Fig. 3.

MTV increases circulating levels of cytokines and chemokines. Cytokines (TNF-α and IL-6) and chemokines (MIP-2 and MCP-1) in plasma detected by ELISA. Mice receiving combination of CLP + MTV (two-hit model) compared to mice subjected to CLP alone or sham operation followed by MTV. Two-hit model in wildtype mice compared to subgroup of TLR4^−/− mice (TLK4 KO) or wildtype (WT) mice that received intratracheally neutralizing antibodies to either integrin β5 (β5 Ab) or WISP1 (WISP1 Ab) or a control antibody (IgG) followed with CLP 12, 14, 16 and 18 h and MTV 0, 2, 4 and 6 h, respectively. CLP alone induced expected increase in circulating cytokines and chemokines but not induced by MTV alone. Two-hit model (CLP + MTV) increased cytokines and chemokines by MTV in time-dependent manner whereas deletion of TLR4 prevented these increases and inhibition of WISP1 or integrin β5 with neutralizing antibodies also blocked increases induced by MTV. *P < 0.05 compared with CLP alone at 16 h; **P < 0.05 compared with two-hit WT at 16 h; ***P < 0.05 compared with two-hit WT; ^#P < 0.05 compared with CLP alone at 18 h; ^##P < 0.05 compared with two-hit WT at 18 h; ^### P < 0.05 compared with two-hit WT at 18 h. CLP cecal ligation and puncture, IL interleukin, MCP-1 monocyte chemoattractant protein-1, MIP-2 macrophage inflammatory protein-1, MTV moderate tidal ventilation, TLR4 toll-like receptor 4, TNF-α tumor necrosis factor alpha, WISP1 WNT1 inducible secreted protein