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 Pseudomonas aeruginosa 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 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-36γ 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-36γ proximally to M(19) at a cleavage site distinct from those generated by NE and CatS, which cleave IL-36γ 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-36γ neutralization ameliorated the hyperinflammatory response and improved lung immunity in Thbs1(−/−) mice during P. aeruginosa 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.