The pET28a plasmid (Addgene), which contains the T7 promoter and terminator, was used to obtain a Vim-expressing vector. The gene encoding mouse Vim (GenBank accession number: NP_035831.2) but with modified codon usage was optimized for expression in Escherichia coli (Cosmogenetech). The insert was amplified using a primer set that added a 6×His tag sequence immediately upstream of the Vim gene, allowing the expressed protein to be fused with a 6×His tag at the N-terminus. Subsequently, both the plasmid and the insert were cleaved with NcoI and XhoI restriction enzymes and then ligated together. The expression vector was transformed into BL21 (DE3) competent E. coli (Enzynomics, Chungcheongnam-do, Korea), which then was cultured at 37°C until the optical density at 600 nm reached 0.6–0.8. Following the addition of 0.2 mM isopropyl β-D-1-thiogalactopyranoside to induce Vim expression, the culture was incubated for an additional 4 h. Subsequently, the cells were harvested, resuspended in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5), and lysed via sonication. Then the total lysate was centrifuged to separate insoluble and soluble proteins in the pellet and supernatant, respectively. The pellet was resuspended in the original volume of the total lysate. Vim expression was confirmed by SDS-PAGE, followed by Western blotting using the primary antibody anti-6×His tag (MA1–135) (Thermo Fisher Scientific, MA, USA) and the secondary antibody anti-rabbit IgG-HRP (A90–116P) (Bethyl Laboratories, TX, USA). Vim was isolated by resuspending the harvested cells in wash buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 6 M urea, pH 7.5) and then purifying the protein using Ni-NTA resin (Qiagen, California, USA) followed by dialysis against 5 mM HCl. The purity of the obtained Vim was analyzed using the ‘plot lanes’ option in ImageJ software.

Six-week-old male BALB/c mice (Orient Bio; n=12, housed 5 per cage) were maintained in polysulfon Individually ventilated cage in a specific-pathogen-free system. All animal experimental procedures were reviewed and approved by Department of Laboratory Animals, Institutional Animal Care and Use Committee (IACUC) of the School of Medicine, the Catholic University of Korea (IACUC No. 2023–0030–05).

To induce SSc, the mice were initially immunized with 50 µg Vim protein (Department of Microbiology, The Catholic University of Korea) and complete Freund’s adjuvant (CFA; Chondrex) via intradermal injection at two sites on their backs. To provide continuous analgesia after intradermal injections, mice were first anesthetized under inhalational isoflurane (3–4% induction, 1.5–2% maintenance via nose cone). Immediately after anesthesia, a single subcutaneous injection of tramadol (25 mg/kg) was administered. Following recovery, acetaminophen was supplied in the drinking water at a concentration of 0.2 mg/mL (26), beginning one hour after tramadol injection and continued for 24 hours (27). All veterinary treatments were performed under the guidance of the attending veterinarian. Subsequently, the mice received daily subcutaneous injections of 50 µg BLM (HY-17565A, MedChemExpress) dissolved in 100 µL phosphate-buffered saline (CBP3071, Dynebio) for 3 weeks. The BLM was injected subcutaneously near the neck region. Two weeks after the first immunization, the mice were immunized again with Vim and incomplete Freund’s adjuvant (Chondrex) for boosting. Control mice consisted of those injected with BLM, either alone or in combination with CFA.

Five weeks after the first immunization, blood was collected from mice by retro-orbital venous plexus puncture at the time of sacrifice, and spleens were harvested aseptically. Red blood cells (RBCs) in the blood samples were lysed using ACK lysis buffer according to the manufacturer’s protocol, and the remaining leukocytes were collected for further analysis. Spleen were gently dissociated using sterilized glass slides with frosted ends to create a single-cell suspension. The cell suspension was then treated with ACK lysis buffer to remove residual RBCs and filtered through a 40 μm cell strainer to obtain a single-cell suspension of splenocytes. The cells were washed and resuspended in complete RPMI 1640 medium containing 5% FBS. Cells were then stained with peridinin-chlorophyll protein (PerCP)-conjugated anti-mouse CD4 antibody (Invitrogen) for 30 min at 4°C. Then the cells were permeabilized, fixed using the CytoPerm/CytoFix kit (BD Biosciences) in accordance with the manufacturer’s protocol, and stained with phycoerythrin (PE)-conjugated anti-mouse IL-4 (BD Pharmingen) and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IL-17 (Invitrogen) antibodies. All samples were analyzed using a cytoFLEX flow cytometer (Beckman Coulter). The flow cytometry data were processed using FlowJo™ software.

Helper T cells were detected by staining spleen and lung tissue sections 7 µm thick with FITC-conjugated anti-mouse CD4 (BD Biosciences), Alexa Fluor 647-conjugated anti-mouse IL-4 (BD Biosciences) and PE-conjugated anti-mouse IL-17 (BD Biosciences) antibodies. Anti-Vim antibody-producing B cells were identified by staining splenic tissue sections 5 µm thick with labeled Vim, prepared using the Lightning‐Link FITC antibody labeling kit (Odenton) following the manufacturer’s instructions (Innova Bioscience). The tissue sections were also stained with PE-conjugated anti-mouse CD19 (BD Biosciences) and Alexa Fluor 647-conjugated anti-mouse B220 (BD Biosciences) antibodies. The stained sections were analyzed using a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss).

Vim-specific total IgG and IgG1 levels in serum were measured via ELISA. Microtiter plates were coated with Vim (5 μg/mL) and incubated at 4°C overnight, followed by a blocking step for 30 min at room temperature. Each serum sample diluted 1:500 in Tris buffered saline (pH 8.0) was added to the microtiter plates for 1 h, after which time the plates were washed three times. Vim-specific total IgG or IgG1 were detected colorimetrically using anti-mouse IgG Fc-HRP for color development. The absorbance at 450 nm was measured with an ELISA microplate reader (Molecular Devices Inc.) and the optical density was determined.

Skin and lung tissues were fixed in 10% (v/v) neutral-buffered formalin and embedded in paraffin. Tissue sections (5 µm thick) were stained with hematoxylin and eosin (H&E) and examined using a photomicroscope (Olympus, Tokyo, Japan). Dermal thickness was measured using the IMT iSolution Lite software v10 (IMT iSolution Inc.). Areas of collagen deposition in Masson’s trichrome (MT; Polysciences, Inc.) stained sections were identified using a photomicroscope (Olympus).

Dermal thickness and lung fibrosis severity were scored as described previously (28, 29). Dermal thickness was measured from the top of the granular layer to the junction between the dermis and the subcutaneous fat. Lung fibrosis severity was evaluated using the modified Ashcroft scoring system on H&E- and MT-stained sections, as previously described (30). The fibrosis score was obtained by analyzing skin and lung cross-sections from H&E- and MT-stained slides. Immunohistochemistry was performed using a Vectastain ABC kit (Vector Laboratories) in sections incubated with specific antibodies (anti-smooth muscle antibody [αSMA; Abcam], anti-cell surface Vim [CSV; Abnova], anti-Col1A1, anit-IL-4, and anti- IL-17 [all from Invitrogen]) overnight at 4°C, then with biotinylated secondary antibody for 30 min at RT, followed by streptavidin-peroxidase complex for 30 min at RT. The final color product was developed using diaminobenzidine (DAKO) as the chromogen. Slides were prepared for each mouse (n=12). Immunoassayed tissue sections were examined under a photomicroscope (Olympus). The number of positive cells in one high-power field (magnification 400×) was determined using Image J software, and average cell numbers were calculated for three randomly selected tissue sections.

The statistical analyses were performed using Prism, version 10 for Mac (GraphPad). Multiple groups were compared using one-way ANOVA, two-way ANOVA, or the Kruskal-Wallis test; for groups with significant differences, a Bonferroni or Dunn’s post hoc test was used to examine the differences. The numerical data of two groups were compared using a nonparametric Mann–Whitney U test (two-tailed). The results are presented as the mean ± standard deviation.

The mouse-Vim-expressing vector pET28a-Vim was generated to produce Vim conjugated with a 6×His tag at the N-terminus (Figures 1A, B). Insertion of the Vim gene was confirmed by amplifying the region between the T7 promoter and the plasmid terminator using specific primers and then verifying the size of the amplicon (Figures 1C, D). As most of the expressed Vim was insoluble, it was solubilized using 6 M urea prior to its purification on a Ni-NTA column. A purity of 94.7% was achieved, and the concentration of the purified Vim protein was 27.7 mg/L.

The conventional mouse model of SSc obtained by BLM injection is pathologically similar to SSc in humans but it does not completely mimic the disease from an immune cell perspective. Thus, a novel mouse model was established by administering homogenized Vim protein and CFA to the mice during their injection with BLM. As in the conventional model, BLM was injected subcutaneously daily for 21 days. A second group of mice was injected with BLM and a third group with BLM and CFA. In the Vim-treated mice, homogenized Vim protein was mixed with CFA at a 1:1 ratio and administered twice through intradermal injections separated by 2 weeks (Figures 2A, B). Morphological examination of the dorsal skin in scleroderma mice from vimentin immunized mice revealed changes at days 0 and 12. The white spots visible on the dorsal surface correspond to intradermal injection sites of vimentin in a CFA-containing solution (Figure 2B). All mice were euthanized 35 days after the first BLM injection, and the frequency of helper T cell subsets was examined in the blood and spleen. Both Th2 and Th17 cell frequencies (as a percentage of CD4+ T cells) increased remarkably in BLM-treated Vim-injected mice (BC+Vim; BCV, Figures 2C, D). An analysis of the immune cells in skin and lung tissues showed increases in infiltrated Th2 and Th17 cells in BCV mice (Figure 2E). These results demonstrated that the combined injection of BLM and Vim+CFA increases Th2 and Th17 cell numbers, not only systemically but also within SSc lesions.

To investigate whether the administration of BLM and Vim activates B cells autoreactive for Vim, autoreactive antibody titers and the number of spleen B cells were determined as follows: FITC-tagged Vim protein was reacted with spleen tissues from the three groups of mice. The number of B cells and the frequency of autoreactive B cells in the spleen were assessed by staining with anti-B220 and anti-CD19 antibodies. The results showed increases in the number of B cells and in the number of Vim-positive cells in the spleens of the BCV group (Figure 3A). The serum concentration of anti-Vim antibodies was measured to determine whether these B cells produce and secrete autoantibodies against Vim (Figure 3B). The concentrations of total IgG and IgG1 against Vim were significantly higher in BCV mice. These results demonstrated that, in the novel SSc animal model, Vim autoantibodies are produced.

The pathological changes in the skin tissues of the mice were investigated by H&E, MT, and immunohistochemistry staining. In the H&E- and MT-stained sections, greater skin thickness and larger areas of collagen deposition were observed in the BCV group, indicating more severe skin sclerosis in these mice. In addition, the expression levels of the fibrosis markers αSMA and Col1A1 were significantly higher in the BCV group than in the BLM and BLM+CFA (BC) mice (Figure 4). Thus, immunization with Vim protein provides an animal model of SSc that more closely resembles SSc in humans than the model based on BLM alone.

Similar to human SSc, skin and lung sclerosis were observed in the mouse models. In Vim-treated mice, lung tissue was examined to determine whether Vim immunization has further effects on lung fibrosis. Pathological symptoms were evaluated based on H&E, MT, and immunohistochemistry staining. In the lung tissues of BCV group, the lung histological score, collagen deposition areas, and expression levels of αSMA and Col1A1 were higher than in the control mice (Figure 5). These results demonstrate the validity of our novel mouse model in studies of lung fibrosis, such as occurs in SSc.

Whether the newly established mouse model of SSc was accompanied by immunological changes was investigated by immunohistochemically staining skin and lung tissues for pro-inflammatory cytokines and CSV. The levels of the pro-inflammatory cytokines IL-4 and IL-17 in skin tissue were significantly higher in BCV mice than in BLM or BC mice (Figures 6A, B). By contrast, their expression did not significantly differ between the latter two groups. CSV expression was also significantly increased in both the skin and the lung tissues of BCV mice. These results imply the presence of activated immune cells in BCV mice, thus suggesting that this novel model is a more effective animal model for studies of the autoimmune features of SSc.