NLRP3 inflammasome inhibitor(Exscientia/University of Oxford)

Salmonella enterica serovar Enteritidis (SE) is a major foodborne pathogen causing severe intestinal diseases in humans and animals, posing significant global public health and economic burdens. The Salmonella protein tyrosine phosphatase (sptP), a type III secretion system effector and critical SE virulence factor, contributes to pathogenesis, but its precise role in SE-induced intestinal injury remains unclear. Specifically, whether sptP modulates the NLRP3/Caspase-1 inflammasome (a central inflammatory regulator) is undefined. To address this, we constructed an sptP gene deficient SE strain (sptP^-) via CRISPR/Cas9, verified by PCR and sequencing. Given that MCC950 is a selective NLRP3 inflammasome inhibitor, we designed an animal study to examine the contribution of sptP to SE pathogenicity in chicks and its potential interplay with NLRP3 activation. Chicks were randomly divided into 5 groups (n = 10/group): SE sptP⁺ (wild-type), sptP⁻ (mutant), sptP⁺+MCC950 (NLRP3 inhibitor), sptP⁻+MCC950, and PBS control. Ileal tissues were collected 24 h post-infection. Histopathological injury was scored via H&E staining. Bacterial burdens in the ileum and spleen were quantified by plate counting. Cytokine levels (IL-1β, IL-18) were measured using ELISA and RT‑PCR. Protein expression (pro‑caspase‑1, cleaved‑caspase‑1, GSDMD, GSDMD‑N) was analyzed by Western blot, and pyroptosis was quantified via LDH release assay. The sptP mutant (sptP^-) was successfully generated without unintended mutations. The sptP⁻ infection reduced IL-1β/IL-18 expression, cleaved-caspase-1 and GSDMD-N levels, and significantly diminished pyroptosis. MCC950 pretreatment markedly attenuated pathology in sptP⁺-infected chicks, including reduced Caspase-1/GSDMD cleavage, lower inflammatory cytokines, decreased pyroptosis, and alleviated intestinal damage. Notably, no significant differences in pathology, cytokines, or pyroptosis markers were observed between sptP⁻ and sptP⁻+MCC950 groups. Importantly, bacterial loads did not differ across groups, excluding colonization differences as a confounding factor. These findings demonstrate that sptP promotes SE-induced intestinal injury by activating the host NLRP3/Caspase-1 inflammasome pathway, driving inflammatory cytokine release and pyroptosis. This study clarifies a previously undefined mechanism by which sptP exacerbates SE-associated intestinal damage, addressing the knowledge gap regarding its regulation of the NLRP3 inflammasome.

Nucleotide-binding oligomerization domain like receptor protein 3 (NLRP3) inflammasome is considered as a critical contributor to pathological angiogenesis and neuronal dysfunction. However, its role in ischemic retinopathies remains unclear. In this study, we aimed to evaluate the role of NLRP3 inflammasome in ischemic retinopathies and the effects of the NLRP3 inflammasome inhibitor OLT1177 on retinal neovascularization (RNV) and neuronal dysfunction in a mouse model of oxygen induced retinopathy (OIR). A single-cell transcriptome analysis was performed using public data from the preretinal proliferative membranes in patients suffering from proliferative diabetic retinopathy (PDR), which was verified by immunofluorescence and western blot. The OIR mouse model was established, with OLT1177 being intravitreally injected on postnatal day 12. The effects of OLT1177 on RNV, neuronal loss and the activation of NLRP3 inflammasome were explored. Our results demonstrated that the NLRP3 inflammasome and associated inflammatory responses were observed within microglia in both the preretinal proliferative membranes of PDR patients and the retinas of OIR mice. OLT1177 attenuated pathological RNV at a concentration of 100 μM. Furthermore, OLT1177 reduced the neuronal loss and maintained the astrocytic framework in the non-perfusion areas of retinas. Microglial activation and NLRP3 inflammasome-associated microglial inflammation were alleviated in the OLT1177-treated mice. In conclusion, NLRP3 inflammasome activation and microglial inflammation occurred in ischemic retinopathies and the inhibitor OLT1177 was well-tolerated and beneficial in attenuating RNV and preventing neuronal death in the retinas of OIR mice, which indicated that OLT1177 might be a promising treatment option for ischemic retinopathies.