Open Access, Peer-reviewed
pISSN 1598-2629 eISSN 2092-6685
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Immune Netw. 2024 Oct;24(5):e37. English.
Published online Oct 16, 2024.
https://doi.org/10.4110/in.2024.24.e37
Copyright © 2024. The Korean Association of Immunologists
Original Article

STING-STAT6 Signaling Pathway Promotes IL-4+ and IFN-α+ Fibrotic T Cell Activation and Exacerbates Scleroderma in SKG Mice

Kun Hee Lee,1,2 Jin Seok Woo,1 Ha Yeon Jeong,1,2 Jeong Won Choi,1,2 Chul Hwan Bang,3 Jeehee Youn,4 view all
    • 1Lab of Translational ImmunoMedicine, Catholic Research Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea.
    • 2Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea.
    • 3Department of Dermatology, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea.
    • 4Department of Anatomy & Cell Biology, College of Medicine, Hanyang University, Seoul 04763, Korea.
    • 5Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea.
    • 6Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea.
  • Correspondence to Sung-Hwan Park. Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea. Email: rapark@catholic.ac.kr
    Correspondence to Mi-La Cho. Department of Pathology, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea. Email: iammila@catholic.ac.kr
Received November 28, 2023; Revised September 21, 2024; Accepted September 25, 2024.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

AbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALSNotesAbbreviationsACKNOWLEDGEMENTSReferences

Abstract

Systemic sclerosis (SS) is an autoimmune disease and pathological mechanisms of SS are unclear. In this study, we investigated the role of T cells in the progression of SS using SKG mice and humanized mice. SKG mice have a spontaneous point mutation in ZAP70. We induced scleroderma in SKG mice and a humanized SS mouse model to assess whether T cell-mediated immune responses induce SS. As a result, we found increased dermal thickness, fibrosis, and lymphocyte infiltration in skin tissue in SKG SS mice compared to BALB/c mice (control). Also, blood cytokine level, including IL-4- and IFN-α which are produced by CD4+ T cells via STIM1/STING/STAT6/IRF3 signaling pathways, were increased in SKG mice. Interestingly, skin fibrosis was reduced by inhibiting STING pathway in skin fibroblast. Next, we demonstrated the pathophysiological role of IL-4 and IFN-α in skin fibrosis using a humanized SS mouse model and found increased IL-4- and IFN-α-producing CD4+ T cells and fibrosis. In this study, we found that STING-induced production of IL-4- and type I IFN by CD4+ T cells is a key factor in mouse model and humanized mouse model of SS. Our findings suggest that the STING/STAT6/IRF3 signaling pathways are potential therapeutic targets in SS.

Keywords
Systemic sclerosis; T cell; STING; STAT6 transcription factor; Cytokine

Scleroderma, also known as systemic sclerosis (SS), is an autoimmune disease characterized by thickening of the skin, fibrosis, and vasculopathy (1). The pathological mechanisms of SS are unclear; however, pro-inflammatory cytokines and the tissue microenvironment are important in its development (2, 3).

Collagen, the main effector molecule in SS, is more abundant in skin with mononuclear cell infiltration (4). Inflammatory cytokines secreted by immune cells enhance the deposition of collagen (5). The levels of Th cells that produce IL-4 (Th2) and IL-17 (Th17) are increased in skin and blood in SS (6, 7, 8). However, IL-17A in SS patients may induce inflammatory responses while protecting against fibrosis (9).

Calcium (Ca2+) signaling plays an important role in the regulation of the immune system (10). Store-operated Ca2+ entry (SOCE) is a major Ca2+ signaling pathway in the immune system. It is regulated by stromal interaction molecule 1 (STIM1) and acts as a Ca2+ sensor at the endoplasmic reticulum (ER) membrane. Activated STIM1 translocates from the ER membrane to the plasma membrane (PM) and interacts with Calcium release-activated calcium channel protein 1 (ORAI1), which acts as a Ca2+ channel at PM (11). STIM1 also regulates the activity of stimulator of interferon genes (STING) (12), an ER-resident protein upstream of type I IFN production in myeloid cells (13, 14). DNA-bound cGAS produces a cyclic dinucleotide, 2′,3′-cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), which is a STING ligand (15). cGAMP provokes translocation of STING from the ER to the ER-Golgi compartment, recruiting TANK-binding kinase 1 (TBK1) (16). Interferon regulatory factor 3 (IRF3) and signal transducer and activator of transcription 6 (STAT6) (17, 18), which are transcription factors that induce type I IFN and IL-4, respectively, are phosphorylated and translocate to the nucleus to produce type I IFN and IL-4 (19, 20).

The SKG mouse has a point mutation of the gene encoding TCR–z chain-associated protein 70 (ZAP70), a key molecule in signal transduction in T cells, and develops spontaneous chronic autoimmune arthritis (21, 22). The expression of IL-1, TNF-α, and IL-6 is increased in arthritic SKG mice (23). Thus, external stimuli may trigger autoimmune diseases in SKG mice by stimulating autoimmune T cells.

To assess the role of T cells in SS, SKG mice were subcutaneously injected with bleomycin (BLM). Next, we evaluated whether BLM-treated SKG mice exhibited more of the SS phenotype than BLM-treated BALB/c, and if so, determined the underlying molecular mechanisms. We investigated whether the STIM1-STING pathway is activated in CD4+ T cells in BLM-treated SKG mice. We also explored whether IL-4- and IFN-α-producing CD4+ T cells exist by increase in phospho-STAT6 (pSTAT6) and phospho-IRF3 (pIRF3) and whether skin fibrosis is induced by these cytokines. Finally, we confirmed that inhibition of STING-STAT6 pathway decreased IL-4- and IFN-α-producing CD4+ T cells and suppressed skin fibrosis.

Animals

Eight-week-old BALB/c and SKG male mice were used. BALB/c mice were purchased from OrientBio (Seongnam, Korea) and SKG mice were from Saeronbio (Uiwang, Korea). Seven-week-old NOD/scid/IL-2Rγ–/– female mice (NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJ; NSG) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). They were fed standard mouse chow (Ralston Purina, St. Louis, MO, USA) and water ad libitum. Experimental procedures were reviewed and approved by the Animal Research Ethics Committee of the Catholic University of Korea (Seoul, Korea).

BLM, H-151 and AS1517499 treatment

The mice were anesthetized with isoflurane, and their backs were shaved. They were given daily subcutaneous injections of 0.5 mg/ml BLM in 100 μl for 2 wk (Dong-A Pharm, Seoul, Korea). They were euthanized 3 weeks after the final BLM treatment. Back skin tissue was fixed in 10% formalin for histological analysis. Spleen tissue was embedded in Optimal Cutting Temperature Compound (LEICA, 3801480) for cryosectioning. H-151 (1 or 10 mg/kg) in 10% DMSO/40% PEG300/5% Tween-80/45% Saline was injected intraperitoneally three times a week for 3 wk after the final injection of BLM. AS1517499 (10 mg/kg) in 20% DMSO in saline was subcutaneously injected three times a week for 3 weeks after the final injection of BLM.

Histological assessment

Harvested tissue was fixed in 10% formalin and embedded in paraffin. The tissue was sliced at 5 µm thickness and placed on standard microscopy slides. Sections were stained with Hematoxylin & Eosin and Masson’s trichrome (MT). MT staining was conducted using the Trichrome Stain (Masson) Kit (HT15, Sigma-Aldrich, St. Louis, MO, USA). After deparaffinization and rehydration, the slides were immersed in Bouin’s solution (HT 10132, Sigma-Aldrich) at 56°C for 1 h. Subsequently, the slides were washed with tap water for 5 min. Next, the tissues were stained with Weigert’s hematoxylin for 5 min, and washed with tap water for 5 min. The slides were stained in Biebrich scarlet-acid fuchsin for 5 min, rinsed in distilled water, incubated in phosphotungstic-phosphomolybdic acid for 5 min, dyed with aniline blue for 5 min, and fixed in 1% acetic acid for 2 min. Finally, the slides were washed in distilled water, dehydrated, and mounted. Dermal thickness was measured as described previously.

Immunohistochemistry

Harvested tissue was fixed in 10% formalin and embedded in paraffin. The tissue was sliced at 5 µm thickness and placed on standard microscopy slides. Immunohistochemistry was performed using the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA). Tissue was incubated with primary Abs against α-smooth muscle actin (α-SMA), collagen 1, IL-1β, IL-6, TNFα, IL-4, IL-17, phospho-STING, phospho-IRF3, phospho-STAT6, and IFNα overnight at 4°C. The primary Abs were detected using a biotinylated secondary Ab, followed by incubation with streptavidin-peroxidase complex for 30 min. The final color was produced using DAB chromogen (Dako, Carpinteria, CA, USA). Stained cells, which were identified according to a dark brown deposit in the nucleus, were enumerated in three randomly selected high-power fields (HPFs; 400×; 2.37 mm2). The sections were counterstained with hematoxylin and photographed with an Olympus photomicroscope (Tokyo, Japan).

Flow cytometry

Cells were isolated from the spleens of BALB/c and SKG mice. Prior to staining, cells were stimulated for 3 days with LPS (1 µg/ml) and treated for 4 h with Golgistop (BD Biosciences, San Jose, CA, USA). The cells were surface-stained with PerCP-conjugated anti-CD4 Abs (eBioscience). Then these cells were permeabilized using Cytofix/Cytoperm solution (BD Pharmingen, Franklin Lakes, NJ, USA), and subjected to intracellular staining using PE-conjugated anti-IL-4 (BD Biosciences), APC-conjugated anti-IL-17 (eBioscience), FITC-conjugated anti-IFNα (R&D Systems Minneapolis, MN, USA), anti-phosphor STING (Invitrogen, Waltham, MA, USA), anti-phosphor IRF3 (Cell Signaling Technology, Danvers, MA, USA), and anti-phosphor STAT6 (Santa Cruz Biotechnology, Dallas, TX, USA) Abs. Next, anti-phosphor STING, anti-phosphor IRF3, and anti-phosphor STAT6 were detected using an Efluor 450-conjugated secondary Ab (Invitrogen) or APC-conjugated secondary Ab (BD Biosciences). Flow cytometry was performed on a cytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

T-cell differentiation

Splenocytes were acquired from spleen tissues of BALB/c mice and SKG mice and sieved through a mesh screen. Next, red blood cells were lysed in hypotonic ACK buffer. The remaining splenocytes were maintained in RPMI 1640 medium containing 5% fetal bovine serum. Before T cell differentiation, the plate was coated with anti-CD3 (0.5 μg/ml; BD Pharmingen) for 2 h at 37°C. Next, splenocytes were cultured with anti-CD28 (1 μg/ml) Ab, anti-IFN-γ (2 μg/ml) Ab, and IL-4 (10 ng/ml) in the absence or presence of H-151 (0.5 μM; InvivoGen), or AS1517499 (10 μM) for 3 days. Recombinant mouse IL-4 and Abs to CD28 and IFN-γ were purchased from R&D Systems.

Confocal microscopy

Skin tissue sections of 5 µm thickness, spleen tissue sections of 7 µm thickness, splenocytes from spleen tissues of BALB/c and SKG mice, and human skin fibroblasts were used for immunostaining. Splenocytes were attached to standard microscopy slides using Cytospin. We used anti-CD4 (Novus Biologicals, Centennial, CO, USA), anti-IL-4 (Invitrogen), anti-IL-17 (Invitrogen), anti-IFNα (Invitrogen), anti-phosphor STING (Invitrogen), anti-phosphor IRF3 (Cell Signaling), anti-phosphor STAT6 (Invitrogen), anti-GM130 (Santa Cruz Biotechnology), anti-STING (Cell Signaling Technology), anti-STIM1 (Invitrogen), anti-ORAI1 (Invitrogen) Abs, α-SMA and fibronectin (Abcam, Cambridge, UK). Slides were visualized under a microscope (LSM 700; Carl Zeiss, Oberkochen, Germany) at ×200 magnification.

SSc patients and the isolation of PBMCs

Peripheral blood was acquired from healthy controls (HC, n=4) and SS patients (n=3) who visited Seoul St. Mary’s Hospital, a tertiary referral hospital in the Republic of Korea. PBMCs were isolated from heparinized blood by Ficoll-Paque density gradient centrifugation.

Humanized scleroderma mouse model

To develop the patient-derived humanized scleroderma mouse model, isolated human PBMCs (5×106/mouse) from SS patients were intraperitoneally injected into NSG mice (24). After 2 wk, the mice were anesthetized with isoflurane, and their backs were shaved. They were given daily subcutaneous injections of 25 µg BLM for 4 weeks (Dong-A Pharm, Seoul, Korea). The mice were euthanized after the final treatment. Back skin tissue was fixed in 10% formalin for histological analysis.

Primary skin fibroblast culture

Normal skin tissues from a healthy donor were washed with PBS, cut into small pieces, and adhered to and cultured on the bottoms of tissue culture flasks. After 3 days, primary skin fibroblasts were collected and cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Gibco), containing 100 μg/ml streptomycin and 100 U/ml penicillin, at 37°C in an atmosphere of 5.0% CO2. Skin fibroblasts were cultured with/without 10 ng/ml IL-4, 1,500 units/ml IFNα, and 0.5 µM H-151 for 1 day. Recombinant human IL-4 and IFNα were purchased from R&D Systems.

Statistical analysis

Data are presented as means ± SEMs. Statistical analysis was performed using Prism ver. 8 software for Windows (GraphPad Software, San Diego, CA, USA). Normally distributed continuous data were analyzed using the parametric Student’s t-test. Among-group differences in means were subjected to one-way ANOVA; p<0.05 was taken to indicate statistical significance.

Ethics approval

Study procedures involving humans were approved by the Institutional Review Board of the Catholic University of Korea (approval number: KC17TNSI0237). All mice experimental procedures were approved by the Department of Laboratory Animals, Institutional Animal Care and Use Committee (IACUC) of the School of Medicine, the Catholic University of Korea and conformed with all National Institutes of Health (Bethesda, MD, USA) guidelines (Approval number: CUMC-2022-0049-05 and CUMC-2020-0351-04).

Fibrosis and inflammatory cytokines are increased in skin of BLM-treated SKG mice

SKG mice spontaneously develop arthritis. To investigate the role of T cells in SS pathogenesis, we injected BLM into BALB/c and SKG mice for 2 wk (Fig. 1A). H&E and MT staining revealed that skin thickness and fibrosis were increased in BLM-treated SKG mice compared to BLM-treated BALB/c mice (Fig. 1B). Immunohistochemistry showed that the expressions of α-SMA and Col1 were significantly increased in the skin tissue of BLM-treated SKG mice compared to BLM-treated BALB/c mice (Fig. 1C). To assess the effects of T cells on SS, we measured the expression level of inflammatory cytokines of skin tissue via immunohistochemistry. BLM-treated SKG mice had increased expression of inflammatory cytokines, such as IL-1β, IL-6, TNF-α, IL-4, IL-17, and IFN-α (Fig. 1D). These data suggest that BLM-induced SS is further developed in SKG mice

Figure 1
Fibrosis and inflammatory cytokines are increased in skin of BLM-treated SKG mice. (A) Mice were given daily subcutaneous injections of BLM for 2 wk (BLM-treated BALB/c mice [n=5] and SKG mice [n=4]). (B) H&E staining and MT images of dermis thickness and collagen density in skin tissues of BLM-treated mice. Scale bars=100 μm. (C) Representative α-SMA and Col1-stained images of skin tissues of BLM-treated mice. Scale bars=100 μm. (D) Immunohistochemistry for IL-1β, IL-6, TNF-α, IL-4, IL-17, and IFN-α in skin tissues of BLM-treated mice. Scale bars=100 μm. Values are means ± SEMs from three independent experiments.
BLM, bleomycin.

*p<0.05, **p<0.01, and ***p<0.001.

Levels of inflammatory cytokines are increased in CD4+ T cells of BLM-treated SKG mice in vivo and the STIM1/STING pathway and its STAT6 and IRF3 downstream pathways are activated in CD4+ T cells of SKG mice in vitro

Th2 (CD4+IL-4+) and Th17 (CD4+IL-17+) cells are important in SS. Flow cytometry showed that the number of Th2 (CD4+IL-4+) cells was increased among splenocytes in BLM-treated SKG mice compared to BLM-treated BALB/c mice (Fig. 2A). The number of Th17(CD4+IL-17+) cells tended to increase in splenocytes from BLM-treated SKG mice compared to splenocytes from BLM-treated BALB/c mice, but this was not statistically significant. Immunofluorescence showed that the numbers of Th2 cells and Th17 cells in spleen tissue were increased in BLM-treated SKG mice (Fig. 2B). Activation of TCR signaling by mutation of ZAP70 activates ORAI1 and STIM1, leading to activation of STING. CD4+ T cells from BALB/c and SKG mice were differentiated under Th2-skewing conditions. After differentiation, these cells were stained with Abs against STIM1 and ORAI1 to measure their co-localization at the PM. The results showed that STIM1 translocated to the PM and co-localized with ORAI1 in cells from SKG mice (Fig. 2C). In addition, the number of pSTING-producing CD4+ T cells was higher in SKG mice than in BALB/c mice by flow cytometry (Fig. 2D and Supplementary Fig. 1). Also, the number of CD4+ T cells expressing pSTAT6 and pIRF3, downstream transcription factors of STING, and the number of CD4+ T cells simultaneously expressing IL-4 and IFN-α were evaluated by flow cytometry (Fig. 2D and Supplementary Fig. 1). Therefore, autoimmune T cells of SKG mice were increased the STIM1/STING pathway, resulting in T cells co-expressing IL-4 and IFN-α.

Figure 2
Levels of inflammatory cytokines are increased in CD4+ T cells of BLM-treated SKG mice in vivo and the STIM1/STING pathway and its STAT6 and IRF3 downstream pathways are activated in CD4+ T cells of SKG mice in vitro. (A) Numbers of CD4+IL-4+ (Th2) and CD4+IL-17+ (Th17) cells in spleens of BLM-treated mice (n=3 per group). Cells were stimulated with LPS for 3 days and Golgistop for the final 4 h. (B) Spleen tissues of BLM-treated mice were stained with anti-CD4 (green), anti-IL-4 (red), and anti-IL-17 (red) Abs to identify Th2 and Th17 cells (BLM-treated BALB/c mice [n=5] and SKG mice [n=4]). Scale bars=20 μm. (C) CD4+ T cells from spleen tissues of mice were stained with anti-ORAI1 (green) and anti-STIM1 (red) Abs. Scale bars=20 μm. (D) Numbers of pSTING+ cells, pSTAT6+ cells, pIRF3+ cells, and IL-4+ IFN-α+ cells of CD4+ T cells in spleens of mice. Cells were stimulated with anti-CD3 Ab, anti-CD28 Ab, anti-IFN-γ Ab, and IL-4 for 3 days. Values are means ± SEMs from three independent experiments.
ns, not significant.

*p<0.05, **p<0.01, and ***p<0.001.

The STING pathway is activated and the production of IL-4 and IFN-α is increased in CD4+ T cells from BLM-treated SKG mice in vivo

The numbers of pSTING-producing CD4+ T cells, pSTAT6-producing CD4+ T cells, and pIRF3-producing CD4+ T cells were higher among splenocytes from BLM-treated SKG mice than BLM-treated BALB/c mice (Fig. 3A and Supplementary Fig. 2A). The number of pSTING, pSTAT6, and pIRF3 of CD4+ T cells in spleen tissue were increased in BLM-treated SKG mice (Fig. 3B). In addition, the numbers of IFN-α+CD4+ T cells and IL-4- and IFN-α-producing CD4+ T cells among splenocytes were in BLM-treated SKG mice than in BLM-treated BALB/c mice according to flow cytometry and immunofluorescence (Fig. 3C, D, and Supplementary Fig. 2B). Immunofluorescence showed that STING was activated and translocated to the Golgi apparatus in splenocytes of BLM-treated SKG mice (Fig. 3E). Next, skin tissue was stained and subjected to immunohistochemistry and immunofluorescence analysis. pSTING, pSTAT6, and pIRF3 were significantly increased in the skin tissue of BLM-treated SKG mice (Fig. 3F). Immunofluorescence revealed more IL-4- and IFN-α-producing CD4+ T cells in the skin of BLM-treated SKG mice than in BLM-treated BALB/c mice (Fig. 3G).

Figure 3
The STING pathway is activated and the production of IL-4 and IFN-α is increased in CD4+ T cells from BLM-treated SKG mice in vivo. (A) Numbers of pSTING+ cells, pSTAT6+ cells, and pIRF3+ cells among CD4+ T cells from spleens of BLM-treated BALB/c mice (n=5) and SKG mice (n=4). (B) Spleen tissues of BLM-treated mice were stained with anti-CD4 (green), anti-pSTING (red), anti-pSTAT6 (red), and anti-pIRF3 (red) Abs. Scale bars=20 μm. (C) Numbers of IFN-α+ cells and IL-4+ IFN-α+ cells among CD4+ T cells from spleens of BLM-treated BALB/c mice (n=5) and SKG mice (n=4). (D) Spleen tissues of BLM-treated mice stained with anti-CD4 (green), anti-IL-4 (red), and anti-IFNα (blue) Abs. Scale bars=20 μm. (E) Splenocytes from spleen tissues of BLM-treated mice stained with anti-GM130 (green) and anti-STING (red) Abs. Scale bars=20 μm. (F) Immunohistochemistry of pSTING, pSTAT6, and pIRF3 in skin tissues in BLM-treated mice. Scale bars=100 μm. (G) Representative immunofluorescence images of CD4 (green), IL-4 (red), IFN-α (white), and DAPI (blue) in skin tissues from BLM-treated mice. Scale bars=20 μm. Values are means ± SEMs from three independent experiments.
GM130, Golgi matrix protein 130.

*p<0.05, **p<0.01, and ***p<0.001.

IL-4 and IFN-α induced fibrosis via the STING pathway, and inhibition of STING reduced fibroblast fibrosis and the production of IL-4 and IFN-α from CD4+ T cells

We treated skin fibroblasts with IL-4 and IFN-α to confirm the effects of IL-4 and IFN-α on skin fibroblasts. As a results, the expression of α-SMA and fibronectin (fibrosis markers) and pSTING, pSTAT6, and pIRF3 in human skin fibroblasts were increased by IL-4 and IFN-α treatment (Fig. 4A). Further, the expression of pSTING, pSTAT6, pIRF3, and fibrosis markers were suppressed by H-151 (STING inhibitor). Therefore, IL-4 and IFN-α increase fibrosis by increasing pSTING, pSTAT6, and pIRF3 expression in skin fibroblasts. In addition, to assess the effects of STING on the expression of IL-4 and IFN-α in CD4+ T cells, we differentiated splenocytes from BALB/c and SKG mice under Th2-skewing conditions in the absence or presence of H-151. The numbers of pSTAT6- and pIRF3-expressing CD4+ T cells and IL-4- and IFN-α-producing CD4+ T cells were increased in SKG mice compared to BALB/c mice, an effect reversed by STING inhibition (Fig. 4B and C, Supplementary Fig. 3A and B). We further confirmed whether fibrosis was inhibited when H-151 was administered to BLM-treated SKG mice. As a result, dermal thickness and the numbers of α-SMA- and Col1-positive cells in skin tissue were reduced by treatment with H-151 (Fig. 4D and E). Additionally, it was confirmed that pSTING, pSTAT6, and pIRF3 were decreased in skin tissue by H-151 treatment (Fig. 4F).

Figure 4
IL-4 and IFN-α induced fibrosis via the STING pathway, and inhibition of STING reduced fibroblast fibrosis and the production of IL-4 and IFN-α from CD4+ T cells. (A) Skin fibroblasts stained with anti-a-SMA (green), anti-fibronectin (green), anti-pSTING (green), anti-pSTAT6 (green) and anti-pIRF3 (green) Abs. Scale bars=20 μm. Skin fibroblasts were stimulated with/without IL-4, IFN-α, and H-151 for 1 days. (B) Numbers of pSTAT6+ cells, pIRF3+ cells, (C) IL-4+ cells, IFN-α+ cells, and IL-4+ IFN-α+ cells among CD4+ T cells isolated from the spleens of mice. Cells were stimulated with anti-CD3 Ab, anti-CD28 Ab, anti-IFN-γ Ab, and IL-4 with/without H-151 for 3 days. Values are means ± SEMs from three independent experiments.
*p<0.05, **p<0.01, and ***p<0.001.

Inhibition of STAT6 reduced skin fibrosis and the production of IL-4 and IFN-α from CD4+ T cells

Next, to assess the role of STAT6 (downstream of STING), we injected AS1517499 (STAT6 inhibitor) into BLM-treated mice. Inhibition of STAT6 reduced dermal thickness and decreased the numbers of α-SMA- and Col1-positive cells in skin tissue (Fig. 5A and B). In addition, there were fewer Th2, and IFN-α+CD4+ T cells, as well as IL-4- and IFN-α-producing CD4+ T cells, among splenocytes in SS mice after STAT6 inhibition (Fig. 5C). Moreover, the number of Th2 and IL-4- and IFN-α-producing CD4+ T cells was reduced by the STAT6 inhibitor in vitro (Fig. 5D). Therefore, the STING and STAT6 pathways are potential therapeutic targets in scleroderma.

Figure 5
Inhibition of STAT6 reduced skin fibrosis and the production of IL-4 and IFN-α from CD4+ T cells. (A) H&E staining of skin tissue. The mice were given daily subcutaneous injections of BLM for 2 wk. Next, 10 mg/kg AS1517499, a STAT6 inhibitor, was subcutaneously injected three times a week for 3 wk (wild type [n=3], BLM-treated mice [n=5] and BLM and STAT6 inhibitor-treated mice [n=5]). Scale bars=100 μm. (B) Representative α-SMA- and Col1-stained images of skin tissues. Scale bars=100 μm. (C) Numbers of CD4+IL-4+ (Th2), IFN-α+ CD4+ T cells, and IL-4+ IFN-α+ cells among CD4+ T cells from the spleens of mice. (D) Numbers of IL-4+ cells and IL-4+ IFN-α+ cells among CD4+ T cells isolated from the spleens of mice. Cells were stimulated with anti-CD3 Ab, anti-CD28 Ab, anti-IFN-γ Ab, and IL-4 with/without AS1517499 for 3 days. Values are means ± SEMs from three independent experiments.
WT, wild type; STAT6 i, STAT6 inhibitor.

*p<0.05, **p<0.01, and ***p<0.001.

IL-4- and IFN-α-producing CD4+ T cells in a humanized SS mice model

We found that the expression of pSTING and IL-4- and IFN-α-producing CD4+ T cells were higher in T cells from SS patients compared to HC (Supplementary Fig. 4). To investigate the role of human T cells in SS pathogenesis, we designed a humanized SS mouse model using PBMCs from a patient with SS. We injected PBMCs from the patient into NSG mice, which lack functional/mature T, B, and NK cells (Fig. 6A). H&E staining revealed that skin thickness was increased in humanized SS mice compared to BLM-treated NSG mice (Fig. 6B). α-SMA and Col1 immunohistochemistry showed that skin fibrosis was significantly increased in humanized SS mice (Fig. 6C). Also, pSTING, pSTAT6, and pIRF3 were significantly increased in the skin tissue of humanized SS mice (Fig. 6D). In addition, IL-4- and IFN-α-producing human CD4+ T cells were identified among splenocytes from humanized SS mice (Fig. 5E). Further, immunofluorescence detected IL-4- and IFN-α-producing human CD4+ T cells in the skin of the humanized SS mouse model (Fig. 5F). These results suggest that IL-4- and IFN-α-producing CD4+ T cells are identified in the patient with SS and that they contribute to inducing SS.

Figure 6
IL-4- and IFN-α-producing CD4+ T cells in a humanized SS mouse model. (A) A patient-derived humanized SS mouse model was developed by injecting human PBMCs and BLMs into NSG mice (normal control [n=3], BLM-treated mice [n=3] and BLM and patient PBMC-treated mice [n=5]). (B) H&E staining of skin tissues from the humanized SS mouse model. Scale bars=100 μm. (C) Representative α-SMA- and Col1-stained images of skin tissues of the humanized SS mouse model. Scale bars=100 μm. (D) Immunohistochemistry of pSTING, pSTAT6, and pIRF3 in skin tissues of the humanized SS mouse model. Scale bars=100 μm. (E) Numbers of IL-4+ IFN-α+ CD4+ T cells in spleen of the humanized SS mouse model. (F) Representative immunofluorescence images of CD4 (green), IL-4 (red), IFN-α (white), and DAPI (blue) in skin tissues from the humanized SS mouse model. Scale bars=20 μm. Values are means ± SEMs from three independent experiments.
hIl-4, human IL-4; hIFN α, human IFN α.

*p<0.05, **p<0.01, and ***p<0.001.

SS is an autoimmune disease involving skin thickening, fibrosis, and vasculopathy (1). SS is categorized as limited or diffuse, which refers to the degree of skin involvement (25). Both types can involve problems in other organs. It has a high mortality rate because of organ-based complications including pulmonary arterial hypertension, lung fibrosis, renal failure, and gastrointestinal tract dysfunction (26).

T-cell activation is significantly associated with SS (27). There are greater numbers of Th2 cells and Th17 cells in the skin and blood of SS patients (6, 7, 8). IL-4 concentrations are also higher in serum and PBMCs (28). IL-4 stimulates fibroblast proliferation and their differentiation to myofibroblasts, increasing the production of extracellular matrix and collagen and playing a key role in the development of fibrosis (29, 30). CD4+ CD8+ double-positive T cells produce a high level of IL-4, which may enhance extracellular matrix deposition by fibroblasts in SS skin (31). Monocyte chemoattractant protein 1 (MCP-1) stimulates IL-4 synthesis in SS fibroblasts, significantly increasing the production of collagen in SS dermal fibroblasts (32). In a putative murine model of scleroderma, stimulation of IL-4 with Tsk/+ fibroblasts increased type I collagen secretion by these cells, and anti-IL-4 treatment prevented the development of dermal fibrosis (33). Moreover, IL-4 promotes the biogenesis of collagen proteins in SS fibroblasts by increasing the stability and transcription of type 1 and 3 procollagens, and fibronectin (28, 34). Thus, IL-4 not only activates fibroblasts to increase the production of extracellular matrix and collagen proteins but also regulates the expression of a-SMA and collagen genes. IL-17A is a pro-inflammatory cytokine produced by Th17 cells and natural killer cells (35). The IL-17 mRNA level is elevated in lesional skin and PBMCs of SS patients (36). In murine models, IL-17A induces fibrosis by promoting fibroblast proliferation and inducing the synthesis of TGF-β, collagen, and connective tissue growth factor (CTGF) (37). In humans, IL-17A induces pro-inflammatory cytokine synthesis and inhibits that of collagen and CTGF, thus having pro-inflammatory and anti-fibrotic effects (38). Therefore, the pro-fibrotic mechanisms of IL-17A in animal models cannot be extrapolated to humans (35).

SKG mice spontaneously develop T cell-mediated autoimmune arthritis. To investigate the role of T cells in SS, we injected BLM into SKG mice, which developed thicker skin and more fibrosis than BLM-treated BALB/c mice. In addition, there were more Th2 and Th17 cells in blood and the levels of cytokines related to SS were elevated in skin lesions of BLM-treated SKG, as in patients with SS. Our data indicate that the increased numbers of inflammatory T cells caused by the mutation of ZAP70 can induce SS, thus showing potential as a therapeutic target.

STIM1, a key component of SOCE, regulates STING activity via physical interactions under resting conditions (12). Activated STIM1 translocates from the ER membrane to the PM, where it dissociates from and thereby activates STING. Dissociation of STIM1 and STING triggers the production of type I IFN and inflammatory cytokines by activating IRF3 and STAT6. We found that STIM1 is activated in T cells from SKG mice, which activates STING signaling. Also, when the STIM1–STING axis is activated, it induces the production of type I IFN and IL-4 and accelerates the development and progression of SS. In this study, activation of STIM1 by the ZAP70 mutation led to the dissociation and activation of STING. Activating STING in SKG mice induced the production of type I IFN and IL-4 and increased disease severity in BLM-induced SKG mice compared to BALB/c mice. This is the first report that STING-induced production of type I IFN and IL-4 in T cells is implicated in the development and progression of SS. Although T cells have been reported to express type I IFN and IL-4, there are no reports of T cells expressing them simultaneously (6, 39). These two cytokines are known to be cytokines that increase fibrosis (30, 40). We confirmed that IL-4- and IFN-α-producing CD4+ T cells were infiltrated in the dermis of skin tissue in the SS mouse model in which fibrosis was induced (Figs. 3G and 6F). To determine whether IL-4- and IFN-α-producing CD4+ T cells affect fibrosis, human skin fibroblasts were treated with IL-4- and IFN-α alone or in combination. As a result, it was confirmed that the expression of fibrosis markers increased statistically significantly in the group treated with the combination treatment compared to the single treatment group (Fig. 4A). Through this, it was found that that IL-4- and IFN-α-producing CD4+ T cells through STING activation strongly promote the progression of tissue fibrosis. Therefore, targeting these cells is thought to be important in the treatment of not only SS but also other autoimmune diseases related to fibrosis. IFN-regulated genes are expressed in the peripheral blood and skin of patients with SS (41). STAT6 induces the expression of IL-4 in T cells and is a transcription factor involved in collagen production by fibroblasts (42, 43). STING has been studied in mainly dendritic cells (DCs) or macrophages. We demonstrate the pathophysiological role of STING in T cells on the SS. Our data provide the possibility of STING as the therapeutic target not only in DCs or macrophages but also T cells.

Previously, we have established a humanized animal model of SS (24). In this study, we used a humanized SS mouse model which represents the pathophysiology of human SS patients better than the mouse SS model. It was confirmed that the expression of pSTING in CD4+ T cells and the number of IL-4- and IFN-α-producing CD4+ T cells were increased in SS patients compared to healthy controls (Supplementary Fig. 4). The results showed enhanced fibrosis and the production of type I IFN and IL-4 in skin of humanized SS mouse model compared to skin of BLM-treated NSG mice.

Therefore, STING-induced type I IFN and IL-4 production is implicated in the development and progression of SS in not only mice but also humans. Our data from humanized SS mouse model provide the strong evidence that STING is one of the candidates for SS treatment. Also, our data suggest that STAT6 is another therapeutic target of SS by regulating IL-4 expression.

Taken together, we found the crucial role of STING in the development and progression of SS. Our data suggest that STING-induced production of IL-4- and type I IFN by CD4+ T cells is a key factor in mouse model and humanized mouse model of SS. Therefore, STING is a potential therapeutic target in SS.

Supplementary Figure 1

The representative flow cytometry plots. The representative flow cytometry plots of pSTING+ cells, pSTAT6+ cells, pIRF3+ cells, and IL-4+ IFN-α+ cells of CD4+ T cells in spleens of mice. Cells were stimulated with anti-CD3 Ab, anti-CD28 Ab, anti-IFN-γ Ab, and IL-4 for 3 days.

Click here to view.(517K, ppt)

Supplementary Figure 2

The representative flow cytometry plots. (A) The representative flow cytometry plots of pSTING+ cells, pSTAT6+ cells, and pIRF3+ cells among CD4+ T cells from spleens of BLM-treated BALB/c mice (n=5) and SKG mice (n=4). (B) The representative flow cytometry plots of IFN-α+ cells and IL-4+ IFN-α+ cells among CD4+ T cells from spleens of BLM-treated BALB/c mice (n=5) and SKG mice (n=4).

Click here to view.(380K, ppt)

Supplementary Figure 3

The representative flow cytometry plots. The representative flow cytometry plots of (A) pSTAT6+ cells, pIRF3+ cells and (B) IL-4+ IFN-α+ cells among CD4+ T cells isolated from the spleens of mice. Cells were stimulated with anti-CD3 Ab, anti-CD28 Ab, anti-IFN-γ Ab, and IL-4 with/without H-151 for 3 days.

Click here to view.(451K, ppt)

Supplementary Figure 4

Expression levels of pSTING and IL-4- and IFN-α-producing CD4+ T cells in SS patients. The numbers of pSTING+ cells and IL-4+ IFN-α+ cells among CD4+ T cells from the PBMCs of HC (n=4) and SS patients (n=3).

Click here to view.(423K, ppt)

AbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALSNotesAbbreviationsACKNOWLEDGEMENTSReferences
Notes

Conflict of Interest:The authors declare no potential conflicts of interest.

Data Availability:All datasets generated for this study are included in the article.

Author Contributions:

  • Conceptualization: Lee KH, Cho ML.

  • Data curation: Lee KH.

  • Formal analysis: Lee KH.

  • Investigation: Lee KH, Woo JS.

  • Methodology: Lee KH, Woo JS, Jeong HY, Choi JW.

  • Resources: Youn J, Park SH.

  • Software: Lee KH, Woo JS.

  • Validation: Lee KH, Woo JS.

  • Writing - original draft: Lee KH, Woo JS.

  • Writing - review & editing: Bang CH, Park SH, Cho ML.

α-SMA α-smooth muscle actin
BLM bleomycin
cGAMP cyclic guanosine monophosphate-adenosine monophosphate
CTGF connective tissue growth factor
DC dendritic cell
ER endoplasmic reticulum
GM130 Golgi matrix protein 130
IRF3 Interferon regulatory factor 3
MT Masson's trichrome
PBMC peripheral blood mononuclear cell
PM plasma membrane
SOCE store-operated Ca2+ entry
SS systemic sclerosis
STAT6 signal transducer and activator of transcription 6
STIM1 stromal interaction molecule 1
STING stimulator of interferon genes
TBK1 TANK-binding kinase 1
TCR T-cell receptor
ZAP70 z chain-associated protein 70

This research was supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (RS-2023-00217274, Republic of Korea) and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: RS-2022-KH127879[HV22C0069]).

AbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALSNotesAbbreviationsACKNOWLEDGEMENTSReferences

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Publication Types
Original Article
MeSH Terms
Animals
Autoimmune Diseases
Bites and Stings
Cytokines
Fibroblasts
Fibrosis
Immunity
Interleukin-4
Interleukin-4*
Lymphocytes
Mice*
Point Mutation
Scleroderma, Systemic
Skin
STAT6 Transcription Factor
STAT6 Transcription Factor*
T-Lymphocytes*
Metrics
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