RESUMO
Current serological tests cannot differentiate between total immunoglobulin A (IgA) and dimeric IgA (dIgA) associated with mucosal immunity. Here, we describe two new assays, dIgA-ELISA and dIgA-multiplex bead assay (MBA), that utilize the preferential binding of dIgA to a chimeric form of secretory component, allowing the differentiation between dIgA and monomeric IgA. dIgA responses elicited through severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were measured in (i) a longitudinal panel, consisting of 74 samples (n = 20 individuals) from hospitalized cases of coronavirus disease 2019 (COVID-19); (ii) a longitudinal panel, consisting of 96 samples (n = 10 individuals) from individuals with mild COVID-19; (iii) a cross-sectional panel with PCR-confirmed SARS-CoV-2 infection with mild COVID-19 (n = 199) and (iv) pre-COVID-19 samples (n = 200). The dIgA-ELISA and dIgA-MBA demonstrated a specificity for dIgA of 99% and 98.5%, respectively. Analysis of dIgA responses in the longitudinal panels revealed that 70% (ELISA) and 50% (MBA) of patients elicited a dIgA response by day 20 after PCR diagnosis with a SARS-CoV-2 infection. Individuals with mild COVID-19 displayed increased levels of dIgA within the first 3 weeks after diagnosis but responses appeared to be short lived, compared with sustained IgA levels. However, in samples from hospitalized patients with COVID-19 we observed high and sustained levels of dIgA, up to 245 days after PCR diagnosis. Our results suggest that severe COVID-19 infections are associated with sustained levels of plasma dIgA compared with mild cases.
Assuntos
COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2/metabolismo , Estudos Transversais , Imunoglobulina A , Anticorpos Antivirais , Imunoglobulina MRESUMO
BACKGROUND: Typical symptoms of uncomplicated dengue fever (DF) include headache, muscle pains, rash, cough, and vomiting. A proportion of cases progress to severe dengue hemorrhagic fever (DHF), associated with increased vascular permeability, thrombocytopenia, and hemorrhages. Progression to severe dengue is difficult to diagnose at the onset of fever, which complicates patient triage, posing a socio-economic burden on health systems. METHODS: To identify parameters associated with protection and susceptibility to DHF, we pursued a systems immunology approach integrating plasma chemokine profiling, high-dimensional mass cytometry and peripheral blood mononuclear cell (PBMC) transcriptomic analysis at the onset of fever in a prospective study conducted in Indonesia. RESULTS: After a secondary infection, progression to uncomplicated dengue featured transcriptional profiles associated with increased cell proliferation and metabolism, and an expansion of ICOS+CD4+ and CD8+ effector memory T cells. These responses were virtually absent in cases progressing to severe DHF, that instead mounted an innate-like response, characterised by inflammatory transcriptional profiles, high circulating levels of inflammatory chemokines and with high frequencies of CD4low non-classical monocytes predicting increased odds of severe disease. CONCLUSIONS: Our results suggests that effector memory T cell activation might play an important role ameliorating severe disease symptoms during a secondary dengue infection, and in the absence of that response, a strong innate inflammatory response is required to control viral replication. Our research also identified discrete cell populations predicting increased odds of severe disease, with potential diagnostic value.
Assuntos
Dengue , Dengue Grave , Humanos , Leucócitos Mononucleares , Estudos Prospectivos , Linfócitos TRESUMO
COVID-19 clinical presentation differs considerably between individuals, ranging from asymptomatic, mild/moderate and severe disease which in some cases are fatal or result in long-term effects. Identifying immune mechanisms behind severe disease development informs screening strategies to predict who are at greater risk of developing life-threatening complications. However, to date clear prognostic indicators of individual risk of severe or long COVID remain elusive. Autoantibodies recognize a range of self-antigens and upon antigen recognition and binding, important processes involved in inflammation, pathogen defence and coagulation are modified. Recent studies report a significantly higher prevalence of autoantibodies that target immunomodulatory proteins including cytokines, chemokines, complement components, and cell surface proteins in COVID-19 patients experiencing severe disease compared to those who experience mild or asymptomatic infections. Here we discuss the diverse impacts of autoantibodies on immune processes and associations with severe COVID-19 disease.
Assuntos
Autoanticorpos/imunologia , Autoanticorpos/metabolismo , COVID-19/complicações , COVID-19/imunologia , SARS-CoV-2/imunologia , Animais , Autoimunidade/fisiologia , COVID-19/metabolismo , Humanos , SARS-CoV-2/metabolismo , Síndrome de COVID-19 Pós-AgudaRESUMO
BACKGROUND: There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. METHODS: The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). RESULTS: None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. CONCLUSIONS: This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp- strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).
Assuntos
Antimaláricos , Vacinas Antimaláricas , Malária Falciparum , Malária , Antimaláricos/uso terapêutico , Artemeter/uso terapêutico , Combinação Arteméter e Lumefantrina/uso terapêutico , Austrália , Humanos , Malária/tratamento farmacológico , Vacinas Antimaláricas/efeitos adversos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/prevenção & controle , Masculino , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Desenvolvimento de Vacinas , Vacinas Atenuadas/efeitos adversosRESUMO
The CXCR3 chemokine CXCL10 or IFN-γ inducible protein 10 (IP-10) has been identified as an important biomarker of cerebral malaria (CM) mortality in children. Studies in mouse malaria infection models have shown that CXCL10 blockade alleviates brain intravascular inflammation and protects infected mice from CM. Despite the key role that CXCL10 plays in the development of CM, the leucocytic sources of CXCL10 in response to human malaria are not known. Here we investigated CXCL10 responses to Plasmodium falciparum in peripheral blood mononuclear cells (PBMCs). We found that PBMCs from malaria-unexposed donors produce CXCL10 in response to P. falciparum and that this response is IFN-γ-dependent. Moreover, CD14+ monocytes were identified as the main leucocytic sources of CXCL10 in peripheral blood, suggesting an important role for innate immune responses in the activation of this pathway involved in the development of symptomatic malaria.
Assuntos
Quimiocina CXCL10/metabolismo , Eritrócitos/imunologia , Interferon gama/metabolismo , Monócitos/imunologia , Plasmodium falciparum/fisiologia , Eritrócitos/parasitologia , HumanosRESUMO
Endocytosis is a major mechanism through which cellular signaling by G protein-coupled receptors (GPCRs) is terminated. However, recent studies demonstrate that GPCRs are internalized in an active state and continue to signal from within endosomes, resulting in effects on cellular function that are distinct to those arising at the cell surface. Endocytosis inhibitors are commonly used to define the importance of GPCR internalization for physiological and pathophysiological processes. Here, we provide the first detailed examination of the effects of these inhibitors on neurogenic contractions of gastrointestinal smooth muscle, a key preliminary step to evaluate the importance of GPCR endocytosis for gut function. Inhibitors of clathrin-mediated endocytosis (Pitstop2, PS2) or G protein-coupled receptor kinase-2/3-dependent phosphorylation (Takeda compound 101, Cmpd101), significantly reduced GPCR internalization. However, they also attenuated cholinergic contractions through different mechanisms. PS2 abolished contractile responses by colonic muscle to SNC80 and morphine, which strongly and weakly internalize δ-opioid and µ-opioid receptors, respectively. PS2 did not affect the increased myogenic contractile activity following removal of an inhibitory neural influence (tetrodotoxin) but suppressed electrically evoked neurogenic contractions. Ca2+ signaling by myenteric neurons in response to exogenous ATP was unaffected by PS2, suggesting inhibitory actions on neurotransmitter release rather than neurotransmission. In contrast, Cmpd101 attenuated contractions to the cholinergic agonist carbachol, indicating direct effects on smooth muscle. We conclude that, although PS2 and Cmpd101 are effective blockers of GPCR endocytosis in enteric neurons, these inhibitors are unsuitable for the study of neurally mediated gut function due to their inhibitory effects on neuromuscular transmission and smooth muscle contractility.NEW & NOTEWORTHY Internalization of activated G protein-coupled receptors is a major determinant of the type and duration of subsequent downstream signaling events. Inhibitors of endocytosis effectively block opioid receptor internalization in enteric neurons. The clathrin-dependent endocytosis inhibitor Pitstop2 blocks effects of opioids on neurogenic contractions of the colon in an internalization-independent manner. These inhibitors also significantly impact cholinergic neuromuscular transmission. We conclude that these tools are unsuitable for examination of the contribution of neuronal G protein-coupled receptor endocytosis to gastrointestinal motility.
Assuntos
Benzamidas/farmacologia , Clatrina/metabolismo , Colo , Endocitose , Músculo Liso , Piridinas/farmacologia , Receptores Opioides delta/metabolismo , Sulfonamidas/farmacologia , Tiazolidinas/farmacologia , Triazóis/farmacologia , Analgésicos Opioides/farmacologia , Animais , Colo/metabolismo , Colo/patologia , Colo/fisiopatologia , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Endossomos/metabolismo , Sistema Nervoso Entérico/metabolismo , Motilidade Gastrointestinal/fisiologia , Camundongos , Músculo Liso/metabolismo , Músculo Liso/patologia , Músculo Liso/fisiopatologia , Fosforilação/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides mu/metabolismo , Transdução de Sinais , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Pathogens can release extracellular vesicles (EVs) for cell-cell communication and host modulation. EVs from Plasmodium falciparum, the deadliest malaria parasite species, can transfer drug resistance genes between parasites. EVs from late-stage parasite-infected RBC (iRBC-EVs) are immunostimulatory and affect endothelial cell permeability, but little is known about EVs from early stage iRBC. We detected the parasite virulence factor PfEMP1, which is responsible for iRBC adherence and a major contributor to disease severity, in EVs, only up to 12-hr post-RBC invasion. Furthermore, using PfEMP1 transport knockout parasites, we determined that EVs originated from inside the iRBC rather than the iRBC surface. Proteomic analysis detected 101 parasite and 178 human proteins in iRBC-EVs. Primary human monocytes stimulated with iRBC-EVs released low levels of inflammatory cytokines and showed transcriptomic changes. Stimulation with iRBC-EVs from PfEMP1 knockout parasites induced more gene expression changes and affected pathways involved in defence response, stress response, and response to cytokines, suggesting a novel function of PfEMP1 when present in EVs. We show for the first time the presence of PfEMP1 in early stage P. falciparum iRBC-EVs and the effects of these EVs on primary human monocytes, uncovering a new mechanism of potential parasite pathogenesis and host interaction.
Assuntos
Malária Falciparum/genética , Plasmodium falciparum/genética , Proteômica , Proteínas de Protozoários/genética , Animais , Adesão Celular/genética , Comunicação Celular/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Eritrócitos/parasitologia , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Interações Hospedeiro-Parasita/genética , Humanos , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Monócitos/metabolismo , Monócitos/parasitologia , Plasmodium falciparum/patogenicidadeRESUMO
Malaria remains a significant worldwide public health problem. To address biological questions, researchers rely on the experimental murine model. For decades, chloroquine (CQ) and pyrimethamine (Pyr) have been used to clear Plasmodium infections in experimental animals using standardised accepted protocols and, because of this, drug-treated controls are rarely included. However, there is limited data available on the modulation of anti-malarial immunity, including generation of memory B cells, when these drugs are administered days after malaria infection. We investigated B cell responses to an important malaria glycolipid, glycosylphosphatidylinositol (GPI), and the hapten nitrophenol (NP), with or without standard CQ and Pyr treatment using the murine model. At day 14, CQ/Pyr treatment significantly suppressed the frequency of NP+IgG1+ memory B cells in NP-KLH-immunised mice. Furthermore, CQ/Pyr-treated NP-KLH-immunised mice did not have significantly higher cellular counts of NP+ B cells, germinal centre B cells, nor NP+IgG1+ memory B cells than naïve mice (CQ/Pyr treated and untreated). CQ/Pyr-treated GPI-KLH-immunised mice did not have significantly higher cellular counts of GPI+ B cells than naïve untreated mice. By day 28, this effect appeared to resolve since all immunised mice, whether treated or untreated, had significantly higher B cell proliferative responses than naïve mice (CQ/Pyr treated and untreated) for the majority of B cell phenotypes. The current study emphasises the potential for drug modulation of antigenic B cell responses when using standardised malaria treatment protocols in the experimental murine model. It is recommended that drug-treated controls are included when using experimental malaria infections to address biological questions.
Assuntos
Anticorpos Antiprotozoários/sangue , Antimaláricos/uso terapêutico , Linfócitos B/imunologia , Cloroquina/uso terapêutico , Glicosilfosfatidilinositóis/imunologia , Malária/tratamento farmacológico , Nitrofenóis/imunologia , Plasmodium/imunologia , Pirimetamina/uso terapêutico , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Antimaláricos/efeitos adversos , Cloroquina/efeitos adversos , Modelos Animais de Doenças , Combinação de Medicamentos , Feminino , Humanos , Imunização , Imunoglobulina G/imunologia , Malária/imunologia , Malária/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pirimetamina/efeitos adversosRESUMO
Immunity to Plasmodium falciparum malaria is slow to develop, and it is often asserted that malaria suppresses host immunity, although this is poorly understood and the molecular basis for such activity remains unknown. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a virulence factor that plays a key role in parasite-host interactions. We investigated the immunosuppressive effect of PfEMP1 on monocytes/macrophages, which are central to the antiparasitic innate response. RAW macrophages and human primary monocytes were stimulated with wild-type 3D7 or CS2 parasites or transgenic PfEMP1-null parasites. To study the immunomodulatory effect of PfEMP1, transcription factor activation and cytokine and chemokine responses were measured. The level of activation of NF-κB was significantly lower in macrophages stimulated with parasites that express PfEMP1 at the red blood cell surface membrane than in macrophages stimulated with PfEMP1-null parasites. Modulation of additional transcription factors, including CREB, also occurred, resulting in reduced immune gene expression and decreased tumor necrosis factor (TNF) and interleukin-10 (IL-10) release. Similarly, human monocytes released less IL-1ß, IL-6, IL-10, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), MIP-1ß, and TNF specifically in response to VAR2CSA PfEMP1-containing parasites than in response to PfEMP1-null parasites, suggesting that this immune regulation by PfEMP1 is important in naturally occurring infections. These results indicate that PfEMP1 is an immunomodulatory molecule that affects the activation of a range of transcription factors, dampening cytokine and chemokine responses. Therefore, these findings describe a potential molecular basis for immune suppression by P. falciparum.
Assuntos
Quimiocinas/metabolismo , Citocinas/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Idoso , Animais , Anticorpos Antiprotozoários/metabolismo , Antígenos de Protozoários/metabolismo , Linhagem Celular , Feminino , Regulação da Expressão Gênica/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Humanos , Macrófagos/microbiologia , Malária Falciparum/metabolismo , Malária Falciparum/microbiologia , Masculino , Camundongos , Pessoa de Meia-Idade , Monócitos/microbiologia , Fatores de Virulência/metabolismo , Adulto JovemRESUMO
Endogenous opioids activate opioid receptors (ORs) in the enteric nervous system to control intestinal motility and secretion. The µ-OR mediates the deleterious side effects of opioid analgesics, including constipation, respiratory depression, and addiction. Although the δ-OR (DOR) is a promising target for analgesia, the function and regulation of DOR in the colon are poorly understood. This study provides evidence that endogenous opioids activate DOR in myenteric neurons that may regulate colonic motility. The DOR agonists DADLE, deltorphin II, and SNC80 inhibited electrically evoked contractions and induced neurogenic contractions in the mouse colon. Electrical, chemical, and mechanical stimulation of the colon evoked the release of endogenous opioids, which stimulated endocytosis of DOR in the soma and proximal neurites of myenteric neurons of transgenic mice expressing DOR fused to enhanced green fluorescent protein. In contrast, DOR was not internalized in nerve fibers within the circular muscle. Administration of dextran sulfate sodium induced acute colitis, which was accompanied by DOR endocytosis and an increased density of DOR-positive nerve fibers within the circular muscle. The potency with which SNC80 inhibited neurogenic contractions was significantly enhanced in the inflamed colon. This study demonstrates that DOR-expressing neurons in the mouse colon can be activated by exogenous and endogenous opioids. Activated DOR traffics to endosomes and inhibits neurogenic motility of the colon. DOR signaling is enhanced during intestinal inflammation. This study demonstrates functional expression of DOR by myenteric neurons and supports the therapeutic targeting of DOR in the enteric nervous system. NEW & NOTEWORTHY DOR is activated during physiologically relevant reflex stimulation. Agonist-evoked DOR endocytosis is spatially and temporally regulated. A significant proportion of DOR is internalized in myenteric neurons during inflammation. The relative proportion of all myenteric neurons that expressed DOR and the overlap with the nNOS-positive population are increased in inflammation. DOR-specific innervation of the circular muscle is increased in inflammation, and this is consistent with enhanced responsiveness to the DOR agonist SNC80.
Assuntos
Colite Ulcerativa/metabolismo , Colo/metabolismo , Sistema Nervoso Entérico/metabolismo , Motilidade Gastrointestinal , Receptores Opioides delta/metabolismo , Animais , Benzamidas/farmacologia , Colo/fisiologia , Colo/fisiopatologia , Endocitose , Leucina Encefalina-2-Alanina/metabolismo , Sistema Nervoso Entérico/fisiologia , Sistema Nervoso Entérico/fisiopatologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular , Oligopeptídeos/metabolismo , Piperazinas/farmacologia , Receptores Opioides delta/agonistas , Receptores Opioides delta/genéticaRESUMO
HIV-1-specific T-cell responses in exposed seronegative subjects suggest that a viral breach of the exposure site is more common than current transmission rates would suggest and that host immunity can extinguish subsequent infection foci. The Preexposure Prophylaxis Initiative (iPrEx) chemoprophylaxis trial provided an opportunity to rigorously investigate these responses in a case-control immunology study; 84 preinfection peripheral blood mononuclear cell samples from individuals enrolled in the iPrEx trial who later seroconverted were matched with 480 samples from enrolled subjects who remained seronegative from both the placebo and active treatment arms. T-cell responses to HIV-1 Gag, Protease, Integrase, Reverse Transcriptase, Vif, and Nef antigens were quantified for all subjects in an IFN-γ enzyme-linked immunospot (ELISpot) assay. IFN-γ responses varied in magnitude and frequency across subjects. A positive response was more prevalent in those who remained persistently HIV-1-negative for Gag (P = 0.007), Integrase (P < 0.001), Vif (P < 0.001), and Nef (P < 0.001). When correlated with outcomes in the iPrEx trial, Vif- and Integrase-specific T-cell responses were associated with reduced HIV-1 infection risk [hazard ratio (HR) = 0.36, 95% confidence interval (95% CI) = 0.19-0.66 and HR = 0.52, 95% CI = 0.28-0.96, respectively]. Antigen-specific responses were independent of emtricitabine/tenofovir disoproxil fumarate use. IFN-γ secretion in the ELISpot was confirmed using multiparametric flow cytometry and largely attributed to effector memory CD4+ or CD8+ T cells. Our results show that HIV-1-specific T-cell immunity can be detected in exposed but uninfected individuals and that these T-cell responses can differentiate individuals according to infection outcomes.
Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Imunidade Celular/imunologia , Leucócitos Mononucleares/imunologia , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Infecções por HIV/sangue , Infecções por HIV/virologia , Soropositividade para HIV/imunologia , HIV-1/metabolismo , HIV-1/fisiologia , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Leucócitos Mononucleares/metabolismo , Modelos Logísticos , Masculino , Análise Multivariada , Adulto JovemRESUMO
BACKGROUND: γδ T cells are important for both protective immunity and immunopathogenesis during malaria infection. However, the immunological processes determining beneficial or detrimental effects on disease outcome remain elusive. The aim of this study was to examine expression and regulatory effect of the inhibitory receptor T-cell immunoglobulin domain and mucin domain 3 (TIM3) on γδ T cells. While TIM3 expression and function on conventional αß T cells have been clearly defined, the equivalent characterization on γδ T cells and associations with disease outcomes is limited. This study investigated the functional capacity of TIM3+ γδ T cells and the underlying mechanisms contributing to TIM3 upregulation and established an association with malaria disease outcomes. METHODS: We analyzed TIM3 expression on γδ T cells in 132 children aged 5-10 years living in malaria endemic areas of Papua New Guinea. TIM3 upregulation and effector functions of TIM3+ γδ T cells were assessed following in vitro stimulation with parasite-infected erythrocytes, phosphoantigen and/or cytokines. Associations between the proportion of TIM3-expressing cells and the molecular force of infection were tested using negative binomial regression and in a Cox proportional hazards model for time to first clinical episode. Multivariable analyses to determine the association of TIM3 and IL-18 levels were conducted using general linear models. Malaria infection mouse models were utilized to experimentally investigate the relationship between repeated exposure and TIM3 upregulation. RESULTS: This study demonstrates that even in the absence of an active malaria infection, children of malaria endemic areas have an atypical population of TIM3-expressing γδ T cells (mean frequency TIM3+ of total γδ T cells 15.2% ± 12). Crucial factors required for γδ T cell TIM3 upregulation include IL-12/IL-18, and plasma IL-18 was associated with TIM3 expression (P = 0.002). Additionally, we show a relationship between TIM3 expression and infection with distinct parasite clones during repeated exposure. TIM3+ γδ T cells were functionally impaired and were associated with asymptomatic malaria infection (hazard ratio 0.54, P = 0.032). CONCLUSIONS: Collectively our data demonstrate a novel role for IL-12/IL-18 in shaping the innate immune response and provide fundamental insight into aspects of γδ T cell immunoregulation. Furthermore, we show that TIM3 represents an important γδ T cell regulatory component involved in minimizing malaria symptoms.
Assuntos
Receptor Celular 2 do Vírus da Hepatite A/fisiologia , Interleucina-12/fisiologia , Interleucina-18/fisiologia , Malária/imunologia , Linfócitos T/imunologia , Animais , Criança , Pré-Escolar , Citocinas , Eritrócitos , Humanos , Interleucina-12/sangue , Interleucina-18/sangue , Camundongos , Papua Nova Guiné , Receptores de Antígenos de Linfócitos T gama-delta , RiscoRESUMO
In the past decade, research on the functions of extracellular vesicles in malaria has expanded dramatically. Investigations into the various vesicle types, from both host and parasite origin, has revealed important roles for extracellular vesicles in disease pathogenesis and susceptibility, as well as cell-cell communication and immune responses. Here, work relating to extracellular vesicles in malaria is reviewed, and the areas that remain unknown and require further investigations are highlighted.
Assuntos
Comunicação Celular , Vesículas Extracelulares/metabolismo , Malária/parasitologia , HumanosRESUMO
It is unclear whether naturally acquired immunity to Plasmodium falciparum results from the acquisition of antibodies to multiple, diverse antigens or to fewer, highly conserved antigens. Moreover, the specific antibody functions required for malaria immunity are unknown, and hence informative immunological assays are urgently needed to address these knowledge gaps and guide vaccine development. In this study, we investigated whether merozoite-opsonizing antibodies are associated with protection from malaria in a strain-specific or strain-transcending manner by using a novel field isolate and an immune plasma-matched cohort from Papua New Guinea with our validated assay of merozoite phagocytosis. Highly correlated opsonization responses were observed across the 15 parasite strains tested, as were strong associations with protection (composite phagocytosis score across all strains in children uninfected at baseline: hazard ratio of 0.15, 95% confidence interval of 0.04 to 0.63). Opsonizing antibodies had a strong strain-transcending component, and the opsonization of transgenic parasites deficient for MSP3, MSP6, MSPDBL1, or P. falciparum MSP1-19 (PfMSP1-19) was similar to that of wild-type parasites. We have provided the first evidence that merozoite opsonization is predominantly strain transcending, and the highly consistent associations with protection against diverse parasite strains strongly supports the use of merozoite opsonization as a correlate of immunity for field studies and vaccine trials. These results demonstrate that conserved domains within merozoite antigens targeted by opsonization generate strain-transcending immune responses and represent promising vaccine candidates.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Merozoítos/imunologia , Proteínas Opsonizantes/imunologia , Plasmodium falciparum/imunologia , Adolescente , Anticorpos Antiprotozoários/sangue , Criança , Pré-Escolar , Humanos , Malária Falciparum/sangue , Avaliação de Resultados da Assistência ao Paciente , Fagocitose/imunologiaRESUMO
Activated G protein-coupled receptors traffic to endosomes and are sorted to recycling or degradative pathways. Endosomes are also a site of receptor signaling of sustained and pathophysiologically important processes, including inflammation. However, the mechanisms of endosomal sorting of receptors and the impact of disease on trafficking have not been fully defined. We examined the effects of inflammation on the subcellular distribution and trafficking of the substance P (SP) neurokinin 1 receptor (NK1R) in enteric neurons. We studied NK1R trafficking in enteric neurons of the mouse colon using immunofluorescence and confocal microscopy. The impact of inflammation was studied in IL10(-/-)-piroxicam and trinitrobenzenesulfonic acid colitis models. NK1R was localized to the plasma membrane of myenteric and submucosal neurons of the uninflamed colon. SP evoked NK1R endocytosis and recycling. Deletion of ß-arrestin2, which associates with the activated NK1R, accelerated recycling. Inhibition of endothelin-converting enzyme-1 (ECE-1), which degrades endosomal SP, prevented recycling. Inflammation was associated with NK1R endocytosis in myenteric but not submucosal neurons. Whereas the NK1R in uninflamed neurons recycled within 60 min, NK1R recycling in inflamed neurons was delayed for >120 min, suggesting defective recycling machinery. Inflammation was associated with ß-arrestin2 upregulation and ECE-1 downregulation, which may contribute to the defective NK1R recycling. We conclude that inflammation evokes redistribution of NK1R from the plasma membrane to endosomes of myenteric neurons through enhanced SP release and defective NK1R recycling. Defective recycling may be secondary to upregulation of ß-arrestin2 and downregulation of ECE-1. Internalized NK1R may generate sustained proinflammatory signals that disrupt normal neuronal functions.
Assuntos
Colite Ulcerativa/metabolismo , Endossomos/metabolismo , Sistema Nervoso Entérico/metabolismo , Receptores da Neurocinina-1/metabolismo , Animais , Arrestinas/genética , Arrestinas/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Endocitose , Enzimas Conversoras de Endotelina , Inflamação/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico , beta-ArrestinasRESUMO
BACKGROUND: Severe malaria (SM) is associated with high levels of cytokines such as tumor necrosis factor (TNF), interleukin 1 (IL-1), and interleukin 6 (IL-6). The role of chemokines is less clear, as is their cellular source. METHODS: In a case-control study of children with SM (n = 200), uncomplicated malaria (UM) (n = 153) and healthy community controls (HC) (n = 162) in Papua, New Guinea, we measured cytokine/chemokine production by peripheral blood mononuclear cells (PBMCs) stimulated with live Plasmodium falciparum parasitized red blood cells (pRBC). Cellular sources were determined. Associations between immunological endpoints and clinical/parasitological variables were tested. RESULTS: Compared to HC and UM, children with SM produced significantly higher IL-10, IP-10, MIP-1ßm and MCP-2. TNF and MIP-1α were significantly higher in the SM compared to the UM group. IL-10, IL-6, MIP-1α, MIP-1ß, and MCP-2 were associated with increased odds of SM. SM syndromes were associated with distinct cytokine/chemokine response profiles compared to UM cases. TNF, MIP-1ß, and MIP-1α were produced predominantly by monocytes and γδ T cells, and IL-10 by CD4(+) T cells. CONCLUSIONS: Early/innate PBMC responses to pRBC in vitro are informative as to cytokines/chemokines associated with SM. Predominant cellular sources are monocytes and γδ T cells. Monocyte-derived chemokines support a role for monocyte infiltrates in the etiology of SM.
Assuntos
Citocinas/metabolismo , Malária Falciparum/imunologia , Monócitos/imunologia , Plasmodium falciparum/imunologia , Linfócitos T/imunologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Receptores de Lipopolissacarídeos/análise , Masculino , Monócitos/química , Papua Nova Guiné , Receptores de Antígenos de Linfócitos T gama-delta/análise , Linfócitos T/químicaRESUMO
Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with ß-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and ß-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/ß-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from ß-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained ß-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.
Assuntos
Sistema Nervoso Entérico/metabolismo , Neurônios/metabolismo , Proteólise , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/metabolismo , Somatostatina-28/metabolismo , Somatostatina/metabolismo , Animais , Arrestinas/genética , Arrestinas/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Linhagem Celular Tumoral , Endossomos/genética , Endossomos/metabolismo , Enzimas Conversoras de Endotelina , Feminino , Fármacos Gastrointestinais/farmacologia , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Masculino , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Camundongos , Octreotida/farmacocinética , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Receptores de Somatostatina/genética , Somatostatina/genética , Somatostatina-28/genética , beta-ArrestinasRESUMO
The substance P neurokinin 1 receptor (NK1R) regulates motility, secretion, inflammation and pain in the intestine. The distribution of the NK1R is a key determinant of the functional effects of substance P in the gut. Information regarding the distribution of NK1R in subtypes of mouse enteric neurons is lacking and is the focus of the present study. NK1R immunoreactivity (NK1R-IR) is examined in whole-mount preparations of the mouse distal colon by indirect immunofluorescence and confocal microscopy. The distribution of NK1R-IR within key functional neuronal subclasses was determined by using established neurochemical markers. NK1R-IR was expressed by a subpopulation of myenteric and submucosal neurons; it was mainly detected in large multipolar myenteric neurons and was colocalized with calcitonin gene-related peptide, neurofilament M, choline acetyltransferase and calretinin. The remaining NK1R-immunoreactive neurons were positive for nitric oxide synthase. NK1R was expressed by most of the submucosal neurons and was exclusively co-expressed with vasoactive intestinal peptide, with no overlap with choline acetyltransferase. Treatment with substance P resulted in the concentration-dependent internalisation of NK1R from the cell surface into endosome-like structures. Myenteric NK1R was mainly expressed by intrinsic primary afferent neurons, with minor expression by descending interneurons and inhibitory motor neurons. Submucosal NK1R was restricted to non-cholinergic secretomotor neurons. These findings highlight key differences in the neuronal distribution of NK1R-IR between the mouse, rat and guinea-pig, with important implications for the functional role of NK1R in regulating intestinal motility and secretion.
Assuntos
Colo/inervação , Sistema Nervoso Entérico/metabolismo , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Animais , Anticorpos/imunologia , Calbindina 2/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Colina O-Acetiltransferase/biossíntese , Colo/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Trato Gastrointestinal/inervação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neurofilamentos/metabolismo , Óxido Nítrico Sintase/metabolismo , Receptores da Neurocinina-1/biossíntese , Receptores da Neurocinina-1/imunologia , Peptídeo Intestinal Vasoativo/biossínteseRESUMO
Sema4D, also known as CD100, is a constitutively expressed immune semaphorin on T cells and NK cells. CD100 has important immune regulatory functions that improve antigen-specific priming by antigen-presenting cells, and can also act as a costimulatory molecule on T cells. We investigated the consequence of HIV-1 infection on CD100 expression by T cells, and whether CD100 expression signifies functionally competent effector cells. CD100 expression on T cells from healthy individuals was compared with HIV-1-infected subjects including elite controllers, noncontrollers, and patients receiving antiretroviral therapy. The frequency and fluorescence intensity of CD100 on CD8(+) and CD4(+) T cells were decreased during HIV-1 infection. Furthermore, the absolute number of CD100-expressing CD8(+) T cells was positively associated with the magnitude of HIV-1-specific T-cell responses. CD8(+) T cells lacking CD100 expression were functionally impaired and present in increased numbers in HIV-1-infected individuals. The number of CD100(-)CD8(+) T cells positively correlated with T-cell immunosenescence, immune activation, and viral load. Loss of CD100 expression appears to result from direct antigen stimulation, as in vitro cytokine exposure and viral replication did not significantly impact CD100 expression. These data suggest that loss of CD100 expression probably plays an important role in dysfunctional immunity in HIV-1 infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Semaforinas/deficiência , Células Apresentadoras de Antígenos , Antígenos CD , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD8-Positivos/metabolismo , Estudos de Casos e Controles , Estudos Transversais , Citocinas/metabolismo , Citometria de Fluxo , Infecções por HIV/tratamento farmacológico , Humanos , Ativação Linfocitária , Carga Viral , Replicação ViralRESUMO
Background: Mechanosensation is an important trigger of physiological processes in the gastrointestinal tract. Aberrant responses to mechanical input are associated with digestive disorders, including visceral hypersensitivity. Transient Receptor Potential Vanilloid 4 (TRPV4) is a mechanosensory ion channel with proposed roles in visceral afferent signaling, intestinal inflammation, and gut motility. While TRPV4 is a potential therapeutic target for digestive disease, current mechanistic understanding of how TRPV4 may influence gut function is limited by inconsistent reports of TRPV4 expression and distribution. Methods: In this study we profiled functional expression of TRPV4 using Ca2+ imaging of wholemount preparations of the mouse, monkey, and human intestine in combination with immunofluorescent labeling for established cellular markers. The involvement of TRPV4 in colonic motility was assessed in vitro using videomapping and contraction assays. Results: The TRPV4 agonist GSK1016790A evoked Ca2+ signaling in muscularis macrophages, enteric glia, and endothelial cells. TRPV4 specificity was confirmed using TRPV4 KO mouse tissue or antagonist pre-treatment. Calcium responses were not detected in other cell types required for neuromuscular signaling including enteric neurons, interstitial cells of Cajal, PDGFRα+ cells, and intestinal smooth muscle. TRPV4 activation led to rapid Ca2+ responses by a subpopulation of glial cells, followed by sustained Ca2+ signaling throughout the enteric glial network. Propagation of these waves was suppressed by inhibition of gap junctions or Ca2+ release from intracellular stores. Coordinated glial signaling in response to GSK1016790A was also disrupted in acute TNBS colitis. The involvement of TRPV4 in the initiation and propagation of colonic motility patterns was examined in vitro. Conclusions: We reveal a previously unappreciated role for TRPV4 in the initiation of distension-evoked colonic motility. These observations provide new insights into the functional role of TRPV4 activation in the gut, with important implications for how TRPV4 may influence critical processes including inflammatory signaling and motility.