Showing papers by "Richard A. Flavell published in 2021"

Potential intestinal infection and faecal-oral transmission of SARS-CoV-2.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to more than 200 countries and regions globally. SARS-CoV-2 is thought to spread mainly through respiratory droplets and close contact. However, reports have shown that a notable proportion of patients with coronavirus disease 2019 (COVID-19) develop gastrointestinal symptoms and nearly half of patients confirmed to have COVID-19 have shown detectable SARS-CoV-2 RNA in their faecal samples. Moreover, SARS-CoV-2 infection reportedly alters intestinal microbiota, which correlated with the expression of inflammatory factors. Furthermore, multiple in vitro and in vivo animal studies have provided direct evidence of intestinal infection by SARS-CoV-2. These lines of evidence highlight the nature of SARS-CoV-2 gastrointestinal infection and its potential faecal-oral transmission. Here, we summarize the current findings on the gastrointestinal manifestations of COVID-19 and its possible mechanisms. We also discuss how SARS-CoV-2 gastrointestinal infection might occur and the current evidence and future studies needed to establish the occurrence of faecal-oral transmission.

Skin-resident innate lymphoid cells converge on a pathogenic effector state.

Abstract: Tissue-resident innate lymphoid cells (ILCs) help sustain barrier function and respond to local signals ILCs are traditionally classified as ILC1, ILC2 or ILC3 on the basis of their expression of specific transcription factors and cytokines1 In the skin, disease-specific production of ILC3-associated cytokines interleukin (IL)-17 and IL-22 in response to IL-23 signalling contributes to dermal inflammation in psoriasis However, it is not known whether this response is initiated by pre-committed ILCs or by cell-state transitions Here we show that the induction of psoriasis in mice by IL-23 or imiquimod reconfigures a spectrum of skin ILCs, which converge on a pathogenic ILC3-like state Tissue-resident ILCs were necessary and sufficient, in the absence of circulatory ILCs, to drive pathology Single-cell RNA-sequencing (scRNA-seq) profiles of skin ILCs along a time course of psoriatic inflammation formed a dense transcriptional continuum—even at steady state—reflecting fluid ILC states, including a naive or quiescent-like state and an ILC2 effector state Upon disease induction, the continuum shifted rapidly to span a mixed, ILC3-like subset also expressing cytokines characteristic of ILC2s, which we inferred as arising through multiple trajectories We confirmed the transition potential of quiescent-like and ILC2 states using in vitro experiments, single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) and in vivo fate mapping Our results highlight the range and flexibility of skin ILC responses, suggesting that immune activities primed in healthy tissues dynamically adapt to provocations and, left unchecked, drive pathological remodelling In studies using mouse models of psoriasis, a spectrum of innate lymphoid cell types is reconfigured and converges via multiple trajectories on a type 3-like state, demonstrating the range and flexibility of innate lymphoid cell responses in the skin

Pooled CRISPR screening identifies m6A as a positive regulator of macrophage activation.

Abstract: m6A RNA modification is implicated in multiple cellular responses. However, its function in the innate immune cells is poorly understood. Here, we identified major m6A “writers” as the top candidate genes regulating macrophage activation by LPS in an RNA binding protein focused CRISPR screening. We have confirmed that Mettl3-deficient macrophages exhibited reduced TNF-α production upon LPS stimulation in vitro. Consistently, Mettl3flox/flox;Lyzm-Cre mice displayed increased susceptibility to bacterial infection and showed faster tumor growth. Mechanistically, the transcripts of the Irakm gene encoding a negative regulator of TLR4 signaling were highly decorated by m6A modification. METTL3 deficiency led to the loss of m6A modification on Irakm mRNA and slowed down its degradation, resulting in a higher level of IRAKM, which ultimately suppressed TLR signaling–mediated macrophage activation. Our findings demonstrate a previously unknown role for METTL3-mediated m6A modification in innate immune responses and implicate the m6A machinery as a potential cancer immunotherapy target.

m6A demethylase ALKBH5 controls CD4+ T cell pathogenicity and promotes autoimmunity

Abstract: N6-methyladenosine (m6A) modification is dynamically regulated by "writer" and "eraser" enzymes. m6A "writers" have been shown to ensure the homeostasis of CD4+ T cells, but the "erasers" functioning in T cells is poorly understood. Here, we reported that m6A eraser AlkB homolog 5 (ALKBH5), but not FTO, maintains the ability of naive CD4+ T cells to induce adoptive transfer colitis. In addition, T cell-specific ablation of ALKBH5 confers protection against experimental autoimmune encephalomyelitis. During the induced neuroinflammation, ALKBH5 deficiency increased m6A modification on interferon-γ and C-X-C motif chemokine ligand 2 messenger RNA (mRNA), thus decreasing their mRNA stability and protein expression in CD4+ T cells. These modifications resulted in attenuated CD4+ T cell responses and diminished recruitment of neutrophils into the central nervous system. Our findings reveal an unexpected specific role of ALKBH5 as an m6A eraser in controlling the pathogenicity of CD4+ T cells during autoimmunity.

METTL3-mediated m6A RNA methylation promotes the anti-tumour immunity of natural killer cells.

Abstract: Natural killer (NK) cells exert critical roles in anti-tumor immunity but how their functions are regulated by epitranscriptional modification (e.g., N6-methyladenosine (m6A) methylation) is unclear. Here we report decreased expression of the m6A “writer” METTL3 in tumor-infiltrating NK cells, and a positive correlation between protein expression levels of METTL3 and effector molecules in NK cells. Deletion of Mettl3 in NK cells alters the homeostasis of NK cells and inhibits NK cell infiltration and function in the tumor microenvironment, leading to accelerated tumor development and shortened survival in mice. The gene encoding SHP-2 is m6A modified, and its protein expression is decreased in METTL3-deficient NK cells. Reduced SHP-2 activity renders NK cells hyporesponsive to IL-15, which is associated with suppressed activation of the AKT and MAPK signaling pathway in METTL3-deficient NK cells. These findings show that m6A methylation safeguards the homeostasis and tumor immunosurveillance function of NK cells. Post transcriptional modifications to RNA may be important in the function of NK cells but this is not fully known. Here the authors show that m6A-methyltransferase METTL3 deletion in NK cells leads to reduced function and protection against tumour challenge through suppressing response to IL-15.

Detection of differentially abundant cell subpopulations in scRNA-seq data.

Abstract: Comprehensive and accurate comparisons of transcriptomic distributions of cells from samples taken from two different biological states, such as healthy versus diseased individuals, are an emerging challenge in single-cell RNA sequencing (scRNA-seq) analysis. Current methods for detecting differentially abundant (DA) subpopulations between samples rely heavily on initial clustering of all cells in both samples. Often, this clustering step is inadequate since the DA subpopulations may not align with a clear cluster structure, and important differences between the two biological states can be missed. Here, we introduce DA-seq, a targeted approach for identifying DA subpopulations not restricted to clusters. DA-seq is a multiscale method that quantifies a local DA measure for each cell, which is computed from its k nearest neighboring cells across a range of k values. Based on this measure, DA-seq delineates contiguous significant DA subpopulations in the transcriptomic space. We apply DA-seq to several scRNA-seq datasets and highlight its improved ability to detect differences between distinct phenotypes in severe versus mildly ill COVID-19 patients, melanomas subjected to immune checkpoint therapy comparing responders to nonresponders, embryonic development at two time points, and young versus aging brain tissue. DA-seq enabled us to detect differences between these phenotypes. Importantly, we find that DA-seq not only recovers the DA cell types as discovered in the original studies but also reveals additional DA subpopulations that were not described before. Analysis of these subpopulations yields biological insights that would otherwise be undetected using conventional computational approaches.

The emerging role of Janus kinase inhibitors in the treatment of autoimmune and inflammatory diseases.

Abstract: Autoimmune and inflammatory diseases are common and diverse, and they can affect nearly any organ system. Much of the pathogenesis of these diseases is related to dysregulated cytokine activity. Historically, autoimmune and inflammatory diseases have been treated with medications that nonspecifically suppress the immune system. mAbs that block the action of pathogenic cytokines emerged 2 decades ago and have become widely useful. More recently, agents that simultaneously block multiple pathogenic cytokines via inhibition of the downstream Janus kinase (JAK)–signal transducer and activator of transcription pathway have emerged and are becoming increasingly important. These small-molecule inhibitors, collectively termed JAK inhibitors, are US Food and Drug Administration–approved in a few autoimmune/inflammatory disorders and are being evaluated in many others. Here, we review the biology of the JAK–signal transducer and activator of transcription pathway and the use of JAK inhibitors to treat autoimmune and inflammatory diseases across medical subspecialties.

MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche.

Abstract: Intestinal stromal cells are known to modulate the propagation and differentiation of intestinal stem cells1,2. However, the precise cellular and molecular mechanisms by which this diverse stromal cell population maintains tissue homeostasis and repair are poorly understood. Here we describe a subset of intestinal stromal cells, named MAP3K2-regulated intestinal stromal cells (MRISCs), and show that they are the primary cellular source of the WNT agonist R-spondin 1 following intestinal injury in mice. MRISCs, which are epigenetically and transcriptomically distinct from subsets of intestinal stromal cells that have previously been reported3–6, are strategically localized at the bases of colon crypts, and function to maintain LGR5+ intestinal stem cells and protect against acute intestinal damage through enhanced R-spondin 1 production. Mechanistically, this MAP3K2 specific function is mediated by a previously unknown reactive oxygen species (ROS)–MAP3K2–ERK5–KLF2 axis to enhance production of R-spondin 1. Our results identify MRISCs as a key component of an intestinal stem cell niche that specifically depends on MAP3K2 to augment WNT signalling for the regeneration of damaged intestine. A subset of intestinal stromal cells that is regulated by the kinase MAP3K2 protects intestinal stem cells against injury by producing the WNT agonist R-spondin 1.

IL-27 signalling promotes adipocyte thermogenesis and energy expenditure.

Abstract: Thermogenesis in brown and beige adipose tissue has important roles in maintaining body temperature and countering the development of metabolic disorders such as obesity and type 2 diabetes1,2. Although much is known about commitment and activation of brown and beige adipose tissue, its multiple and abundant immunological factors have not been well characterized3–6. Here we define a critical role of IL-27–IL-27Rα signalling in improving thermogenesis, protecting against diet-induced obesity and ameliorating insulin resistance. Mechanistic studies demonstrate that IL-27 directly targets adipocytes, activating p38 MAPK–PGC-1α signalling and stimulating the production of UCP1. Notably, therapeutic administration of IL-27 ameliorated metabolic morbidities in well-established mouse models of obesity. Consistently, individuals with obesity show significantly decreased levels of serum IL-27, which can be restored after bariatric surgery. Collectively, these findings show that IL-27 has an important role in orchestrating metabolic programs, and is a highly promising target for anti-obesity immunotherapy. Therapeutic administration of IL-27—serum levels of which are decreased in individuals with obesity—improves thermogenesis, protects against diet-induced obesity and ameliorates insulin resistance in mouse models of obesity.

Treatment of granuloma annulare and suppression of proinflammatory cytokine activity with tofacitinib.

Abstract: Background Granuloma annulare (GA) is a common cutaneous inflammatory disorder characterized by macrophage accumulation and activation in skin. Its pathogenesis is poorly understood, and there are no effective treatments. The potential health implications of severe GA are unknown. Objective We sought to better understand GA pathogenesis and evaluate a molecularly targeted treatment approach for this disease. Methods We used single-cell RNA sequencing to study the immunopathogenesis of GA and also evaluated the efficacy of tofacitinib (a Janus kinase 1/3 inhibitor) in 5 patients with severe, long-standing GA in an open-label clinical trial. Results Using single-cell RNA sequencing, we found that in GA lesions IFN-γ production by CD4+ T cells is upregulated and is associated with inflammatory polarization of macrophages and fibroblasts. In particular, macrophages upregulate oncostatin M, an IL-6 family cytokine, which appears to act on fibroblasts to alter extracellular matrix production, a hallmark of GA. IL-15 and IL-21 production appears to feed back on CD4+ T cells to sustain inflammation. Treatment of 5 patients with recalcitrant GA with tofacitinib inhibited IFN-γ and oncostatin M, as well as IL-15 and IL-21, activity and resulted in clinical and histologic disease remission in 3 patients and marked improvement in the other 2. Inhibition of these effects at the molecular level paralleled the clinical improvement. Evidence of systemic inflammation is also present in some patients with severe GA and is mitigated by tofacitinib. Conclusions The Janus kinase-signal transducer and activator of transcription pathway is activated in GA, likely in part through the activity of IFN-γ and oncostatin M, and Janus kinase inhibitors appear to be an effective treatment.

IL-4-BATF signaling directly modulates IL-9 producing mucosal mast cell (MMC9) function in experimental food allergy

Abstract: Background This study group has previously identified IL-9–producing mucosal mast cell (MMC9) as the primary source of IL-9 to drive intestinal mastocytosis and experimental IgE-mediated food allergy. However, the molecular mechanisms that regulate the expansion of MMC9s remain unknown. Objectives This study hypothesized that IL-4 regulates MMC9 development and MMC9-dependent experimental IgE-mediated food allergy. Methods An epicutaneous sensitization model was used and bone marrow reconstitution experiments were performed to test the requirement of IL-4 receptor α (IL-4Rα) signaling on MMC9s in experimental IgE-mediated food allergy. Flow cytometric, bulk, and single-cell RNA-sequencing analyses on small intestine (SI) MMC9s were performed to illuminate MMC9 transcriptional signature and the effect of IL-4Rα signaling on MMC9 function. A bone marrow–derived MMC9 culture system was used to define IL-4–BATF signaling in MMC9 development. Results Epicutaneous sensitization– and bone marrow reconstitution–based models of IgE-mediated food allergy revealed an IL-4 signaling-dependent cell-intrinsic effect on SI MMC9 accumulation and food allergy severity. RNA-sequencing analysis of SI-MMC9s identified 410 gene transcripts reciprocally regulated by IL-4 signaling, including Il9 and Batf. In silico analyses identified a 3491-gene MMC9 transcriptional signature and identified 2 transcriptionally distinct SI MMC9 populations enriched for metabolic or inflammatory programs. Employing an in vitro MMC9-culture model system showed that generation of MMC9-like cells was induced by IL-4 and this was in part dependent on BATF. Conclusions IL-4Rα signaling directly modulates MMC9 function and exacerbation of experimental IgE-mediated food allergic reactions. IL-4Rα regulation of MMC9s is in part BATF-dependent and occurs via modulation of metabolic transcriptional programs.

Viral replication in human macrophages enhances an inflammatory cascade and interferon driven chronic COVID-19 in humanized mice.

Abstract: Chronic COVID-19 is characterized by persistent viral RNA and sustained interferon (IFN) response which is recapitulated and required for pathology in SARS-CoV-2 infected MISTRG6-hACE2 humanized mice. As in the human disease, monocytes, and macrophages in SARS-CoV-2 infected MISTRG6-hACE2 are central to disease pathology. Here, we describe SARS-CoV-2 uptake in tissue resident human macrophages that is enhanced by virus specific antibodies. SARS-CoV-2 replicates in these human macrophages as evidenced by detection of double-stranded RNA, subgenomic viral RNA and expression of a virally encoded fluorescent reporter gene; and it is inhibited by Remdesivir, an inhibitor of viral replication. Although early IFN deficiency leads to enhanced disease, blocking either viral replication with Remdesivir or the downstream IFN stimulated cascade by injecting anti-IFNAR2 in vivo in the chronic stages of disease attenuates many aspects of the overactive immune-inflammatory response, especially the inflammatory macrophage response, and most consequentially, the chronic disease itself.

Combined liver-cytokine humanization comes to the rescue of circulating human red blood cells.

Abstract: In vivo models that recapitulate human erythropoiesis with persistence of circulating red blood cells (RBCs) have remained elusive. We report an immunodeficient murine model in which combined human liver and cytokine humanization confer enhanced human erythropoiesis and RBC survival in the circulation. We deleted the fumarylacetoacetate hydrolase (Fah) gene in MISTRG mice expressing several human cytokines in place of their murine counterparts. Liver humanization by intrasplenic injection of human hepatocytes (huHep) eliminated murine complement C3 and reduced murine Kupffer cell density. Engraftment of human sickle cell disease (SCD)–derived hematopoietic stem cells in huHepMISTRGFah−/− mice resulted in vaso-occlusion that replicated acute SCD pathology. Combined liver–cytokine–humanized mice will facilitate the study of diseases afflicting RBCs, including bone marrow failure, hemoglobinopathies, and malaria, and also preclinical testing of therapies.

The RNA helicase Dhx15 mediates Wnt-induced antimicrobial protein expression in Paneth cells.

Abstract: RNA helicases play roles in various essential biological processes such as RNA splicing and editing. Recent in vitro studies show that RNA helicases are involved in immune responses toward viruses, serving as viral RNA sensors or immune signaling adaptors. However, there is still a lack of in vivo data to support the tissue- or cell-specific function of RNA helicases owing to the lethality of mice with complete knockout of RNA helicases; further, there is a lack of evidence about the antibacterial role of helicases. Here, we investigated the in vivo role of Dhx15 in intestinal antibacterial responses by generating mice that were intestinal epithelial cell (IEC)-specific deficient for Dhx15 (Dhx15 f/f Villin1-cre, Dhx15ΔIEC). These mice are susceptible to infection with enteric bacteria Citrobacter rodentium (C. rod), owing to impaired α-defensin production by Paneth cells. Moreover, mice with Paneth cell-specific depletion of Dhx15 (Dhx15 f/f Defensinα6-cre, Dhx15ΔPaneth) are more susceptible to DSS (dextran sodium sulfate)-induced colitis, which phenocopy Dhx15ΔIEC mice, due to the dysbiosis of the intestinal microbiota. In humans, reduced protein levels of Dhx15 are found in ulcerative colitis (UC) patients. Taken together, our findings identify a key regulator of Wnt-induced α-defensins in Paneth cells and offer insights into its role in the antimicrobial response as well as intestinal inflammation.

Interleukin-10 improves stroke outcome by controlling the detrimental Interleukin-17A response.

Abstract: Background Lymphocytes have dichotomous functions in ischemic stroke. Regulatory T cells are protective, while IL-17A from innate lymphocytes promotes the infarct growth. With recent advances of T cell-subtype specific transgenic mouse models it now has become possible to study the complex interplay of T cell subpopulations in ischemic stroke. Methods In a murine model of experimental stroke we analyzed the effects of IL-10 on the functional outcome for up to 14 days post-ischemia and defined the source of IL-10 in ischemic brains based on immunohistochemistry, flow cytometry, and bone-marrow chimeric mice. We used neutralizing IL-17A antibodies, intrathecal IL-10 injections, and transgenic mouse models which harbor a deletion of the IL-10R on distinct T cell subpopulations to further explore the interplay between IL-10 and IL-17A pathways in the ischemic brain. Results We demonstrate that IL-10 deficient mice exhibit significantly increased infarct sizes on days 3 and 7 and enlarged brain atrophy and impaired neurological outcome on day 14 following tMCAO. In ischemic brains IL-10 producing immune cells included regulatory T cells, macrophages, and microglia. Neutralization of IL-17A following stroke reversed the worse outcome in IL-10 deficient mice and intracerebral treatment with recombinant IL-10 revealed that IL-10 controlled IL-17A positive lymphocytes in ischemic brains. Importantly, IL-10 acted differentially on αβ and γδ T cells. IL-17A producing CD4+ αβ T cells were directly controlled via their IL-10-receptor (IL-10R), whereas IL-10 by itself had no direct effect on the IL-17A production in γδ T cells. The control of the IL-17A production in γδ T cells depended on an intact IL10R signaling in regulatory T cells (Tregs). Conclusions Taken together, our data indicate a key function of IL-10 in restricting the detrimental IL-17A-signaling in stroke and further supports that IL-17A is a therapeutic opportunity for stroke treatment.

JUN Amino-Terminal Kinase 1 Signaling in the Proximal Tubule Causes Cell Death and Acute Renal Failure in Rat and Mouse Models of Renal Ischemia/Reperfusion Injury.

Abstract: Activation of the JUN amino-terminal kinase (JNK) pathway is prominent in most forms of acute and progressive tubulointerstitial damage, including acute renal ischemia/reperfusion injury (IRI). Two forms of JNK, JNK1 and JNK2, are expressed in the kidney. Systemic administration of pan-JNK inhibitors suppresses renal IRI; however, the contribution of JNK1 versus JNK2, and the specific role of JNK activation in the proximal tubule in IRI, remains unknown. These questions were addressed in rat and mouse models of acute bilateral renal IRI. Administration of the JNK inhibitor, CC-930, substantially reduced the severity of renal failure, tubular damage, and inflammation at 24 hours in a rat IRI model. Additionally, Jnk1−/− mice, but not Jnk2−/− mice, were shown to be significantly protected against acute renal failure, tubular damage, and inflammation in the IRI model. Furthermore, mice with conditional Jnk1 deletion in the proximal tubule also showed considerable protection from IRI-induced renal failure, tubular damage, and inflammation. Finally, primary cultures of Jnk1−/−, but not Jnk2−/−, tubular epithelial cells were protected from oxidant-induced cell death, in association with preventing phosphorylation of proteins (receptor interacting serine/threonine kinase 3 and mixed lineage kinase domain-like pseudokinase) in the necroptosis pathway. In conclusion, JNK1, but not JNK2, plays a specific role in IRI-induced cell death in the proximal tubule, leading to acute renal failure.

A humanized mouse model of chronic COVID-19 to evaluate disease mechanisms and treatment options

Abstract: Coronavirus-associated acute respiratory disease, called coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More than 90 million people have been infected with SARS-CoV-2 and more than 2 million people have died of complications due to COVID-19 worldwide. COVID-19, in its severe form, presents with an uncontrolled, hyperactive immune response and severe immunological injury or organ damage that accounts for morbidity and mortality. Even in the absence of complications, COVID-19 can last for several months with lingering effects of an overactive immune system. Dysregulated myeloid and lymphocyte compartments have been implicated in lung immunopathology. Currently, there are limited clinically-tested treatments of COVID-19 with disparities in the apparent efficacy in patients. Accurate model systems are essential to rapidly evaluate promising discoveries but most currently available in mice, ferrets and hamsters do not recapitulate sustained immunopathology described in COVID19 patients. Here, we present a comprehensively humanized mouse COVID-19 model that faithfully recapitulates the innate and adaptive human immune responses during infection with SARS-CoV-2 by adapting recombinant adeno-associated virus (AAV)-driven gene therapy to deliver human ACE2 to the lungs 1 of MISTRG6 mice. Our unique model allows for the first time the study of chronic disease due to infection with SARS-CoV-2 in the context of patient-derived antibodies to characterize in real time the potential culprits of the observed human driving immunopathology; most importantly this model provides a live view into the aberrant macrophage response that is thought to be the effector of disease morbidity and ARDS in patients. Application of therapeutics such as patient-derived antibodies and steroids to our model allowed separation of the two aspects of the immune response, infectious viral clearance and immunopathology. Inflammatory cells seeded early in infection drove immune-patholgy later, but this very same early anti-viral response was also crucial to contain infection.

Cerebellar Kv3.3 potassium channels activate TANK-binding kinase 1 to regulate trafficking of the cell survival protein Hax-1.

Abstract: Mutations in KCNC3, which encodes the Kv3.3 potassium channel, cause degeneration of the cerebellum, but exactly how the activity of an ion channel is linked to the survival of cerebellar neurons is not understood. Here, we report that Kv3.3 channels bind and stimulate Tank Binding Kinase 1 (TBK1), an enzyme that controls trafficking of membrane proteins into multivesicular bodies, and that this stimulation is greatly increased by a disease-causing Kv3.3 mutation. TBK1 activity is required for the binding of Kv3.3 to its auxiliary subunit Hax-1, which prevents channel inactivation with depolarization. Hax-1 is also an anti-apoptotic protein required for survival of cerebellar neurons. Overactivation of TBK1 by the mutant channel leads to the loss of Hax-1 by its accumulation in multivesicular bodies and lysosomes, and also stimulates exosome release from neurons. This process is coupled to activation of caspases and increased cell death. Our studies indicate that Kv3.3 channels are directly coupled to TBK1-dependent biochemical pathways that determine the trafficking of cellular constituents and neuronal survival.

A hierarchical GBP network promotes cytosolic LPS recognition and sepsis

Abstract: Bacterial lipopolysaccharide (LPS) is one of the most bioactive substances known. Trace amounts trigger robust immunity to infection but also life-threatening sepsis causing millions of deaths each year. LPS contamination of the cytosol elicits a caspase-dependent inflammasome pathway promoting cytokine release and host cell death. Here, we report an immune GTPase network controls multiple steps in this pathway by genome-engineering mice to lack 7 different guanylate-binding proteins (GBPs). Gbp2-/- and Gbp3-/- mice had severe caspase-11-driven defects that protected them from septic shock. Gbp2 recruited caspase-11 for LPS recognition whereas Gbp3 assembled and trafficked the pyroptotic pore-forming protein, gasdermin D, after caspase-11 cleavage. Together, our results identify a new functional hierarchy wherein different GBPs choreograph sequential steps in the non-canonical inflammasome pathway to control Gram-negative sepsis. One-Sentence Summary Immune GTPase network orchestrates hierarchical immunity to bacterial products in vivo

CFTR is a negative regulator of γδ T cell IFN-γ production and antitumor immunity.

Abstract: CFTR, a chloride channel and ion channel regulator studied mostly in epithelial cells, has been reported to participate in immune regulation and likely affect the risk of cancer development. However, little is known about the effects of CFTR on the differentiation and function of γδ T cells. In this study, we observed that CFTR was functionally expressed on the cell surface of γδ T cells. Genetic deletion and pharmacological inhibition of CFTR both increased IFN-γ release by peripheral γδ T cells and potentiated the cytolytic activity of these cells against tumor cells both in vitro and in vivo. Interestingly, the molecular mechanisms underlying the regulation of γδ T cell IFN-γ production by CFTR were either TCR dependent or related to Ca2+ influx. CFTR was recruited to TCR immunological synapses and attenuated Lck-P38 MAPK-c-Jun signaling. In addition, CFTR was found to modulate TCR-induced Ca2+ influx and membrane potential (Vm)-induced Ca2+ influx and subsequently regulate the calcineurin-NFATc1 signaling pathway in γδ T cells. Thus, CFTR serves as a negative regulator of IFN-γ production in γδ T cells and the function of these cells in antitumor immunity. Our investigation suggests that modification of the CFTR activity of γδ T cells may be a potential immunotherapeutic strategy for cancer.