells was sharply attenuated by LILRA3.
CCL2, CCL3, CXCL8 and CXCL10 are the main chemokines secreted by
monocytes, and LILRA3 was able to conspicuously reduce expression of
these molecules on U937 cells. This finding is in agreement with our
transwell assay, which demonstrated impaired migration capacity of U937
cells when LILRA3 was overexpressed. Based on these results, we
speculate that LILRA3 might prevent monocyte migration from the
circulation into tissues, thereby suppressing inflammation.
In this study, LILRA3-overexpressing cells engulfed more latex beads
than cells harboring the null vector. This result indicates that LILRA3
can promote the phagocytosis capacity of U937 cells. Phagocytosis is
predominantly mediated by endocytic receptors present on the phagocyte
membrane[37]. SR and MR are important receptors
associated with endocytosis. SR consists of a family of receptors with
many ligands and can be divided into two subgroups: class A and class B.
Class A and class B can be further divided into many types including
SR-AI, SR-AII, SR-AIII, SR-BI, SR-BII, and
SRBIII[29,37]. SRBIII, also termed CD36, is mainly
expressed on cells of lymphoid and hematopoietic lineages, such as
monocytes, Mø, platelets, endothelial cells and a variety of cultured
cell lines[38,39]. MR is a carbohydrate-binding
receptor mainly expressed by Mø and DCs[32]. In
our study, both CD36 and CD206 were detected on U937 cells, and LILRA3
increased CD36 expression but had a limited effect on CD206 expression.
These results are consistent with our phagocytosis assay.
Similar to previous studies reporting that recombinant LILRA3 could
induce the proliferation of CD8+ T-cells and NK cells
in mixed lymphocyte reactions (MLR)[21], we
observed that increased LILRA3 expression could enhance U937 cells
proliferation in vitro. PI3K/Akt and PI3K/MEK/Erk signaling are two
essential pathways involved in cell survival and
growth[40,41], and we found that LILRA3 activated
these two pathways to regulate cell proliferation. Foxo3a, a
tumor-suppressive transcriptional factor, is known to regulate various
cellular events such as metabolism, proliferation, tumorigenesis, cell
cycle arrest, apoptosis and longevity[42-44].
Previous studies have shown that the two key kinases Akt and Erk have
the ability to regulate the transcriptional activity of Foxo3a via
specific phosphorylation[45-47]. Akt- or
MEK-mediated phosphorylation causes Foxo3a to bind to the 14-3-3
protein, followed by translocation from the nucleus to the cytoplasm,
with inhibition of transcriptional
activity[43,44,48]. The Ras/Erk pathway also
regulates cell growth by inhibiting Foxo3a via MDM2-mediated
degradation[44]. In our study, we observed that
Foxo3a phosphorylation was decreased when LILRA3 was overexpressed. In
contrast, phosphorylation of Foxo3a was conspicuously reduced when
LILRA3-overexpressing cells were pre-incubated with specific inhibitors
of MEK, Akt and PI3K. One possible explanation for this inconsistency is
that LILRA3 might directly/indirectly affect Foxo3a phosphorylation
through an unknown mechanism. Further experiments are needed to
elucidate this mechanism.
Taken together, we found that LILRA3 might function as an
anti-inflammatory molecule in U937 cells based on the following: 1)
LILRA3 directly inhibited inflammation by decreasing proinflammatory
cytokine secretion; 2) LILRA3 restrained monocytes in circulation system
by attenuating their migration ability and accordingly reduced Mø and
DCs in tissues, thereby restricting activation of adaptive immunity; 3)
increased LILRA3 enabled monocytic cells to engulf more pathogens in
circulation; 4) LILRA3 increased the proliferation of immunocytes, such
as monocytes, CD8+ T-cells and NK cells, thus allowing
these cells to kill more pathogens and microorganisms.
Collectively, for the first time, we report that LILRA3 expression is
markedly increased in IBD patients. LILRA3 might function as an
anti-inflammatory modulator in innate immune responses and regulate cell
proliferation through a combination of Akt and MEK/Erk signaling
pathways. Further efforts are needed to explore the exact role of LILRA3
in adaptive immunity.