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.

Single-Cell Analysis of Patients with Axial Spondyloarthritis After Anti-TNFα Treatment: Experimental Data and Review of the Literature.

Axial spondyloarthritis (Ax-SpA) is a chronic inflammatory disease that predominantly affects the axial joints and is most common in young men. However, the precise immune cell subset involved in Ax-SpA remains unclear. Our study characterized the periphery immune landscape of Ax-SpA patients before and after anti-TNFα treatment using single-cell transcriptomics and proteomics sequencing and elucidated the effects of anti-TNFα treatment at the single-cell level. First, we found that peripheral granulocytes and monocytes significantly increased in Ax-SpA patients. Second, we identified a more functional subtype of regulatory T cells, which was present in synovial fluid and increased in patients after treatment. Third, we identified a cluster of inflammatory monocyte subset with stronger inflammatory and chemotactic characteristics. A potential interaction between classical monocytes and granulocytes via the CXCL8/2-CXCR1/2 signaling pathway was observed, which decreased after treatment. Together, these results defined the complex expression profiles and advanced our understanding of the immune atlas in Ax-SpA patients before and after anti-TNFα treatment.

Disulfiram alleviates pristane-induced lupus via inhibiting GSDMD-mediated pyroptosis.

Activation of multiple inflammasomes in monocytes/macrophages is associated with the pathogenesis of systemic lupus erythematosus (SLE). Gasdermin D (GSDMD)-mediated pyroptosis, a common consequence of multiple activated inflammasomes, is a programmed cell death with strong inflammatory responses. This suggested that targeting monocyte/macrophage pyroptosis might provide an opportunity to cure SLE. Here, we aimed to investigate the effect of disulfiram (DSF), a small molecule inhibitor of pyroptosis, and its potential therapeutic mechanism for SLE. The mRNA expression of GSDMD and IL-1ß were significantly increased in peripheral blood mononuclear cells (PBMCs) from SLE patients. Importantly, we found serum from SLE patients rather than healthy controls induced GSDMD-mediated pyroptosis in THP-1 cells, as evidenced by enhanced LDH release, increased number of PI-positive cells, and high expression of full-length GSDMD and N-terminal GSDMD. Interestingly, treatment with DSF obviously inhibited pyroptosis of THP-1 cells induced by serum from SLE patients. Of note, DSF administration reduced proteinuria, serum anti-dsDNA level, and renal immune complex. It also attenuated renal damage in PIL mice. Further research found that the high level of serum IL-ß and GSDMD-mediated pyroptosis of glomerular macrophages in PIL mice were rescued with DSF treatment. These data implied that GSDMD-mediated monocytes/macrophages pyroptosis played an important role in the pathogenesis of SLE and DSF might be a potential alternative therapeutic agent for SLE.

Diabetic nephropathy execrates epithelial-to-mesenchymal transition (EMT) via miR-2467-3p/Twist1 pathway.

Although diabetic nephropathy (DN) is induced by a complicate interplay of multiple factors, the underlying mechanisms remain poorly characterized, even the treatment. Herein, we show that both of DN patients and STZ-induced type 1 diabetic rat exhibit the reduction both of urinary and circulating miR-2467-3p. We identify a negative correlation between miR-2467-3p levels and renal dysfunction. Administration of miR-2467-3p prevents diabetes-induced renal dysfunction and represses renal fibrosis in STZ-induced type 1 diabetic rats. Conversely, anti-miR-2467 overexpression exacerbates renal dysfunction and fibrosis in STZ-induced rats. In diabetic condition, the reduction of miR-2467-3p promotes expression of Twist1, inducing epithelial-to-mesenchymal transition (EMT), resulting in renal fibrosis and kidney dysfunction. Together, our study presents miR-2467/Twist1/EMT as a regulatory axis of renal dysfunction in DN.