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 entichment 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 (d7), 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) Quantification of fibroblasts/myofibroblasts in non-immune cell subset. (E) Representative images of lung sections at day 21 after treatment stained with Sirius Red, Scale bar= 100 µm. (F) Collagen levels in whole lung of mice assessed on Sirius Red-polarized images. (G) 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. (H) Representative images of lung sections at day 14 after treatment stained with H&E, Scale bar= 100 µm. (I) Fibrosis score assessed by the Ashcroft method. (J) Quantification of fibroblasts/myofibroblasts in non-immune cell subset. (K) Representative images of lung sections at day 14 after treatment stained with Sirius Red, Scale bar= 100 µm. (L) Collagen levels in whole lung of mice assessed on Sirius Red-polarized images. (M) P2rx7 KO 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. Fibrosis score assessed by the Ashcroft method is showed. 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, 1.n.: intranasal, H&E: hematoxylin & eosin, AU: arbitrary units.

HEI3090 favors an anti-fibrotic immune signature in the lungs

(A) 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 TGF 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 text, *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,E,H) 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, (C) nlrp3-/- splenocytes (F) and (I) il18-/- splenocytes. Collagen fiber intensity of adoptive transfer of WT splenocytes, (D) nlrp3-/- splenocytes (G) and (J) il18-/- splenocytes. (K) IL-18 levels in sera of WT BLM-induced mice at day 14 (L) 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<0.0.5. WT: Wildtype, BLM: bleomycin, i.p: intraperitoneal, i.n.: intranasal, i.v.: intravenous, H&E: hematoxylin & eosin.

used in this study

The components of the NLRP3 inflammasome are dampened in IPF

(A) mRNA expression of NLRP3, PYCARD, CASP1 and GSDMD between control and IPF patients. Each point represents one patient. Two-tailed unpaired t-test with Welch’s correction, ***p < 0.001, ****p < 0.0001. (B) Gene set enrichment analysis (GSEA) plot associating IL-18 and IL-18RAP 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 limits lung fibrosis progression

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. (A) Whole lung images at day 21 after treatment stained with H&E, Scale bar at 10X and 20X= 100 µm. (B) Whole lungs were divided in their entirety in several fields as shown to evaluate fibrosis severity using the Ashcroft method. The fibrosis score is the mean of all fields. (C) BLM-treated p2rx7-/- or p2rx7-/- adoptively transferred with p2rx7-/- splenocytes mice received HEI3090 for 14 days started at day 1. Whole lungs were removed, fixed and stained with Sirius red. The percentage of fibrosis in whole lung (up panel) was quantified concomitantly with the collagen quantification (lower panel) as described in the Material and Method section. Right: p2rx7 KO mice treated vehicle (n=6) or HEI3090 (n=5). Left: p2rx7 KO mice adoptively transferred with p2rx7 KO splenocytes treated with vehicle (n=6) or HEI3090 (n=4). Each point represents one mouse.Two-tailed Mann-Whitney test shows no significance. (E) BLM-inhaled WT and P2rx7 KO mice were treated or not with HEI3090. At D14, whole lungs were removed, fixed in formol, and stained with H&E and lung fibrosis was scored. The Dunnett’s multiple comparison test is shown. p values: * p. < 0.05, **p < 0.01.

Activation of P2RX7 with HEI3090 reshapes immune infiltration in the lungs

WT mice were given 2.5 U/kg at day 1 i.n. and treated for 14 days with 1.5 mg/kg of HEI3090 or Vehicle. At Day 14 lungs were analyzed by flow cytometry. (A) Percentage of IFN-γ producing and IL17A of TCD3 cells (CD45+CD3+). (B) Ratio of IFN-γ over IL- 17A in lung immune cells (CD45+) or CD4+T cells of WT mice (C) Percentage of IFN-γ producing NK cells (NK1.1+CD3-) (D) Percentage of cells in lungs of mice (E) Percentage of Tregs (CD3+CD4+Foxp3+NK1.1-) of CD4+T cells (F) GMFI of TGFβ+ NK cells (NK1.1+CD3-), T cells (CD3+NK1.1-), CD4+Foxp3- T cells, Tregs and CD8+ T cells. (G) CD45+, inflammatory monocytes and eosinophils cell count assessed by flow cytometry. Each point represents one mouse, data represented as mean±SEM, two-tailed Mann-Whitney test, *p < 0.05, **p < 0.01. GMFI: geometric mean fluorescence intensity, Tregs: regulatory T cells, NK: Natural Killer, NKT: Natural Killer T cells, DC: Dendritic cells, PMN: polymorphonuclear cells.

HEI3090 reactivates an immune response

(A) Experimental design. WT mice were given 2.5 U/kg at day 1 i.n. and treated for 14 days with 1.5 mg/kg of HEI3090 or Vehicle. (B) Percentage of immune cells of CD45+ in spleen assessed by flow cytometry at day 14. (C) 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 7 days after BLM administration until day 21. (D) Percentage of immune cells of CD45+ in spleen assessed by flow cytometry at day 21. Data represented as mean±SEM, two-tailed Mann-Whitney test, *p < 0.05, **p < 0.01. WT: Wildtype, BLM: bleomycin, i.p.: intraperitoneal, i.n.: intranasal, NK: Natural Killer, NKT: Natural Killer T cells, DC: Dendritic cells, PMN: polymorphonuclear cells.

HEI3090 activity requires P2RX7’s expressing immune cells.

(A) Experimental design. 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. Mice were given i.v. 3.106 p2rx7-/- splenocytes from male age-matched mice at day 1,4,7,9 and 11. (B) Representative images of lung sections at day 14 after treatment stained with H&E and Sirius Red, scale bar= 100 µm. (C) Fibrosis score assessed by the Ashcroft method.

HEI3090 activity requires P2RX7’s expressing immune cells.

(A) Experimental design. p2rx7-/- mice were given 3.106 p2rx7-/- or il1β-/- 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) Representative images of lung sections at day 14 after treatment stained with H&E and Sirius Red with p2rx7-/- splenocytes, scale bar= 100 µm (left) and fibrosis score assessed by the Ashcroft method (right) (C) Representative images of lung sections at day 14 after treatment stained with H&E and Sirius Red with il1β-/- splenocytes, bar= 100 µm (left) and fibrosis score assessed by the Ashcroft method (right) (D) IL-1β levels in sera of WT mice at day 14 after treatment. (E) Percentage of P2RX7+ cells in spleens of WT and various KO mice assessed by flow cytometry (F) NLRP3 and IL-18 expression in spleens of WT and various KO mice assessed by Western Blot. Each point represents one mouse, data represented as violin plot or mean±SEM, 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, KO: knock-out