C3 deficiency promotes pulmonary inflammation in AT1R-induced mouse model for systemic sclerosis

Abstract

Introduction: Autoantibody-mediated complement activation plays an essential role in a variety of autoimmune disorders. However, the role of complement in systemic sclerosis (SSc) remains largely unknown. In this study, we aimed to determine the role of complement C3 in the development of a recently described SSc mouse model based on autoimmunity to angiotensin II receptor type 1 (AT1R).

Methods: Mice were immunized with cell membrane extract isolated from Chinese hamster ovary (CHO) cells overexpressing AT1R or non-transfected CHO cells as a control. Peripheral blood, dorsal skin and the lung were then collected to evlauate disease characteristics. Apoptotic cells in the lung of mice were detected using the DeadEnd™ Fluorometric TUNEL System.

Results: Our results showed that experimental SSc in this model was featured by the deposition of IgG, but not of complement C3, in the lung. After immunization with AT1R, C3-deficient mice developed more severe pulmonary inflammations than wild type controls, whereas skin inflammation and fibrosis were not different as well as the anti-AT1R ab levels. Further, C3-deficient mice showed an increased rate of pulmonary cell apoptosis as compared to controls. The apoptosis rate correlated with the corresponding degree of lung inflammation.

Discussion: Taken together, our findings suggest an anti-apoptotic and anti-inflammatory role of complement C3 in pulmonary autoimmune inflammation.

Keywords: complement; inflammation; mouse model; pulmonary inflammation; systemic sclerosis.

Figure 1

Deposition of IgG but not complement 3 in lung tissue of hAT1R-immunized mice. (A). Cryosections of lung tissue from hAT1R-immunized mice or control mice were incubated with DyLight 649-labeled goat anti-mouse IgG antibody to detect IgG deposition (red), complement 3 was stained with rat anti-mouse C3 antibody and Alexa488 labeled-goat anti-rat IgG antibody (green), nucleus was stained with DAPI (blue). The representative micrographs were shown (630x, scale bar= 25mm). (B, C). The quantitative analysis of the fluorescent signals of IgG and complement 3 was performed with pictures of representative fields of each group (n=4~5) using ImageJ software. The results are presented as the mean ± SD, and the statistical analysis was performed by using Student’s t-test (*: p<0.05).

Figure 2

C3 deficiency promotes the AT1R immunization-induced lung inflammation. (A). Schematic diagram of immunization protocol and groups. Mice were immunized at week 0 with membrane extract (AT1R ME or control ME) and boosted 3 weeks later. Nine weeks after the first immunization, mice were sacrificed, and blood and tissue samples were collected for evaluation. Five to six mice were analysed in each group. (B). Levels of anti-AT1R IgG were determined in sera by ELISA. (C). Representative micrographs of H&E-stained lung sections of control ME- or AT1R ME-immunized mice. Black arrows indicate inflammatory cell infiltration in the lung. Scale bar = 100 µm. (D). Quantitative analysis of pulmonary inflammation in mice. Statistical analysis was performed by using two-way ANOVA test. * p<0.05, ***, p<0.001.

Figure 3

C3 deficiency does not affect skin inflammation. Wild type and C3-deficient (C3 KO) mice were immunized with control ME or AT1R ME. (A). Representative micrographs of H&E-stained skin sections of control ME- or AT1R ME-immunized mice. Black arrows indicate inflammatory cell infiltration around blood vessels. Scale bar = 50 µm. (B). Incidence of skin inflammation in mice. The numbers of mice with skin inflammation/all mice are presented on the top of corresponding bars. (C). Representative micrographs of Masson’s Trichrome stained skin sections of control ME- or AT1R ME-immunized mice. Double-headed arrows indicate the collagen layer of the skin. Scale bar = 100 µm. (D). Quantitative analysis of skin thickness. Thickness of the skin was defined as the thickness of the collagen layer stained in blue from the Masson’s Trichrome staining. (E). Quantitative analysis of collagen content Collagen content was determined using Sircol collagen detection kit and expressed as μg per mm² of the skin. Statistical analysis was performed by using two-way ANOVA test. *, p<0.05, **, p<0.01 ***, p<0.001.

Figure 4

Increased apoptosis of lung epithelial cells in C3-deficient mice. (A). Representative micrographs of apoptotic cells in lung cryosections from WT or C3 deficient mice visualized by TUNEL assay. Positive apoptotic cells are labelled in green fluorescence. Scale bar= 25mm. (B). Quantitative analysis of apoptotic cells as determined in the TUNEL assay. The percentage of apoptotic cells in the total cells were presented as mean ± SD. Statistical analysis was performed by using two-way ANOVA test. *, p<0.05, ***, p<0.001. (C). The correlation of severity of lung inflammation and apoptotic cell rate from the AT1R immunized C3 deficient mice. Blue line shows the simple linear regression line. R square and p values were presented in the plot.