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. 2016 Jun;30(6):2336-50.
doi: 10.1096/fj.201500044. Epub 2016 Mar 8.

Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis

Hongmei Gu  1 , Amanda J Fisher  1 , Elizabeth A Mickler  1 , Frank Duerson 3rd  1 , Oscar W Cummings  2 , Marc Peters-Golden  3 , Homer L Twigg 3rd  4 , Trent M Woodruff  5 , David S Wilkes  4 , Ragini Vittal  6
Affiliations

Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis

Hongmei Gu et al. FASEB J. 2016 Jun.

Abstract

Complement activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins-complement component 3a (C3a) and complement component 5a (C5a)-in IPF, which interact with TGF-β and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C5a to their respective receptors (C3aR and C5aR) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C5a induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C5aR in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C5a, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson's trichrome, hydroxyproline, collagen type I α 1 chain, and collagen type I α 2 chain). Pharmacologic or RNA interference-specific interventions suppressed complement activation (C3a and C5a) and soluble terminal complement complex formation (C5b-9) locally and active TGF-β1 systemically. C3aR/C5aR antagonists suppressed local mRNA expressions of tgfb2, tgfbr1/2, ltbp1/2, serpine1, tsp1, bmp1/4, pdgfbb, igf1, but restored the proteoglycan, dcn Clinically, compared with pathologically normal human subjects, patients with IPF presented local induction of C5aR, local and systemic induction of soluble C5b-9, and amplified expression of C3aR/C5aR in lesions. The blockade of C3aR and C5aR arrested the progression of fibrosis by attenuating local complement activation and TGF-β/bone morphologic protein signaling as well as restoring decorin, which suggests a promising therapeutic strategy for patients with IPF.-Gu, H., Fisher, A. J., Mickler, E. A., Duerson, F., III, Cummings, O. W., Peters-Golden, M., Twigg, H. L., III, Woodruff, T. M., Wilkes, D. S., Vittal, R. Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.

Keywords: BMP; C5b-9; IPF; TGF-β1; decorin.

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Figures

Figure 1.
Figure 1.
C3a and C5a induce mesenchymal activation in normal primary human fetal lung fibroblast cultures. IMR-90 cells were grown to 65–70% confluence and were serum starved for ∼ 36 h. A) Cells were treated with varying doses of human recombinant C3a or C5a (10, 50, or 100 nM × 24 h). Cell lysates were immunoblotted with antibodies recognizing α-SMA and β-actin (loading control). B) Immunoblots described in panel A were analyzed by densitometry. Values are expressed as means ± sem of triplicate experiments; statistics: 1-way ANOVA, Bonferroni. C) Mesenchymal activation observed in panel A was confirmed by immunofluorescence staining with anti–α-SMA and DAPI. D) Triplicate results of the immunofluorescent labeling were quantitated by assessing the percent positively labeled cells. Values are expressed as means ± sem; statistics: 1-way ANOVA, Bonferroni. E) Cell lysates from panel A were immunoblotted against fibronectin, collagen type I [Col(I)], and β-actin (loading control). F) Temporal analyses of the respective receptors for C3a and C5a and mesenchymal induction were observed at the indicated time points. Immunoblotting results indicate concomitant induction of C3aR and C5aR with α-SMA. Results are representative of 3 independent experiments. Original magnification, ×20. Scale bars, 100 μm.
Figure 2.
Figure 2.
Pharmacologic blockade of receptors specific to C3a and C5a (C3aR and C5aR) arrest the progression of bleomycin-mediated lung fibrosis. C57-BL/6 mice were subjected to an intratracheal instillation of PBS or bleomycin (0.025 U) on d 0. A) C3a and C5a were analyzed in the BALF collected at the indicated time points after bleomycin injury. Values are given as means ± sem (n = 4 mice per group); unpaired Student's t test. B) Expressions of C3aR and C5aR were assessed in the PBS or bleomycin-instilled mice at d 28 by using immunostained using 3,3′-diaminobenzidine (brown) with corresponding secondary IgG. Nuclei were counterstained by using hematoxylin. C) In the clinically relevant therapeutic model, wherein at d 14, (period of significant collagen deposition), antagonists against C3aR (C3aRA, SB290157; 300 μg/20 g mouse) or C5aR (C5aRA, PMX-205; 200 μg/20 g mouse) were administered intraperitoneally 3 times/wk for 2 wk. Mice were euthanized at d 28. D) Histopathologic exam using hematoxylin and eosin (H&E) and trichrome staining showed that bleomycin-induced fibrosis and collagen deposition were attenuated by the inhibitors. E, F) Analysis of hydroxyproline (E) and col1a1 and col1a2 (F) mRNA expression in the lung. Values are given as means ± sem (n = 10–12 mice/group); 1-way ANOVA, Bonferroni. Results are representative of 3 independent experiments. AW, airway; BLEO, bleomycin; FF, fibroblastic foci. Original magnification, ×20 (B), ×10 (D). Scale bar, 100 μm.
Figure 3.
Figure 3.
Pharmacologic blockade of receptors specific to C3a and C5a suppresses complement activation in bleomycin-mediated lung fibrosis. BALF collected from Fig. 2 were analyzed for C3a (A), C5a (B), and C5b-9 (C) levels in the lung by ELISA. Values are given as means ± sem (n = 5–6 mice per group); 1-way ANOVA, Newman-Keuls (A) and Bonferroni (B, C). BLEO, bleomycin.
Figure 4.
Figure 4.
Local and systemic C5b-9, a soluble TCC, in patients with IPF. A, E) Patients who were diagnosed with IPF or normal volunteers were analyzed for C5b-9 levels by ELISA in BALF (A) or plasma (E). Values are given as means ± sem; unpaired t test. B–D, F–H) Correlation between BALF (B–D) and plasma (F–H) analyses of C5b-9 and percentage forced vital capacity (FVC%) (B, F), percentage carbon monoxide lung diffusing capacity (DLCO%) (C, G), and composite physiologic index (CPI) (D, H) in patients with IPF. Statistics are provided in the text.
Figure 5.
Figure 5.
Suppression of TGF-β1 activity in the plasma as a result of pharmacologic blockade of C3aR and C5aR in bleomycin-induced lung fibrosis. A) Plasma collected from Fig. 2 was analyzed for active TGF-β1 by ELISA. B) mRNA expression for tgfb1 was analyzed in the lung by quantitative RT-PCR. Values are given as means ± sem (n = 5–6 mice per group); 1-way ANOVA, Newman-Keuls (A). BLEO, bleomycin; RQ, relative quantitation.
Figure 6.
Figure 6.
Regulation of genes belonging to the TGF-β superfamily (ligands, receptors, and modulators) resulting from pharmacologic blockade of C3aR and C5aR in bleomycin-induced lung fibrosis. RNA was isolated from the right lungs, and cDNA was subjected to real-time PCR reactions by using the Mouse TGF-β BMP Signaling PCR array (Qiagen). Specific genes analyzed were isoform tgfb2 (A), receptors tgbr1 (B) and tgbr2 (C), binding proteins ltbp1 (D) and ltbp2 (E), and modulators serpine1 (F), tsp1 (G), and dcn (H). Values are given as means ± sem (n = 5–6 per group); 1-way ANOVA, Bonferroni (A, C, E, F), Newman-Keuls (B, D, G, H). BLEO, bleomycin; RQ, relative quantitation.
Figure 7.
Figure 7.
Suppression of genes belonging to the BMP superfamily and profibrotic mediators that interact with TGF-β as a result of pharmacologic blockade of C3aR and C5aR in bleomycin-induced lung fibrosis. RNA was isolated from the right lungs, and cDNA was subjected to real-time PCR reactions by using the Mouse TGF-β BMP Signaling PCR array (Qiagen). Specific genes analyzed were bmp1 (A), bmp4 (B), pdgfbb (C), and igf1 (E). Values are given as means ± sem (n = 5–6 per group); 1-way ANOVA, Newman-Keuls (A, B), Bonferroni (C, D). BLEO, bleomycin; RQ, relative quantitation.
Figure 8.
Figure 8.
RNAi-mediated gene silencing of C3aR and C5aR arrests the progression of bleomycin-induced lung fibrosis. A) C57-BL/6 mice were subjected to an intratracheal instillation of PBS or bleomycin (0.025 U) on d 0, followed by intratracheal instillation of 50 μg RNAi at d 14. Tissues were harvested on d 28. B) Protein expressions of C3aR and C5aR were assessed by immunostaining in bleomycin-injured mice instilled with siRNA specific to C3ar or C5ar or nontargeting controls at d 28 to confirm efficacy by 3,3′-diaminobenzidine (brown) with corresponding secondary IgG. Nuclei were counterstained by using hematoxylin. Abbreviations: AW: Airway, FF: Fibroblastic foci. C) Histopathologic exam using hematoxylin and eosin (H&E) and trichrome staining showed that bleomycin-induced fibrotic lung and collagen deposition was attenuated by silencing C3ar and C5ar. D, E) Analysis of hydroxyproline (D) and col1a1 and col1a2 (E) mRNA expression in the lung. Values are given as means ± sem (n = 5–7 per group); 1-way ANOVA, Bonferroni. AW, airway; BLEO, bleomycin; FF, fibroblastic foci. Compared with bleomycin: ***P < 0.001; **P < 0.01; *P < 0.05. Results are representative of 2 independent experiments. Original magnification, ×20 (B), ×10 (C). Scale bar, 100 μm.
Figure 9.
Figure 9.
C3aR and C5aR expression profiles in the lungs of patients with IPF. A) Comparative immunohistochemical staining was performed on paraffin-embedded human IPF lung biopsy explants obtained during lung transplantation and tissue resected from normal (non-IPF) lung tissue using C3aR or C5aR. Trichrome staining shows areas of fibrotic foci with rabbit IgG antibodies. (3,3′-diaminobenzidine, brown; hematoxylin, blue). Representative lesions are presented subsequent to examining lung biopsies from 5 different normal subjects and patients with IPF. B) Double immunofluorescent staining of IPF lung tissue sections with C3aR or C5aR with α-SMA shows costaining in the inset. C) Fibroblasts cultured ex vivo from normal and IPF lungs were immunoblotted for C3aR, C5aR, and β-actin. Densitometric analyses of C3aR and C5aR compared with β-actin are expressed as means ± sem; unpaired t test. AW, airway; FF, fibroblastic foci. Original magnification, ×40. Scale bar, 600 μm.
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