GSDME-mediated keratinocyte pyroptosis participates in the pathogenesis of psoriasis by promoting skin inflammation.

OBJECTIVE: Psoriasis is a common, chronic inflammatory disease with unclear etiology. Keratinocytes in psoriasis are susceptible to exogenous triggers that induce inflammatory cell death. This study investigated whether GSDME-mediated pyroptosis in keratinocytes contributes to the pathogenesis of psoriasis. METHODS: Skin samples from patients with psoriasis and healthy controls were collected to evaluate the expression of GSDME, cleaved-caspase-3, and inflammatory factors. We then analyzed the data series, GSE41662, to further compare the expression of GSDME between lesional and non-lesional skin samples in those with psoriasis. In vivo, caspase-3 inhibitor and GSDME deficiency mice (Gsdme-/-) were applied to block caspase-3/GSDME activation in the imiquimod-induced psoriasis model. Skin inflammation, disease severity, and pyroptosis-related proteins were analyzed. In vitro, tumor necrosis factor-α (TNF-α)-induced caspase-3/GSDME-mediated pyroptosis in the HACAT cell line was explored. RESULTS: Our analysis of the GSE41662 data series found that GSDME were upregulated in psoriasis lesions, compared to normal skin. High levels of inflammatory cytokines such as IL-1ß, IL-6, and TNF-α were also found in psoriasis lesions. In mice of Gsdme-/- and caspase-3 inhibitor groups, the severity of skin inflammation was attenuated, and GSDME and C-caspase-3 levels decreased after imiquimod treatment. Similarly, IL-1ß, IL-6, and TNF-α were decreased in Gsdme-/- and caspase-3 inhibitor groups. In vitro, TNF-α induced HACAT cell pyroptosis through caspase-3/GSDME pathway activation, which was suppressed by blocking caspase-3 or silencing GSDME. CONCLUSION: Our study provides a novel explanation that TNF-α/caspase-3/GSDME-mediated keratinocyte pyroptosis is highly responsible for the initiation and acceleration of skin inflammation and progression of psoriasis.

Cyclophosphamide induced intestinal injury is alleviated by blocking the TLR9/caspase3/GSDME mediated intestinal epithelium pyroptosis.

OBJECTIVES: Cyclophosphamide (CYC) was commonly used to treat autoimmune disorders, and it could also cause side effects such as intestinal damage. This study aimed to explore the mechanism of CYC-induced intestinal cytotoxicity and provide evidence for protecting from intestinal damage by blocking TLR9/caspase3/GSDME mediated pyroptosis. METHODS: Intestinal epithelial cells (IEC-6) were treated with 4-hydroxycyclophosphamide (4HC), a key active metabolite of CYC. The pyroptotic rate of IEC-6 cells was detected by Annexin V/PI-Flow cytometry, microscopy imaging, and PI staining. The expression and activation of TLR9, caspase3 and GSDME in IEC-6 cells were detected by western blot and immunofluorescence staining. In addition, hydroxychloroquine (HCQ) and ODN2088 were used to inhibit TLR9 to investigate the role of TLR9 on caspase3/GSDME-mediated pyroptosis. Finally, mice lacking Gsdme or TLR9 or pretreating with HCQ were injected intraperitoneally with CYC, and the incidence and severity of intestinal damage were assessed. RESULTS: CYC induced lytic cell death in IEC-6 cells and increased the expression of TLR9, activated caspase3, and GSDME-N. Besides, both ODN2088 and HCQ could inhibit CYC-induced pyroptosis in IEC-6 cells. In vivo, CYC-induced intestinal injury was characterized by a large amount of intestinal villi abscission and structural disordered. Gsdme or TLR9 deficiency, or pretreatment of HCQ effectively attenuated intestinal damage in CYC-induced model mice. CONCLUSIONS: These results indicate an alternative mechanism for CYC-induced intestinal damage, which actives TLR9/caspase3/GSDME signaling pathway, leading to pyroptosis of intestinal epithelial cells. And targeting pyroptosis might be a potential therapeutic approach for CYC-induced intestinal damage.

Polydatin protects against gouty nephropathy by inhibiting renal tubular cell pyroptosis.

OBJECTIVE: To investigate the protective effect and mechanism of polydatin (PD) against gouty nephropathy (GN) in mice. METHODS: Twenty-four mice were randomly divided into three groups: the control group (no treatment), the GN group (300 mg/kg hypoxanthine + 150 mg/kg potassium oxonate), and the GN + PD group (300 mg/kg hypoxanthine + 150 mg/kg potassium oxonate + 50 mg/kg PD). Histological changes in the kidneys and the levels of uric acid (UA), blood urea nitrogen (BUN), and serum creatinine (SCr) in the sera were measured. In addition, the expression of gasdermin D (GSDMD) protein in renal tubular epithelial cells, and the expression of NOD-like receptor protein 3 (NLRP3), GSDMD, and caspase-1 proteins in the kidney tissues were determined by immunohistochemistry, immunofluorescence, and Western blot. RESULTS: In vitro, PD inhibited the expression of NLRP3, caspase-1, and GSDMD and protected the renal tubular epithelial cells from pyroptosis. In vivo, PD treatment significantly ameliorated the pathological changes in kidney tissue, and reversed the decrease of serum UA and BUN in GN model mice. The expression of NLRP3, GSDMD, and caspase-1 proteins was also decreased in the PD-treated GN mice. CONCLUSION: The results suggest that PD has a protective effect on mice with GN, which may be related to the downregulation of NLRP3, GSDMD, and caspase-1 proteins and the inhibition of renal tubular epithelial cells pyroptosis.

Blocking GSDME-mediated pyroptosis in renal tubular epithelial cells alleviates disease activity in lupus mice.

An increase in apoptosis and/or defects in the clearance of apoptotic cells resulting in massive secondary necrosis have been recognized as the main causes of systemic lupus erythematosus (SLE). Recent findings have revealed that gasdermin E (GSDME)-mediated pyroptosis is a mechanism associated with secondary necrosis. We aimed to investigate the effects of GSDME-mediated pyroptosis on disease activity in lupus mice. In vivo, high levels of GSDME expression were observed in the renal tubules of pristane-induced lupus (PIL) mice and SLE patients. In lupus mice, GSDME knockout or SP600125 administration effectively ameliorated lupus-like features by inhibiting GSDME-mediated renal tubular epithelial cell pyroptosis. In vitro, treatment with tumour necrosis factor-α (TNF-α) plus cycloheximide (CHX) or SLE sera induced HK2 cells to undergo pyroptosis in a caspase-3- and GSDME-dependent manner. Likewise, SP600125 significantly reduced GSDME expression and decreased pyroptosis in HK2 cells. GSDME-mediated pyroptosis may be associated with SLE pathogenesis, and targeting GSDME may be a potential strategy for treating SLE.

Attenuation of Rheumatoid Arthritis Through the Inhibition of Tumor Necrosis Factor-Induced Caspase 3/Gasdermin E-Mediated Pyroptosis.

OBJECTIVE: To determine the role of gasdermin E (GSDME)-mediated pyroptosis in the pathogenesis and progression of rheumatoid arthritis (RA), and to explore the potential of GSDME as a therapeutic target in RA. METHODS: The expression and activation of caspase 3 and GSDME in the synovium, macrophages, and monocytes of RA patients were determined by immunohistochemistry, immunofluorescence, and Western blot analysis. The correlation of activated GSDME with RA disease activity was evaluated. The pyroptotic ability of monocytes from RA patients was tested, and the effect of tumor necrosis factor (TNF) on caspase 3/GSDME-mediated pyroptosis of monocytes and macrophages was investigated. In addition, collagen-induced arthritis (CIA) was induced in mice lacking Gsdme, and the incidence and severity of arthritis were assessed. RESULTS: Compared to cells from healthy controls, monocytes and synovial macrophages from RA patients showed increased expression of activated caspase 3, GSDME, and the N-terminal fragment of GSDME (GSDME-N). The expression of GSDME-N in monocytes from RA patients correlated positively with disease activity. Monocytes from RA patients with higher GSDME levels were more susceptible to pyroptosis. Furthermore, TNF induced pyroptosis in monocytes and macrophages by activating the caspase 3/GSDME pathway. The use of a caspase 3 inhibitor and silencing of GSDME significantly blocked TNF-induced pyroptosis. Gsdme deficiency effectively alleviated arthritis in a mouse model of CIA. CONCLUSION: These results support the notion of a pathogenic role of GSDME in RA and provide an alternative mechanism for RA pathogenesis involving TNF, which activates GSDME-mediated pyroptosis of monocytes and macrophages in RA. In addition, targeting GSDME might be a potential therapeutic approach for RA.

Bronchial epithelial pyroptosis promotes airway inflammation in a murine model of toluene diisocyanate-induced asthma.

Airway epithelial injury in response to allergens such as toluene diisocyanate (TDI) leads to persistent airway inflammation. Pyroptosis is recognized as a strong proinflammatory cell death process. However, the role of pyroptosis in bronchial epithelial injury and airway inflammation in TDI-induced asthma remains unknown. In this study, cytotoxic effect of TDI on 16HBE cells (a human bronchial epithelial cell line) was detected. Then a TDI-induced experimental asthma mouse model was established for in vivo study. Here we found that TDI induced pyroptosis in 16HBE cells, as evidenced by enhanced expressions of caspase-1 and elevated levels of LDH, IL-1ß and HMGB1. As expected, TDI-induced inflammatory cell death was significantly blocked by a specific NLRP3 inflammasome inhibitor. Intriguingly, in asthmatic mice, the increased cleavages of caspase-1 and pyroptotic executioner gasdermin D (GSDMD) in bronchial epithelial cells were decreased by NLRP3 inflammasome inhibitor. Furthermore, inhibition of NLRP3 inflammasome attenuated airway hyper-responsiveness and airway inflammation, accompanied by lower levels of IL-1ß, IgE and Th2-related cytokines. Our data suggest that bronchial epithelial pyroptosis exacerbates airway inflammation and hyper-responsiveness in TDI-induced asthma via NLRP3 inflammasome activation and GSDND cleavage. Therefore, NLRP3 inflammasome-mediated pyroptosis may be a potential treatment target for TDI-induced asthma.