Thrombospondin-1 Restricts Interleukin-36γ-Mediated Neutrophilic Inflammation during Pseudomonas aeruginosa Pulmonary Infection

Interleukin-36 gamma (IL-36 gamma), a member of the IL-1 cytokine superfamily, amplifies lung inflammation and impairs host defense during acute pulmonary Pseudomonas aeruginosa infection. To be fully active, IL-36 gamma is cleaved at its N-terminal region by pro teases such as neutrophil elastase (NE) and cathepsin S (CatS). However, it remains unclear whether limiting extracellular proteolysis restrains the inflammatory cascade triggered by IL-36 gamma during P. aeruginosa infection. Thrombospondin-1 (TSP-1) is a matricellular protein with inhibitory activity against NE and the pathogen-secreted Pseudomonas elastase LasB-both proteases implicated in amplifying inflammation. We hypothesized that TSP-1 tempers the inflammatory response during lung P. aeruginosa infection by inhibiting the proteolytic environment required for IL-36 gamma activation. Compared to wild type (WT) mice, TSP-1-deficient (Thbs1(-/-)) mice exhibited a hyperinflammatory response in the lungs during P. aeruginosa infection, with increased cytokine production and an unrestrained extracellular proteolytic environment characterized by higher free NE and LasB, but not CatS activity. LasB cleaved IL-36 gamma proximally to M-19 at a cleavage site distinct from those generated by NE and CatS, which cleave IL-36 gamma proximally to Y-16 and S-18, respectively. N-terminal truncation experiments in silico predicted that the M-19 and the S-18 isoforms bind the IL-36R complex almost identically. IL-36 gamma neutralization ameliorated the hyperinflammatory response and improved lung immunity in Thbs1(-/-) mice during P. aeruginosa infection. Moreover, administration of cleaved IL-36 gamma induced cytokine production and neutrophil recruitment and activation that was accentuated in Thbs1(-/-) mice lungs. Collectively, our data show that TSP-1 regulates lung neutrophilic inflammation and facilitates host defense by restraining the extracellular proteolytic environment required for IL-36 gamma activation. IMPORTANCE Pseudomonas aeruginosa pulmonary infection can lead to exaggerated neutrophilic inflammation and tissue destruction, yet host factors that regulate the neutrophilic response are not fully known. IL-36 gamma is a proinflammatory cytokine that dramatically increases in bioactivity following N-terminal processing by proteases. Here, we demonstrate that thrombospondin-1, a host matricellular protein, limits Nterminal processing of IL-36 gamma by neutrophil elastase and the Pseudomonas aeruginosa-secreted protease LasB. Thrombospondin-1-deficient mice (Thbs1(-/-)) exhibit a hyperinflammatory response following infection. Whereas IL-36 gamma neutralization reduces inflammatory cytokine production, limits neutrophil activation, and improves host defense in Thbs1(-/-) mice, cleaved IL-36 gamma administration amplifies neutrophilic inflammation in Thbs1(-/-) mice. Our findings indicate that thrombospondin-1 guards against feed-forward neutrophilic inflammation mediated by IL-36 gamma in the lung by restraining the extracellular proteolytic environment.

Automated summary

IL-36γ is a proinflammatory cytokine that requires proteolytic activation, while TSP-1 limits this activation by inhibiting extracellular proteolysis. TSP-1 deficiency leads to exaggerated inflammation during P. aeruginosa infection.