Critical role for the lung endothelial nonmuscle myosin light-chain kinase isoform in the severity of inflammatory murine lung injury

Abstract

Global knockout of the nonmuscle isoform of myosin light-chain kinase (nmMLCK), a primary cellular regulator of cytoskeletal machinery, is strongly protective in preclinical murine models of inflammatory lung injury. The current study was designed to assess the specific contribution of endothelial cell (EC) nmMLCK to the severity of murine inflammatory lung injury produced by lipopolysaccharide (LPS) and mechanical ventilation ventilator-induced lung injury or ventilation (VILI). Responses to combined LPS/VILI exposure were assessed in: (i) wild-type (WT) C57BL/6J mice; (ii) transgenic mice with global deletion of nmMLCK (nmMylk ^-/-); (iii) transgenic nmMylk ^-/- mice with overexpression of nmMLCK restricted to the endothelium (nmMylk ^-/-/ec-tg+). Lung inflammation indices included lung histology, bronchoalveolar lavage (BAL) polymorphonuclear leukocytes (PMNs), lung protein biochemistry, tissue albumin levels, Evans blue dye (EBD) lung extravasation, and plasma cytokines (interleukin-6 [IL-6], keratinocyte chemoattractant [KC]/IL-8, IL-1bβ, extracellular nicotinamide phosphoribosyltransferase, tumor necrosis factor-α). Compared to WT C57BL/6J mice, the severity of LPS/VILI-induced lung injury was markedly reduced in mice with global nmMLCK deletion reflected by reductions in histologic inflammatory lung injury, BAL PMN counts, mitogen-activated protein kinase, and NF-kB pathway activation in lung homogenates, plasma cytokine levels, and parameters of lung permeability (increased BAL protein, tissue albumin levels, EBD lung extravasation). In contrast, mice with restricted overexpression of nmMLCK in EC (nmMylk ^-/-/ec-tg+) showed significant persistence of LPS/VILI-induced lung injury severity compared to WT mice. In conclusion, these studies strongly endorse the role of EC nmMLCK in driving the severity of preclinical inflammatory lung injury. Precise targeting of EC nmMLCK may represent an attractive therapeutic strategy to reduce lung inflammation and both lung and systemic vascular permeability.

Keywords: ARDS; MLCK.

Figure 1

Endothelial‐specific overexpression of nmMLCK abolishes the protection against LPS/VILI lung injury observed innmMYLK ^−/−mice. (a/b) Compared to PBS‐challenged wild‐type (WT) C57BL6 mice (inset), H&E staining WT mice exposed to a “two‐hit” injury model of LPS (18 h) and ventilator‐induced lung injury (4 h) (LPS/VILI) reveals significant increases in alveolar edema, leukocyte infiltration, and inflammatory lung injury (first panel in a). In contrast, LPS/VILI‐challenged nmMylk ^−/− mice were protected with significantly reduced PMN infiltration and decreases in vascular leakage (second panel in a). This reduction in lung injury severity was nearly entirely eliminated in nmMylk ^{−/−/ec‐tg+} mice with global nmMLCK KO, except for overexpression of nmMLCK restricted to the endothelium (third panel in a). (b) Image J quantification of H&E staining expressed as the % area of staining. * p < 0.05 comparisons. (c) Total BAL PMN counts corroborate H&E staining findings, showing that compared to either the LPS/VILI‐challenged WT or nmMylk ^{−/−/ec‐tg+} mice, the nmMylk ^−/− mice have a significant reduction in PMN's found in the alveolar space, indicating a higher level of EC barrier integrity. There is no significant difference in barrier integrity via PMN infiltration between the wild‐type mice and the nmMylk ^{−/−/ec‐tg+} mice. *p < 0.05 comparisons. Ctrl, control; H&E, hematoxylin and eosin; KO, knockout; LPS, lipopolysaccharide; nmMLCK, nonmuscle isoform of myosin light‐chain kinase; PMN, polymorphonuclear leukocyte; VILI, ventilator‐induced lung injury or ventilation; WT, wild type.

Figure 2

Inflammatory cytokine plasma levels are reduced or differentially expressed in nmMylk ^–/− compared to the nmMylk ^{−/−/ec‐tg+} . The Meso Scale ELISA‐based Discovery platform was utilized to assess plasma cytokine levels in the LPS/VILI injury model. Depicted are the mouse plasma levels of inflammatory cytokines of IL‐6 (a), IL‐1β (b), KC (c), TNF‐α (d), and eNAMPT (e) obtained in control spontaneous breathing mice (n = 3) for each genotype/group and in mice exposed to LPS/VILI (n = 3) for each genotype/group. Cytokine levels in all animals exposed to LPS/VILI show a drastic increase compared to their control counterparts. nmMylk ^−/− mice exhibited significantly lower levels of cytokines (a, c, d, e) compared to WT mice, except for plasma levels of IL‐1β (b) were unaffected by nmMylk genotypes. The reduced plasma cytokine levels observed in LPS/VILI‐challenged nmMylk ^−/− mice were restored in nmMylk ^{−/−/ec‐tg+} mice to levels approximating WT mice. Baseline levels of all five cytokines in each genotype group were comparable, except for the levels of IL‐1β in nmMylk ^{−/−/ec‐tg+} mice, which exhibited an elevated baseline level compared to either the WT or nmMylk ^−/− control mice. *p < 0.05. Ctrl, control; ELISA, enzyme‐linked immunoassay; eNAMPT, extracellular nicotinamide phosphoribosyltransferase; IL, interleukin; KC, keratinocyte‐derived cytokine; LPS, lipopolysaccharide; VILI, ventilator‐induced lung injury or ventilation; WT, wild type.

Figure 3

Effect of nmMylk genotypes on murine lung vascular integrity. (a–d) Representative photos of murine lungs exposed to PBS or LPS/VILI via an intratracheal instillation (1 mg/kg, harvest timepoint 20 h post LPS instillation, VILI 20 ml/kg, 4 h). Sixty minutes before harvest, mice (n = 3/group) were injected intravenously with 30 mg/kg of Evans blue dye (EBD) conjugated to albumin as we have previously described. Lungs were flushed with PBS before removal. Comparisons of EBD extravasation in each group were compared with EBD leakage minimal in PBS‐exposed mice. All genotypes exhibited similar EBD levels for the PBS‐exposed mice (a). EBD leakage was markedly increased in each LPS/VILI‐exposed group with greatest increases in EBD extravasation observed in nmMylk ^{−/−/ec‐tg+} mice (b) with marked reductions in nmMylk ^−/− mice (c). EBD leakage in nmMylk ^{−/−/ec‐tg+} mice showed increased EBD extravasation approximating WT mice (d). (e/f) Consistent with the findings of EC barrier integrity disruption shown in EBD pictures, LPS/VILI‐exposed WT, and nmMylk ^{−/−/ec‐tg+} mice had similar values for BAL protein levels and lung tissue albumin levels compared to PBS‐exposed animals. In contrast, by comparison, LPS/VILI‐exposed nmMylk ^−/− mice had significantly lower values in both readouts of vascular permeability (* p < 0.05 comparisons). Ctrl, control; LPS, lipopolysaccharide; PBS, phosphate‐buffered saline; VILI, ventilator‐induced lung injury or ventilation; WT, wild type.

Figure 4

(a) Western blots in mouse lung tissues exposed to LPS/VILI showing significantly increased phosphorylation/expression of p‐NF‐kB, p‐ERK, NOX4, NRF2, and p‐p90RSK compared to PBS‐exposed control mice. (b–d) Quantifications of panel (a) western blots showing significant reductions in levels of p‐NF‐ƘB, p‐ERK and p‐p90RSK in LPS/VILI nmMylk ^−/− mice, approximating levels of PBS‐exposed controls. In contrast, LPS/VILI‐exposed nmMylk ^{−/−/ec‐tg+} mice exhibited significantly greater phosphorylation/expression levels, comparable to LPS/VILI‐exposed WT mice. *p  < 0.05 vs control; **p <0.05 vs WT; ***p<0.05 vs nmMylk‐/‐. Ctrl, control; LPS, lipopolysaccharide; NF‐kB, nuclear factor‐kB; VILI, ventilator‐induced lung injury or ventilation; WT, wild type.