Mechanism Of IBD Susceptibility Gene CARINH To Maintain Intestinal Immunity And Commensal Bacterial Homeostasis

Inflammatory bowel disease(IBD)is a chronic disease with complex etiology influenced by genetic factors,dysregulation of the interaction between the intestinal immune system and microbiota is an important cause of IBD.In this research paper,we elucidate a long non-coding RNA located at an inflammatory bowel disease risk locus-CARINH(Colitis Associated IRF1 antisense Regulator of Intestinal Homeostasis),and further explore the molecular mechanism that CARINH transcript maintains intestinal homeostasis and inhibits the occurrence and development of IBD.We found in mice that Carinh and its neighboring gene encoding the transcription factor Irf1 together form a positive feedback loop in myeloid cells,and the Carinht/Irf1 positive feedback loop is regulated by the gut microbiota,and maintains the intestinal host-commensal homeostasis through the anti-inflammatory factor IL-18BP and anti-microbial factors called guanylate-binding proteins(GBPs).Meanwhile,we demonstrated that the function of the CARINH/IRF1 loop was conserved between humans and mice.And SNP rs2188962 is the IBD susceptibility locus with the highest risk factor located at the CARINH locus from the human genetics study,its mutation impairs the inducible expression of the CARINH/IRF1 loop and thus increases genetic predisposition to IBD.Therefore,our study demonstrates the mechanism that how an IBD-associated IncRNA CARINH maintains intestinal homeostasis and protects the host against colitis.The main results of this paper are as follows:1.lncRNA Carinh alleviates colonic inflammationWe demonstrated that IncRNA Carinh has no protein-coding potential by using gene transcription and translation reporter plasmid system.By using DSS to induce the colitis model in CarinhKO mice,we found that CarinhKO mice exhibited a significantly aggravated colitis phenotype with more severe intestinal damage compared with control mice.These results suggest that the lncRNA Carinh alleviates DSS-induced colonic inflammation.2.Myeloid cell-derived Carinh alleviates colonic inflammationWe then explored which cell-derived Carinh played a role in the development of colitis.Bone marrow chimeric-mice induced colitis model showed that hematopoietic cell-derived Carinh alleviates DSS-induced colonic inflammation.The DSS-induced colitis model in Rag1-/-mice transferred CD3+T cells and CD4+CD45RBhighT cells transfer colitis model both indicated that T cell-derived Carinh could not alleviates colonic inflammation.Clodronate liposome clearance and anti-CSF-1R antibody clearance experimentally induced colitis models both showed that myeloid cell-derived Carinh could alleviate colonic inflammation.These results suggest that myeloid cell-derived Carinh alleviates DSS-induced colonic inflammation.3.Carinh alleviates colonic inflammation by promoting Irf1 transcription in myeloid cellsRNA transcriptome sequencing of BMDM cells revealed that Carinh’s neighboring gene Irf1 was significantly down-regulated in CarinhKO mice.We then modeled colitis in Irf1KO mice and found that Irf1 gene deficiency aggravates DSS-induced colitis.Furthermore,we found that only Irf1Δmyeloid cells mice had aggravated colitis,but no difference was found in Irf1ΔIEC mice or Irf1ΔTcells mice,suggesting that myeloid cells derived Irf1 alleviates DSS-induced colonic inflammation.Mechanistically,we found that Carinh regulates the transcriptional level of Irf1 by recruiting CBP/p300 to promote H3K27 acetylation in the promoter region of Irf1.We also found that replenishment of the IL-18BP restored colitis in CarinhKO mice.These results suggest that Carinh could alleviate DSS-induced colonic inflammation by promoting Irf1 transcription in myeloid cells.4.Gut microbiota promotes the expression of the Carinh/Irf1 positive feedback loop to maintain intestinal homeostasisMeanwhile,we first found that the mRNA levels of Carinh,Irf1,and Il18bp were significantly down-regulated in intestinal tissues from GF mice and ABx-treated mice.We then found that Carinh and Irf1 together form a positive feedback loop in myeloid cells,and the loop is regulated by the gut microbiota.16S rDNA sequencing showed that Carinh or Irf1 gene deficiency can lead to gut microbiota changes in mice,and the altered gut microbiota aggravates DSS-induced colitis in turn.Furthermore,we found that Carinh affects the gut microbiota by regulating IRF1 downstream GBPs.These results suggest that gut microbiota promotes the expression of the Carinh/Trf1 positive feedback loop to maintain intestinal homeostasis.5.CARINH is a highly conserved inflammatory regulator geneWe found that CARINH was highly expressed in EBV-infected lymphocytes and intestinal tissues by analyzing GTEx data.CARINH mRNA levels were significantly up-regulated in colon tissue of IBD patients,while CARINH mRNA levels were positively correlated with IRF1 mRNA levels in PBMCs of IBD patients.We found that CARINH mRNA and IRF1 were specifically accumulated in CD11b+myeloid cells in human intestinal tissue.Furthermore,we found that the CARINH/IRF1 positive feedback loop is conserved in humans by genetic intervention in THP-1 cells.These results suggest that CARINH is a highly conserved inflammatory regulator gene.6.IBD-associated SNP rs2188962 impairs the inducible expression of CARINHA fine-mapping study of IBD identified SNP rs2188962 as the most likely susceptibility locus in the Chr5:131.2MB-132.2MB chromosomal region.We found that cells with neither C/T nor T/T mutations at SNP rs2188962 could induce the expression of CARINH,IRF1,and IL18BP normally,suggesting that SNP rs2188962 may increase the risk of IBD by affecting the inducible expression of CARINH and its downstream target genes.These results suggest that the IBD-associated SNP rs2188962 impairs the inducible expression of CARINH.In conclusion,our study elucidates that the lncRNA CARINH from an inflammatory bowel disease risk locus alleviates colonic inflammation by maintaining intestinal homeostasis.This is the first in-depth exploration of the role of IBD-related lncRNAs in intestinal homeostasis and inflammation,and explains the pathophysiology function of SNP rs2188962,a possible causal variant of IBD.This study provides a potential molecular target for the clinical diagnosis of IBD and helps to deepen the understanding of the genetic associated autoimmune diseases.