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

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