Figure Legends
Figure 1: LILRA3 is increased in IBD patients compared with healthy controls. (A) Impact of the 6.7-kb deletion on LILRA3 mRNA expression in peripheral blood. mRNA expression was evaluated by qRT-PCR assay. Data are expressed as the mean ± SD. Sample groups and genotypes were indicated. ** p < 0.01,*** p < 0.001 versus same genotypes in healthy controls (HC). (B) mRNA expression of LILRA3 in blood when samples homozygous for the deletion (-/-) were excluded. (C) mRNA expression of LILRA3 in the intestine. Subjects possessing undetectable LILRA3 levels were excluded. (D) Protein expression of LILRA3 in the intestine. Protein was extracted from 8 non-inflammatory bowel disease (NIBD), 7 CD and 10 UC samples. GAPDH was used as a loading control. (E) H&E staining (the left two columns) and immunohistochemistry for LILRA3 (the right column) were performed on 3 μm colon sections, and representative pictures are shown. Scale bar is 50 μm for the left column, 30 μm for the right two columns. (F) Negative control staining for immunohistochemistry. Sections were treated with PBS instead of the primary antibody. Scale bar is 30 μm. (G) Quantification of LILRA3+ cells in intestinal biopsies. A total of 20 NIBD, 14 CD and 14 UC samples were enrolled. Each experiment was repeated for at least three times. *p < 0.05,**p < 0.01, ***p < 0.001 versus HC or NIBD groups.
Figure 2: LILRA3 is mainly expressed on CD68+macrophages in human intestinal. (A) Localization of LILRA3 expression by Immunofluorescence. CD68 monoclonal antibody was used to mark macrophages. LILRA3 protein were expressed in CD68+macrophages and CD patients possessed more CD68+LILRA3+ cells than Non-IBD group. Scale bar is 20 μm for each column. (B) Negative control staining for immunofluorescence. Sections were treated with PBS instead of the primary antibody. (C) Quantification of LILRA3+CD68+ cells in intestinal biopsies. Each group includes 5 samples. Each experiment was repeated for at least three times. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3: Effect of LILRA3 on U937 cells and related mechanisms. (A) Verification of LILRA3 expression in the two established cells by qRT-PCR, Elisa and western blotting. (B) Effect of LILRA3 on cytokine secretion by U937 cells. Expression of IFN-γ, TNF-α and IL-6 was assessed by Elisa. Data are expressed as mean ± SD. (C) Supernate collected from the LILRA3-overexpressing U937 cell lines was used to culture the U937 cells, and secretion of IFN-γ, TNF-α as well as IL-6 were significantly decreased. Cells cultured for 12 hours showed a more significant effect of inhibition. (D) Representative flow cytometry analysis of apoptosis stained with annexin V-PE and 7-AAD among LILRA3-overexpressing U937 cells and U937 cells expressing the null vector. (E) Statistical analysis of apoptotic rate (%) in the two established cell lines. (F) Effect of LILRA3 on U937 cell migration. Cell counts migrated into the lower chamber were analyzed between the two cell lines. (G) Impact of LILRA3 on chemokine secretion in U937 cells by Elisa assay. CCL2, CCL3, IL-8 and CXCL10 were dramatically decreased by LILRA3. (H) Recombinant CCL2 and CXCL8 were added to the upper chamber in the migration assay, and LILRA3 induced impaired cell migration was reversed by the exogenous IL-8. Data are expressed as mean ± SD. Each experiment was repeated for at least three times.*p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4: LILRA3 enhances the phagocytosis ability of U937 cells by upregulating CD36 plasma membrane expression . (A) Representative FCM analysis of phagocytosis by U937 cells at 0.5, 6, 12 and 24 hours. The percentage of GFP+ Texas Red+ cells was analyzed. (B) Phagocytosis rates were statistically analyzed between LILRA3-overexpressing U937 cells and U937 cells expressing the null vector. (C, D and E) Effects of LILRA3 on CD36 and CD206 expression according to qRT-PCR and western blotting. (F) The specific antibody of CD36 could significantly decrease the phagocytosis ability of LILRA3-overexpression cell lines at a concentration dependent manner. Each experiment was repeated for at least three times.*p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5: LILRA3 increases U937 cell proliferation and the possible signaling pathway . (A) Proliferation of the LILRA3-overexpressing U937 cells and null vector U937 cells were analyzed by CCK-8 assay. OD at 450 nm was detected at 0, 12, 24, 48 and 72 hours. (B, C and D) Western blot analysis of p-Akt, Akt, p-MEK, MEK, p-Erk, Erk, p-P38 MAPK and P38 MAPK in LILRA3 overexpressing cells (OE) and null vector cells (NV). (E, F and G) Western blot analysis of p-PDK1, PDK1, p-c-Raf, p-PI3K, PI3K, p-Foxo3a and Foxo3a, p-Foxo4, p-Foxo1 in LILRA3 overexpressing cells (OE) and null vector cells (NV). Each experiment was repeated for at least three times.*p < 0.05, **p < 0.01, ***p < 0.001 versus null vector cells. GAPDH abundance was used as a control.
Figure 6: LILRA3 might regulate U937 cells proliferation through PI3K/Akt and PI3K/MEK/Erk signaling pathways . (A-C) Western blot analysis for the inhibitory efficiency of GSK1120212, MK-2206-2HCL and LY294002 and the expression of the related downstream proteins (p-Foxo3a and Foxo3a for (A) and (B), p-Akt, Akt, p-MEK, MEK, p-Foxo3a and Foxo3a for (C)). GAPDH abundance was used as a control. (D) CCK-8 assay to detect the proliferation of LILRA3-overexpressing U937 cells (OE) after pretreated with three specific inhibitors for indicated time. NV: Null vector U937 cells. Each experiment was repeated for at least three times. *p < 0.05, #p < 0.01 versus LILRA3-overexpressing U937 cells pretreated with DMSO.
Figure 7: A schematic of the possible signaling pathway of LILRA3 . LILRA3 might interact with one of the listed or unknown receptors and trigger PI3K/Akt and PI3K/MEK/Erk signaling to regulate U937 cell proliferation. LILRA3 might directly/indirectly inhibit Foxo3a phosphorylation. Whether Foxo3a plays a role in LILRA3-induced cell proliferation needs to be further investigated.