Pyroptosis in sepsis induced organ dysfunction.

As an uncontrolled inflammatory response to infection, sepsis and sepsis induced organ dysfunction are great threats to the lives of septic patients. Unfortunately, the pathogenesis of sepsis is complex and multifactorial, which still needs to be elucidated. Pyroptosis is a newly discovered atypical form of inflammatory programmed cell death, which depends on the Caspase-1 dependent classical pathway or the non-classical Caspase-11 (mouse) or Caspase-4/5 (human) dependent pathway. Many studies have shown that pyroptosis is related to sepsis. The Gasdermin proteins are the key molecules in the membrane pores formation in pyroptosis. After cut by inflammatory caspase, the Gasdermin N-terminal fragments with perforation activity are released to cause pyroptosis. Pyroptosis is closely related to the occurrence and development of sepsis induced organ dysfunction. In this review, we summarized the molecular mechanism of pyroptosis, the key role of pyroptosis in sepsis and sepsis induced organ dysfunction, with the aim to bring new diagnostic biomarkers and potential therapeutic targets to improve sepsis clinical treatments.

Macrophages regulates the transition of pericyte to peritoneal fibrosis through the GSDMD/IL-1ß axis.

BACKGROUND: End stage renal disease (ESRD) has caused public health problem with high prevalence worldwide. Peritoneum from peritoneal dialysis patients with ESRD can induce pathological changes of the peritoneum, including fibrosis. The trans-differentiation of pericytes has been found to be closely associated with inflammatory diseases, such as organ fibrosis. However, the function of macrophages in regulating the transition of pericyte to peritoneal fibrosis is unclear. METHODS: Histological examination was conducted using Hematoxylin and eosin (HE) staining and Masson's trichrome staining. The protein levels were determined via western blot. Enzyme-linked immunosorbent assay (ELISA) was used to examine IL-1ß concentrations. Gasdermin D (GSDMD) was knocked out in mice by Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9). RESULTS: Mice receiving dextrose peritoneal dialysate displayed mesothelial cell monolayer loss and thickness of submesothelial compact zone increase. Moreover, dextrose peritoneal dialysate treatment up-regulated GSDMD expression. GSDMD knockdown inhibited IL-1ß production in macrophages. Further, pericytes were treated with cultural supernatant from macrophages. We found that GSDMD knockdown suppressed fibrosis and vascular endothelial growth factor (VEGF)/phosphoinositide 3-kinase (PI3K) pathway in pericytes. In addition, GSDMD were knocked out in mice using CRISPR/Cas9. The histological examinations revealed that GSDMD-/- alleviated the damage of peritoneal tissue and thickness of submesothelial compact zone. GSDMD-/- attenuated interleukin-1beta (IL-1ß) level and peritoneal fibrosis induced by dextrose peritoneal dialysate treatment in pericytes in vivo. CONCLUSION: These results demonstrated that macrophages can regulate the transition of pericyte to peritoneal fibrosis via the GSDMD/IL-1ß axis, which provides a new therapeutic target.

Tks5 Regulates Synaptic Podosome Formation and Stabilization of the Postsynaptic Machinery at the Neuromuscular Junction.

Currently, the etiology of many neuromuscular disorders remains unknown. Many of them are characterized by aberrations in the maturation of the neuromuscular junction (NMJ) postsynaptic machinery. Unfortunately, the molecular factors involved in this process are still largely unknown, which poses a great challenge for identifying potential therapeutic targets. Here, we identified Tks5 as a novel interactor of αdystrobrevin-1, which is a crucial component of the NMJ postsynaptic machinery. Tks5 has been previously shown in cancer cells to be an important regulator of actin-rich structures known as invadosomes. However, a role of this scaffold protein at a synapse has never been studied. We show that Tks5 is crucial for remodeling of the NMJ postsynaptic machinery by regulating the organization of structures similar to the invadosomes, known as synaptic podosomes. Additionally, it is involved in the maintenance of the integrity of acetylcholine receptor (AChR) clusters and regulation of their turnover. Lastly, our data indicate that these Tks5 functions may be mediated by its involvement in recruitment of actin filaments to the postsynaptic machinery. Collectively, we show for the first time that the Tks5 protein is involved in regulation of the postsynaptic machinery.

Gasdermins mediate cellular release of mitochondrial DNA during pyroptosis and apoptosis.

Pyroptosis and intrinsic apoptosis are two forms of regulated cell death driven by active caspases where plasma membrane permeabilization is induced by gasdermin pores. Caspase-1 induces gasdermin D pore formation during pyroptosis, whereas caspase-3 promotes gasdermin E pore formation during apoptosis. These two types of cell death are accompanied by mitochondrial outer membrane permeabilization due to BAK/BAX pore formation in the external membrane of mitochondria, and to some extent, this complex also affects the inner mitochondrial membrane facilitating mitochondrial DNA relocalization from the matrix to the cytosol. However, the detailed mechanism responsible for this process has not been investigated. Herein, we reported that gasdermin processing is required to induce mitochondrial DNA release from cells during pyroptosis and apoptosis. Gasdermin targeted at the plasma membrane promotes a fast mitochondrial collapse along with the initial accumulation of mitochondrial DNA in the cytosol and then facilitates the DNA's release from the cell when the plasma membrane ruptures. These findings demonstrate that gasdermin action has a critical effect on the plasma membrane and facilitates the release of mitochondrial DNA as a damage-associated molecular pattern.

Gasdermin D restricts Burkholderia cenocepacia infection in vitro and in vivo.

Burkholderia cenocepacia (B. cenocepacia) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including cystic fibrosis patients. The lack of gasdermin D (GSDMD) protects mice against endotoxin lipopolysaccharide (LPS) shock. On the other hand, GSDMD promotes mice survival in response to certain bacterial infections. However, the role of GSDMD during B. cenocepacia infection is not yet determined. Our in vitro study shows that GSDMD restricts B. cenocepacia replication within macrophages independent of its role in cell death through promoting mitochondrial reactive oxygen species (mROS) production. mROS is known to stimulate autophagy, hence, the inhibition of mROS or the absence of GSDMD during B. cenocepacia infections reduces autophagy which plays a critical role in the restriction of the pathogen. GSDMD promotes inflammation in response to B. cenocepacia through mediating the release of inflammasome dependent cytokine (IL-1ß) and an independent one (CXCL1) (KC). Additionally, different B. cenocepacia secretory systems (T3SS, T4SS, and T6SS) contribute to inflammasome activation together with bacterial survival within macrophages. In vivo study confirmed the in vitro findings and showed that GSDMD restricts B. cenocepacia infection and dissemination and stimulates autophagy in response to B. cenocepacia. Nevertheless, GSDMD promotes lung inflammation and necrosis in response to B. cenocepacia without altering mice survival. This study describes the double-edged functions of GSDMD in response to B. cenocepacia infection and shows the importance of GSDMD-mediated mROS in restriction of B. cenocepacia.

CAVIN2 is frequently silenced by CpG methylation and sensitizes lung cancer cells to paclitaxel and 5-FU.

Aim: To explore the biological functions and clinical significance of CAVIN2 in lung cancer. Materials & methods: Methylation-specific PCR was used to measure promoter methylation of CAVIN2. The function of CAVIN2 was tested by Cell Counting Kit-8, colony formation, Transwell, flow cytometric analysis, acridine orange/ethidium bromide, chemosensitivity assay and xenograft assay. Results: CAVIN2 is significantly downregulated by promoter methylation in lung cancer. CAVIN2 overexpression inhibits lung cancer cell migration and invasion. Furthermore, ectopic expression of CAVIN2 inhibits cell proliferation in vivo and in vitro by inducing G2/M cell cycle arrest, which sensitizes the chemosensitivity of lung cancer cells to paclitaxel and 5-fluorouracil, but not cisplatin. Conclusion: CAVIN2 is a tumor suppressor in non-small-cell lung cancer and can sensitize lung cancer cells to paclitaxel and 5-fluorouracil.

Metabolic competition between host and pathogen dictates inflammasome responses to fungal infection.

The NLRP3 inflammasome has emerged as a central immune regulator that senses virulence factors expressed by microbial pathogens for triggering inflammation. Inflammation can be harmful and therefore this response must be tightly controlled. The mechanisms by which immune cells, such as macrophages, discriminate benign from pathogenic microbes to control the NLRP3 inflammasome remain poorly defined. Here we used live cell imaging coupled with a compendium of diverse clinical isolates to define how macrophages respond and activate NLRP3 when faced with the human yeast commensal and pathogen Candida albicans. We show that metabolic competition by C. albicans, rather than virulence traits such as hyphal formation, activates NLRP3 in macrophages. Inflammasome activation is triggered by glucose starvation in macrophages, which occurs when fungal load increases sufficiently to outcompete macrophages for glucose. Consistently, reducing Candida's ability to compete for glucose and increasing glucose availability for macrophages tames inflammatory responses. We define the mechanistic requirements for glucose starvation-dependent inflammasome activation by Candida and show that it leads to inflammatory cytokine production, but it does not trigger pyroptotic macrophage death. Pyroptosis occurs only with some Candida isolates and only under specific experimental conditions, whereas inflammasome activation by glucose starvation is broadly relevant. In conclusion, macrophages use their metabolic status, specifically glucose metabolism, to sense fungal metabolic activity and activate NLRP3 when microbial load increases. Therefore, a major consequence of Candida-induced glucose starvation in macrophages is activation of inflammatory responses, with implications for understanding how metabolism modulates inflammation in fungal infections.

Extended subsite profiling of the pyroptosis effector protein gasdermin D reveals a region recognized by inflammatory caspase-11.

Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1ß (pro-IL18/pro-IL1ß). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1'-P4', the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1'-P4' region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1ß cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1'-P4' region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, i.e. caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1'-P4'-based inhibitors could be exploited to specifically target inflammatory caspases.

The CHORD protein CHP-1 regulates EGF receptor trafficking and signaling in C. elegans and in human cells.

The intracellular trafficking of growth factor receptors determines the activity of their downstream signaling pathways. Here, we show that the putative HSP-90 co-chaperone CHP-1 acts as a regulator of EGFR trafficking in C. elegans. Loss of chp-1 causes the retention of the EGFR in the ER and decreases MAPK signaling. CHP-1 is specifically required for EGFR trafficking, as the localization of other transmembrane receptors is unaltered in chp-1(lf) mutants, and the inhibition of hsp-90 or other co-chaperones does not affect EGFR localization. The role of the CHP-1 homolog CHORDC1 during EGFR trafficking is conserved in human cells. Analogous to C. elegans, the response of CHORDC1-deficient A431 cells to EGF stimulation is attenuated, the EGFR accumulates in the ER and ERK2 activity decreases. Although CHP-1 has been proposed to act as a co-chaperone for HSP90, our data indicate that CHP-1 plays an HSP90-independent function in controlling EGFR trafficking through the ER.

Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy.

Membrane targeting of autophagy-related complexes is an important step that regulates their activities and prevents their aberrant engagement on non-autophagic membranes. ATG16L1 is a core autophagy protein implicated at distinct phases of autophagosome biogenesis. In this study, we dissected the recruitment of ATG16L1 to the pre-autophagosomal structure (PAS) and showed that it requires sequences within its coiled-coil domain (CCD) dispensable for homodimerisation. Structural and mutational analyses identified conserved residues within the CCD of ATG16L1 that mediate direct binding to phosphoinositides, including phosphatidylinositol 3-phosphate (PI3P). Mutating putative lipid binding residues abrogated the localisation of ATG16L1 to the PAS and inhibited LC3 lipidation. On the other hand, enhancing lipid binding of ATG16L1 by mutating negatively charged residues adjacent to the lipid binding motif also resulted in autophagy inhibition, suggesting that regulated recruitment of ATG16L1 to the PAS is required for its autophagic activity. Overall, our findings indicate that ATG16L1 harbours an intrinsic ability to bind lipids that plays an essential role during LC3 lipidation and autophagosome maturation.

The high-affinity phosphate-binding protein PstS is accumulated under high fructose concentrations and mutation of the corresponding gene affects differentiation in Streptomyces lividans.

The secreted protein pattern of Streptomyces lividans depends on the carbon source present in the culture media. One protein that shows the most dramatic change is the high-affinity phosphate-binding protein PstS, which is strongly accumulated in the supernatant of liquid cultures containing high concentrations (>3 %) of certain sugars, such as fructose, galactose and mannose. The promoter region of this gene and that of its Streptomyces coelicolor homologue were used to drive the expression of a xylanase in S. lividans that was accumulated in the culture supernatant when grown in the presence of fructose. PstS accumulation was dramatically increased in a S. lividans polyphosphate kinase null mutant (Deltappk) and was impaired in a deletion mutant lacking phoP, the transcriptional regulator gene of the two-component phoR-phoP system that controls the Pho regulon. Deletion of the pstS genes in S. lividans and S. coelicolor impaired phosphate transport and accelerated differentiation and sporulation on solid media. Complementation with a single copy in a S. lividans pstS null mutant returned phosphate transport and sporulation to levels similar to those of the wild-type strain. The present work demonstrates that carbon and phosphate metabolism are linked in the regulation of genes and that this can trigger the genetic switch towards morphogenesis.