RESUMO
The neurotrophic tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB) and its ligands brain derived neurotrophic factor (Bdnf), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) are known primarily for their multiple effects on neuronal differentiation and survival. Here, we provide evidence that Ntrk2 plays a role in the pathologic remodeling of the spleen that accompanies chronic infection. We show that in Leishmania donovani-infected mice, Ntrk2 is aberrantly expressed on splenic endothelial cells and that new maturing blood vessels within the white pulp are intimately associated with F4/80(hi)CD11b(lo)CD11c(+) macrophages that express Bdnf and NT-4/5 and have pro-angiogenic potential in vitro. Furthermore, administration of the small molecule Ntrk2 antagonist ANA-12 to infected mice significantly inhibited white pulp neovascularization but had no effect on red pulp vascular remodeling. We believe this to be the first evidence of the Ntrk2/neurotrophin pathway driving pathogen-induced vascular remodeling in lymphoid tissue. These studies highlight the therapeutic potential of modulating this pathway to inhibit pathological angiogenesis.
Assuntos
Leishmania donovani/patogenicidade , Leishmaniose Visceral/patologia , Glicoproteínas de Membrana/metabolismo , Neovascularização Fisiológica/fisiologia , Proteínas Tirosina Quinases/metabolismo , Baço/irrigação sanguínea , Animais , Azepinas/farmacologia , Benzamidas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Linhagem Celular , Células Endoteliais/metabolismo , Feminino , Leishmaniose Visceral/parasitologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores de Fator de Crescimento Neural/biossíntese , Transdução de Sinais/fisiologia , Baço/metabolismo , Esplenomegalia/parasitologia , Esplenomegalia/patologiaRESUMO
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
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
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
Vascular cell adhesion molecule-1 (VCAM-1) interacts with its major ligand very late antigen-4 (VLA-4) to mediate cell adhesion and transendothelial migration of leukocytes. We report an important role for VCAM-1/VLA-4 interactions in the generation of immune responses during experimental visceral leishmaniasis caused by Leishmania donovani. Our studies demonstrate that these molecules play no direct role in the recruitment of leukocytes to the infected liver, but instead contribute to IL-12p40-production by splenic CD8(+) dendritic cells (DC). Blockade of VCAM-1/VLA-4 interactions using whole antibody or anti-VCAM-1 Fab' fragments reduced IL-12p40 mRNA accumulation by splenic DC 5 hours after L. donovani infection. This was associated with reduced anti-parasitic CD4(+) T cell activation in the spleen and lowered hepatic IFNgamma, TNF and nitric oxide production by 14 days post infection. Importantly, these effects were associated with enhanced parasite growth in the liver in studies with either anti-VCAM-1 or anti-VLA-4 antibodies. These data indicate a role for VCAM-1 and VLA-4 in DC activation during infectious disease.
Assuntos
Células Dendríticas/metabolismo , Integrina alfa4beta1/imunologia , Subunidade p40 da Interleucina-12/biossíntese , Leishmaniose Visceral/imunologia , Molécula 1 de Adesão de Célula Vascular/imunologia , Animais , Anticorpos Antiprotozoários/biossíntese , Linfócitos T CD4-Positivos/imunologia , Leishmania donovani , Fígado/parasitologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos , Baço/imunologiaRESUMO
Increasing evidence suggests that the infectiousness of patients for the sand fly vector of visceral leishmaniasis is linked to parasites found in the skin. Using a murine model that supports extensive skin infection with Leishmania donovani, spatial analyses at macro-(quantitative PCR) and micro-(confocal microscopy) scales indicate that parasite distribution is markedly skewed. Mathematical models accounting for this heterogeneity demonstrate that while a patchy distribution reduces the expected number of sand flies acquiring parasites, it increases the infection load for sand flies feeding on a patch, increasing their potential for onward transmission. Models representing patchiness at both macro- and micro-scales provide the best fit with experimental sand fly feeding data, pointing to the importance of the skin parasite landscape as a predictor of host infectiousness. Our analysis highlights the skin as a critical site to consider when assessing treatment efficacy, transmission competence and the impact of visceral leishmaniasis elimination campaigns.Parasitemia has been considered the main determinant of visceral leishmaniasis transmission. By combining imaging, qPCR and experimental xenodiagnoses with mathematical models, Doehl et al. argue that the patchy landscape of parasites in the skin is necessary to explain infectiousness.
Assuntos
Insetos Vetores/parasitologia , Leishmania donovani/fisiologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/transmissão , Psychodidae/parasitologia , Pele/parasitologia , Animais , Humanos , Camundongos , Modelos Biológicos , ParasitemiaRESUMO
BACKGROUND: Wound infections are a common complication of surgery that add significantly to the morbidity of patients and costs of treatment. The global trend towards reducing length of hospital stay post-surgery and the increase in day case surgery means that surgical site infections (SSI) will increasingly occur after hospital discharge. Surveillance of SSIs is important because rates of SSI are viewed as a measure of hospital performance, however accurate detection of SSIs post-hospital discharge is not straightforward. METHODS: We conducted a systematic review of methods of post discharge surveillance for surgical wound infection and undertook a national audit of methods of post-discharge surveillance for surgical site infection currently used within United Kingdom NHS Trusts. RESULTS: Seven reports of six comparative studies which examined the validity of post-discharge surveillance methods were located; these involved different comparisons and some had methodological limitations, making it difficult to identify an optimal method. Several studies evaluated automated screening of electronic records and found this to be a useful strategy for the identification of SSIs that occurred post discharge. The audit identified a wide range of relevant post-discharge surveillance programmes in England, Scotland and Wales and Northern Ireland; however, these programmes used varying approaches for which there is little supporting evidence of validity and/or reliability. CONCLUSION: In order to establish robust methods of surveillance for those surgical site infections that occur post discharge, there is a need to develop a method of case ascertainment that is valid and reliable post discharge. Existing research has not identified a valid and reliable method. A standardised definition of wound infection (e.g. that of the Centres for Disease Control) should be used as a basis for developing a feasible, valid and reliable approach to defining post discharge SSI. At a local level, the method used to ascertain post discharge SSI will depend upon the purpose of the surveillance, the nature of available routine data and the resources available.
Assuntos
Alta do Paciente , Infecção da Ferida Cirúrgica/diagnóstico , Hospitais , Humanos , Controle de Infecções , Auditoria Médica , Reprodutibilidade dos Testes , Infecção da Ferida Cirúrgica/epidemiologiaRESUMO
Recent thymic emigrants (RTEs) represent a source of antigen-naïve T cells that enter the periphery throughout life. However, whether RTEs contribute to the control of chronic parasitic infection and how their potential might be harnessed by therapeutic intervention is currently unclear. Here, we show that CD4+ recent thymic emigrants emerging into the periphery of mice with ongoing Leishmania donovani infection undergo partial activation and are recruited to sites of granulomatous inflammation. However, CD4+ RTEs displayed severely restricted differentiation either into IFNγ+ or IFNγ+TNFα+ effectors, or into IL-10-producing regulatory T cells. Effector cell differentiation in the chronically infected host was not promoted by adoptive transfer of activated dendritic cells or by allowing extended periods of post-thymic differentiation in the periphery. Nevertheless, CD4+ RTEs from infected mice retained the capacity to transfer protection into lymphopenic RAG2-/- mice. Taken together, our data indicate that RTEs emerging into a chronically inflamed environment are not recruited into the effector pool, but retain the capacity for subsequent differentiation into host protective T cells when placed in a disease-free environment.
RESUMO
The high level of functional diversity and plasticity in monocytes/macrophages has been defined within in vitro systems as M1 (classically activated), M2 (alternatively activated) and deactivated macrophages, of which the latter two subtypes are associated with suppression of cell mediated immunity, that confers susceptibility to intracellular infection. Although the Leishmania parasite modulates macrophage functions to ensure its survival, what remains an unanswered yet pertinent question is whether these macrophages are deactivated or alternatively activated. This study aimed to characterize the functional plasticity and polarization of monocytes/macrophages and delineate their importance in the immunopathogenesis of Post kala-azar dermal leishmaniasis (PKDL), a chronic dermatosis of human leishmaniasis. Monocytes from PKDL patients showed a decreased expression of TLR-2/4, along with an attenuated generation of reactive oxidative/nitrosative species. At disease presentation, an increased mRNA expression of classical M2 markers CD206, ARG1 and PPARG in monocytes and lesional macrophages indicated M2 polarization of macrophages which was corroborated by increased expression of CD206 and arginase-1. Furthermore, altered vitamin D signaling was a key feature in PKDL, as disease presentation was associated with raised plasma levels of monohydroxylated vitamin D3 and vitamin D3- associated genes, features of M2 polarization. Taken together, in PKDL, monocyte/macrophage subsets appear to be alternatively activated, a phenotype that might sustain disease chronicity. Importantly, repolarization of these monocytes to M1 by antileishmanial drugs suggests that switching from M2 to M1 phenotype might represent a therapeutic opportunity, worthy of future pharmacological consideration.
Assuntos
Leishmaniose Cutânea/imunologia , Leishmaniose Visceral/complicações , Macrófagos/imunologia , Monócitos/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular , Criança , Feminino , Perfilação da Expressão Gênica , Humanos , Terapia de Imunossupressão , Índia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Post kala-azar dermal leishmaniasis (PKDL), a cutaneous sequela of visceral leishmaniasis (VL), develops in some patients alongside but more commonly after apparent cure from VL. In view of the pivotal role of PKDL patients in the transmission of VL, here we review clinical, epidemiological, parasitological, and immunological perspectives of this disease, focusing on five hypotheses to explain the development of PKDL: (i) the role of antimonial drugs; (ii) UV-induced skin damage; (iii) reinfection; (iv) organ specific failure of memory T cell responses; and (v) genetic susceptibility of the host. This review will enable researchers and clinicians to explore the unresolved mystery of PKDL and provide a framework for future application of 'omic' approaches for the control and eventual elimination of VL.
Assuntos
Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Antimônio/farmacologia , Antiprotozoários/efeitos adversos , Antiprotozoários/uso terapêutico , Predisposição Genética para Doença , Humanos , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/epidemiologia , Linfócitos T/imunologia , Raios UltravioletaRESUMO
Antimicrobial proteins influence intestinal microbial ecology and limit proliferation of pathogens, yet the regulation of their expression has only been partially elucidated. Here, we have identified a putative pathway involving epithelial cells and intestinal intraepithelial lymphocytes (iIELs) that leads to antimicrobial protein (AMP) production by Paneth cells. Mice lacking γδ iIELs (TCRδ(-/-)) express significantly reduced levels of the AMP angiogenin 4 (Ang4). These mice were also unable to up-regulate Ang4 production following oral challenge by Salmonella, leading to higher levels of mucosal invasion compared to their wild type counterparts during the first 2 hours post-challenge. The transfer of γδ iIELs from wild type (WT) mice to TCRδ(-/-) mice restored Ang4 production and Salmonella invasion levels were reduced to those obtained in WT mice. The ability to restore Ang4 production in TCRδ(-/-) mice was shown to be restricted to γδ iIELs expressing Vγ7-encoded TCRs. Using a novel intestinal crypt co-culture system we identified a putative pathway of Ang4 production initiated by exposure to Salmonella, intestinal commensals or microbial antigens that induced intestinal epithelial cells to produce cytokines including IL23 in a TLR-mediated manner. Exposure of TCR-Vγ7(+) γδ iIELs to IL-23 promoted IL22 production, which triggered Paneth cells to secrete Ang4. These findings identify a novel role for γδ iIELs in mucosal defence through sensing immediate epithelial cell cytokine responses and influencing AMP production. This in turn can contribute to the maintenance of intestinal microbial homeostasis and epithelial barrier function, and limit pathogen invasion.
Assuntos
Comunicação Celular , Enterócitos/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Linfócitos/metabolismo , Celulas de Paneth/metabolismo , Ribonuclease Pancreático/biossíntese , Animais , Linhagem Celular , Interleucina-23/biossíntese , Interleucinas/farmacologia , Mucosa Intestinal/microbiologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Salmonella/imunologia , Estresse Fisiológico , Interleucina 22RESUMO
Hepatic resistance to Leishmania donovani infection in mice is associated with the development of granulomas, in which a variety of lymphoid and non-lymphoid populations accumulate. Although previous studies have identified B cells in hepatic granulomas and functional studies in B cell-deficient mice have suggested a role for B cells in the control of experimental visceral leishmaniasis, little is known about the behaviour of B cells in the granuloma microenvironment. Here, we first compared the hepatic B cell population in infected mice, where ≈60% of B cells are located within granulomas, with that of naïve mice. In infected mice, there was a small increase in mIgM(lo)mIgD(+) mature B2 cells, but no enrichment of B cells with regulatory phenotype or function compared to the naïve hepatic B cell population, as assessed by CD1d and CD5 expression and by IL-10 production. Using 2-photon microscopy to quantify the entire intra-granuloma B cell population, in conjunction with the adoptive transfer of polyclonal and HEL-specific BCR-transgenic B cells isolated from L. donovani-infected mice, we demonstrated that B cells accumulate in granulomas over time in an antigen-independent manner. Intra-vital dynamic imaging was used to demonstrate that within the polyclonal B cell population obtained from L. donovani-infected mice, the frequency of B cells that made multiple long contacts with endogenous T cells was greater than that observed using HEL-specific B cells obtained from the same inflammatory environment. These data indicate, therefore, that a subset of this polyclonal B cell population is capable of making cognate interactions with T cells within this unique environment, and provide the first insights into the dynamics of B cells within an inflammatory site.
Assuntos
Linfócitos B/citologia , Granuloma/parasitologia , Leishmaniose Visceral/metabolismo , Fígado/parasitologia , Linfócitos T/citologia , Transferência Adotiva , Animais , Antígenos CD1d/biossíntese , Antígenos CD5/biossíntese , Modelos Animais de Doenças , Feminino , Granuloma/patologia , Interleucina-10/biossíntese , Leishmania donovani/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , FenótipoRESUMO
Receptor tyrosine kinases are involved in multiple cellular processes, and drugs that inhibit their action are used in the clinic to treat several types of cancer. However, the value of receptor tyrosine kinase inhibitors (RTKIs) for treating infectious disease has yet to be explored. Here, we have shown in mice that administration of the broad-spectrum RTKI sunitinib maleate (Sm) blocked the vascular remodeling and progressive splenomegaly associated with experimental visceral leishmaniasis. Furthermore, Sm treatment restored the integrity of the splenic microarchitecture. Although restoration of splenic architecture was accompanied by an increase in the frequency of IFN-gamma+CD4+ T cells, Sm treatment alone was insufficient to cause a reduction in tissue parasite burden. However, preconditioning by short-term Sm treatment proved to be successful as an adjunct therapy, increasing the frequency of IFN-gamma+ and IFN-gamma+TNF+CD4+ T cells, enhancing NO production by splenic macrophages, and providing dose-sparing effects when combined with a first-line immune-dependent anti-leishmanial drug. We propose, therefore, that RTKIs may prove clinically useful as agents to restore immune competence before the administration of chemo- or immunotherapeutic drugs in the treatment of visceral leishmaniasis or other diseases involving lymphoid tissue remodeling, including cancer.
Assuntos
Imunocompetência/efeitos dos fármacos , Indóis/farmacologia , Leishmaniose Visceral/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Actinas/análise , Animais , Linfócitos T CD4-Positivos/imunologia , Interferon gama/biossíntese , Leishmania donovani , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/patologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Esplenomegalia/patologia , SunitinibeRESUMO
Although gammadelta T cells are a common feature of many pathogen-induced immune responses, the factors that influence, promote, or regulate the response of individual gammadelta T-cell subsets to infection is unknown. Here we show that in the absence of Vgamma1+ T cells, novel subsets of gammadelta T cells, expressing T-cell receptor (TCR)-Vgamma chains that normally define TCRgammadelta+ dendritic epidermal T cells (DETCs) (Vgamma5+), intestinal intraepithelial lymphocytes (iIELs) (Vgamma7+), and lymphocytes associated with the vaginal epithelia (Vgamma6+), are recruited to the spleen in response to bacterial infection in TCR-Vgamma1-/- mice. By comparison of phenotype and structure of TCR-Vgamma chains and/or -Vdelta chains expressed by these novel subsets with those of their epithelium-associated counterparts, the Vgamma6+ T cells elicited in infected Vgamma1-/- mice were shown to be identical to those found in the reproductive tract, from where they are presumably recruited in the absence of Vgamma1+ T cells. By contrast, Vgamma5+ and Vgamma7+ T cells found in infected Vgamma1-/- mice were distinct from Vgamma5+ DETCs and Vgamma7+ iIELs. Functional analyses of the novel gammadelta T-cell subsets identified for infected Vgamma1-/- mice showed that whereas the Vgamma5+ and Vgamma7+ subsets may compensate for the absence of Vgamma1+ T cells by producing similar cytokines, they do not possess cytocidal activity and they cannot replace the macrophage homeostasis function of Vgamma1+ T cells. Collectively, these findings identify novel subsets of gammadelta T cells, the recruitment and activity of which is under the control of Vgamma1+ T cells.
Assuntos
Listeria monocytogenes/patogenicidade , Listeriose/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Genes Codificadores da Cadeia gama de Receptores de Linfócitos T , Homeostase , Humanos , Listeria monocytogenes/imunologia , Listeriose/microbiologia , Ativação Linfocitária , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T gama-delta/deficiência , Receptores de Antígenos de Linfócitos T gama-delta/genética , Análise de Sequência de DNA , Baço/citologia , Baço/imunologiaRESUMO
BACKGROUND & AIMS: Intestinal epithelial integrity and permeability is dependent on intercellular tight junction (TJ) complexes. How TJ integrity is regulated remains unclear, although phosphorylation and dephosphorylation of the integral membrane protein occludin is an important determinant of TJ formation and epithelial permeability. We have investigated the role intestinal intraepithelial lymphocytes (iIELs) play in regulating epithelial permeability in response to infection. METHODS: Recombinant strains of Toxoplasma gondii were used to assess intestinal epithelial barrier function and TJ integrity in mice with intact or depleted populations of iIELs. Alterations in epithelial permeability were correlated with TJ structure and the state of phosphorylation of occludin. iIEL in vivo reconstitution experiments were used to identify the iIELs required to maintain epithelial permeability and TJ integrity. RESULTS: In the absence of gammadelta+ iIELs, intestinal epithelial barrier function and the ability to restrict epithelial transmigration of Toxoplasma and the unrelated intracellular bacterial pathogen Salmonella typhimurium was severely compromised. Leaky epithelium in gammadelta+ iIEL-deficient mice was associated with the absence of phosphorylation of serine residues of occludin and lack of claudin 3 and zona occludens-1 proteins in TJ complexes. These deficiencies were attributable to the absence of a single subset of gammadelta T-cell receptor (TCR-Vgamma7+) iIELs that, after reconstituting gammadelta iIEL-deficient mice, restored epithelial barrier function and TJ complexes, resulting in increased resistance to infection. CONCLUSIONS: These findings identify a novel role for gammadelta+ iIELs in maintaining TJ integrity and epithelial barrier function that have implications for understanding the pathogenesis of intestinal inflammatory diseases associated with disruption of TJ complexes.
Assuntos
Permeabilidade da Membrana Celular/imunologia , Junções Intercelulares/metabolismo , Mucosa Intestinal/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/imunologia , Animais , Western Blotting , Modelos Animais de Doenças , Imuno-Histoquímica , Imunoprecipitação , Mucosa Intestinal/imunologia , Mucosa Intestinal/parasitologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ocludina , Fosforilação , RNA/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Toxoplasma/isolamento & purificação , Toxoplasma/patogenicidade , Toxoplasmose Animal/imunologia , Toxoplasmose Animal/metabolismo , Toxoplasmose Animal/patologiaRESUMO
Gammadelta T cells play important but poorly defined roles in pathogen-induced immune responses and in preventing chronic inflammation and pathology. A major obstacle to defining their function is establishing the degree of functional redundancy and heterogeneity among gammadelta T cells. Using mice deficient in Vgamma1+ T cells which are a major component of the gammadelta T cell response to microbial infection, a specific immunoregulatory role for Vgamma1+ T cells in macrophage and gammadelta T cell homeostasis during infection has been established. By contrast, Vgamma1+ T cells play no significant role in pathogen containment or eradication and cannot protect mice from immune-mediated pathology. Pathogen-elicited Vgamma1+ T cells also display different functional characteristics at different stages of the host response to infection that involves unique and different populations of Vgamma1+ T cells. These findings, therefore, identify distinct and nonoverlapping roles for gammadelta T cell subsets in infection and establish the complexity and adaptability of a single population of gammadelta T cells in the host response to infection that is not predetermined, but is, instead, shaped by environmental factors.
Assuntos
Listeriose/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/microbiologia , Animais , Movimento Celular/genética , Movimento Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica/genética , Feminino , Imunofenotipagem , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Listeriose/genética , Listeriose/patologia , Cirrose Hepática/genética , Cirrose Hepática/imunologia , Cirrose Hepática/microbiologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/deficiência , Receptores de Antígenos de Linfócitos T gama-delta/genética , Subpopulações de Linfócitos T/metabolismo , Fatores de TempoRESUMO
gammadelta T cells are a diverse population of T cells that are widely distributed and are a common feature of pathogen-induced immune responses. It is not clear, however, whether different populations of gammadelta T cells have specific functions, and what factors determine the functional properties of individual populations. A murine model of peroral Toxoplasma gondii infection was used to determine the contribution Vgamma1+ intestinal intraepithelial lymphocytes (IELs) vs systemic Vgamma1+ T cells make to the acute and chronic stages of the host immune response, and whether the macrophage cytocidal activity of Vgamma1+ T cells described in bacterial infections is seen in other, unrelated infectious disease models. In response to oral infection with virulent type 1 or avirulent type II strains of T. gondii, TCR-delta-/- mice rapidly developed severe ileitis. In contrast, in mice deficient in Vgamma1+ T cells and IELs and wild-type mice, inflammation was delayed in onset and less severe. The protective effect of (Vgamma1-) IELs to Toxoplasma infection was unrelated to their cytolytic and cytokine (Th1)-producing capabilities. Systemic Vgamma1+ T cells were shown to play an essential role in limiting parasite growth and inflammation in peripheral tissues and, in particular, in the CNS, that was associated with their ability to efficiently kill parasite-elicited and infected macrophages. These findings suggest that macrophage cytocidal activity of Vgamma1+ T cells may be a universal feature of pathogen-induced immune responses and that microenvironmental factors influence the involvement and function of gammadelta T cells in the host response to infection.
Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/parasitologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose Animal/patologia , Animais , Citotoxicidade Imunológica , Ileíte/etiologia , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/deficiência , Toxoplasmose Animal/imunologiaRESUMO
Gammadelta T cells have a direct role in resolving the host immune response to infection by eliminating populations of activated macrophages. Macrophage reactivity resides within the Vgamma1/Vdelta6.3 subset of gammadelta T cells, which have the ability to kill activated macrophages following infection with Listeria monocytogenes (Lm). However, it is not known how gammadelta T cell macrophage cytocidal activity is regulated, or what effector mechanisms gammadelta T cells use to kill activated macrophages. Using a macrophage-T cell coculture system in which peritoneal macrophages from naive or Lm-infected TCRdelta-/- mice were incubated with splenocytes from wild-type and Fas ligand (FasL)-deficient mice (gld), the ability of Vgamma1 T cells to bind macrophages was shown to be dependent upon Fas-FasL interactions. Combinations of anti-TCR and FasL Abs completely abolished binding to and killing of activated macrophages by Vgamma1 T cells. In addition, confocal microscopy showed that Fas and the TCR colocalized on Vgamma1 T cells at points of contact with macrophages. Collectively, these studies identify an accessory or coreceptor-like function for Fas-FasL that is essential for the interaction of Vgamma1 T cells with activated macrophages and their elimination during the resolution stage of pathogen-induced immune responses.
Assuntos
Citotoxicidade Imunológica/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Glicoproteínas de Membrana/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Subpopulações de Linfócitos T/imunologia , Receptor fas/fisiologia , Animais , Adesão Celular/genética , Adesão Celular/imunologia , Comunicação Celular/genética , Comunicação Celular/imunologia , Morte Celular/genética , Morte Celular/imunologia , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica/genética , Proteína Ligante Fas , Listeria monocytogenes/imunologia , Ativação de Macrófagos/genética , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/microbiologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores de Antígenos de Linfócitos T gama-delta/deficiência , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Receptor fas/biossíntese , Receptor fas/metabolismoRESUMO
Immunoregulation is an emerging paradigm of gammadelta T cell function. The mechanisms by which gammadelta T cells mediate this function, however, are not clear. Studies have identified a direct role for gammadelta T cells in resolving the host immune response to infection, by eliminating populations of activated macrophages. The aim of this study was to identify macrophage-reactive gammadelta T cells and establish the requirements/outcomes of macrophage-gammadelta T cell interactions during the immune response to the intracellular bacterium, Listeria monocytogenes (Lm). Using a macrophage-T cell coculture system in which peritoneal macrophages from naive or Lm-infected TCRdelta(-/-) mice were incubated with splenocytes from naive and Lm-infected alphabeta/gammadelta T cell-deficient and wild-type mice, the ability to bind macrophages was shown to be restricted to gammadelta T cells and the GV5S1 (Vgamma1) subset of gammadelta T cells. Macrophage adherence resulted in a 4- to 10-fold enrichment of Vgamma1(+) T cells. Enrichment of Vgamma1 T cells was dependent upon the activation status of macrophages, but independent of the activation status of gammadelta T cells. Vgamma1 T cells were cytotoxic for activated macrophages with both the binding to and killing of macrophages being TCR dependent because anti-TCRgammadelta Abs inhibited both Vgamma1 binding and killing activities. These studies establish the identity of macrophage cytotoxic gammadelta T cells, the conditions under which this interaction occurs, and the outcome of this interaction. These findings are concordant with the involvement of Vgamma1 T cells in macrophage homeostasis during the resolution of pathogen-mediated immune responses.