PAMPs and DAMPs in IBD

Abstract

An aberrant innate immune response against the enteric microbial flora plays an essential role in the pathogenesis of inflammatory bowel disease (IBD). The innate immune response is driven by myeloid cells through recognition of Pathogen Associated Pattern Molecules (PAMPs). In addition to these “exogenous” danger signals, it has been recently recognized that the “endogenous” danger signals, Damage Associated Molecular Pattern Molecules (DAMPs), can initiate or perpetuate the innate immune response. The prototype DAMP is high mobility group box 1 (HMGB1). While HMGB1 is a chromatin-associated protein and binds to DNA, it is released to extracellular space by cells undergoing necrosis. HMGB1 is also secreted from activated macrophages. Once released extracellulaly, HMGB1 triggers inflammatory signaling pathways in neighboring cells through the receptor for advanced glycation end products (RAGE), as well as toll-like receptor (TLR) 2 and TLR4. The purpose of this study is to elucidate immunologic effects of HMGB1 at the molecular level in macrophages and in vivo in a mouse model of IBD. The murine macrophage cell line, RAW264.7 cells, murine peritoneal and bonemarrow (BM) derived macrophages were stimulated with HMGB1 alone and combination with IFNγ. BM derived macrophages were also stimulated with HMGB1 and various PAMPs in combination. Mucosal HMGB1 in the inflamed colon of IL-10−/− mice was examined by immunohistochemisty. Fecal HMGB-1 levels were determined by a specific ELISA. HMGB1 stimulated IL-12 p40 protein and mRNA expression in RAW264.7 cells, peritoneal and BM derived macrophages. IFNγ in combination with HMGB1 synergistically activated IL-12 p40 expression. Gene expression patterns induced by HMGB1 in macrophages differed from the prototype PAMP, LPS, as HMGB1 did not induce IL-10 expression, while LPS induced IL-10 as well as IL-12 p40. Muramyl-dipeptide (MDP), but not other PAMPs, synergistically en hance IL-12 p40 and TNF expression induced by HMGB1 in BM derived macrophages. In vivo, a marked upregulation of HMGB1 protein was observed in intestinal epithelial cells and lamina propria cells of IL-10−/− mice compared to wild type mice. HMGB1 expression was prominent in the cytoplasm, consistent with the nuclear to cytoplasmic redistribution of HMGB1. Fecal HMGB1 was much more abundant in IL-10−/− mice compared to wild type mice. Fecal HMGB1 levels correlated with histological severity in IL-10−/− mice. HMGB1 stimulates proinflammatory cytokine production from macrophages, likely via distinct signaling pathways from that of PAMPs. HMGB1 specifically synergies with MDP to produce proinflammatory cytokines. HMGB1 is up-regulated in the inflamed colon of IL-10−/− mice and is detected in feces of mice with active colitis suggesting that HMGB1 could play a role in the pathogenesis of IBD.