Introduction Inflammatory bowel disease (IBD) is a collection of disease entities characterized by chronic, relapsing, and remitting inflammation within the gastrointestinal tract. IBD comprises Crohn’s disease (CD) and ulcerative colitis (UC). IBD has multiple causes, including genetics, gut alteration of the microbiota, and environmental factors [ 1 ].
The disease course typically starts with chronic, aberrant immune response, which triggers extensive tissue damage at the inflammatory site [ 2 ]. With an ongoing, unbalanced wound healing response, chronic tissue damage and fibrosis occur, ultimately resulting in organ dysfunction [ 3 ]. Vimentin, an intermediate filament protein, regulates key cellular functions such as morphology, adhesion, polarization, stiffness, and migration [ 4 ].
Knockout studies in the 1980s revealed that knockout mice ( Vim-/- ) exhibit impaired wound healing, artery remodeling, and vasoactivity due to these cellular defects. Notably, loss of vimentin reduces inflammation and infection, highlighting its role in various human diseases [ 5 – 7 ]. Additional studies revealed that vimentin, both intracellular (iVm) and extracellular (eVm), plays a synergistic role in inflammation. iVm mediates biochemical and mechanical signals from the cell surroundings [ 5 ].
It also binds to nucleotide oligomerization domain protein 2 ( NOD2 ), activating the nuclear factor kappa-light-chain-enhancer of activated B cells ( NFκB) pathway and inducing inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNFα and induce autophagy [ 8 , 9 ]. IL-6 then activates Janus kinase (JAK) 1/2—signal transducer and activator of transcription 3 ( STAT3 ) axis, further amplifying inflammation. NOD2 -induced NFκB signaling also primes NLRP3 inflammasome activation through upregulation of NLRP3 and pro-IL-1β, leading to Caspase-1 -mediated inflammation and cell death [ 10 ]. eVm exists as a soluble or cell surface vimentin (CSV) and is released under cellular stress, inflammation, or cell death [ 11 – 13 ].
Elevated levels of eVim and its modified forms have been observed in inflammatory conditions such as sepsis, pulmonary fibrosis, and rheumatoid arthritis [ 14 – 16 ]. Acting as matrix damage-associated molecular patterns (DAMPs), they bind to pattern recognition receptors (PRRs) such as Toll-like receptor (TLR) 2/4 and integrins, promoting neutrophil migration activating NFκB and STAT3 signaling, which further drives NLRP3 inflammasome activation [ 17 ]. eVm may also indirectly contribute to necroptosis by fostering a pro-inflammatory environment. Necroptosis, mediated by receptor-interacting protein kinase 1/3 ( RIPK1/RIPK3 )-dependent phosphorylation of mixed lineage kinase domain-like protein ( MLKL ), leads to plasma membrane rupture and the release of DAMPs.
This process further increases eVim release, creating a vicious inflammatory cycle. Although not directly triggering necroptosis, eVm may promote it by sustaining cytokine-rich conditions [ 18 ]. Given these roles, vimentin likely contributes to IBD inflammation.
NOD2 mutations disrupt its interaction with iVm due to alterations in the leucine-rich repeat [ 19 ]. This impairs bacterial clearance and dysregulates NFκB signaling, leading to chronic inflammation [ 8 , 20 ]. Stress-induced reorganization and extracellular release of vimentin further amplify secondary inflammatory pathways, including inflammasome activation and necroptosis.
Studies with Caspase-8 knockout mice showed that RIP3 knockdown reduces intestinal inflammation, while RIP3 -driven necroptosis increases IL-8, IL-1β, and IL-33 levels [ 21 ]. Consistently, vimentin expression is elevated in the colonic tissues of CD patients compared to healthy individuals [ 22 ]. Vimentin also appears to contribute to IBD-associated fibrosis.
Although extracellular matrix deposition is part of normal repair, it becomes excessive in IBD, resulting in submucosal fibrosis and tissue stiffening [ 23 ]. Epithelial-mesenchymal transition (EMT), a fibrosis-driving process linked to vimentin, has been implicated in CD pathogenesis [ 24 ]. Similarly, UC patients show increased collagen deposition and upregulation of vimentin and other fibrosis markers [ 25 ].
Therapeutic targeting of vimentin has shown promise. ALD-R491, a small molecule that binds iVm, significantly reduced colitis severity in dextran sodium sulfate (DSS) models, outperforming tofacitinib and 5-aminosalicylic acid [ 26 ]. However, targeting eVm in IBD remains unexplored.
Previous studies have used the eVm-targeting monoclonal antibody humanized virus-suppressing factor (hzVSF) to inhibit viral entry in hepatitis B and reduce inflammation in COVID-19 patients [ 27 , 28 ]. Given eVm’s pro-inflammatory role alongside iVm, we hypothesized that its inhibition could attenuate inflammation and fibrosis. This study aimed to evaluate the therapeutic effects of anti-vimentin antibody treatment on IBD in both in vivo and in vitro settings.
Materials and methods Animals Eight-week-old male C57BL/6 mice were purchased from Orient Bio Inc. (Seongnam, Korea). The mice were maintained under specific pathogen-free conditions at the Institute of Medical Science of the Catholic University of Korea with ad libitum access to water and were fed standard mouse chow (Ralston Purina, St.
Louis, MO, USA). The experimental procedures were performed in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experiments provided by the Department of Laboratory Animals of the Institutional Animal Care and Use Committee of the Catholic University of Korea, and conformed to all the guidelines of the National Institutes of Health (USA) (permit numbers: 2017–0067–02). To minimize animal suffering, mice were fully anesthetized under isoflurane gas (2–2.5%) and were euthanized by cervical dislocation.
DSS-induced colitis and administration of anti-vimentin Ab In this study, three groups of mice—normal control, IBD group, and anti-vimentin group, were used to examine the therapeutic implication of anti-vimentin Ab in IBD. Four mouse were allocated to the IBD group, five to the anti-vimentin group, and two to the normal control. IBD was mimicked for both the IBD group and the anti-vimentin Ab group by mixing 3% DSS (MP Biomedicals, Santa Ana, CA, USA) in autoclaved drinking water for five days, whereas the normal control received drinking water without DSS.
Anti-vimentin Ab group, unlike the IBD group, received intraperitoneal injection of monoclonal IgG4 anti-vimentin antibody hzVSF-V13 (3 mg/kg; ImmuneMed, Seoul, Korea) in a three-day interval while being fed with DSS mixed drinking water. This antibody is a monoclonal IgG4 antibody that specifically binds to extracellular vimentin. Previous studies have demonstrated its binding specificity, cross-reactivity to both mouse and human vimentin, and therapeutic effects in murine models of viral infection [ 27 , 29 ].
Importantly, hzVSF-v13 has shown cross-reactivity with both human and non-human vimentin, and its IgG4 isotype indicates a non-cytotoxic, immunomodulatory role [ 30 ]. This particular concentration was chosen because previous studies have indicated that low to intermediate dose in the range of 1–4 mg/kg yields best clinical results both in animal and human studies [ 27 , 30 ]. The mice were monitored daily for body weight and the presence of blood in the stool.
On the eleventh day after DSS administration, mice were euthanized and colons were separated from the small intestine at the ileocecal junction and from the anus at the distal end of rectum. Gross photographs of the colons were taken and their lengths were measured. Colon tissues were fixed in 10% formalin in a cassette for histologic analysis.
Data are representative of two independent experiments. Assessment of colitis severity During the DSS treatment cycles, a disease activity index (DAI) score was assessed to evaluate the progression of colitis. The DAI was a composite score determined by factors such as relative body weight loss, stool softness, and blood in the rectum or stool71.
Each parameter was scored from 0 to 4, with a maximum cumulative score of 12. Body weight loss was scored as follows: score 0, no weight loss compared to initial weight; 1, weight loss within 1–5%; 2, weight loss within 5–10%; 3, weight loss within 10–20%; 4, greater than 20% weight loss. Stool consistency was determined as follows: score 0, normal (solid pellet); 1, soft but in pellet shape; 2, loose stool but with some solidity; 3, loose stool with signs of liquid consistency; 4, watery diarrhea.
Rectal bleeding was scored as follows: score 0, no sign of blood; score 1, no bleeding; 2, slight bleeding; 3, bloody diarrhea; 4, gross bleeding. Mice that reached a DAI score of 12 were considered to have met the humane endpoint criteria and were euthanized within 24 hours. No animals died before meeting the euthanasia criteria.
Intracellular staining and flow cytometry Cells were isolated from the spleen stimulated with 25 ng/mL phorbol myristate acetate and 250 ng/mL ionomycin (Sigma-Aldrich, St. Louis, MO, USA) in the presence of GolgiStop (BD Biosciences, San Jose, CA, USA) for 4 hours. Cells were reacted with anti-CD4 (PerCP Cy5.5, 45-0042-82) and anti-CD25 (APC, 102012) antibodies at 4 °C for 30 minutes, permeabilized with Cytofix/Cytoperm solution (BD Pharmingen, Franklin Lakes, NJ, USA), and stained intracellularly with anti-IL-17 (FITC, 11-7177-81) or anti–Foxp3 (PE, 12-5773-82) antibodies.
Samples were analyzed with a FACSCalibur (BD Pharmingen) fluorescence-activated cell sorting (FACS) instrument, and data were analyzed with FlowJo (Tree Star, Ashland, OR, USA). Histopathological analysis Colon tissue sections (5 μm thick) were fixed and embedded in 10% (v/v) neutral-buffered formalin (Sigma-Aldrich, St. Louis, MO).
Histological analysis was performed on H&E-stained colitis tissue and scored by two experimenters in a blinded fashion. Stained sections were examined under a photomicroscope (Olympus, Tokyo, Japan) (magnification 200×). The histological scoring system was used for evaluation of the degree of colitis [ 31 ].
Loss of epithelium, crypt damage, depletion of goblet cells, and infiltration of inflammatory cells were assessed in the sections. Histological evaluation and scoring of loss of epithelium (0, no loss of epithelium; 1, 0–5% loss of epithelium; 2, 5–10% loss of epithelium; and 3, > 10% loss of epithelium), crypt damage (0, no damage; 1, 0–10% loss of crypt; 2, 10–20% loss of crypt; 3, > 20% loss of crypt), depletion of goblet cells (0, none; 1, mild; 2, moderate; and 3, severe), and infiltration of inflammatory cells (0, none; 1, mild; 2, moderate; and 3, severe) were performed. The total histological score ranged from 0 to 12.
Immunohistochemistry (IHC) Formalin-fixed colon sections were paraffin-embedded and 5 μm sections were deparaffinized using xylene and dehydrated in an alcohol series. At least four sections from each colon were analyzed. The sections were treated with proteinase K in tris-ethylenediaminetetraacetic acid buffer for antigen retrieval and washed in 1 × phosphate-buffered saline (PBS; pH 7.5).
Endogenous peroxidase activity was quenched with methanol and 3% H 2 O 2 . IHC was performed using the Envision Detection System (Dako, Denmark). The tissues were incubated with primary anti-vimentin (ab92547), anti-CSV (H00007431-M08), anti-IL-17 (PA5–79470), anti-IL-1β (NB600–633), anti-IL-6 (NB600–1131), anti- TNFα (ab6671), anti-phosphorylated STAT3 (pSTAT3 ; ab76315), anti- NFκB (ab16502), anti-alpha smooth muscle actin (αSMA; ab7817), anti-collagen-1 (MT; PA5–89281), anti-transforming growth factor beta (TGFβ; BS-0086R), anti- pMLKL (PA5–105677), anti- RIP1 , anti- RIP3 (PA5–19956) and anti- Caspase-1 (PA5–38099) Abs overnight at 4°C followed by a horseradish peroxidase-conjugated secondary Ab for 30 minutes.
The final color product was developed using 3,3-diaminobenzidine (Dako). The sections were counterstained with Mayer’s hematoxylin and examined by photomicroscopy (Olympus). Intracellular staining and flow cytometry Cells were isolated from the spleen, stimulated with 25 ng/mL phorbol myristate acetate and 250 ng/mL ionomycin (Sigma-Aldrich) in the presence of GolgiStop (BD Biosciences, San Jose, CA, USA) for 4 h.
Cells were reacted with anti-CD4 (PerCP-Cy5.5, 45-0042-82) and anti-CD25 (APC, 102012) Abs at 4°C for 30 min; permeabilized with Cytofix/Cytoperm solution (BD Pharmingen, Franklin Lakes, NJ, USA); and stained intracellularly with anti-IL-17(FITC, 11-7177-81) or anti-Foxp3 (PE, 12-5773-82) Abs. Samples were analyzed using the Calibur (BD Pharmingen) fluorescence-activated cell sorting (FACS) instrument, and data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Stimulation of CCD-18CO cells To confirm the effect of vimentin and anti-vimentin Ab in vitro , CCD-18CO cells from Korean Cell Line Bank (Seoul, Korea) were used.
The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, 12,800,017; Gibco, New York, NY, USA), supplemented with 10% (v/v) fetal bovine serum (FBS, #16,000,044; Gibco), and maintained at 37 °C in a 5% CO 2 atmosphere. CCD-18CO cells were seeded in six-well plates at an initial density of 5 × 10 4 cells per well and allowed to adhere for 24 hours. Next, the medium was replaced with serum-free DMEM with 1x insulin-transferrin-selenium-sodium pyruvate (ITS-A) solution for 24 hours.
The medium was replaced with a fresh medium and the cells were treated with the anti-vimentin Ab (10 μg/mL) or recombinant vimentin (10 ng/mL) with or without 10 ng/mL human recombinant TGFβ for 24 hours. Finally, the cells were harvested for western blotting. Protein fractionation Proteins in the cells were extracted using radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Cat.
89,901; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with complete protease inhibitor (Cat. 78,438; Thermo) and 0.5 M ethylenediaminetetraacetic acid (EDTA; 78,438; Fisher). After incubating the cocktail solution on ice for 20 minutes, the samples were centrifuged at 14,000 g for 15 minutes at 4 °C.
The supernatants were collected into new tubes. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Cat. 23,225, Thermo).
Immunoblot analysis Proteins (10 µg) were separated via 10% gradient SDS-PAGE, transferred to Hybond enhanced chemiluminescence (ECL) membranes (10,600,001; Cytiva), and incubated with Abs against anti-alpha smooth muscle actin (α-SMA) (ab7817[1A4]; Abcam, 1:1,000), anti-Col1a1 (PA5–89281; Invitrogen, 1:1,000), anti-fibronectin (ab2414; Abcam, 1:1,000), anti-pSTAT3 (Y705) (#9145; Cell signaling, 1:1000), anti-STAT3 (#9139; Cell signaling, 1:1000), anti-NF-κB p65 (#3033s; Cell signaling, 1:1000), and β-actin (SC-47778; Santa Cruz,:1000) using the SNAP i.d. Protein Detection System (Millipore, Billerica, IL, USA). After incubation with the corresponding secondary anti-mouse (SC-2005; Santa Cruz) and anti-rabbit (SC-2357; Santa Cruz) Abs, protein bands were visualized using X-film (100NIF; EA8EC).
Data are representative of two independent experiments. Statistical analysis Statistical analysis was performed using Prism (GraphPad Software, USA). Normally distributed continuous data were analyzed by parametric Student’s t -test.
Differences in means among groups were subjected to analysis of variance followed by Bonferroni post hoc test. Values are means ± standard error of the mean (SEM). Value of p < 0.05 was indicative of statistical significance.
Ethics approval and consent to participate The animal study was reviewed and approved by the Laboratory Animals Welfare Act (permit number 2024-0183-01], the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experiments of the Department of Laboratory Animals of the Institutional Animal Care and Use Committee of the Catholic University of Korea (permit number 2024-0183-01). Results The anti-vimentin Ab improved the symptoms of DSS-induced colitis To investigate the effect of vimentin blockade on the development of IBD, anti-vimentin Ab was intraperitoneally injected repeatedly at 3-day intervals, simultaneously with 3% DSS administration. In DSS-induced colitis mice, the anti-vimentin Ab improved weight loss ( Fig 1a ).
The anti-vimentin Ab also restored colon length on day 11 after treatment, as compared to the control group (6.11 ± 0.12 vs. 5.57 ± 0.06, p < 0.05; Fig 1b ). The anti-vimentin Ab treated group had a significantly lower histological score than the DSS group ( Fig 1c ).
Compared to the DSS group, the vimentin Ab-treated group had a smaller fraction of IL-17 + cells (12.14 ± 5.56 vs. 6.67 ± 3.68) and a larger fraction of CD25 + Foxp3 + cells (1.88 ± 0.30 vs. 2.0 ± 0.59) among total ex vivo splenic CD4 + cells ( Fig 1d ).
Immunohistochemical analysis was performed on colon tissues to determine the levels of vimentin and CSV. Compared with normal mice, colon tissue sections of DSS-induced colitis mice showed significantly higher total vimentin (21.29 ± 2.37 vs. 10.62 ± 0.87, p < 0.005) and CSV levels (24.52 ± 0.69 vs.
11.08 ± 1.10, p < 0.0001). Treatment with the anti-vimentin Ab reduced the increased total vimentin and CSV levels in colon tissues of DSS-induced colitis mice to levels similar to those in normal mice ( Fig 1e ). 10.1371/journal.pone.0352641.g001 Fig 1 Anti-vimentin Ab improved the symptoms of dextran sulfate sodium-induced ulcerative colitis.
Acute colitis was induced in C57BL/6 mice by oral administration of 3% DSS in distilled water for 5 days, followed by regular drinking water for 7 days. Anti-vimentin Ab 3 mg/kg was injected intraperitoneally, simultaneously with DSS injection at 3-day intervals (DSS induced mice, n = 4, Anti-vimentin ab with DSS induced mice, n = 5, normal mice, n = 2). (a) Weight expressed as a percentage from day 0.
(b) Colon length of DSS-induced mice evaluated on day 12 after treatment. (c) On day 11, colon tissue sections were stained with hematoxylin and eosin. Representative images are shown (original magnification 200× , scale bar 100 μm).
(d) Frequencies of IL-17 + CD4 + cells and CD25 + Foxp3 + CD4 + cells in mouse spleen analyzed by flow cytometry. (e) On day 11, colon tissue sections were stained with anti-vimentin and anti-cell surface vimentin Abs. Representative images are shown (original magnification 400 × , scale bar 100 μm).
Percentages of positive cells among total cells are shown. Data are means ± SEM (*p < 0.05, **p < 0.005, ****p < 0.001). DSS: Dextran sodium sulfate; Ab: Antibody; NC: Normal control; CSV: Cell surface vimentin; H&E: Hematoxylin and eosin; FACS: Fluorescence-Activated Cell Sorting; Treg: Regulatory T cells.
The anti-vimentin Ab suppressed inflammation-related transcriptional activity in murine colon tissues with colitis To determine the effect of the anti-vimentin Ab on the transcriptional activities of pro-inflammatory pathways, pSTAT3 (Y705) and NFκB were examined by immunohistochemical analysis. Compared to normal mice, the colon tissues of DSS-induced colitis mice had larger numbers of pSTAT3 + (91.50 ± 6.00 vs. 31.40 ± 5.14, p < 0.0005) and NFκB + (92.25 ± 9.58 vs.
39.80 ± 5.05, p < 0.005) cells. The anti-vimentin Ab significantly reduced the numbers of these cells, as compared to DSS-treated mice ( Fig 2a ). Confocal immunofluorescence microscopy was performed on colonic tissue sections, staining for CD4, IL-17, CD25, and Foxp3 to identify Th17 and regulatory T (Treg) cells, respectively.
Quantification was based on multiple high-power fields per sample. Quantitative analysis revealed that, compared to the DSS group, the anti-vimentin antibody-treated group exhibited fewer IL-17 ⁺ cells (62.75 ± 7.96 vs. 15.40 ± 2.89, p < 0.0005) and a higher number of CD25 ⁺ Foxp3 ⁺ cells (11.50 ± 3.28 vs.
34.80 ± 6.36, p < 0.05) among CD4 ⁺ cells ( Fig 2b ). These results indicate that blockade of vimentin by an anti-vimentin Ab may influence the transcriptional activities of pro-inflammatory pathways while also modulating intestinal inflammation through interaction with Th17 and Treg cells. 10.1371/journal.pone.0352641.g002 Fig 2 Anti-vimentin Ab suppressed inflammation-related transcriptional activity in DSS-induced colitis mice.
Acute colitis was induced in C57BL/6 mice by oral administration of 3% DSS in distilled water for 5 days, followed by regular drinking water for 7 days. Anti-vimentin Ab 3 mg/kg was injected intraperitoneally, simultaneously with DSS injection at 3-day intervals (DSS induced mice, n = 4, Anti-vimentin ab with DSS induced mice, n = 5, normal mice, n = 2). (a) On day 11 after DSS administration, colon tissue sections were stained with antibodies for pSTAT3 and NFκB .
Representative images are shown (original magnification 400 × , scale bar 100 μm). Percentages of positive cells among total cells are shown. (b) Confocal immunofluorescence microscopy was performed on colonic tissue sections, staining for CD4, IL-17, CD25, and Foxp3 to identify Th17 and regulatory T (Treg) cells, respectively.
Quantification was based on multiple high-power fields per sample. Data are means ± SEM (*p < 0.05, **p < 0.005, ***p < 0.0005). Ab: Antibody; DSS: Dextran sodium sulfate; IBD: inflammatory bowel disease; No.: Number; NC: Normal control; pSTAT3 : Phosphorylated signal transducer and activator of transcription 3; NFκB : Nuclear factor kappa B; Treg: regulatory T cells.
The anti-vimentin Ab decreased the number of inflammatory cytokine-secreting cells in murine colon tissue with colitis Immunohistochemical analysis of colon tissues was performed to determine whether the anti-vimentin Ab reduces the number of inflammatory cytokine-secreting cells in colon tissues of mice with DSS-induced colitis. The anti-vimentin Ab significantly reduced the increased numbers of IL-17 (133.75 ± 15.89 vs. 85.20 ± 7.23, p < 0.05), IL-6 (165.25 ± 16.00 vs.
76.40 ± 5.38, p < 0.0005), TNFα (101.00 ± 20.75 vs. 63.60 ± 9.36, p < 0.05), and IL-1β+ (231.50 ± 21.83 vs. 134.80 ± 14.95, p < 0.05) cells in the intestinal tissues of mice with DSS-induced colitis ( Fig 3 ).
The results indicate that the anti-vimentin Ab influenced the levels of inflammatory cytokines in inflamed colon tissue. 10.1371/journal.pone.0352641.g003 Fig 3 Anti-vimentin Ab decreased the number of inflammatory cells in murine colon tissue with colitis. Acute colitis was induced in C57BL/6 mice by oral administration of 3% DSS in distilled water for 5 days, followed by regular drinking water for 7 days.
Anti-vimentin Ab 3 mg/kg was injected intraperitoneally, simultaneously with DSS injection at 3-day intervals (DSS induced mice, n = 4, Anti-vimentin ab with DSS induced mice, n = 5, normal mice, n = 2). On day 11 after DSS administration, colon tissue sections were stained with antibodies for IL-17, IL-6, TNFα , and IL-1β. Percentages of positive cells among total cells are shown.
Data are means ± SEM (*p < 0.05, ***p < 0.0005). DSS: Dextran sodium sulfate; NC: Normal control; Ab: Antibody; No.: Number; TNFα : Tumor necrosis factor alpha. The anti-vimentin Ab decreased the levels of necroptosis-related marker in murine colon tissues with colitis Markers associated with necroptosis- and inflammasome-related inflammatory signaling, including pMLKL, RIPK1, and RIPK3, were detected in colon tissues from mice with DSS-induced colitis.
Anti-vimentin Ab treatment was associated with a significant reduction in the numbers of pMLKL⁺ (184.50 ± 11.32 vs. 78.00 ± 4.58, p < 0.0001), RIPK1⁺ (253.75 ± 13.31 vs. 139.20 ± 7.51, p < 0.0001), RIPK3⁺ (189.50 ± 11.06 vs.
81.00 ± 6.58, p < 0.0001), and Caspase-1⁺ (198.75 ± 11.84 vs. 126.80 ± 4.58, p < 0.001) cells compared with DSS-treated mice ( Fig 4 ). 10.1371/journal.pone.0352641.g004 Fig 4 Anti-vimentin Ab decreased the number of inflammatory necroptotic cells in murine colon tissues with colitis.
Acute colitis was induced in C57BL/6 mice by oral administration of 3% DSS in distilled water for 5 days, followed by regular drinking water for 7 days. Anti-vimentin Ab 3 mg/kg was injected intraperitoneally, simultaneously with DSS injection at 3-day intervals (DSS induced mice, n = 4, Anti-vimentin ab with DSS induced mice, n = 5, normal mice, n = 2). On day 11 after DSS administration, colon tissue sections were stained with antibodies for pMLKL , RIP3 , RIP1 , and Caspase-1 .
Percentages of positive cells among total cells are shown. Data are means ± SEM (***p < 0.0005, ****p < 0.001). Ab: Antibody; NC: Normal control; IBD: Inflammatory bowel disease; DSS: Dextran sodium sulfate; No.: Number; pMLKL : Phosphorylated mixed lineage kinase domain like pseudokinase; RIP3 : Receptor-interacting protein kinase-3; RIP1 : Receptor-interacting protein kinase-1.
These markers are known to be upregulated in inflammatory conditions and are associated with necroptosis- and inflammasome-related signaling pathways. Therefore, the observed reduction following anti-vimentin Ab treatment was interpreted as attenuation of inflammation-associated signaling environments rather than direct inhibition of necroptotic or pyroptotic cell death execution. The anti-vimentin Ab reduced the levels of fibrosis-related markers in mice with colitis To investigate the antifibrotic effect of the anti-vimentin Ab in mice with DSS-induced colitis, colon sections were stained with Masson’s trichrome.
In mice with colitis, increased collagen deposition and severe intestinal structure damage due to infiltration of inflammatory cells were observed. These effects were in part reversed by the anti-vimentin Ab. Compared to normal mice, the colon tissue of mice with DSS-induced colitis had larger numbers of cells expressing fibrosis-related markers in αSMA (153.00 ± 9.57 vs.
98.80 ± 7.81, p < 0.005), Col1α1 (165.25 ± 16.00 vs. 74.20 ± 7.39, p < 0.0005), and TGFβ (101.00 ± 20.75 vs. 69.4 ± 12.11).
The anti-vimentin Ab significantly reduced the numbers of cells expressing fibrosis-related markers, compared to DSS-treated mice ( Fig 5 ). 10.1371/journal.pone.0352641.g005 Fig 5 Anti-vimentin Ab reduced fibrosis in mice with colitis. Acute colitis was induced in C57BL/6 mice by oral administration of 3% DSS in distilled water for 5 days, followed by regular drinking water for 7 days.
Anti-vimentin Ab 3 mg/kg was injected intraperitoneally, simultaneously with DSS injection at 3-day intervals (DSS induced mice, n = 4, Anti-vimentin ab with DSS induced mice, n = 5, normal mice, n = 2). (a) On day 11 after DSS administration, colon sections were stained with Masson’s trichrome. The percentage collagen-positive area was analyzed as ImageJ.
(b) Colon tissue sections were stained with antibodies for α-SMA, Col1A1, and TGFβ. Numbers of antibody-positive cells are shown. Data are means ± SEM (**p < 0.005, ***p < 0.0005).
DSS: dextran sodium sulfate; Ab: Antibody; NC: Normal control; IBD: Inflammatory bowel disease; αSMA: Alpha smooth muscle actin; Col1α1: Collagen type I alpha 1; TGFβ: Transforming growth factor beta; MT: Masson’s trichrome stain for collagen. The anti-vimentin Ab reduced fibrosis-related response and increased inflammation in human colon epithelial cells in vitro We investigated the effect of vimentin on fibrosis using CCD-18CO human colon epithelial cells. When cells were treated with TGF-β and cultured for 24 hours, the levels of fibrosis-related factors such as α-SMA (1.75 ± 0.17 vs.
1.07 ± 0.20), Col1a1 (1.20 ± 0.49 vs. 2.30 ± 0.37), and fibronectin (3.24 ± 1.22 vs. 2.30 ± 0.37) increased, with the change in fibronectin showing statistical significance.
Treatment with vimentin further increased the levels of fibrosis-related factors induced by TGF-β ( Fig 6a ). To determine whether blocking vimentin improves fibrosis in human colon epithelial cells, TGF-β and the anti-vimentin antibody were added to CCD-18CO cells. In cells concomitantly administered the anti-vimentin antibody, the levels of fibrosis markers decreased, and the reduction in fibronectin expression was statistically significant (α-SMA: 1.21 ± 0.49 vs.
1.76 ± 0.64, Col1α1: 1.20 ± 0.49 vs. 2.30 ± 0.37, fibronectin 1.61 ± 0.51 vs. 3.34 ± 0.49; Fig 6b ).
Lastly, the effect of anti-vimentin antibody treatment on the transcriptional activity of pro-inflammatory pathways—pSTAT3 and NF-κB—was evaluated by western blot analysis. Upon treatment with TGF-β for 24 hours, both pSTAT3 and NF-κB protein levels were elevated. The addition of anti-vimentin antibody significantly reduced the expression of pSTAT3 (1.57 ± 0.18 vs.
1.12 ± 0.28, p < 0.05) and NF-κB (1.56 ± 0.11 vs. 1.00 ± 0.18, p < 0.05) protein levels ( Fig 6c ). 10.1371/journal.pone.0352641.g006 Fig 6 The anti-vimentin Ab reduced fibrosis and increased inflammation in human colon epithelial cells in vitro.
CCD-18CO cells were starved for 24 hours with serum-free DMEM and stimulated with recombinant vimentin (10 ng/mL) (a) or anti-vimentin Ab (10 μg/mL) (b) with or without TGFβ (10 ng/mL) for 24 hours. The levels of αSMA, Col1a1, and fibronectin were analyzed by western blotting. (c) the effect of anti-vimentin antibody treatment on the transcriptional activity of pro-inflammatory pathways— pSTAT3 (Y705) and NFκB —was evaluated by western blot analysis.
Data are means ± SEM (*p < 0.05**p < 0.005). Ab: Antibody; αSMA: Alpha smooth muscle actin; Col1α1: Collagen type I alpha 1; TGFβ: Transforming growth factor beta; ng: Nanogram; ml: Milliliter; rh: Recombinant human; pSTAT3 : Phosphorylated signal transducer and activator of transcription 3; NFκB : Nuclear factor kappa B. Discussion Vimentin is a potential therapeutic target in IBD, as indicated by its upregulation following tissue injury and its involvement in multiple signaling pathways linked to cell migration during wound repair.
It also modulates the functions of immunocytes and participates in the pro-inflammatory responses required to fend off infection by interacting with inflammasome NLRP3 and inducing cytokine production [ 32 ]. This process is also in part mediated by necroptosis, which is a result of continued mucosal barrier disruption in IBD. We aimed to recreate the inflammatory environment of IBD in mouse and determine the effect of controlling the eVm level using an anti-vimentin Ab.
DSS-induced colitis mice displayed clinical characteristics that resembled IBD. The overall weight, length, and histological score of colon samples were severely impaired, possibly due to extensive colonic inflammation. The mice treated with the anti-vimentin Ab showed better clinical outcomes than those with DSS-induced colitis ( Fig 1a , b , c ).
In ex vivo spleen FACS, the anti-vimentin Ab-treated mice with DSS-induced colitis had a lower percentage of cells with IL-17 and a higher percentage of Treg cells ( Fig 1d ). Likewise, confocal immunofluorescence microscopy on colonic tissue sections revealed that compared to the DSS group, the anti-vimentin antibody-treated group exhibited fewer IL-17 ⁺ cells and a higher number of CD25 ⁺ Foxp3 ⁺ cells among CD4 ⁺ cells ( Fig 2b ). Based on the roles of IL-17 and Tregs in innate immunity, it can be inferred that inhibiting eVim can modulate intestinal inflammation.
This correlation was confirmed by immunochemical analysis of the vimentin levels in normal mice, mice with DSS-induced colitis, and anti-vimentin Ab-treated mice with DSS-induced colitis. Colon tissues from the DSS-induced colitis group had the highest total vimentin and CSV; tissue in the vimentin Ab treated DSS-induced colitis group was similar to that of normal mice ( Fig 1e ). Intraperitoneal injection of the anti-vimentin Ab to mice treated with different DSS concentrations resulted in similar weights, colon lengths, and histologic scores.
Our findings implicate vimentin in multiple pathogenic mechanisms of IBD, namely, inflammation, fibrosis-related response, and necroptosis. Both in vivo and in vitro studies showed that the levels of fibrosis-related response markers were elevated in the mice with DSS-induced colitis, as compared to anti-vimentin Ab treated mice with DSS-induced colitis ( Figs 5 , 6a , 6b ). This is in agreement with reports of a link between increased collagen deposition and fibrosis with upregulation of vimentin.
Given the role of vimentin intermediate filaments in wound healing and fibrosis, this is unsurprising. This dependency of fibrosis on the eVm level suggests that the severity of fibrosis in IBD can be ameliorated by an anti-vimentin Ab that targets eVim including CSV. Although differences in the expression of fibrosis-associated markers such as αSMA, Col1a1, and fibronectin between the DSS group and the anti-vimentin Ab group were not statistically significant in an in vitro study using CCD-18CO human colon cell line, we believe this may be attributed to the limited number of replicates rather than a lack of biological effect.
This is further supported by the significant anti-fibrotic effects observed in our in vivo murine models. Follow-up studies with larger sample sizes and optimized in vitro conditions to further assess the therapeutic potential of anti-vimentin antibody in human colon-derived cells is needed. Controlling the vimentin level suppressed several pro-inflammatory pathways and inflammatory cytokine production.
The activation levels of pro-inflammatory pathways in pSTAT3 and NFκB were higher in the mice with DSS-induced colitis than in the anti-vimentin Ab-treated mice with DSS-induced colitis ( Fig 2a ). Similar results were obtained for the inflammatory cytokines IL-17, IL-6, TNFα , and IL-1β ( Fig 3 ). The DSS-induced colitis group had higher levels of these cytokines than the anti-vimentin Ab-treated DSS-induced colitis group.
In vitro western blot analysis of colon tissue lysates revealed a similar pattern. Upon treatment with TGFβ, both pSTAT3 and NFκB protein levels were elevated. The addition of anti-vimentin antibody appeared to reduce the expression of both proteins; however, these reductions did not reach statistical significance, which we believe has to do with the limited number of replicates rather than an absence of biological effect ( Fig 6c ).
The fact that in vitro study displayed a similar trend in pSTAT3 and NFκB upon administration of anti-vimentin Ab is nonetheless a noteworthy finding. Considering eVm’s ability to activate NFκB / STAT3 pathway and further trigger NLRP3 inflammasome activation, our results are in accordance with previous findings and demonstrate the importance of vimentin in the inflammatory pathogenesis of IBD [ 8 , 9 ]. Cell necroptosis is a key driver of inflammation initiation and aggravation [ 33 ].
Damage to the intestinal mucosal barrier, and the subsequent higher permeability of intestinal epithelial cells, may contribute to the development of IBD [ 34 ]. Necroptosis, a process of cell death that is normally regulated, is implicated in the development of intestinal inflammation [ 20 ]. Studies using caspase-8 knockout mice have shown that RIP3 knockdown ameliorates intestinal inflammation, whereas necroptosis induced by RIP3 increased the expression of the inflammatory cytokines IL-8, IL-1β, and IL-33, among others [ 21 ].
Necroptosis induces translocation of NFKβ and assembly of the NLRP3 inflammasome [ 35 ]. These mouse studies have shown that necroptosis of intestinal epithelial cells aggravates intestinal inflammation. Regulation of necroptosis can suppress UC, but the serum level of TNFα and the mucosal tissue level of RIP3 are increased in patients with CD [ 20 ].
Although this study was not aimed at suppressing necroptosis, we found that regulating eVm with the anti-vimentin Ab suppressed the levels of necroptotic markers. The anti-vimentin Ab-treated DSS-induced colitis group showed decreased levels of necroptotic markers ( pMLKL, RIP3, RIP1, and Caspase1 ), as compared to the DSS-induced colitis group ( Fig 5 ). While eVm does not directly exert influence on neroptosis, blocking eVm indirectly attenuate necroptosis by reducing inflammation, lowering TNFα levels, and disrupting DAMP-induced signaling loops [ 36 ].
A limitation of this study is the lack of analysis on the dose-dependent effects of anti-vimentin antibody treatment. We used a single dosage of 3 mg/kg, based on its demonstrated efficacy in prior studies. In a woodchuck hepatitis virus model, Korolowicz et al.
(2021) tested multiple doses (0.1, 4.0, and 16.0 mg/kg) and found that an intermediate dose yielded the strongest therapeutic response. Similarly, in a clinical trial for COVID-19, investigators reported better outcomes with a lower dose (200 mg) compared to a higher dose (400 mg) (Kang, Choe et al. 2020, Prasenohadi, Burhan et al.
2022). In our preliminary tests, a higher dose (9 mg/kg) produced comparable therapeutic outcomes but was associated with increased mortality. Based on these findings, we proceeded with the 3 mg/kg dosage.
Future studies are needed to determine whether anti-vimentin antibody efficacy is dose-dependent within a lower dose range. In summary, an Ab that reduced the extracellular vimentin level improved the clinical symptoms of IBD in a mouse model. These improved symptoms were correlated with decreased fibrosis, inflammation, and necroptosis in vivo and in vitro .
To our knowledge, this is the first attempt to use an Ab targeting vimentin in IBD. Currently, IBD requires multimodal therapy that includes a wide spectrum of medications from aminosalicylates to biologics such as anti-TNF agents or anti-IL-23 antibodies. Encompassing anti-vimentin Ab, which offers a symptomatic aid by effecting a novel pathway, as a part of this multimodal approach would provide a more comprehensive method of attacking IBD pathophysiology.
Further studies should analyze whether the effect is dose dependent, and whether the results would be replicated in human.