RESUMEN
Most individuals who consume foods contaminated with the bacterial pathogen Listeria monocytogenes (Lm) develop mild symptoms, while others are susceptible to life-threatening systemic infections (listeriosis). Although it is known that the risk of severe disease is increased in certain human populations, including the elderly, it remains unclear why others who consume contaminated food develop listeriosis. Here, we used a murine model to discover that pulmonary coinfections can impair the host's ability to adequately control and eradicate systemic Lm that cross from the intestines to the bloodstream. We found that the resistance of mice to oral Lm infection was dramatically reduced by coinfection with Streptococcus pneumoniae (Spn), a bacterium that colonizes the respiratory tract and can also cause severe infections in the elderly. Exposure to Spn or microbial products, including a recombinant Lm protein (L1S) and lipopolysaccharide (LPS), rendered otherwise resistant hosts susceptible to severe systemic Lm infection. In addition, we show that this increase in susceptibility was dependent on an increase in the production of interleukin-10 (IL-10) from Ncr1+ cells, including natural killer (NK) cells. Lastly, the ability of Ncr1+ cell derived IL-10 to increase disease susceptibility correlated with a dampening of both myeloid cell accumulation and myeloid cell phagocytic capacity in infected tissues. These data suggest that efforts to minimize inflammation in response to an insult at the respiratory mucosa render the host more susceptible to infections by Lm and possibly other pathogens that access the oral mucosa.
Asunto(s)
Listeria monocytogenes/inmunología , Listeriosis/inmunología , Neumonía/inmunología , Animales , Progresión de la Enfermedad , Susceptibilidad a Enfermedades , Femenino , Interleucina-10/metabolismo , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/fisiología , Lipopolisacáridos , Listeria monocytogenes/patogenicidad , Listeriosis/complicaciones , Listeriosis/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedades de la Boca/complicaciones , Enfermedades de la Boca/inmunología , Enfermedades de la Boca/microbiología , Enfermedades de la Boca/patología , Neumonía/complicaciones , Neumonía/etiología , Neumonía/patologíaRESUMEN
Toll-like receptor (TLR) stimulation activates macrophages to resist intracellular pathogens. Yet, the intracellular bacterium Listeria monocytogenes (Lm) causes lethal infections in spite of innate immune cell activation. Lm uses direct cell-cell spread to disseminate within its host. Here, we have shown that TLR-activated macrophages killed cell-free Lm but failed to prevent infection by spreading Lm. Instead, TLR signals increased the efficiency of Lm spread from "donor" to "recipient" macrophages. This enhancement required nitric oxide (NO) production by nitric oxide synthase-2 (NOS2). NO increased Lm escape from secondary vacuoles in recipient cells and delayed maturation of phagosomes containing membrane-like particles that mimic Lm-containing pseudopods. NO also promoted Lm spread during systemic in vivo infection, as shown by the fact that inhibition of NOS2 with 1400W reduced spread-dependent Lm burdens in mouse livers. These findings reveal a mechanism by which pathogens capable of cell-cell spread can avoid the consequences of innate immune cell activation by TLR stimuli.
Asunto(s)
Listeria monocytogenes/inmunología , Listeriosis/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Receptores Toll-Like/metabolismo , Animales , Células Cultivadas , Inmunidad Innata/inmunología , Listeria monocytogenes/metabolismo , Listeriosis/metabolismo , Activación de Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/inmunología , Óxido Nítrico Sintasa de Tipo II/inmunología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fagosomas/inmunología , Fagosomas/metabolismo , Receptores Toll-Like/inmunologíaRESUMEN
Interferons (IFNs) target macrophages to regulate inflammation and resistance to microbial infections. The type II IFN (IFNγ) acts on a cell surface receptor (IFNGR) to promote gene expression that enhance macrophage inflammatory and anti-microbial activity. Type I IFNs can dampen macrophage responsiveness to IFNγ and are associated with increased susceptibility to numerous bacterial infections. The precise mechanisms responsible for these effects remain unclear. Type I IFNs silence macrophage ifngr1 transcription and thus reduce cell surface expression of IFNGR1. To test how these events might impact macrophage activation and host resistance during bacterial infection, we developed transgenic mice that express a functional FLAG-tagged IFNGR1 (fGR1) driven by a macrophage-specific promoter. Macrophages from fGR1 mice expressed physiologic levels of cell surface IFNGR1 at steady state and responded equivalently to WT C57Bl/6 macrophages when treated with IFNγ alone. However, fGR1 macrophages retained cell surface IFNGR1 and showed enhanced responsiveness to IFNγ in the presence of type I IFNs. When fGR1 mice were infected with the bacterium Listeria monocytogenes their resistance was significantly increased, despite normal type I and II IFN production. Enhanced resistance was dependent on IFNγ and associated with increased macrophage activation and antimicrobial function. These results argue that down regulation of myeloid cell IFNGR1 is an important mechanism by which type I IFNs suppress inflammatory and anti-bacterial functions of macrophages.
Asunto(s)
Listeria monocytogenes/fisiología , Listeriosis/inmunología , Macrófagos/inmunología , Receptores de Interferón/genética , Animales , Regulación hacia Abajo , Femenino , Humanos , Interferón Tipo I/inmunología , Listeriosis/genética , Listeriosis/microbiología , Activación de Macrófagos , Masculino , Ratones , Ratones Noqueados , Receptores de Interferón/inmunología , Receptor de Interferón gammaRESUMEN
Natural killer (NK) cells produce interferon (IFN)-γ and thus have been suggested to promote type I immunity during bacterial infections. Yet, Listeria monocytogenes (Lm) and some other pathogens encode proteins that cause increased NK cell activation. Here, we show that stimulation of NK cell activation increases susceptibility during Lm infection despite and independent from robust NK cell production of IFNγ. The increased susceptibility correlated with IL-10 production by responding NK cells. NK cells produced IL-10 as their IFNγ production waned and the Lm virulence protein p60 promoted induction of IL-10 production by mouse and human NK cells. NK cells consequently exerted regulatory effects to suppress accumulation and activation of inflammatory myeloid cells. Our results reveal new dimensions of the role played by NK cells during Lm infection and demonstrate the ability of this bacterial pathogen to exploit the induction of regulatory NK cell activity to increase host susceptibility.
Asunto(s)
Interleucina-10/inmunología , Células Asesinas Naturales/inmunología , Listeriosis/inmunología , Traslado Adoptivo , Animales , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Interleucina-10/biosíntesis , Listeria monocytogenes/inmunología , Listeriosis/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Changes made in the 8th edition of the Guide for the Care and Use of Laboratory Animals included new recommendations for the amount of space for breeding female mice. Adopting the new recommendations required, in essence, the elimination of trio breeding practices for all institutions. Both public opinion and published data did not readily support the new recommendations. In response, the National Jewish Health Institutional Animal Care and Use Committee established a program to directly compare the effects of breeding format on mouse pup survival and growth. Our study showed an overall parity between trio and pairwise breeding formats on the survival and growth of the litters, suggesting that the housing recommendations for breeding female mice as stated in the current Guide for the Care and Use of Laboratory Animals should be reconsidered.
Asunto(s)
Cruzamiento/métodos , Vivienda para Animales/ética , Animales , Autoinmunidad , Peso Corporal , Cruzamiento/legislación & jurisprudencia , Femenino , Guías como Asunto , Vivienda para Animales/legislación & jurisprudencia , Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Ratones , Ratones Transgénicos , EmbarazoRESUMEN
Listeria monocytogenes (Lm) is both a life-threatening pathogen of humans and a model organism that is widely used to dissect the mechanisms of innate and adaptive immune resistance to infection. Specific aspects of the immune response to systemic Lm infection can be protective, neutral, or in some cases deleterious. In this review, we seek to provide an overview of the early events during Lm infection that dictate or regulate host innate and adaptive immune responses. We highlight several recent developments that add to our understanding of the complex interplay between inflammatory responses, host susceptibility to infection, and the development of protective immunity.
Asunto(s)
Listeria monocytogenes/inmunología , Listeriosis/inmunología , Inmunidad Adaptativa , Animales , Células Dendríticas/inmunología , Humanos , Inmunidad Innata , Células Asesinas Naturales/inmunologíaRESUMEN
The ability of type I IFNs to increase susceptibility to certain bacterial infections correlates with downregulation of myeloid cell surface IFNGR, the receptor for the type II IFN (IFN-γ), and reduced myeloid cell responsiveness to IFN-γ. In this study, we show that the rapid reductions in mouse and human myeloid cell surface IFNGR1 expression that occur in response to type I IFN treatment reflect a rapid silencing of new ifngr1 transcription by repressive transcriptional regulators. Treatment of macrophages with IFN-ß reduced cellular abundance of ifngr1 transcripts as rapidly and effectively as actinomycin D treatment. IFN-ß treatment also significantly reduced the amounts of activated RNA polymerase II (pol II) and acetylated histones H3 and H4 at the ifngr1 promoter and the activity of an IFNGR1-luc reporter construct in macrophages. The suppression of IFNGR1-luc activity required an intact early growth response factor (Egr) binding site in the proximal ifngr1 promoter. Three Egr proteins and two Egr/NGFI-A binding (Nab) proteins were found to be expressed in bone macrophages, but only Egr3 and Nab1 were recruited to the ifngr1 promoter upon IFN-ß stimulation. Knockdown of Nab1 in a macrophage cell line prevented downregulation of IFNGR1 and prevented the loss of acetylated histones from the ifngr1 promoter. These data suggest that type I IFN stimulation induces a rapid recruitment of a repressive Egr3/Nab1 complex that silences transcription from the ifngr1 promoter. This mechanism of gene silencing may contribute to the anti-inflammatory effects of type I IFNs.
Asunto(s)
Proteína 3 de la Respuesta de Crecimiento Precoz/metabolismo , Silenciador del Gen/fisiología , Interferón Tipo I/metabolismo , Receptores de Interferón/metabolismo , Proteínas Represoras/metabolismo , Animales , Western Blotting , Inmunoprecipitación de Cromatina , Regulación hacia Abajo , Proteína 3 de la Respuesta de Crecimiento Precoz/inmunología , Citometría de Flujo , Humanos , Interferón Tipo I/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Mieloides/inmunología , Células Mieloides/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Interferón/inmunología , Proteínas Represoras/inmunología , Transcripción Genética , Receptor de Interferón gammaRESUMEN
An acquired immune deficiency due to interferon gamma (IFN-γ) autoantibodies was diagnosed in a 78-year-old Japanese man with treatment-refractory disseminated nontuberculous mycobacterial infection. In addition to standard antimycobacterial therapy, he was successfully treated with rituximab to eliminate B cells and thereby the autoantibody. Subsequently, he obtained a sustained remission from infection.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Autoanticuerpos/inmunología , Factores Inmunológicos/uso terapéutico , Interferón gamma/inmunología , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/inmunología , Anciano , Autoanticuerpos/sangre , Humanos , Masculino , RituximabRESUMEN
The generation of reactive oxygen species (ROS) is inherent to immune responses. ROS are crucially involved in host defense against pathogens by promoting bacterial killing, but also as signaling agents coordinating the production of cytokines. Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable channel gated via binding of ADP-ribose, a metabolite formed under conditions of cellular exposure to ROS. Here, we show that TRPM2-deficient mice are extremely susceptible to infection with Listeria monocytogenes (Lm), exhibiting an inefficient innate immune response. In a comparison with IFNγR-deficient mice, TRPM2(-/-) mice shared similar features of uncontrolled bacterial replication and reduced levels of inducible (i)NOS-expressing monocytes, but had intact IFNγ responsiveness. In contrast, we found that levels of cytokines IL-12 and IFNγ were diminished in TRPM2(-/-) mice following Lm infection, which correlated with their reduced innate activation. Moreover, TRPM2(-/-) mice displayed a higher degree of susceptibility than IL-12-unresponsive mice, and supplementation with recombinant IFNγ was sufficient to reverse the unrestrained bacterial growth and ultimately the lethal phenotype of Lm-infected TRPM2(-/-) mice. The severity of listeriosis we observed in TRPM2(-/-) mice has not been reported for any other ion channel. These findings establish an unsuspected role for ADP-ribose and ROS-mediated cation flux for innate immunity, opening up unique possibilities for immunomodulatory intervention through TRPM2.
Asunto(s)
Inmunidad Innata/fisiología , Listeria monocytogenes/inmunología , Canales Catiónicos TRPM/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Citocinas/biosíntesis , Femenino , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/genética , Interferón gamma/farmacología , Interleucina-12/deficiencia , Interleucina-12/genética , Interleucina-12/inmunología , Subunidad beta 2 del Receptor de Interleucina-12/deficiencia , Subunidad beta 2 del Receptor de Interleucina-12/genética , Subunidad beta 2 del Receptor de Interleucina-12/inmunología , Listeria monocytogenes/patogenicidad , Listeriosis/inmunología , Listeriosis/prevención & control , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Receptores de Interferón/inmunología , Proteínas Recombinantes , Canales Catiónicos TRPM/deficiencia , Canales Catiónicos TRPM/genética , Receptor de Interferón gammaRESUMEN
Listeria monocytogenes (Lm) infection induces rapid and robust activation of host natural killer (NK) cells. Here we define a region of the abundantly secreted Lm endopeptidase, p60, that potently but indirectly stimulates NK cell activation in vitro and in vivo. Lm expression of p60 resulted in increased IFNγ production by naïve NK cells co-cultured with treated dendritic cells (DCs). Moreover, recombinant p60 protein stimulated activation of naive NK cells when co-cultured with TLR or cytokine primed DCs in the absence of Lm. Intact p60 protein weakly digested bacterial peptidoglycan (PGN), but neither muropeptide recognition by RIP2 nor the catalytic activity of p60 was required for NK cell activation. Rather, the immune stimulating activity mapped to an N-terminal region of p60, termed L1S. Treatment of DCs with a recombinant L1S polypeptide stimulated them to activate naïve NK cells in a cell culture model. Further, L1S treatment activated NK cells in vivo and increased host resistance to infection with Francisella tularensis live vaccine strain (LVS). These studies demonstrate an immune stimulating function for a bacterial LysM domain-containing polypeptide and suggest that recombinant versions of L1S or other p60 derivatives can be used to promote NK cell activation in therapeutic contexts.
Asunto(s)
Proteínas Bacterianas/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Listeria monocytogenes/inmunología , Animales , Proteínas Bacterianas/biosíntesis , Células Cultivadas , Células Dendríticas/inmunología , Francisella tularensis/inmunología , Interferón gamma/biosíntesis , Interleucina-18/biosíntesis , Interleucina-18/genética , Listeriosis/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas RecombinantesRESUMEN
Cyclic-di-GMP and cyclic-di-AMP are second messengers produced by bacteria and influence bacterial cell survival, differentiation, colonization, biofilm formation, virulence, and bacteria-host interactions. In this study, we show that in both RAW264.7 macrophage cells and primary bone marrow-derived macrophages, the production of IFN-ß and IL-6, but not TNF, in response to cyclic-di-AMP and cyclic-di-GMP requires MPYS (also known as STING, MITA, and TMEM173). Furthermore, expression of MPYS was required for IFN response factor 3 but not NF-κB activation in response to these bacterial metabolites. We also confirm that MPYS is required for type I IFN production by cultured macrophages infected with the intracellular pathogens Listeria monocytogenes and Francisella tularensis. However, during systemic infection with either pathogen, MPYS deficiency did not impact bacterial burdens in infected spleens. Serum IFN-ß and IL-6 concentrations in the infected control and MPYS(-/-) mice were also similar at 24 h postinfection, suggesting that these pathogens stimulate MPYS-independent cytokine production during in vivo infection. Our findings indicate that bifurcating MPYS-dependent and -independent pathways mediate sensing of cytosolic bacterial infections.
Asunto(s)
AMP Cíclico/inmunología , GMP Cíclico/análogos & derivados , Factor 3 Regulador del Interferón/inmunología , Interferón Tipo I/inmunología , Macrófagos/inmunología , Proteínas de la Membrana/inmunología , Animales , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Línea Celular , AMP Cíclico/metabolismo , GMP Cíclico/inmunología , GMP Cíclico/metabolismo , Citocinas/biosíntesis , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/biosíntesis , Macrófagos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
For many intracellular pathogens, their virulence depends on an ability to spread between cells of an epithelial layer. For intercellular spread to occur, these pathogens deform the plasma membrane into a protrusion structure that is engulfed by the neighboring cell. Although the polymerization of actin is essential for spread, how these pathogens manipulate the actin cytoskeleton in a manner that enables protrusion formation is still incompletely understood. Here, we identify the mammalian actin binding protein synaptopodin as required for efficient intercellular spread. Using a model cytosolic pathogen, Shigella flexneri , we show that synaptopodin contributes to organization of actin around bacteria and increases the length of the actin tail at the posterior pole of the bacteria. We show that synaptopodin presence enables protrusions to form and to resolve at a greater rate, indicating that greater stability of the actin tail enables the bacteria to push against the membrane with greater force. We demonstrate that synaptopodin recruitment around bacteria requires the bacterial protein IcsA, and we show that this recruitment is further enhanced in a type 3 secretion system dependent manner. These data establish synaptopodin as required for intracellular bacteria to reprogram the actin cytoskeleton in a manner that enables efficient protrusion formation and enhance our understanding of the cellular function of synaptopodin. Authors Summary: Intercellular spread is essential for many cytosolic dwelling pathogens during their infectious life cycle. Despite knowing the steps required for intercellular spread, relatively little is known about the host-pathogen interactions that enable these steps to occur. Here, we identify a requirement for the actin binding protein synaptopodin during intercellular spread by cytosolic bacteria. We show synaptopodin is necessary for the stability and recruitment of polymerized actin around bacteria. We also demonstrate synaptopodin is necessary to form plasma membrane structures known as protrusions that are necessary for the movement of these bacteria between cells. Thus, these findings implicate synaptopodin as an important actin-binding protein for the virulence of intracellular pathogens that require the actin cytoskeleton for their spread between cells.
RESUMEN
The mechanisms for NK cell activation during infection by intracellular bacterial pathogens are not clearly defined. To dissect how Listeria monocytogenes infection elicits NK cell activation, we evaluated the requirements for activation of naive splenic NK cells by infected bone marrow-derived dendritic cells (BMDCs). We found that NK cell activation in this setting required infection of BMDCs by live wild type bacteria. NK cells were not activated when BMDCs were infected with a live hemolysin deficient (Deltahly) strain. Neutralization of IL-12, TNF-alpha, or caspase-1 each dramatically reduced NK cell IFN-gamma production in response to live wt L. monocytogenes infection. Addition of recombinant IL-18, but not IL-1beta, reversed the effects of caspase-1 inhibition. Recombinant IL-18 also restored NK cell activation by BMDCs infected with Deltahly L. monocytogenes, which produced IL-12 but not IL-18. IL-18 acted on NK cells because MyD88 expression was required in responding NK cells, but not infected BMDC. However, secreted cytokines were not sufficient for activation of naive NK cells by infected BMDCs. Rather, NK cell activation additionally required contact between infected BMDCs and NK cells. These data suggest that the activation of NK cells during L. monocytogenes infection requires both secreted cytokines and ligation of NK activating receptors during direct contact with infected DCs.
Asunto(s)
Comunicación Celular/inmunología , Células Dendríticas/inmunología , Interleucina-18/fisiología , Células Asesinas Naturales/inmunología , Listeria monocytogenes/inmunología , Activación de Linfocitos/inmunología , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/microbiología , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/microbiología , Femenino , Interferón gamma/biosíntesis , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/microbiología , Listeriosis/inmunología , Listeriosis/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/biosíntesis , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/fisiologíaRESUMEN
Bacterial and viral pathogens are predominant causes of pulmonary infections and complications. Morbidity and mortality from these infections is increased in populations that include the elderly, infants, and individuals with genetic disorders such as Down syndrome. Immune senescence, concurrent infections, and other immune alterations occur in these susceptible populations, but the underlying mechanisms that dictate increased susceptibility to lung infections are not fully defined. Here, we review unique features of the lung as a mucosal epithelial tissue and aspects of inflammatory and immune responses in model pulmonary infections and co-infections by influenza virus and Streptococcus pneumoniae. In these models, lung inflammatory responses are a double-edged sword: recruitment of immune effectors is essential to eliminate bacteria and virus-infected cells, but inflammatory cytokines drive changes in the lung conducive to increased pathogen replication. Excessive accumulation of inflammatory cells also hinders lung function, possibly causing death of the host. Some animal studies have found that targeting host modulators of lung inflammatory responses has therapeutic or prophylactic effects in these infection and co-infection models. However, conflicting results from other studies suggest microbiota, sequence of colonization, or other unappreciated aspects of lung biology also play important roles in the outcome of infections. Regardless, a predisposition to excessive or aberrant inflammatory responses occurs in susceptible human populations. Hence, in appropriate contexts, modulation of inflammatory responses may prove effective for reducing the frequency or severity of pulmonary infections. However, there remain limitations in our understanding of how this might best be achieved-particularly in diverse human populations.
Asunto(s)
Coinfección/inmunología , Interacciones Huésped-Patógeno/inmunología , Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones Neumocócicas/inmunología , Neumonía Neumocócica/inmunología , Neumonía Viral/inmunología , Streptococcus pneumoniae/inmunología , Anciano , Animales , Coinfección/virología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/inmunología , Humanos , Inmunidad Innata , Lactante , Inflamación/inmunología , Gripe Humana/virología , Infecciones por Orthomyxoviridae/virología , Infecciones Neumocócicas/microbiología , Neumonía Neumocócica/microbiología , Neumonía Viral/virologíaRESUMEN
Lung inflammation is tightly controlled to balance microbial clearance with the tissue damage that accompanies this response. Bacterial pathogens including Streptococcus pneumoniae (S. pneumoniae) modulate immune regulation by promoting secretion of the anti-inflammatory cytokine IL-10. The important cellular sources of IL-10 that impact protection against different bacterial infections are not well characterized. We find that S. pneumoniaeactivates IL-10 secretion from natural killer (NK) cells in the lung, which restrict host protection in a mouse model of sublethal infection. Direct transfer of wild-type NK cells into the lungs of IL-10-deficient mice drives bacterial expansion, identifying NK cells as a critical source of IL-10 promoting S. pneumoniae infection. The S. pneumoniae virulence protein Spr1875 was found to elicit NK cell IL-10 production in purified cells and in the lungs of live animals. These findings reveal therapeutic targets to combat bacterial-driven immune regulation in the lung.
Asunto(s)
Interleucina-10/biosíntesis , Células Asesinas Naturales/metabolismo , Enfermedades Pulmonares/inmunología , Infecciones Estreptocócicas/inmunología , Streptococcus pneumoniae/patogenicidad , Animales , Vacunas Bacterianas/inmunología , Femenino , Inmunidad Innata , Células Asesinas Naturales/inmunología , Enfermedades Pulmonares/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Infecciones Estreptocócicas/microbiología , Streptococcus pneumoniae/inmunologíaRESUMEN
The type II interferon (IFNγ) promotes resistance to intracellular pathogens. Most immune and somatic cells also express the IFNγ receptor (IFNGR) and respond to IFNγ. While myeloid cell have been implicated as important targets of IFNγ, it remains unknown if IFNγ signaling to myeloid cell types suffices for resistance to infection. Here, we addressed this question by generating mice in which IFNGR1 is selectively expressed by myeloid cells. These "MSGR1" (myeloid selective IFNGR1) mice express an epitope-tagged Ifngr1 transgene (fGR1) from the myeloid-specific c-fms promoter in a background lacking endogenous Ifngr1. IFNGR staining was selectively observed on myeloid cells in the MSGR1 mice and correlated with responsiveness of these cells to IFNγ. During systemic infection by the bacterium Listeria monocytogenes, activation marker staining was comparable on monocytes from MSGR1 and control B6 mice. Bacterial burdens and survival were also equivalent in MSGR1 and wildtype B6 animals at a timepoint when B6.Ifngr1 -/- mice began to succumb. These data confirm that activation of inflammatory monocytes and neutrophils is a key mechanism by which IFNγ promotes innate anti-bacterial immunity and suggest that IFNγ targeting of myeloid cells is largely sufficient to mediate protection against systemic L. monocytogenes.
RESUMEN
Stimulator of interferon genes (STING) plays a central role in innate immune responses to viral and intracellular bacterial infections, and cellular damage. STING is a cytosolic sensor of cyclic dinucleotides (CDNs) including those produced by pathogenic bacteria and those arising endogenously as products of the DNA sensor cGAS (e.g., 2'3' cGAMP). The two most common alternative allelic variants of STING in humans are STING-R71H-G230A-R293Q (STING-HAQ) and STING-R232H that are found in 20.4% and 13.7-17.6% of the population, respectively. To determine the biologic consequences of these genotypic variations, we generated knock-in mice containing the murine equivalents of each variant and studied their responsiveness to CDNs. Homozygous STING-HAQ (R71H-I229A-R292Q) and STING-R231H mice were found to be unresponsive to all exogenous CDNs tested (ci-di-GMP, ci-di-AMP, 3'3' cGAMP and Rp,Rp-CDA). Responses of homozygous STING-HAQ mice to endogenous 2'3' cGAMP was also greatly impaired. However, homozygous STING-R231H mice are fully responsive to 2'3' cGAMP. Analysis of heterozygous mice revealed reduced responsiveness to exogenous and endogenous CDNs in mice carrying a single copy of STING-HAQ, while STING-R231H heterozygous mice exhibit reduced responsiveness to exogenous but not endogenous CDNs. These findings confirm and extend previous reports by demonstrating differing impact of allelic variation of STING on the ability to sense and respond to exogenous vs. endogenous CDNs. Finally, the STING-R231H variant mouse represents a useful tool with which to examine the relative contributions of STING sensing of exogenous and endogenous CDNs in the context of bacterial infections and CDN-based cancer immunotherapeutics.
Asunto(s)
Mordeduras y Picaduras/metabolismo , Genotipo , Macrófagos/inmunología , Alelos , Animales , Mordeduras y Picaduras/genética , Técnicas de Sustitución del Gen , Ratones , Ratones Transgénicos , Nucleótidos Cíclicos/metabolismo , Polimorfismo GenéticoRESUMEN
Kinase activity plays an essential role in the regulation of immune cell defenses against pathogens. The protein kinase CK2 (formerly casein kinase II) is an evolutionarily conserved kinase with hundreds of identified substrates. CK2 is ubiquitously expressed in somatic and immune cells, but the roles of CK2 in regulation of immune cell function remain largely elusive. This reflects the essential role of CK2 in organismal development and limited prior work with conditional CK2 mutant murine models. Here, we generated mice with a conditional (floxed) allele of Csnk2a, which encodes the catalytic CK2α subunit of CK2. When crossed to Lyz2-cre mice, excision of Csnk2a sequence impaired CK2α expression in myeloid cells but failed to detectably alter myeloid cell development. By contrast, deficiency for CK2α increased inflammatory myeloid cell recruitment, activation, and resistance following systemic Listeria monocytogenes (Lm) infection. Results from mixed chimera experiments indicated that CK2α deficiency in only a subset of myeloid cells was not sufficient to reduce bacterial burdens. Nor did cell-intrinsic deficiency for CK2α suffice to alter accumulation or activation of monocytes and neutrophils in infected tissues. These data suggest that CK2α expression by Lyz2-expressing cells promotes inflammatory and anti-bacterial responses through effects in trans. Our results highlight previously undescribed suppressive effects of CK2 activity on inflammatory myeloid cell responses and illustrate that cell-extrinsic effects of CK2 can shape inflammatory and protective innate immune responses.
Asunto(s)
Quinasa de la Caseína II/inmunología , Listeria monocytogenes , Listeriosis/inmunología , Células Mieloides/inmunología , Animales , Quinasa de la Caseína II/genética , Femenino , Inflamación/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Legionella pneumophila is an opportunistic human pathogen and causative agent of the acute pneumonia known as Legionnaire's disease. Upon inhalation, the bacteria replicate in alveolar macrophages (AM), within an intracellular vacuole termed the Legionella-containing vacuole. We recently found that, in vivo, IFNγ was required for optimal clearance of intracellular L. pneumophila by monocyte-derived cells (MC), but the cytokine did not appear to influence clearance by AM. Here, we report that during L. pneumophila lung infection, expression of the IFNγ receptor subunit 1 (IFNGR1) is down-regulated in AM and neutrophils, but not MC, offering a possible explanation for why AM are unable to effectively restrict L. pneumophila replication in vivo. To test this, we used mice that constitutively express IFNGR1 in AM and found that prevention of IFNGR1 down-regulation enhanced the ability of AM to restrict L. pneumophila intracellular replication. IFNGR1 down-regulation was independent of the type IV Dot/Icm secretion system of L. pneumophila indicating that bacterial effector proteins were not involved. In contrast to previous work, we found that signaling via type I IFN receptors was not required for IFNGR1 down-regulation in macrophages but rather that MyD88- or Trif- mediated NF-κB activation was required. This work has uncovered an alternative signaling pathway responsible for IFNGR1 down-regulation in macrophages during bacterial infection.
Asunto(s)
Legionella pneumophila/crecimiento & desarrollo , Enfermedad de los Legionarios/microbiología , Pulmón/microbiología , Macrófagos Alveolares/microbiología , FN-kappa B/metabolismo , Receptores de Interferón/antagonistas & inhibidores , Animales , Regulación hacia Abajo , Interferón Tipo I/metabolismo , Legionella pneumophila/metabolismo , Enfermedad de los Legionarios/metabolismo , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Ratones , Ratones Transgénicos , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Transducción de Señal , Receptor de Interferón gammaRESUMEN
The type II IFN (IFNγ) enhances antimicrobial activity yet also drives expression of genes that amplify inflammatory responses. Hence, excessive IFNγ stimulation can be pathogenic. Here, we describe a previously unappreciated mechanism whereby IFNγ itself dampens myeloid cell activation. Staining of monocytes from Listeria monocytogenes-infected mice provided evidence of type I IFN-independent reductions in IFNGR1. IFNγ was subsequently found to reduce surface IFNGR1 on cultured murine myeloid cells and human CD14+ peripheral blood mononuclear cells. IFNγ-driven reductions in IFNGR1 were not explained by ligand-induced receptor internalization. Rather, IFNγ reduced macrophage Ifngr1 transcription by altering chromatin structure at putative Ifngr1 enhancer sites. This is a distinct mechanism from that used by type I IFNs. Ligand-induced reductions in IFNGR1 altered myeloid cell sensitivity to IFNγ, blunting activation of STAT1 and 3. Our data, thus, reveal a mechanism by which IFNGR1 abundance and myeloid cell sensitivity to IFNγ can be modulated in the absence of type I IFNs. Multiple mechanisms, thus, exist to calibrate macrophage IFNGR1 abundance, likely permitting the fine tuning of macrophage activation and inflammation.