The Gene Polymorphisms And Expression Of LILRA3 In Inflammatory Bowel Diseases And Its Roles In Regulating Functions Of Human Monocytes

Backgrounds and Objectives Inflammatory bowel diseases(IBDs),including Crohn’s disease(CD)and Ulcerative colitis(UC),are chronic inflammatory diseases mainly involve the gastrointestinal tract.It was characterized by recurring episodes of relapsing and remitting inflammation and its main clinical manifestations are abdominal pain,diarrhea as well as hematochezia.The internal mechanism of IBD is still obscure,yet a complex interaction between predisposing genes,environmental factors,and dysregulated mucosal immune responses to the commensal gut microbiome has been widely accepted as the pathogenesis of IBD.LILRA3(Leukocyte Immunoglobulin Like Receptor A3,LILRA3)also termed as ILT-6/CD85e,is a special member of LILRs(Leukocyte Lmmunoglobulin like Receptors,LILRs).Studies about LILRA3 are limited and most of which reported that LILRA3 was associated with many autoimmune diseases such as systemic lupus erythematosus,rheumatoid arthritis,multiple sclerosis and lymphoma.Besides,increased LILRA3 was detected in some autoimmune diseases which indicating a crucial rule of it in human immune response.Until now,there are limited studies about LILRA3 in China.Since dysregulated immune responses are critical cause of IBD,and the rules of LILRA3 in immune response,we speculate that LILRA3 might associate with IBD development.To verify our speculation,we detected LILRA3 expression systematically(in the peripheral blood)and locally(in the colon)in IBD patients,and further explore its possible mechanism in IBD pathogenesis.Our study will offer a unique insight into the theoretical basis of whether LILRA3 can be a potential therapeutic target for IBD.Methods 185 CD patients,193 UC patients and 509 healthy controls were collected from the gastroenterology department of Zhongnan Hospital of Wuhan University from September 2014 to January 2016.1ml peripheral blood samples were obtained from each subject and the basic information together with clinical data was recorded.Total DNA was extracted from the peripheral blood and PCR-LDR(Polymerase Chain Reaction-Ligation Detection Reaction)was applied to genotype the polymorphism of 6.7kb deficiency on its DNA sequence and two SNPs including rs 103294 and rs410852.Relationships between genotypes and disease developing,genotypes and phenotypes of disease were analyzed.Total RNA was extracted from 36 CD patients,48 UC patients and 53 healthy controls and qRT-PCR was used to determine systematically LILRA3 mRNA expression in blood.Besides,the influence of genotyping variants on LILRA3 mRNA expression was further investigated.Non-inflamed colonic mucosa samples from 20 patients with polyps or neoplasms and inflamed colonic of 14 CD and UC patients were obtained to explore LILRA3 site expression by IHC(Immunohistochemistry,IHC).Total RNA was extracted from inflamed colonic of 36 CD,52 UC and normal biopsies of 14 healthy controls to detect locally LILRA3 mRNA expression in colon.Another inflamed colon biopsies from 7 CD,10 UC patients and 8 normal biopsies of NIBD patients were collected.Total protein was extracted from the samples.Western-blotting was applied to detect LILRA3 protein expression in the colon.LILRA3 over-expressing U937 cell line and U937 cells transfected with null plasmids were established.The potential function of LILRA3 on U937 cytokine secretion,cell migration,cell apoptosis and cell phagocytosis,cell proliferation were then investigated.Results Polymorphism of 6.7kb deletion and rs410852 has no association with CD or UC development(P>0.05).At the genotype level,rs103294 has no association with CD or UC development while at the allelic level,rs 103294 was related with CD(P=100.04,OR=1.32,95%CI=1.01-1.73).No significant association was found between 6.7kb deletion,rs103294 genotypes and phenotypes of CD or UC(P>0.05).CD patients carrying rs410852 genotype were less likely to develop intestinal stricturing or penetrating(stricturing,P=0.03,OR=0.23,95%CI=0.07-0.75;penetrating,P=0.04,OR=0.20,95%CI=0.05-0.74).Variants of 6.7kb affected LILRA3 expression with that of homozygous deletion subjects were almost undetectable.IBD patients possessed increased LILRA3 mRNA and protein levels compared with those of control group.IHC tests revealed that LILRA3 existed in a soluble form,and was mainly located in the LP(lamina Propria,LP)of the mucosa.These data reflects important roles of LILRA3 in inflammation.The two cell lines were successfμlly established and we observe that(1)LILRA3 could markedly decrease secretion of proinflammatory cytokines such as IFN-y,TNF-a and IL-6;(2)LILRA3 abated monocyte migration by reducing the expression of several chemokines and chemokine receptors on U937 cells;(3)LILRA3 enhanced phagocytosis of U937 cells by increasing CD36 expression;(4)LILRA3 promoted U937 cell proliferation through activating a combination of Akt and MEK/Erk signaling pathways;(5)LILRA3 had limited effect on U937 cell apoptosis.Conclusions(1)LILRA3 gene was a susceptibility locus for IBD in Chinese Han popμlation.Its genotype polymorphisms were associated with IBD clinical phenotype.These results need to be verified in a different popμlation.(2)6.7kb has no association with IBD development,while its variation affects LILRA3 expression.(3)The mRNA and protein levels of LILRA3 are significantly elevated in both CD and UC patients compared with those of control group.More LILRA3-positive cells were detected in the LP of CD and UC colon samples.(4)LILRA3 significantly decrease IFN-y,TNF-a and IL-6 secretion.(5)LILRA3 abated monocyte migration by reducing the expression of several chemokines and chemokine receptors on U937 cells.(6)LILRA3 could not influnce the apoptosis of U937 cells.(7)LILRA3 increased CD36 expression on U937 cell membrane thus enhanced the phagocytosis ability of U937 cells.(8)LILRA3 could promote proliferation of U937 cells by activating Akt and MEK/Erk signaling pathways.