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.