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Missense mutations in PLCG2 can cause autoinflammation with phospholipase C gamma 2-associated antibody deficiency and immune dysregulation (APLAID). Here, we generated a mouse model carrying an APLAID mutation (p.Ser707Tyr) and found that inflammatory infiltrates in the skin and lungs were only partially ameliorated by removing inflammasome function via the deletion of caspase-1. Also, deleting interleukin-6 or tumor necrosis factor did not fully prevent APLAID mutant mice from autoinflammation. Overall, these findings are in accordance with the poor response individuals with APLAID have to treatments that block interleukin-1, JAK1/2 or tumor necrosis factor. Cytokine analysis revealed increased granulocyte colony-stimulating factor (G-CSF) levels as the most distinct feature in mice and individuals with APLAID. Remarkably, treatment with a G-CSF antibody completely reversed established disease in APLAID mice. Furthermore, excessive myelopoiesis was normalized and lymphocyte numbers rebounded. APLAID mice were also fully rescued by bone marrow transplantation from healthy donors, associated with reduced G-CSF production, predominantly from non-hematopoietic cells. In summary, we identify APLAID as a G-CSF-driven autoinflammatory disease, for which targeted therapy is feasible.

Oxysterols (i.e., oxidized cholesterol species) have complex roles in biology. 25-hydroxycholesterol (25HC), a product of activity of cholesterol-25-hydroxylase (CH25H) upon cholesterol, has recently been shown to be broadly antiviral, suggesting therapeutic potential against SARS-CoV-2. However, 25HC can also amplify inflammation and tissue injury and be converted by CYP7B1 to 7α,25HC, a lipid with chemoattractant activity via the G protein-coupled receptor, EBI2/GPR183. Here, using in vitro studies and two different murine models of SARS-CoV-2 infection, we investigate the effects of these two oxysterols on SARS-CoV-2 pneumonia. We show that while 25HC and enantiomeric-25HC are antiviral in vitro against human endemic coronavirus-229E, they did not inhibit SARS-CoV-2; nor did supplemental 25HC reduce pulmonary SARS-CoV-2 titers in the K18-human ACE2 mouse model in vivo. 25HC treatment also did not alter immune cell influx into the airway, airspace cytokines, lung pathology, weight loss, symptoms, or survival but was associated with increased airspace albumin, an indicator of microvascular injury, and increased plasma pro-inflammatory cytokines. Conversely, mice treated with the EBI2/GPR183 inhibitor NIBR189 displayed a modest increase in lung viral load only at late time points, but no change in weight loss. Consistent with these findings, although Ch25h was upregulated in the lungs of SARS-CoV-2-infected WT mice, lung viral titers and weight loss in Ch25h−/– and Gpr183−/– mice infected with the beta variant were similar to control animals. Taken together, endogenous 25-hydroxycholesterols do not significantly regulate early SARS-CoV-2 replication or pathogenesis and supplemental 25HC may have pro-injury rather than therapeutic effects in SARS-CoV-2 pneumonia.

Proteasome dysfunction can lead to autoinflammatory disease associated with elevated type I interferon (IFN-αβ) and NF-κB signaling; however, the innate immune pathway driving this is currently unknown. Here, we identified protein kinase R (PKR) as an innate immune sensor for proteotoxic stress. PKR activation was observed in cellular models of decreased proteasome function and in multiple cell types from patients with proteasome-associated autoinflammatory disease (PRAAS). Furthermore, genetic deletion or small-molecule inhibition of PKR in vitro ameliorated inflammation driven by proteasome deficiency. In vivo, proteasome inhibitor–induced inflammatory gene transcription was blunted in PKR-deficient mice compared with littermate controls. PKR also acted as a rheostat for proteotoxic stress by triggering phosphorylation of eIF2α, which can prevent the translation of new proteins to restore homeostasis. Although traditionally known as a sensor of RNA, under conditions of proteasome dysfunction, PKR sensed the cytoplasmic accumulation of a known interactor, interleukin-24 (IL-24). When misfolded IL-24 egress into the cytosol was blocked by inhibition of the endoplasmic reticulum–associated degradation pathway, PKR activation and subsequent inflammatory signaling were blunted. Cytokines such as IL-24 are normally secreted from cells; therefore, cytoplasmic accumulation of IL-24 represents an internal danger-associated molecular pattern. Thus, we have identified a mechanism by which proteotoxic stress is detected, causing inflammation observed in the disease PRAAS.

BCG when administered by the intravenous but not subcutaneous route provides protection against lethal SARS-CoV-2 infection in mice, reducing immunopathology in addition to viral load and thus providing an experimental model for delineating innate mechanisms of resistance against COVID-19., In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Guérin (BCG) has been reported to confer nonspecific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here, we demonstrate that intravenous, but not subcutaneous, inoculation of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 (SCV2) and results in reduced viral loads in non-transgenic animals infected with an α variant. The observed increase in host resistance was associated with reductions in SCV2-induced tissue pathology, inflammatory cell recruitment, and cytokine production that multivariate analysis revealed as only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and ensuing immunopathology. While intravenous BCG vaccination is not a clinically acceptable practice, our findings provide an experimental model for identifying mechanisms by which nonspecific stimulation of the pulmonary immune response promotes host resistance to SCV2 lethality.

Host resistance to Mycobacterium tuberculosis infection requires the activities of multiple leukocyte subsets, yet the roles of the different innate effector cells during tuberculosis are incompletely understood. Here we uncover an unexpected association between eosinophils and Mtb infection. In humans, eosinophils are decreased in the blood but enriched in resected human tuberculosis lung lesions and autopsy granulomas. Influx of eosinophils is also evident in infected zebrafish, mice, and nonhuman primate granulomas, where they are functionally activated and degranulate. Importantly, employing complementary genetic models of eosinophil deficiency, we demonstrate that, in mice, eosinophils are required for optimal pulmonary bacterial control and host survival after Mtb infection. Collectively, our findings uncover an unexpected recruitment of eosinophils to the infected lung tissue and a protective role for these cells in the control of Mtb infection in mice.

Andrea C. Bohrer (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Inflammation and Innate Immunity Unit. Bethesda, MD, United States of America.); Ehydel Castro (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Inflammation and Innate Immunity Unit. Bethesda, MD, United States of America.); Zhidong Hu (Fudan University. Shanghai Public Health Clinical Center. Department of Scientific Research. Shanghai, China / Fudan University. Shanghai Emerging and Re-emerging Infectious Disease Institute. Tuberculosis Center. Shanghai, China.); Artur T. L. Queiroz (The KAB Group / Multinational Organization Network Sponsoring Translational and Epidemiological Research. Salvador, BA, Brasil / Fundacao Oswaldo Cruz. Instituto Goncalo Moniz. Salvador, BA, Brasil.); Claire E. Tocheny (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Inflammation and Innate Immunity Unit. Bethesda, MD, United States of America.); Maike Assmann (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Inflammation and Innate Immunity Unit. Bethesda, MD, United States of America.); Shunsuke Sakai (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases. T Lymphocyte Biology Section. Bethesda, MD, United States of America.); Christine Nelson (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases. T Lymphocyte Biology Section. Bethesda, MD, United States of America.); Paul J. Baker (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Inflammation and Innate Immunity Unit. Bethesda, MD, United States of America.); Hui Ma (Fudan University. Shanghai Public Health Clinical Center. Department of Scientific Research. Shanghai, China / Fudan University. Shanghai Emerging and Re-emerging Infectious Disease Institute. Tuberculosis Center. Shanghai, China.); Lin Wang (Fudan University. Shanghai Emerging and Re-emerging Infectious Disease Institute. Tuberculosis Center. Shanghai, China / Fudan University. Shanghai Public Health Clinical Center. Department of Thoracic Surgery. Shanghai, China.); Wen Zilu (Fudan University. Shanghai Emerging and Re-emerging Infectious Disease Institute. Tuberculosis Center. Shanghai, China / Fudan University. Shanghai Public Health Clinical Center. Department of Thoracic Surgery. Shanghai, China.); Elsa du Bruyn (University of Cape Town. Institute of Infectious Disease and Molecular Medicine. Wellcome Centre for Infectious Diseases Research in Africa. Cape Town, South Africa.); Catherine Riou (University of Cape Town. Institute of Infectious Disease and Molecular Medicine. Wellcome Centre for Infectious Diseases Research in Africa. Cape Town, South Africa.); Keith D. Kauffman (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases. T Lymphocyte Biology Section. Bethesda, MD, United States of America.); Tuberculosis Imaging Program (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Division of Intramural Research. Tuberculosis Imaging Program. Bethesda, MD, United States of America.); Ian N. Moore (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Comparative Medicine Branch. Infectious Disease Pathogenesis Section. Bethesda, MD, United States of America.); Franca Del Nonno (Istituto Di Ricovero e Cura a Carattere Scientifico. National Institute for Infectious Diseases "L. Spallanzani". Pathology Unit. Rome, Italy.); Linda Petrone (Istituto Di Ricovero e Cura a Carattere Scientifico. Department of Epidemiology and Preclinical Research National Institute for Infectious Diseases. Translational Research Unit. Rome, Italy.); Delia Goletti (Istituto Di Ricovero e Cura a Carattere Scientifico. Department of Epidemiology and Preclinical Research National Institute for Infectious Diseases. Translational Research Unit. Rome, Italy.); Adrian R. Martineau (University College London. Institute of Immunity and Transplantation. London, United Kingdom.); David M. Lowe (University College London. Institute of Immunity and Transplantation. London, United Kingdom.); Mark R. Cronan (Max Planck Institute for Infection Biology. In Vivo Cell Biology of Infection Unit. Berlin, Germany / Duke University School of Medicine. Department of Molecular Genetics and Microbiology. Durham, NC, United States of America.); Robert J. Wilkinson (University of Cape Town. Institute of Infectious Disease and Molecular Medicine. Wellcome Centre for Infectious Diseases Research in Africa. Cape Town, South Africa / Imperial College London. Department of Infectious Diseases. London, United Kingdom.); Clifton E. Barry, III (University of Cape Town. Institute of Infectious Disease and Molecular Medicine. Wellcome Centre for Infectious Diseases Research in Africa. Cape Town, South Africa / National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Tuberculosis Research Section. Bethesda, MD, United States of America.); Laura E. Via (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Division of Intramural Research. Tuberculosis Imaging Program. Bethesda, MD, United States of America / National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Tuberculosis Research Section. Bethesda, MD, United States of America.); Daniel L. Barber (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases. T Lymphocyte Biology Section. Bethesda, MD, United States of America.); Amy D. Klion (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases. Human Eosinophil Section. Bethesda, MD, United States of America.); Bruno B. Andrade (The KAB Group / Multinational Organization Network Sponsoring Translational and Epidemiological Research. Salvador, BA, Brasil / Fundacao Oswaldo Cruz. Instituto Goncalo Moniz. Salvador, BA, Brasil.); Yanzheng Song (Fudan University. Shanghai Emerging and Re-emerging Infectious Disease Institute. Tuberculosis Center. Shanghai, China / Fudan University. Shanghai Public Health Clinical Center. Department of Thoracic Surgery. Shanghai, China.); Ka-Wing Wong (Fudan University. Shanghai Public Health Clinical Center. Department of Scientific Research. Shanghai, China / Fudan University. Shanghai Emerging and Re-emerging Infectious Disease Institute. Tuberculosis Center. Shanghai, China.); Katrin D. Mayer-Barber (National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Immunology and Microbiology. Inflammation and Innate Immunity Unit. Bethesda, MD, United States of America.).

Boosting immune cell function by targeting the coinhibitory receptor PD-1 may have applications in the treatment of chronic infections. Here, we examine the role of PD-1 during Mycobacterium tuberculosis (Mtb) infection of rhesus macaques. Animals treated with anti-PD-1 monoclonal antibody developed worse disease and higher granuloma bacterial loads compared with isotype control-treated monkeys. PD-1 blockade increased the number and functionality of granuloma Mtb-specific CD8 T cells. In contrast, Mtb-specific CD4 T cells in anti-PD-1-treated macaques were not increased in number or function in granulomas, expressed increased levels of CTLA-4, and exhibited reduced intralesional trafficking in live imaging studies. In granulomas of anti-PD-1-treated animals, multiple proinflammatory cytokines were elevated, and more cytokines correlated with bacterial loads, leading to the identification of a role for caspase 1 in the exacerbation of tuberculosis after PD-1 blockade. Last, increased Mtb bacterial loads after PD-1 blockade were found to associate with the composition of the intestinal microbiota before infection in individual macaques. Therefore, PD-1-mediated coinhibition is required for control of Mtb infection in macaques, perhaps because of its role in dampening detrimental inflammation and allowing for normal CD4 T cell responses.

Boosting immune cell function by targeting the co-inhibitory receptor PD-1 may have applications in the treatment of chronic infections. Here we examine the role of PD-1 during Mycobacterium tuberculosis (Mtb) infection of rhesus macaques. Animals treated with anti-PD-1 monoclonal antibody developed worse disease and higher granuloma bacterial loads compared to isotype control treated monkeys. PD-1 blockade increased the number and functionality of granuloma Mtb-specific CD8 T cells. In contrast, Mtb-specific CD4 T cells in anti-PD-1 treated macaques were not increased in number or function in granulomas, expressed increased levels of CTLA-4 and exhibited reduced intralesional trafficking in live imaging studies. In granulomas of anti-PD-1 treated animals, multiple proinflammatory cytokines were elevated, and more cytokines correlated with bacterial loads, leading to the identification of a role for caspase 1 in the exacerbation of tuberculosis after PD-1 blockade. Lastly, increased Mtb bacterial loads after PD-1 blockade were found to associate with the composition of the intestinal microbiota prior to infection in individual macaques. Therefore, PD-1-mediated co-inhibition is required for control of Mtb infection in macaques, perhaps due to its role in dampening detrimental inflammation as well as allowing for normal CD4 T cell responses.

Boosting immune cell function by targeting the co-inhibitory receptor PD-1 may have applications in the treatment of chronic infections. Here we examine the role of PD-1 duringMycobacterium tuberculosis(Mtb) infection of rhesus macaques. Animals treated with αPD-1 mAb developed worse disease and higher granuloma bacterial loads compared to isotype control treated monkeys. PD-1 blockade increased the number and functionality of granuloma Mtb-specific CD8 T cells. In contrast, Mtb-specific CD4 T cells in αPD-1 treated macaques were not increased in number or function in granulomas, upregulated high levels of CTLA-4 and exhibited reduced intralesional trafficking in live imaging studies. In granulomas of αPD-1 treated animals, multiple pro-inflammatory cytokines were elevated, and more cytokines correlated with bacterial loads, leading to the identification of a role for caspase 1 in the exacerbation of tuberculosis after PD-1 blockade. Lastly, increased Mtb bacterial loads after PD-1 blockade were found to associate with the composition of the intestinal microbiota prior to infection in individual macaques. Therefore, PD-1-mediated co-inhibition is required for control of Mtb infection in macaques, perhaps due to its role in dampening detrimental inflammation as well as allowing for normal CD4 T cell responses.

Inflammasomes are signaling hubs that activate inflammatory caspases to drive cytokine maturation and cell lysis. Inflammasome activation by Salmonella Typhimurium infection or Salmonella-derived molecules is extensively studied in murine myeloid cells. Salmonella-induced inflammasome signaling in human innate immune cells, is however, poorly characterized. Here, we show that Salmonella mutation to inactivate the Salmonella pathogenicity island-2 type III secretion system (SPI2 T3SS) potentiates S. Typhimurium-induced inflammasome responses from primary human macrophages, resulting in strong IL-1β production and macrophage death. Inactivation of the SPI1 T3SS diminished human macrophage responses to WT and ΔSPI2 Salmonella. Salmonella ΔSPI2 elicited a mixed inflammasome response from human myeloid cells, in which NLR family CARD-domain containing protein 4 (NLRC4) and NLR family PYRIN-domain containing protein 3 (NLRP3) perform somewhat redundant functions in generating IL-1β and inducing pyroptosis. Our data suggest that Salmonella employs the SPI2 T3SS to subvert SPI1-induced NLRP3 and NLRC4 inflammasome responses in human primary macrophages, in a species-specific immune evasion mechanism. The Gram-negative bacterium Salmonella Typhimurium suppresses human macrophage inflammasome responses via the Salmonella Pathogenicity Island 2.

Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria both in vivo and in vitro. These lipid-bound structures carry a range of immunogenic components derived from the parent cell, which are transported into host target cells and activate the innate immune system. Recent advances in the field have shed light on some of the multifaceted roles of OMVs in host-pathogen interactions. In this study, we investigated the ability of OMVs from two clinically important pathogens, Pseudomonas aeruginosa and Helicobacter pylori, to activate canonical and noncanonical inflammasomes. P. aeruginosa OMVs induced inflammasome activation in mouse macrophages, as evidenced by "speck" formation, as well as the cleavage and secretion of interleukin-1β and caspase-1. These responses were independent of AIM2 and NLRC4 canonical inflammasomes, but dependent on the noncanonical caspase-11 pathway. Moreover, P. aeruginosa OMVs alone were able to activate the inflammasome in a TLR-dependent manner, without requiring an exogenous priming signal. In contrast, H. pylori OMVs were not able to induce inflammasome activation in macrophages. Using CRISPR/Cas9 knockout THP-1 cells lacking the human caspase-11 homologs, caspase-4 and -5,we demonstrated that caspase-5 but not caspase-4 is required for inflammasome activation by P. aeruginosa OMVs in human monocytes. In contrast, free P. aeruginosa lipopolysaccharide (LPS) transfected into cells induced inflammasome responses via caspase-4. This suggests that caspase-4 and caspase-5 differentially recognize LPS depending on its physical form or route of delivery into the cell. These findings have relevance to Gram-negative infections in humans and the use of OMVs as novel vaccines.

ObjectivePyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND) is a recently described monogenic autoinflammatory disease. The causal p.S242RMEFVmutation disrupts a binding motif of the regulatory 14-3-3 proteins within pyrin. Here, we investigate a family with clinical features consistent with PAAND in whom the novel p.E244KMEFVmutation, located in the +2 site of the 14-3-3 binding motif in pyrin, has been found.MethodsMultiplex cytokine analyses were performed on p.E244K patient and control serum. Peripheral blood mononuclear cells were stimulated ex vivo with lipopolysaccharide (LPS). In vitro, inflammasome complex formation was evaluated by flow cytometry of Apoptosis-associated Speck-like protein containing a Caspase recruitment domain (ASC) specks. Interleukin-1β (IL-1β) and IL-18 production was quantified by ELISA. The ability of the p.E244K pyrin mutation to interact with 14-3-3 was assessed by immunoprecipitation.ResultsPAAND p.E244K patient serum displayed a different cytokine profile compared with patients with Familial Mediterranean Fever (FMF). In overexpression models, p.E244K pyrin was associated with decreased 14-3-3 binding and increased ASC speck formation. THP-1 monocytes expressing PAAND pyrin mutations demonstrated spontaneous caspase-1-dependent IL-1β and IL-18 secretion, as well as cell death, which were significantly greater than those of wild-type and the FMF-associated mutation p.M694V.ConclusionIn PAAND, disruption of the +2 position of a 14-3-3 binding motif in pyrin results in its constitutive activation, with spontaneous production of IL-1β and IL-18, associated with inflammatory cell death. The altered serum cytokine profile may explain the different clinical features exhibited by PAAND patients compared with those with FMF.

Evidence is accumulating that protein tyrosine phosphorylation plays a crucial role in the ability of important human bacterial pathogens to cause disease. While most works have concentrated on its role in the regulation of a major bacterial virulence factor, the polysaccharide capsule, recent studies have suggested a much broader role for this post-translational modification. This prompted us to investigate protein tyrosine phosphorylation in the human pathogen Shigella flexneri. We first completed a tyrosine phosphoproteome, identifying 905 unique tyrosine phosphorylation sites on at least 573 proteins (approximately 15% of all proteins). This is the most tyrosine-phosphorylated sites and proteins in a single bacterium identified to date, substantially more than the level seen in eukaryotic cells. Most had not previously been identified and included proteins encoded by the virulence plasmid, which is essential for S. flexneri to invade cells and cause disease. In order to investigate the function of these phosphorylation sites in important virulence factors, phosphomimetic and ablative mutations were constructed in the type 3 secretion system ATPase Spa47 and the master virulence regulator VirB. This revealed that tyrosine residues phosphorylated in our study are critical for Spa47 and VirB activity, and tyrosine phosphorylation likely regulates their functional activity and subsequently the virulence of this major human pathogen. This study suggests that tyrosine phosphorylation plays a critical role in regulating a wide variety of virulence factors in the human pathogen S. flexneri and serves as a base for future studies defining its complete role.

The innate immune system is our first line of defense against viral pathogens. Host cell pattern recognition receptors sense viral components and initiate immune signaling cascades that result in the production of an array of cytokines to combat infection. Retinoic acid-inducible gene-I (RIG-I) is a pattern recognition receptor that recognizes viral RNA and, when activated, results in the production of type I and III interferons (IFNs) and the upregulation of IFN-stimulated genes. Ubiquitination of RIG-I by the E3 ligases tripartite motif-containing 25 (TRIM25) and Riplet is thought to be requisite for RIG-I activation; however, recent studies have questioned the relative importance of these two enzymes for RIG-I signaling. In this study, we show that deletion of Trim25 does not affect the IFN response to either influenza A virus (IAV), influenza B virus, Sendai virus or several RIG-I agonists. This is in contrast to deletion of either Rig-i or Riplet, which completely abrogated RIG-I-dependent IFN responses. This was consistent in both mouse and human cell lines, as well as in normal human bronchial cells. With most of the current TRIM25 literature based on exogenous expression, these findings provide critical evidence that Riplet, and not TRIM25, is required endogenously for the ubiquitination of RIG-I. Despite this, loss of TRIM25 results in greater susceptibility to IAV infection in vivo, suggesting that it may have an alternative role in host antiviral defense. This study refines our understanding of RIG-I signaling in viral infections and will inform future studies in the field.

Lung endothelial cell apoptosis and injury occur throughout all stages of acute lung injury/acute respiratory distress syndrome and impact disease progression. Caspases 1, 4, and 5 are essential for completion of the apoptotic program known as pyroptosis that also involves proinflammatory cytokines. Because gasdermin D (GSDMD) mediates pyroptotic death and is essential for pore formation, we hypothesized that it might direct caspase 1-encapsulated microparticle (MP) release and mediate endothelial cell death. Our present work provides evidence that GSDMD is released by LPS-stimulated THP-1 monocytic cells, where it is packaged into microparticles together with active caspase 1. Furthermore, only MP released from stimulated monocytic cells that contain both cleaved GSDMD and active caspase 1 induce endothelial cell apoptosis. MPs pretreated with caspase 1 inhibitor Y-VAD or pan-caspase inhibitor Z-VAD do not contain cleaved GSDMD. MPs from caspase 1-knockout cells are also deficient in p30 active GSDMD, further confirming that caspase 1 regulates GSDMD function. Although control MPs contained cleaved GSDMD without caspase 1, these fractions were unable to induce cell death, suggesting that encapsulation of both caspase 1 and GSDMD is essential for cell death induction. Release of microparticulate active caspase 1 was abrogated in GSDMD knockout cells, although cytosolic caspase 1 activation was not impaired. Last, higher concentrations of microparticulate GSDMD were detected in the plasma of septic patients with acute respiratory distress syndrome than in that of healthy donors. Taken together, these findings suggest that GSDMD regulates the release of microparticulate active caspase 1 from monocytes essential for induction of cell death and thereby may play a critical role in sepsis-induced endothelial cell injury.

Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase-4 and caspase-5. When activated, these trigger pyroptotic cell death and caspase-1-dependent IL-1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase-4/5-dependent IL-1β production elicited by transfected LPS. Given that both caspase-4 and caspase-5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase-4 and caspase-5 were genetically deleted either individually or together. We found that the deletion of caspase-4 suppressed cell death and IL-1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase-5 did not confer protection against transfected LPS, cell death and IL-1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase-4 and caspase-5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL-1β maturation, downstream of cytoplasmic LPS detection by caspase-4/5. We also show that both caspase-4 and caspase-5 are functionally important for appropriate responses to intracellular Gram-negative bacteria.

CRISPR/Cas9-based gene targeting allows deletion of a gene of interest from cultured cell lines. Due to difficulty in transiently transfecting hematopoetic cells with components required for this process, we have adopted a lentiviral system for delivery of the CRISPR/Cas9 components into myeloid cell lines. Here, we detail the process of knocking out genes from pools of cultured myeloid cells using this CRISPR/Cas9 system and describe methods of validating these knockout pools.

CRISPR/Cas9-based gene targeting allows deletion of a gene of interest from cultured cell lines. Due to difficulty in transiently transfecting hematopoetic cells with components required for this process, we have adopted a lentiviral system for delivery of the CRISPR/Cas9 components into myeloid cell lines. Here, we detail the process of knocking out genes from pools of cultured myeloid cells using this CRISPR/Cas9 system and describe methods of validating these knockout pools.

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined.

International audience; Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

Cell surface innate immune receptors can directly detect a variety of extracellular pathogens to which cytoplasmic innate immune sensors are rarely exposed. Instead, within the cytoplasm, the environment is rife with cellular machinery and signaling pathways that are indirectly perturbed by pathogenic microbes to activate intracellular sensors, such as pyrin, NLRP1, NLRP3, or NLRC4. Therefore, subtle changes in key intracellular processes such as phosphorylation, ubiquitination, and other pathways leading to posttranslational protein modification are key determinants of innate immune recognition in the cytoplasm. This concept is critical to establish the “guard hypothesis” whereby otherwise homeostatic pathways that keep innate immune sensors at bay are released in response to alterations in their posttranslational modification status. Originally identified in plants, evidence that a similar guardlike mechanism exists in humans has recently been identified, whereby a mutation that prevents phosphorylation of the innate immune sensor pyrin triggers a dominantly inherited autoinflammatory disease. It is also noteworthy that even when a cytoplasmic innate immune sensor has a direct ligand, such as bacterial peptidoglycan (NOD1 or NOD2), RNA (RIG-I or MDA5), or DNA (cGAS or IFI16), it can still be influenced by posttranslational modification to dramatically alter its response. Therefore, due to their existence in the cytoplasmic milieu, posttranslational modification is a key determinant of intracellular innate immune receptor functionality.

International audience; Pyrin responds to pathogen signals and loss of cellular homeostasis by forming an inflammasome complex that drives the cleavage and secretion of interleukin-1β (IL-1β). Mutations in the B30.2/SPRY domain cause pathogen-independent activation of pyrin and are responsible for the autoinflammatory disease familial Mediterranean fever (FMF). We studied a family with a dominantly inherited autoinflammatory disease, distinct from FMF, characterized by childhood-onset recurrent episodes of neutrophilic dermatosis, fever, elevated acute-phase reactants, arthralgia, and myalgia/myositis. The disease was caused by a mutation in MEFV, the gene encoding pyrin (S242R). The mutation results in the loss of a 14-3-3 binding motif at phosphorylated S242, which was not perturbed by FMF mutations in the B30.2/SPRY domain. However, loss of both S242 phosphorylation and 14-3-3 binding was observed for bacterial effectors that activate the pyrin inflammasome, such as Clostridium difficile toxin B (TcdB). The S242R mutation thus recapitulated the effect of pathogen sensing, triggering inflammasome activation and IL-1β production. Successful therapy targeting IL-1β has been initiated in one patient, resolving pyrin-associated autoinflammation with neutrophilic dermatosis. This disease provides evidence that a guard-like mechanism of pyrin regulation, originally identified for Nod-like receptors in plant innate immunity, also exists in humans.

Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase-4 and caspase-5. When activated, these trigger pyroptotic cell death and caspase-1-dependent IL-1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase-4/5-dependent IL-1β production elicited by transfected LPS. Given that both caspase-4 and caspase-5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase-4 and caspase-5 were genetically deleted either individually or together. We found that the deletion of caspase-4 suppressed cell death and IL-1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase-5 did not confer protection against transfected LPS, cell death and IL-1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase-4 and caspase-5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL-1β maturation, downstream of cytoplasmic LPS detection by caspase-4/5. We also show that both caspase-4 and caspase-5 are functionally important for appropriate responses to intracellular Gram-negative bacteria.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of anti-viral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2 deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV) and herpes simplex virus 1 (HSV-1) show impaired production of anti-viral cytokines and – in the case of EMCV and HSV-1 – reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

Systemic juvenile idiopathic arthritis (sJIA) shares clinical features with classic monogenic autoinflammatory diseases, characterised by fevers, arthritis and evanescent rashes. Disease exacerbations are associated with elevated serum cytokine levels including 1L-1β, IL-6, and IL-18; and clinical response to anakinra, canakinumab and tocilizumab suggests that cytokine dysregulation is a key pathophysiological mechanism. Macrophage activation syndrome (MAS) may complicate sJIA, rendering individuals clinically indistinguishable from their familial haemophagocytic lymphohistiocytosis (fHLH) counterparts; with NK and CD8+ cell dysfunction leading to sustained immune cell activation and cytokine storm. Whilst there have been HLA associations and polymorphisms noted on sJIA genome-wide association studies, in rare cases mutations have been found in genes encoding key components of the inflammatory response, which may contribute to disease pathogenesis.

Combustion in the cylinder of a spark ignition engine, particularly under low load conditions, is subject to cycle-by-cycle variations due to factors such as mixture quality and quantity and internal exhaust gas recirculation. The major result of this phenomenon is an increase in the variability of indicated engine torque at a given engine operating point. Automotive control problems dealing with torque production at low engine loads, particularly the control of idle speed, rely on accurate information about the transfer functions of different engine subsystems, however combustion variability and the effect it has on torque production is often overlooked. In this paper we illustrate the effects that combustion variability at idle has on different transfer functions related to indicated torque, and propose new models for torque production at constant operating points. We also present a model of an in-line, six-cylinder, 4.0 litre Ford engine for use in idle speed simulations and control applications. The integration of combustion variability into an idle speed control framework is also discussed. Copyright © 2002 SAE International.

This analysis is concerned with the horizontal and vertical consensus of definitions of work appropriate to accomplish the goals of university administrators and faculty, as well as the congruency of ideal (intent) and actual (action/behavior) work of faculty. Data-gathering techniques include the following: historical analysis of the university; interviews with the provost, 5 college deans, and 32 chairpersons; and a survey of 503 faculty (69% return rate). Faculty work perceived to achieve departmental goals lacked horizontal consensus, especially among chairpersons in the same college and among faculty in the same department. Vertical consensus was weak, especially between chairpersons and their faculty, and in most colleges between deans and chairpersons. Greatest incongruencies were between faculty intentions and reported actions. There was declining support for administrative goals as one moved down the hierarchical structure within the university. Perspectives were explored that account for the lack of goal consensus and the incongruency between intentions and reported actions.

The large lecture section is often perceived as a homogeneous mass of students who can be taught satisfactorily by a single teaching method — usually lectures, textbooks, and objective exams. An attempt to improve the quality of teaching and learning was made by offering three distinct options to 695 students enrolled in two large classes: (1) conventional lecture/objective exam format; (2) small groups of five to eight students; (3) independent study. The students responded with 86% selecting the conventional method, 11% choosing small groups, and 3% deciding upon independent study. Students in the conventional classes tended to have high scores on Rotter's External Control Scale and high scores on personality need scales (Personality Research Form) for harm avoidance, order, social recognition, and succorence; they also tended to be less intellectually inclined and more vocationally oriented. The students in nonconventional options tended to have high personality need scores for cognitive structure, achievement, autonomy, and understanding.

In 1927 Luther L. Bernard wrote to several eminent sociologists concerning his plans for a cooperative work on the History of Sociology in the United States. He felt the need to bring together "the facts regarding the development of sociology in each of the universities in which there has been for some time an outstanding department or chair in the subject." Bernard went on to write, "Fortunately, almost all of the founders of these older departments are still actively teaching and it should be possible for them severally to tell the stories of the founding and development of their chairs or departments first hand."' Hundreds of letters were sent to sociologists throughout the United States asking them to provide information regarding their careers and the history of sociology in the institutions with which they had been affiliated. Each sociologist was asked to write his life history in narrative form; in addition a 16-point outline was provided to offer a framework for the autobiographical statement. The outline included such items as educational background, occupations before and after entering sociology, various influential teachers in sociology and other fields, other significant intellectual influences, various research projects conducted, and "any other influences, vocational, professional, individual, general, political, social, economic, etc., which have helped to mould your career as a sociologist, giving the details."2 W. I. Thomas and Robert Park, along with 256 other sociologists,3 responded to Bernard's query; however, the autobiographies and departmental histories were never published. Luther Bernard's wife, Jessie, explains her husband's unfinished work: "Once gathered, he seemed satisfied. There would always be time to assemble and edit them for publication. Further, other concerns engaged a good deal of his time and the sheer physical labor involved in processing the mass of material for presentation to the profession seemed overwhelming."4

audience and present the wrong content. The authors challenge sociology teachers to recognize the shifting demographic trends in the student body that include more older students, women, and racial and ethnic minorities. The content of the introductory course must be pared down to reflect afew critical principles that are elaborated throughout the course. The authors suggest the use of monographs as appropriate readings to illustrate key concepts and excite students about sociology.