The P2RX7/IL-18/IFN-γ pathway is downregulated in IPF

(A) Heatmap of mRNA expression of P2RX7 in control and IPF patients with a cluster of fibrosis-associated genes. Raw p-values are shown (Limma). (B) mRNA expression of P2RX7, IL18, IL18R1, IL18RAP and IFNG between control and IPF patients from 213 individuals, corresponding to 91 controls and 122 IPF patients. Two-tailed unpaired t-test with Welch’s correction, ***p < 0.001, ****p < 0.0001. (C) Gene set enrichment analysis (GSEA) plot associating P2RX7 mRNA levels from IPF patients with three immunological signatures. The green line represents the enrichment score and the black lines the specific signature-associated genes. NES: Normalized enrichment score, FDR: False discovery rate. Pearson’s correlation test.

Activation of P2RX7 with HEI3090 inhibits lung fibrosis progression

(A) Experimental design. WT mice were given 2.5 U/kg of bleomycin by i.n. route. At the end of the inflammatory phase, 1.5 mg/kg of HEI3090 or vehicle were given daily until day 21. (B) Representative images of lung sections at day 21 after treatment stained with H&E and Sirius Red, Scale bar= 100 µm. (C) Fibrosis score assessed by the Ashcroft method. (D) Collagen levels in whole lung of mice assessed on Sirius Red-polarized images. (E) Experimental design. WT mice were given 2.5 U/kg of bleomycin by i.n. route. 1.5 mg/kg of HEI3090 or vehicle were given daily until day 14. (F) Representative images of lung sections at day 14 after treatment stained with H&E and Sirius Red, Scale bar= 100 µm. (G) Fibrosis score assessed by the Ashcroft method. (H) Collagen levels in whole lung of mice assessed on Sirius Red-polarized images. Each point represents one mouse, two-tailed Mann-Whitney test, p values: *p < 0.05, **p < 0.01. WT: Wildtype, BLM: bleomycin, i.p.: intraperitoneal, i.n.: intranasal, H&E: hematoxylin & eosin, AU: arbitrary units.

HEI3090 favors an anti-fibrotic immune signature in the lungs

WT mice were given 2.5 U/kg of bleomycin by i.n. route and treated daily i.p. with 1.5 mg/kg of HEI3090 or Vehicle. Lungs were analyzed by flow cytometry at day 14. (A) Contour plot of IFN-γ and IL-17A producing T cells (CD3+NK1.1-) (left) and ratio of IFN-γ over IL-17A in T cells (CD3+NK1.1-) (right). (B) Percentage of IFN-γ producing CD4+ and CD8+ T cells. (C) Percentage and GMFI of IL-17A+ cells of CD4+ T cells (CD3+CD4+NK1.1-). (D) Percentage and GMFI of TGFβ in CD45+ cells. (E) Dotplot showing lung inflammatory monocytes, gated on lineage-CD11c-CD11b+ cells (left) and percentage of lung inflammatory monocytes (Ly6ChighLy6G-) (right) (F) Percentage of alveolar macrophages (CD11c+SiglecF+) and (G) lung eosinophils (CD11b+SiglecF+CD11c-). Each point represents one mouse, data represented as violin plots or mean±SEM, two-tailed Mann-Whitney test, *p < 0.05, **p < 0.01. GMFI: geometric mean fluorescence intensity. i.n.: intranasal, i.p.: intraperitoneal.

The P2RX7/NLRP3/IL-18 pathway in immune cells is required for HEI3090’s antifibrotic effect

(A) Experimental design. p2rx7-/-mice were given 3.106 WT, nlrp3-/-or il18-/-splenocytes i.v. one day prior to BLM delivery (i.n. 2.5 U/kg). Mice were treated daily i.p. with 1.5 mg/kg HEI3090 or vehicle for 14 days. (B,D,F) Representative images of lung sections at day 14 after treatment stained with H&E and Sirius Red, scale bar= 100 µm. Fibrosis score assessed by the Ashcroft method of adoptive transfer of WT splenocytes, (E) nlrp3-/-splenocytes and (G) il18-/-splenocytes. (H) IL-18 levels in sera of WT BLM-induced mice at day 14 (I) Ratio of IFN-γ over IL-17A in lung T cells (CD3+NK1.1-) of WT mice neutralized for IL-18 or not (isotype control) every 3 days. Each point represents one mouse, two-tailed Mann-Whitney test, *p < 0.05. WT: Wildtype, BLM: bleomycin, i.p.: intraperitoneal, i.n.: intranasal, i.v.: intravenous, H&E: hematoxylin & eosin.

Antibodies used in this study