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  • O12 - TNFR1 Level in Sepsis is Dependent on MyD88-dependent Upregulation of iNOS in Hepatocytes

    Author(s):

    Meihong Deng, Patricia Loughran, Liyong Zhang, Melanie Scott, Timothy Billiar, UPMC



    Background: Our previous work showed thatTLR4-dependent TNFR1 shedding from mouse hepatocyts (HCs) occurs via iNOS signaling in vivo and in vitro. However, the upstream signal for iNOS upregulation and TNFR1 shedding is unclear. MyD88 and Trif are two well-known signaling molecules downstream of TLR4.

    Hypothesis: Therefore, we hypothesized that HC-TNFR1 shedding might be MyD88 or Trif dependent in sepsis.

    Methods: WT (C57BL/6), MyD88KO, TrifKO mice were injected with LPS (5mg/kg, ip) for 6, and 12h (n=6 mice/group).

    Results: TNFR1-shedding was completely suppressed in the MyD88KO but not in TrifKO mice after LPS treatment. INOS mRNA level in the liver was significant lower in MyD88KO than in WT and TrifKO mice. These findings using LPS as the activator show that HC-TNFR1 shedding and iNOS upregulation are MyD88-dependent. Several other relevant ligands can signal through MyD88. TNFR1 levels increased significantly in the media after 24 hrs treatment with Myddosome signals (HKLM-TLR2 ligand, LPS-TLR4 ligand, HMGB1-TLR4 ligand, ST-FLA -TLR5 ligand, OD1669 -TLR9 ligand and IL-1-IL-1R ligand) compared with the no treatment controls. To further investigate the role of MyD88 in hepatocyte TNFR1 shedding, WT, MyD88KO, TrifKO, HC-MyD88KO (HC MyD88 specific KO) and MyD88-flox mice (cell-specific KO controls) were subjected to a clinically relevant polymircrobial sepsis model (CLP with 5mg/kg imipenem, 8h, n=6 mice/group). Likewise, TNFR1-shedding was MyD88-dependent (334+/-83ng/mL in WT vs 179+/-36ng/mL in MyD88KO, p<0.05) but not Trif-dependent (284+/-114ng/mL) after CLP. Importantly, plasma TNFR1 level and iNOS mRNA level in the liver was significantly lower in HCMyD88KO than in MyD88-flox mice.

    Conclusions: These data link systemic TNFR1 levels and TNFR1 shedding in sepsis to signaling pathways that upregulate iNOS through MyD88 in HCs.