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  • O13 - Hepatic High Motility Group Box-1 (HMGB1) levels are affected by invariant Natural Killer T-cells in polymicrobial sepsis

    Author(s):

    John Young, Whitney Young, Shiang Chung, Yaping Chen, William Cioffi, Alfred Ayala, Daithi Heffernan, Brown University, Rhode Island Hospital



    Background: Invariant natural killer T-cells (iNKT) contribute to mortality in the murine model of polymicrobial sepsis. They play important roles in dampening the inflammatory response to sepsis, specifically neutrophils and macrophages. High motility group box 1 (HMGB1) is a highly conserved ubiquitous protein that has roles as a cytokine. It has been shown to be an early mediator of ischemic injury and a late mediator of experimental sepsis and is a potential target for immunomodulatory therapies. This study sought to clarify the role iNKT-cells in expression of hepatic HMGB1.

    Hypothesis: Hepatic HMGB1 will rise following CLP in WT mice with iNKT deficient mice showing a dampened HMGB1 response to sepsis.

    Methods: C57BL/6j (WT) mice or Jα18 -/- (iNK-/-) mice were subjected to either sham laparotomy (sham) or cecal ligation and puncture (CLP). 24 (for WT mice) or 48 hours (for WT and iNKT-/- mice) later, liver samples were prepared and HMGB1 was assessed using western blot analysis. The integrated density value was measured and normalized by GAPDH. Results are expressed as a normalized HMGB1: GAPDH ratio. Circulating TNF-α and IFN-γ (known stimulants of HMGB1 production) were measured using CBA cytometric analysis. Student’s t and ANOVAs were used for statistical analysis.

    Results: 24h post CLP, WT mice displayed no statistically significant difference in HMGB1 expression as compared to their sham counterparts (0.61+/-0.09 vs. 0.49+/-0.08; p>0.05). However, at 48h post CLP, hepatic HMGB1 expression was increased in WT mice (1.01+/-0.07 vs. 0.49+/-0.08; p<0.05). In contrast, iNKT-/- mice subjected to CLP did not show any change in expression of hepatic HMGB1 as compared to their sham counterparts (1.076+/-0.15 vs. 1.011 +/-0.1; p=0.7). iNKT-/- displayed significantly lower levels of TNF-α(15.3 +/-1.4 vs 49.2 +/-2.1 p=0.01) and IFN-γ (2.1+/-0.7 vs 12.3+/-1.7 ; p<0.001).

    Conclusions: Congruent with our current understanding of HMGB1, we have shown that CLP leads to a late increased hepatic HMGB1 expression. iNKT-/- mice display lower cytokine levels of known drivers of HMGB1 expression in sepsis. Both baseline and the septic response of hepatic HMGB1 appears to be modulated by iNKT-cells.