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In recent years, increasing evidence has highlighted the profound connection between DNA damage repair and the activation of immune responses. We spoke with researchers about their mechanistic interplays and the implications for cancer and other diseases.
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Daño del ADN , Reparación del ADN , Transducción de Señal , InmunidadRESUMEN
Bacterial membrane vesicles have emerged as gadgets allowing remote communication between the microbiota and distal host organs. Here we describe a protocol for enriching vesicles from serum and colon that could widely be adapted for other tissues. We detail pre-clearing of serum or colon fluids using 0.2-µm syringe filters and their concentration by centrifugal filter devices. We also describe vesicle isolation with qEV size exclusion columns and finally the concentration of isolated vesicle fractions for downstream analyses. For complete details on the use and execution of this protocol, please refer to Erttmann et al. (2022).1.
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Microbiota , Animales , Ratones , Membranas , ColonRESUMEN
Introduction: Typhoid toxin-expressing Salmonella enterica causes DNA damage in the intestinal mucosa in vivo, activating the DNA damage response (DDR) in the absence of inflammation. To understand whether the tissue microenvironment constrains the infection outcome, we compared the immune response and DDR patterns in the colon and liver of mice infected with a genotoxigenic strain or its isogenic control strain. Methods: In situ spatial transcriptomic and immunofluorescence have been used to assess DNA damage makers, activation of the DDR, innate immunity markers in a multiparametric analysis. Result: The presence of the typhoid toxin protected from colonic bacteria-induced inflammation, despite nuclear localization of p53, enhanced co-expression of type-I interferons (IfnbI) and the inflammasome sensor Aim2, both classic features of DNA-break-induced DDR activation. These effects were not observed in the livers of either infected group. Instead, in this tissue, the inflammatory response and DDR were associated with high oxidative stress-induced DNA damage. Conclusions: Our work highlights the relevance of the tissue microenvironment in enabling the typhoid toxin to suppress the host inflammatory response in vivo.
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Salmonella enterica , Fiebre Tifoidea , Ratones , Animales , Salmonella enterica/genética , Mutágenos , Daño del ADN , Inflamación , Reparación del ADNRESUMEN
The microbiota are vital for immune homeostasis and provide a competitive barrier to bacterial and fungal pathogens. Here, we investigated how gut commensals modulate systemic immunity and response to viral infection. Antibiotic suppression of the gut microbiota reduced systemic tonic type I interferon (IFN-I) and antiviral priming. The microbiota-driven tonic IFN-I-response was dependent on cGAS-STING but not on TLR signaling or direct host-bacteria interactions. Instead, membrane vesicles (MVs) from extracellular bacteria activated the cGAS-STING-IFN-I axis by delivering bacterial DNA into distal host cells. DNA-containing MVs from the gut microbiota were found in circulation and promoted the clearance of both DNA (herpes simplex virus type 1) and RNA (vesicular stomatitis virus) viruses in a cGAS-dependent manner. In summary, this study establishes an important role for the microbiota in peripheral cGAS-STING activation, which promotes host resistance to systemic viral infections. Moreover, it uncovers an underappreciated risk of antibiotic use during viral infections.
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Microbioma Gastrointestinal , Herpesvirus Humano 1 , Interferón Tipo I , Virosis , Antibacterianos , Antivirales , Humanos , Inmunidad Innata , Proteínas de la Membrana/genética , Nucleotidiltransferasas/genéticaRESUMEN
Radiation is an essential preparative procedure for bone marrow (BM) transplantation and cancer treatment. The therapeutic efficacy of radiation and associated toxicity varies from patient to patient, making it difficult to prescribe an optimal patient-specific irradiation dose. The molecular determinants of radiation response remain unclear. AIM2-like receptors (ALRs) are key players in innate immunity and determine the course of infections, inflammatory diseases, senescence, and cancer. Here it is reported that mice lacking ALRs are resistant to irradiation-induced BM injury. It is shown that nuclear ALRs are inhibitors of DNA repair, thereby accelerate genome destabilization, micronuclei generation, and cell death, and that this novel function is uncoupled from their role in innate immunity. Mechanistically, ALRs bind to and interfere with chromatin decompaction vital for DNA repair. The finding uncovers ALRs as targets for new interventions against genotoxic tissue injury and as possible biomarkers for predicting the outcome of radio/chemotherapy.
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Muerte Celular/genética , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Animales , Muerte Celular/inmunología , Reparación del ADN/inmunología , Proteínas de Unión al ADN/inmunología , Modelos Animales de Enfermedad , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Ratones , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
The gut epithelium serves to maximize the surface for nutrient and fluid uptake, but at the same time must provide a tight barrier to pathogens and remove damaged intestinal epithelial cells (IECs) without jeopardizing barrier integrity. How the epithelium coordinates these tasks remains a question of significant interest. We used imaging and an optical flow analysis pipeline to study the dynamicity of untransformed murine and human intestinal epithelia, cultured atop flexible hydrogel supports. Infection with the pathogen Salmonella Typhimurium (STm) within minutes elicited focal contractions with inward movements of up to â¼1,000 IECs. Genetics approaches and chimeric epithelial monolayers revealed contractions to be triggered by the NAIP/NLRC4 inflammasome, which sensed type-III secretion system and flagellar ligands upon bacterial invasion, converting the local tissue into a contraction epicenter. Execution of the response required swift sublytic Gasdermin D pore formation, ion fluxes, and the propagation of a myosin contraction pulse across the tissue. Importantly, focal contractions preceded, and could be uncoupled from, the death and expulsion of infected IECs. In both two-dimensional monolayers and three-dimensional enteroids, multiple infection-elicited contractions coalesced to produce shrinkage of the epithelium as a whole. Monolayers deficient for Caspase-1(-11) or Gasdermin D failed to elicit focal contractions but were still capable of infected IEC death and expulsion. Strikingly, these monolayers lost their integrity to a markedly higher extent than wild-type counterparts. We propose that prompt NAIP/NLRC4/Caspase-1/Gasdermin D/myosin-dependent contractions allow the epithelium to densify its cell packing in infected regions, thereby preventing tissue disintegration due to the subsequent IEC death and expulsion process.
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Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiología , Proteína Inhibidora de la Apoptosis Neuronal/metabolismo , Animales , Infecciones Bacterianas/fisiopatología , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Unión al Calcio/metabolismo , Caspasa 1/metabolismo , Caspasas/metabolismo , Células Epiteliales/metabolismo , Epitelio/metabolismo , Humanos , Inflamasomas , Mucosa Intestinal/microbiología , Intestinos , Ratones , Contracción Muscular/fisiología , Cultivo Primario de Células , Receptores de Reconocimiento de Patrones/metabolismo , Salmonella typhimurium/patogenicidad , Sistemas de Secreción Tipo III/metabolismoRESUMEN
DNA repair via homologous recombination (HR) is indispensable for genome integrity and cell survival but if unrestrained can result in undesired chromosomal rearrangements. The regulatory mechanisms of HR are not fully understood. Cyclic GMP-AMP synthase (cGAS) is best known as a cytosolic innate immune sensor critical for the outcome of infections, inflammatory diseases, and cancer. Here, we report that cGAS is primarily a chromatin-bound protein that inhibits DNA repair by HR, thereby accelerating genome destabilization, micronucleus generation, and cell death under conditions of genomic stress. This function is independent of the canonical STING-dependent innate immune activation and is physiologically relevant for irradiation-induced depletion of bone marrow cells in mice. Mechanistically, we demonstrate that inhibition of HR repair by cGAS is linked to its ability to self-oligomerize, causing compaction of bound template dsDNA into a higher-ordered state less amenable to strand invasion by RAD51-coated ssDNA filaments. This previously unknown role of cGAS has implications for understanding its involvement in genome instability-associated disorders including cancer.
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Muerte Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Inestabilidad Genómica , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/fisiología , Reparación del ADN por Recombinación , Animales , Núcleo Celular/genética , Cromatina/genética , Daño del ADN , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleotidiltransferasas/genética , Transducción de SeñalRESUMEN
Hydrogen peroxide (H2O2) has a major function in host-microbial interactions. Although most studies have focused on the endogenous H2O2 produced by immune cells to kill microbes, bacteria can also produce H2O2. How microbial H2O2 influences the dynamics of host-microbial interactions is unclear. Here we show that H2O2 released by Streptococcus pneumoniae inhibits inflammasomes, key components of the innate immune system, contributing to the pathogen colonization of the host. We also show that the oral commensal H2O2-producing bacteria Streptococcus oralis can block inflammasome activation. This study uncovers an unexpected role of H2O2 in immune suppression and demonstrates how, through this mechanism, bacteria might restrain the immune system to co-exist with the host.
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Coinfección/inmunología , Peróxido de Hidrógeno/metabolismo , Tolerancia Inmunológica , Inmunidad Innata , Inflamasomas/inmunología , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Coinfección/microbiología , Modelos Animales de Enfermedad , Interacciones Microbiota-Huesped/inmunología , Humanos , Peróxido de Hidrógeno/inmunología , Inflamasomas/metabolismo , Ratones , Ratones Noqueados , Streptococcus oralis/inmunología , Streptococcus oralis/metabolismo , Streptococcus oralis/patogenicidad , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidadRESUMEN
Inflammasomes are multiprotein signaling platforms responsible for the maturation of pro-IL-1ß and pro-IL-18 as well as the induction of an inflammatory cell death termed pyroptosis. Most inflammasomes consist of an upstream sensor, in most cases an adaptor protein (ASC) and inflammatory caspases such as caspase-1. Upon activation, sensor proteins oligomerize with adaptor proteins, forming large complexes called specks. These complexes can be stabilized and detected by Western blotting or fluorescence microscopy providing a direct evidence of inflammasome activation. Here we describe protocols for two complementary methods for detecting inflammasome complexes: (1) biochemical isolation and detection of ASC oligomers by Western blot analysis and (2) microscopic visualization of active caspase-1-ASC complexes. These protocols have successfully been applied in our recent study to unveil new regulatory mechanisms for different inflammasomes including the DNA sensor AIM2 (Erttmann et al., 2016).
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Inflamasomas/metabolismo , Animales , Caspasa 1/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Inmunidad Innata/fisiología , Interleucina-1beta/metabolismoRESUMEN
Detection of DNA damage in cells is fundamental for the study of DNA repair and genome-instability associated processes including carcinogenesis. Many studies often rely on cytotoxicity assays to estimate genotoxicity. However, measurements of cytotoxicity, a delayed outcome requiring high threshold genotoxicity to induce, does not provide information about the subtle, early genotoxic effects relevant for mechanistic understanding of DNA repair processes. Here describe how to combine two simple procedures for monitoring the presence of DNA damage in individual eukaryotic cells using: (1) the Comet assay for measuring initial DNA breaks and (2) the Micronucleus assay for detecting delayed outcome DNA breaks in dividing cells. We discuss the principles, experimental design considerations and troubleshooting tips for optimizing these methods. They require standard molecular biology instruments and a fluorescent microscope.
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Ensayo Cometa/métodos , Pruebas de Micronúcleos/métodos , Animales , Daño del ADN/genética , Genoma de Planta/genética , Inestabilidad Genómica/genética , HumanosRESUMEN
Ubiquitination is a reversible posttranslational modification that regulates nearly all cellular processes. The ubiquitin polypeptide is conjugated via its C-terminus to amine groups of lysine residues on target protein. Additionally, ubiquitins moieties can be conjugated in tandem to the initial ubiquitin via any of its internal lysine residues or N terminal methionine residue, resulting in the formation of polyubiquitin chains with distinct biophysical properties and biological functions. Elucidating the types of polyubiquitin chains present in proteins is essential for understanding their function and mechanism of regulation. Traditionally, ubiqutin modifications have been elucidated by exogenously co-expressing proteins of interest with epitope-tagged ubiquitins mutated in specific lysine residues. However, this strategy is prone experimental artifacts. In this protocol, we describe how to elucidate endogenous ubiquitin modifications. This procedure combines TUBE (Tandem Ubiquitin Binding Entity)-based isolation of ubiquitin conjugates, digestion with linkage specific deubiquitinases and immunoblotting. This procedure is very robust can be applied to profile types and architectural organization polyubiquitin chains present on the any proteins of interest and has been instrumental in elucidating ubiquitin modifications in NOD2 signaling in our recent study (Panda & Gekara, 2018).
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Poliubiquitina/metabolismo , Proteínas/química , Proteínas/metabolismo , Animales , Bioensayo/métodos , Enzimas Desubicuitinizantes/metabolismo , Humanos , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
Cyclic cGMP-AMP synthase (cGAS) alerts the innate immune system to the presence of foreign or damaged self-DNA inside the cell and is critical for the outcome of infections, inflammatory diseases, and cancer. Two studies now demonstrate that cGAS activation is regulated by differential subcellular localization through its non-enzymatic, N-terminal domain.
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ADN/inmunología , Inmunidad Innata/inmunología , Nucleótidos Cíclicos/inmunología , Nucleotidiltransferasas/inmunología , Transducción de Señal/inmunología , Animales , Sitios de Unión/genética , Membrana Celular/enzimología , Núcleo Celular/enzimología , Citosol/enzimología , ADN/genética , ADN/metabolismo , Humanos , Nucleótidos Cíclicos/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Unión ProteicaRESUMEN
NOD2 is essential for antimicrobial innate immunity and tissue homeostasis, but require tight regulation to avert pathology. A focal point of NOD2 signaling is RIP2, which upon polyubiquitination nucleates the NOD2:RIP2 complex, enabling signaling events leading to inflammation, yet the precise nature and the regulation of the polyubiquitins coordinating this process remain unclear. Here we show that NOD2 signaling involves conjugation of RIP2 with lysine 63 (K63), K48 and M1 polyubiquitin chains, as well as with non-canonical K27 chains. In addition, we identify MYSM1 as a proximal deubiquitinase that attenuates NOD2:RIP2 complex assembly by selectively removing the K63, K27 and M1 chains, but sparing the K48 chains. Consequently, MYSM1 deficient mice have unrestrained NOD2-mediated peritonitis, systemic inflammation and liver injury. This study provides a complete overview of the polyubiquitins in NOD2:RIP2 signaling and reveal MYSM1 as a central negative regulator restricting these polyubiquitins to prevent excessive inflammation.
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Endopeptidasas/metabolismo , Inflamación/patología , Hígado/metabolismo , Hígado/patología , Proteína Adaptadora de Señalización NOD2/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Citocinas/metabolismo , Endopeptidasas/química , Lisina/metabolismo , Ratones , Poliubiquitina/metabolismo , Unión Proteica , Dominios Proteicos , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Transducción de Señal , Transactivadores , Proteasas Ubiquitina-Específicas , UbiquitinaciónRESUMEN
DNA damage-induced activation of the cytoplasmic DNA sensor cGAS influences the outcome of infections, autoinflammation, and cancer. Recent studies by Harding et al. (2017. Nature. http://dx.doi.org/10.1038/nature23470), Mackenzie et al. (2017. Nature. http://dx.doi.org/10.1038/nature23449), and Bartsch et al. (2017. Human Molecular Genetics. https://doi.org/10.1093/hmg/ddx283) demonstrate a role for micronuclei formation in DNA damage-induced immune activation.
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Daño del ADN , HumanosRESUMEN
The success of pathogens depends on their ability to circumvent immune defences. Francisella tularensis is one of the most infectious bacteria known. The remarkable virulence of Francisella is believed to be due to its capacity to evade or subvert the immune system, but how remains obscure. Here, we show that Francisella triggers but concomitantly inhibits the Toll-like receptor, RIG-I-like receptor, and cytoplasmic DNA pathways. Francisella subverts these pathways at least in part by inhibiting K63-linked polyubiquitination and assembly of TRAF6 and TRAF3 complexes that control the transcriptional responses of pattern recognition receptors. We show that this mode of inhibition requires a functional type VI secretion system and/or the presence of live bacteria in the cytoplasm. The ability of Francisella to enter the cytosol while simultaneously inhibiting multiple pattern recognition receptor pathways may account for the notable capacity of this bacterium to invade and proliferate in the host without evoking a self-limiting innate immune response.
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Francisella tularensis/inmunología , Evasión Inmune/inmunología , Inmunidad Innata/inmunología , Factor 3 Asociado a Receptor de TNF/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Francisella tularensis/patogenicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Receptores de Reconocimiento de Patrones/antagonistas & inhibidores , Tularemia/inmunología , Tularemia/microbiología , Tularemia/patología , Sistemas de Secreción Tipo VI/metabolismo , Ubiquitinación/inmunologíaRESUMEN
The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and non-infectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1ß processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1ß processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand-cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.
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Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/fisiología , Inflamasomas/inmunología , Macrófagos/inmunología , Proteínas de la Membrana/inmunología , Animales , Chlamydia trachomatis/genética , Chlamydia trachomatis/inmunología , AMP Cíclico/inmunología , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Femenino , Humanos , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Macrófagos/microbiología , Masculino , Proteínas de la Membrana/genética , Ratones , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunologíaRESUMEN
BACKGROUND: Neurotropic flaviviruses such as tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) are causative agents of severe brain-related diseases including meningitis, encephalitis, and microcephaly. We have previously shown that local type I interferon response within the central nervous system (CNS) is involved in the protection of mice against tick-borne flavivirus infection. However, the cells responsible for mounting this protective response are not defined. METHODS: Primary astrocytes were isolated from wild-type (WT) and interferon alpha receptor knock out (IFNAR-/-) mice and infected with neurotropic flaviviruses. Viral replication and spread, IFN induction and response, and cellular viability were analyzed. Transcriptional levels in primary astrocytes treated with interferon or supernatant from virus-infected cells were analyzed by RNA sequencing and evaluated by different bioinformatics tools. RESULTS: Here, we show that astrocytes control viral replication of different TBEV strains, JEV, WNV, and ZIKV. In contrast to fibroblast, astrocytes mount a rapid interferon response and restrict viral spread. Furthermore, basal expression levels of key interferon-stimulated genes are high in astrocytes compared to mouse embryonic fibroblasts. Bioinformatic analysis of RNA-sequencing data reveals that astrocytes have established a basal antiviral state which contributes to the rapid viral recognition and upregulation of interferons. The most highly upregulated pathways in neighboring cells were linked to type I interferon response and innate immunity. The restriction in viral growth was dependent on interferon signaling, since loss of the interferon receptor, or its blockade in wild-type cells, resulted in high viral replication and virus-induced cytopathic effects. Astrocyte supernatant from TBEV-infected cells can restrict TBEV growth in astrocytes already 6 h post infection, the effect on neurons is highly reinforced, and astrocyte supernatant from 3 h post infection is already protective. CONCLUSIONS: These findings suggest that the combination of an intrinsic constitutive antiviral response and the fast induction of type I IFN production by astrocytes play an important role in self-protection of astrocytes and suppression of flavivirus replication in the CNS.
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Astrocitos , Flavivirus/fisiología , Regulación Viral de la Expresión Génica/fisiología , Receptor de Interferón alfa y beta/metabolismo , Animales , Animales Recién Nacidos , Antivirales/farmacología , Astrocitos/metabolismo , Astrocitos/patología , Astrocitos/virología , Biología Computacional , Inmunoglobulina G/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/virología , Oxazinas/farmacología , ARN Mensajero/metabolismo , Receptor de Interferón alfa y beta/genética , Xantenos/farmacologíaRESUMEN
The ATM kinase is a central component of the DNA damage repair machinery and redox balance. ATM dysfunction results in the multisystem disease ataxia-telangiectasia (AT). A major cause of mortality in AT is respiratory bacterial infections. Whether ATM deficiency causes innate immune defects that might contribute to bacterial infections is not known. Here we have shown that loss of ATM impairs inflammasome-dependent anti-bacterial innate immunity. Cells from AT patients or Atm(-/-) mice exhibited diminished interleukin-1ß (IL-1ß) production in response to bacteria. In vivo, Atm(-/-) mice were more susceptible to pulmonary S. pneumoniae infection in a manner consistent with inflammasome defects. Our data indicate that such defects were due to oxidative inhibition of inflammasome complex assembly. This study reveals an unanticipated function of reactive oxygen species (ROS) in negative regulation of inflammasomes and proposes a theory for the notable susceptibility of AT patients to pulmonary bacterial infection.