Nonmuscle myosin light chain kinase regulates murine asthmatic inflammation

Abstract

Myosin light chain kinase (MLCK; gene code, MYLK) is a multifunctional enzyme involved in isoform-specific nonmuscle (nm) and smooth muscle contraction, inflammation, and vascular permeability, processes directly relevant to asthma pathobiology. In this report, we highlight the contribution of the nm isoform (nmMLCK) to asthma susceptibility and severity, supported by studies in two lines of transgenic mice with knocking out nmMLCK or selectively overexpressing nmMLCK in endothelium. These mice were sensitized to exhibit ovalbumin-mediated allergic inflammation. Genetically engineered mice with targeted nmMLCK deletion (nmMLCK(-/-)) exhibited significant reductions in lung inflammation and airway hyperresponsiveness. Conversely, mice with overexpressed nmMLCK in endothelium (nmMLCK(ec/ec)) exhibited elevated susceptibility and severity in asthmatic inflammation. In addition, reduction of nmMLCK expression in pulmonary endothelium by small interfering RNA results in reduced asthmatic inflammation in wild-type mice. These pathophysiological assessments demonstrate the positive contribution of nmMLCK to asthmatic inflammation, and a clear correlation of the level of nmMLCK with the degree of experimental allergic inflammation. This study confirms MYLK as an asthma candidate gene, and verifies nmMLCK as a novel molecular target in asthmatic pathobiology.

Figure 1.

Effect of nonmuscle myosin light chain kinase (nmMLCK) expression on airway reactivity and inflammation in a murine asthma model. (A) Airway hyperreactivity was assessed in wild-type (WT), nmMLCK^−/−, and nmMLCK^ec/ec mice using relative airway pressure time index (APTI), a measurement of acetylcholine-induced changes in airway pressures. (B) Airway reactivity to methacholine in ovalbumin (OVA)-challenged mice. (C) Effects of nmMLCK expression on bronchoalveolar lavage (BAL) protein leakage. (D) Effects of nmMLCK expression on inflammatory leukocyte infiltration. (E–G) Effects of nmMLCK expression on Th2 cytokine secretion: IL-5 (E), IL-13 (F), and eotaxin (G) into the alveolar space (BAL) in all murine groups. *P < 0.05 compared with OVA-challenged WT mice; n ≥ 5 for all groups.

Figure 2.

Histological Evaluation of the influence of nmMLCK expression on lung inflammation and airway remodeling. Histology (hematoxylin and eosin [H&E]) sections in WT, nmMLCK^−/−, and nmMLCK^ec/ec mice demonstrating leukocyte infiltration and recruitment to the (A) peribronchial and (B) perivascular areas, respectively, after OVA challenge. (C) Lung sections stained with periodic acid-Schiff (PAS) to detect goblet cell (arrows) presence and activity in PBS or OVA-challenged mice. (D) Major basic protein (MBP) staining in tissue microarray samples derived from the OVA-challenged murine lines. All histological evaluations were quantified and summarized in the bar graphs accompanying each analysis shown in the last column. *P < 0.05; n = 5–6. OD, optical density.

Figure 3.

Effect of nmMLCK silencing on asthmatic inflammation in mice. WT mice received nmMLCK small interfering RNA (siRNA) to reduce nmMLCK expression in the lungs, and were challenged by OVA to activate asthmatic inflammation. (A) Effects of nmMLCK siRNA on airway hyperreactivity reflected by acetylcholine-induced APTI. (B) Effects of nmMLCK siRNA on BAL protein leakage. (C–E) Effects of nmMLCK expression on inflammatory leukocyte infiltration (macrophage, eosinophil, and neutrophil). *P < 0.05 compared with control siRNA–PBS group; **P < 0.05 compared with control siRNA–OVA group; n = 5 or 6.