O-034 Novel Isoforms of Leukocyte Immunoglobulin Like Receptor B3 in Pediatric UC (2014)

The inflammatory bowel diseases (IBD) Crohn’s disease (CD) and ulcerative (UC) colitis are chronic relapsing gastrointestinal (GI) inflammatory diseases that are driven by an aberrant intestinal inflammatory response associated with exaggerated T-cell and macrophage (MØ’s) activation directed against luminal antigens. Recently a novel family of molecules known as inhibitory receptors that negatively-regulate the activating signal associated with inflammatory responses has been identified. To define the involvement of inhibitory receptors in IBD, we examined RNAseq datasets derived from rectal biopsy samples from pediatric patients with confirmed persisting diagnosis of CD, UC, or non-IBD control (Ctl) (Crohn's & Colitis Foundation of America (CCFA) sponsored RISK study) and determined the susceptibility of mice deficient in the murine homologue of Lilrb3, Paired Immunoglobulin receptor B (Pirb) in the NSAID (piroxicam)-induced Il10−/−-colitis model. We demonstrate differential expression of the human inhibitory receptor members “LILRB” LILRB2, LILRB3 and LILRB4 between CD, UC and non-IBD. Notably, LILRB1 and LILRB3 were significantly increased in UC and not CD compared with non-IBD. Importantly, LILRB3 positively correlated with S100A9 and IL1B expression (P < 0.001 r2 = 0.766 and P < 0.0001 r2 = 0.69, respectively) suggesting that levels positively correlate with disease severity and mucosal inflammation. This was somewhat counterintuitive; as the LILRB3 receptor constrains inflammation and we would predict that increased expression would negatively correlate with inflammation and injury. Examination of the RNAseq datasets for LILRB3 isoforms in non-IBD and UC groups identified 9 different LILRB3 isoforms present in rectal biopsy samples from both non-IBD and pediatric UC. Bioinformatic analyses revealed that the majority of these isoforms were expressed at low levels and were not differently expressed between groups. However, we identified 2 novel LILRB3 isoforms that were increased 1.6- and 2.4-fold in the pediatric UC group compared with non-IBD. Novel LILRB3 isoform 1 lacked exons 1-4 (LILRB3Ex1-4) encoding the 5’-UTR, signal peptide and the C-terminal end proximal to C2-type immunoglobulin like domains. Isoform 2 lacked exons 1–7 (LILRB3Ex1-7) which encoded for the 5’-UTR, signal peptide and complete extracellular domains but still possessed the transmembrane and intracellular regions. Structural analysis of LILRB3Ex1-7 isoform predicts that variant would be a dominant negative “form” of LILRB3 and hypothesized that this isoform (LILRB3Ex1-7) could counteract the full length “FL” LILRB3 inhibitory activity leading to unconstrained immune activation. Consistent with this concept, the ratio of FL LILRB3 and LILRB3Ex1-7 RNA transcripts in rectal biopsy samples from pediatric UC patients was 2.75-fold lower than that observed in non-IBD controls. This was specific to the LILRB3Ex1-7 isoform as the ratios of other isoforms of LILRB3 to FL LILRB3 were not significantly different between non-IBD and pediatric UC patients. Mice deficient in both Pirb and Il10 had increased susceptibility to piroxicam-induced colitis further corroborating a role for Lilrb3 in the negative regulation of colonic inflammation and the colitic phenotype. Lilrb3 negatively regulates innate colonic inflammation and development of IBD. We are currently defining the importance of Lilrb3 isoforms in the negative regulation of innate immune intestinal inflammation in IBD.