DNA from macrophages induces fibrosis and vasculopathy through POLR3A/STING/type I interferon axis in systemic sclerosis.

OBJECTIVE: To clarify the role of RNA polymerase III A (POLR3A)/type I IFN in the pathogenesis of SSc. METHODS: Cytosolic DNA and stimulator of IFN genes (STING) pathway in skin or serum of SSc patients were detected by immunofluorescence, immunohistochemistry and western blotting. DNA from human macrophages was transfected to SSc fibroblasts or human umbilical vein endothelial cells (HUVECs) and then markers of POLR3A/STING pathway were detected by real-time qPCR, western blotting and confocal microscopy. After H151 treatment or knocking down POLR3A/STING, type I IFN response, monocytes adhesion and activation of fibroblasts and HUVECs were evaluated. Regulation of IFN regulatory factor 3 (IRF3) on monocyte chemoattractant protein-1 (MCP-1) was determined by chromatin immunoprecipitation. In bleomycin (BLM)-induced SSc mice, the effect of STING knockout or H151 on vasculopathy and fibrosis was assessed. RESULTS: Cytosolic DNA, colocalization of STING with alpha-smooth muscle actin (α-SMA) or CD31 in the skin, and STING pathway in the serum of SSc patients were increased. Macrophage-derived DNA stimulated the translocation of POLR3A from nucleus to the perinuclear region near STING and activated POLR3A/STING/type I IFN response, monocytes adhesion and MCP-1 expression in fibroblasts/HUVECs and collagen overproduction of fibroblasts. The activated IRF3 bound to the promoter of MCP-1. STING deficiency or H151 administration ameliorated fibrosis and vasculopathy both in vitro and in BLM-induced SSc mice. CONCLUSIONS: SSc presented increased DNA leakage and STING pathway activation. DNA from macrophages induced type I IFN signature of fibroblasts and ECs through POLR3A/STING pathway. Blocking POLR3A/STING axis provides a new therapeutic target for SSc.

Palmar erythema and palmar papules as predictors for dermatomyositis-related acute/subacute interstitial lung disease: a retrospective study.

OBJECTIVES: DM-related acute/subacute interstitial lung disease (A/S-ILD) remains a big therapeutic challenge due to its insidious onset and rapid development. In the present study, we aimed to investigate the association between clinical features of DM and ILD. METHODS: We retrospectively assessed skin manifestations, muscle damage, laboratory tests, concurrent ILD and malignancy in 207 patients with DM and analysed the high-risk factors for ILD. RESULTS: In the 207 DM patients, 153 patients had ILD, in which 131 had chronic ILD (CILD) and 22 had A/S-ILD. The proportions of mechanic's hands, palmar papules and muscle weakness, as well as anti-melanoma differentiation-associated gene 5 (MDA5) antibody and lactic dehydrogenase (LDH), alanine aminotransferase (ALT) and ferritin in the ILD group were significantly higher compared with the non-ILD group. The onset age over 56 years, mechanic's hands and muscle weakness were independent predictive factors for ILD. The proportions of palmar papules, palmar erythema, anti-MDA5 antibody, ALT, aspartate aminotransferase (AST), LDH and ESR in the A/S-ILD group were higher compared with the CILD group. Palmar erythema and palmar papules were independent predictive factors for A/S-ILD. Palmar papules were positively correlated with anti-MDA5 antibody. CONCLUSION: The onset age over 56 years, mechanic's hands or muscle weakness predicted the incidence of DM-related ILD, while palmar erythema or palmar papules could predict potential DM-related A/S-ILD.

Autophagy mediates 2-methoxyestradiol-inhibited scleroderma collagen synthesis and endothelial-to-mesenchymal transition induced by hypoxia.

OBJECTIVES: To investigate whether autophagy mediates 2-methoxyestradiol (2-ME)-inhibited hypoxia-induced fibrosis and endothelial-to-mesenchymal transition (endoMT) in SSc. METHODS: Autophagy in the skin of SSc patients was assessed by transmission electron microscopy. SSc skin fibroblasts and human umbilical vein endothelial cells (HUVECs) were cultured under hypoxic (1% O2) conditions with 2-ME or autophagy inhibitor. Collagen I and connective tissue growth factor (CTGF) in fibroblasts and vascular endothelial (VE)-cadherin, CD31, vimentin and α-smooth muscle actin (α-SMA) in HUVECs were examined by western blotting. Autophagic markers were evaluated by confocal microscopy and immunofluorescence. RESULTS: SSc skins presented increased autolysosomes, LC3-II, collagen I and CTGF. Hypoxia-challenged fibroblasts and HUVECs formed more autophagosomes and autolysosomes, with increased LC3 and decreased P62. Meanwhile, hypoxia increased collagen I and CTGF in fibroblasts and increased vimentin and α-SMA but decreased VE-cadherin and CD31 in HUVECs. Bafilomycin A1 increased LC3-II and P62 in fibroblasts and HUVECs and decreased collagen I and CTGF in fibroblasts and vimentin and α-SMA in HUVECs, while upregulating VE-cadherin and CD31. 3-methyladenine decreased autophagy and fibrosis in fibroblasts and endothelial-to-mesenchymal transition in HUVECs. 2-ME-treated HUVECs showed more autophagosomes and fewer autolysosomes while 2-ME-treated fibroblasts showed fewer of both. Moreover, 2-ME decreased LC3-II and increased P62 in fibroblasts and increased both in HUVECs. Inhibition of autophagy by 2-ME showed the same effect with bafilomycin A1 on fibroblast collagen synthesis as well as endothelial and mesenchymal markers in HUVECs. CONCLUSION: Autophagy mediated hypoxia-induced fibroblast collagen synthesis and endoMT in SSc, which could be reversed by 2-ME.

2-Methoxyestradiol inhibits hypoxia-induced scleroderma fibroblast collagen synthesis by phosphatidylinositol 3-kinase/Akt/mTOR signalling.

Objectives: To investigate the mechanism of 2-methoxyestradiol (2-ME) in inhibiting hypoxia-induced collagen synthesis of fibroblasts in SSc. Methods: The expressions of hypoxia-inducible factor 1 alpha (HIF-1α) and connective tissue growth factor (CTGF) in skin specimens derived from SSc patients and healthy volunteers were examined by immunohistochemistry. HIF-1α was knocked down by lentiviral transduction, and SSc dermal fibroblasts cultured under normoxic (21% O2) or hypoxic (1% O2) condition were treated with PI3K inhibitor LY294002, rapamycin or 2-ME (25 µM). The protein levels of HIF-1α, CTGF, collagen I, p-Akt and p-mTOR were examined by western blotting or immunofluorescence. Apoptosis and cell cycle of fibroblasts were assessed by flow cytometry and by measuring caspase 3 activity, and cell proliferation was evaluated by Cell Counting Kit-8. Results: The expressions of HIF-1α and CTGF were increased in skins of SSc patients compared with healthy controls. Hypoxia up-regulated the protein levels of HIF-1α, CTGF and collagen I in SSc fibroblasts. In contrast, 2-ME inhibited PI3K/Akt/mTOR pathway and down-regulated protein levels of HIF-1α, CTGF and collagen I. Knockdown of HIF-1α reduced expressions of CTGF and collagen I, which were further down-regulated by 2-ME intervention. Moreover, 2-ME promoted the apoptosis and inhibited the proliferation of SSc fibroblasts by arresting the cell cycle at the G2/M phase. Conclusion: 2-ME reduced the production of CTGF and collagen I in SSc fibroblasts induced by hypoxia through PI3K/Akt/mTOR/HIF-1α signalling and inhibited the proliferation of fibroblasts. These findings suggested that 2-ME could be employed as a promising antifibrotic therapy for SSc.

Interferon-gamma Inhibits Melanogenesis and Induces Apoptosis in Melanocytes: A Pivotal Role of CD8+ Cytotoxic T Lymphocytes in Vitiligo.

Increased expression of the cytokine interferon (IFN)-γ plays a pivotal role in vitiligo-induced depigmentation. However, the major source of IFN-γ in vitiligo patients and the mechanisms underlying melanocyte destruction are unknown. In this study, a large number of skin infiltrating IFN-γ+ cells and CD8+ T cells were detected in progressive vitiligo. Among the peripheral blood mononuclear cells (PBMCs) of vitiligo patients, CD8+ cytotoxic T lymphocytes (CTLs) that express IFN-γ exhibited significant expansion, which suggests that activated CTLs are the main source of increased IFN-γ in progressive vitiligo. An in vitro analysis demonstrated that IFN-γ inhibits melanogenesis in primary cultured human melanocytes by altering melanogenic enzyme mRNA expression and, more importantly, that IFN-γ directly induces melanocyte apoptosis. Our data indicate that vitiligo pathophysiology may be linked to globally activated CD8+ CTL subpopulations, which produce increased IFN-γ and induce melanocyte dysfunction and apoptosis.

VISTA antibody-loaded Fe3O4@TiO2 nanoparticles for sonodynamic therapy-synergistic immune checkpoint therapy of pancreatic cancer.

Breaking the poor permeability of immune checkpoint inhibitors (ICIs) caused by the stromal barrier and reversing the immunosuppressive microenvironment are significant challenges in pancreatic cancer immunotherapy. In this study, we synthesized core-shell Fe3O4@TiO2 nanoparticles to act as carriers for loading VISTA monoclonal antibodies to form Fe3O4@TiO2@VISTAmAb (FTV). The nanoparticles are designed to target the overexpressed ICIs VISTA in pancreatic cancer, aiming to improve magnetic resonance imaging-guided sonodynamic therapy (SDT)-facilitated immunotherapy. Laser confocal microscopy and flow cytometry results demonstrate that FTV nanoparticles are specifically recognized and phagocytosed by Panc-2 cells. In vivo experiments reveal that ultrasound-triggered TiO2 SDT can induce tumor immunogenic cell death (ICD) and recruit T-cell infiltration within the tumor microenvironment by releasing damage-associated molecular patterns (DAMPs). Furthermore, ultrasound loosens the dense fibrous stroma surrounding the pancreatic tumor and increases vascular density, facilitating immune therapeutic efficiency. In summary, our study demonstrates that FTV nanoparticles hold great promise for synergistic SDT and immunotherapy in pancreatic cancer.

Cathepsin B/NLRP3/GSDMD axis-mediated macrophage pyroptosis induces inflammation and fibrosis in systemic sclerosis.

BACKGROUND: Pyroptosis is a newly discovered type of programmed cell death associated with inflammatory and fibrotic diseases. Macrophages play an important role in inducing early immune inflammation in systemic sclerosis (SSc). OBJECTIVE: To investigate the effect of macrophages pyroptosis on fibrosis of SSc. METHODS: Pyroptosis/inflammatory markers in serum and skin of SSc patients were detected. Bleomycin (BLM) was subcutaneously injected to establish SSc mouse model. The levels of pyroptosis markers, dermal thickness and collagen deposition in skin were assessed before and after the administration of pyroptosis inhibitors, including MCC950, Disulfiram and necrosulfonamide (NSA). Human-derived monocyte-macrophage cell line (THP-1) or mouse bone marrow-derived macrophages (BMDMs) were primed with lipopolysaccharide (LPS) and stimulated by silicon dioxide (SiO2) to induce cell pyroptosis. Fibroblasts from patients with SSc were co-cultured with pyroptotic THP-1 cells, and the collagen production was assessed. RESULTS: Pyroptotic/inflammatory proteins, including NLRP3, cleaved-Caspase (CASP)1, GSDMD-N terminal and IL-18 were increased in the serum, and ASC aggregation and GSDMD were elevated in macrophages in the skin of SSc patients. SSc mice showed increased pyroptosis markers, dermal thickness and collagen deposition in skins, which were alleviated by MCC950, Disulfiram and NSA. Pyroptosis of THP-1 cells and BMDMs was induced by LPS/SiO2, and it was reduced by the inhibitors of Cathepsin B, NLRP3, CASP1 and GSDMD. Co-culture with pyroptotic THP-1 cells increased the fibrotic proteins in fibroblasts, which were alleviated by pyroptosis inhibitors. CONCLUSIONS: SSc patients and BLM-induced mouse model presented increased pyroptosis. LPS/SiO2-induced macrophage pyroptosis promoted fibrosis of SSc through Cathepsin B/NLRP3/GSDMD pathway.

Inhibition of JAK1/STAT3 pathway by 2-methoxyestradiol ameliorates psoriatic features in vitro and in an imiquimod-induced psoriasis-like mouse model.

Psoriasis is characterized by hyperproliferative keratinocytes, dilated capillaries and leukocyte infiltration. 2-Methoxyestradiol (2-ME) has shown significant inhibition on proliferation, angiogenesis and inflammation. To evaluate the anti-psoriatic potential of 2-ME, psoriasis-like dermatitis was induced by topical application of imiquimod (IMQ) on the dorsal skin of C57BL/6 mice for seven consecutive days, followed by treatment of vehicle or 2-ME ointment from Day 4 on. The psoriasis area and severity index (PASI) was assessed daily. On Day 8, skin histology and spleen index were assessed. The effects of 2-ME on the proliferation, apoptosis, cell cycle, vascular endothelial growth factor A (VEGFA), and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways of HaCaT cells stimulated by interleukin-17 (IL-17A) were detected, together with its effect on the proliferation, tube formation and VEGF receptor expression of human umbilical vein endothelial cells (HUVECs). We found that topical 2-ME treatment significantly improved IMQ-induced psoriasis-like dermatitis and decreased the PASI scores, the activation of STAT3 in the skin (P < 0.05), and the spleen index in mice (P < 0.01). In vitro, 2-ME inhibited the proliferation of HaCaT cells by inducing apoptosis and G2/M phase arrest (P < 0.01). Moreover, 2-ME suppressed IL-17A-induced VEGFA (2.5 µM: P < 0.05; 5 µM: P < 0.01) and phosphorylation of STAT3 by blocking p-JAK1 in HaCaT cells and prevented tube formation (P < 0.01) and proliferation by targeting VEGF receptors 1 (VEGFR1) and 2 (VEGFR2) in HUVECs. We conclude that 2-ME alleviated psoriasis in vivo and in vitro by inhibiting JAK1/STAT3 pathway and was a promising therapeutic agent for psoriasis.

2-Methoxyestradiol inhibits bleomycin-induced systemic sclerosis through suppression of fibroblast activation.

BACKGROUND: The most dominant feature of systemic sclerosis (SSc) is fibrosis, which is caused by overproduction of collagen by fibroblasts. 2-Methoxyestradiol (2-ME) has exhibited disease-modifying activity in animal models of rheumatoid arthritis and autoimmune encephalomyelitis and inhibitory effect in cell proliferation and collagen synthesis. Therefore, we hypothesized that 2-ME may exhibit antifibrotic effect in SSc. OBJECTIVE: To investigate the antifibrotic effect of 2-ME in SSc. METHODS: We established a bleomycin-induced SSc mice model by injection with bleomycin daily for 21 days. 2-ME (100mg/kg/d) was simultaneously administered for 14 days. On the end of Week1 (W1), W2, W3 and W4, skins and lungs were collected for histological examination and analysis of hydroxyproline content and mRNA level of α1(I) procollagen (COL1A1) and COL1A2. In skin fibroblasts derived from SSc patients and healthy subjects treated with 2-ME (1, 5, or 25 µM), we examined cell proliferation, expression of α-smooth muscle actin (SMA) and mRNA level of COL1A1, COL1A2, COL3A1, matrix metalloproteinase(MMP)-1 and tissue inhibitors of MMP (TIMP)-1. RESULTS: We found reduced dermal thickness and lung fibrosis and decreased hydroxyproline content and mRNA level of COL1A1 and COL1A2 in skin and lung in SSc mice treated with 2-ME. In cell study, we observed a dose- and time-dependent inhibitory effect on proliferation of SSc fibroblasts by 2-ME. We also detected reduced α-SMA expression, decreased mRNA level of COL1A1, COL1A2, COL3A1 and TIMP-1, and increased mRNA level of MMP-1 in SSc fibroblasts treated with 2-ME. CONCLUSION: 2-ME could suppress SSc tissue fibrosis, which may be attributable to its inhibitory effect on the excessive proliferation, differentiation and production of collagen in fibroblasts. 2-ME is rising as a prospective agent for control of fibrosis in SSc.

Antifibrotic effects of crocetin in scleroderma fibroblasts and in bleomycin-induced sclerotic mice.

OBJECTIVE: To investigate the antifibrotic effects of crocetin in scleroderma fibroblasts and in sclerotic mice. METHODS: Skin fibroblasts that were isolated from three systemic scleroderma (SSc) patients and three healthy subjects were treated with crocetin (0.1, 1 or 10 µM). Cell proliferation was measured with an MTT assay. Alpha-smooth muscle actin was detected via an immunohistochemical method. Alpha 1 (I) procollagen (COL1A1), alpha 1 (III) procollagen (COL3A1), matrix metalloproteinase (MMP)-1 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA levels were measured using real-time PCR. SSc mice were established by the subcutaneous injection of bleomycin. Crocetin (50 mg/kg/d) was injected intraperitoneally for 14 days. Dermal thickness and lung fibrosis were assessed with Masson's trichrome staining. Plasma ET-1 was detected with an enzyme-linked immunosorbent assay (ELISA). Skin and lung ET-1 and COL1A1 mRNA levels were measured via real-time PCR. RESULTS: Crocetin inhibited the proliferation of SSc and normal fibroblasts, an effect that increased with crocetin concentration and incubation time. Crocetin decreased the expression of α-SMA and the levels of mRNA for COL1A1, COL3A1 and matrix metalloproteinase-1, while crocetin increased TIMP-1 mRNA levels in both SSc and normal fibroblasts. Skin and lung fibrosis was induced, and the levels of ET-1 in the plasma, skin and lungs were elevated in bleomycin-injected mice. Crocetin alleviated the thickening of the dermis and lung fibrosis; decreased COL1A1 mRNA levels in the skin and lung; and simultaneously decreased ET-1 concentrations in the plasma and ET-1 mRNA levels in the skin and lungs of the bleomycin-induced sclerotic mice, especially during the early phase (weeks 1-3). CONCLUSION: Crocetin inhibits cell proliferation, differentiation and collagen production in SSc fibroblasts. Crocetin alleviates skin and lung fibrosis in a bleomycin-induced SSc mouse model, in part due to a reduction in ET-1.

IL-17A induces endothelial inflammation in systemic sclerosis via the ERK signaling pathway.

Recent reports have demonstrated that endothelial cells are involved in vascular inflammatory injury in systemic sclerosis (SSc) and interleukin-17A (IL-17A) plays a crucial role in the pathogenesis of SSC. However, little is known about the effects of IL-17A on endothelial cell inflammation in SSC. The aim of our study was to investigate the role of IL-17A in endothelial inflammation. Here, we showed that IL-17A mRNA and protein levels were augmented in the peripheral blood and more IL-17⁺ lymphocytes infiltrated in the perivascular areas in the involved skin of SSC patients. SSC patient serum induced chemokine and adhesion molecule expression in HUVECs, which was blocked by IL-17A neutralization. IL-17A alone induced chemokine and adhesion molecule expression and promoted T cell-HUVEC adhesion. Extracellular signal-regulated kinase (ERK) inhibition and IL-17A neutralization prominently inhibited chemokine and adhesion molecule expression and blocked T cell-HUVEC adhesion. IL-17A derived from SSC patient serum mediated endothelial cells inflammation by up-regulating chemokines and adhesion molecules, which was blocked by ERK inhibition. These data imply that ERK signal pathway might play a key role in the progression of endothelial injury induced by IL-17A in SSC.

Th17 and natural Treg cell population dynamics in systemic lupus erythematosus.

OBJECTIVE: To investigate the relative abundance and activities of Th17 cells and natural Treg cells in systemic lupus erythematosus (SLE). METHODS: Blood samples were collected from 50 adult patients with SLE. Samples were processed to detect Th17 cells and natural Treg cells by flow cytometry, and related gene expression was assessed by real-time reverse transcription-polymerase chain reaction. Skin biopsy specimens were collected for histologic assessment. The function of Th17 cells in relation to human umbilical vein endothelial cells (HUVECs) was studied in vitro. Th17 cells were also examined in lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice. RESULTS: We demonstrated the presence of Th17 cells among the peripheral blood mononuclear cells (PBMCs) and in the involved organs of patients with active SLE. Both the percentage of circulating Th17 cells and the ability to produce interleukin-17A (IL-17A) were increased in samples derived from patients with active SLE. The number of Th17 cells increased during SLE flare, especially in patients with vasculitis, and decreased following certain treatments. We observed that IL-17A from patients with SLE could induce adhesion molecule messenger RNA expression in HUVECs and adhesion of T cells to HUVECs. An increase in the percentage of Th17 cells was correlated with natural Treg cell depletion during disease flare. Finally, expansion of the Th17 cell population was detected in MRL/lpr mice. CONCLUSION: SLE flare might be linked to the expansion of the Th17 cell population and the depletion of natural Treg cell subpopulations. Expansion of the Th17 cell population might be related to a distinct cytokine environment in active SLE. Th17 cells and microenvironmental IL-17A are involved in vascular inflammation in SLE. Antagonism of Th17 cells by IL-17A-blocking antibodies should be explored as a treatment of SLE.

Antifibrotic effects of crocetin in scleroderma fibroblasts and in bleomycin-induced sclerotic mice

OBJECTIVE: To investigate the antifibrotic effects of crocetin in scleroderma fibroblasts and in sclerotic mice. METHODS: Skin fibroblasts that were isolated from three systemic scleroderma (SSc) patients and three healthy subjects were treated with crocetin (0.1, 1 or 10 μM). Cell proliferation was measured with an MTT assay. Alpha-smooth muscle actin was detected via an immunohistochemical method. Alpha 1 (I) procollagen (COL1A1), alpha 1 (III) procollagen (COL3A1), matrix metalloproteinase (MMP)-1 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA levels were measured using real-time PCR. SSc mice were established by the subcutaneous injection of bleomycin. Crocetin (50 mg/kg/d) was injected intraperitoneally for 14 days. Dermal thickness and lung fibrosis were assessed with Masson's trichrome staining. Plasma ET-1 was detected with an enzyme-linked immunosorbent assay (ELISA). Skin and lung ET-1 and COL1A1 mRNA levels were measured via real-time PCR. RESULTS: Crocetin inhibited the proliferation of SSc and normal fibroblasts, an effect that increased with crocetin concentration and incubation time. Crocetin decreased the expression of α-SMA and the levels of mRNA for COL1A1, COL3A1 and matrix metalloproteinase-1, while crocetin increased TIMP-1 mRNA levels in both SSc and normal fibroblasts. Skin and lung fibrosis was induced, and the levels of ET-1 in the plasma, skin and lungs were elevated in bleomycin-injected mice. Crocetin alleviated the thickening of the dermis and lung fibrosis; decreased COL1A1 mRNA levels in the skin and lung; and simultaneously decreased ET-1 concentrations in the plasma and ET-1 mRNA levels in the skin and lungs of the bleomycin-induced sclerotic mice, especially during the early phase (weeks 1-3). CONCLUSION: Crocetin inhibits cell proliferation, differentiation and collagen production in SSc fibroblasts. Crocetin alleviates skin and lung fibrosis in a bleomycin-induced SSc ...