Lentivirus-mediated overexpression of suppressor of cytokine signaling-3 reduces neutrophilic airway inflammation by suppressing T-helper 17 responses in mice with chronic Pseudomonas aeruginosa lung infections.

The aim of the present study was to explore the effect of overexpressed suppressor of cytokine signaling­3 (SOCS3) on T-helper (Th)17 cell responses and neutrophilic airway inflammation in mice with chronic Pseudomonas aeruginosa (PA) infections. SOCS3 expression was enhanced via the administration of tail vein injections of therapeutic lentivirus in mice with chronic PA lung infections. SOCS3 expression in the blood and lung tissue was assessed using reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analysis. Total and differential cell numbers and myeloperoxidase levels in the bronchoalveolar lavage (BAL) fluid were assessed, as well as the number of bacterial colonies in the lungs. Histological analysis of lung tissue was performed using hematoxylin and eosin staining and phosphorylated­signal transducer and activator of transcription­3 (p­STAT3) expression was measured by western blot analysis and immunohistochemistry. The expression of STAT3 mRNA and retinoid­related orphan receptor (ROR)γt were measured by RT­qPCR. The percentage of interleukin (IL)­17+ cells among cluster of differentiation (CD)4+ cells was calculated using flow cytometry and levels of IL­17A and IL­6 were assessed by ELISA. The expression of SOCS3 was significantly increased in CD4+ T cells following lentivirus injection and the inflammation of neutrophilic airways was notably ameliorated. Enhanced SOCS3 expression was associated with a significant decrease in the expression of p­STAT3 and RORγt in CD4+ T cells. Additionally, the percentage of IL­17+ cells among CD4+ T cells and the IL­17 contents in the BAL fluid were significantly decreased. Lentivirus­mediated overexpression of SOCS3 was revealed to ameliorate neutrophilic airway inflammation by inhibiting pulmonary Th17 responses in mice with chronic PA lung infections.

Upregulation of SOCS3 in lung CD4+ T cells in a mouse model of chronic PA lung infection and suppression of Th17­mediated neutrophil recruitment in exogenous SOCS3 transfer in vitro.

Neutrophilic airway inflammation in chronic lung infections caused by Pseudomonas aeruginosa (PA) is associated with T helper (Th)17 responses. Suppressor of cytokine signaling 3 (SOCS3) is the major negative modulator of Th17 function through the suppression of signal transducer and activator of transcription (STAT)3 activation. The aim of the present study was to investigate the expression of SOCS3 in lung CD4+ T cells in a mouse model of chronic PA lung infection and the effect of exogenous SOCS3 on Th17­mediated neutrophil recruitment in vitro. A mouse model of chronic PA lung infection was established and the activation of STAT3 and Th17 response in lung tissues and lung CD4+ T cells was assessed. The protein and mRNA expression of SOCS3 in lung CD4+ T cells was analyzed by western blotting and reverse transcription­quantitative polymerase chain reaction. The authors constructed a recombinant lentivirus carrying the SOCS3 gene and transferred it into lung CD4+ T cells isolated from a mouse model. These transfected cells were stimulated with interleukin (IL)­23 in vitro and the protein level of p­STAT3 and retinoid­related orphan receptor (ROR)γt was determined by western blotting. The expression of IL­17A+ cells was analyzed by flow cytometry and the level of IL­17A in cell culture supernatant was measured by ELISA. The mouse lung epithelial cell line, MLE­12, was cocultured with lung CD4+ T cells that overexpressed the SOCS3 gene and the culture supernatant was harvested and used for a chemotaxis assay. Compared with control mice, mice with chronic PA lung infection had significantly higher level of p­STAT3 and Th17 response in both lung tissues and lung CD4+ T cells. The protein and mRNA level of SOCS3 in lung CD4+ T cells increased as the chronic PA lung infection developed. Exogenous SOCS3 gene transfer in PA­infected lung CD4+ T cells decreased p­STAT3 and RORγt expression and suppressed the level of IL­17A+ cells in vitro. MLE­12 cells cocultured with SOCS3­overexpressing lung CD4+ T cells expressed a significantly lower level of neutrophil chemoattractants chemokine (C­X­C motif) ligand (CXCL) 1 and CXCL5, and recruited significantly smaller numbers of migrating neutrophils than those cocultured with control cells. SOCS3 was upregulated in lung CD4+ T cells following the activation of STAT3/Th17 axis in a mouse model of chronic PA lung infection. Exogenous SOCS3 transfer in PA­infected lung CD4+ T cells suppresses Th17­mediated neutrophil recruitment in vitro.