Seven-week old female wild type C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, US). Complement C3-deficient mice (B6.129S4-C3^tm1Crr/J, C3 KO) were obtained from University of Lübeck, Lübeck, Germany. All mice were housed under specific pathogen free conditions with 12-hour light/darkness cycles at the animal facility of Research Center Borstel, Germany. All animal experiments were reviewed and approved by the Animal Research Ethics Board of the Ministry of Energy Change, Agriculture, Environment, Nature, and Digitalization, Kiel, Germany (Number 104-8/17).

Cell membrane extract (ME) isolated from Chinese Hamster Ovary (CHO) cells overexpressing AT1R or non-transfected CHO cells were kindly provided by CellTrend (Luckenwalde, Berlin, Germany). Eight to ten weeks old female mice were immunized with 0.2 mg of control or AT1R ME in 50 µl of PBS emulsified with complete Freund adjuvant (CFA, Sigma-Aldrich, USA) by subcutaneous injection into the footpad as described previously (9). Three weeks after the immunization, the mice were boosted with the same amount of antigen emulsified with incomplete Freund adjuvant (IFA, Sigma-Aldrich, USA). Nine weeks after the first immunization, peripheral blood, dorsal skin and the lung were harvested for the evaluation of disease characteristics.

The deposition of IgG and C3 in the murine lung were determined by immunofluorescence staining. Briefly, the lung samples were collected from mice and cryosection was performed at thickness of 3 um, after fixation in pre-cold acetone and blocking in blocking buffer (Carl Roth, Germany), IgG binding was detected using DyLight 649-labeled goat anti-mouse IgG antibody (polyclonal, BioLegend, USA), complement 3 was detected with rat anti-mouse C3 antibody (RMC11H9, Cedarlane, Canada) and Alexa488 labled-goat anti-rat IgG antibody (polyclonal, Initrogen, USA). Coverslips were mounted with Gold mounting agent (Thermo Fisher, USA) that contains 4`, 6-diamidino-2-phenylindole (DAPI) for nuclear staining, the fluorescence was evaluated by confocal microscopy at a magnification indicated in the legends to figures (Leica SP5, Germany). The intensity of the fluorescent signals was analyzed and quantified using Image J software (version 1.53t, USA).

The level of anti-AT1R autoantibodies in the mouse serum samples were detected by using enzyme-linked immunosorbent assay (ELISA) as previously described (9). Levels of anti-AT1R antibodies were defined as the dilution at which the OD value reached the half of maximal OD values of the curve.

H&E staining was performed to determine the inflammation in tissues. Paraffin-embedded sections from mouse skin and lung were deparaffinized in xylene and rehydrated in ethanol series with decreasing concentrations, then stained with hematoxylin and eosin solutions. Thereafter, the stained sections were dehydrated and then mounted with mount medium. Severity of pulmonary and skin inflammation was quantified by the size and number of infiltrates in the lung and skin, and scored in a double-blinded fashion as described previously (9).

To evaluate tissue fibrosis, paraffin-embedded tissue sections of mouse skin and lung were stained with Masson’s Trichrome staining kit (Sigma-Aldrich, USA). Tissue sections were deparaffinized in xylene and rehydrated in ethanol series with decreasing concentrations. Rehydrated sections were fixed in Bouin’s solution at RT overnight, counterstained with Hematoxylin Gill II solution, washed and incubated with Biebrich scarlet-Acid Fuchsin, and subjected to phosphotungstic/phosphomolybdic acid solution. Phosphoric acid treated sections were then incubated with aniline blue solution, differentiated in 1% acetic acid, rinsed with deionized water, dehydrated in ethanol, and finally mounted with mounting medium. Skin thickness was measured as the thickness of the blue collagen layer.

The collagen content of the skin was determined by using the Sircol collagen detection kit (Biocolor, UK) according to the manufacture’s instruction. Briefly, punches of skin tissues (9 mm²) were cut into pieces and digested in 0.5 M acetic acid solution containing porcine pepsin (Sigma, USA) at 4°C overnight on the shaker. In the next day, supernatant was collected and was mixed with Sircol dye solution and incubated on shaker. Afterwards, samples were centrifuged and pellets were washed with ice cold acid salt wash reagent to remove unbound dyes. Dye bound collagen pellets were dissolved in Alkali buffer was added to each sample to release collagen. To record the OD values, samples were transferred into a plate and measured on TECAN microplate reader at a wavelength of 550 nm. Amount of collagen in samples were calculated according to the standard curve generated with gradient diluted collagen.

Immunohistochemistry staining was conducted on murine tissue sections to assess the cellularity of infiltrated cells. Lung paraffin-embedded sections underwent deparaffinization in xylene followed by rehydration in a gradient of ethanol. Subsequently, antigen retrieval was achieved by microwave heating with 1× citrate buffer (pH=6) (ZytoMed, BE, Germany, Cat#ZUC028-500), and sections were blocked using 3% H₂O₂. They were then incubated with specific primary antibodies for 1 hour at room temperature, including rabbit anti-CD20 (Invitrogen, MA, USA, Cat# PA5-16701, 1:500), rabbit anti-CD3 (ZytoMed, Cat# RBK024-05, 1:100), or rat anti-mouse neutrophil antibodies (Cedarlane, ON, Canada, Cat# CL8993AP, 1:800). Following washing steps, the sections were treated with ImmPRESS-HRP goat anti-rat IgG polymer kit (VECTOR, Cat# MP-7404-50) or ZytoChem Plus-HRP polymer kit (ZytoMed, Cat#POLHRP-100), followed by incubation with 3-Amino-9-ethylcarbazole (AEC) (ZytoMed, Cat#ZUC054-200) to visualize immunoreactivity. Finally, counterstaining with hematoxylin was performed, and bright-field microscopy was used to capture images.

The apoptotic cells in the lung of mice were detected with DeadEnd™ Fluorometric TUNEL System (Promega, USA) according to the manufacturer recommended procedures. Briefly, the deparaffinize and rehydrated lung sections were permeabilized with 0.2% Triton X-100 and equilibrated in equilibration buffer. Immediately before the reaction, the TUNEL mixture was freshly prepared and added to the sections. After incubation with rTdT incubation buffer, the reactions were stopped by SSC buffer. Sections were finally mounted with mounting medium containing DAPI for counterstaining. To quantify the level of apoptotic cells, six to ten random confocal images were recorded and the number of apoptotic cells and total cells were measured, and the rate of apoptotic cells were calculated as number of apoptotic cells divided by total number of cells.

All data was analyzed by using Graphpad Prism software (version: prism 10.2). Statistical differences of quantitative data between two groups were determined by two-tail unpaired Student’s t test. Two-way ANOVA was used to determine the statistical significance among multiple groups with two category variables. Significant differences between qualitative datasets were determined by Fisher-exact test. A p value below 0.05 was considered as statistically significant.

To investigate the role of complement activation in the AT1R-induced mouse model for SSc, we first determined the deposition of IgG and complement C3 in the lung. Direct Immunofluorescence staining revealed that mice immunized with AT1R showed significantly higher levels of IgG deposition in the lung than mice immunized with control ME ( Figures 1A, B ). By contrast, no significant difference in C3 deposition was observed between AT1R-immunized mice and controls suggesting the lack of IgG-mediated complement activation ( Figures 1C ). In contrast, C3 deposition was effectively detected in a mouse model of epidermolysis bullosa acquisita ( Supplementary Figure 1 ), where complement activation is a crucial step in disease progression (23).

To better examine the role of complement C3 in this mouse model, C3 deficient mice and wild type (WT) control mice were immunized with AT1R ME or control ME. Nine weeks after the first immunization, blood, lung and skin were collected for further evaluation ( Figure 2A ). As shown in Figure 2B , immunizations with AT1R induced comparable levels of anti-AT1R IgG in C3 deficient and WT mice, suggesting that C3 deficiency does not affect the production of abs against AT1R.

Histological examination of lung tissue showed that AT1R immunization induced pulmonary inflammation in both C3 deficient mice and WT controls. Notably, C3-deficient mice developed a more severe AT1R-induced inflammation in the lung than WT mice ( Figures 2C, D , p<0.001). Immunohistochemistry analysis revealed that lung inflammation primarily consisted of T cells, with neutrophils and B cells also present, and the composition of lung infiltrates was comparable between AT1R-immunized C3 deficient and WT mice ( Supplementary Figure 2 ). In addition, a trend of increased pulmonary inflammation, although not significant, in C3-deficient mice as compared to WT mice was also observed after immunization with control ME.

We next examined the histopathological changes in the skin, another tissue involved in this experimental model (9). AT1R immunization induced comparable incidence of skin inflammation between WT controls and C3 deficient mice ( Figures 3A, B ). Furthermore, both skin thickness and collagen content were not significantly different between C3-deficient mice and WT controls immunized with AT1R ( Figures 3C–E ), indicating that C3 is dispensable for disease manifestations in the skin and indicate organ-specificity of the protective C3 effects for the lung.

In the next step we aimed to explore the mechanism underlying the effect C3 on pulmonary inflammation. Since it has been reported that C3 plays a protective role in apoptosis human airway epithelial cells and β-cells (24, 25), a common trigger of inflammation, we determined apoptosis of pulmonary cells in immunized mice. Compared to WT controls, C3-deficient mice showed a significantly increased pulmonary cell apoptosis rate after AT1R immunization (p<0.01) ( Figures 4A, B ). However, increased pulmonary cell apoptosis rate in C3-deficient mice as compared to corresponding WT controls was also observed in animals which received control ME (p<0.01). Finally, we evaluated the relationship between cell apoptosis and pulmonary inflammation. As shown in Figure 4C , a strong correlation was observed between pulmonary cell apoptosis rate and pulmonary inflammation in AT1R-immunized C3-deficient mice (R² = 0.74, p=0.029), while such correlation was much weaker in the other three groups (data not shown).