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

TSP-1 does not alter IL-36 cytokines expression but downregulates the proteolytic environment required for activation. Thbs1^−/− and WT mice were i.t. inoculated with P. aeruginosa at an inoculum of 10⁶ CFU, and lung tissue (A) Il36a, (B) Il36b, and (C) Il36g transcripts were measured at 5 hpi and 1 dpi by quantitative reverse transcription-PCR (qRT-PCR) using gadph as the internal housekeeping gene. (D and E) IL-36γ expression in the lungs measured by Western blot at 1 dpi. Density expression of IL-36γ is normalized to β-actin. (F) IL-6 and CXCL-1 production by bone marrow-derived dendritic cells (BMDCs) after stimulation with full-length (fIL-36γ) or cleaved IL-36γ (cIL-36γ, S¹⁸ isoform). (G) Cathepsin S (CatS), (H) neutrophil elastase (NE), and (I) LasB activity were measured in the BALF of WT and Thbs1^−/− mice at 5 hpi and 1 dpi using the specific substrates 2-aminobenzoyl-l-alanyl-glycyl-l-leucyl-l-alanyl-para-nitro-benzyl-amide, N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide, and Mca-GRWPPMG∼LPWEK(Dnp)-D-R-NH2, respectively. *, P < 0.05, for single comparisons, the Shapiro-Wilk test was used to assess normal distribution, followed by a Mann-Whitney U test or a parametric t test. A two-way ANOVA test was followed by a post hoc test for multiple comparisons over time. Each data point represents an individual mouse, combined from two independent experiments, except for the Western blot and in vitro BMDC stimulation, which were performed once. Lines indicate the median.

PA14 LasB cleaves IL-36γ proximally to M¹⁹, and sequential N-terminal truncation models in silico predict the bioactivity of the M¹⁹ isoform. (A) Full-length IL-36γ was incubated with wild-type PA14 (PA WT) or lasB::Tn5 mutant supernatant in the presence or absence of LasB inhibitor. (B) Full-length IL-36γ was incubated with PA WT, purified LasB (pLasB), or recombinant NE and visualized by SDS-PAGE. Teal arrow points to M¹⁹ IL-36γ, and yellow arrows point to Y¹⁶ IL-36γ. (C) N-terminal sequencing of IL-36γ NE and IL-36γ LasB were analyzed by Edman degradation, with arrowheads indicating the site of cleavage. (D) IL-36γ S¹⁸ (salmon) and M¹⁹ (teal) isoforms associate in a similar orientation to a model of the IL-1Rrp2/IL1RAcP receptor complex (gray and orange, respectively). Parts of D2 and D3 of IL-1Rrp2 and the D2 domain of IL1RAcP contribute to the identified cytokine-binding site. Helix I¹⁰⁴-G¹⁰⁹ (red star) is close to the N termini of both isoforms pointed toward the IL-1Rrp2 D3 domain. The single black arrow indicates loop L¹⁵⁵-N¹⁶⁰ (red), which makes favorable electrostatic contact with the IL1RAcP D2 domain. Loop T⁶¹-D⁷² (double black arrow, red loop) faces the D3 domain of the accessory protein. (E) The electrostatic pattern of M¹⁹ isoform binding may influence the IL-1Rrp2 D3 domain. The black star denotes a predicted concentration of electrostatic repulsions (basic charge in blue) exist at the lower contact interface between the isoform and the IL-1Rrp2 D3 domain. (F) Y¹⁶ isoform (yellow) binds in the upside-down fashion, but there are differences compared to the M¹⁹ isoform (see position shift of the landmark helix [red star]). For reference, the single and double black arrows again indicate the loops shown in in panel D. (G) The Y¹⁶ isoform binds in the upside-down arrangement, but the intermolecular packing is less efficient between the cytokine and IL-36R complex. The figures were prepared and electrostatic potential surfaces calculated using PyMol Molecular Graphics System v1.3 (Schrodinger, LLC).

IL-36γ neutralization reduces lung bacterial burden and the inflammatory response during P. aeruginosa infection in Thbs1^−/− mice. Thbs1^−/− and WT mice were i.t. inoculated with P. aeruginosa at an inoculum of 10⁶ CFU. After 5 hpi, Thbs1^−/− mice were treated with a rabbit anti-mouse IL-36γ neutralizing antibody. Thbs1^−/− and WT mice treated with a rabbit-IgG served as control. At 1 dpi, (A) lung bacterial burden, (B) CXCL-1, (C) CXCL-2, (D) GM-CSF, (E) IL-1β, and (F) IL-17A production in lung tissue homogenates, (G) BALF free NE activity, (H) lung tissue MPO activity, and (I and J) immune cell composition in the BALF were measured. *, P < 0.05 by one-way ANOVA test followed by a post hoc test. Each data point represents an individual mouse; the experiment was performed once without excluding any data. Lines indicate the median.