RESUMO
Despite extensive mapping of long noncoding RNAs in immune cells, their function in vivo remains poorly understood. In this study, we identify over 100 long noncoding RNAs that are differentially expressed within 24 h of Th1 cell activation. Among those, we show that suppression of Malat1 is a hallmark of CD4+ T cell activation, but its complete deletion results in more potent immune responses to infection. This is because Malat1-/- Th1 and Th2 cells express lower levels of the immunosuppressive cytokine IL-10. In vivo, the reduced CD4+ T cell IL-10 expression in Malat1-/- mice underpins enhanced immunity and pathogen clearance in experimental visceral leishmaniasis (Leishmania donovani) but more severe disease in a model of malaria (Plasmodium chabaudi chabaudi AS). Mechanistically, Malat1 regulates IL-10 through enhancing expression of Maf, a key transcriptional regulator of IL-10 Maf expression correlates with Malat1 in single Ag-specific Th cells from P. chabaudi chabaudi AS-infected mice and is downregulated in Malat1-/- Th1 and Th2 cells. The Malat1 RNA is responsible for these effects, as antisense oligonucleotide-mediated inhibition of Malat1 also suppresses Maf and IL-10 levels. Our results reveal that through promoting expression of the Maf/IL-10 axis in effector Th cells, Malat1 is a nonredundant regulator of mammalian immunity.
Assuntos
Interleucina-10/metabolismo , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , Proteínas Proto-Oncogênicas c-maf/metabolismo , RNA Longo não Codificante/genética , Células Th1/imunologia , Células Th2/imunologia , Animais , Feminino , Regulação da Expressão Gênica , Humanos , Tolerância Imunológica , Imunidade/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-maf/genética , Regulação para CimaRESUMO
Determining the mechanisms that distinguish protective immunity from pathological chronic inflammation remains a fundamental challenge. miR-132 has been shown to play largely immunoregulatory roles in immunity; however, its role in CD4+ T cell function is poorly understood. Here, we show that CD4+ T cells express high levels of miR-132 and that T cell activation leads to miR-132 up-regulation. The transcriptomic hallmark of splenic CD4+ T cells lacking the miR-132/212 cluster during chronic infection is an increase in mRNA levels of ribosomal protein (RP) genes. BTAF1, a co-factor of B-TFIID and novel miR-132/212-3p target, and p300 contribute towards miR-132/212-mediated regulation of RP transcription. Following infection with Leishmania donovani, miR-132-/- CD4+ T cells display enhanced expression of IL-10 and decreased IFNγ. This is associated with reduced hepatosplenomegaly and enhanced pathogen load. The enhanced IL-10 expression in miR-132-/- Th1 cells is recapitulated in vitro following treatment with phenylephrine, a drug reported to promote ribosome synthesis. Our results uncover that miR-132/212-mediated regulation of RP expression is critical for optimal CD4+ T cell activation and protective immunity against pathogens.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Interferência de RNA , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Animais , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/biossíntese , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Ligação Proteica , Baço/imunologia , Baço/metabolismo , Baço/microbiologia , Fator de Transcrição TFIID/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Visceral leishmaniasis is associated with significant changes in hematological function but the mechanisms underlying these changes are largely unknown. In contrast to naïve mice, where most long-term hematopoietic stem cells (LT-HSCs; LSK CD150+ CD34- CD48- cells) in bone marrow (BM) are quiescent, we found that during Leishmania donovani infection most LT-HSCs had entered cell cycle. Loss of quiescence correlated with a reduced self-renewal capacity and functional exhaustion, as measured by serial transfer. Quiescent LT-HSCs were maintained in infected RAG2 KO mice, but lost following adoptive transfer of IFNγ-sufficient but not IFNγ-deficient CD4+ T cells. Using mixed BM chimeras, we established that IFNγ and TNF signalling pathways converge at the level of CD4+ T cells. Critically, intrinsic TNF signalling is required for the expansion and/or differentiation of pathogenic IFNγ+CD4+ T cells that promote the irreversible loss of BM function. These findings provide new insights into the pathogenic potential of CD4+ T cells that target hematopoietic function in leishmaniasis and perhaps other infectious diseases where TNF expression and BM dysfunction also occur simultaneously.
Assuntos
Células da Medula Óssea/citologia , Linfócitos T CD4-Positivos/citologia , Células-Tronco Hematopoéticas/citologia , Leishmania donovani/fisiologia , Leishmaniose Visceral/fisiopatologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células da Medula Óssea/metabolismo , Ciclo Celular , Proliferação de Células , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND & AIMS: Kupffer cells (KCs), the resident tissue macrophages of the liver, play a crucial role in the clearance of pathogens and other particulate materials that reach the systemic circulation. Recent studies have identified KCs as a yolk sac-derived resident macrophage population that is replenished independently of monocytes in the steady state. Although it is now established that following local tissue injury, bone marrow derived monocytes may infiltrate the tissue and differentiate into macrophages, the extent to which newly differentiated macrophages functionally resemble the KCs they have replaced has not been extensively studied. METHODS: We studied the two populations of KCs using intravital microscopy, morphometric analysis and gene expression profiling. An ion homeostasis gene signature, including genes associated with scavenger receptor function and extracellular matrix deposition, allowed discrimination between these two KC sub-types. RESULTS: Bone marrow derived "KCs" accumulating as a result of genotoxic injury, resemble but are not identical to their yolk sac counterparts. Reflecting the differential expression of scavenger receptors, yolk sac-derived KCs were more effective at accumulating acetylated low density lipoprotein, whereas surprisingly, they were poorer than bone marrow-derived KCs when assessed for uptake of a range of bacterial pathogens. The two KC populations were almost indistinguishable in regard to i) response to lipopolysaccharide challenge, ii) phagocytosis of effete red blood cells and iii) their ability to contain infection and direct granuloma formation against Leishmania donovani, a KC-tropic intracellular parasite. CONCLUSIONS: Bone marrow-derived KCs differentiate locally to resemble yolk sac-derived KC in most but not all respects, with implications for models of infectious diseases, liver injury and bone marrow transplantation. In addition, the gene signature we describe adds to the tools available for distinguishing KC subpopulations based on their ontology. LAY SUMMARY: Liver macrophages play a major role in the control of infections in the liver and in the pathology associated with chronic liver diseases. It was recently shown that liver macrophages can have two different origins, however, the extent to which these populations are functionally distinct remains to be fully addressed. Our study demonstrates that whilst liver macrophages share many features in common, regardless of their origin, some subtle differences in function exist. DATA REPOSITORY: Gene expression data are available from the European Bioinformatics Institute ArrayExpress data repository (accession number E-MTAB-4954).
Assuntos
Medula Óssea , Humanos , Células de Kupffer , Fígado , Macrófagos , MonócitosRESUMO
It is now recognized that innate immunity to intestinal microflora plays a significant role in mediating immune health, and modulation of microbial sensing may underpin the impact of plant natural products in the diet or when used as nutraceuticals. In this context, we have examined five classes of plant-derived flavonoids (flavonols, flavones, flavanones, catechins, and cyanidin) for their ability to regulate cytokine release induced by the Toll-like receptor 2 (TLR2) agonist Pam3CSK4. We found that the flavonols selectively co-stimulated IL-1ß secretion but had no impact on the secretion of IL-6. Importantly, this costimulation of TLR2-induced cytokine secretion was dependent on regiospecific methylation of the flavonol scaffold with a rank order of quercetin-3,4'-dimethylether > quercetin-3-methylether > casticin. The mechanism underpinning this costimulation did not involve enhanced inflammasome activation. In contrast, the methylated flavonols enhanced IL-1ß gene expression through transcriptional regulation, involving mechanisms that operate downstream of the initial NF-κB and STAT1 activation events. These studies demonstrate an exquisite level of control of scaffold bioactivity by regiospecific methylation, with important implications for understanding how natural products affect innate immunity and for their development as novel immunomodulators for clinical use.
Assuntos
Flavonoides/química , Interleucina-1beta/biossíntese , Monócitos/metabolismo , Receptor 2 Toll-Like/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Cicloeximida/farmacologia , Sinergismo Farmacológico , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopeptídeos/farmacologia , Metilação/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Fosforilação/efeitos dos fármacos , Quercetina/análogos & derivados , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estereoisomerismo , Receptor 2 Toll-Like/agonistas , Transcrição Gênica/efeitos dos fármacosRESUMO
IL-10 is a critical regulatory cytokine involved in the pathogenesis of visceral leishmaniasis caused by Leishmania donovani and clinical and experimental data indicate that disease progression is associated with expanded numbers of CD4⺠IFNγ⺠T cells committed to IL-10 production. Here, combining conditional cell-specific depletion with adoptive transfer, we demonstrate that only conventional CD11c(hi) DCs that produce both IL-10 and IL-27 are capable of inducing IL-10-producing Th1 cells in vivo. In contrast, CD11c(hi) as well as CD11c(int/lo) cells isolated from infected mice were capable of reversing the host protective effect of diphtheria toxin-mediated CD11c⺠cell depletion. This was reflected by increased splenomegaly, inhibition of NO production and increased parasite burden. Thus during chronic infection, multiple CD11c⺠cell populations can actively suppress host resistance and enhance immunopathology, through mechanisms that do not necessarily involve IL-10-producing Th1 cells.
Assuntos
Antígeno CD11c/análise , Interleucina-10/biossíntese , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Células Th1/imunologia , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Toxina Diftérica , Progressão da Doença , Interleucina-17/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Baço/parasitologiaRESUMO
Therapeutic vaccines, when used alone or in combination therapy with antileishmanial drugs, may have an important place in the control of a variety of forms of human leishmaniasis. Here, we describe the development of an adenovirus-based vaccine (Ad5-KH) comprising a synthetic haspb gene linked to a kmp11 gene via a viral 2A sequence. In nonvaccinated Leishmania donovani-infected BALB/c mice, HASPB- and KMP11-specific CD8(+) T cell responses were undetectable, although IgG1 and IgG2a antibodies were evident. After therapeutic vaccination, antibody responses were boosted, and IFNγ(+)CD8(+) T cell responses, particularly to HASPB, became apparent. A single vaccination with Ad5-KH inhibited splenic parasite growth by â¼66%, a level of efficacy comparable to that observed in early stage testing of clinically approved antileishmanial drugs in this model. These studies indicate the usefulness of adenoviral vectors to deliver leishmanial antigens in a potent and host protective manner to animals with existing L. donovani infection.
Assuntos
Antígenos de Protozoários/imunologia , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Vacinas de DNA/uso terapêutico , Adenoviridae , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Linfócitos T CD8-Positivos , Mapeamento de Epitopos , Epitopos de Linfócito T , Feminino , Citometria de Fluxo , Imunoglobulina G/sangue , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Baço/parasitologia , Vacinas de DNA/genéticaRESUMO
Highly phagocytic macrophages line the marginal zone (MZ) of the spleen and the lymph node subcapsular sinus. Although these macrophages have been attributed with a variety of functions, including the uptake and clearance of blood and lymph-borne pathogens, little is known about the effector mechanisms they employ after pathogen uptake. Here, we have combined gene expression profiling and RNAi using a stromal macrophage cell line with in situ analysis of the leishmanicidal activity of marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM) in wild type and gene targeted mice. Our data demonstrate a critical role for interferon regulatory factor-7 (IRF-7) in regulating the killing of intracellular Leishmania donovani by these specialised splenic macrophage sub-populations. This study, therefore, identifies a new role for IRF-7 as a regulator of innate microbicidal activity against this, and perhaps other, non-viral intracellular pathogens. This study also highlights the importance of selecting appropriate macrophage populations when studying pathogen interactions with this functionally diverse lineage of cells.
Assuntos
Fator Regulador 7 de Interferon/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Animais , Expressão Gênica/imunologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Interferon-alfa/imunologia , Interferon-alfa/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Fagossomos/imunologia , RNA Interferente Pequeno , Baço/imunologia , Baço/parasitologiaRESUMO
Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+) T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+) T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.
Assuntos
Linfócitos T CD8-Positivos/parasitologia , Granuloma/parasitologia , Células de Kupffer/parasitologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Movimento Celular/imunologia , Granuloma/imunologia , Células de Kupffer/imunologia , Leishmania donovani/genética , Leishmania donovani/crescimento & desenvolvimento , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/parasitologia , Fígado/citologia , Fígado/imunologia , Fígado/parasitologia , Macrófagos/imunologia , Macrófagos/parasitologia , Complexo Principal de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fagocitose/imunologiaRESUMO
Progressive splenomegaly is a hallmark of visceral leishmaniasis in humans, canids, and rodents. In experimental murine visceral leishmaniasis, splenomegaly is accompanied by pronounced changes in microarchitecture, including expansion of the red pulp vascular system, neovascularization of the white pulp, and remodeling of the stromal cell populations that define the B-cell and T-cell compartments. Here, we show that Ly6C/G(+) (Gr-1(+)) cells, including neutrophils and inflammatory monocytes, accumulate in the splenic red pulp during infection. Cell depletion using monoclonal antibody against either Ly6C/G(+) (Gr-1; RB6) or Ly6G(+) (1A8) cells increased parasite burden. In contrast, depletion of Ly6C/G(+) cells, but not Ly6G(+) cells, halted the progressive remodeling of Meca-32(+) and CD31(+) red pulp vasculature. Strikingly, neither treatment affected white pulp neovascularization or the remodeling of the fibroblastic reticular cell and follicular dendritic cell networks. These findings demonstrate a previously unrecognized compartment-dependent selectivity to the process of splenic vascular remodeling during experimental murine visceral leishmaniasis, attributable to Ly6C(+) inflammatory monocytes.
Assuntos
Antígenos Ly/metabolismo , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Neovascularização Patológica , Baço/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Linfócitos B/imunologia , Linfócitos B/parasitologia , Linfócitos B/patologia , Western Blotting , Células Cultivadas , Cricetinae , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Células Dendríticas/patologia , Feminino , Fibroblastos/imunologia , Fibroblastos/parasitologia , Fibroblastos/patologia , Citometria de Fluxo , Técnicas Imunoenzimáticas , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Macrófagos/imunologia , Macrófagos/parasitologia , Macrófagos/patologia , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Monócitos/parasitologia , Monócitos/patologia , Neutrófilos/imunologia , Neutrófilos/parasitologia , Neutrófilos/patologia , Baço/parasitologia , Baço/patologia , EsplenomegaliaRESUMO
Optimal hepatic resistance to Leishmania donovani in mice requires the coordinated effort of a variety of leukocyte populations that together induce activation of local macrophages to a leishmanicidal state. Although nitric oxide and reactive oxygen intermediates are potent leishmanicidal effector molecules operating in the acquired phase of immunity, there have long been suggestions that other mechanisms of leishmanicidal activity exist. We recently discovered that Irf-7 regulates a novel innate leishmanicidal response in resident splenic macrophages that line the marginal zone. Here, we tested whether this mechanism also operates in Kupffer cells, the resident macrophage population of the liver and the major target for hepatic infection by L. donovani. Comparing the Kupffer cell responses in situ in B6 and B6.Irf-7(-/-) mice, we found no evidence that Irf-7 affected amastigote uptake or early survival. However, we did find that Irf-7-deficient mice had impaired acquired resistance to hepatic L. donovani infection. This phenotype was attributable to a reduction in the capacity of hepatic CD4(+) T cells, NK cells, and NKT cells to produce gamma interferon (IFN-γ) and also to defective induction of NOS2 in infected Kupffer cells. Our data therefore add interferon regulatory factor 7 (IRF-7) to the growing list of interferon regulatory factors that have effects on downstream events in the acquired cellular immune response to nonviral pathogens.
Assuntos
Fator Regulador 7 de Interferon/imunologia , Células de Kupffer/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Fígado/imunologia , Animais , Separação Celular , Citometria de Fluxo , Fator Regulador 7 de Interferon/metabolismo , Células de Kupffer/metabolismo , Leishmania donovani/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Signal regulatory protein alpha (SIRPalpha) and its cognate ligand CD47 have been documented to have a broad range of cellular functions in development and immunity. Here, we investigated the role of SIRPalpha-CD47 signalling in invariant NKT (iNKT) cell responses. We found that CD47 was required for the optimal production of IFN-gamma from splenic iNKT cells following exposure to the alphaGalCer analogue PBS-57 and in vivo infection of mice with Leishmania donovani. Surprisingly, although SIRPalpha was undetectable in the liver of uninfected mice, the hepatic iNKT-cell response to infection was also impaired in CD47-/- mice. However, we found that SIRPalpha was rapidly induced on Kupffer cells following L. donovani infection, via a mechanism involving G-protein-coupled receptors. Thus, we describe a novel amplification pathway affecting cytokine production by hepatic iNKT cells, which may facilitate the breakdown of hepatic tolerance after infection.
Assuntos
Células de Kupffer/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Fígado/imunologia , Ativação Linfocitária , Células T Matadoras Naturais/imunologia , Receptores Imunológicos/imunologia , Animais , Antígeno CD47/genética , Antígeno CD47/imunologia , Tolerância Imunológica , Camundongos , Camundongos KnockoutRESUMO
Background: Polyparasitism is commonplace in countries where endemicity for multiple parasites exists, and studies in animal models of coinfection have made significant inroads into understanding the impact of often competing demands on the immune system. However, few studies have addressed how previous exposure to and treatment for one infection impacts a subsequent heterologous infection. Methods: We used a C57BL/6 mouse model of drug-treated Leishmania donovani infection followed by experimental Plasmodium chabaudi AS malaria, focusing on hematological dysfunction as a common attribute of both infections. We measured parasite burden, blood parameters associated with anemia and thrombocytopenia, and serum thrombopoietin. In addition, we quantified macrophage iNOS expression through immunohistological analysis of the liver and spleen. Results: We found that the thrombocytopenia and anemia that accompanies primary L. donovani infection was rapidly reversed following single dose AmBisome® treatment, along with multiple other markers associated with immune activation (including restoration of tissue microarchitecture and reduced macrophage iNOS expression). Compared to naive mice, mice cured of previous VL showed comparable albeit delayed clinical responses (including peak parasitemia and anemia) to P. chabaudi AS infection. Thrombocytopenia was also evident in these sequentially infected mice, consistent with a decrease in circulating levels of thrombopoietin. Architectural changes to the spleen were also comparable in sequentially infected mice compared to those with malaria alone. Conclusions: Our data suggest that in this sequential infection model, previously-treated VL has limited impact on the subsequent development of malaria, but this issue deserves further attention in models of more severe disease or through longitudinal population studies in humans.
RESUMO
Visceral leishmaniasis is an important yet neglected parasitic disease caused by infection with Leishmania donovani or L infantum. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulation, and extensive hematological complications. Thrombocytopenia is a dominant hematological feature seen in both humans and experimental models, but the mechanisms behind this infection-driven thrombocytopenia remain poorly understood. Using a murine model of experimental visceral leishmaniasis (EVL), we demonstrated a progressive decrease in platelets from day 14 after infection, culminating in severe thrombocytopenia by day 28. Plasma thrombopoietin (TPO) levels were reduced in infected mice, at least in part because of the alterations in the liver microenvironment associated with granulomatous inflammation. Bone marrow (BM) megakaryocyte cytoplasmic maturation was significantly reduced. In addition to a production deficit, we identified significant increases in platelet clearance. L donovani-infected splenectomized mice were protected from thrombocytopenia compared with sham operated infected mice and had a greater response to exogenous TPO. Furthermore, infection led to higher levels of platelet opsonization and desialylation, both associated with platelet clearance in spleen and liver, respectively. Critically, these changes could be reversed rapidly by drug treatment to reduce parasite load or by administration of TPO agonists. In summary, our findings demonstrate that the mechanisms underpinning thrombocytopenia in EVL are multifactorial and reversible, with no obvious residual damage to the BM microenvironment.
Assuntos
Leishmaniose Visceral , Trombocitopenia , Animais , Modelos Animais de Doenças , Leishmaniose Visceral/complicações , Leishmaniose Visceral/tratamento farmacológico , Megacariócitos , Camundongos , TrombopoetinaRESUMO
Increasing evidence suggests that in hosts infected with parasites of the Leishmania donovani complex, transmission of infection to the sand fly vector is linked to parasite repositories in the host skin. However, a detailed understanding of the dispersal (the mechanism of spread) and dispersion (the observed state of spread) of these obligatory-intracellular parasites and their host phagocytes in the skin is lacking. Using endogenously fluorescent parasites as a proxy, we apply image analysis combined with spatial point pattern models borrowed from ecology to characterize dispersion of parasitized myeloid cells (including ManR+ and CD11c+ cells) and predict dispersal mechanisms in a previously described immunodeficient model of L. donovani infection. Our results suggest that after initial seeding of infection in the skin, heavily parasite-infected myeloid cells are found in patches that resemble innate granulomas. Spread of parasites from these initial patches subsequently occurs through infection of recruited myeloid cells, ultimately leading to self-propagating networks of patch clusters. This combination of imaging and ecological pattern analysis to identify mechanisms driving the skin parasite landscape offers new perspectives on myeloid cell behavior following parasitism by L. donovani and may also be applicable to elucidating the behavior of other intracellular tissue-resident pathogens and their host cells.
Assuntos
Processamento de Imagem Assistida por Computador , Leishmania donovani/patogenicidade , Leishmaniose Visceral/parasitologia , Microscopia Confocal , Microscopia de Fluorescência , Células Mieloides/parasitologia , Pele/parasitologia , Análise Espacial , Animais , Antígenos CD11/metabolismo , Análise por Conglomerados , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Interações Hospedeiro-Parasita , Insetos Vetores/parasitologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/transmissão , Receptor de Manose/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Teóricos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Phlebotomus/parasitologia , Pele/imunologia , Pele/metabolismoRESUMO
Ly6Chi inflammatory monocytes develop in the bone marrow and migrate to the site of infection during inflammation. Upon recruitment, Ly6Chi monocytes can differentiate into dendritic cells or macrophages. According to the tissue environment they can also acquire different functions. Several studies have described pre-activation of Ly6Chi monocytes in the bone marrow during parasitic infection, but whether this process occurs during experimental visceral leishmaniasis and, if so, the mechanisms contributing to their activation are yet to be established. In wild type C57BL/6 (B6) mice infected with Leishmania donovani, the number of bone marrow Ly6Chi monocytes increased over time. Ly6Chi monocytes displayed a highly activated phenotype from 28 days to 5 months post infection (p.i), with >90% expressing MHCII and >20% expressing iNOS. In comparison, in B6.Rag2-/- mice <10% of bone marrow monocytes were MHCII+ at day 28 p.i., an activation deficiency that was reversed by adoptive transfer of CD4+ T cells. Depletion of CD4+ T cells in B6 mice and the use of mixed bone marrow chimeras further indicated that monocyte activation was driven by IFNγ produced by CD4+ T cells. In B6.Il10-/- mice, L. donovani infection induced a faster but transient activation of bone marrow monocytes, which correlated with the magnitude of CD4+ T cell production of IFNγ and resolution of the infection. Under all of the above conditions, monocyte activation was associated with greater control of parasite load in the bone marrow. Through reinfection studies in B6.Il10-/- mice and drug (AmBisome®) treatment of B6 mice, we also show the dependence of monocyte activation on parasite load. In summary, these data demonstrate that during L. donovani infection, Ly6Chi monocytes are primed in the bone marrow in a process driven by CD4+ T cells and whereby IFNγ promotes and IL-10 limits monocyte activation and that the presence of parasites/parasite antigen plays a crucial role in maintaining bone marrow monocyte activation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Interferon gama/imunologia , Leishmaniose Visceral/imunologia , Monócitos/imunologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Lentiviral vectors deliver antigens to dendritic cells (DCs) in vivo, but they do not trigger DC maturation. We therefore expressed a viral protein that constitutively activates NF-kappaB, vFLIP from Kaposi's sarcoma-associated herpesvirus (KSHV), in a lentivector to mature DCs. vFLIP activated NF-kappaB in mouse bone marrow-derived DCs in vitro and matured these DCs to a similar extent as lipopolysaccharide; costimulatory markers CD80, CD86, CD40, and ICAM-1 were upregulated and tumor necrosis factor alpha and interleukin-12 secreted. The vFLIP-expressing lentivector also matured DCs in vivo. When we coexpressed vFLIP in a lentivector with ovalbumin (Ova), we found an increased immune response to Ova; up to 10 times more Ova-specific CD8(+) T cells secreting gamma interferon were detected in the spleens of vFLIP_Ova-immunized mice than in the spleens of mice immunized with GFP_Ova. Furthermore, this increased CD8(+) T-cell response correlated with improved tumor-free survival in a tumor therapy model. A single immunization with vFLIP_Ova also reduced the parasite load when mice were challenged with OVA-Leishmania donovani. In conclusion, vFLIP from KSHV is a DC activator, maturing DCs in vitro and in vivo. This demonstrates that NF-kappaB activation is sufficient to induce many aspects of DC maturation and that expression of a constitutive NF-kappaB activator can improve the efficacy of a vaccine vector.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/virologia , Lentivirus/genética , NF-kappa B/biossíntese , Proteínas Virais/imunologia , Vacinas Virais/genética , Animais , Vacinas Anticâncer/imunologia , Citocinas/biossíntese , Leishmania donovani/imunologia , Leishmaniose/prevenção & controle , Lentivirus/imunologia , Camundongos , Neoplasias/imunologia , Ovalbumina/imunologia , Receptores Imunológicos/biossíntese , Baço/imunologia , Análise de Sobrevida , Proteínas Virais/genética , Vacinas Virais/imunologiaRESUMO
Background: Liposomal amphotericin B (AmBisome®) as a treatment modality for visceral leishmaniasis (VL) has had significant impact on patient care in some but not all regions where VL is endemic. As the mode of action of AmBisome® in vivo is poorly understood, we compared the tissue-specific transcriptome in drug-treated vs untreated mice with experimental VL. Methods: BALB/c mice infected with L. donovani were treated with 8mg/kg AmBisome®, resulting in parasite elimination from liver and spleen over a 7-day period. At day 1 and day 7 post treatment (R x+1 and R x+7), transcriptomic profiling was performed on spleen and liver tissue from treated and untreated mice and uninfected mice. BALB/c mice infected with M. bovis BCG (an organism resistant to amphotericin B) were analysed to distinguish between direct effects of AmBisome® and those secondary to parasite death. Results: AmBisome® treatment lead to rapid parasitological clearance. At R x+1, spleen and liver displayed only 46 and 88 differentially expressed (DE) genes (P<0.05; 2-fold change) respectively. In liver, significant enrichment was seen for pathways associated with TNF, fatty acids and sterol biosynthesis. At R x+7, the number of DE genes was increased (spleen, 113; liver 400). In spleen, these included many immune related genes known to be involved in anti-leishmanial immunity. In liver, changes in transcriptome were largely accounted for by loss of granulomas. PCA analysis indicated that treatment only partially restored homeostasis. Analysis of BCG-infected mice treated with AmBisome® revealed a pattern of immune modulation mainly targeting macrophage function. Conclusions: Our data indicate that the tissue response to AmBisome® treatment varies between target organs and that full restoration of homeostasis is not achieved at parasitological cure. The pathways required to restore homeostasis deserve fuller attention, to understand mechanisms associated with treatment failure and relapse and to promote more rapid restoration of immune competence.
RESUMO
Human visceral leishmaniasis, a parasitic disease of major public health importance in developing countries, is characterized by variable degrees of severity of anemia, but the mechanisms underlying this change in peripheral blood have not been thoroughly explored. Here, we used an experimental model of visceral leishmaniasis in C57BL/6 mice to explore the basis of anemia following infection with Leishmania donovani. 28 days post-infection, mice showed bone marrow dyserythropoiesis by myelogram, with a reduction of TER119+ CD71-/+ erythroblasts. Reduction of medullary erythropoiesis coincided with loss of CD169high bone marrow stromal macrophages and a reduction of CXCL12-expressing stromal cells. Although the spleen is a site of extramedullary erythropoiesis and erythrophagocytosis, splenectomy did not impact the extent of anemia or affect the repression of medullary hematopoiesis that was observed in infected mice. In contrast, these changes in bone marrow erythropoiesis were not evident in B6.Rag2-/- mice, but could be fully reconstituted by adoptive transfer of IFNγ-producing but not IFNγ-deficient CD4+ T cells, mimicking the expansion of IFNγ-producing CD4+ T cells that occurs during infection in wild type mice. Collectively, these data indicate that anemia during experimental murine visceral leishmaniasis can be driven by defects associated with the bone marrow erythropoietic niche, and that this represents a further example of CD4+ T cell-mediated immunopathology affecting hematopoietic competence.
Assuntos
Anemia/etiologia , Linfócitos T CD4-Positivos/imunologia , Eritropoese/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Transferência Adotiva , Animais , Medula Óssea/fisiologia , Linfócitos T CD4-Positivos/transplante , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Leishmaniose Visceral/complicações , Leishmaniose Visceral/parasitologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Estromais/fisiologiaRESUMO
Background: Human visceral leishmaniasis, caused by infection with Leishmania donovani or L. infantum, is a potentially fatal disease affecting 50,000-90,000 people yearly in 75 disease endemic countries, with more than 20,000 deaths reported. Experimental models of infection play a major role in understanding parasite biology, host-pathogen interaction, disease pathogenesis, and parasite transmission. In addition, they have an essential role in the identification and pre-clinical evaluation of new drugs and vaccines. However, our understanding of these models remains fragmentary. Although the immune response to Leishmania donovani infection in mice has been extensively characterized, transcriptomic analysis capturing the tissue-specific evolution of disease has yet to be reported. Methods: We provide an analysis of the transcriptome of spleen, liver and peripheral blood of BALB/c mice infected with L. donovani. Where possible, we compare our data in murine experimental visceral leishmaniasis with transcriptomic data in the public domain obtained from the study of L. donovani-infected hamsters and patients with human visceral leishmaniasis. Digitised whole slide images showing the histopathology in spleen and liver are made available via a dedicated website, www.leishpathnet.org. Results: Our analysis confirms marked tissue-specific alterations in the transcriptome of infected mice over time and identifies previously unrecognized parallels and differences between murine, hamster and human responses to infection. We show commonality of interferon-regulated genes whilst confirming a greater activation of type 2 immune pathways in infected hamsters compared to mice. Cytokine genes and genes encoding immune checkpoints were markedly tissue specific and dynamic in their expression, and pathways focused on non-immune cells reflected tissue specific immunopathology. Our data also addresses the value of measuring peripheral blood transcriptomics as a potential window into underlying systemic disease. Conclusions: Our transcriptomic data, coupled with histopathologic analysis of the tissue response, provide an additional resource to underpin future mechanistic studies and to guide clinical research.