IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis

Abstract

Interleukin 17A (IL-17A) and complement (C') activation have each been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). We have reported that IL-17A induces epithelial injury via TGF-β in murine bronchiolitis obliterans; that TGF-β and the C' cascade present signaling interactions in mediating epithelial injury; and that the blockade of C' receptors mitigates lung fibrosis. In the present study, we investigated the role of IL-17A in regulating C' in lung fibrosis. Microarray analyses of mRNA isolated from primary normal human small airway epithelial cells indicated that IL-17A (100 ng/ml; 24 h; n = 5 donor lungs) induces C' components (C' factor B, C3, and GPCR kinase isoform 5), cytokines (IL8, -6, and -1B), and cytokine ligands (CXCL1, -2, -3, -5, -6, and -16). IL-17A induces protein and mRNA regulation of C' components and the synthesis of active C' 3a (C3a) in normal primary human alveolar type II epithelial cells (AECs). Wild-type mice subjected to IL-17A neutralization and IL-17A knockout (il17a^-/- ) mice were protected against bleomycin (BLEO)-induced fibrosis and collagen deposition. Further, BLEO-injured il17a^-/- mice had diminished levels of circulating Krebs Von Den Lungen 6 (alveolar epithelial injury marker), local caspase-3/7, and local endoplasmic reticular stress-related genes. BLEO-induced local C' activation [C3a, C5a, and terminal C' complex (C5b-9)] was attenuated in il17a^-/- mice, and IL-17A neutralization prevented the loss of epithelial C' inhibitors (C' receptor-1 related isoform Y and decay accelerating factor), and an increase in local TUNEL levels. RNAi-mediated gene silencing of il17a in fibrotic mice arrested the progression of lung fibrosis, attenuated cellular apoptosis (caspase-3/7) and lung deposition of collagen and C' (C5b-9). Compared to normals, plasma from IPF patients showed significantly higher hemolytic activity. Our findings demonstrate that limiting complement activation by neutralizing IL-17A is a potential mechanism in ameliorating lung fibrosis.-Cipolla, E., Fisher, A. J., Gu, H., Mickler, E. A., Agarwal, M., Wilke, C. A., Kim, K. K., Moore, B. B., Vittal, R. IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis.

Keywords: C3a; C5a; C5b-9; DAF; ER stress.

Figure 1.

IL-17A activates C′ by modulating the mRNA and protein expression of the C′ components in vitro. A) Normal human primary AECs were exposed to recombinant human IL-17A (100 ng/ml; 24 h). Subsequent to RNA isolation, cDNA was generated and subjected to real-time quantitative PCR analyses. Means ± sem; Student’s t test. B) Protein lysates from AECs exposed to recombinant human IL-17A (100 ng/ml; 4 h) were subjected to immunoblot analysis against C3aR, C5aR, DAF, and vinculin. C) Conditioned medium collected from AECs exposed to the indicated doses of IL-17A for 4 h was analyzed for active C3a by ELISA. Means ± sem. D) AECs exposed to IL-17A (100 ng/ml; 4 h) were methanol fixed, immunostained for DAF, and counterstained with DAPI. Results represent 3 independent experiments. Scale bars, 100 µm.

Figure 2.

IL-17A deficiency protects against BLEO-induced lung fibrosis and collagen deposition. A) C57-BL6 wild-type mice were subjected to an intratracheal instillation of PBS or BLEO (0.025 U) on d 0 and euthanized at the indicated time points. RNA isolated from lung homogenates was subjected to real-time quantitative PCR. Means ± sem; Student’s t test. B) Time line of exposure of mice to PBS or BLEO until euthanasia. C) Left lungs were analyzed for hydroxyproline (n = 11–12/group). Means ± sem; 1-way ANOVA and Bonferroni. D) Histopathologic examination showed that BLEO-induced fibrosis and collagen deposition were lower in the il17a^−/− mice. Results are representative of 3 independent experiments. Scale bars, 100 μm.

Figure 3.

IL-17A deficiency protects against BLEO-induced cellular apoptosis and ER stress in the lung. A) Plasma from lungs described in Fig. 2B was analyzed for the AEC marker KL6/MUC-1 by ELISA (n = 11–12/group). Means ± sem; 1-way ANOVA and Bonferroni. B) Supernatants of lung homogenates from the right lungs were subjected to caspase-3/7 luciferase assay (n = 11–12/group). Means ± sem; 1-way ANOVA and Bonferroni. C–K) RNA was isolated from right lung homogenates, and cDNA was subjected to real-time PCR reactions. The specific genes were atf4 (C), atf6 (D), atf6b (E), eif2a (F), serp1 (G), pdia3 (H), vimp1 (I), os9 (J) and scap (K) (n = 5–6/group). Means ± sem; 1-way ANOVA and Bonferroni (F–I, K), Newman-Keuls (C, E, J).

Figure 4.

IL-17A deficiency protects against BLEO-induced local C′ activation. The BALF samples collected for the analysis shown in Fig. 2 were also analyzed for C3a (A), C5a (B), and C5b-9 (C) levels by ELISA (n = 7–8/group). Means ± sem. One-way ANOVA, Newman-Keuls (A, C), and Bonferroni (B).

Figure 5.

Neutralization of IL-17A prevents BLEO-induced lung fibrosis, apoptosis, and loss of C′ inhibitors. A) C57-BL6 mice received tail vein injections of the adenoviral vectors 3 d before BLEO injury and then were euthanized 21 d after injury. B) Histopathologic examination with hematoxylin and eosin indicates scarred lung in the Ad-LUC groups, which is prevented with administration of the Ad-IL-17R:Fc. C) Tissues were immunostained with antibodies against IL-17A, using secondary alkaline red phosphatase and counterstained with hematoxylin and eosin. D) Top: colocalization of ZO-1 and α-SMA using fluorescent labeling in the lungs of Ad-LUC and Ad-IL-17R:Fc groups. Nuclei were counterstained with DAPI. Bottom: TUNEL staining with the analyses of number of cells staining TUNEL⁺ (n = 5/group). Means ± sem; 1-way ANOVA and Bonferroni. E) Immunostaining of the C′ inhibitors DAF and Crry expression in the epithelial cells of lungs of Ad-LUC and Ad-IL-17R:Fc groups. Normal expression of these proteins are diminished or lost in the Ad-LUC group with BLEO injury, and this is presented in the Ad-IL-17R:Fc despite BLEO injury.

Figure 6.

RNAi-mediated gene silencing of il17a mitigates progression of fibrosis and lung deposition of collagen and C′. A) PBS or BLEO (0.025 U) was instilled intratracheally in C57-BL6 mice on d 0, followed by oropharyngeal instillation of 50 μg RNAi on d 14. Tissues were harvested on d 28. B) Formalin-fixed paraffin-embedded tissues were immunostained with antibodies against IL-17A using secondary alkaline red phosphatase and counterstained with hematoxylin. C) Left lungs were analyzed for hydroxyproline (n = 11–12/group). Means ± sem; 1-way ANOVA and Bonferroni. D) Histopathological examination showed that BLEO-injured fibrotic lung and collagen deposition were reduced by silencing il17a. Scale bars, 100 μm. E, F) We analyzed caspase-3/7 (E) and C5b-9 expressions (F) in the supernatants of the lung homogenates by the luciferase assay and ELISA, respectively (n = 10–12/group). Means ± sem; 1-way ANOVA and Bonferroni. Results are representative of 3 independent experiments.

Figure 7.

Hemolytic activity in IPF. Hemolytic activity was assessed in 7 normal patients and 23 with IPF, with or without heat inactivation. Means ± sem; 1-way ANOVA and post hoc Bonferroni.