Increased Collagen V Triggers Inflammation and Fibrosis in Early Pulmonary Disease of Systemic Sclerosis: Insights from the IMU-COLV Model

Increased Collagen V Triggers Inflammation and Fibrosis in Early Pulmonary Disease of Systemic Sclerosis: Insights from the IMU-COLV Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Increased Collagen V Triggers Inflammation and Fibrosis in Early Pulmonary Disease of Systemic Sclerosis: Insights from the IMU-COLV Model Vitória Elias Contini, Zelita Aparecida de Jesus Queiroz, Sérgio Catanozi, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6814249/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Understanding the early stages of pulmonary disease pathogenesis in systemic sclerosis (SSc) is crucial for developing therapeutic strategies to mitigate pulmonary fibrosis progression. Our aim is to evaluate the early stages of pulmonary fibrosis in the IMU-COLV model. This study used the SSc IMU-COLV model in female C57BL/6 mice immunized with collagen V (COLV) emulsified in Freund's adjuvant. Mice were categorized into groups based on immunization duration (15, 30, and 45 days) to assess lymphocyte populations, Factor VIII, VEGF, caspase-3, α-AML, and collagen expression through immunostaining and image analysis. Total collagen content was quantified using 4-hydroxyproline, while α-AML, TGF-β1, and collagen gene expressions were evaluated via RT-qPCR. At day 15, significant increases in CD3+, CD4+, CD8+, and CD20 + lymphocytes, Factor VIII, VEGF, and α-AML were observed, alongside enhanced COLV and Col5α1/Col5α2 gene expressions. Inflammation decreased at day 30; however, by day 45, a nonspecific interstitial pneumonia pattern emerged, with intrapulmonary artery thickening, increased Caspase-3, α-AML, collagen types I and III, Col1α1 and Col3α1 genes, and total collagen levels. Furthermore, vimentin, α-AML, and TGF-β1 gene expressions were higher in the 45-day group. The IMU-COLV model exhibits an early inflammatory phase at day 15, with COLV deposition, leading to pulmonary remodeling and fibrosis at days 30 and 45, mediated by TGF-β1 activation. The increased COLV expression and associated inflammatory infiltrates observed in this model suggest that COLV contributes to pulmonary fibrosis progression. Health sciences/Medical research Health sciences/Pathogenesis Pulmonary Fibrosis Collagen V Experimental model of SSc Systemic Sclerosis lung Collagen Fibrillogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Systemic sclerosis (SSc) is a complex autoimmune disease characterized by a combination of vascular dysfunction, fibrosis, and immune system dysregulation. This multifactorial nature of SSc leads to diverse symptoms and complications, making its management and treatment challenging [ 1 , 2 ]. The most severe SSc manifestations are fibrotic and vascular pulmonary complications, particularly interstitial lung disease (ILD). ILD results in progressive lung-tissue scarring, which impairs lung function and can lead to respiratory failure. In addition, vascular complications such as pulmonary arterial hypertension further worsen the disease prognosis. Thus, early diagnosis and effective management strategies aimed at mitigating these pulmonary manifestations are critical for improving patient outcomes and quality of life [ 1 – 3 ]. The exact initiating events of SSc remain unclear, complicating the understanding of the underlying mechanisms of the disease. Consequently, animal models have proven invaluable in advancing our knowledge of SSc pathology [ 4 , 5 ]. In previous studies, we demonstrated that the IMU-COLV mouse model, induced by immunization with collagen V (COLV), mirrors key human SSc features, including cutaneous, vascular, and pulmonary remodeling. This model has therefore become an essential preclinical tool for investigating the mechanisms of disease progression and exploring potential therapeutic approaches [ 6 ]. Moreover, the IMU-COLV model suggests that COLV is a critical factor in SSc pathogenesis. In the pulmonary tissue, COLV is located in the interstitium and capillary basement membranes and is surrounded by vascular smooth cells [ 7 ]. The COL V isoform [α1(V)2, α2(V)] contains a fibrillary portion embedded within the heterotypic collagen I/III fibrils. The NH3-terminal region is project through the fibril surface hinders further addition of new molecules, thereby influencing subsequent growth of fibril diameter [ 8 ]. Thus, COLV plays a role in the organization, growth and dimensioning of the diameter of heterotypic fibrils, and is currently considered important in nucleating collagen I-containing fibrils in vivo [ 9 ]. In addition, COLV can modify the stiffness of the extracellular matrix (ECM), which in turn affects basic cellular functions. It can play a role in modifying the cell phenotypes during organogenesis and ECM remodeling when soft tissues are prevalent [ 10 ]. Additionally, COLV is considered a sequestered antigen, since its immunogenic properties allow it to be recognized as an autoantigen in chronic pathological conditions when exposed to the immune system [ 11 – 19 ]. Recent studies have revealed that autoimmunity against COLV primarily targets the α1(V) chain [ 20 – 23 ], with anti-COLV antibodies more frequently detected in patients with early SSc, particularly those directed against the α1(V) chain peptides [ 24 , 25 ]. Furthermore, studies have shown that patients with SSc exhibit increased COLV, COL5A2 gene, and α2(V) chain expression in the skin and lungs, especially during the early stages of the disease [ 26 – 30 ]. Notably, abnormal COLV deposition in the skin of patients with SSc is associated with skin thickening and disease activity [ 30 ]. As previously described, the IMU-COLV model induces pulmonary alterations, including increased tissue elastance and airway resistance, along with a nonspecific interstitial pneumonia histologic pattern. These changes are accompanied by thickening of small and medium intrapulmonary arteries, fibrotic septal thickening, and elevated COLV expression [ 6 ]. However, the early stages of pulmonary fibrosis in this model require further investigation, and understanding these early events could provide crucial insights into the mechanisms driving SSc-related pulmonary fibrosis. We hypothesize that COLV is crucial in the establishment and progression of pulmonary fibrosis in SSc. To test this hypothesis, this study aimed to assess the early stages of pulmonary fibrosis in the IMU-COLV model, focusing on histological, molecular, and immunological changes during pulmonary fibrosis onset. Given that pulmonary manifestations are the primary cause of morbidity and mortality in patients with SSc, understanding the early events in pulmonary fibrosis development could offer new therapeutic strategies targeting these critical processes. Materials and methods Experimental model Female C57BL/6 mice (n = 54), aged 6–7 weeks, were obtained from the animal facility of our institution. The mice were housed in specific pathogen-free conditions with free access to food and fresh water in a temperature-controlled room (22–24°C) and maintained on a 12-h light/dark cycle. All experimental procedures were approved by The Committee on Ethical Use of Laboratory Animals of the Faculty of Medicine at the University of São Paulo (CEUA-protocol number: 1543/2020; 1416/2019. To induce experimental SSc, mice (n = 30) were subcutaneously immunized with 150 µg of human placental COLV (Col V; Sigma) dissolved in 10 mM acetic acid and emulsified with an equal volume of complete Freund's adjuvant. At day 20 post-immunization, two booster doses of 150 µg/200 µL COLV mixed with incomplete Freund's adjuvant were administered via intramuscular injection at 15-day intervals (IMU-COLV) (Fig. 1 ). One hour before immunization, tramadol hydrochloride (40 mg/kg body weight) was administered subcutaneously as preventive analgesia. The control group (CT; n = 24) was inoculated with 10 mM acetic acid mixed with complete or incomplete Freund's adjuvant, following the same immunization protocol. To investigate early pulmonary changes associated with experimental SSc, animals were euthanized at 15 (n = 10), 30 (n = 10), and 45 (n = 10) days after the initial COLV immunization. Before euthanasia, the mice were anesthetized with an intraperitoneal injection of ketamine hydrochloride (100 mg/kg body weight) and xylazine (10 mg/kg body weight), followed by euthanasia through nuchal dislocation. Preparation Lung tissue samples were collected and fixed in 10% buffered formalin for morphometric and immunohistochemical analysis. Additional lung fragments were stored at -80°C for subsequent molecular analysis. Lung Morphological Analysis For histological examination, lung samples were fixed in 10% buffered formalin for 24 h and subsequently embedded in paraffin. Sections of 3–4 µM thickness were cut and stained with hematoxylin and eosin (H&E) for general histological assessment, and picrosirius red for collagen fiber detection. Immunofluorescence for Collagen For collagen immunostaining (Col I, III, and V), 4 µm lung tissue sections were adhered to glass slides pre-coated with 3-aminopropyltriethoxy silane (Sigma, St. Louis, MO, USA). Antigen retrieval was performed by incubating the slides with bovine pepsin (8 mg/mL in 0.5 N acetic acid) (Sigma) for 30 min at 37°C. After several washes with phosphate-buffered saline (PBS), the slides were blocked with 5% bovine serum albumin (BSA) diluted in PBS (pH 7.0). Subsequently, the slides were incubated overnight at 4°C with rabbit polyclonal antibodies: anti-Col I (1:1,200; Rockland), anti-Col III (1:1,400; Rockland), and anti-Col V (1:1,000). After washing with PBS containing 0.05% Tween 20, the slides were incubated for 1 h at 25°C with goat anti-rabbit IgG Alexa Fluor 488 (Invitrogen, Life Technologies) at a 1:200 dilution in PBS containing 0.006% Evans blue. The slides were mounted with buffered glycerin and analyzed under a fluorescence microscope (Olympus BX-51, Olympus Co, Tokyo, Japan). Immunohistochemistry For immunohistochemical analysis, 3–4 µm lung tissue sections were deparaffinized and incubated in a 0.3% hydrogen peroxide solution for 5 min to inhibit endogenous peroxidase activity. Afterward, sections underwent a cycle of four 5-min washes. The following primary antibodies were used for the immunostaining reactions: smooth muscle α-actin (α-SMA, mouse monoclonal, 1:50; Santa Cruz Biotechnology Inc., USA), vascular endothelial growth factor (VEGF, 1:400; Santa Cruz Biotechnology Inc., USA), Factor VIII (1:1200; Santa Cruz Biotechnology Inc., USA), Caspase-3 (1:4,000; Novus Biologicals, USA), CD3 (1:1,000; Santa Cruz Biotechnology Inc., USA), CD4 (1:1,000; Santa Cruz Biotechnology Inc., USA), CD8 (1:1,200; Santa Cruz Biotechnology Inc., USA), and CD20 (1:100; Santa Cruz Biotechnology Inc., USA). Antigen retrieval was performed using either bovine pepsin (4 mg/mL in 0.01 N glycine buffer, Sigma) for 30 min at 37°C, or citrate buffer (pH 6.0) at high temperature (125°C for 1 min) in a pressure cooker (Pascal). Subsequently, the sections were incubated with the primary antibodies overnight at 4°C. Immunoreactions were detected using a biotin-streptavidin-peroxidase kit (Vector) per manufacturer instructions. The chromogen used was 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO), and the sections were counterstained with Harris hematoxylin (Merck, Darmstadt, Germany). Morphometric Analysis Collagen fibers of types I, III, and V were quantified in the lung parenchyma using Image-Pro Plus 6.0 software [ 31 ]. In addition, two blinded observers analyzed 10 randomly selected microscopic fields at 400x magnification. Collagen fiber thresholds were set on all slides after enhancing the contrast, ensuring that the fibers were easily distinguishable as green bands. Collagen fiber density was calculated as the ratio of the measured collagen fibers to the total tissue area analyzed, with the result expressed as a percentage. For immunostained cell analysis, 10 random lung tissue fields were evaluated at 1,000x magnification using Image-Pro Plus 6.0 software. α-SMA, VEGF, Factor VIII, Caspase-3, CD3, CD4, CD8, and CD20 expressions were determined by counting the number of positive cells and expressed as the proportion of total cells in the field analyzed. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) The total RNA was isolated according to a standard Trizol® (Invitrogen, Life Techologies Co., Carlsbad, CA, USA) RNA isolation protocol. Col1a1 (Col I α1 chain), Col3a1 (Col III α1 chain), Col5a1 (Col V α1 chain), Col5a2 (Col V α2 chain), TGF-β, Vimentin, and α-SMA expressions were assessed via qRT-PCR. For cDNA synthesis, total RNA from each sample was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). qRT-PCR reactions were prepared using the Platinum SYBR Green qPCR SuperMix-UDG Kit (Invitrogen, Life Technologies, USA). Amplification and cDNA production were performed using a Step One thermal cycler (Applied Biosystems), with 1,000 ng of total RNA per sample. Relative gene expression was calculated using the 2-ΔΔCT method, with β-2 microglobulin used as the housekeeping gene for normalization. Collagen Quantification by Measuring 4-Hydroxyproline Total lung collagen deposition was quantified by measuring the 4-hydroxyproline content, as previously described with modifications [ 24 ]. Briefly, lung samples were freeze-dried (Edwards, Modulyo), weighed, and subsequently hydrolyzed in 6 N HCl for 22 h at 100°C. The hydroxyproline content was determined spectrophotometrically by measuring absorbance at 560 nm. The results were expressed as nanograms of 4-hydroxyproline per milligram of protein [ 24 ]. Anti-nuclear Antibody (ANA) Detection Sera obtained from IMU-COLV and CT groups of mice from 15, 30, and 45 days were tested for ANAs using slides with HEp-2 cells (Nova-Lite HEp-2 ANA Kit), per manufacturer instructions. Slides were incubated for 30 min at room temperature with sera diluted at 1:80 in PBS with Tween20. After a washing cycle with PBS/Tween20, the slides were incubated for 30 min with conjugated goat anti-mouse IgG antibody ALEXA FLUOR 488 (INVITROGEN) (1:200). Subsequently, slides were counterstained with 0.006% Evans blue, mounted on coverslips with buffered glycerin and examined under a fluorescence microscope (Olympus BX51). Statistical Analysis Data were analyzed for normality of distribution and categorized as parametric or non-parametric accordingly, using GraphPad Prism version 8.0.2 (CA, USA). Results for each variable are presented as means ± standard deviation. One-way analysis of variance was used to compare the means between groups, with Tukey or Sidak’s post-test applied for normally distributed variables. The Kruskal–Wallis test was used for non-normally distributed variables, followed by the Dunn test. Results Temporal Dynamics of Inflammation and Collagen Deposition in the Pulmonary Fibrotic Process of the IMU-COLV Model Fifteen days post-immunization with COLV, animals in the IMU-COLV15 group exhibited a prominent inflammatory cell infiltrate around vessels and bronchi (Fig. 2 A). This infiltrate expanded around the alveolar septa and vessels compared to the CT group (Fig. 2 D), with notable presence around small vessels and bronchioles in the pulmonary interstitium (Fig. 2 E vs. Figure 2 H). At 30 days post-immunization (IMU-COLV30), the inflammatory infiltrate decreased, and collagen deposition began in the perivascular region (Fig. 2 B). This process was more widespread, with thick collagen fibers in the peribronchial region, a feature absents in the CT group (Fig. 2 D). In Fig. 2 F, intense collagen deposition in the peribronchovascular region extending into the lung parenchyma was visible in the IMU-COLV30 group, which was not observed in the CT group (Fig. 2 H). At 45 days post-immunization (IMU-COLV45), lung parenchyma, vessel, and bronchi remodeling wer evident (Fig. 2 C), with almost no inflammatory cells present, replaced by substantial collagen deposition in the vessel wall and interstitium (Fig. 2 G). Lung interstitial destruction was more pronounced in the IMU-COLV45 group than in the CT group (Figs. 2 D and H). Quantification of immune cell populations confirmed increased TCD3 + lymphocytes in the IMU-COLV15 group compared with both IMU-COLV30 (12.65 ± 4.78 vs. 8.47 ± 2.14, p = 0.0058) and IMU-COLV45 groups (12.65 ± 4.78 vs. 8.39 ± 1.68, p = 0.0047) (Fig. 3 E). TCD4 + lymphocytes were also significantly higher in the IMU-COLV15 group than in the IMU-COLV30 (33.99 ± 2.01 vs. 23.38 ± 2.03, p = 0.0001) and IMU-COLV45 groups (33.99 ± 2.01 vs. 23.77 ± 0.74, p = 0.0001) (Fig. 3 J). Similarly, TCD8 + lymphocytes were elevated in the IMU-COLV15 group compared with both the IMU-COLV30 (39.40 ± 2.43 vs. 22.87 ± 2.09, p = 0.0001) and IMU-COLV45 groups (39.40 ± 2.43 vs. 23.07 ± 2.47, p = 0.0001) (Fig. 3 O). Finally, the number of CD20 + B lymphocytes was significantly higher in the IMU-COLV15 group than in the IMU-COLV30 (34.14 ± 3.81 vs. 27.50 ± 3.22, p = 0.0011) and IMU-COLV45 groups (34.14 ± 3.81 vs. 28.24 ± 1.64, p = 0.0044) (Fig. 3 T). Temporal Changes in Vascular Reactivity and Endothelial Markers in the IMU-COLV Model Immunostaining for Factor VIII + endothelial cells revealed intense expression, particularly in the perivascular region of the IMU-COLV15 group (Fig. 4 A), in contrast to the lower expression observed in the IMU-COLV30, IMU-COLV45, and CT groups (Figs. 4 B, C, and D). Quantitative analysis confirmed a significant increase in Factor VIII + expression in the IMU-COLV15 group compared with the IMU-COLV30 (13.87 ± 0.426 vs. 5.16 ± 0.0983, p = 0.0001) and IMU-COLV45 groups (13.87 ± 0.426 vs. 4.99 ± 0.90, p = 0.0001) (Fig. 4 E). VEGF + endothelial cells were also increased in the IMU-COLV15 group compared with the IMU-COLV30 and IMU-COLV45 groups, as confirmed by quantitative analysis (IMU-COLV15 vs. IMU-COLV30: 3.81 ± 0.78 vs. 2.64 ± 0.57, p = 0.0008; IMU-COLV15 vs. IMU-COLV45: 3.81 ± 0.78 vs. 2.45 ± 0.73, p = 0.0001) (Fig. 4 J). α-AML + myofibroblast immunostaining revealed a significant increase in the IMU-COLV45 group (Fig. 4 F-H) compared with the IMU-COLV15 (29.45 ± 5.39 vs. 23.43 ± 2.52, p = 0.0051) and IMU-COLV30 groups (29.45 ± 5.39 vs. 24.54 ± 2.31, p = 0.0421) (Fig. 4 O). Finally, Caspase-3 + cells in the endothelial cells of the pulmonary microvasculature were significantly increased in the IMU-COLV45 group compared with the IMU-COLV30 group (13.60 ± 2.76 vs. 9.89 ± 1.79, p = 0.0161). Histological Characterization of Collagen Deposition and Fibrosis Progression in the IMU-COLV Model Histological analysis of collagen fibers in the lung tissue was performed using Masson’s trichrome and picrosirius red staining, confirming the morphological patterns observed in H&E staining (Fig. 2 A-H). In the IMU-COLV15 group (Fig. 5 A and E), collagen fibers were predominantly distributed around the vessels and bronchi, consistent with the typical, organized pattern observed in the CT group (Fig. 5 D and H). This arrangement suggests minimal disruption of normal lung tissue structure at this early stage. In contrast, the IMU-COLV30 group exhibited a more heterogeneous histological pattern, characterized by the presence of both thinner and thicker collagen fibers surrounding the perivascular region (Fig. 5 B and F). This variation in collagen fiber thickness suggests the onset of lung tissue remodeling, a hallmark of the early stages of fibrosis. In the IMU-COLV45 group (Fig. 5 C and G), collagen deposition had significantly intensified, leading to a disorganized fibrotic pattern, particularly in the peribronchial and perivascular regions, where intense blue staining indicated dense collagen accumulation compared with the CT group (Fig. 5 D and H). This progressive collagen fiber disorganization suggests progressive fibrosis. These histological observations were further corroborated by biochemical quantification of 4-hydroxyproline, a collagen content marker. The analysis revealed a significant increase in total collagen in the lung tissue of the IMU-COLV45 group (5.203 ± 1.350) compared with the IMU-COLV15 (3.773 ± 1.006, p = 0.0091) and IMU-COLV30 groups (3.213 ± 0.4715, p = 0.0001) (Fig. 5 I). This data provides quantitative evidence of increased collagen deposition in the progressive stages of fibrosis, supporting the histological findings. Spatiotemporal Distribution and Collagen Type Composition in Fibrosis Progression in the IMU-COLV Model Regarding collagen type composition in the lung tissue, Fig. 6 A illustrates a subtle type I collagen (Col I) expression in the IMU-COLV15 group, primarily surrounding the vessels and bronchi. In the IMU-COLV30 group (Fig. 6 B), Col I deposition increased, particularly around the bronchi and blood vessel walls, compared with the CT group (Fig. 6 D). In the IMU-COLV45 group (Fig. 6 C), a notable increase in Col I expression was observed, extending throughout the lung tissue. This deposition extended to the alveolar septa and caused vessel wall thickening, indicating more advanced fibrosis than that of the IMU-COLV15 group (Fig. 6 A). Quantitative analysis confirmed these observations, with significant differences between the IMU-COLV45 and IMU-COLV15 groups (39.76 ± 7.18 vs. 20.29 ± 4.66, p = 0.0240), indicating established fibrosis in the former group. For type III collagen (Col III), initial expression at day 15 (Fig. 6 F and I) was confined to the vessels and bronchi, following a pattern similar to that seen in CT animals. In the IMU-COLV30 group (Fig. 6 G), Col III deposition increased around the vessels and bronchi, and extended into the lung parenchyma. By day 45 (Fig. 6 H), Col III expression had expanded throughout the lung interstitium, surpassing the levels observed in both the IMU-COLV15 and CT groups (Fig. 6 F and I). Quantitative analysis confirmed the morphological findings, revealing significant differences between the IMU-COLV30 and IMU-COLV15 groups (31.04 ± 7.20 vs. 21.67 ± 4.41, p = 0.0059), as well as between the IMU-COLV45 and IMU-COLV15 groups (30.53 ± 4.01 vs. 21.67 ± 4.41, p = 0.0107) (Fig. 6 J). Upon analyzing COLV expression, fine fibers were observed around the pulmonary interstitium as well as on the walls of the pulmonary microvasculature and bronchi across the IMU-COLV15, IMU-COLV30, IMU-COLV45, and CT groups (Fig. 6 K-N). These patterns were consistent across all groups, indicating COLV presence in the structural components of the lung tissue. However, quantitative analysis revealed a significant decrease in COLV expression in the IMU-COLV30 and IMU-COLV45 groups compared with the IMU-COLV15 group. Specifically, the IMU-COLV30 group demonstrated a reduction in COLV expression (11.26 ± 1.24 vs. 9.24 ± 1.28, p = 0.0181), as did the IMU-COLV 45-day group (11.26 ± 1.24 vs. 8.65 ± 1.34, p = 0.0031). These findings suggest that the initial increase in COLV observed at day 15 was followed by a significant decrease at progressive stages of fibrosis, as confirmed by the statistical comparison across the groups. Gene Expression Profile of Collagen and Fibrosis-Related Markers in Lung Tissues of the IMU-COLV Model Total mRNA extracted from the lung tissues of both IMU-COLV and CT groups was used to assess mRNA expression for the α1 chains of Col I and Col III, as well as the α1 and α2 chains of COLV using RT-PCR. Analysis of TGF-β gene expression, a key protein involved in SSc pathogenesis, revealed significant upregulation in the IMU-COLV45 group compared with both the IMU-COLV15 (2.46 ± 0.81 vs. 0.93 ± 0.64, p < 0.0001) and IMU-COLV30 groups (2.46 ± 0.81 vs. 1.16 ± 0.84, p = 0.0003) (Fig. 7 A). Similarly, α-AML gene expression showed a significant increase in the IMU-COLV45 group (2.79 ± 0.78 vs. 1.03 ± 0.45, p < 0.0001; 2.79 ± 0.78 vs. 1.43 ± 0.36, p < 0.0001) compared with both the IMU-COLV15 and IMU-COLV30 groups (Fig. 7 B). Vimentin expression followed a similar trend (2.24 ± 0.38 vs. 1.09 ± 0.48, p < 0.0001; 2.24 ± 0.38 vs. 1.41 ± 0.37, p < 0.0001) (Fig. 7 C), indicating a progressive increase in fibrosis-related gene expression as the disease progresses. Regarding collagen gene expression, Col1a1, encoding the α1 chain of collagen I, was significantly upregulated in the IMU-COLV45 group compared with both the IMU-COLV15 (2.98 ± 1.01 vs. 1.65 ± 0.38) and IMU-COLV30 groups (2.98 ± 1.01 vs. 1.66 ± 0.31) (Fig. 7 D). Similarly, the expression of Col3a1, which encodes the α1 chain of collagen III, was significantly increased in the IMU-COLV45 group compared with the IMU-COLV15 (3.26 ± 0.76 vs. 1.58 ± 0.36) and IMU-COLV30 groups (3.26 ± 0.76 vs. 1.38 ± 0.13) (Fig. 7 E), further supporting the notion of ongoing collagen deposition in the later stages of fibrosis. In contrast, the expression of Col5a1, which encodes the α1 chain of COLV, showed a significant decrease in the IMU-COLV30 and IMU-COLV45 groups compared with the IMU-COLV15 group (2.78 ± 0.76 vs. 1.29 ± 0.48, p < 0.0001; 2.78 ± 0.76 vs. 1.86 ± 0.30, p = 0.0093) (Fig. 7 F). Similarly, the expression of Col5a2, which encodes the α2 chain of COLV, was also significantly lower in the IMU-COLV30 and IMU-COLV45 groups than in the IMU-COLV15 group (2.94 ± 0.88 vs. 1.02 ± 0.73, p < 0.0001; 2.94 ± 0.88 vs. 1.81 ± 0.52, p = 0.0065) (Fig. 7 G). These findings highlight distinct changes in collagen gene expression during fibrosis progression in the IMU-COLV model, with significant Col I and III upregulation and COLV downregulation as fibrosis advances. ANA Presence during Fibrosis Progression in the IMU-COLV Model The analysis of the indirect immunofluorescence reaction with HEp-2 cells for ANA detection in the sera of the IMU-COLV model showed no significant difference in immunostaining between the IMU-COLV15 and CT groups. In contrast, the frequency of ANA in the sera of the IMU-COLV model increased with disease progression: days 30 (p = 0.0017) and 45 (p = 0.0006) (Fig. 8 A). Figure 8 B shows the negative ANA, predominant in CT animal sera, and positive ANA in the IMU-COLV model, revealed by fluorescent staining. Discussion This study highlights the progressive stages of pulmonary fibrosis in a COLV-induced SSc model, with emphasis on the key mechanistic processes driving disease progression. Early Onset and Progressive Evolution of Pulmonary Fibrosis in the IMU-COLV Model of SSc One of the major strengths of our study is the use of the IMU-COLV model for investigating pulmonary fibrosis evolution in SSc, particularly in its early stages. Although several experimental SSc models have been developed to examine immune dysregulation and fibrosis, a comprehensive understanding of fibrosis progression over time in these models remains elusive [ 32 ]. In this regard, the IMU-COLV model offers a unique advantage, as it demonstrates early immune infiltration and pulmonary fibrosis within 15 days post-induction—an observation that distinguishes it from other commonly used models. Furthermore, in a study by Meng et al. (2019), Hypochlorous Acid (HOCl) was used to induce pulmonary fibrosis, with immune cell infiltrates (CD4+, CD8+, and CD19+) observed 6 weeks post-induction [ 33 ]. Conversely, in the IMU-COLV model, we observed similar immune infiltration and early signs of fibrosis much sooner, at 15 days. Moreover, the establishment of fibrosis in the HOCl model was associated with increased TGF-β and α-SMA expression at 6 weeks, while in our IMU-COLV model, similar fibrotic markers were evident at 30 and 45 days post-induction, indicating a faster disease progression. In addition to the HOCl model [ 33 ], the topoisomerase I (TopoI) mouse model, another widely used model for studying lung interstitial fibrosis in SSc, has shown evidence of fibrosis only after 8 weeks of induction [ 34 ]. Our findings underscore the unique value of the IMU-COLV model, which allows for the investigation of earlier stages of fibrosis and immune dysregulation. This early onset and the dynamic progression observed in the IMU-COLV model make it an excellent tool for understanding the mechanisms of pulmonary fibrosis in SSc and evaluating potential therapeutic interventions. The modifications in the constitution of pulmonary fibrosis in the IMU-COLV model were previously observed by Teodoro et al. [ 6 ]; these changes closely resemble the morphological pattern of interstitial fibrosis observed in patients with SSc. In both animals and humans with SSc, in addition to the increased collagen synthesis, structurally anomalous COLV was identified, characterized by shorter, thicker fibers with heterogeneous distribution [ 26 ]. Studies have demonstrated that patients with SSc exhibit increased COLV, Col5a2 gene, and α2(V) chain expressions in both skin and lung tissues during the early stages of the disease [ 28 – 30 ]. Moreover, anomalous COLV deposition in the skin of patients with SSc is associated with skin thickening and disease activity [ 30 ]. These findings, coupled with the observation that nasal COLV tolerance in a rabbit scleroderma model reduces both the inflammatory and fibrotic processes in the lung and skin—along with a decrease in TGF-β expression—further support the hypothesis that COLV plays a pivotal role in SSc pathogenesis. COLV acts as a neoantigen that triggers an autoimmune response, contributing to disease progression [ 35 , 36 ]. Vascular Reactivity and Angiogenesis (15 Days): In the early phase of SSc, vascular changes were predominant, marked by increased expression of angiogenic factors such as VEGF and Factor VIII. These factors contribute to vascular reactivity and angiogenesis, which are crucial in fibrotic process initiation [ 37 ]. VEGF is a known promoter of blood vessel formation and endothelial cell proliferation, while Factor VIII plays a critical role in coagulation and vascular integrity [ 38 ]. The upregulation of these angiogenic markers suggests that vascular dysfunction is one of the earliest triggers of fibrosis in SSc. This early vascular injury may promote subendothelial fibrosis, an SSc pathogenesis hallmark, by facilitating inflammatory cell and immune response influx into the affected tissues [ 39 ]. Immune Response Activation (15 Days): At 15 days post-immunization, marked infiltration of immune cells, including T lymphocytes (CD3+, CD4+, CD8+) and B lymphocytes (CD20+), was observed within the lung tissue, indicating an active immune response that exacerbates fibrosis. The early presence of activated immune cells, particularly CD4 + Th2 and Th17 cells, plays a pivotal role in driving inflammation and fibrosis [ 40 , 41 ]. Th2 cells secrete cytokines like IL-4 and IL-13, promoting fibroblast activation and extracellular matrix (ECM) production, while Th17 cells produce IL-17, contributing to tissue inflammation [ 40 ]. The infiltration of B cells, which produce autoantibodies, further amplifies the immune response, driving chronic fibrosis via the secretion of TGF-β, a potent fibrogenic cytokine [ 42 ]. Early Collagen Synthesis and the Role of COLV (15 Days): At the gene expression level, the upregulation of Col5a1 and Col5a2 genes—responsible for encoding COLV alpha chains—indicates that COLV contributes to early pulmonary matrix remodeling. COLV is a minor fibrillar collagen that interacts with Col I and Col III to regulate collagen fiber assembly [ 43 ]. Its overexpression early in the disease correlates with fibrosis initiation, suggesting that COLV may act as a key neoantigen in the autoimmune response that triggers fibrosis in SSc [ 24 ]. The structural changes in COLV fibers (thicker and more irregularly distributed) could contribute to disorganized collagen deposition, which is a hallmark of SSc-associated fibrosis. Among the mechanisms potentially activated through immunization with COLV that contribute to the development of pulmonary interstitial fibrosis in SSc, the role of the endothelium is particularly noteworthy. Endothelial damage early in the disease initiates and perpetuates the SSc-associated vasculopathy [ 44 ]. This damage leads to vasoconstriction and subendothelial fibrosis, processes that foster intraluminal thrombosis development and thickening of the vascular smooth muscle layers [ 44 ]. In addition, abnormal angiogenesis, characterized by increased expression of angiogenic factors, such as PDGF, VEGF, ET-1, and TGF-β, is a hallmark of SSc pathology [ 45 ]. Notably, in the IMU-COLV model, we observed heightened VEGF and Factor VIII expression 15 days following the first COLV immunization. These findings indicate that vascular alterations occur earlier in this SSc model than previously recognized, providing insight into the early events that contribute to disease progression. Vasculopathy is considered a primary trigger for the exaggerated immune response observed in patients with SSc [ 46 ]. In the early stages of the disease, lymphocytic infiltration of affected tissues becomes evident [ 47 ]. In our IMU-COLV model, pulmonary vascular alterations concurrently occurred with immune cell infiltration at 15 days post-immunization. This infiltration was marked by an abundance of T and B lymphocytes within the pulmonary tissue. In patients with SSC, T lymphocytes show heightened expression of activation markers; moreover, this immune dysregulation is particularly prominent in the Th2 and Th17 subsets [ 40 , 41 , 48 ]. Th2 cells (which produce IL-4 and IL-13) and Th17 cells (which produce IL-17) are significantly increased in both the skin and peripheral blood, especially in patients with the diffuse form of the disease [ 48 ]. These findings underscore the early and pronounced immune activation associated with vasculopathy in SSc. Transition to Regenerative and Progression to Fibrotic Phase in SSc Early Pulmonary Disease is Simultaneous to the ANA-increased Frequency (30–45 Days) : At day 30, the immune infiltrate began to decrease, signaling a shift from inflammation to fibrosis. The increase in α-SMA expression and ECM remodeling (increase in Col I and Col III) suggests that fibroblasts are becoming more differentiated into myofibroblasts, which actively participate in ECM production. This transition from inflammation to fibrosis is a key step in the disease, where the role of the immune system in tissue injury is superseded by the fibrotic repair process. However, the pathological collagen deposition leads to tissue scarring, impairing lung function. At day 45, the disease was marked by increased collagen deposition in the interstitium and around the broncho-vascular regions. Col1a1, Col3a1, Vimentin, and TGF-β1 upregulation further confirmed the presence of fibrosis, indicating that the fibrotic process is not only sustained but also progressing. TGF-β1 plays a central role in the fibrotic process by promoting fibroblast differentiation into myofibroblasts and inducing the synthesis of collagen and other ECM proteins [ 49 ]. The rise in α-SMA expression in myofibroblasts further supports the idea that these cells are crucial fibrosis mediators in SSc, participating in the contractile force that contributes to tissue remodeling and scarring. Notably, in the IMU-COLV model, the autoimmunity is more significant from day 30 onwards and the frequency of ANA increases with the fibrosis progression in this model. As mentioned above, the presence of ANA is marked by fibrosis progression, suggesting that vascular alterations and extracellular matrix remodeling likely expose autoantigens that trigger autoimmunity. In this aspect, COLV is considered a hidden antigen, as the fibrillar region of this molecule is found embedded in the heterotypic collagen fibrils [ 11 – 18 , 20 ]. Considering the increased COLV expression at 15 days in the IMU-COLV model and its immunologic properties, we suggested that COLV itself could contribute to autoimmunity in this SSc model [ 11 – 18 , 20 ]. In addition, although other SSc models have shown the presence of autoantibodies [ 33 , 50 – 52 ], our study stands out for having evaluated autoimmunity in the early stages of the model. Furthermore, approximately 90% of patients with SSc have a positive ANA serum test, which is usually an early finding in the disease [ 53 , 54 ], corroborating what was shown in our IMU-COLV model. Role of COLV in Pulmonary Fibrosis : The increased COLV expression in the early stages of the disease suggests its role as a mediator of immune activation and fibrosis initiation. COLV interacts with Col I to regulate collagen fibril formation, and its abnormal deposition and structural alterations are implicated in SSc pathogenesis [ 43 ]. The role of COLV as a neoantigen has been previously noted in SSc, where its presence triggers an autoimmune response that contributes to fibrosis [ 24 ]. In this model, COLV upregulation in the lungs correlates with both early inflammatory infiltrates and collagen deposition initiation, emphasizing its involvement in the initial stages of lung fibrosis. Col5a2 and TGF-β1 have common points of interaction; however, no data are available on their inhibition or stimulatory effects on their expression [ 55 ]. TGF-β1 and Fibrotic Signaling : The sustained TGF-β1 expression during the later stages of the disease suggests its pivotal role in fibrosis progression. TGF-β1 is known to regulate various cellular processes, including fibroblast differentiation into myofibroblasts, collagen synthesis, and ECM organization [ 49 ]. This cytokine also facilitates the angiogenesis and vascular remodeling observed early in the disease, further linking immune activation, vascular alterations, and fibrosis [ 56 ]. As the disease progresses, the effects of TGF-β1 on fibroblast activation and ECM deposition become more pronounced, reinforcing the fibrotic tissue architecture. However, knowledge of the signaling pathway that regulates this process is necessary to explain the presence of cellular activation, as well as the organization of the extracellular matrix in this disease. Conclusion The mechanisms driving fibrosis in this SSc model are complex, involving vascular changes, immune cell infiltration, and collagen remodeling, with COLV playing a central role in the initial stages of the disease. The early vascular reactivity driven by angiogenic factors such as VEGF and Factor VIII leads to immune activation, resulting in inflammation and subsequent fibrosis. Over time, the immune response transitions into a fibrotic response, with increased collagen deposition, myofibroblast activation, and ECM remodeling, primarily driven by TGF-β1 signaling. Understanding these mechanistic pathways is crucial for developing targeted therapies aimed at modulating immune responses, preventing collagen deposition, and slowing fibrosis progression in patients with SSc. Declarations Author contributions: VC - Abstract, Introduction, Materials and methods, Results, Discussion, Conclusion; ZQ - Materials and Methods, Results, Figures; SC and AF - Schematic of the Col V immunization protocol for inducing the ES model in C57BL/6 mice and controls; LS - Materials and Methods, Results, Figures; AT - Materials and Methods SF - Materials and Methods; DF - Materials and Methods; TL - Materials and Methods, Results, Legends; JA - Materials and Methods; CA - Statistical analysis; AV – Discussion; PD – Abstract; WT – Discussion Funding: This research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, and by the funds from grant 2018/00415-0, 218/20403-6 São Paulo Research Foundation (FAPESP) and Sociedade Brasileira de Reumatologia, Brazil, 2023-16. Data Availability: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 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Brazil","correspondingAuthor":false,"prefix":"","firstName":"Percival","middleName":"Degrava","lastName":"Sampaio-Barros","suffix":""},{"id":466499458,"identity":"5e01c2a2-47a5-47aa-a7c9-b2b08ef25215","order_by":13,"name":"Vera Luiza Capelozzi","email":"","orcid":"","institution":"Department of Pathology of the Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brazil","correspondingAuthor":false,"prefix":"","firstName":"Vera","middleName":"Luiza","lastName":"Capelozzi","suffix":""},{"id":466499460,"identity":"83bf07f2-aeb9-4e45-a20d-95343e4beea0","order_by":14,"name":"Walcy Rosolia Teodoro","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYFAC5gYoIwGIK+QgbMYGrGoZkGQNoFrOGJOqhbGNCC0Gxw82fuZh+CPHz5587MPHeQbRDBLpzx4w7riHW8uZxGZpHgYDY8meZ8kzZ24zyG2QyDE3YDxTjFOL2YHENmaglsQNN3KMmXm3/QFpYZNgbEvAreX8Q7CWeoiWOSBb0p/h13IDYkuCAVhLA0hLghleLfY3HjZLzjEwNpwJ9AvjjGMGuW08b8wNEs/g1iLZn3zww5sKOXlgiB1m+FBjkNvPDgyxjztwawEBJh4DJB4bCOHXAIy3H2gCbAQ0jIJRMApGwQgDAEztUH1iYvZ0AAAAAElFTkSuQmCC","orcid":"","institution":"Rheumatology Division of the Hospital das Clinicas FMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brazil","correspondingAuthor":true,"prefix":"","firstName":"Walcy","middleName":"Rosolia","lastName":"Teodoro","suffix":""}],"badges":[],"createdAt":"2025-06-03 19:53:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6814249/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6814249/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84197795,"identity":"4c648beb-ddaf-4027-ab32-0a7c9dbfb488","added_by":"auto","created_at":"2025-06-09 08:01:53","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1331749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiagram of the Col V immunization protocol used to induce the SSc model in C57BL/6 mice and control groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic of the Col V immunization protocol for inducing the ES model in C57BL/6 mice and controls. Study groups were euthanized at 15, 30 and 45 days IMU-COLV: immunized with Col V.\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6814249/v1/860cf3f41c8f460eb5bc1ce2.jpeg"},{"id":84197796,"identity":"07813b0e-f972-400f-89cf-7c6ec48c4ad0","added_by":"auto","created_at":"2025-06-09 08:01:53","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5623844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the Progression of Pulmonary Remodeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrographs of transverse histological sections of lung tissue from C57BL/6 mice after 15, 30 and 45 days of immunization with Col V and control, stained with Hematoxylin and Eosin (HE).\u003c/p\u003e","description":"","filename":"Figure2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6814249/v1/5e924cf7416074b7477a4e9d.jpeg"},{"id":84198807,"identity":"e74bf0dd-a9dc-4b2b-b647-bfc3710f6059","added_by":"auto","created_at":"2025-06-09 08:09:54","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9461059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistomorphometric Analysis of Inflammatory Cell Infiltration in Lung Tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression of CD3+, CD4+, CD8+ and CD20+ in the lung tissue of C57BL/6 mice after 15, 30 and 45 days of immunization with Col V and control. Positive marking in brown. GraphPad Prism version 8; One-way ANOVA, followed by Tukey and Sidak post-tests.\u003c/p\u003e","description":"","filename":"Figure3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6814249/v1/498c30ce2b63aad4f031320e.jpeg"},{"id":84197800,"identity":"8fd7ab7c-41ae-4c2d-a2b4-1303a3479a09","added_by":"auto","created_at":"2025-06-09 08:01:53","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8594826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of Pulmonary Tissue Vascular Reactivity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression of cells positive for Factor VIII, VEGF+, α-AML and Caspase-3 in the lung tissue of C57BL/6 mice after 15, 30 and 45 days of immunization with Col V and control. Positive marking in brown. GraphPad Prism version 8; one-way ANOVA, followed by Tukey and Sidak post-tests.\u003c/p\u003e","description":"","filename":"Figure4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6814249/v1/a2ccee35898e94035a7c6f7b.jpeg"},{"id":84198806,"identity":"8dac55c9-1620-4ee9-a804-96d58cd24b2e","added_by":"auto","created_at":"2025-06-09 08:09:53","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7181977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the Remodeling of the Interstitium in Lung Tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative images of the morphological profile of the collagen in the bronchial region of the lungs of mice from the 15, 30 and 45-day immunization groups with Col V (IMU-COLV: Panels A, B, C, E, F and G) and controls (Panels D and H). Staining with Masson's Trichrome and Picrosirius, acquired under polarization. The arrows indicate the location and histoarchitecture of the collagen fibers in the tissue. Magnification 400x.\u003c/p\u003e","description":"","filename":"Figure5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6814249/v1/bf4af46ba82531c72ed4e575.jpeg"},{"id":84197801,"identity":"886ca9d4-5708-4ecd-b748-a21c0bf51322","added_by":"auto","created_at":"2025-06-09 08:01:53","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13714025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the Remodeling of the Interstitium in Lung Tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression of Collagen I, III and V in the lung tissue of C57BL/6 mice after 15, 30 and 45 days of immunization with Col V (IMU-COLV) and control (CT). Positive fluorescent green staining. GraphPad Prism version 8; One-way ANOVA, followed by Turkey and Sidak post-tests.\u003c/p\u003e","description":"","filename":"Figure6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6814249/v1/a851fa67167da3e01587667f.jpeg"},{"id":84198805,"identity":"a0cbc876-49b6-4665-a5f0-e48992383a6c","added_by":"auto","created_at":"2025-06-09 08:09:53","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2817379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of Col1a1, Col3a1, Col5a1, Col5a2, Tgf-β, α-Sma, and Vimentin genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelative expression of the \u003cem\u003eTgf-β\u003c/em\u003e, \u003cem\u003eα-Sma\u003c/em\u003e, \u003cem\u003eVimentin\u003c/em\u003e, \u003cem\u003eCol1a1\u003c/em\u003e, \u003cem\u003eCol3a1\u003c/em\u003e, \u003cem\u003eCol5a1\u003c/em\u003e and \u003cem\u003eCol5a2 \u003c/em\u003egenes in the lung tissue of C57BL/6 mice after 15, 30 and 45 days of immunization with Col V and control. GraphPad Prism version 8; one way ANOVA, followed by Tukey and Sidak post-tests.\u003c/p\u003e","description":"","filename":"Figure7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6814249/v1/46f03107e5f11bff7ccf6c62.jpeg"},{"id":84197820,"identity":"285691da-930a-42eb-930e-e185b68fe543","added_by":"auto","created_at":"2025-06-09 08:01:54","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":85097,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eANA Presence during Fibrosis Progression in the IMU-COLV Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eANA research by immunofluorescence with Hep-2 cells in the progression of the IMU-COLV model, where it was predominantly negative in the serum of the control group, while ANA research was positive in the 30 and 45 days groups. ANA showed a dotted pattern as the model progressed. Original magnification: 400X. GraphPad Prism version 8; One-way ANOVA, followed by Turkey and Sidak post-tests.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6814249/v1/d98c3f26b335639b8d6fe331.jpg"},{"id":90881687,"identity":"8ecf1531-026f-4942-aa34-f8066b50a4cf","added_by":"auto","created_at":"2025-09-09 09:47:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28954343,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6814249/v1/1c4a2d34-b0d1-4720-b3a0-8791f0d6edc4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Increased Collagen V Triggers Inflammation and Fibrosis in Early Pulmonary Disease of Systemic Sclerosis: Insights from the IMU-COLV Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSystemic sclerosis (SSc) is a complex autoimmune disease characterized by a combination of vascular dysfunction, fibrosis, and immune system dysregulation. This multifactorial nature of SSc leads to diverse symptoms and complications, making its management and treatment challenging [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The most severe SSc manifestations are fibrotic and vascular pulmonary complications, particularly interstitial lung disease (ILD). ILD results in progressive lung-tissue scarring, which impairs lung function and can lead to respiratory failure. In addition, vascular complications such as pulmonary arterial hypertension further worsen the disease prognosis. Thus, early diagnosis and effective management strategies aimed at mitigating these pulmonary manifestations are critical for improving patient outcomes and quality of life [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe exact initiating events of SSc remain unclear, complicating the understanding of the underlying mechanisms of the disease. Consequently, animal models have proven invaluable in advancing our knowledge of SSc pathology [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In previous studies, we demonstrated that the IMU-COLV mouse model, induced by immunization with collagen V (COLV), mirrors key human SSc features, including cutaneous, vascular, and pulmonary remodeling. This model has therefore become an essential preclinical tool for investigating the mechanisms of disease progression and exploring potential therapeutic approaches [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Moreover, the IMU-COLV model suggests that COLV is a critical factor in SSc pathogenesis.\u003c/p\u003e \u003cp\u003eIn the pulmonary tissue, COLV is located in the interstitium and capillary basement membranes and is surrounded by vascular smooth cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The COL V isoform [α1(V)2, α2(V)] contains a fibrillary portion embedded within the heterotypic collagen I/III fibrils. The NH3-terminal region is project through the fibril surface hinders further addition of new molecules, thereby influencing subsequent growth of fibril diameter [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, COLV plays a role in the organization, growth and dimensioning of the diameter of heterotypic fibrils, and is currently considered important in nucleating collagen I-containing fibrils in vivo [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition, COLV can modify the stiffness of the extracellular matrix (ECM), which in turn affects basic cellular functions. It can play a role in modifying the cell phenotypes during organogenesis and ECM remodeling when soft tissues are prevalent [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, COLV is considered a sequestered antigen, since its immunogenic properties allow it to be recognized as an autoantigen in chronic pathological conditions when exposed to the immune system [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Recent studies have revealed that autoimmunity against COLV primarily targets the α1(V) chain [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], with anti-COLV antibodies more frequently detected in patients with early SSc, particularly those directed against the α1(V) chain peptides [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, studies have shown that patients with SSc exhibit increased COLV, COL5A2 gene, and α2(V) chain expression in the skin and lungs, especially during the early stages of the disease [\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Notably, abnormal COLV deposition in the skin of patients with SSc is associated with skin thickening and disease activity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs previously described, the IMU-COLV model induces pulmonary alterations, including increased tissue elastance and airway resistance, along with a nonspecific interstitial pneumonia histologic pattern. These changes are accompanied by thickening of small and medium intrapulmonary arteries, fibrotic septal thickening, and elevated COLV expression [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the early stages of pulmonary fibrosis in this model require further investigation, and understanding these early events could provide crucial insights into the mechanisms driving SSc-related pulmonary fibrosis.\u003c/p\u003e \u003cp\u003eWe hypothesize that COLV is crucial in the establishment and progression of pulmonary fibrosis in SSc. To test this hypothesis, this study aimed to assess the early stages of pulmonary fibrosis in the IMU-COLV model, focusing on histological, molecular, and immunological changes during pulmonary fibrosis onset. Given that pulmonary manifestations are the primary cause of morbidity and mortality in patients with SSc, understanding the early events in pulmonary fibrosis development could offer new therapeutic strategies targeting these critical processes.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental model\u003c/h2\u003e \u003cp\u003eFemale C57BL/6 mice (n\u0026thinsp;=\u0026thinsp;54), aged 6\u0026ndash;7 weeks, were obtained from the animal facility of our institution. The mice were housed in specific pathogen-free conditions with free access to food and fresh water in a temperature-controlled room (22\u0026ndash;24\u0026deg;C) and maintained on a 12-h light/dark cycle. All experimental procedures were approved by The Committee on Ethical Use of Laboratory Animals of the Faculty of Medicine at the University of S\u0026atilde;o Paulo (CEUA-protocol number: 1543/2020; 1416/2019.\u003c/p\u003e \u003cp\u003eTo induce experimental SSc, mice (n\u0026thinsp;=\u0026thinsp;30) were subcutaneously immunized with 150 \u0026micro;g of human placental COLV (Col V; Sigma) dissolved in 10 mM acetic acid and emulsified with an equal volume of complete Freund's adjuvant. At day 20 post-immunization, two booster doses of 150 \u0026micro;g/200 \u0026micro;L COLV mixed with incomplete Freund's adjuvant were administered via intramuscular injection at 15-day intervals (IMU-COLV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). One hour before immunization, tramadol hydrochloride (40 mg/kg body weight) was administered subcutaneously as preventive analgesia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe control group (CT; n\u0026thinsp;=\u0026thinsp;24) was inoculated with 10 mM acetic acid mixed with complete or incomplete Freund's adjuvant, following the same immunization protocol. To investigate early pulmonary changes associated with experimental SSc, animals were euthanized at 15 (n\u0026thinsp;=\u0026thinsp;10), 30 (n\u0026thinsp;=\u0026thinsp;10), and 45 (n\u0026thinsp;=\u0026thinsp;10) days after the initial COLV immunization. Before euthanasia, the mice were anesthetized with an intraperitoneal injection of ketamine hydrochloride (100 mg/kg body weight) and xylazine (10 mg/kg body weight), followed by euthanasia through nuchal dislocation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation\u003c/h3\u003e\n\u003cp\u003eLung tissue samples were collected and fixed in 10% buffered formalin for morphometric and immunohistochemical analysis. Additional lung fragments were stored at -80\u0026deg;C for subsequent molecular analysis.\u003c/p\u003e\n\u003ch3\u003eLung Morphological Analysis\u003c/h3\u003e\n\u003cp\u003eFor histological examination, lung samples were fixed in 10% buffered formalin for 24 h and subsequently embedded in paraffin. Sections of 3\u0026ndash;4 \u0026micro;M thickness were cut and stained with hematoxylin and eosin (H\u0026amp;E) for general histological assessment, and picrosirius red for collagen fiber detection.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence for Collagen\u003c/h3\u003e\n\u003cp\u003eFor collagen immunostaining (Col I, III, and V), 4 \u0026micro;m lung tissue sections were adhered to glass slides pre-coated with 3-aminopropyltriethoxy silane (Sigma, St. Louis, MO, USA). Antigen retrieval was performed by incubating the slides with bovine pepsin (8 mg/mL in 0.5 N acetic acid) (Sigma) for 30 min at 37\u0026deg;C. After several washes with phosphate-buffered saline (PBS), the slides were blocked with 5% bovine serum albumin (BSA) diluted in PBS (pH 7.0).\u003c/p\u003e \u003cp\u003eSubsequently, the slides were incubated overnight at 4\u0026deg;C with rabbit polyclonal antibodies: anti-Col I (1:1,200; Rockland), anti-Col III (1:1,400; Rockland), and anti-Col V (1:1,000). After washing with PBS containing 0.05% Tween 20, the slides were incubated for 1 h at 25\u0026deg;C with goat anti-rabbit IgG Alexa Fluor 488 (Invitrogen, Life Technologies) at a 1:200 dilution in PBS containing 0.006% Evans blue.\u003c/p\u003e \u003cp\u003eThe slides were mounted with buffered glycerin and analyzed under a fluorescence microscope (Olympus BX-51, Olympus Co, Tokyo, Japan).\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eFor immunohistochemical analysis, 3\u0026ndash;4 \u0026micro;m lung tissue sections were deparaffinized and incubated in a 0.3% hydrogen peroxide solution for 5 min to inhibit endogenous peroxidase activity. Afterward, sections underwent a cycle of four 5-min washes.\u003c/p\u003e \u003cp\u003eThe following primary antibodies were used for the immunostaining reactions: smooth muscle α-actin (α-SMA, mouse monoclonal, 1:50; Santa Cruz Biotechnology Inc., USA), vascular endothelial growth factor (VEGF, 1:400; Santa Cruz Biotechnology Inc., USA), Factor VIII (1:1200; Santa Cruz Biotechnology Inc., USA), Caspase-3 (1:4,000; Novus Biologicals, USA), CD3 (1:1,000; Santa Cruz Biotechnology Inc., USA), CD4 (1:1,000; Santa Cruz Biotechnology Inc., USA), CD8 (1:1,200; Santa Cruz Biotechnology Inc., USA), and CD20 (1:100; Santa Cruz Biotechnology Inc., USA).\u003c/p\u003e \u003cp\u003eAntigen retrieval was performed using either bovine pepsin (4 mg/mL in 0.01 N glycine buffer, Sigma) for 30 min at 37\u0026deg;C, or citrate buffer (pH 6.0) at high temperature (125\u0026deg;C for 1 min) in a pressure cooker (Pascal). Subsequently, the sections were incubated with the primary antibodies overnight at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eImmunoreactions were detected using a biotin-streptavidin-peroxidase kit (Vector) per manufacturer instructions. The chromogen used was 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO), and the sections were counterstained with Harris hematoxylin (Merck, Darmstadt, Germany).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMorphometric Analysis\u003c/h2\u003e \u003cp\u003eCollagen fibers of types I, III, and V were quantified in the lung parenchyma using Image-Pro Plus 6.0 software [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In addition, two blinded observers analyzed 10 randomly selected microscopic fields at 400x magnification. Collagen fiber thresholds were set on all slides after enhancing the contrast, ensuring that the fibers were easily distinguishable as green bands. Collagen fiber density was calculated as the ratio of the measured collagen fibers to the total tissue area analyzed, with the result expressed as a percentage.\u003c/p\u003e \u003cp\u003eFor immunostained cell analysis, 10 random lung tissue fields were evaluated at 1,000x magnification using Image-Pro Plus 6.0 software. α-SMA, VEGF, Factor VIII, Caspase-3, CD3, CD4, CD8, and CD20 expressions were determined by counting the number of positive cells and expressed as the proportion of total cells in the field analyzed.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantitative Real-Time Polymerase Chain Reaction (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eThe total RNA was isolated according to a standard Trizol\u0026reg; (Invitrogen, Life Techologies Co., Carlsbad, CA, USA) RNA isolation protocol. Col1a1 (Col I α1 chain), Col3a1 (Col III α1 chain), Col5a1 (Col V α1 chain), Col5a2 (Col V α2 chain), TGF-β, Vimentin, and α-SMA expressions were assessed via qRT-PCR.\u003c/p\u003e \u003cp\u003eFor cDNA synthesis, total RNA from each sample was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). qRT-PCR reactions were prepared using the Platinum SYBR Green qPCR SuperMix-UDG Kit (Invitrogen, Life Technologies, USA). Amplification and cDNA production were performed using a Step One thermal cycler (Applied Biosystems), with 1,000 ng of total RNA per sample.\u003c/p\u003e \u003cp\u003eRelative gene expression was calculated using the 2-ΔΔCT method, with β-2 microglobulin used as the housekeeping gene for normalization.\u003c/p\u003e\n\u003ch3\u003eCollagen Quantification by Measuring 4-Hydroxyproline\u003c/h3\u003e\n\u003cp\u003eTotal lung collagen deposition was quantified by measuring the 4-hydroxyproline content, as previously described with modifications [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, lung samples were freeze-dried (Edwards, Modulyo), weighed, and subsequently hydrolyzed in 6 N HCl for 22 h at 100\u0026deg;C. The hydroxyproline content was determined spectrophotometrically by measuring absorbance at 560 nm. The results were expressed as nanograms of 4-hydroxyproline per milligram of protein [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnti-nuclear Antibody (ANA) Detection\u003c/h2\u003e \u003cp\u003eSera obtained from IMU-COLV and CT groups of mice from 15, 30, and 45 days were tested for ANAs using slides with HEp-2 cells (Nova-Lite HEp-2 ANA Kit), per manufacturer instructions. Slides were incubated for 30 min at room temperature with sera diluted at 1:80 in PBS with Tween20. After a washing cycle with PBS/Tween20, the slides were incubated for 30 min with conjugated goat anti-mouse IgG antibody ALEXA FLUOR 488 (INVITROGEN) (1:200). Subsequently, slides were counterstained with 0.006% Evans blue, mounted on coverslips with buffered glycerin and examined under a fluorescence microscope (Olympus BX51).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003e Data were analyzed for normality of distribution and categorized as parametric or non-parametric accordingly, using GraphPad Prism version 8.0.2 (CA, USA). Results for each variable are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. One-way analysis of variance was used to compare the means between groups, with Tukey or Sidak\u0026rsquo;s post-test applied for normally distributed variables. The Kruskal\u0026ndash;Wallis test was used for non-normally distributed variables, followed by the Dunn test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eTemporal Dynamics of Inflammation and Collagen Deposition in the Pulmonary Fibrotic Process of the IMU-COLV Model\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFifteen days post-immunization with COLV, animals in the IMU-COLV15 group exhibited a prominent inflammatory cell infiltrate around vessels and bronchi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This infiltrate expanded around the alveolar septa and vessels compared to the CT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), with notable presence around small vessels and bronchioles in the pulmonary interstitium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE vs. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt 30 days post-immunization (IMU-COLV30), the inflammatory infiltrate decreased, and collagen deposition began in the perivascular region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This process was more widespread, with thick collagen fibers in the peribronchial region, a feature absents in the CT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, intense collagen deposition in the peribronchovascular region extending into the lung parenchyma was visible in the IMU-COLV30 group, which was not observed in the CT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eAt 45 days post-immunization (IMU-COLV45), lung parenchyma, vessel, and bronchi remodeling wer evident (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), with almost no inflammatory cells present, replaced by substantial collagen deposition in the vessel wall and interstitium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Lung interstitial destruction was more pronounced in the IMU-COLV45 group than in the CT group (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and H).\u003c/p\u003e \u003cp\u003eQuantification of immune cell populations confirmed increased TCD3\u0026thinsp;+\u0026thinsp;lymphocytes in the IMU-COLV15 group compared with both IMU-COLV30 (12.65\u0026thinsp;\u0026plusmn;\u0026thinsp;4.78 vs. 8.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14, p\u0026thinsp;=\u0026thinsp;0.0058) and IMU-COLV45 groups (12.65\u0026thinsp;\u0026plusmn;\u0026thinsp;4.78 vs. 8.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68, p\u0026thinsp;=\u0026thinsp;0.0047) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). TCD4\u0026thinsp;+\u0026thinsp;lymphocytes were also significantly higher in the IMU-COLV15 group than in the IMU-COLV30 (33.99\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01 vs. 23.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03, p\u0026thinsp;=\u0026thinsp;0.0001) and IMU-COLV45 groups (33.99\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01 vs. 23.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74, p\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Similarly, TCD8\u0026thinsp;+\u0026thinsp;lymphocytes were elevated in the IMU-COLV15 group compared with both the IMU-COLV30 (39.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43 vs. 22.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09, p\u0026thinsp;=\u0026thinsp;0.0001) and IMU-COLV45 groups (39.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43 vs. 23.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47, p\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO). Finally, the number of CD20\u0026thinsp;+\u0026thinsp;B lymphocytes was significantly higher in the IMU-COLV15 group than in the IMU-COLV30 (34.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81 vs. 27.50\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22, p\u0026thinsp;=\u0026thinsp;0.0011) and IMU-COLV45 groups (34.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81 vs. 28.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64, p\u0026thinsp;=\u0026thinsp;0.0044) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eT).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTemporal Changes in Vascular Reactivity and Endothelial Markers in the IMU-COLV Model\u003c/h2\u003e \u003cp\u003eImmunostaining for Factor VIII\u0026thinsp;+\u0026thinsp;endothelial cells revealed intense expression, particularly in the perivascular region of the IMU-COLV15 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), in contrast to the lower expression observed in the IMU-COLV30, IMU-COLV45, and CT groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C, and D). Quantitative analysis confirmed a significant increase in Factor VIII\u0026thinsp;+\u0026thinsp;expression in the IMU-COLV15 group compared with the IMU-COLV30 (13.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.426 vs. 5.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0983, p\u0026thinsp;=\u0026thinsp;0.0001) and IMU-COLV45 groups (13.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.426 vs. 4.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90, p\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eVEGF\u0026thinsp;+\u0026thinsp;endothelial cells were also increased in the IMU-COLV15 group compared with the IMU-COLV30 and IMU-COLV45 groups, as confirmed by quantitative analysis (IMU-COLV15 vs. IMU-COLV30: 3.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 vs. 2.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57, p\u0026thinsp;=\u0026thinsp;0.0008; IMU-COLV15 vs. IMU-COLV45: 3.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 vs. 2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73, p\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eα-AML\u0026thinsp;+\u0026thinsp;myofibroblast immunostaining revealed a significant increase in the IMU-COLV45 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-H) compared with the IMU-COLV15 (29.45\u0026thinsp;\u0026plusmn;\u0026thinsp;5.39 vs. 23.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52, p\u0026thinsp;=\u0026thinsp;0.0051) and IMU-COLV30 groups (29.45\u0026thinsp;\u0026plusmn;\u0026thinsp;5.39 vs. 24.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31, p\u0026thinsp;=\u0026thinsp;0.0421) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO). Finally, Caspase-3\u0026thinsp;+\u0026thinsp;cells in the endothelial cells of the pulmonary microvasculature were significantly increased in the IMU-COLV45 group compared with the IMU-COLV30 group (13.60\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76 vs. 9.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79, p\u0026thinsp;=\u0026thinsp;0.0161).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHistological Characterization of Collagen Deposition and Fibrosis Progression in the IMU-COLV Model\u003c/h2\u003e \u003cp\u003eHistological analysis of collagen fibers in the lung tissue was performed using Masson\u0026rsquo;s trichrome and picrosirius red staining, confirming the morphological patterns observed in H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-H). In the IMU-COLV15 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and E), collagen fibers were predominantly distributed around the vessels and bronchi, consistent with the typical, organized pattern observed in the CT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and H). This arrangement suggests minimal disruption of normal lung tissue structure at this early stage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, the IMU-COLV30 group exhibited a more heterogeneous histological pattern, characterized by the presence of both thinner and thicker collagen fibers surrounding the perivascular region (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and F). This variation in collagen fiber thickness suggests the onset of lung tissue remodeling, a hallmark of the early stages of fibrosis.\u003c/p\u003e \u003cp\u003eIn the IMU-COLV45 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and G), collagen deposition had significantly intensified, leading to a disorganized fibrotic pattern, particularly in the peribronchial and perivascular regions, where intense blue staining indicated dense collagen accumulation compared with the CT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and H). This progressive collagen fiber disorganization suggests progressive fibrosis.\u003c/p\u003e \u003cp\u003eThese histological observations were further corroborated by biochemical quantification of 4-hydroxyproline, a collagen content marker. The analysis revealed a significant increase in total collagen in the lung tissue of the IMU-COLV45 group (5.203\u0026thinsp;\u0026plusmn;\u0026thinsp;1.350) compared with the IMU-COLV15 (3.773\u0026thinsp;\u0026plusmn;\u0026thinsp;1.006, p\u0026thinsp;=\u0026thinsp;0.0091) and IMU-COLV30 groups (3.213\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4715, p\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). This data provides quantitative evidence of increased collagen deposition in the progressive stages of fibrosis, supporting the histological findings.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSpatiotemporal Distribution and Collagen Type Composition in Fibrosis Progression in the IMU-COLV Model\u003c/h2\u003e \u003cp\u003eRegarding collagen type composition in the lung tissue, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA illustrates a subtle type I collagen (Col I) expression in the IMU-COLV15 group, primarily surrounding the vessels and bronchi. In the IMU-COLV30 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), Col I deposition increased, particularly around the bronchi and blood vessel walls, compared with the CT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In the IMU-COLV45 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), a notable increase in Col I expression was observed, extending throughout the lung tissue. This deposition extended to the alveolar septa and caused vessel wall thickening, indicating more advanced fibrosis than that of the IMU-COLV15 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Quantitative analysis confirmed these observations, with significant differences between the IMU-COLV45 and IMU-COLV15 groups (39.76\u0026thinsp;\u0026plusmn;\u0026thinsp;7.18 vs. 20.29\u0026thinsp;\u0026plusmn;\u0026thinsp;4.66, p\u0026thinsp;=\u0026thinsp;0.0240), indicating established fibrosis in the former group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor type III collagen (Col III), initial expression at day 15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF and I) was confined to the vessels and bronchi, following a pattern similar to that seen in CT animals. In the IMU-COLV30 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG), Col III deposition increased around the vessels and bronchi, and extended into the lung parenchyma. By day 45 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), Col III expression had expanded throughout the lung interstitium, surpassing the levels observed in both the IMU-COLV15 and CT groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF and I). Quantitative analysis confirmed the morphological findings, revealing significant differences between the IMU-COLV30 and IMU-COLV15 groups (31.04\u0026thinsp;\u0026plusmn;\u0026thinsp;7.20 vs. 21.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.41, p\u0026thinsp;=\u0026thinsp;0.0059), as well as between the IMU-COLV45 and IMU-COLV15 groups (30.53\u0026thinsp;\u0026plusmn;\u0026thinsp;4.01 vs. 21.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.41, p\u0026thinsp;=\u0026thinsp;0.0107) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eUpon analyzing COLV expression, fine fibers were observed around the pulmonary interstitium as well as on the walls of the pulmonary microvasculature and bronchi across the IMU-COLV15, IMU-COLV30, IMU-COLV45, and CT groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK-N). These patterns were consistent across all groups, indicating COLV presence in the structural components of the lung tissue. However, quantitative analysis revealed a significant decrease in COLV expression in the IMU-COLV30 and IMU-COLV45 groups compared with the IMU-COLV15 group. Specifically, the IMU-COLV30 group demonstrated a reduction in COLV expression (11.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24 vs. 9.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28, p\u0026thinsp;=\u0026thinsp;0.0181), as did the IMU-COLV 45-day group (11.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24 vs. 8.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34, p\u0026thinsp;=\u0026thinsp;0.0031). These findings suggest that the initial increase in COLV observed at day 15 was followed by a significant decrease at progressive stages of fibrosis, as confirmed by the statistical comparison across the groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGene Expression Profile of Collagen and Fibrosis-Related Markers in Lung Tissues of the IMU-COLV Model\u003c/h2\u003e \u003cp\u003eTotal mRNA extracted from the lung tissues of both IMU-COLV and CT groups was used to assess mRNA expression for the α1 chains of Col I and Col III, as well as the α1 and α2 chains of COLV using RT-PCR.\u003c/p\u003e \u003cp\u003eAnalysis of TGF-β gene expression, a key protein involved in SSc pathogenesis, revealed significant upregulation in the IMU-COLV45 group compared with both the IMU-COLV15 (2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 vs. 0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and IMU-COLV30 groups (2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 vs. 1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84, p\u0026thinsp;=\u0026thinsp;0.0003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Similarly, α-AML gene expression showed a significant increase in the IMU-COLV45 group (2.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 vs. 1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; 2.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 vs. 1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared with both the IMU-COLV15 and IMU-COLV30 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Vimentin expression followed a similar trend (2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 vs. 1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; 2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 vs. 1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), indicating a progressive increase in fibrosis-related gene expression as the disease progresses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding collagen gene expression, Col1a1, encoding the α1 chain of collagen I, was significantly upregulated in the IMU-COLV45 group compared with both the IMU-COLV15 (2.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01 vs. 1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38) and IMU-COLV30 groups (2.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01 vs. 1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Similarly, the expression of Col3a1, which encodes the α1 chain of collagen III, was significantly increased in the IMU-COLV45 group compared with the IMU-COLV15 (3.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 vs. 1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36) and IMU-COLV30 groups (3.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 vs. 1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE), further supporting the notion of ongoing collagen deposition in the later stages of fibrosis.\u003c/p\u003e \u003cp\u003eIn contrast, the expression of Col5a1, which encodes the α1 chain of COLV, showed a significant decrease in the IMU-COLV30 and IMU-COLV45 groups compared with the IMU-COLV15 group (2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 vs. 1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; 2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 vs. 1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30, p\u0026thinsp;=\u0026thinsp;0.0093) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Similarly, the expression of Col5a2, which encodes the α2 chain of COLV, was also significantly lower in the IMU-COLV30 and IMU-COLV45 groups than in the IMU-COLV15 group (2.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 vs. 1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; 2.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 vs. 1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52, p\u0026thinsp;=\u0026thinsp;0.0065) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eThese findings highlight distinct changes in collagen gene expression during fibrosis progression in the IMU-COLV model, with significant Col I and III upregulation and COLV downregulation as fibrosis advances.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eANA Presence during Fibrosis Progression in the IMU-COLV Model\u003c/h2\u003e \u003cp\u003eThe analysis of the indirect immunofluorescence reaction with HEp-2 cells for ANA detection in the sera of the IMU-COLV model showed no significant difference in immunostaining between the IMU-COLV15 and CT groups. In contrast, the frequency of ANA in the sera of the IMU-COLV model increased with disease progression: days 30 (p\u0026thinsp;=\u0026thinsp;0.0017) and 45 (p\u0026thinsp;=\u0026thinsp;0.0006) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB shows the negative ANA, predominant in CT animal sera, and positive ANA in the IMU-COLV model, revealed by fluorescent staining.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study highlights the progressive stages of pulmonary fibrosis in a COLV-induced SSc model, with emphasis on the key mechanistic processes driving disease progression.\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEarly Onset and Progressive Evolution of Pulmonary Fibrosis in the IMU-COLV Model of SSc\u003c/h2\u003e \u003cp\u003eOne of the major strengths of our study is the use of the IMU-COLV model for investigating pulmonary fibrosis evolution in SSc, particularly in its early stages. Although several experimental SSc models have been developed to examine immune dysregulation and fibrosis, a comprehensive understanding of fibrosis progression over time in these models remains elusive [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this regard, the IMU-COLV model offers a unique advantage, as it demonstrates early immune infiltration and pulmonary fibrosis within 15 days post-induction\u0026mdash;an observation that distinguishes it from other commonly used models.\u003c/p\u003e \u003cp\u003eFurthermore, in a study by Meng et al. (2019), Hypochlorous Acid (HOCl) was used to induce pulmonary fibrosis, with immune cell infiltrates (CD4+, CD8+, and CD19+) observed 6 weeks post-induction [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Conversely, in the IMU-COLV model, we observed similar immune infiltration and early signs of fibrosis much sooner, at 15 days. Moreover, the establishment of fibrosis in the HOCl model was associated with increased TGF-β and α-SMA expression at 6 weeks, while in our IMU-COLV model, similar fibrotic markers were evident at 30 and 45 days post-induction, indicating a faster disease progression.\u003c/p\u003e \u003cp\u003eIn addition to the HOCl model [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], the topoisomerase I (TopoI) mouse model, another widely used model for studying lung interstitial fibrosis in SSc, has shown evidence of fibrosis only after 8 weeks of induction [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Our findings underscore the unique value of the IMU-COLV model, which allows for the investigation of earlier stages of fibrosis and immune dysregulation. This early onset and the dynamic progression observed in the IMU-COLV model make it an excellent tool for understanding the mechanisms of pulmonary fibrosis in SSc and evaluating potential therapeutic interventions.\u003c/p\u003e \u003cp\u003eThe modifications in the constitution of pulmonary fibrosis in the IMU-COLV model were previously observed by Teodoro et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]; these changes closely resemble the morphological pattern of interstitial fibrosis observed in patients with SSc. In both animals and humans with SSc, in addition to the increased collagen synthesis, structurally anomalous COLV was identified, characterized by shorter, thicker fibers with heterogeneous distribution [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Studies have demonstrated that patients with SSc exhibit increased COLV, Col5a2 gene, and α2(V) chain expressions in both skin and lung tissues during the early stages of the disease [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Moreover, anomalous COLV deposition in the skin of patients with SSc is associated with skin thickening and disease activity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These findings, coupled with the observation that nasal COLV tolerance in a rabbit scleroderma model reduces both the inflammatory and fibrotic processes in the lung and skin\u0026mdash;along with a decrease in TGF-β expression\u0026mdash;further support the hypothesis that COLV plays a pivotal role in SSc pathogenesis. COLV acts as a neoantigen that triggers an autoimmune response, contributing to disease progression [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eVascular Reactivity and Angiogenesis (15 Days):\u003c/h2\u003e \u003cp\u003eIn the early phase of SSc, vascular changes were predominant, marked by increased expression of angiogenic factors such as VEGF and Factor VIII. These factors contribute to vascular reactivity and angiogenesis, which are crucial in fibrotic process initiation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. VEGF is a known promoter of blood vessel formation and endothelial cell proliferation, while Factor VIII plays a critical role in coagulation and vascular integrity [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The upregulation of these angiogenic markers suggests that vascular dysfunction is one of the earliest triggers of fibrosis in SSc. This early vascular injury may promote subendothelial fibrosis, an SSc pathogenesis hallmark, by facilitating inflammatory cell and immune response influx into the affected tissues [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eImmune Response Activation (15 Days):\u003c/h2\u003e \u003cp\u003eAt 15 days post-immunization, marked infiltration of immune cells, including T lymphocytes (CD3+, CD4+, CD8+) and B lymphocytes (CD20+), was observed within the lung tissue, indicating an active immune response that exacerbates fibrosis. The early presence of activated immune cells, particularly CD4\u0026thinsp;+\u0026thinsp;Th2 and Th17 cells, plays a pivotal role in driving inflammation and fibrosis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Th2 cells secrete cytokines like IL-4 and IL-13, promoting fibroblast activation and extracellular matrix (ECM) production, while Th17 cells produce IL-17, contributing to tissue inflammation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The infiltration of B cells, which produce autoantibodies, further amplifies the immune response, driving chronic fibrosis via the secretion of TGF-β, a potent fibrogenic cytokine [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eEarly Collagen Synthesis and the Role of COLV (15 Days):\u003c/h2\u003e \u003cp\u003eAt the gene expression level, the upregulation of Col5a1 and Col5a2 genes\u0026mdash;responsible for encoding COLV alpha chains\u0026mdash;indicates that COLV contributes to early pulmonary matrix remodeling. COLV is a minor fibrillar collagen that interacts with Col I and Col III to regulate collagen fiber assembly [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Its overexpression early in the disease correlates with fibrosis initiation, suggesting that COLV may act as a key neoantigen in the autoimmune response that triggers fibrosis in SSc [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The structural changes in COLV fibers (thicker and more irregularly distributed) could contribute to disorganized collagen deposition, which is a hallmark of SSc-associated fibrosis.\u003c/p\u003e \u003cp\u003eAmong the mechanisms potentially activated through immunization with COLV that contribute to the development of pulmonary interstitial fibrosis in SSc, the role of the endothelium is particularly noteworthy. Endothelial damage early in the disease initiates and perpetuates the SSc-associated vasculopathy [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This damage leads to vasoconstriction and subendothelial fibrosis, processes that foster intraluminal thrombosis development and thickening of the vascular smooth muscle layers [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In addition, abnormal angiogenesis, characterized by increased expression of angiogenic factors, such as PDGF, VEGF, ET-1, and TGF-β, is a hallmark of SSc pathology [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Notably, in the IMU-COLV model, we observed heightened VEGF and Factor VIII expression 15 days following the first COLV immunization. These findings indicate that vascular alterations occur earlier in this SSc model than previously recognized, providing insight into the early events that contribute to disease progression.\u003c/p\u003e \u003cp\u003eVasculopathy is considered a primary trigger for the exaggerated immune response observed in patients with SSc [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In the early stages of the disease, lymphocytic infiltration of affected tissues becomes evident [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In our IMU-COLV model, pulmonary vascular alterations concurrently occurred with immune cell infiltration at 15 days post-immunization. This infiltration was marked by an abundance of T and B lymphocytes within the pulmonary tissue. In patients with SSC, T lymphocytes show heightened expression of activation markers; moreover, this immune dysregulation is particularly prominent in the Th2 and Th17 subsets [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Th2 cells (which produce IL-4 and IL-13) and Th17 cells (which produce IL-17) are significantly increased in both the skin and peripheral blood, especially in patients with the diffuse form of the disease [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. These findings underscore the early and pronounced immune activation associated with vasculopathy in SSc.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransition to Regenerative and Progression to Fibrotic Phase in SSc Early Pulmonary Disease is Simultaneous to the ANA-increased Frequency (30\u0026ndash;45 Days)\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eAt day 30, the immune infiltrate began to decrease, signaling a shift from inflammation to fibrosis. The increase in α-SMA expression and ECM remodeling (increase in Col I and Col III) suggests that fibroblasts are becoming more differentiated into myofibroblasts, which actively participate in ECM production. This transition from inflammation to fibrosis is a key step in the disease, where the role of the immune system in tissue injury is superseded by the fibrotic repair process. However, the pathological collagen deposition leads to tissue scarring, impairing lung function.\u003c/p\u003e \u003cp\u003eAt day 45, the disease was marked by increased collagen deposition in the interstitium and around the broncho-vascular regions. Col1a1, Col3a1, Vimentin, and TGF-β1 upregulation further confirmed the presence of fibrosis, indicating that the fibrotic process is not only sustained but also progressing. TGF-β1 plays a central role in the fibrotic process by promoting fibroblast differentiation into myofibroblasts and inducing the synthesis of collagen and other ECM proteins [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The rise in α-SMA expression in myofibroblasts further supports the idea that these cells are crucial fibrosis mediators in SSc, participating in the contractile force that contributes to tissue remodeling and scarring.\u003c/p\u003e \u003cp\u003eNotably, in the IMU-COLV model, the autoimmunity is more significant from day 30 onwards and the frequency of ANA increases with the fibrosis progression in this model. As mentioned above, the presence of ANA is marked by fibrosis progression, suggesting that vascular alterations and extracellular matrix remodeling likely expose autoantigens that trigger autoimmunity. In this aspect, COLV is considered a hidden antigen, as the fibrillar region of this molecule is found embedded in the heterotypic collagen fibrils [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16 CR17\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Considering the increased COLV expression at 15 days in the IMU-COLV model and its immunologic properties, we suggested that COLV itself could contribute to autoimmunity in this SSc model [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16 CR17\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, although other SSc models have shown the presence of autoantibodies [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], our study stands out for having evaluated autoimmunity in the early stages of the model. Furthermore, approximately 90% of patients with SSc have a positive ANA serum test, which is usually an early finding in the disease [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], corroborating what was shown in our IMU-COLV model.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eRole of COLV in Pulmonary Fibrosis\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eThe increased COLV expression in the early stages of the disease suggests its role as a mediator of immune activation and fibrosis initiation. COLV interacts with Col I to regulate collagen fibril formation, and its abnormal deposition and structural alterations are implicated in SSc pathogenesis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The role of COLV as a neoantigen has been previously noted in SSc, where its presence triggers an autoimmune response that contributes to fibrosis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this model, COLV upregulation in the lungs correlates with both early inflammatory infiltrates and collagen deposition initiation, emphasizing its involvement in the initial stages of lung fibrosis. Col5a2 and TGF-β1 have common points of interaction; however, no data are available on their inhibition or stimulatory effects on their expression [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003eTGF-β1 and Fibrotic Signaling\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eThe sustained TGF-β1 expression during the later stages of the disease suggests its pivotal role in fibrosis progression. TGF-β1 is known to regulate various cellular processes, including fibroblast differentiation into myofibroblasts, collagen synthesis, and ECM organization [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This cytokine also facilitates the angiogenesis and vascular remodeling observed early in the disease, further linking immune activation, vascular alterations, and fibrosis [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. As the disease progresses, the effects of TGF-β1 on fibroblast activation and ECM deposition become more pronounced, reinforcing the fibrotic tissue architecture. However, knowledge of the signaling pathway that regulates this process is necessary to explain the presence of cellular activation, as well as the organization of the extracellular matrix in this disease.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":" \u003cp\u003eThe mechanisms driving fibrosis in this SSc model are complex, involving vascular changes, immune cell infiltration, and collagen remodeling, with COLV playing a central role in the initial stages of the disease. The early vascular reactivity driven by angiogenic factors such as VEGF and Factor VIII leads to immune activation, resulting in inflammation and subsequent fibrosis. Over time, the immune response transitions into a fibrotic response, with increased collagen deposition, myofibroblast activation, and ECM remodeling, primarily driven by TGF-β1 signaling. Understanding these mechanistic pathways is crucial for developing targeted therapies aimed at modulating immune responses, preventing collagen deposition, and slowing fibrosis progression in patients with SSc.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVC - Abstract, Introduction, Materials and methods, Results, Discussion, Conclusion; ZQ - Materials and Methods, Results, Figures; SC and AF - Schematic of the Col V immunization protocol for inducing the ES model in C57BL/6 mice and controls; LS - Materials and Methods, Results, Figures; AT - Materials and Methods SF - Materials and Methods; DF - Materials and Methods; TL - Materials and Methods, Results, Legends; JA - Materials and Methods; CA - Statistical analysis; AV \u0026ndash; Discussion; PD \u0026ndash; Abstract; WT \u0026ndash; Discussion\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brasil (CAPES) - Finance Code 001, and by the funds from grant 2018/00415-0, 218/20403-6 S\u0026atilde;o Paulo Research Foundation (FAPESP) and Sociedade Brasileira de Reumatologia, Brazil, 2023-16.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of Committee on Ethical Use of Laboratory Animals of Faculty of Medicine at the University of S\u0026atilde;o Paulo (protocol number 1416/2019 date of approval 27.11.2019 and 1543/2020 date of approval 31.07.2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVolkmann ER, Andr\u0026eacute;asson K, Smith V. 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Curr Opin Physiol. 2023; 34:100678. doi: 10.1016/j.cophys.2023.100678.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pulmonary Fibrosis, Collagen V, Experimental model of SSc, Systemic Sclerosis lung, Collagen, Fibrillogenesis","lastPublishedDoi":"10.21203/rs.3.rs-6814249/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6814249/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding the early stages of pulmonary disease pathogenesis in systemic sclerosis (SSc) is crucial for developing therapeutic strategies to mitigate pulmonary fibrosis progression. Our aim is to evaluate the early stages of pulmonary fibrosis in the IMU-COLV model. This study used the SSc IMU-COLV model in female C57BL/6 mice immunized with collagen V (COLV) emulsified in Freund's adjuvant. Mice were categorized into groups based on immunization duration (15, 30, and 45 days) to assess lymphocyte populations, Factor VIII, VEGF, caspase-3, α-AML, and collagen expression through immunostaining and image analysis. Total collagen content was quantified using 4-hydroxyproline, while α-AML, TGF-β1, and collagen gene expressions were evaluated via RT-qPCR. At day 15, significant increases in CD3+, CD4+, CD8+, and CD20\u0026thinsp;+\u0026thinsp;lymphocytes, Factor VIII, VEGF, and α-AML were observed, alongside enhanced COLV and Col5α1/Col5α2 gene expressions. Inflammation decreased at day 30; however, by day 45, a nonspecific interstitial pneumonia pattern emerged, with intrapulmonary artery thickening, increased Caspase-3, α-AML, collagen types I and III, Col1α1 and Col3α1 genes, and total collagen levels. Furthermore, vimentin, α-AML, and TGF-β1 gene expressions were higher in the 45-day group. The IMU-COLV model exhibits an early inflammatory phase at day 15, with COLV deposition, leading to pulmonary remodeling and fibrosis at days 30 and 45, mediated by TGF-β1 activation. The increased COLV expression and associated inflammatory infiltrates observed in this model suggest that COLV contributes to pulmonary fibrosis progression.\u003c/p\u003e","manuscriptTitle":"Increased Collagen V Triggers Inflammation and Fibrosis in Early Pulmonary Disease of Systemic Sclerosis: Insights from the IMU-COLV Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 08:01:47","doi":"10.21203/rs.3.rs-6814249/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"22b01923-bee8-4b1c-bcbd-4b69353694c0","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49528934,"name":"Health sciences/Medical research"},{"id":49528935,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-09-09T09:39:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-09 08:01:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6814249","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6814249","identity":"rs-6814249","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}