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. 2018 Jul 1;315(1):L1-L10.
doi: 10.1152/ajplung.00395.2017. Epub 2018 Mar 22.

The relationship between complement C3 expression and the MUC5B genotype in pulmonary fibrosis

Tsukasa Okamoto  1 , Susan K Mathai  1 , Corinne E Hennessy  1 , Laura A Hancock  1 , Avram D Walts  1 , Adrianne L Stefanski  1 , Kevin K Brown  2 , David A Lynch  2 , Gregory P Cosgrove  2 , Steve D Groshong  2 , Carlyne D Cool  1 , Marvin I Schwarz  1 , Nirmal K Banda  1 , Joshua M Thurman  1 , Ivana V Yang  1 , V Michael Holers  1 , David A Schwartz  1   2
Affiliations

The relationship between complement C3 expression and the MUC5B genotype in pulmonary fibrosis

Tsukasa Okamoto et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

The common gain-of-function MUC5B promoter variant ( rs35705950 ) is the strongest risk factor for the development of idiopathic pulmonary fibrosis (IPF). While the role of complement in IPF is controversial, both MUC5B and the complement system play a role in lung host defense. The aim of this study was to evaluate the relationship between complement component 3 (C3) and MUC5B in patients with IPF and in bleomycin-induced lung injury in mice. To do this, we evaluated C3 gene expression in whole lung tissue from 300 subjects with IPF and 175 healthy controls. Expression of C3 was higher in IPF than healthy controls {1.40-fold increase [95% confidence interval (CI) 1.31-1.50]; P < 0.0001} and even greater among IPF subjects with the highest-risk IPF MUC5B promoter genotype [TT vs. GG = 1.59-fold (95% CI 1.15-2.20); P < 0.05; TT vs. GT = 1.66-fold (95% CI 1.20-2.30); P < 0.05]. Among subjects with IPF, C3 expression was significantly higher in the lung tissue without microscopic honeycombing than in the lung tissue with microscopic honeycombing [1.40-fold increase (95% CI 1.23- 1.59); P < 0.01]. In mice, while bleomycin exposure increased Muc5b protein expression, C3-deficient mice were protected from bleomycin-induced lung injury. In aggregate, our findings indicate that the MUC5B promoter variant is associated with higher C3 expression and suggest that the complement system may contribute to the pathogenesis of IPF.

Keywords: MUC5B; complement component 3; host defense; idiopathic pulmonary fibrosis.

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Figures

Fig. 1.
Fig. 1.
Relative complement component 3 (C3) expression in human lung tissues from 175 controls and 300 idiopathic pulmonary fibrosis (IPF) cases by quantitative PCR. A: IPF lung tissue had significantly higher C3 expression than control lung. Relative C3 expression in human lung tissue compared with genotypes at rs35705950 from 175 controls and 300 IPF cases by quantitative PCR. B: in control lungs, there is no significant difference in C3 expression between genotypes. C: in IPF lung tissue, the TT genotype had significantly higher C3 expression than GG or GT. Statistical tests were performed on Δcycle threshold (ΔCT) values (C3 CT values normalized to housekeeping gene GAPDH), and relative expression is visually depicted as (10 – ΔCT) on this graph. Lines indicate mean values with bars denoting ± SE.
Fig. 2.
Fig. 2.
The relationship of the complement component 3 (C3) expression based on the MUC5B promoter rs35705950 genotypes in the control and idiopathic pulmonary fibrosis (IPF) lung tissues by quantitative PCR. A: IPF lung tissue with GG had significantly higher C3 expression than control lung with GG. B: IPF lung tissue with T allele had significantly higher C3 expression than control lung with T allele. Statistical tests were performed on Δcycle threshold (ΔCT) values (C3 ΔCT values normalized to housekeeping gene GAPDH), and relative expression is visually depicted as (10 – ΔCT) on this graph. Lines indicate mean values with bars denoting ± SE.
Fig. 3.
Fig. 3.
The comparison of complement component 3 (C3) expression related to microscopic honeycombing scores. Regarding microscopic honeycombing scores, 0, absent; 1, rare; 2, present. The C3 expression was significantly higher for 0 than 1 or 2 score. Statistical tests wwere performed on Δcycle threshold (ΔCT) values (C3 Ct values normalized to housekeeping gene GAPDH), and relative expression is visually depicted as (10 – ΔCT) on this graph. The data of GG, GT, and TT subjects are plotted in red, green, and blue colors, respectively. Lines indicate mean values with bars denoting ± SE.
Fig. 4.
Fig. 4.
Immunohistochemistry of complement component 3 (C3), C3d, and C4d. Normal lungs are stained by C3, C3d, and C4d (A, E, and I). Idiopathic pulmonary fibrosis (IPF) lungs are stained by C3 (B and C), C3d (F and G), and C4d (J and K) (arrows). Isotype controls for C3, C3d, and C4d are shown (D, H, and L). Airway epithelial cells (B, F, and J) and interstitial lesions (C, G, and K) are shown. In IPF lung tissue C3 was expressed in airway epithelial cells, vascular walls, and interstitial lesions (B and C). C3d and C4d were expressed in the vascular walls and interstitial lesions but not in airway epithelial cells (F, G, J, and K). Brown color, DAB; blue color, hematoxylin. Magnification: ×4.
Fig. 5.
Fig. 5.
Immunofluorescence double staining of complement component 3 (C3) and MUC5B in human lung tissue. Double positive cells (arrows) denote airway epithelial cells (A, C, and D). There is no C3 expression in honeycomb cysts (E). B and F are isotype controls for A and E, respectively. Magnifications: A, B, E, and F: ×10; C: ×20; D: ×40. Fluorescent colors: green, C3 staining; red, MUC5B staining: yellow, overlay; blue, DAPI.
Fig. 6.
Fig. 6.
The phenotype of complement component 3-deficient (C3−/−) mice compared with wild-type (WT) mice in the repeat bleomycin model. A: body weight increased more significantly in C3−/− mice with bleomycin treatment compared with WT mice with bleomycin treatment. B: in bronchoalveolar lavage (BAL) profile, total cells, macrophages, and lymphocytes were significantly lower in C3−/− mice with bleomycin treatment than WT mice with bleomycin treatment. C: the hydroxyprolin concentration was significantly lower in C3−/− mice with bleomycin treatment than WT mice with bleomycin treatment. D: Muc5b expression in BAL was significantly lower in C3−/− mice compared with WT mice treated with bleomycin. E: there was no significant difference of C3a in BAL between WT mice and Muc5b−/− mice treated with bleomycin. Values are presented as maximum, upper quartile, mean, lower quartile, and minimum. Each group: n = 9–16. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 7.
Fig. 7.
A: the histology of mouse lungs in the repeat bleomycin mice model after staining with hematoxylin and eosin (H&E) and picro-sirius red staining (×4). B: immunostaining for MUC5B in bleomycin-induced lung injury. MUC5B is expressed in airway epithelial cells and interstitial lesions. Muc5b expression in lung tissue of complement component 3-deficient (C3−/−) mice treated with bleomycin is diminished compared with WT treated with bleomycin. There are double positive cells as arrows (C3 and MUC5B). C: C3 and C4 staining in mice lungs. C3 and C4 are expressed in airway epithelial cells and injured interstitial lesions.
Fig. 8.
Fig. 8.
The association between C3a and Muc5b in A549 cells. Muc5b gene expression increased 8 h after exposure of C3a in a dose-dependent manner. The experiment was performed twice with the same result. Values are presented as maximum, upper quartile, mean, lower quartile, and minimum. ΔCT, Δcycle threshold. *P < 0.05.
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