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Pyroptosis is a programmed necrotic cell death executed by gasdermins, a family of pore-forming proteins. The cleavage of gasdermins by specific proteases enables their pore-forming activity. The activation of the prototype member of the gasdermin family, gasdermin D (GSDMD), is linked to innate immune monitoring by inflammasomes. Additional gasdermins such as GSDMA, GSDMB, GSDMC, and GSDME are activated by inflammasome-independent mechanisms. Pyroptosis is emerging as a key host defense strategy against pathogens. However, excessive pyroptosis causes cytokine storm and detrimental inflammation leading to tissue damage and organ dysfunction. Consequently, dysregulated pyroptotic responses contribute to the pathogenesis of various diseases, including sepsis, atherosclerosis, acute respiratory distress syndrome, and neurodegenerative disorders. This review will discuss the inflammatory consequences of pyroptosis and the mechanisms of pyroptosis-induced tissue damage and disease pathogenesis., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Reports in recent years have shown that pancreatic β-cell pyroptosis represents a critical mechanism involved with the progressive failure of pancreatic function. Previous research from our laboratory has indicated that artemether can increase the number of cells in pancreatic islets of db/db mice. In this study, we further examined whether artesunate (ART) protects pancreatic β-cells from the damage of streptozotocin (STZ) by inhibiting pyroptosis., Materials and Methods: In vitro, MIN6 cells exposed to 1 mM STZ were treated with ART (0.8 or 1.6 μM). The effects of ART on STZ-treated cells were evaluated through CCK-8 assay, flow cytometry and western blot, and further compared the effects of ART with the NLRP3 inhibitor, Mcc950 upon pyroptosis pathway proteins using western blot. In vivo, Male C57 mice were administered with a single intraperitoneal injection of STZ, and those with confirmed diabetes mellitus were given ART (0.5 or 1.0 mg/ml in drinking water) for 18 days. The effects of ART on STZ-induced diabetes were assessed by the observation of the general situation, glucose tolerance test, hematoxylin-eosin (HE) staining and immunohistochemistry., Results: In MIN6 cells treated with STZ, we found that ART increased cell viability, decreased the number of late apoptotic cells (including pyroptosis cells) and inhibited the expression of proteins associated with the pyroptosis pathway. In STZ-induced animal model, the administration of ART reduced blood glucose levels, improved the consumption status within this diabetic mouse model and inhibited the expression of proteins include in the pyroptosis pathway in mice pancreats., Conclusions: Inhibition of pyroptosis may be a critical mechanism through which artesunate exerts protective effects upon pancreatic β cells., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The pathological mechanism of corneal injuries mediated by alkali burns are associated with Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain containing 3 protein (NLRP3)-related corneal sterile inflammation. Whether the executive protein gasdermin D (GSDMD) of pyroptosis mediated by the NLRP3 inflammasome is present in alkali-induced corneal lesions remains unclear. Dexamethasone (Dex) is a commonly used drug for ocular surface diseases that can maintain corneal transparency and anti-inflammatory effects by topical administration. Here, we presented evidence that the effect of Dex on the pyroptosis-related caspase-1/GSDMD pathway in corneal alkali burns (CABs). We assessed the clinical manifestations and histological characteristics of the placebo group, 0.05% Dex group, 0.1% Dex group on day 3 or day 7 postburn and the control group (healthy corneas). The expression of factors (including NLRP3, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N termini, pro-interleukin(IL)-1β, IL-1β, pro-IL-18 and IL-18) involved in the pyroptosis related caspase-1/GSDMD signaling pathway was demonstrated by molecular experiments in CAB. Alkali burns can upregulate the originally relatively dim expression of NLRP3, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, pro-IL-1β, pro-IL-18, IL-1β and IL-18 in the healthy corneal epithelium and stroma. However, Dex can reverse the enhanced expression at the two timepoints. Corneal sterile inflammation can activate the NLRP3 inflammasome through the innate immune response mechanism and then activate the pyroptosis-related caspase-1/GSDMD signaling pathway. In addition, Dex can inhibit pyroptosis through this pathway., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Gasdermin-D (GSDMD) is a member of the gasdermin (Gsdm) protein family, and its cleavage by inflammatory cysteine proteases (caspases, CASPs) is a critical event in cell pyroptosis. The role and functions of GSDMD on mice and humans are widely studied, but its expression, structure, and function in other species are less known. In the present work, rabbit anti-porcine GSDMD (pGSDMD) polyclonal antibody was prepared by immunizing New Zealand white rabbits with prokaryotic expressed recombinant pGSDMD (rpGSDMD). The prepared polyclonal antibody showed good specificity in Western blot and indirect immunofluorescence (IIF) assays. Western blot results showed that the polyclonal antibody could recognize overexpressed pGSDMD in human embryonic kidney cells (HEK293T) and endogenously expressed pGSDMD in cultured intestinal porcine enterocytes (IPEC-J2) and porcine kidney cells (PK-15). Western blot also revealed that pGSDMD was expressed in the heart, liver, lung, kidney, gallbladder, and jejunum of pigs. HEK293T cells overexpressing GSDMD showed green fluorescence in the IIF assay only after being treated with 0.3% Triton-X 100, which indicated that the full-length pGSDMD was located in the plasma but not on the cell membrane. This work provides a useful tool and basic information for further studies on pGSDMD., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The aim of this study was to identify and characterize, at the molecular and transcriptional levels, sequences encoding the different members of the four families of shrimp antimicrobial peptides (AMPs) in species of the genus Farfantepenaeus. The identification of the AMP sequences was performed by in silico analysis as well as by molecular cloning and nucleotide sequencing. We identified all seven shrimp ALFs (ALF-A to ALF-G), both Type IIa and Type IIb crustins as well as two stylicins (STY1 and STY2) in Farfantepenaeus. Only two genes (PEN1/2 and PEN4) of the four-member penaeidin family (PEN1/2 to PEN5) were found and this is the first report of stylicins as well as of several additional members of ALFs, crustins and penaeidins in species of the genus Farfantepenaeus. All AMP genes have shown to be constitutively transcribed in the shrimp immune cells (hemocytes), except for ALF-G. Finally, the transcriptional profile of the different AMPs was assessed in the hemocytes of F. paulensis (pink shrimp) following an experimental infection with the opportunistic filamentous fungus Fusarium solani. We found that while the expression of ALF-B was induced at 24 h, the STY2 gene was down-regulated at 48 h post-challenge. These results provide evidence of the molecular diversity of AMPs from shrimp of the genus Farfantepenaeus in terms of sequences, biochemical properties and expression profiles in response to infectious diseases., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In mammals, interleukin (IL)-17A and IL-17F, mainly produced by Th17 cells, are hallmark inflammatory cytokines that play important roles in the intestinal mucosal immune response. In contrast, three mammalian IL-17A and IL-17F counterparts (IL-17A/F1-3) have been identified in teleosts, and most of their functions have been described in the lymphoid organs. However, their function in the intestinal mucosal immune response is poorly understood. In this study, a recombinant (r) tiger puffer fish fugu (Takifugu rubripes) IL-17A/F1 was produced and purified using a mammalian expression system, and was used to stimulate cells isolated from fugu head kidney and intestines. The gene expression levels of TNF-α, IL-1β, IL-6, and β-defensin-like protein-1 (BD-1) genes were evaluated at 0, 3, 6 and 12 h post-stimulation (hps). Phagocytic activity and superoxide anion production were evaluated at the same time points using an NBT assay. The rIL-17A/F1 protein was shown to induce the expression of pro-inflammatory cytokines and antimicrobial peptides in both head kidney and intestinal cells. Expression levels for IL-1β, TNF-α, and IL-6 were all up-regulated between 3 and 12 hps. In addition, stimulation with rIL-17A/F1 enhanced phagocytic activity at 24 hps. Superoxide anion production was increased at 48 hps in the head kidney cells and moderately increased at 48 hps in intestinal cells. This study suggests that fugu IL-17A/F1 plays an important role in promoting the innate immune response and may act as a bridge between innate and adaptive immunity in the head kidney and intestine., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The Wnt signal transduction pathway is involved in a wide variety of cellular processes, including cell proliferation, differentiation, apoptosis, and immunity against microbial infection. In the current study, we cloned and characterized two Wnt homologues (Mn-Wnt4 and Mn-Wnt16) in Macrobrachium nipponense. The full length cDNA of Mn-Wnt4 was 3144 bp with a 1074 bp open reading frame (ORF) that encoded a protein containing 358 amino acid residues. The full length cDNA of Mn-Wnt16 transcript was 2893 bp with a 1281 bp ORF that encoded a 427 amino acid protein. Mn-Wnt4 and Mn-Wnt16 proteins contained a highly conserved WNT1 domain. Tissue distribution analysis showed that Mn-Wnt4 and Mn-Wnt16 were highly expressed in the stomach. The transcriptional levels of Mn-Wnt4 and Mn-Wnt16 in the stomach were upregulated at most tested time points after bacterial (Staphylococcus aureus and Vibrio parahaemolyticus) and viral (White spot syndrome virus) infection. Moreover, the expression levels of some antimicrobial peptides (AMPs) (including anti-lipopolysaccharide factor [ALF] and crustin [CRU]) were upregulated after V. parahaemolyticus infection. We further used dsRNA-mediated RNA interference technology to explore the relationship between these two Wnt genes and the expression levels of AMPs during V. parahaemolyticus infection. Mn-Wnt4 knockdown could significantly inhibit the expression of ALF1 and CRU4 in the stomach of V. parahaemolyticus-injected prawns, whereas Mn-Wnt16 silencing could result in the inhibition of the expression level of CRU3 and CRU4 in the stomach of V. parahaemolyticus-infected prawns. These findings indicated that the Wnt gene family might participate in the body's innate immune response to Vibrio infection by regulating the synthesis of a variety of AMPs. Our study will help to understand the role of the Wnt signaling pathway in the immune response of crustaceans., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The ancient phylum of Cnidaria contains many aquatic species with peculiar lifestyle. In order to survive, these organisms have evolved attack and defense mechanisms that are enabled by specialized cells and highly developed venoms. Pore-forming toxins are an important part of their venomous arsenal. Along some other types, the most representative are examples of four protein families that are commonly found in other kingdoms of life: actinoporins, Cry-like proteins, aerolysin-like toxins and MACPF/CDC toxins. Some of the homologues of pore-forming toxins may serve other functions, such as in food digestion, development and response against pathogenic organisms. Due to their interesting physico-chemical properties, the cnidarian pore-forming toxins may also serve as tools in medical research and nanobiotechnological applications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pore formation is a common mechanism of action for many bacterial toxins. More than one third of clostridial toxins are pore-forming toxins (PFTs) belonging to the β-PFT class. They are secreted as soluble monomers rich in β-strands, which recognize a specific receptor on target cells and assemble in oligomers. Then, they undergo a conformational change leading to the formation of a β-barrel, which inserts into the lipid bilayer forming functional pore. According to their structure, clostridial β-PFTs are divided into several families. Clostridial cholesterol-dependent cytolysins form large pores, which disrupt the plasma membrane integrity. They are potent virulence factors mainly involved in myonecrosis. Clostridial heptameric β-PFTs (aerolysin family and staphylococcal α-hemolysin family) induce small pores which trigger signaling cascades leading to different cell responses according to the cell types and toxins. They are mainly responsible for intestinal diseases, like necrotic enteritis, or systemic diseases/toxic shock from intestinal origin. Clostridial intracellularly active toxins exploit pore formation through the endosomal membrane to translocate the enzymatic component or domain into the cytosol. Single chain protein toxins, like botulinum and tetanus neurotoxins, use hydrophobic α-helices to form pores, whereas clostridial binary toxins encompass binding components, which are structurally and functionally related to β-PFTs, but which have acquired the specific activity to internalize their corresponding enzymatic components. Structural analysis suggests that β-PFTs and binding components share a common evolutionary origin., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

LcrV, a component of the type III secretion system (T3SS) translocon in Yersinia pestis, has been concerned in suppressing inflammation through Toll-like receptor 2 (TLR2) by inducing expression of the anti-inflammatory cytokine interleukin-10 (IL-10). Previous studies have reported that LcrV aa E33, E34, K42 and/or E204 and E205 were important for interactions with TLR2 in vitro. While, recently there have been conflicting reports doubting this interaction and its importance in vivo. To further investigate the role of these residues, we replaced the wild-type lcrV gene on the pCD1Ap virulence plasmid of Y. pestis with lcrV2345 gene, which encodes a mutant protein by substituting all five of the amino acid residues with glutamine. The characteristics of the wild-type LcrV and mutant LcrV2345 were evaluated in tissue culture and mice. When purified protein was incubated with HEK293 cells synthesizing human TLR2 with or without CD14, LcrV2345 induced higher levels of IL-8 than wild-type LcrV, indicating that the LcrV2345 was not impaired in its ability to interact with TLR2. LcrV2345 stimulated higher levels of tumor necrosis factor-alpha (TNF-α) production than LcrV in J774A.1 cells, while neither protein elicited significant levels of IL-10. We also found there was no statistically significant difference in virulence between strains with wild-type LcrV and with mutated LcrV2345 administered by either subcutaneous or intranasal route in mice. Additionally, there were no discernible differences in survival kinetics. Serum levels of cytokines, such as IL-10 and TNF-α, bacterial burden, and the extent of organ inflammation were also indistinguishable in both strains. Our data confirm that immunomodulation mediated by LcrV/TLR2 interactions does not play a significant role in the pathogenicity of Y. pestis., (Published by Elsevier Ltd.)

Although Haliotis midae is the most economically important cultured abalone species in South Africa, infectious diseases have the potential to severely limit the production of this shellfish. Consequently, it is becoming increasingly important to characterise the abalone immune system in order to better understand their ability to combat infection. This study reports the identification and characterisation of a perforin-like protein, designated hmMpeg1, which is believed to be involved in the H. midae immune system. hmMpeg1 encodes for a 78 kDa protein that has significant sequence similarity to Mpeg proteins from other abalone species and includes the conserved cytolytic membrane attack complex/perforin (MACPF) domain of perforin. Real-time quantitative PCR (qPCR) analysis demonstrated expression of hmMpeg1 mRNA in haemocytes and epipodia samples from H. midae exposed to a heat-killed, Gram-negative bacterial pathogen, Vibrio anguillarum 5676. hmMpeg1 mRNA in haemocytes increased significantly 48 h post-infection (h.p.i) (8.2 fold; P < 0.05), coinciding with a decrease in the total number of circulating haemocytes, and reached a maximum at 96 h.p.i (17.2 fold; P < 0.05). Similarly, a significant increase in the level of hmMpeg1 mRNA occurred at 24 h.p.i in epipodia samples (3.8 fold; P < 0.05), reaching a maximum at 48 h.p.i (4.5 fold; P < 0.05). In addition, western blot analysis detected a significant increase in hmMpeg1 between 24 h.p.i (4.2 fold; P < 0.05) and 48 h.p.i (3.1 fold; P < 0.05) in the epipodia, and between 48 h.p.i (1.7 fold; P < 0.05) and 96 h.p.i (1.9 fold; P < 0.05) in haemocytes, sampled from abalone exposed to the abalone pathogen V. anguillarum 5676. The importance of hmMpeg1, in terms of its function and importance in the H. midae immune response, is discussed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The pore-forming ability of the Bacillus thuringiensis toxin Cry9Ca, its two single-site mutants R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at R164 was evaluated under a variety of experimental conditions using an electrophysiological assay. All four toxin preparations depolarized the apical membrane of freshly isolated third-instar Manduca sexta midguts bathing in a solution containing 122 mM KCl at pH 10.5, but the 55-kDa fragment was considerably more active than Cry9Ca and its mutants. The activity of the latter toxins was greatly enhanced, however, when the experiments were conducted in the presence of fifth-instar M. sexta midgut juice. This effect was also observed after midgut juice proteins had been denatured by heating at 95 degrees C or after inorganic ions and small molecules had been removed from the midgut juice by extensive dialysis. A similar stimulation of toxin activity was also observed when the experiments were carried out in the presence of the lipids extracted from an equivalent volume of midgut juice. Depolarization of the cell membrane was also greatly enhanced, in the absence of midgut juice, by the addition of a cocktail of water-soluble protease inhibitors. These results indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut., ((c) 2010 Elsevier Inc. All rights reserved.)

LcrV is a key Yersinia pestis antigen, immune regulator, and component of the type III secretion system (T3SS). Researchers have shown that N-acyl-homoserine lactones (AHLs) can down-regulate the expression of the LcrV homolog, PcrV, in Pseudomonas aeruginosa. Using ELISA, western blot, DNA microarray analysis, and real time PCR we demonstrate that the addition of AHL molecules N-octanoyl-homoserine lactone (C8) or N-(3-oxooctanoyl)-homoserine lactone (oxo-C8) to Y. pestis cultures down-regulates LcrV protein expression. DNA microarray analysis shows 10 additional T3SS genes are consistently down-regulated by C8 or oxo-C8. This is the first report demonstrating that AHLs regulate Y. pestis virulence factor expression.

A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular characterization of an aexT-like toxin gene (designated as aexU) from a diarrheal isolate SSU of A. hydrophila. The aexU gene was 1539bp in length and encoded a protein of 512 amino acid (aa) residues. The NH(2)-terminus of AexU (aa residues 1-231) exhibited a 67% homology with the NH(2)-terminus of AexT from A. salmonicida. Importantly, its COOH-terminus (aa residues 232-512) had no homology with any known functional proteins in the database; however, the full-length AexU retained ADP-ribosyltransferase activity. The expression and subsequent secretion of AexU was T3SS dependent, as inactivation of the ascV gene that codes for an inner-membrane component of the T3SS channel from the wild-type (WT) bacterium, blocked translocation of AexU in HT-29 human colonic epithelial cells. We provided evidence that inactivation of acrV and axsE genes (homologs of lcrV and exsE in Yersinia species and P. aeruginosa, respectively) from A. hydrophila SSU, altered expression and/or secretion of AexU. We deleted an aexU gene from the WT, as well as from the DeltaaopB mutant, of A. hydrophila, generating a single knockout (DeltaaexU) and a double knockout mutant, DeltaaopB/DeltaaexU. Increased phagocytosis was observed in RAW264.7 murine macrophages infected with the DeltaaopB/DeltaaexU mutant, as compared to macrophages when infected with the parental DeltaaopB strain. Further, mice infected with the DeltaaexU mutant had a 60% survival rate, compared to animals infected with the WT or the DeltaaexU-complemented strain that caused 90-100% of the animals to die at a 2-3 LD(50s) dose. Immunization of mice with the recombinant AexU protected them from subsequent lethal challenge dose by the WT bacterium. Finally, we detected specific anti-AexU antibodies in the sera of mice that survived challenge by the WT bacterium, which may indicate that AexU plays an important role in the pathogenesis of Aeromonas infections.

We recently identified a novel type III secretion system (T3SS) effector, AexU, from a diarrheal isolate SSU of Aeromonas hydrophila, and demonstrated that mice infected with the DeltaaexU mutant were significantly protected from mortality. Although the NH(2)-terminal domain of this toxin exhibits homology to AexT of A. salmonicida, a fish pathogen, and ExoT/S of Pseudomonas aeruginosa, the COOH-terminal domain of AexU is unique, with no homology to any known proteins in the NCBI database. In this study, we purified the full-length AexU and its NH(2)-terminal (amino acid residues 1-231) and COOH-terminal (amino acid residues 232-512) domains after expression of their corresponding genes in Escherichia coli as histidine-tag fusion proteins using the bacteriophage T7 RNA polymerase/promoter-based pET-30a vector system. The full-length and NH(2)- and COOH-terminal domains of AexU exhibited ADP-ribosyltransferase activity, with the former two exhibiting much higher activity than the latter. These different forms of AexU were also successfully expressed and produced in the HeLa Tet-Off cell system using a pBI-EGFP vector, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and intracellular staining of the toxin using flow cytometric analysis. Production of AexU in HeLa cells resulted in possible actin reorganization and cell rounding, as determined by phalloidin staining and confocal microscopy. Based on electron microscopy, the toxin also caused chromatin condensation, which is indicative of apoptosis. Apoptosis of HeLa cells expressing and producing AexU was confirmed by 7-amino actinomycin D (7-AAD) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] assays, by detection of cytoplasmic histone-associated DNA fragments, and by activation of caspases 3 and 9. These effects were much more pronounced in host cells that expressed and produced the full-length or NH(2)-terminal domain of AexU, compared to those that expressed and produced the COOH-terminal domain or the vector alone. This study represents the first characterization of this novel T3SS effector.

The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.

We investigated the proportion, phenotype, and cytotoxicity of CD8+CD57+ and CD57- T cells in peripheral blood from 20 tuberculosis (TB)-patients and 20 healthy tuberculin skin test-positive donors. Our results showed an increase in CD8+CD57+ T cells from TB-patients as compared with those from age-matched healthy donors (p<0.0001). CD8+CD57+ T cells from TB-patients expressed CD69, perforin, granzyme-A, and a CD28-CD62L-CD161- phenotype without recognition for the alpha-galactosylceramide-CD1d complex. This cell subset also expressed TNF-alpha and IFN-gamma, under phorbol-myristate-acetate/ionomycin stimulation. Interestingly, the cytotoxicity against autologous monocytes was higher in CD57- cells from TB-patients and donors than their CD57+ counterparts, in the presence of Mycobacterium tuberculosis H37Rv culture filtrate. However, only CD8+CD57+ T cells from TB-patients exhibited spontaneous cytotoxicity against monocytes in the absence of antigen. Our results suggest that CD8+CD57+ T cells are a subset of effector cells that could be helpful to evaluate the cell-mediated immune response to M. tuberculosis.