Abstract
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [NOX] enzymes serve several hemostatic and host defense functions in various lung diseases, but the role of NOX4 signaling in tuberculous pleurisy is not well understood. The role of NOX4 signaling in tuberculous pleural fibrosis was studied using invitro pleural mesothelial cell (PMC) experiments and a murine model of Mycobacterium bovis bacillus Calmette–Guérin (BCG) pleural infection. The production of NOX4 reactive oxygen species (NOX4–ROS) and the epithelial mesenchymal transition (EMT) in PMCs were both induced by heat-killed mycobacterium tuberculosis (HKMT). In cultured PMCs, HKMT-induced collagen-1 synthesis and EMT were blocked by pretreatment with small interfering RNA (siRNA) NOX4. Moreover, NOX4–ROS production and subsequent fibrosis were reduced by treatment with losartan and the toll-like receptor 4 (TLR4) inhibitor TAK-242. The HKMT-induced EMT and intracellular ROS production were mediated by NOX4 via the activation of extracellular signal-regulated kinase (ERK) signaling. Finally, in a BCG-induced pleurisy model, recruitment of inflammatory pleural cells, release of inflammatory cytokines, and thickened mesothelial fibrosis were attenuated by SiNOX4 compared to SiCon. Our study identified that HKMT-induced pleural fibrosis is mediated by NOX4–ERK–ROS via TLR4 and Angiotensin II receptor type1 (AT1R). There results suggest that NOX4 may be a novel therapeutic target for intervention in tuberculous pleural fibrosis.
Highlights
Tuberculosis (TB) is the ninth leading cause of death worldwide and the leading cause from a single infectious agent [1]
It was hypothesized that NADPH oxidase 4 (NOX4) activation plays a critical role in collagen synthesis and cell proliferation in pleural mesothelial cell (PMC) after TB infection and that the inhibition of NOX4 signaling using small interfering RNA after TB infection may reduce pleural fibrosis
50 μg of proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Thermo Scientific, Waltham, MA, USA).Target proteins were detected with primary antibodies as follows: anti-NOX4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-toll-like receptor 4 (TLR4) (Santa Cruz Biotechnology, Santa Cruz, USA), anti-SNAIL (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti Zo-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Collagen 1A (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Sigma, St Loise, MO, USA), anti-E-cadherin (Cell Signaling, Danvers, MA, USA), anti-LC3II (Cell Signaling, Danvers, MA, USA), anti-P62 (Cell Signaling, Danvers, MA, USA) and anti-phosphorylated extracellular signal-regulated kinase (ERK) (Cell Signaling Technology, Danvers, MA, USA).HRP-conjugated goat anti-mouse and anti-rabbit IgG (Thermo Scientific, Waltham, MA, USA) were used as secondary antibodies
Summary
Tuberculosis (TB) is the ninth leading cause of death worldwide and the leading cause from a single infectious agent [1]. Tuberculous pleurisy, the most common extrapulmonary TB, can cause exudative pleural effusions and pleural fibrosis [2]. Pleural mesothelial cells (PMCs) play an important role in pleural fibrosis and in the immune response associated with tuberculous pleurisy [4]. Several studies have investigated the related signaling in PMCs in response to Mycobacterium bovis bacillus Calmette-Guérin (BCG) [4,12,13]. A PMC cell model after heat-killed M. tuberculosis (HKMT) treatment and a BCG-induced pleurisy mouse model were used to explore the NOX4-related signaling pathway and its underlying mechanism. It was hypothesized that NOX4 activation plays a critical role in collagen synthesis and cell proliferation in PMCs after TB infection and that the inhibition of NOX4 signaling using small interfering RNA (siRNA) after TB infection may reduce pleural fibrosis
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