Periodontitis is an immune inflammatory disease that leads to progressive destruction of bone and connective tissue, accompanied by the dysfunction and even loss of periodontal ligament stem cells (PDLSCs). Pyroptosis mediated by gasdermin-D (GSDMD) participates in the pathogenesis of inflammatory diseases. However, whether pyroptosis mediates PDLSC loss, and inflammation triggered by pyroptosis is involved in the pathological progression of periodontitis remain unclear. Here, we found that PDLSCs suffered GSDMD-dependent pyroptosis to release interleukin-1β (IL-1β) during human periodontitis. Importantly, the increased IL-1β level in gingival crevicular fluid was significantly correlated with periodontitis severity. The caspase-4/GSDMD-mediated pyroptosis caused by periodontal bacteria and cytoplasmic lipopolysaccharide (LPS) dominantly contributed to PDLSC loss. By releasing IL-1β into the tissue microenvironment, pyroptotic PDLSCs inhibited osteoblastogenesis and promoted osteoclastogenesis, which exacerbated the pathological damage of periodontitis. Pharmacological inhibition of caspase-4 or IL-1β antibody blockade in a rat periodontitis model lead to the significantly reduced loss of alveolar bone and periodontal ligament damage. Furthermore, Gsdmd deficiency alleviated periodontal inflammation and bone loss in mouse experimental periodontitis. These findings indicate that GSDMD-driven PDLSC pyroptosis and loss plays a pivotal role in the pathogenesis of periodontitis by increasing IL-1β release, enhancing inflammation, and promoting osteoclastogenesis.

Pyroptosis is a type of acute cell death that mainly occurs in immune cells. It is characterized with robust release of inflammatory cytokines and has emerged to play a critical role in the pathogenesis of sepsis-associated immune disorders. In this study, we screened for pyroptotic inhibitors with the ultimate goal to benefit sepsis treatments. Accidentally, we identified that nitrosonisoldipine (NTS), a photodegradation product of calcium channel inhibitor nisoldipine, inhibits noncanonical pyroptosis. Using murine immortalized BM-derived macrophage and human THP-1 cell line, we further discovered that NTS not only inhibits noncanonical pyroptosis mediated by caspase-11 or caspase-4 but also canonical pyroptosis mediated by caspase-1. Mechanistically, NTS directly inhibits the enzyme activities of these inflammatory caspases, and these inhibitory effects persist despite extensive washout of the drug. By contrast, apoptosis mediated by caspase-3/-7 was not affected by NTS. Mice pretreated with NTS intraperitoneally displayed improved survival rate and extended survival time in LPS- and polymicrobe-induced septic models, respectively. In conclusion, NTS is a selective inhibitor of inflammatory caspases that blocks both the noncanonical and canonical pyroptotic pathways. It is safe for intraperitoneal administration and might be used as a prototype to develop drugs for sepsis treatments.

Background: Alteration of commensal bacterial composition is associated with many inflammatory diseases. However, few studies have pinpointed the specific bacterial genes that may suppress host immune responses against microbes and maintain homeostasis in the host intestine. Methods: High-throughput screening was performed in Caenorhabditis elegans with a single gene knockout ut screening was performed in Caenorhabditis elegans with a single gene knockout Escherichia coli (E. coli) library and identified the immune suppression gene blc. The coding sequences of blc among different kinds of E. coli strains were aligned to identify the single nucleotide polymorphisms (SNPs). Physiological and biochemical experiments were performed in C. elegans and mice to explore the function of the blc variant. Findings: By screening 3983 E. coli mutants, we discovered that 9 bacterial genes, when deleted, activate innate immunity in the host C. elegans. Among these 9 genes, the gene encoding blc showed a distinctive SNP in many clinically pathogenic bacteria. We found that bacteria with this SNP, which converts Blc (G84) to Blc (E84), are highly enriched in the faeces of patients with inflammatory bowel disease (IBD). Exposure to B1c(E84)-encoding bacteria resulted in epithelial barrier disruption and immune activation in both worms and mice. Detailed analysis indicated that infection with B1c(E84)-encoding bacteria causes a significant decrease in LPE levels in the intestine and subsequently disrupts gut epithelial integrity in mice. Consistently, the levels of LPE in patients with IBD are significantly lower than those in healthy people. Finally, supplementation with LPE, which activates LPA(1)/PLC beta/PKC signaling, reversed the defects induced by B1c(E84)-encoding bacteria. Interpretation: Our results identified a novel bacterial gene, blc, in E. coli that regulates host gut integrity and immunity. (C) 2020 The Authors. Published by Elsevier B.V.

Pyroptosis is a type of programmed lytic cell death that could be activated by either the canonical or noncanonical inflammasome pathway. In this study, we aimed to examine the effect of hypertonic solution on noncanonical pyroptosis in macrophage. We found that although hypertonic solution had a general inhibitory effect on noncanonical pyroptosis, the underlying mechanism varied by the solute causing hypertonicity. Specifically, hypertonic NaCl or KCl solution inhibited the cleavage of gasdermin D, the pore-forming protein in pyroptosis, whereas hypertonic saccharide solution did not affect the cleavage or membrane binding of gasdermin D. In this case, nevertheless, pyroptosis was still inhibited as evidenced by the preserved mitochondria activity and cell membrane permeability.

Missense mutations in the gasdermin-A3 (Gsdma3) gene are associated with skin inflammation and hair loss in mice. However, the physiological function of Gsdma3 remains unclear. Herein, we reported that mice carrying the Gsdma3 Y344H mutation that encodes a presumptive activated form of Gsdma3 show increased heat production along with lower body fat percentages. Detailed analysis indicated that this metabolic phenotype is mediated by serum IL-6-induced up-regulation of thermogenesis in brown adipose tissue. The mutant form of Gsdma3 promotes the expression of IL-6 in the epidermis in a c-Jun N-terminal kinase (JNK) signaling-dependent manner. The higher whole-body heat production in alopecia and excoriation mice could be suppressed by an IL-6 receptor/GP130 inhibitor. Our results uncovered Gsdma3/IL-6-dependent cross talk between the skin and brown adipose tissue.

Background: Gut integrity is compromised in abdominal sepsis with increased cellular apoptosis and altered barrier permeability. Intestinal epithelial cells (IEC) form a physiochemical barrier that separates the intestinal lumen from the host's internal milieu and is strongly involved in the mucosal inflammatory response and immune response. Recent research indicates the involvement of the stimulator of interferons genes (STING) pathway in uncontrolled inflammation and gut mucosal immune response. Methods: We investigated the role of STING signaling in sepsis and intestinal barrier function using intestinal biopsies from human patients with abdominal sepsis and with an established model of abdominal sepsis in mice. Findings: In human abdominal sepsis, STING expression was elevated in peripheral blood mononuclear cells and intestinal biopsies compared with healthy controls, and the degree of STING expression in the human intestinal lamina propria correlated with the intestinal inflammation in septic patients. Moreover, elevated STING expression was associated with high levels of serum intestinal fatty acid binding protein that served as a marker of enterocyte damage. Inmice, the intestinal STING signaling pathway was markedly activated following the induction of sepsis induced by cecal ligation perforation (CLP). STING knockout mice showed an alleviated inflammatory response, attenuated gut permeability, and decreased bacterial translocation. Whereas mice treated with a STING agonist (DMXAA) following CLP developed greater intestinal apoptosis and a more severe systemic inflammatory response. We demonstrated thatmitochondrial DNA (mtDNA) was released during sepsis, inducing the intestinal inflammatory response through activating the STING pathway. We finally investigated DNase I administration at 5 hours post CLP surgery, showing that it reduced systemic mtDNA and inflammatory cytokines levels, organ damage, and bacterial translocation, suggesting that inhibition of mtDNA-STING signaling pathway protects against CLP-induced intestinal barrier dysfunction. Interpretation: Our results indicate that the STING signaling pathway can contribute to lethal sepsis by promoting IEC apoptosis and through disrupting the intestinal barrier. Our findings suggest that regulation of the mtDNA STING pathway may be a promising therapeutic strategy to promote mucosal healing and protect the intestinal barrier in septic patients. Fund: National Natural Science Foundation of China. (c) 2019 Published by Elsevier B. V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Hypomagnesemia is a significant risk factor for critically ill patients to develop sepsis, a life-threatening disease with a mortality rate over 25%. Our clinic data analysis showed that hypomagnesemia is associated with a decreased monocyte count in septic patients. At the cellular level, we found that Mg2+ inhibits pyroptosis. Specifically, Mg2+ limits the oligomerization and membrane localization of gasdermin D N-terminal (GSDMD-NT) upon the activation of either the canonical or noncanonical pyroptotic pathway. Mechanistically, we demonstrated that Ca2+ influx is a prerequisite for the function of GSDMD-NT. Mg2+ blocks Ca2+ influx by inhibiting the ATP-gated Ca2+ channel P2X7, thereby impeding the function of GSDMD-NT and inhibiting lipopolysaccharide (LPS)-induced noncanonical pyroptosis. Furthermore, Mg2+ administration protects mice from LPS-induced lethal septic shock. Together, our data reveal the underlying mechanism of how Mg2+ inhibits pyroptosis and suggest potential clinic applications of magnesium supplementation for sepsis prevention and treatment.

Gasdermin B (GSDMB) has been reported to be associated with immune diseases in humans, but the detailed molecular mechanisms remain unsolved. The N-terminus of GSDMB by itself, unlike other gasdermin family proteins, does not induce cell death. Here, we show that GSDMB is highly expressed in the leukocytes of septic shock patients, which is associated with increased release of the gasdermin D (GSDMD) N-terminus. GSDMB expression and the accumulation of the N-terminal fragment of GSDMD are induced by the activation of the non-canonical pyroptosis pathway in a human monocyte cell line. The downregulation of GSDMB alleviates the cleavage of GSDMD and cell death. Consistently, the overexpression of GSDMB promotes GSDMD cleavage, accompanied by increased LDH release. We further found that GSDMB promotes caspase-4 activity, which is required for the cleavage of GSDMD in non-canonical pyroptosis, by directly binding to the CARD domain of caspase-4. Our study reveals a GSDMB-mediated novel regulatory mechanism for non-canonical pyroptosis and suggests a potential new strategy for the treatment of inflammatory diseases.