BACKGROUND: Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) is the most severe form of cutaneous adverse drug reaction and is characterized by extensive epidermal destruction of the skin and mucosal surfaces. Controversy remains regarding the immunopathogenesis of the disease. It has long been assumed that CD8 cytotoxic T cells mediate cell death by releasing cytotoxic granules and soluble granulysin that trigger keratinocyte apoptosis. However, this does not explain the massive cell death or inflammation that is observed clinically. We have preliminary evidence from transcriptional profiling of patient skin samples suggesting that the cell death pathways necroptosis and pyroptosis may mediate SJS/TEN. Herein we utilize retrospectively and prospectively collected patient samples to investigate these cell death pathways. OBJECTIVE: The goals of this study are two-fold: (i) to investigate cell death pathways in retrospectively-collected (SJS/TEN) patient skin samples and (ii) to directly test the cell death mediators and pathways mediating SJS/TEN using a novel in vitro model. METHODS: Clinically and histopathologically confirmed SJS/TEN skin specimens and control skin specimens from non-blistering T cell mediated drug reactions and healthy skin were obtained following retrospective analysis from a multi-centered patient database. Gene expression profiling is being performed using the NanoString nCounter® System on these samples as a second patient cohort to confirm and expand on preliminary study findings. In parallel, we have optimized the use of a novel human skin platform for an in vitro model of SJS/TEN. We also collected human serum from a prospective study of SJS/TEN and control patients and have optimized and are actively collecting blister fluid from SJS/TEN and control patients in an ongoing prospective study for use in this model. RESULTS: Through an extensive pathology database and medical record search of potential cases at Brigham and Women's Hospital, we identified a second patient cohort of SJS/TEN, non-blistering delayed-type drug hypersensitivity reactions and healthy controls. We identified and are collecting thorough demographic, clinical and laboratory data on 61 potential candidates for SJS/TEN, 4 for Drug Reaction with Eosinophilia Syndrome (DRESS), and 200 for Morbilliform Drug Eruptions (MDE). This second cohort is in the final step of analysis with review by an expert clinician to confirm cases. In parallel, we have designed an expansive gene panel to confirm cell death mediator and marker transcription in our bank of skin samples. This 815 gene panel uses the pre-designed panel from Nanostring®, spiked with an additional 30 genes specific to apoptosis, pyroptosis, and necroptosis. We reviewed multiple potential in vitro skin models and identified GenoSkin® as the most suitable human skin platform for our in vitro model. We collected serum from 6 SJS/TEN patients and 6 non-blistering drug reaction patients and 3 healthy controls, and are actively collecting blister fluid from SJS/TEN and thermal burn control patients for analysis in this model. CONCLUSIONS: Our preliminary data suggest necroptosis and pyroptosis induced by soluble death mediators tumor necrosis factor (TNF) alpha and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as the main cell death pathways responsible for SJS/TEN. We have successfully identified a large number of potential patient samples of both cases and controls to perform transcriptional profiling using a self-designed gene panel to confirm and expand upon our preliminary data. We have successfully collected prospectively patient serum and are actively collecting patient blister fluid for analysis in an optimized in vitro model using GenoSkin®. SJS/TEN is severely understudied and lacks a standard protocol for care. This stems from uncertainty surrounding disease pathobiology. It is critical that we use innovative approaches to interrogate the mechanism mediating disease to advance the field, and, most importantly, to improve the quality of care for these patients.

Aim: This study aims to identify pyroptosis-related genes (PRGs), their functional immune characteristics, and distinct pyroptosis-related clusters in periodontitis. Methods: Differentially expressed (DE)-PRGs were determined by merging the expression profiles of GSE10334, GSE16134, and PRGs obtained from previous literatures and Molecular Signatures Database (MSigDB). Least absolute shrinkage and selection operator (LASSO) regression was applied to screen the prognostic PRGs and develop a prognostic model. Consensus clustering was applied to determine the pyroptosisrelated clusters. Functional analysis and single-sample gene set enrichment analysis (ssGSEA) were performed to explore the biological characteristics and immune activities of the clusters. The hub pyroptosis-related modules were defined using weighted correlation network analysis (WGCNA). Results: Of the 26 periodontitis-related DE-PRGs, the highest positive relevance was for High-Mobility Group Box 1 (HMGB1) and SR-Related CTD Associated Factor 11 (SCAF11). A 14-PRG-based signature was developed through the LASSO model. In addition, three pyroptosis-related clusters were obtained based on the 14 prognostic PRGs. Caspase 3 (CASP3), Granzyme B (GZMB), Interleukin 1 Alpha (IL1A), IL1Beta (B), IL6, Phospholipase C Gamma 1 (PLCG1) and PYD And CARD Domain Containing (PYCARD) were dysregulated in the three clusters. Distinct biological functions and immune activities, including human leukocyte antigen (HLA) gene expression, immune cell infiltration, and immune pathway activities, were identified in the three pyroptosisrelated clusters of periodontitis. Furthermore, the pink module associated with endoplasmic stress-related functions was found to be correlated with cluster 2 and was suggested as the hub pyroptosis-related module. Conclusion: The study identified 14 key pyroptosis-related genes, three distinct pyroptosis-related clusters, and one pyroptosis-related gene module describing several molecular aspects of pyroptosis in the pathogenesis and immune micro-environment regulation of periodontitis and also highlighted functional heterogeneity in pyroptosisrelated mechanisms.

Doxorubicin (DOX) is an incessantly used chemotherapeutic drug that can cause detrimental dose-dependent effects such as cardiotoxicity and congestive heart failure. Studies have focused on therapeutic strategies such as exosomes derived from embryonic stem cell (ES-Exos) and antioxidants for example resveratrol; however, the function of mesenchymal stem cell-derived exosomes (MSC-Exos) have never been examined in DOX-induced pyroptosis. MSC-Exos maintains the therapeutic potential of exosome therapy without the ethical concerns. Hence, the current study focuses on determining whether MSC-Exos has the potential to ameliorate inflammation-induced cell death pyroptosis in our established in vitro DOX-induced cardiotoxicity (DIC) model. Rat embryonic cardiomyocytes (H9c2) were first exposed to DOX to stimulate pyroptosis, followed by subsequent treatment with MSC-Exos, with further analysis performed through immunocytochemistry, western blotting, and RT-PCR. We evaluated the therapeutic potential of MSC-Exos by investigating the pyroptotic initiator HMGB1 which binds to TLR4 resulting in the formation of the NLRP3 inflammasome that initiates pyroptosis by activating the pyroptotic markers, caspase-1, IL-1β and IL-18, and the pyroptotic executioner GSDMD. Our data depicted that treatment with MSC-Exos significantly (p

Microbiota colonisation of the intestinal tract makes it difficult for pattern recognition receptors (PRR) to discriminate between beneficial microbes and harmful pathogens. We aim to define the roles of cytosolic Nod-like receptors (NLR) in intestinal immunity and homeostasis. Upon activation, some NLR form inflammasomes that mediate the release of inflammatory cytokines and pyroptosis, an inflammatory form of cell death. NLR activation in the non-hematopoietic compartment was shown to be protective during acute intestinal infection. To identify the cell type responsible for this protection, we generated transgenic mice in which the key inflammasome adaptor molecule Asc is selectively ablated in intestinal epithelial cells (IEC) (Asc^ΔVC) and observed that inflammasomes are important for controlling Citrobacter rodentium clearance in these mice. To further dissect the importance of pathogen clearance by IEC inflammasome, ex vivo cultures of primary IEC organoids were established. Thus far this system has revealed profound differences in inflammasome regulation between IEC organoids and bone marrow-derived macrophages (BMDM). This research will inform our understanding of cell type-specific regulation of inflammasomes.

No description available.

Stem cells, with the ability to self-renew and differentiate into intended cell types, are a valuable tool for disease modeling and mechanistic study. CRISPR-Cas9 has been widely used for genome editing due to its high efficiency and convenience. However, CRISPR-Cas9 has large-deletion safety issues that dramatically restrict its applications. Wiskott-Aldrich syndrome (WAS) is an inborn immunological disorder caused by WASP deficiency. WASP functions in the nucleus, which may help to understand WAS pathology, are poorly defined. Pannexin 1 (PANX1) forms large plasma membrane pores to exchange intracellular small molecules with the extracellular environment and functions in inflammatory processes. The regulatory mechanisms of the PANX1 channel remain obscure. In this dissertation, I focused on mechanistic studies of CRISPR-Cas9 genome editing, and two immune disease relevant genes, WASP and PANX1 using stem cell-derived immune cells. We first found that CRISPR-induced large deletions (LDs) are predominantly mediated by the MMEJ repair pathway through statistical studies. Further, we found POLQ and RPA play vital roles in CRISPR-induced LDs. Modulation of POLQ and RPA can decrease CRISPR-induced LDs and increase HDR efficiency. Using three isogenic WAS iPSC models generated via gene editing, we successfully recapitulated WAS phenotypes, and for the first time, revealed that WASP regulates RNA splicing via epigenetically controlling the transcription of splicing factors and directly participating in the splicing machinery through a liquid-liquid phase separation process. We established a full-length human PANX1 (hPANX1) channel model via cryo-electron microscopy experiments and molecular dynamics simulation study, and found that hPANX1 channel is a homo-heptamer with both the N- and C-termini stretching deeply into the pore funnel. Functional studies of three selected residues support the new hPANX1 channel model and suggest the potential regulatory role of hPANX1 in pyroptosis upon immune responses. Overall, the mechanistic studies of WASP, PANX1 and CRISPR genome editing revealed new roles of WASP in regulating RNA splicing, new functional insights of PANX1 in pyroptosis, and uncovered two critical players POLQ and RPA in CRISPR-induced LDs.

Pseudomonas aeruginosa est un pathogène opportuniste responsable d’infections nosocomiales sévères associées à un taux élevé de mortalité. Le système de sécrétion de Type III (SST3) et les effecteurs qu’il injecte sont considérés comme des facteurs de virulence prépondérants de P. aeruginosa. Récemment nous avons caractérisé, un groupe de souches ne possédant pas les gènes du SST3, mais dont la virulence repose sur la sécrétion d’une nouvelle toxine de 172 kDa, nommée Exolysine (ExlA) qui provoque la perméabilisation de la membrane des cellules hôtes. ExlA est sécrétée dans le milieu par une porine de la membrane externe, nommée ExlB, formant ainsi un nouveau système de sécrétion à deux partenaires (TPS), ExlBA. Outre le domaine TPS du coté N-terminal de la protéine, impliqué dans sa sécrétion, ExlA possède différents domaines ; des répétitions hémagglutinines, cinq motifs Arginine-Glycine-Acide Aspartique (RGD) et un domaine C-Terminal faiblement conservé. Des tests de cytotoxicité sur des cellules eucaryotes ont montrés que la délétion du domaine C-terminal abolissait l’activité toxique d’ExlA. En utilisant un modèle de liposomes et différents types de cellules eucaryotes, comme les globules rouges, nous avons démontré qu’ExlA forme des pores membranaires de 1.6 nm. De plus, par un criblage cellulaire à haut-débit d’une banque de mutants obtenus par une mutagenèse de transposition, nous avons montré qu’un facteur bactérien additionnel était requis dans la toxicité d’ExlA. En effet, parmi les 7 400 mutants, nous avons identifiés 3 transposons insérés dans des gènes codant pour le pili de type IV, démontrant ainsi que cet appendice impliqué dans l’adhésion des bactéries participe à la toxicité d’ExlA, en permettant un contact rapproché entre la bactérie et les cellules hôtes. Un criblage de macrophages primaires de souris KO pour différentes protéines impliquées dans la voie de l’activation de l’inflammasome, nous a permis de démontrer que le pore formé par ExlA est responsable de l’activation de la Caspase-1 par l’inflammasome NLRP3 conduisant à la maturation de l’interleukine-1ß. Une étude bio-informatique a révélé la présence de gènes homologues à exlA chez d’autres espèces de Pseudomonas non pathogènes, comme P. putida, P. protegens, P. entomophila. Nous avons montré que ces bactéries environnementales sont aussi capables de provoquer une mort cellulaire dépendante de la Caspase-1. Finalement, un criblage d’une banque de macrophages dont les gènes ont été invalidés par la technologie CRISPR/cas9 a révélé que plusieurs protéines du système immunitaire, indirectement liées à l’activation de la Caspase-1 sont impliquées dans la mort cellulaire médiée par ExlA. De plus, nous avons montré que plusieurs sgRNAs ciblant un microARN, mir-741, était grandement enrichi dans les macrophages ayant résisté à une infection avec ExlA. Mir-741 régule l’expression d’enzymes (St8sIa1 et Agpat5) impliquées dans la voie de biosynthèse des sphingolipides et des glycérophospholipides, suggérant ainsi que l’activité d’ExlA requiert un environnement lipidique particulier. / Pseudomonas aeruginosa is a human opportunistic pathogen responsible for nosocomial infections associated with high mortality. The type III secretion system (T3SS) and T3SS-exported toxins have been considered as key infectivity virulence factors. Our team recently characterized a group of strains lacking T3SS, but employing a new pore-forming toxin of 172 kDa, named Exolysin (ExlA) that provokes cell membrane disruption. In this work we demonstrated that the ExlA secretion requires ExlB, a predicted outer membrane protein encoded in the same operon, showing that ExlA-ExlB define a new active Two-Partner Secretion (TPS) system. In addition to the TPS secretion signals, ExlA harbors several distinct domains, which comprise hemagglutinin domains, five Arginine-Glycine-Aspartic acid (RGD) motifs and a non-conserved C-terminal region lacking any identifiable sequence motifs. Cytotoxic assays showed that the deletion of the C-terminal region abolishes host-cell cytolysis. Using liposomes and eukaryotic cells, including red blood cells, we demonstrated that ExlA forms membrane pores of 1.6 nm. Based on a transposon mutagenesis strategy and a high throughput cellular live-dead screen, we identified additional bacterial factors required for ExlA-mediated cell lysis. Among 7 400 mutants, we identified three transposons inserted in genes encoding components of the Type IV pili, which are adhesive extracellular appendices. Type IV pili probably mediate close contact between bacteria and host cells and facilitate ExlA cytotoxic activity. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages to achieve host cell intoxication. Using mice primary bone marrow macrophages we showed that ExlA pores provoke activation of Caspase-1 via the NLRP3-inflamasomme followed by the maturation of the pro-interleukin-1ß. Mining of microbial genomic databases revealed the presence of exlA-like genes in other Pseudomonas species rarely associated with human infections P. putida, P. protegens and P. entomophila. Interestingly, we showed that these environmental bacteria are also able to provoke Caspase-1 cleavage and pro-inflammatory cell death of macrophages. Finally, genome-wide loss-of-function CRISPR/cas9 RAW library screen revealed that several components of the immune system response, indirectly linked to Caspase-1 are involved in the ExlA-mediated cell lysis. Moreover, we found at least three sgRNAs targeting miRNA, mir-741 were highly enriched in resistant macrophages challenged by ExlA. This miRNA regulates enzymes (St8sIa1 and Agpat5) in the sphingolipids and glycerophololipids biosynthesis pathways, suggesting that ExlA activity may require proper lipid environment.

Cancer remains the second most common cause of death worldwide affecting around 10 million patients every year. Among the therapeutic options, chemotherapeutic drugs are widely used but often associated with side effects. In addition, toxicity against immune cells may hamper anti-tumor immune responses. Some chemotherapeutic drugs, however, preserve immune functions and some can even stimulate anti-tumor immune responses through the induction of immunogenic cell death (ICD) rather than apoptosis. ICD stimulates the immune system by several mechanisms including the release of damage-associated molecular patterns (DAMPs) from dying cells. In this review, we will discuss the consequences of inducing two recently characterized forms of ICD, i.e., pyroptosis and necroptosis, in the tumor microenvironment (TME) and the perspectives they may offer to increase the immunogenicity of the so-called cold tumors and to stimulate effective anti-tumor immune responses.

Nlrp1b is a NOD-like receptor of the innate immune system that upon sensing of anthrax lethal toxin oliogmerizes and forms a protein scaffold that binds to and activates pro-caspase-1; this complex is called an inflammasome. Nlrp1b is highly polymorphic and different alleles display an all or none ability to sense lethal toxin. Here I show that Nlrp1b is cleaved in the FIIND domain, and that the cleaved fragments remain associated even after activation by lethal toxin. The inflammasome activity of an inactive allele was restored by three mutations, one of which also restored cleavage. A heterologous cleavage site was inserted into an uncleaved mutant of Nlrp1b; induced proteolysis of the cleavage site rescued inflammasome activity. An uncleaved mutant of Nlrp1b showed no deficiency in FIIND self-association, but did have reduced recruitment of pro-caspase-1. These data provide evidence that cleavage of Nlrp1b is required for proper recruitment and activation of caspase-1.

Nlrp1b is a NOD-like receptor of the innate immune system that upon sensing of anthrax lethal toxin oliogmerizes and forms a protein scaffold that binds to and activates pro-caspase-1; this complex is called an inflammasome. Nlrp1b is highly polymorphic and different alleles display an all or none ability to sense lethal toxin. Here I show that Nlrp1b is cleaved in the FIIND domain, and that the cleaved fragments remain associated even after activation by lethal toxin. The inflammasome activity of an inactive allele was restored by three mutations, one of which also restored cleavage. A heterologous cleavage site was inserted into an uncleaved mutant of Nlrp1b; induced proteolysis of the cleavage site rescued inflammasome activity. An uncleaved mutant of Nlrp1b showed no deficiency in FIIND self-association, but did have reduced recruitment of pro-caspase-1. These data provide evidence that cleavage of Nlrp1b is required for proper recruitment and activation of caspase-1.