Fig 2: Leukocyte immunoglobulin-like receptor A3 (LILRA3) is increased in inflammatory bowel disease (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 quantitative real time polymerase chain reaction (qRT–PCR) assay. Data are expressed as mean ± standard deviation (s.d.);** P< 0·01,*** P< 0·001versusthe same genotypes in healthy controls (HC). (b) mRNA expression of LILRA3 in blood. (c,d) mRNA and protein expression of LILRA3 in the intestine. Protein was extracted from eight non-inflammatory bowel disease (NIBD), seven Crohn’s disease (CD) and 10 ulcerative colitis (UC) samples. (e) Hematoxylin and eosin (H&E) staining (left two columns) and immunohistochemistry for LILRA3 (right column). Representative pictures are shown. Scale bar is 50 µm for the left column, 30 µm for the two right columns. (f) Negative control staining for immunohistochemistry. 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; * P < 0·05, ** P < 0·01, *** P < 0·001 versus HC or NIBD groups.

Fig 3: Leukocyte immunoglobulin-like receptor A3 (LILRA3) increases U937 cell proliferation and the possible signaling pathway. (a) Proliferation of the LILRA3 over-expressing U937 cells and null vector U937 cells were analyzed by CCK-8 assay. Optical density (OD) at 450 nm was detected at 0, 12, 24, 48 and 72 h. (b–d) Western blot analysis of p-protein kinase B (Akt), Akt, p-mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), MEK, p-Erk, Erk, p-P38 (MAPK) and P38 MAPK in LILRA3 over-expressing cells (OE) and null vector cells (NV). (e,f,g) Western blot analysis of p-3-phosphoinositide-dependent protein kinase 1 (PDK1), PDK1, p-c-Raf, p-phosphoinositide 3-kinase/protein kinase (PI3K), PI3K, p-class O forkhead box transcription factors (Foxo3a) and Foxo3a, p-Foxo4 and p-Foxo1 in LILRA3 over-expressing cells (OE) and null vector cells (NV). Each experiment was repeated for at least three times; * P < 0·05,** P < 0·01,*** P < 0·001versusnull vector cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) abundance was used as a control.

Fig 4: A schematic of the possible signaling pathway of leukocyte immunoglobulin-like receptor A3 (LILRA3) . LILRA3 might interact with one of the listed or unknown ligands and trigger phosphoinositide 3-kinase/protein kinase (PI3K/Akt) and PI3K/mitogen-activated protein kinase/extracellular signal-regulated kinase (PI3K/MEK/Erk) signaling to regulate U937 cell proliferation. LILRA3 might directly/indirectly inhibit class O forkhead box transcription factors (Foxo3a) phosphorylation. Whether Foxo3a plays a role in LILRA3-induced cell proliferation needs to be further investigated.

Fig 5: Effect of leukocyte immunoglobulin-like receptor A3 (LILRA3) on U937 cells and related mechanisms. (a) 6.7-kb deletion and genotypes of rs103294, rs410852 for the U937 cell line. U937 cell line is homozygous for LILRA3 deletion (-/-). rs103294 is CC (wild-type), rs410852 is AA (wild-type). (b) Verification of LILRA3 expression in the two established cells by quantitative real time polymerase chain reaction (qRT–PCR), enzyme-linked immunosorbent assay (ELISA) and Western blotting. (c) Effect of LILRA3 on cytokine secretion by U937 cells. Expression of interferon (IFN)-?, tumor necrosis factor (TNF)-a, interleukin (IL)-6 and IL-10 was assessed by ELISA. Data are expressed as mean ± standard deviation (s.d.). (d) Supernate collected from the LILRA3 over-expressing U937 cell lines was used to culture the U937 cells, and secretion of IFN-?, TNF-a and IL-6 were significantly decreased. Cells cultured for 12 h showed a more significant effect of inhibition. (e) Representative flow cytometry analysis of apoptosis and statistical analysis of apoptotic rate (%) in the two established cell lines. (f) Effect of LILRA3 on U937 cell migration. (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 ± s.d.; * P < 0·05,** P < 0·01,*** P < 0·001.