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The pathologic consequences of Coronavirus Disease-2019 (COVID-19) include elevated inflammation and dysregulated vascular functions associated with thrombosis. In general, disruption of vascular homeostasis and ensuing prothrombotic events are driven by activated platelets, monocytes, and macrophages, which form aggregates (thrombi) attached to the endothelium lining of vessel walls. However, molecular pathways underpinning the pathological interactions between myeloid cells and endothelium during COVID-19 remain undefined. Here, we tested the hypothesis that modulations in the expression of cellular receptors angiotensin-converting enzyme 2 (ACE2), CD147, and glucose-regulated protein 78 (GRP78), which are involved in homeostasis and endothelial performance, are the hallmark responses induced by SARS-CoV-2 infection. Cultured macrophages and lungs of hamster model systems were used to test this hypothesis. The results indicate that while macrophages and endothelial cells are less likely to support SARS-CoV-2 proliferation, these cells may readily respond to inflammatory stimuli generated by the infected lung epithelium. SARS-CoV-2 induced modulations of tested cellular receptors correlated with corresponding changes in the mRNA expression of coagulation cascade regulators and endothelial integrity components in infected hamster lungs. Among these markers, tissue factor (TF) had the best correlation for prothrombotic events during SARS-CoV-2 infection. Furthermore, the single-molecule fluorescence in situ hybridization (smFISH) method alone was sufficient to determine the peak and resolution phases of SARS-CoV-2 infection and enabled screening for cellular markers co-expressed with the virus. These findings suggest possible molecular pathways for exploration of novel drugs capable of blocking the prothrombotic shift events that exacerbate COVID-19 pathophysiology and control the disease.
Assuntos
COVID-19 , Trombose , Humanos , COVID-19/patologia , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2 , Chaperona BiP do Retículo Endoplasmático , Células Endoteliais/metabolismo , Hibridização in Situ Fluorescente , Peptidil Dipeptidase A/metabolismo , Pulmão/metabolismo , Trombose/patologia , Endotélio/metabolismo , HomeostaseRESUMO
Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169-mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and subgenomic RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral genomic and subgenomic RNA in CD169+ macrophages elicited a pro-inflammatory cytokine expression (TNFα, IL-6 and IL-1ß) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response.
Assuntos
COVID-19 , Humanos , Enzima de Conversão de Angiotensina 2/genética , Citocinas/metabolismo , Macrófagos , RNA Viral/metabolismo , SARS-CoV-2RESUMO
Establishing diagnosis of latent and active histoplasmosis is challenging. Interferon gamma-release assays (IGRAs) may provide evidence of latent and active infection. An enzyme-linked immunospot (ELISpot) assay was developed using yeast cell lysate (YCL) antigen prepared from a representative North American Histoplasma capsulatum strain. Assay parameters were optimized by measuring responses in healthy volunteers with and without Histoplasma infection. Assay performance as an aid for diagnosing histoplasmosis was assessed in a prospective cohort of 88 people with suspected or confirmed infection, and 44 healthy controls enrolled in two centers in North America (2013 to 2018). Antigen specificity of IFN-γ release was demonstrated using ELISpot and enzyme-linked immunosorbent assay (ELISA). Antigen-evoked, single-cell mRNA expression by memory T cells was shown using flow cytometry. The area under the receiver operating characteristic curve (AUC) was estimated at 0.89 (95% confidence interval [CI]: 78.5% to 99.9%). At optimal cutoff, sensitivity was 77.2% (95% CI: 54.6% to 92.2%) and specificity was 100% (95% CI: 89.7% to 100%). Sixteen of 44 healthy volunteers (36.4%) from a region of hyperendemicity had positive responses, suggesting detection of previously unrecognized (latent) infection. The ELISpot assay is sensitive and specific as an aid to diagnose H. capsulatum infection and disease, supporting proof of concept and further development.
Assuntos
Histoplasmose , Testes de Liberação de Interferon-gama , Humanos , Histoplasmose/diagnóstico , Interferon gama , Estudos Prospectivos , ELISPOT , Antígenos de Fungos , Ensaio de Imunoadsorção Enzimática , RNA Mensageiro , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the rapidly evolving SARS-CoV-2 virus and for development of prophylactic and therapeutic strategies to combat emerging mutants. Studies show that the spike proteins of SARS-CoV and SARS-CoV-2 bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which are clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: (1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients; (2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and (3) hACE2Tg mice do not mimic the natural course of SARS-CoV-2 infection in humans. Moreover, one of most outstanding features of coronavirus infection is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a possible co-receptor for SARS-CoV-2 entry. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. RESULTS: Here we successfully generated a hCD147 knock-in mouse model (hCD147KI) in the NOD-scid IL2Rgammanull (NSG) background. In this hCD147KI-NSG mouse model, the hCD147 genetic sequence was placed downstream of the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of hCD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 (JAX) model. In addition, the hCD147KI-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations in immunosuppressed states. Our data show (1) the human CD147 protein is expressed in various organs (including bronchiolar epithelial cells) in hCD147KI-NSG mice by immunohistochemical staining and flow cytometry; (2) hCD147KI-NSG mice are marginally sensitive to SARS-CoV-2 infection compared to WT-NSG littermates characterized by increased viral copies by qRT-PCR and moderate body weight decline compared to baseline; (3) a significant increase in leukocytes in the lungs of hCD147KI-NSG mice, compared to infected WT-NSG mice. CONCLUSIONS: hCD147KI-NSG mice are more sensitive to COVID-19 infection compared to WT-NSG mice. The hCD147KI-NSG mouse model can serve as an additional animal model for further interrogation whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.
RESUMO
Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169- mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and sub-genomic (sg) RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral gRNA and sgRNA in CD169 + macrophages elicited a pro-inflammatory cytokine expression (TNFα, IL-6 and IL-1ß) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir pre- treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response. Author Summary: Over-exuberant production of pro-inflammatory cytokine expression by macrophages has been hypothesized to contribute to severity of COVID-19 disease. Molecular mechanisms that contribute to macrophage-intrinsic immune activation during SARS- CoV-2 infection are not fully understood. Here we show that CD169, a macrophage- specific sialic-acid binding lectin, facilitates abortive SARS-CoV-2 infection of macrophages that results in innate immune sensing of viral replication intermediates and production of proinflammatory responses. We identify an ACE2-independent, CD169- mediated endosomal viral entry mechanism that results in cytoplasmic delivery of viral capsids and initiation of virus replication, but absence of infectious viral production. Restricted viral replication in CD169 + macrophages and detection of viral genomic and sub-genomic RNAs by cytoplasmic RIG-I-like receptor family members, RIG-I and MDA5, and initiation of downstream signaling via the adaptor protein MAVS, was required for innate immune activation. These studies uncover mechanisms important for initiation of innate immune sensing of SARS-CoV-2 infection in macrophages, persistent activation of which might contribute to severe COVID-19 pathophysiology.
RESUMO
Background: An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the rapidly evolving SARS-CoV-2 virus and for development of prophylactic and therapeutic strategies to combat emerging mutants. Studies show that the spike proteins of SARS-CoV and SARS-CoV-2 bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which are clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: 1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients; 2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and 3) hACE2Tg mice do not mimic the natural course of SARS-CoV-2 infection in humans. Moreover, one of most outstanding features of coronavirus infection is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a possible co-receptor for SARS-CoV-2 entry. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. Results: Here we successfully generated a hCD147 knock-in mouse model (hCD147KI) in the NOD- scid IL2Rgamma null (NSG) background. In this hCD147KI-NSG mouse model, the hCD147 genetic sequence was placed downstream of the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of hCD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 (JAX) model. In addition, the hCD147KI-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations in immunosuppressed states. Our data show 1) the human CD147 protein is expressed in various organs (including bronchiolar epithelial cells) in hCD147KI-NSG mice by immunohistochemical staining and flow cytometry; 2) hCD147KI-NSG mice are marginally sensitive to SARS-CoV-2 infection compared to WT-NSG littermates characterized by increased viral copies by qRT-PCR and moderate body weight decline compared to baseline; 3) a significant increase in leukocytes in the lungs of hCD147KI-NSG mice, compared to infected WT-NSG mice. Conclusions: hCD147KI-NSG mice are more sensitive to COVID-19 infection compared to WT-NSG mice. The hCD147KI-NSG mouse model can serve as an additional animal model for further interrogation whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.
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Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) remains as a leading killer among infectious diseases worldwide. The nature of the host immune response dictates whether the initial Mtb infection is cleared or progresses toward active disease, and is ultimately determined by intricate host-pathogen interactions that are yet to be fully understood. The early immune response to infection is mediated by innate immune cells, including macrophages and neutrophils that can phagocytose Mtb and mount an antimicrobial response. However, Mtb can exploit these innate immune cells for its survival and dissemination. Recently, it has become clear that the immune response and metabolic remodeling are interconnected, which is highlighted by the rapid evolution of the interdisciplinary field of immunometabolism. It has been proposed that the net outcome to Mtb infection-clearance or chronic disease-is likely a result of combined immunologic and metabolic activities of the immune cells. Indeed, host cells activated by Mtb infection have strikingly different metabolic requirements than naïve/non-infected cells. Macrophages activated by Mtb-derived molecules or upon phagocytosis acquire a phenotype similar to M1 with elevated production of pro-inflammatory molecules and rely on glycolysis and pentose phosphate pathway to meet their bioenergetic and metabolic requirements. In these macrophages, oxidative phosphorylation and fatty acid oxidation are dampened. However, the non-infected/naive, M2-type macrophages are anti-inflammatory and derive their energy from oxidative phosphorylation and fatty acid oxidation. Similar metabolic adaptations also occur in other phagocytes, including dendritic cells, neutrophils upon Mtb infection. This metabolic reprogramming of innate immune cells during Mtb infection can differentially regulate their effector functions, such as the production of cytokines and chemokines, and antimicrobial response, all of which can ultimately determine the outcome of Mtb-host interactions within the granulomas. In this review, we describe key immune cells bolstering host innate response and discuss the metabolic reprogramming in these phagocytes during Mtb infection. We focused on the major phagocytes, including macrophages, dendritic cells and neutrophils and the key regulators involved in metabolic reprogramming, such as hypoxia-inducible factor-1, mammalian target of rapamycin, the cellular myelocytomatosis, peroxisome proliferator-activator receptors, sirtuins, arginases, inducible nitric acid synthase and sphingolipids.
RESUMO
Amplification of signals by the hybridization chain reaction (HCR) is a powerful approach for increasing signal strength in single-molecule fluorescence in situ hybridization, but probes tagged with an HCR initiator sequence are prone to producing false signals. Here we describe a system of interacting hairpin binary probes in which the HCR initiator sequence is conditionally sequestered. The binding of these probes to a perfectly complementary target unmasks the initiator, enabling the generation of an amplified signal. This probe system can distinguish single-nucleotide variations within single mRNA molecules and produces amplified signals in situ for both mutant and wild-type variants, each in a distinguishable color. This technology will augment studies of imbalanced allelic expression and will be useful for the detection of somatic mutations in cancer biopsies. By tiling these probes along the length of an mRNA target, enhanced signals can be obtained, thereby enabling the scanning of tissue sections for gene expression utilizing lower magnification microscopy, overcoming tissue autofluorescence, and allowing the detection of low-abundance biomarkers in flow cytometry.
Assuntos
Citometria de Fluxo , Neoplasias/diagnóstico , RNA Mensageiro/genética , Imagem Individual de Molécula , Alelos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/isolamento & purificação , Corantes Fluorescentes/química , Genótipo , Humanos , Hibridização in Situ Fluorescente/métodos , Mutação/genética , Neoplasias/genética , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/química , RNA Mensageiro/isolamento & purificaçãoRESUMO
Macrophages are the primary targets of Mycobacterium tuberculosis infection; the early events of macrophage interaction with M. tuberculosis define subsequent progression and outcome of infection. M. tuberculosis can alter the innate immunity of macrophages, resulting in suboptimal Th1 immunity, which contributes to the survival, persistence, and eventual dissemination of the pathogen. Recent advances in immunometabolism illuminate the intimate link between the metabolic states of immune cells and their specific functions. In this review, we describe the little-studied biphasic metabolic dynamics of the macrophage response during progression of infection by M. tuberculosis and discuss their relevance to macrophage immunity and M. tuberculosis pathogenicity. The early phase of macrophage infection, which is marked by M1 polarization, is accompanied by a metabolic switch from mitochondrial oxidative phosphorylation to hypoxia-inducible factor 1 alpha (HIF-1α)-mediated aerobic glycolysis (also known as the Warburg effect in cancer cells), as well as by an upregulation of pathways involving oxidative and antioxidative defense responses, arginine metabolism, and synthesis of bioactive lipids. These early metabolic changes are followed by a late adaptation/resolution phase in which macrophages transition from glycolysis to mitochondrial oxidative metabolism, with a consequent dampening of macrophage proinflammatory and antimicrobial responses. Importantly, the identification of upregulated metabolic pathways and/or metabolic regulatory mechanisms with immunomodulatory functions during M1 polarization has revealed novel mechanisms of M. tuberculosis pathogenicity. These advances can lead to the development of novel host-directed therapies to facilitate bacterial clearance in tuberculosis by targeting the metabolic state of immune cells.
Assuntos
Interações Hospedeiro-Patógeno , Macrófagos/imunologia , Macrófagos/metabolismo , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Aerobiose , Animais , Glicólise , Humanos , Macrófagos/microbiologia , Fosforilação OxidativaRESUMO
Despite advances in diagnosing latent Mycobacterium tuberculosis infection (LTBI), we still lack a diagnostic test that differentiates LTBI from active tuberculosis (TB) or predicts the risk of progression to active disease. One reason for the absence of such a test may be the failure of current assays to capture the dynamic complexities of the immune responses associated with various stages of TB, since these assays measure only a single parameter (release of IFN-γ) and rely on prolonged (overnight) T cell stimulation. We describe a novel, semi-automated RNA flow cytometry assay to determine whether immunological differences can be identified between LTBI and active TB. We analyzed antigen-induced expression of Th1 cytokine mRNA after short (2- and 6-h) stimulation with antigen, in the context of memory T cell immunophenotyping. IFNG and TNFA mRNA induction was detectable in CD4+ T cells after only 2 h of ex vivo stimulation. Moreover, IFNG- and TNFA-expressing CD4+ T cells (Th1 cells) were more frequent in active TB than in LTBI, a difference that is undetectable with conventional, protein-based cytokine assays. We also found that active TB was associated with higher ratios of effector memory to central memory Th1 cells than LTBI. This effector memory phenotype of active TB was associated with increased T cell differentiation, as defined by loss of the CD27 marker, but not with T cell exhaustion, as determined by PD-1 abundance. These results indicate that single-cell-based, mRNA measurements may help identify time-dependent, quantitative differences in T cell functional status between latent infection and active tuberculosis.
Assuntos
Diferenciação Celular/imunologia , Memória Imunológica/imunologia , Tuberculose Latente/imunologia , Células Th1/imunologia , Tuberculose/imunologia , Adulto , Idoso , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Feminino , Humanos , Testes Imunológicos , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Tuberculose Latente/diagnóstico , Tuberculose Latente/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/fisiologia , Células Th1/metabolismo , Tuberculose/diagnóstico , Tuberculose/microbiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes â¼30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described.
Assuntos
Citometria de Fluxo/métodos , Hibridização in Situ Fluorescente/métodos , Proteínas/análise , RNA Mensageiro/análise , Análise de Célula Única/métodos , Automação Laboratorial/métodos , Humanos , Leucócitos Mononucleares/químicaRESUMO
Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitative, high-throughput platform allowing precise quantification of total mRNA transcripts in single cells. In undiagnosed infections posing a significant health burden worldwide, such as latent tuberculosis or asymptomatic recurrent malaria, an important challenge is to develop accurate diagnostic tools. Antigen-specific T cells create a persistent memory to pathogens, making them useful for diagnosis of infection. Stimulation of memory response initiates T-cell transitions between functional states. Numerous studies have shown that changes in protein levels lag real-time T-cell transitions. However, analysis at the single-cell transcriptional level can determine the differences. FISH-Flow is a powerful tool with which to study the functional states of T-cell subsets and to identify the gene expression profiles of antigen-specific T cells during disease progression. Advances in instrumentation, fluorophores, and FISH methodologies will broaden and deepen the use of FISH-Flow, changing the immunological field by allowing determination of functional immune signatures at the mRNA level and the development of new diagnostic tools.
Assuntos
Citometria de Fluxo/métodos , Doenças do Sistema Imunitário/diagnóstico , Hibridização in Situ Fluorescente/métodos , Infecções/diagnóstico , RNA/análise , Subpopulações de Linfócitos T/fisiologia , Linfócitos T/fisiologia , Animais , Antígenos/imunologia , Separação Celular , Ensaios de Triagem em Larga Escala , Humanos , Doenças do Sistema Imunitário/imunologia , Memória Imunológica , Infecções/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , TranscriptomaRESUMO
RNA flow cytometry (FISH-Flow) achieves high-throughput measurement of single-cell gene expression by combining in-situ nucleic acid hybridization with flow cytometry. We tested whether antigen-specific T-cell responses detected by FISH-Flow correlated with latent tuberculosis infection (LTBI), a condition affecting one-third of the world population. Peripheral-blood mononuclear cells from donors, identified as positive or negative for LTBI by current medical practice, were stimulated ex vivo with mycobacterial antigen. IFNG and IL2 mRNA production was assayed by FISH-Flow. Concurrently, immunophenotypes of the cytokine mRNA-positive cells were characterized by conventional, antibody-based staining of cell-surface markers. An association was found between donor LTBI status and antigen-specific induction of IFNG and IL2 transcripts. Induction of these cytokine genes, which was detected by FISH-Flow in a quarter the time required to see release of the corresponding proteins by ELISA, occurred primarily in activated CD4+ T cells via T-cell receptor engagement. Moreover, NK cells contributed to IFNG gene induction. These results show that antigen-driven induction of T-cell cytokine mRNA is a measurable single-cell parameter of the host responses associated with latent tuberculosis. FISH-Flow read-outs contribute a multi-scale dimension to the immunophenotyping afforded by antibody-based flow cytometry. Multi-scale, single-cell analyses may satisfy the need to determine disease stage and therapy response for tuberculosis and other infectious pathologies.
Assuntos
Citocinas/sangue , Tuberculose Latente/sangue , Leucócitos Mononucleares/metabolismo , Estudos de Casos e Controles , Citocinas/biossíntese , Citometria de Fluxo/métodos , Regulação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente/métodos , Interferon gama/sangue , Interleucina-2/sangue , Tuberculose Latente/imunologia , Ativação Linfocitária , Linfócitos T/metabolismoRESUMO
Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein analytes. Shifting the analysis to detection of RNA would provide several significant advantages, which we illustrate by developing a new host immunity-based platform for detection of infections. Cytokine mRNAs synthesized in response to ex vivo stimulation with pathogen-specific Ags are detected in T cells with single-molecule fluorescence in situ hybridization followed by flow cytometry. Background from pre-existing in vivo analytes is lower for RNAs than for proteins, allowing greater sensitivity for detection of low-frequency cells. Moreover, mRNA analysis reveals kinetic differences in cytokine expression that are not apparent at the protein level but provide novel insights into gene expression programs expected to define different T cell subsets. The utility of probing immunological memory of infections is demonstrated by detecting T cells that recognize mycobacterial and viral Ags in donors exposed to the respective pathogens.
Assuntos
Citometria de Fluxo/métodos , Hibridização in Situ Fluorescente/métodos , Ativação Linfocitária , Linfócitos T/imunologia , Tuberculose Pulmonar/imunologia , Adulto , Idoso , Antígenos de Bactérias/imunologia , Citocinas/sangue , Citocinas/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/sangue , RNA Mensageiro/imunologia , Linfócitos T/patologia , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/patologiaRESUMO
It has become clear that soluble MHC I (sMHC I) and soluble MIC (sMIC), which are highly elevated in sera of cancer patients, can be viewed to be tolerogenic, and that metalloproteinases are involved in their generation process. In this review, an overview is provided of the recent progress made in the sMHC I and sMIC fields, with emphasis on their structure, formation, and function, and the key-questions that still await answers are addressed. Understanding better their formation mechanism, it will become more feasible to modulate the immune responses in cancer patients by targeting molecules involved in their generation process.
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Antígenos de Histocompatibilidade Classe I/imunologia , Neoplasias/imunologia , Antígenos de Histocompatibilidade Classe I/sangue , Humanos , Imunomodulação , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Metaloproteases/imunologia , Metaloproteases/metabolismo , Terapia de Alvo Molecular , Neoplasias/sangue , Solubilidade , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
BACKGROUND: NK cells are able to kill tumor and virus-infected cells without the need of prior antigen stimulation. The killing of these target cells is regulated by inhibitory, lysis and co-stimulatory receptors that are expressed on the surface of NK cells. PRINCIPAL FINDINGS: CD100 (Semaphorin 4D), a 150kD transmembrane protein, is expressed on the surface of activated NK cells as a homodimer, mediates the killing of target cells by binding to CD72. CD100 is not involved directly in the killing process but is rather increases NK cytotoxicity by enhancing the adhesion between NK cells and their targets. This increased adhesion leads to a more efficient killing and enhanced IFNgamma secretion. SIGNIFICANCE: Since CD72 is expressed on antigen presenting cells (APC) and the CD100-CD72 interaction lead to the shading of CD100, we suggest that NK interacting with APC cells could be the early source of soluble CD100 which is crucial for the formation of antigen specific immune response. CD100-CD72 interaction can be the mechanism by which NK cell communicate with B cells.
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Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Citotoxicidade Imunológica , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Semaforinas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Adesão Celular , Linhagem Celular , Humanos , Células Matadoras Naturais/citologia , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Peso Molecular , Fosfosserina/metabolismo , Ligação Proteica , Multimerização Proteica , Regulação para Cima/genéticaRESUMO
Although cytomegalovirus (CMV) interferes with major histocompatibility expression in infected cells, both host and donor soluble human leukocyte antigen class I (sHLA-I) are often released into the serum of transplant recipients during CMV infection and may contribute to anti-HLA antibody production and graft rejection. We hypothesized that CMV infection of endothelial cells (EC) induces host T cells to release interferon (IFN)-gamma, which in turn drives the metalloproteinase (MPase)-cleavage pathway of sHLA-I generation in "bystander" uninfected ECs. To test this hypothesis, cultures of peripheral blood mononuclear cells (PBMCs) and either uninfected ECs or CMV-infected ECs (EC/CMV) were established and supernatants were tested in enzyme-linked immunosorbent assay for sHLA-I. Responder PBMC became activated and released sHLA-I via the MPase pathway when stimulated with allogeneic EC/CMV; the sHLA-I release was contact dependent and cytokine independent. In transwell cultures, IFN-gamma released by PBMCs in response to EC/CMV stimulated a release of sHLA-I from uninfected allogeneic ECs across the transwell; this release was also MPase dependent. This implies that CMV infection within the transplanted allograft will not only stimulate the release of self HLA from responding PBMCs, but will also stimulate the release of donor sHLA-I from uninfected bystander ECs, both via the class I MPase-pathway.
Assuntos
Infecções por Citomegalovirus/metabolismo , Células Endoteliais/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Metaloproteases/metabolismo , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Células Endoteliais/enzimologia , Células Endoteliais/imunologia , Rejeição de Enxerto/enzimologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/metabolismoRESUMO
Major histocompatibility complex class I-restricted CD8(+) cytotoxic T lymphocytes (CTL) are implicated in protective Th1 immunity to Mycobacterium tuberculosis infection. We report the identification of three novel HLA-A*0201-restricted CTL epitopes within mycobacterial superoxide dismutase (SodA), L-alanine dehydrogenase (AlaDH), and L-glutamine synthetase (GlnS) proteins.
Assuntos
Aminoácido Oxirredutases/química , Epitopos de Linfócito T , Glutamato-Amônia Ligase/química , Antígeno HLA-A2/metabolismo , Mycobacterium tuberculosis/imunologia , Superóxido Dismutase/química , Linfócitos T Citotóxicos/imunologia , Alanina Desidrogenase , Aminoácido Oxirredutases/imunologia , Sequência de Aminoácidos , Glutamato-Amônia Ligase/imunologia , Humanos , Epitopos Imunodominantes , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , Superóxido Dismutase/imunologia , Tuberculose Pulmonar/imunologiaRESUMO
Soluble human leukocyte antigens (HLA-A, -B, and -C) proteins can be generated by a membrane-bound metalloproteinase (MPase). The MPase-mediated pathway produces soluble nonconformed HLA proteins susceptible to further degradation, and also HLA proteins with high affinity peptides stable at physiologic temperatures. Accessibility of classical HLA to the MPase cleavage inversely correlates with stability of heavy chain (HC) interactions with beta2-microglobulin (beta(2)m). Whether a MPase is involved in release of soluble nonclassical HLA or CD1 proteins is unknown. We have investigated this question with transfectants expressing full-length HLA proteins. Native surface HLA-E and -G complexes, similar to HLA-A2, were unstable at low pH and dissociated giving rise to beta(2)m-free HC. Furthermore, HLA-E and -G proteins, similar to HLA-A2, were readily released from cell surface into supernatants as soluble 37-kilodalton beta(2)m-free HC. However, the stability of surface CD1d complexes was not affected by pH changes and no soluble CD1d was detected. Because beta(2)m-free CD1d HC were expressed on cells, the lack of cleaved soluble products cannot be explained by high stability of native complexes. Instead, absence of a CD1d-specific MPase in these cells or its impaired interactions with substrate HC may be responsible.
Assuntos
Antígenos HLA/metabolismo , Metaloendopeptidases/metabolismo , Antígenos CD1/metabolismo , Antígenos CD1d , Linhagem Celular Tumoral , Membrana Celular/enzimologia , Membrana Celular/imunologia , Antígenos HLA/química , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Ligação Proteica , Conformação Proteica , Solubilidade , Transfecção , Microglobulina beta-2/química , Microglobulina beta-2/metabolismo , Antígenos HLA-ERESUMO
Activation of bronchial epithelial cells (BEC) and disruption of an intact epithelial barrier in a lung transplant recipient can lead to acute or chronic rejection, events that are associated with release of soluble human leukocyte antigen (sHLA) class I. Although we know that HLA is released from mitogen-activated lymphocytes in a metalloproteinase (MPase)-dependent fashion, the mechanism of release from nonlymphoid tissue is not well understood. To this end, we stimulated primary BEC with increasing amounts of the T-helper cell-1 cytokines, interferon gamma (IFNgamma), and/or tumor necrosis factor alpha (TNFalpha) and measured the quantity and forms of HLA class I release. We found that IFNgamma, but not TNFalpha, was able to stimulate a time- and concentration-dependent release of HLA/beta(2)m and beta(2)m-free heavy chain (HC) from the BEC. A portion (50%) of the HLA/beta(2)m release and >90% of the beta(2)m-free HC release was mediated by a MPase. Western blot analysis supported the conclusion that a MPase-sensitive pathway produced 36 and 37 kDa cleaved forms, whereas the secreted 39 kDa form of beta(2)m-associated soluble HLA class I (sHLA/beta(2)m) was MPase-resistant. This adds to the growing understanding of the extracellular processing pathways of major histocompatibility complex class I that may be critical for both chronic rejection as well as immune regulation.