Cell culture and transfection with SARS-CoV-2 spike protein and ACE2 protein

HEK293 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, 12,800,017; Gibco), supplemented with 10% (v/v) fetal bovine serum (FBS, #16,000,044; Gibco), and maintained at 37 °C in a 5% CO2 atmosphere. The cells were seeded at a density of 3.5 × 105 cells per well in 6-well culture plates and allowed to grow for 20–24 h. Subsequently, the cells were transfected with 2 µg plasmid encoding either SARS-CoV-2 spike protein (pBOB-CAG-SARS-CoV-2-Spike-HA, #12,260; Addgene) or ACE2 protein (pLEX307-ACE2-puro, #158,448; Addgene) using 6 µL X-treme GENE HP DNA transfection reagent (6,366,236,001; Roche), following the manufacturer’s instructions. HEK293 cells overexpressing SARS-CoV-2 spike protein and ACE2 were starved in 1X insulin-Transferrin-Selenium (GIB-51,300–044; Gibco) DMEM for 12 h. Then the cells were stimulated with 10 ng/mL TGF-beta1 for 24 h in 1X ITSA DMEM; 48 h post-transfection, the cells were harvested for Western blotting analysis.

Animals

Seven-week-old male C57BL/6 mice were procured from Orient Bio Inc. (Seongnam, Korea). The mice were housed under specific-pathogen-free conditions at the Institute of Medical Science of the Catholic University of Korea. They had ad libitum access to water and were provided with standard mouse chow (Ralston Purina, St. Louis, MO, USA). All experimental procedures involving animals were conducted in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experiments as provided by the Institutional Animal Care and Use Committee of the Catholic University of Korea. Furthermore, the procedures conformed to all National Institutes of Health (USA) guidelines (permit number: [2023-0064]). To minimize any distress or suffering, all mice were anesthetized with isoflurane (2–2.5%) and euthanized by cervical dislocation.

Bleomycin and spike S and ACE2 induction

Bleomycin was administered over a period of 2 weeks. To induce SSc in mice, bleomycin was dissolved in PBS. Male C57BL/6 mice aged 8 weeks were injected subcutaneously with 50 µg bleomycin dissolved in 100 µL PBS daily for 2 weeks. To develop a COVID-19 infection model, mice were intramuscularly injected with 100 µg pLEX307-ACE2-puro (#158,448; Addgene) in 50 µL saline twice every 5 days for 35 days. In addition, 100 µg pcDNA3.1-SARS-2-Spike (#145,032; Addgene) was intramuscularly injected into the Spike group twice every 5 days for 30 days.

Subcellular fractionation

Proteins in the cells were extracted using RIPA lysis and extraction buffer (Cat. 89,901; Thermo) supplemented with complete protease inhibitor (Cat. 78,438; Thermo) and 0.5 M EDTA (Cat. 78,438; Thermo). After incubating the cocktail solution on ice for 20 min, the samples were centrifuged at 14,000 g for 15 min at 4 °C. The supernatants were collected into new tubes. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Cat. 23,225, Thermo).

Western blotting

Each sample containing 20 µg protein was electrophoresed on 10% polyacrylamide gradient gels and then transferred to nitrocellulose membranes. The proteins were separated via SDS-PAGE, transferred to Hybond enhanced chemiluminescence (ECL) membranes (10,600,001; Cytiva), and incubated with antibodies against anti-ACE2 (ab108252; Abcam, 1:1,000), anti-2019-nCoV spike (40,591-MM42; Sino Biological Inc., 1:1,000), anti-alpha smooth muscle actin (α-SMA) (ab7817[1A4]; Abcam, 1:1,000), anti-Col1a1 (PA5–89,281; Invitrogen, 1:1,000), and GAPDH (ab181602[EPR16891]; Abcam, 1:2,000) using the SNAP i.d. Protein Detection System (Millipore, Billerica, IL, USA). After incubation with the corresponding secondary anti-mouse (SC-2005; Santa Cruz) and anti-rabbit (SC-2357; Santa Cruz) antibodies, the protein bands were visualized using X-film (100NIF ; EA8EC).

Enzyme-linked immunosorbent assay (ELISA)

Human SARS-CoV-2 spike protein and mouse anti-phospholipid antibody levels in the serum were measured via ELISA. Serum was isolated from mice injected with or without SARS-CoV-2 spike plasmid DNA. SARS-CoV-2 spike (40,591-MM42; Sino Biological Inc.) and mouse anti-phospholipid antibody (MBS742389; MBS) levels were measured via ELISA according to the manufacturer’s instructions. Absorbance was measured at 405 nm on an ELISA microplate reader (Molecular Devices Inc., Sunnyvale, CA, USA).

Isolation of spleen, blood, skin, and lung T cells

Splenocytes and blood cells circulating in the vasculature were treated with ammonium-chloride-potassium (ACK) buffer to lyse red blood cells. Single-cell suspensions were incubated with phorbol 12-myristate 13-acetate (P8139; Sigma), ionomycin (I0634; Sigma), and protein transport inhibitor (51–2091KZ; BD) in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 5% fetal bovine serum (FBS) (#16,000,044; Gibco) at 37℃ and 5% CO2 for 4 h. Then the cells were washed with phosphate buffer solution containing 0.2% bovine serum albumin and 0.02% sodium azide. Skin from the fibrosis-induced area of 5 mice was placed into PBS media containing 5 mM EDTA (EDS-500G; Sigma), 10 mM HEPES (H0396; TCI), and 10% FBS. Skin fragments were incubated on a rotating plate for 30 min in a dry, non-gassed incubator maintained at 37℃. Then the contents were vortexed vigorously for 10 s and strained through a 40 μm strainer (SPL93040; SPL Life Sciences) to collect the tissue. The skin was transferred into a new tube containing digestion media (containing 0.7 mg/mL collagenase IV (17,104,019; Gibco), 0.7 mg/mL DNase I (11,284,932,001; Roche), and 0.7 mg/mL Dispase II (D4693; Sigma)). Then this tube was placed on a rotating plate for 30 min in a dry, non-gassed incubator maintained at 37℃. The contents were vortexed for 10 s and filtered through a 40 μm strainer into a new tube. Lungs with induced fibrosis from 5 mice were placed into a Petri dish (93,060; TPP) and chopped into small pieces using a scalpel. The lung pieces were incubated in a digestion solution (containing 2 mg/mL collagenase IV and 0.1 mg/mL DNase I) for 1 h. Subsequently, 10% DMEM media was added, followed by centrifugation at 2,000 rpm for 5 min. The contents were filtered through a 40 μm strainer into a new tube. Cells were resuspended in 4 mL 40% Percoll in 5% RPMI media. The cell suspension in 40% Percoll was layered over 4 mL 80% Percoll in a new tube and centrifuged at 2,000 rpm for 30 min at 4℃.

Flow cytometry

For effector T cell analysis, cells were initially stained with PerCP-Cy5.5-conjugated CD4 (45–0042–82; eBioscience) at 4℃ for 30 min and then washed with buffer solution. To stain intracellular cytokines, cells were fixed and permeabilized with a kit (554,715; BD) at 4℃ for 30 min. After washing with buffer solution, the cells were stained with PE-conjugated IL-4 (554,435; BD) and FITC-conjugated IL-17 (11–7177–81; eBioscience) at 4℃ for 30 min. After a final wash with buffer solution, the cells were analyzed by flow cytometry (CytoFLEX; Beckman Coulter).

Histological assessmentHematoxylin and eosin staining and masson’s trichrome staining

To assess the severity of fibrosis and inflammation, the thickness of the skin dermis was measured after sacrificing the animals. Isolated tissues were fixed in 10% (v/v) neutral-buffered formalin (HT501320; Sigma) and embedded in paraffin. µThe Sect. 5 μm thick were deparaffinized using xylene and dehydrated using an alcohol gradient. The sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome.

To stain H&E, sections were stained hematoxylin (S2-5; yd-diagnostics) for 5 min. Wash to tap water 3 times. And then for bluing except to nuclear, sections were incubated 1% HCL for 30s. Wash in tap water. For staining cytosol, the sections were stained eosin (#32,002; MUTO). Wash in tap water. The sections were dehydrated using an alcohol gradient and mounting (S3023; DAKO).

To Masson’s trichrome stain (25088-1; Polysciences), For formalin fixed tissue, re-fix in Bouin’s solution (BOU-OT-L; BIOGNOST) at 60℃ for 1 h. wash in flowing water for 5 min. Stain in weigert’s iron hematoxylin for 10 min. Wash in running water for 5 min. Rinse in distilled water. Stain in biecrich scarlet-acid fuchsin solution for 5 min. Rinse in distilled water. Differenctiated in phosphomolybdic-phosphotungstic acid solution for 10 min. Discard solution, stain in aniline blue for 5 min. Rinse in a tap water. Differentiated in 1% acetic acid for 1 min. Wash in tap water. The sections were dehydrated using an alcohol gradient and mounting.

Immunohistochemistry

After deparaffinization, tissue Sect. 5 μm thick underwent antigen retrieval using proteinase K (S3020; Dako) in Tris-EDTA buffer (93,283; Sigma). Then the sections were incubated with 3% hydrogen peroxide (H0300; Samchun Chemical) in methyl alcohol (59; Duksan) to block endogenous peroxidase activity. Sections were stained with reagents (S0809, K4001, K400311–2, K346811–2, and CS703; Dako) according to the manufacturer’s instructions. Primary antibodies against alpha-SMA (ab7817; Abcam, 1:5,000), Col1a1 (PA5–29,569; Invitrogen, 1:800), IL-6 (NB600–1131; NOVUS, 1:600), IL-4 (PA5-25165; Invitrogen, 1:200), IL-17 A (ab79056; Abcam, 1:600), TNF-alpha (Ab6671; Abcam, 1:150), and IL-1 beta (NB600–633; NOVUS, 1:400) were used for staining.

Immunofluorescence

Tissue Sect. 5 μm thick were deparaffinized and underwent antigen retrieval using proteinase K (S3020; Dako) in Tris-EDTA buffer (93,283; Sigma). The sections were incubated with 3% hydrogen peroxide (H0300; Samchun Chemical) in methyl alcohol (59; Duksan) to block endogenous peroxidase activity. Sections were stained with primary antibodies, including anti-CD4 (14–9766–82; eBioscience), anti-IL-4 conjugated with PE (12–7041–82; eBioscience), and anti-IL-17 A conjugated with FITC (11–7177–81; eBioscience), in 10% normal goat serum in PBS. In addition, sections were stained with anti-mouse IgG secondary antibody conjugated with APC (A-865; Thermo). Nuclei were stained with DAPI (D35271; Invitrogen) in PBS. All stained sections were imaged using a confocal laser scanning microscope (LSM700).

Statistical analysis

To analyze histology in skin and lung, the positive area of DAB-stained were assessed in IHC staining using ImageJ software. In confocal staining, the number of fluorescent cells was counted.

The results are presented as the mean ± SEM. Statistical analysis between two groups was performed using an unpaired t-test. Statistical significance was determined using a p-value cut-off of 0.05. GraphPad Prism version 9.50 (GraphPad Software, San Diego, California, USA) for Windows was used to plot the data.