Characterization of the pathoimmunology of necrotizing enterocolitis reveals novel therapeutic opportunities.

Necrotizing enterocolitis (NEC) is a severe, currently untreatable intestinal disease that predominantly affects preterm infants and is driven by poorly characterized inflammatory pathways. Here, human and murine NEC intestines exhibit an unexpected predominance of type 3/TH17 polarization. In murine NEC, pro-inflammatory type 3 NKp46-RORγt+Tbet+ innate lymphoid cells (ILC3) are 5-fold increased, whereas ILC1 and protective NKp46+RORγt+ ILC3 are obliterated. Both species exhibit dysregulation of intestinal TLR repertoires, with TLR4 and TLR8 increased, but TLR5-7 and TLR9-12 reduced. Transgenic IL-37 effectively protects mice from intestinal injury and mortality, whilst exogenous IL-37 is only modestly efficacious. Mechanistically, IL-37 favorably modulates immune homeostasis, TLR repertoires and microbial diversity. Moreover, IL-37 and its receptor IL-1R8 are reduced in human NEC epithelia, and IL-37 is lower in blood monocytes from infants with NEC and/or lower birthweight. Our results on NEC pathomechanisms thus implicate type 3 cytokines, TLRs and IL-37 as potential targets for novel NEC therapies.

Gp96 Peptide Antagonist gp96-II Confers Therapeutic Effects in Murine Intestinal Inflammation.

BACKGROUND: The expression of heat shock protein gp96 is strongly correlated with the degree of tissue inflammation in ulcerative colitis and Crohn's disease, thereby leading us to the hypothesis that inhibition of expression via gp96-II peptide prevents intestinal inflammation. METHODS: We employed daily injections of gp96-II peptide in two murine models of intestinal inflammation, the first resulting from five daily injections of IL-12/IL-18, the second via a single intrarectal application of TNBS (2,4,6-trinitrobenzenesulfonic acid). We also assessed the effectiveness of gp96-II peptide in murine and human primary cell culture. RESULTS: In the IL-12/IL-18 model, all gp96-II peptide-treated animals survived until day 5, whereas 80% of placebo-injected animals died. gp96-II peptide reduced IL-12/IL-18-induced plasma IFNγ by 89%, IL-1ß by 63%, IL-6 by 43% and tumor necrosis factor (TNF) by 70% compared to controls. The clinical assessment Disease Activity Index of intestinal inflammation severity was found to be significantly lower in the gp96-II-treated animals when compared to vehicle-injected mice. gp96-II peptide treatment in the TNBS model limited weight loss to 5% on day 7 compared with prednisolone treatment, whereas placebo-treated animals suffered a 20% weight loss. Histological disease severity was reduced equally by prednisolone (by 40%) and gp96-II peptide (35%). Mice treated with either gp96-II peptide or prednisolone exhibited improved endoscopic scores compared with vehicle-treated control mice: vascularity, fibrin, granularity, and translucency scores were reduced by up to 49% by prednisolone and by up to 30% by gp96-II peptide. In vitro, gp96-II peptide reduced TLR2-, TLR4- and IL-12/IL-18-induced cytokine expression in murine splenocytes, with declines in constitutive IL-6 (54%), lipopolysaccharide-induced TNF (48%), IL-6 (81%) and in Staphylococcus epidermidis-induced TNF (67%) and IL-6 (81%), as well as IL-12/IL-18-induced IFNγ (75%). gp96-II peptide reduced IL-1ß, IL-6, TNF and GM-CSF in human peripheral blood mononuclear cells to a similar degree without affecting cell viability, whereas RANTES, IL-25 and MIF were twofold to threefold increased. CONCLUSION: gp96-II peptide protects against murine intestinal inflammation by regulating inflammation in vivo and in vitro, pointing to its promise as a novel treatment for inflammatory bowel disease.