Tu1854 The Alarmin High Mobility Group Box 1 (HMGB1) Is Released From Enterocytes During Intestinal Inflammation and Causes Increased Interferon-γ and Decreased Na+/H+ Exchanger Isoform 3 Expression

Aims: The alarmin HMGB1 is released during cell damage and acts as an early or late stage inflammatory cytokine at multiple levels of target cells. The Na+/H+ exchanger Isoform 3 (NHE3) is the major intestinal salt and fluid absorptive transporter and its function is defective in intestinal inflammation. Aim of the study: We wanted to know if HMGB1 can affect NHE3 mRNA or protein expression as well as its localization in mucosal tissue form patients with ulcerative colitis and in NHE3-expressing Caco2bbe colonic cells. Methods and Results: We studied the mRNA and protein expression and localization of HMGB1 and NHE3 in rectal biopsies from the inflamed and noninflamed mucosa of patients with ulcerative colitis by quantitative qPCR and immunohistochemistry. TNF-α and IFN-γmRNA level was significantly elevated in lesions compared to that in its adjacent normal tissue samples of UC patient's rectal, suggesting a reliable endoscopic sampling of inflamed vs noninflamed tissue. HMGB1 mRNA and protein expression was increased in the inflamed as compared to adjacent normal tissues, and it was mostly distributed in cytoplasm and extracellular region. NHE3 protein was markedly reduced in inflamed compared to adjacent normal tissues, though its mRNA level was not significantly different between inflamed and noninflamed mucosa. When Caco2bbe cells overexpressing human NHE3 (Caco2bbe/ hNHE3) were incubated with high concentration of rh-HMGB1 (10μg/ml), NHE3 mRNA was found to be decreased at 1h (0.385±0.015 vs. 1.011±0.011, p 0.05). NHE3 protein, measured byWestern analysis, was not significantly changed when co-cultured with 10μg/ml rhHMGB1 for 1h or 6h, however, it was dramatically decreased at 24h of co-culture with 10μg/ml rhHMGB1. IFN-gamma levels were markedly increased when Caco2bbe/hNHE3 cells were incubated with 10 μg/ml rhHMGB1 for 1h as compared with normal group (29.855±0.055 vs. 29.525±0.050, p 0.05) and 24h (29.066±0.181 vs. 29.525±0.050, p>0.05) of co-culture with 10μg/ml rhHMGB1 as compared to normal group. This suggests that incubation with rhHMGB1 may down-regulate NHE3 mRNA expression via elevated IFN-gamma level at early stage (1h), resulting in a decrease in NHE3 protein expression at late stage (24h). Conclusion: Decreased NHE3 protein level was accompanied by elevated cytoplasm and extracellular HMGB1 in human ulcerative rectitis tissue. This may result from the regulatory effect of cytokines such as IFNgamma induced by exogenous HMGB1 at late stage of inflammation.