ABSTRACT
Ongoing, early-stage clinical trials illustrate the translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, an unresolved challenge is the extensive cell death following transplantation. Here, we performed a pooled CRISPR-Cas9 screen to enhance postmitotic dopamine neuron survival in vivo. We identified p53-mediated apoptotic cell death as a major contributor to dopamine neuron loss and uncovered a causal link of tumor necrosis factor alpha (TNF-α)-nuclear factor κB (NF-κB) signaling in limiting cell survival. As a translationally relevant strategy to purify postmitotic dopamine neurons, we identified cell surface markers that enable purification without the need for genetic reporters. Combining cell sorting and treatment with adalimumab, a clinically approved TNF-α inhibitor, enabled efficient engraftment of postmitotic dopamine neurons with extensive reinnervation and functional recovery in a preclinical PD mouse model. Thus, transient TNF-α inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic hPSC-derived dopamine neurons in PD.
Subject(s)
Cell Survival , Dopaminergic Neurons , NF-kappa B , Tumor Necrosis Factor-alpha , Tumor Suppressor Protein p53 , Dopaminergic Neurons/metabolism , Animals , Humans , NF-kappa B/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Necrosis Factor-alpha/metabolism , Mice , Cell Survival/drug effects , Signal Transduction , Parkinson Disease/metabolism , Pluripotent Stem Cells/metabolism , Apoptosis , Disease Models, Animal , CRISPR-Cas SystemsABSTRACT
NLRs constitute a large, highly conserved family of cytosolic pattern recognition receptors that are central to health and disease, making them key therapeutic targets. NLRC5 is an enigmatic NLR with mutations associated with inflammatory and infectious diseases, but little is known about its function as an innate immune sensor and cell death regulator. Therefore, we screened for NLRC5's role in response to infections, PAMPs, DAMPs, and cytokines. We identified that NLRC5 acts as an innate immune sensor to drive inflammatory cell death, PANoptosis, in response to specific ligands, including PAMP/heme and heme/cytokine combinations. NLRC5 interacted with NLRP12 and PANoptosome components to form a cell death complex, suggesting an NLR network forms similar to those in plants. Mechanistically, TLR signaling and NAD+ levels regulated NLRC5 expression and ROS production to control cell death. Furthermore, NLRC5-deficient mice were protected in hemolytic and inflammatory models, suggesting that NLRC5 could be a potential therapeutic target.
Subject(s)
Inflammation , Intracellular Signaling Peptides and Proteins , NAD , Animals , Mice , Inflammation/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , NAD/metabolism , Humans , Immunity, Innate , Mice, Inbred C57BL , Reactive Oxygen Species/metabolism , Mice, Knockout , Signal Transduction , HEK293 Cells , Inflammasomes/metabolism , Apoptosis Regulatory Proteins/metabolism , Apoptosis Regulatory Proteins/genetics , Toll-Like Receptors/metabolism , Male , Cytokines/metabolism , Calcium-Binding ProteinsABSTRACT
Neutrophils are abundant immune cells in the circulation and frequently infiltrate tumors in substantial numbers. However, their precise functions in different cancer types remain incompletely understood, including in the brain microenvironment. We therefore investigated neutrophils in tumor tissue of glioma and brain metastasis patients, with matched peripheral blood, and herein describe the first in-depth analysis of neutrophil phenotypes and functions in these tissues. Orthogonal profiling strategies in humans and mice revealed that brain tumor-associated neutrophils (TANs) differ significantly from blood neutrophils and have a prolonged lifespan and immune-suppressive and pro-angiogenic capacity. TANs exhibit a distinct inflammatory signature, driven by a combination of soluble inflammatory mediators including tumor necrosis factor alpha (TNF-É) and Ceruloplasmin, which is more pronounced in TANs from brain metastasis versus glioma. Myeloid cells, including tumor-associated macrophages, emerge at the core of this network of pro-inflammatory mediators, supporting the concept of a critical myeloid niche regulating overall immune suppression in human brain tumors.
ABSTRACT
Cell proliferation and cell death are integral elements in maintaining homeostatic balance in metazoans. Disease pathologies ensue when these processes are disturbed. A plethora of evidence indicates that malfunction of cell death can lead to inflammation, autoimmunity, or immunodeficiency. Programmed necrosis or necroptosis is a form of nonapoptotic cell death driven by the receptor interacting protein kinase 3 (RIPK3) and its substrate, mixed lineage kinase domain-like (MLKL). RIPK3 partners with its upstream adaptors RIPK1, TRIF, or DAI to signal for necroptosis in response to death receptor or Toll-like receptor stimulation, pathogen infection, or sterile cell injury. Necroptosis promotes inflammation through leakage of cellular contents from damaged plasma membranes. Intriguingly, many of the signal adaptors of necroptosis have dual functions in innate immune signaling. This unique signature illustrates the cooperative nature of necroptosis and innate inflammatory signaling pathways in managing cell and organismal stresses from pathogen infection and sterile tissue injury.
Subject(s)
Inflammation/metabolism , Inflammation/pathology , Necrosis/metabolism , Signal Transduction , Animals , Bacterial Infections/genetics , Bacterial Infections/metabolism , Bacterial Infections/pathology , Biological Evolution , Cell Death , Humans , Inflammasomes/metabolism , Inflammation/genetics , Interleukin-1beta/metabolism , NF-kappa B/metabolism , Parasitic Diseases/genetics , Parasitic Diseases/metabolism , Parasitic Diseases/pathology , Phosphorylation , Protein Binding , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Ubiquitination , Virus Diseases/genetics , Virus Diseases/metabolism , Virus Diseases/pathologyABSTRACT
TANK binding kinase 1 (TBK1) regulates IFN-I, NF-κB, and TNF-induced RIPK1-dependent cell death (RCD). In mice, biallelic loss of TBK1 is embryonically lethal. We discovered four humans, ages 32, 26, 7, and 8 from three unrelated consanguineous families with homozygous loss-of-function mutations in TBK1. All four patients suffer from chronic and systemic autoinflammation, but not severe viral infections. We demonstrate that TBK1 loss results in hypomorphic but sufficient IFN-I induction via RIG-I/MDA5, while the system retains near intact IL-6 induction through NF-κB. Autoinflammation is driven by TNF-induced RCD as patient-derived fibroblasts experienced higher rates of necroptosis in vitro, and CC3 was elevated in peripheral blood ex vivo. Treatment with anti-TNF dampened the baseline circulating inflammatory profile and ameliorated the clinical condition in vivo. These findings highlight the plasticity of the IFN-I response and underscore a cardinal role for TBK1 in the regulation of RCD.
Subject(s)
Inflammation/enzymology , Protein Serine-Threonine Kinases/deficiency , Tumor Necrosis Factor-alpha/pharmacology , A549 Cells , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis , Autoimmunity/drug effects , Brain/diagnostic imaging , Cell Death/drug effects , Cytokines/metabolism , Deubiquitinating Enzyme CYLD/metabolism , Female , HEK293 Cells , Homozygote , Humans , I-kappa B Kinase/metabolism , Immunophenotyping , Inflammation/pathology , Interferon Type I/metabolism , Interferon-gamma/metabolism , Loss of Function Mutation/genetics , Male , Pedigree , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptors, Pattern Recognition/metabolism , Toll-Like Receptor 3/metabolism , Transcriptome/genetics , Vesiculovirus/drug effects , Vesiculovirus/physiologyABSTRACT
COVID-19 is characterized by excessive production of pro-inflammatory cytokines and acute lung damage associated with patient mortality. While multiple inflammatory cytokines are produced by innate immune cells during SARS-CoV-2 infection, we found that only the combination of TNF-α and IFN-γ induced inflammatory cell death characterized by inflammatory cell death, PANoptosis. Mechanistically, TNF-α and IFN-γ co-treatment activated the JAK/STAT1/IRF1 axis, inducing nitric oxide production and driving caspase-8/FADD-mediated PANoptosis. TNF-α and IFN-γ caused a lethal cytokine shock in mice that mirrors the tissue damage and inflammation of COVID-19, and inhibiting PANoptosis protected mice from this pathology and death. Furthermore, treating with neutralizing antibodies against TNF-α and IFN-γ protected mice from mortality during SARS-CoV-2 infection, sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other infectious and autoinflammatory diseases by limiting tissue damage/inflammation.
Subject(s)
COVID-19/immunology , COVID-19/pathology , Cytokine Release Syndrome/immunology , Cytokine Release Syndrome/pathology , Interferon-gamma/immunology , Tumor Necrosis Factor-alpha/immunology , Animals , Antibodies, Neutralizing/administration & dosage , Cell Death , Disease Models, Animal , Female , Human Umbilical Vein Endothelial Cells , Humans , Inflammation/immunology , Inflammation/pathology , Lymphohistiocytosis, Hemophagocytic/chemically induced , Male , Mice , Mice, Transgenic , THP-1 CellsABSTRACT
Checkpoint blockade with antibodies specific for the PD-1 and CTLA-4 inhibitory receptors can induce durable responses in a wide range of human cancers. However, the immunological mechanisms responsible for severe inflammatory side effects remain poorly understood. Here we report a comprehensive single-cell analysis of immune cell populations in colitis, a common and severe side effect of checkpoint blockade. We observed a striking accumulation of CD8 T cells with highly cytotoxic and proliferative states and no evidence of regulatory T cell depletion. T cell receptor (TCR) sequence analysis demonstrated that a substantial fraction of colitis-associated CD8 T cells originated from tissue-resident populations, explaining the frequently early onset of colitis symptoms following treatment initiation. Our analysis also identified cytokines, chemokines, and surface receptors that could serve as therapeutic targets for colitis and potentially other inflammatory side effects of checkpoint blockade.
Subject(s)
CD8-Positive T-Lymphocytes/cytology , CTLA-4 Antigen/immunology , Colitis/metabolism , Immune Checkpoint Inhibitors/adverse effects , Immunotherapy/adverse effects , Myeloid Cells/metabolism , Receptors, Chemokine/metabolism , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , CTLA-4 Antigen/metabolism , Chemokines/metabolism , Colitis/drug therapy , Colitis/genetics , Colitis/immunology , Cytokines/metabolism , Flow Cytometry , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Humans , Inflammation/drug therapy , Inflammation/genetics , Inflammation/metabolism , Melanoma/genetics , Melanoma/immunology , Melanoma/metabolism , Multigene Family , Myeloid Cells/cytology , RNA-Seq , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Receptors, CXCR3/genetics , Receptors, CXCR3/metabolism , Receptors, CXCR6/genetics , Receptors, CXCR6/metabolism , Receptors, Chemokine/genetics , Single-Cell Analysis , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/metabolismABSTRACT
Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult.
Subject(s)
Coronavirus Infections/immunology , Germinal Center/immunology , Pneumonia, Viral/immunology , T-Lymphocytes, Helper-Inducer/immunology , Aged , Aged, 80 and over , B-Lymphocytes/immunology , COVID-19 , Female , Germinal Center/pathology , Humans , Male , Middle Aged , Pandemics , Proto-Oncogene Proteins c-bcl-6/genetics , Proto-Oncogene Proteins c-bcl-6/metabolism , Spleen/immunology , Spleen/pathology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
New opportunities are needed to increase immune checkpoint blockade (ICB) benefit. Whereas the interferon (IFN)γ pathway harbors both ICB resistance factors and therapeutic opportunities, this has not been systematically investigated for IFNγ-independent signaling routes. A genome-wide CRISPR/Cas9 screen to sensitize IFNγ receptor-deficient tumor cells to CD8 T cell elimination uncovered several hits mapping to the tumor necrosis factor (TNF) pathway. Clinically, we show that TNF antitumor activity is only limited in tumors at baseline and in ICB non-responders, correlating with its low abundance. Taking advantage of the genetic screen, we demonstrate that ablation of the top hit, TRAF2, lowers the TNF cytotoxicity threshold in tumors by redirecting TNF signaling to favor RIPK1-dependent apoptosis. TRAF2 loss greatly enhanced the therapeutic potential of pharmacologic inhibition of its interaction partner cIAP, another screen hit, thereby cooperating with ICB. Our results suggest that selective reduction of the TNF cytotoxicity threshold increases the susceptibility of tumors to immunotherapy.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunotherapy , Tumor Necrosis Factor-alpha/metabolism , Animals , Apoptosis/drug effects , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Humans , Inhibitor of Apoptosis Proteins/metabolism , Interferon-gamma/metabolism , Kaplan-Meier Estimate , Male , Mice , Mice, Inbred C57BL , Neoplasms/mortality , Neoplasms/therapy , RNA, Guide, Kinetoplastida/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptors, Interferon/deficiency , Receptors, Interferon/genetics , Signal Transduction/drug effects , TNF Receptor-Associated Factor 2/deficiency , TNF Receptor-Associated Factor 2/genetics , Tumor Necrosis Factor-alpha/pharmacology , Interferon gamma ReceptorABSTRACT
Necrosis of infected macrophages constitutes a critical pathogenetic event in tuberculosis by releasing mycobacteria into the growth-permissive extracellular environment. In zebrafish infected with Mycobacterium marinum or Mycobacterium tuberculosis, excess tumor necrosis factor triggers programmed necrosis of infected macrophages through the production of mitochondrial reactive oxygen species (ROS) and the participation of cyclophilin D, a component of the mitochondrial permeability transition pore. Here, we show that this necrosis pathway is not mitochondrion-intrinsic but results from an inter-organellar circuit initiating and culminating in the mitochondrion. Mitochondrial ROS induce production of lysosomal ceramide that ultimately activates the cytosolic protein BAX. BAX promotes calcium flow from the endoplasmic reticulum into the mitochondrion through ryanodine receptors, and the resultant mitochondrial calcium overload triggers cyclophilin-D-mediated necrosis. We identify ryanodine receptors and plasma membrane L-type calcium channels as druggable targets to intercept mitochondrial calcium overload and necrosis of mycobacterium-infected zebrafish and human macrophages.
Subject(s)
Macrophages/microbiology , Macrophages/pathology , Mitochondria/metabolism , Mycobacterium Infections, Nontuberculous/metabolism , Tuberculosis/immunology , Tuberculosis/pathology , Tumor Necrosis Factor-alpha/metabolism , Animals , Apoptosis , Calcium/metabolism , Endoplasmic Reticulum/microbiology , Humans , Lysosomes/microbiology , Membrane Potential, Mitochondrial , Mycobacterium Infections, Nontuberculous/pathology , Mycobacterium marinum , Mycobacterium tuberculosis , Necrosis , Reactive Oxygen Species/metabolism , THP-1 Cells , ZebrafishABSTRACT
Pediatric-onset colitis and inflammatory bowel disease (IBD) have significant effects on the growth of infants and children, but the etiopathogenesis underlying disease subtypes remains incompletely understood. Here, we report single-cell clustering, immune phenotyping, and risk gene analysis for children with undifferentiated colitis, Crohn's disease, and ulcerative colitis. We demonstrate disease-specific characteristics, as well as common pathogenesis marked by impaired cyclic AMP (cAMP)-response signaling. Specifically, infiltration of PDE4B- and TNF-expressing macrophages, decreased abundance of CD39-expressing intraepithelial T cells, and platelet aggregation and release of 5-hydroxytryptamine at the colonic mucosae were common in colitis and IBD patients. Targeting these pathways by using the phosphodiesterase inhibitor dipyridamole restored immune homeostasis and improved colitis symptoms in a pilot study. In summary, comprehensive analysis of the colonic mucosae has uncovered common pathogenesis and therapeutic targets for children with colitis and IBD.
Subject(s)
Inflammatory Bowel Diseases/pathology , Inflammatory Bowel Diseases/therapy , Intestinal Mucosa/pathology , Antigens, CD/metabolism , Apyrase/metabolism , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Cell Death/drug effects , Cellular Microenvironment/drug effects , Child , Cohort Studies , Colon/pathology , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Dipyridamole/pharmacology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation/drug effects , Genetic Predisposition to Disease , Homeostasis/drug effects , Humans , Immunoglobulin G/blood , Immunologic Memory , Inflammation/pathology , Inflammatory Bowel Diseases/blood , Inflammatory Bowel Diseases/genetics , Interferon Type I/metabolism , Macrophages/drug effects , Macrophages/metabolism , Methylprednisolone/pharmacology , Myeloid Cells/drug effects , Myeloid Cells/metabolismABSTRACT
Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 cells from the colon mucosa of 18 UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4+ enterocytes, microfold-like cells, and IL13RA2+IL11+ inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and T cells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways.
Subject(s)
Colitis, Ulcerative/pathology , Colon/metabolism , Adult , Aged , Antibodies, Monoclonal/therapeutic use , Bestrophins/metabolism , CD8 Antigens/metabolism , Case-Control Studies , Colitis, Ulcerative/drug therapy , Colitis, Ulcerative/metabolism , Colon/pathology , Enterocytes/cytology , Enterocytes/metabolism , Female , Genetic Loci , Genome-Wide Association Study , Humans , Interleukin-17/metabolism , Male , Middle Aged , Risk Factors , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Thrombospondins/metabolism , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism , Young AdultABSTRACT
Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.
Subject(s)
Immunogenic Cell Death , Proteolysis , Receptor-Interacting Protein Serine-Threonine Kinases , Signal Transduction , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Humans , Animals , Mice , Proteolysis/drug effects , Cell Line, Tumor , Signal Transduction/drug effects , Immunogenic Cell Death/drug effects , Necroptosis/drug effects , Necroptosis/immunology , Neoplasms/immunology , Neoplasms/drug therapy , Mice, Inbred C57BL , Antineoplastic Agents/pharmacology , Immunotherapy/methodsABSTRACT
In the healthy adult liver, most hepatocytes proliferate minimally. However, upon physical or chemical injury to the liver, hepatocytes proliferate extensively in vivo under the direction of multiple extracellular cues, including Wnt and pro-inflammatory signals. Currently, liver organoids can be generated readily in vitro from bile-duct epithelial cells, but not hepatocytes. Here, we show that TNFα, an injury-induced inflammatory cytokine, promotes the expansion of hepatocytes in 3D culture and enables serial passaging and long-term culture for more than 6 months. Single-cell RNA sequencing reveals broad expression of hepatocyte markers. Strikingly, in vitro-expanded hepatocytes engrafted, and significantly repopulated, the injured livers of Fah-/- mice. We anticipate that tissue repair signals can be harnessed to promote the expansion of otherwise hard-to-culture cell-types, with broad implications.
Subject(s)
Antigens, Differentiation/biosynthesis , Cell Culture Techniques , Cell Proliferation/drug effects , Hepatocytes/metabolism , Spheroids, Cellular/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Cell Line, Transformed , Hep G2 Cells , Hepatocytes/transplantation , Human Umbilical Vein Endothelial Cells , Humans , Liver/injuries , Liver/metabolism , Mice, Knockout , Spheroids, Cellular/transplantation , Time FactorsABSTRACT
Interleukin (IL)-22 is central to immune defense at barrier sites. We examined the contributions of innate lymphoid cell (ILC) and T cell-derived IL-22 during Citrobacter rodentium (C.r) infection using mice that both report Il22 expression and allow lineage-specific deletion. ILC-derived IL-22 activated STAT3 in C.r-colonized surface intestinal epithelial cells (IECs) but only temporally restrained bacterial growth. T cell-derived IL-22 induced a more robust and extensive activation of STAT3 in IECs, including IECs lining colonic crypts, and T cell-specific deficiency of IL-22 led to pathogen invasion of the crypts and increased mortality. This reflected a requirement for T cell-derived IL-22 for the expression of a host-protective transcriptomic program that included AMPs, neutrophil-recruiting chemokines, and mucin-related molecules, and it restricted IFNγ-induced proinflammatory genes. Our findings demonstrate spatiotemporal differences in the production and action of IL-22 by ILCs and T cells during infection and reveal an indispensable role for IL-22-producing T cells in the protection of the intestinal crypts.
Subject(s)
Citrobacter rodentium , Enterobacteriaceae Infections , Animals , Anti-Bacterial Agents , Immunity, Innate , Interleukins/metabolism , Intestinal Mucosa , Lymphocytes/metabolism , Mice , Mice, Inbred C57BL , T-Lymphocytes/metabolism , Interleukin-22ABSTRACT
Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.
Subject(s)
COVID-19/immunology , Caspase 8/metabolism , Interferon-gamma/metabolism , Lymphohistiocytosis, Hemophagocytic/immunology , Macrophages/immunology , Mitochondria/metabolism , SARS-CoV-2/physiology , Animals , Caspase 8/genetics , Cells, Cultured , Cytotoxicity, Immunologic , Humans , Interferon-gamma/genetics , Macrophage Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide Synthase Type II/metabolism , Pathogen-Associated Molecular Pattern Molecules/immunology , Signal Transduction , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Understanding the drivers and markers of clonally expanding HIV-1-infected CD4+ T cells is essential for HIV-1 eradication. We used single-cell ECCITE-seq, which captures surface protein expression, cellular transcriptome, HIV-1 RNA, and TCR sequences within the same single cell to track clonal expansion dynamics in longitudinally archived samples from six HIV-1-infected individuals (during viremia and after suppressive antiretroviral therapy) and two uninfected individuals, in unstimulated conditions and after CMV and HIV-1 antigen stimulation. Despite antiretroviral therapy, persistent antigen and TNF responses shaped T cell clonal expansion. HIV-1 resided in Th1-polarized, antigen-responding T cells expressing BCL2 and SERPINB9 that may resist cell death. HIV-1 RNA+ T cell clones were larger in clone size, established during viremia, persistent after viral suppression, and enriched in GZMB+ cytotoxic effector memory Th1 cells. Targeting HIV-1-infected cytotoxic CD4+ T cells and drivers of clonal expansion provides another direction for HIV-1 eradication.
Subject(s)
HIV Infections , HIV-1 , CD4-Positive T-Lymphocytes , Clone Cells , Humans , RNA , ViremiaABSTRACT
Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.
Subject(s)
COVID-19/immunology , Interferon-alpha/immunology , Killer Cells, Natural/immunology , SARS-CoV-2/immunology , Tumor Necrosis Factor-alpha/metabolism , Base Sequence , Humans , Immunity, Innate/immunology , Inflammation/immunology , Interferon-alpha/blood , Pulmonary Fibrosis/pathology , RNA-Seq , Severity of Illness Index , Transcriptome/genetics , United Kingdom , United StatesABSTRACT
The vaccine strain against smallpox, vaccinia virus (VACV), is highly immunogenic yet causes relatively benign disease. These attributes are believed to be caused by gene loss in VACV. Using a targeted small interfering RNA (siRNA) screen, we identified a viral inhibitor found in cowpox virus (CPXV) and other orthopoxviruses that bound to the host SKP1-Cullin1-F-box (SCF) machinery and the essential necroptosis kinase receptor interacting protein kinase 3 (RIPK3). This "viral inducer of RIPK3 degradation" (vIRD) triggered ubiquitination and proteasome-mediated degradation of RIPK3 and inhibited necroptosis. In contrast to orthopoxviruses, the distantly related leporipoxvirus myxoma virus (MYXV), which infects RIPK3-deficient hosts, lacks a functional vIRD. Introduction of vIRD into VACV, which encodes a truncated and defective vIRD, enhanced viral replication in mice. Deletion of vIRD reduced CPXV-induced inflammation, viral replication, and mortality, which were reversed in RIPK3- and MLKL-deficient mice. Hence, vIRD-RIPK3 drives pathogen-host evolution and regulates virus-induced inflammation and pathogenesis.
Subject(s)
Cowpox virus/physiology , Cowpox/immunology , RNA, Small Interfering/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Vaccinia virus/metabolism , Viral Proteins/metabolism , Animals , Evolution, Molecular , HEK293 Cells , Host-Pathogen Interactions , Humans , Inflammation , Mice , Mice, Knockout , Necroptosis/genetics , Orthopoxvirus , Phylogeny , Protein Kinases/genetics , Proteolysis , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Sequence Analysis, RNA , Viral Proteins/genetics , Virus ReplicationABSTRACT
The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.