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. 2011;6(8):e23185.
doi: 10.1371/journal.pone.0023185. Epub 2011 Aug 16.

IL-1 and IL-23 mediate early IL-17A production in pulmonary inflammation leading to late fibrosis

Paméla Gasse  1 , Nicolas Riteau, Rachel Vacher, Marie-Laure Michel, Alain Fautrel, Franco di Padova, Lizette Fick, Sabine Charron, Vincent Lagente, Gérard Eberl, Marc Le Bert, Valérie F J Quesniaux, François Huaux, Maria Leite-de-Moraes, Bernhard Ryffel, Isabelle Couillin
Affiliations

IL-1 and IL-23 mediate early IL-17A production in pulmonary inflammation leading to late fibrosis

Paméla Gasse et al. PLoS One. 2011.

Abstract

Background: Idiopathic pulmonary fibrosis is a devastating as yet untreatable disease. We demonstrated recently the predominant role of the NLRP3 inflammasome activation and IL-1β expression in the establishment of pulmonary inflammation and fibrosis in mice.

Methods: The contribution of IL-23 or IL-17 in pulmonary inflammation and fibrosis was assessed using the bleomycin model in deficient mice.

Results: We show that bleomycin or IL-1β-induced lung injury leads to increased expression of early IL-23p19, and IL-17A or IL-17F expression. Early IL-23p19 and IL-17A, but not IL-17F, and IL-17RA signaling are required for inflammatory response to BLM as shown with gene deficient mice or mice treated with neutralizing antibodies. Using FACS analysis, we show a very early IL-17A and IL-17F expression by RORγt(+) γδ T cells and to a lesser extent by CD4αβ(+) T cells, but not by iNKT cells, 24 hrs after BLM administration. Moreover, IL-23p19 and IL-17A expressions or IL-17RA signaling are necessary to pulmonary TGF-β1 production, collagen deposition and evolution to fibrosis.

Conclusions: Our findings demonstrate the existence of an early IL-1β-IL-23-IL-17A axis leading to pulmonary inflammation and fibrosis and identify innate IL-23 and IL-17A as interesting drug targets for IL-1β driven lung pathology.

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Conflict of interest statement

Competing Interests: Franco di Padova is a full-time employee of Novartis. The remaining authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. IL-23p19, IL-17A or IL-17F expression upon BLM or rmIL-1β.
mRNA expression for IL-23p19, Pro-IL-1β and HPRT1 in lung of 4 individual mice treated with BLM (10 mg/kg) or rmIL-1β (50 µg/kg) or saline were assessed using semi-quantitative PCR at 24 h in wild-type mice (A). Semi-quantitative IL-1β expression in the lung, evaluated with IL-1β/HPRT1 PCR ratio, was reduced in absence of IL-23p19 signaling (B). mRNA expression for IL-17A, IL-17F and HPRT1 in lung of 5 individual mice treated with BLM (10 mg/kg) or rmIL-1β (50 µg/kg) or saline wild-type mice or IL-1R1−/− mice were assessed using semi-quantitative PCR at 24 h (C). IL-17A and IL-17F mRNA levels were compared to the housekeeping gene HPRT1 mRNA levels and expressed as IL-17/HPRT ratio by measuring band densities using a densitometric analyzer (ImageJ) (D). Data represent in FiguremRNA expression Data represent mean values ± SD from 3 ratio independent experiments (n = 4 or 5 mice per group; *, p<0.05; **, p<0.01; ***, p<0.001).
Figure 2
Figure 2. Acute inflammation and remodeling upon BLM are dependent on IL-23p19.
Analysis of acute inflammation and remodeling was performed 24 h after BLM instillation (7.5 mg/kg). IL-23p19 deficient mice showed reduced neutrophil recruitment in BALF (A) and lung tissue (B) and reduced TIMP-1 production in lung (c) in comparison to wild-type mice Data represent mean values ± SD from 2 independent experiments (n = 5 mice per group; **, p<0.01; ***, p<0.001).
Figure 3
Figure 3. Impaired acute lung inflammation upon BLM in IL-17RA deficient mice.
IL-17RA deficient mice showed reduced neutrophil recruitment in BALF (A) and lung tissue (B) in comparison to wild-type mice. BALs and myeloperoxidase (MPO) activity were performed 24 h after BLM instillation (7.5 mg/kg). KC (D), IL-6 (E) and TIMP-1 (F) levels in the lung were also reduced in IL-17RA deficient mice at 24 h but not IL-1β levels (C). Cytokines, chemokine and TIMP-1 quantification in lung homogenates were performed by ELISA. Data represent mean values ± SD from 4 independent experiments (n = 5 mice per group; ns, not significant, *, p<0.05; **, p<0.01).
Figure 4
Figure 4. Acute lung inflammation and remodeling upon BLM depends on IL-17A.
Inflammatory and remodeling responses of wild-type mice treated with neutralizing antibody against IL-17A (150 µg/mouse, i.p. just after BLM or saline i.n. instillation). Neutrophil recruitment in BALF (A) and myeloperoxidase (MPO) activity in lung tissue (B) induced by BLM (7.5 mg/kg) were reduced after mice treatment with anti-IL-17A. IL-1β (C), KC (D), IL-6 (E) and TIMP-1 (F) levels in lung homogenates assessed by ELISA were also reduced 24 h after mice treatment neutralizing antibody against IL-17A. Data represent mean values ± SD from 3 independent experiments (n = 4 mice per group; *, p<0.05; **, p<0.01).
Figure 5
Figure 5. Acute lung inflammation and remodeling upon BLM are independent of IL-17F.
Inflammatory and remodeling responses upon BLM (7.5 mg/kg) of wild-type mice treated with neutralizing antibody against IL-17F (100–150 µg/mouse, i.p. just after BLM or saline i.n. instillation). Neutrophil recruitment in BALF (A) and myeloperoxidase (MPO) activity (B) upon BLM (7.5 mg/kg) were not reduced 24 h after treatment with high doses of neutralizing antibody against IL-17F. IL-1β (C), KC (D), IL-6 (E) and TIMP-1 (F) in lung homogenates assessed by ELISA were also reduced in BLM mice 24 h after treatment with neutralizing antibody against IL-17F. Treatments with anti-IL-17F had not significant effect on all these parameters. Data represent mean values ± SD from 2 independent experiments (n = 6 mice per group).
Figure 6
Figure 6. Early IL-17A and F are produced mainly by γδ T cells.
Lymphocytes from the lung of Rorc(γt)-Gfp TG mice were prepared 24 h after BLM or saline administration, and stimulated in vitro with PMA/ionomycin for 4 h. Specific staining of cell markers (CD1d-tetramer-APC, anti-NK1.1-PerCP-Cy5.5, anti-CD4-APCalexa750, anti-CD8-PB, anti-TCRγδ-APC, anti-TCRαβ-APC), or isotype control and intracellular staining for IL-17 (anti-IL-17A-PE, anti-IL-17F-PE, anti-GFP alexa488 or isotype controls) were performed. FACS analysis showed that BLM administration enhanced the total number of IL-17AposRORγtpos and IL-17FposRORγtpos lung mononuclear cells (A). IL-17-producing cells positive for RORγt were essentially γδ T cells and CD4pos αβ T cells but few iNKT cells (B). Among γδ T cells after bleomycin administration, at least 30% produce IL-17A and 5% produce IL-17F (C). Results are mean values ± SD from 4 independent experiments, with pools of 5 mice/group for each experiment, * p<0.05; **, p<0.01).
Figure 7
Figure 7. Remodeling and fibrosis are dependent on IL-23p19.
Analysis of late pulmonary remodeling and fibrosis was performed 14 days after BLM administration (5 mg/kg). MMP-2 activity (A) and TIMP-1 production (B) were reduced in IL-23p19 deficient mice. Active MMP-2 was expressed as relative intensity (RI). Latent TGF-β1 detected in BALF from wild-type mice was reduced in BALF from IL-23p19 deficient mice (C). Total collagen content in lung was significantly reduced in IL-23p19 deficient mice in comparision to wild-type mice (D). Collagen was measured using the Sircol collagen dye binding assay. Lung microscopic sections showed extensive fibrotic areas with collagen deposition in wild-type mice treated with BLM which was attenuated in IL-23p19−/− mice (e). Sirius red (SR) staining, scale bars 1 mm and 200 µm, n = 5 mice. Data represent mean values ± SD from 2 independent experiments (n = 5 mice per group; *, p<0.05; **, p<0.01; ***, p<0.001).
Figure 8
Figure 8. Attenuation of bleomycin-induced lung fibrosis in IL-17RA−/− mice.
Pulmonary remodeling and fibrotic response of wild-type mice or IL-17RA deficient mice 14 days after BLM or saline i.n. instillation. MMP-2 (72 Kd) activity in BALF was analyzed by zymography 14 days after administration of BLM (5 mg/kg i.n.). Active MMP-2 was upregulated at day 14 after BLM in the BALF of wild-type mice but only partially in IL-17RA−/− mice (A). TIMP-1 as indicator of a fibrotic process was upregulated in the lungs of B6, but to a lesser extent in IL-17RA−/− mice on day 14 (B). The latent form of TGF-β1 was present in BALF from B6 mice, 14 days after BLM, but was reduced in BALF from IL-17RA deficient mice as determined by ELISA assay (C). Active MMP-2 was expressed as relative intensity (RI). TIMP-1 and TGF-β1 levels were assessed by ELISA. Total collagen content enhanced 14 days after bleomycin instillation was significantly reduced in lung of IL-17RA deficient mice (D). Collagen was measured using the Sircol collagen dye binding assay. Data represent mean values ± SD from 2 independent experiments (n = 4 mice per group; *, p<0.05; **, p<0.01; ***p<0.001). Lung microscopic sections showed extensive fibrotic areas at day 14 with collagen deposition in wild-type mice treated with BLM (5 mg/kg i.n.) which was attenuated in IL-17RA−/− mice (E). Sirius red (SR) staining, scale bars 1 mm and 200 µm, n = 5 mice).
Figure 9
Figure 9. Attenuation of bleomycin-induced lung fibrosis upon anti-IL-17A treatment.
Pulmonary remodeling and fibrotic responses of wild-type mice treated with neutralizing antibody against IL-17A (150 µg/mouse, i.p. just after BLM and 3 times per week) 14 days after BLM (5 mg/kg i.n.) or saline. MMP-2 (72 Kd) activity in BALF was analyzed by zymography 14 days after administration of BLM. Active MMP-2 upregulated upon BLM in the BALF of wild-type mice was greatly reduced after 6 treatments with neutralizing antibody against IL-17A (A). TIMP-1 in lung was also significantly decreased after treatment with neutralizing antibody against IL-17A in comparison to untreated BLM mice (B). Latent TGF-β1 detected in BALF from wild-type mice was reduced in BALF from wild-type mice treated with anti-IL-17A antibodies (C). Active MMP-2 was expressed as relative intensity (RI). TIMP-1 and TGF-β1 levels were assessed by ELISA. In order to quantify lung collagen deposition, all the slides were numerised by Qimaging and analyzed by imageJ - NIH software. % of SR by lung were shown. Collagen content enhanced after BLM was significantly reduced when the mice were treated with anti-IL-17A antibody (D). Data represent mean values ± SD from 2 independent experiments (n = 5 mice per group; *, p<0.05; ***, p<0.001). Lung microscopic sections showed extensive fibrotic areas at day 14 with collagen deposition in wild-type mice administrated with BLM which was attenuated in mice treated with neutralizing antibodies against IL-17A (E). Sirius red (SR) staining, scale bars 1 mm and 200 µm, n = 5 mice.
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