WISP1 and TLR4 on Macrophages Contribute to Ventilator-Induced Lung Injury

Abstract

Injurious mechanical ventilation has been shown to directly affect pulmonary and systemic immune responses. How these responses propagate or attenuate remains unknown. The goal of this study was to further determine whether toll-like receptor (TLR) 4 and WNT1-inducible signaling pathway protein 1 (WISP1) could contribute to injurious mechanical ventilation, especially focusing on the role of macrophages during experimental ventilator-induced lung injury. A prospective, randomized, and controlled animal study was designed, and male, wild-type (WT) C57BL/6 mice, TLR4 knockout (TLR4^-/-), and lyzTLR4 knockout (lyzTLR4^-/-) mice aging 8~12 weeks were used. Animals were anesthetized and randomized to spontaneous breathing (SB) group or to high tidal volume (VT, 20 ml/kg) mechanical ventilation (HTV) group. Histological evaluation, alveolar-capillary permeability of Evan's blue albumin (EBA), WISP1 protein levels, macrophage inflammatory protein-2 (MIP-2), and interleukin-6 (IL-6) in plasma and bronchoalveolar lavage fluid (BALF) concentrations were analyzed. HTV group was associated with a significant increase of WISP1 and EBA ratio in C57BL/6 mice, a significant decrease of WISP1 protein levels, and a significant decrease of IL-6, MIP-2 in plasma, and BALF concentrations of pro-inflammatory cytokines in TLR4^-/- and lyzTLR4^-/- knockout mice. In TLR4^-/- mice and lyzTLR4^-/- mice, there were also significant differences between SB group and HTV group in terms of H&E score and EBA ratio and level of pro-inflammation cytokines. The entire TLR4-targeted mice could further improve various inflammatory changes and damages when compared with lyzTLR4-targeted mice. What is more, TLR4^-/- mice and lyzTLR4^-/- mice reacted differently to rWISP1 and/or BMMC treated. TLR4^-/- mice had no response to rWISP1, while lyzTLR4^-/- mice still showed drastic response to both treatments. TLR4 and WISP1, especially the former one, on macrophages could contribute to releasing of pro-inflammatory cytokines during ventilator-induced lung injury. Injurious mechanical ventilation may result in an immune response which is similar to that of infection.

Keywords: TLR4; WISP1; lung injury; mechanical ventilation.

Fig. 1

TLR4 significantly promotes HTV-induced lung injury. WT mice and different levels of TLR4-targeted mice were treated with or without HTV. a Lung H&E staining and the histological alterations of lung parenchyma were shown about graded on a scale from 0 to 4. b EBA depicts the permeability of alveolar capillary in each group. ELISA was performed with the standard protocol, and cytokine profiles of IL-6 and MIP-2 (c, d) were tested at all control and HTV groups (^*P < 0.05; ^**P < 0.01; ^***P < 0.001).

Fig. 2

TLR4 would mediate WISP1 expression level in VILI. Western blotting shows the effects of mechanical ventilation on WISP1 protein levels in several groups of HTV animals vs control groups: spontaneous breathing and ventilated with high tidal volume (among C57, TLR4^−/−, lyzTLR4^−/− sub-groups) for 4 h (^*P < 0.05, ^**P < 0.01, ^***P < 0.001).

Fig. 3

rWISP1 could accelerate inflammatory injury in WT and lyzTLR4-targeted but not entire TLR4-targeted. Isotype control IgG via intratracheal (i.t.) injection together with HTV or rWISP1 protein (20 μg per mouse) i.t. a Lung H&E staining shows structure changes among different groups. b EBA shows the alveolar–capillary permeability after HTV with/without rWISP1. Cytokine profiles of IL-6 and MIP-2 (c, d) were tested by ELISA after HTV with/without rWISP1 (^*P < 0.05; ^**P < 0.01; ^***P < 0.001).

Fig. 4

rWISP1 and TLR4 on BMMC-driven inflammation and lung injury after HTV. LyzTLR4^−/− mice were chosen and injected with BMMC (10⁶/ml) and/or rWISP1 from respiratory tract. a Lung H&E staining shows structure changes among different groups. b EBA depicts the permeability of alveolar capillary in each group with different methods tackled. LyzTLR4^−/− mice were chosen and injected with BMMC (10⁶/ml) and/or rWISP1 from respiratory tract. Finally, IL-6 and MIP-2 concentrations were tested in plasma and BALF (c, d) (^*P < 0.05; ^**P < 0.01; ^***P < 0.001).