ROS-dependent S-palmitoylation activates cleaved and intact gasdermin D.

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores after cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT)1-10. Here we report that GSDMD Cys191 is S-palmitoylated and that palmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS). Cleavage-deficient GSDMD (D275A) is also palmitoylated after inflammasome stimulation or treatment with ROS activators and causes pyroptosis, although less efficiently than palmitoylated GSDMD-NT. Palmitoylated, but not unpalmitoylated, full-length GSDMD induces liposome leakage and forms a pore similar in structure to GSDMD-NT pores shown by cryogenic electron microscopy. ZDHHC5 and ZDHHC9 are the major palmitoyltransferases that mediate GSDMD palmitoylation, and their expression is upregulated by inflammasome activation and ROS. The other human gasdermins are also palmitoylated at their N termini. These data challenge the concept that cleavage is the only trigger for GSDMD activation. They suggest that reversible palmitoylation is a checkpoint for pore formation by both GSDMD-NT and intact GSDMD that functions as a general switch for the activation of this pore-forming family.

Parathyroid hormone-induced down-regulation of miR-532-5p for matrix metalloproteinase-13 expression in rat osteoblasts.

Parathyroid hormone (PTH) acts on osteoblasts and functions as an essential regulator of calcium homeostasis and as a mediator of bone remodeling. We previously reported that PTH stimulates the expression of matrix metalloproteinase-13 (MMP-13) in rat osteoblasts and that MMP-13 plays a key role in bone remodeling, endochondral bone formation, and bone repair. Recent evidence indicated that microRNAs (miRNAs) have regulatory functions in bone metabolism. In this study, we hypothesized that the down-regulation of miRNAs that target MMP-13 by PTH leads to the stimulation of MMP-13 expression in osteoblasts. We used various bioinformatic tools to identify miRNAs that putatively target rat MMP-13. Among these miRNAs, the expression of miR-532-5p in rat osteoblasts decreased at 4 h of PTH-treatment, whereas MMP-13 mRNA expression was maximal at the same time point. When an miR-532-5p mimic was transiently transfected into UMR-106-01 cells, MMP-13 mRNA and protein expression decreased. Using a luciferase reporter assay system, we also identified that miR-532-5p directly targeted the 3' UTRs of MMP-13 gene. Based on these results, we suggest that PTH-induced down-regulation of miR-532-5p resulted in the stimulation of MMP-13 expression in rat osteoblasts. This study identified a significant role of miRNA in controlling bone remodeling via PTH-stimulated MMP-13 expression. This finding enhances our understanding of bone metabolism and bone-related diseases and it could provide information regarding the usage of miRNAs as therapeutic agents or biomarkers.

Gasdermin E dictates inflammatory responses by controlling the mode of neutrophil death.

Both lytic and apoptotic cell death remove senescent and damaged cells in living organisms. However, they elicit contrasting pro- and anti-inflammatory responses, respectively. The precise cellular mechanism that governs the choice between these two modes of death remains incompletely understood. Here we identify Gasdermin E (GSDME) as a master switch for neutrophil lytic pyroptotic death. The tightly regulated GSDME cleavage and activation in aging neutrophils are mediated by proteinase-3 and caspase-3, leading to pyroptosis. GSDME deficiency does not alter neutrophil overall survival rate; instead, it specifically precludes pyroptosis and skews neutrophil death towards apoptosis, thereby attenuating inflammatory responses due to augmented efferocytosis of apoptotic neutrophils by macrophages. In a clinically relevant acid-aspiration-induced lung injury model, neutrophil-specific deletion of GSDME reduces pulmonary inflammation, facilitates inflammation resolution, and alleviates lung injury. Thus, by controlling the mode of neutrophil death, GSDME dictates host inflammatory outcomes, providing a potential therapeutic target for infectious and inflammatory diseases.

The palmitoylation of gasdermin D directs its membrane translocation and pore formation during pyroptosis.

Plasma membrane perforation elicited by caspase cleavage of the gasdermin D (GSDMD) N-terminal domain (GSDMD-NT) triggers pyroptosis. The mechanisms underlying GSDMD membrane translocation and pore formation are not fully understood. Here, using a proteomic approach, we identified fatty acid synthase (FASN) as a GSDMD-binding partner. S-palmitoylation of GSDMD at Cys191/Cys192 (human/mouse), catalyzed by palmitoyl acyltransferases ZDHHC5 and ZDHHC9 and facilitated by reactive oxygen species (ROS), directly mediated membrane translocation of GSDMD-NT but not full-length GSDMD (GSDMD-FL). Palmitoylation of GSDMD-FL could be induced before inflammasome activation by stimuli such as lipopolysaccharide (LPS), consequently serving as an essential molecular event in macrophage priming. Inhibition of GSDMD palmitoylation suppressed macrophage pyroptosis and IL-1ß release, mitigated organ damage, and enhanced the survival of septic mice. Thus, GSDMD-NT palmitoylation is a key regulatory mechanism controlling GSDMD membrane localization and activation, which may offer an additional target for modulating immune activity in infectious and inflammatory diseases.

Palmitoylation of gasdermin D directs its membrane translocation and pore formation in pyroptosis.

Gasdermin D (GSDMD)-mediated macrophage pyroptosis plays a critical role in inflammation and host defense. Plasma membrane perforation elicited by caspase-cleaved GSDMD N-terminal domain (GSDMD-NT) triggers membrane rupture and subsequent pyroptotic cell death, resulting in release of pro-inflammatory IL-1ß and IL-18. However, the biological processes leading to its membrane translocation and pore formation are not fully understood. Here, using a proteomics approach, we identified fatty acid synthase (FASN) as a GSDMD-binding partner and demonstrated that post-translational palmitoylation of GSDMD at Cys191/Cys192 (human/mouse) led to membrane translocation of GSDMD-NT but not full-length GSDMD. GSDMD lipidation, mediated by palmitoyl acyltransferases ZDHHC5/9 and facilitated by LPS-induced reactive oxygen species (ROS), was essential for GSDMD pore-forming activity and pyroptosis. Inhibition of GSDMD palmitoylation with palmitate analog 2-bromopalmitate or a cell permeable GSDMD-specific competing peptide suppressed pyroptosis and IL-1ß release in macrophages, mitigated organ damage, and extended the survival of septic mice. Collectively, we establish GSDMD-NT palmitoylation as a key regulatory mechanism controlling GSDMD membrane localization and activation, providing a novel target for modulating immune activity in infectious and inflammatory diseases. One Sentence Summary: LPS-induced palmitoylation at Cys191/Cys192 is required for GSDMD membrane translocation and its pore-forming activity in macrophages.

Perioperative antimicrobial prophylaxis in clean-contaminated head and neck squamous cell cancer surgeries: Is less better?

Introduction: Judicious use of antibiotics and stringent adherence to practice guidelines is the need of the hour as antibiotic resistance is a rampant problem. Despite several reports in the literature describing the optimal duration of antibiotics, there is no consensus. A "one for all" protocol may be impractical and hence the guidelines need to be tweaked to take into consideration local factors. We designed a protocol for prophylactic antibiotics in clean-contaminated head and neck cancer squamous cell carcinoma (HNSCC) surgeries to prevent unchecked abuse and evaluated its feasibility. Materials and Methods: Two hundred consecutive patients who underwent a clean-contaminated surgery for HNSCC between January 2017 and December 2019 were included. Single-dose intravenous amoxicillin-clavulanate at induction followed by three doses of amoxicillin-clavulanate, metronidazole, and amikacin in the postoperative period was used. Adherence to the antibiotic protocol was assessed from a prospectively maintained database. Results: The mean age was 55.99 ± 11.71 years. The protocol was effective in 70% of the patients with an acceptable surgical site infection (SSI) rate of 12%. Flap-related complications (9.5%) and oro-cutaneous fistula (5%) were common causes of prolonged antibiotics. On univariate analysis, blood transfusion (P = .014), clinical stage at presentation (P = .028), patients undergoing reconstruction (P = .001), longer operative time (P = .009), and pathological T stage (P = 0.03) were at higher chance of deviating from the protocol. On multivariate analysis, age more than 50 years (OR: 2.14, 95% CI: (1.01, 4.52); P value = 0.047) and reconstruction (OR: 3.36, 95% CI: (1.21, 9.32); P value = 0.020) were found to be significant. Conclusions: A three-dose perioperative antibiotic prophylaxis in clean-contaminated HNSCC surgeries is feasible. Similar protocols should be developed and validated at other major centers to limit the unnecessary use of antibiotics and prevent the emergence of antibiotic resistance.

Successful treatment of Strongyloides stercoralis hyperinfection in a case of systemic lupus erythematosus - review of the literature regarding various treatment schedules.

Strongyloides stercoralis hyperinfection poses a great challenge to physicians in the tropics due to their non-specific symptoms and signs. Early identification and initiation of treatment improves mortality rates. Reports of Strongyloides infection in Hansens disease, systemic lupus erythematosus (SLE), organ transplant recipients and malnourished individuals have been reported in the published literature. The outcomes were variable, which may be attributed to a lack of treatment protocol. Treatment has been individualized and reports available are based on individual case reports and small case series. We report a successfully treated case of hyperinfection in SLE and discuss the various treatment options available.