RESUMEN
Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum-infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria-γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs.
Asunto(s)
Antígenos de Protozoos/inmunología , Citotoxicidad Inmunológica , Eritrocitos/inmunología , Linfocitos Intraepiteliales/inmunología , Activación de Linfocitos , Malaria Falciparum/inmunología , Fagocitosis , Plasmodium falciparum/microbiología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos de Protozoos/sangre , Boston , Brasil , Butirofilinas/metabolismo , Células Cultivadas , Eritrocitos/metabolismo , Eritrocitos/parasitología , Femenino , Granzimas/metabolismo , Interacciones Huésped-Parásitos , Humanos , Sinapsis Inmunológicas/metabolismo , Sinapsis Inmunológicas/parasitología , Linfocitos Intraepiteliales/metabolismo , Linfocitos Intraepiteliales/parasitología , Malaria Falciparum/sangre , Malaria Falciparum/parasitología , Masculino , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
Trypanosoma cruzi, the protozoan that causes Chagas disease, has a complex life cycle involving several morphologically and biochemically distinct stages that establish intricate interactions with various insect and mammalian hosts. It has also a heterogeneous population structure comprising strains with distinct properties such as virulence, sensitivity to drugs, antigenic profile and tissue tropism. We present a comparative transcriptome analysis of two cloned T. cruzi strains that display contrasting virulence phenotypes in animal models of infection: CL Brener is a virulent clone and CL-14 is a clone that is neither infective nor pathogenic in in vivo models of infection. Gene expression analysis of trypomastigotes and intracellular amastigotes harvested at 60 and 96 hours post-infection (hpi) of human fibroblasts revealed large differences that reflect the parasite's adaptation to distinct environments during the infection of mammalian cells, including changes in energy sources, oxidative stress responses, cell cycle control and cell surface components. While extensive transcriptome remodeling was observed when trypomastigotes of both strains were compared to 60 hpi amastigotes, differences in gene expression were much less pronounced when 96 hpi amastigotes and trypomastigotes of CL Brener were compared. In contrast, the differentiation of the avirulent CL-14 from 96 hpi amastigotes to extracellular trypomastigotes was associated with considerable changes in gene expression, particularly in gene families encoding surface proteins such as trans-sialidases, mucins and the mucin associated surface proteins (MASPs). Thus, our comparative transcriptome analysis indicates that the avirulent phenotype of CL-14 may be due, at least in part, to a reduced or delayed expression of genes encoding surface proteins that are associated with the transition of amastigotes to trypomastigotes, an essential step in the establishment of the infection in the mammalian host. Confirming the role of members of the trans-sialidase family of surface proteins for parasite differentiation, transfected CL-14 constitutively expressing a trans-sialidase gene displayed faster kinetics of trypomastigote release in the supernatant of infected cells compared to wild type CL-14.
Asunto(s)
Enfermedad de Chagas/parasitología , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidad , Animales , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Genes Protozoarios , Glicoproteínas/genética , Interacciones Huésped-Parásitos , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Neuraminidasa/genética , Proteínas Protozoarias/genética , Proteínas de Unión al ARN/genética , Trypanosoma cruzi/crecimiento & desarrollo , Virulencia/genéticaRESUMEN
A systems analysis was conducted to determine the potential molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole-sporozoite PfSPZ vaccine in African infants. Innate immune activation and myeloid signatures at prevaccination baseline correlated with protection from P. falciparum parasitemia in placebo controls. These same signatures were associated with susceptibility to parasitemia among infants who received the highest and most protective PfSPZ vaccine dose. Machine learning identified spliceosome, proteosome, and resting DC signatures as prevaccination features predictive of protection after highest-dose PfSPZ vaccination, whereas baseline circumsporozoite protein-specific (CSP-specific) IgG predicted nonprotection. Prevaccination innate inflammatory and myeloid signatures were associated with higher sporozoite-specific IgG Ab response but undetectable PfSPZ-specific CD8+ T cell responses after vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against infection by sporozoite injection in malaria-naive mice while diminishing the CD8+ T cell response to radiation-attenuated sporozoites. These data suggest a dichotomous role of innate stimulation for malaria protection and induction of protective immunity by whole-sporozoite malaria vaccines. The uncoupling of vaccine-induced protective immunity achieved by Abs from more protective CD8+ T cell responses suggests that PfSPZ vaccine efficacy in malaria-endemic settings may be constrained by opposing antigen presentation pathways.
Asunto(s)
Inmunidad Innata , Vacunas contra la Malaria , Malaria Falciparum , Plasmodium falciparum , Esporozoítos , Vacunas Atenuadas , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/administración & dosificación , Inmunidad Innata/inmunología , Humanos , Animales , Malaria Falciparum/prevención & control , Malaria Falciparum/inmunología , Plasmodium falciparum/inmunología , Ratones , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/administración & dosificación , Esporozoítos/inmunología , Esporozoítos/efectos de la radiación , Linfocitos T CD8-positivos/inmunología , Lactante , Proteínas Protozoarias/inmunología , Anticuerpos Antiprotozoarios/inmunología , Femenino , Parasitemia/inmunología , Parasitemia/prevención & control , Inmunoglobulina G/inmunología , Inmunoglobulina G/sangre , Eficacia de las VacunasRESUMEN
Influenza A virus causes annual epidemics which affect millions of people worldwide. A recent Influenza pandemic brought new awareness over the health impact of the disease. It is thought that a severe inflammatory response against the virus contributes to disease severity and death. Therefore, modulating the effects of inflammatory mediators may represent a new therapy against Influenza infection. Platelet activating factor (PAF) receptor (PAFR) deficient mice were used to evaluate the role of the gene in a model of experimental infection with Influenza A/WSN/33 H1N1 or a reassortant Influenza A H3N1 subtype. The following parameters were evaluated: lethality, cell recruitment to the airways, lung pathology, viral titers and cytokine levels in lungs. The PAFR antagonist PCA4248 was also used after the onset of flu symptoms. Absence or antagonism of PAFR caused significant protection against flu-associated lethality and lung injury. Protection was correlated with decreased neutrophil recruitment, lung edema, vascular permeability and injury. There was no increase of viral load and greater recruitment of NK1.1(+) cells. Antibody responses were similar in WT and PAFR-deficient mice and animals were protected from re-infection. Influenza infection induces the enzyme that synthesizes PAF, lyso-PAF acetyltransferase, an effect linked to activation of TLR7/8. Therefore, it is suggested that PAFR is a disease-associated gene and plays an important role in driving neutrophil influx and lung damage after infection of mice with two subtypes of Influenza A. Further studies should investigate whether targeting PAFR may be useful to reduce lung pathology associated with Influenza A virus infection in humans.
Asunto(s)
Apoptosis , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Lesión Pulmonar/metabolismo , Lesión Pulmonar/virología , Infecciones por Orthomyxoviridae/prevención & control , Glicoproteínas de Membrana Plaquetaria/fisiología , Receptores Acoplados a Proteínas G/fisiología , Animales , Western Blotting , Pollos , Dihidropiridinas/farmacología , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Subtipo H1N1 del Virus de la Influenza A/genética , Lesión Pulmonar/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Factor de Activación Plaquetaria/genética , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , ARN Mensajero/genética , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Carga ViralRESUMEN
Most SARS-CoV2 infections will not develop into severe COVID-19. However, in some patients, lung infection leads to the activation of alveolar macrophages and lung epithelial cells that will release proinflammatory cytokines. IL-6, TNF, and IL-1ß increase expression of cell adhesion molecules (CAMs) and VEGF, thereby increasing permeability of the lung endothelium and reducing barrier protection, allowing viral dissemination and infiltration of neutrophils and inflammatory monocytes. In the blood, these cytokines will stimulate the bone marrow to produce and release immature granulocytes, that return to the lung and further increase inflammation, leading to acute respiratory distress syndrome (ARDS). This lung-systemic loop leads to cytokine storm syndrome (CSS). Concurrently, the acute phase response increases the production of platelets, fibrinogen and other pro-thrombotic factors. Systemic decrease in ACE2 function impacts the Renin-Angiotensin-Kallikrein-Kinin systems (RAS-KKS) increasing clotting. The combination of acute lung injury with RAS-KKS unbalance is herein called COVID-19 Associated Lung Injury (CALI). This conservative two-hit model of systemic inflammation due to the lung injury allows new intervention windows and is more consistent with the current knowledge.
Asunto(s)
Lesión Pulmonar Aguda/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Pulmón/inmunología , Neumonía Viral/inmunología , Síndrome Respiratorio Agudo Grave/inmunología , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/terapia , COVID-19 , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/terapia , Humanos , Pulmón/patología , Pandemias , Neumonía Viral/patología , Neumonía Viral/terapia , SARS-CoV-2 , Síndrome Respiratorio Agudo Grave/patología , Síndrome Respiratorio Agudo Grave/terapia , Síndrome de Respuesta Inflamatoria Sistémica/patología , Síndrome de Respuesta Inflamatoria Sistémica/terapiaRESUMEN
How innate T cells (ITC), including invariant natural killer T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells, maintain a poised effector state has been unclear. Here we address this question using low-input and single-cell RNA-seq of human lymphocyte populations. Unbiased transcriptomic analyses uncover a continuous 'innateness gradient', with adaptive T cells at one end, followed by MAIT, iNKT, γδ T and natural killer cells at the other end. Single-cell RNA-seq reveals four broad states of innateness, and heterogeneity within canonical innate and adaptive populations. Transcriptional and functional data show that innateness is characterized by pre-formed mRNA encoding effector functions, but impaired proliferation marked by decreased baseline expression of ribosomal genes. Together, our data shed new light on the poised state of ITC, in which innateness is defined by a transcriptionally-orchestrated trade-off between rapid cell growth and rapid effector function.
Asunto(s)
Proliferación Celular/fisiología , Linfocitos/metabolismo , Femenino , Ontología de Genes , Humanos , Inmunidad Innata/fisiología , Inmunofenotipificación , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/fisiología , Masculino , Células T Asesinas Naturales/metabolismo , Subgrupos de Linfocitos T/metabolismoRESUMEN
Protozoan infections are a serious global health problem. Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) eliminate pathogen-infected cells by releasing cytolytic granule contents--granzyme (Gzm) proteases and the pore-forming perforin (PFN)--into the infected cell. However, these cytotoxic molecules do not kill intracellular parasites. CD8(+) CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-γ. However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterol-poor microbial membranes, and GNLY, PFN and Gzms rapidly kill intracellular bacteria. Here we show that GNLY delivers Gzms into three protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii and Leishmania major), in which the Gzms generate superoxide and inactivate oxidative defense enzymes to kill the parasite. PFN delivers GNLY and Gzms into infected cells, and GNLY then delivers Gzms to the intracellular parasites. Killer cell-mediated parasite death, which we term 'microbe-programmed cell death' or 'microptosis', is caspase independent but resembles mammalian apoptosis, causing mitochondrial swelling, transmembrane potential dissipation, membrane blebbing, phosphatidylserine exposure, DNA damage and chromatin condensation. GNLY-transgenic mice are protected against infection by T. cruzi and T. gondii, and survive infections that are lethal to wild-type mice. Thus, GNLY-, PFN- and Gzm-mediated elimination of intracellular protozoan parasites is an unappreciated immune defense mechanism.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Granzimas/inmunología , Células Asesinas Naturales/inmunología , Leishmania major , Perforina/inmunología , Linfocitos T Citotóxicos/inmunología , Toxoplasma , Trypanosoma cruzi , Animales , Antígenos de Diferenciación de Linfocitos T/genética , Enfermedad de Chagas/inmunología , Humanos , Leishmaniasis Cutánea/inmunología , Ratones , Ratones Transgénicos , Toxoplasmosis/inmunologíaRESUMEN
The capacity for rapid localization of epitope-tagged or fluorescent fusion proteins in cells is an important tool for biological discovery and functional analysis. For Trypanosoma cruzi, the protozoan parasite that causes human Chagas disease, visualization of ectopically-expressed proteins in the clinically-relevant mammalian stages is hindered by the necessity to first perform transfection and lengthy selection procedures in the insect vector form of the parasite. Here, we demonstrate the ability to by-pass the insect stage with the delivery of plasmid DNA to non-dividing, tissue culture trypomastigotes such that upon host cell infection, transgenes are expressed and rapidly localized in intracellular T. cruzi amastigotes. The inclusion of a sorting step prior to host cell infection by trypomastigotes greatly enriches (>90%) the number of transgene-expressing amastigotes observed in mammalian host cells. This is a significant methodological advance that has the potential to accelerate the pace of discovery in the Chagas disease field.