RESUMO
Natural killer (NK) cells include different subsets with diverse effector capacities that are poorly understood in the context of parasitic diseases. Here, we investigated inhibitory and activating receptor expression on NK cells in patients with cutaneous leishmaniasis (CL) and explored their phenotypic and functional heterogeneity based on CD57 and NKG2C expression. The expression of CD57 identified NK cells that accumulated in CL patients and exhibited features of senescence. The CD57+ cells exhibited heightened levels of the activating receptor NKG2C and diminished expression of the inhibitory receptor NKG2A. RNA sequencing analyses based on NKG2C transcriptome have revealed two distinct profiles among CL patients associated with cytotoxic and functional genes. The CD57+NKG2C+ subset accumulated in the blood of patients and presented conspicuous features of senescence, including the expression of markers such as p16, yH2ax, and p38, as well as reduced proliferative capacity. In addition, they positively correlated with the number of days until lesion resolution. This study provides a broad understanding of the NK cell biology during Leishmania infection and reinforces the role of senescent cells in the adverse clinical outcomes of CL.
Assuntos
Antígenos CD57 , Senescência Celular , Células Matadoras Naturais , Leishmaniose Cutânea , Subfamília C de Receptores Semelhantes a Lectina de Células NK , Humanos , Leishmaniose Cutânea/imunologia , Células Matadoras Naturais/imunologia , Antígenos CD57/metabolismo , Antígenos CD57/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Senescência Celular/imunologia , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Adulto JovemRESUMO
There are no approved vaccines yet for human visceral leishmaniasis (VL), the most severe form of the leishmaniasis clinical manifestations that is fatal in over 95 % of untreated cases. It is well-accepted that immunological changes during aging have deleterious impact on the efficacy of vaccines and response to infections. In this work, we compared the response of young and aged mice to intranasal vaccination with killed Leishmania amazonensis promastigote antigens (LaAg) that were then challenged with L. infantum infection, a species that causes visceral leishmaniasis. Intranasal vaccination with LaAg induced a similar reduction in parasitism and hepatosplenomegaly in both young and aged mice compared to their unvaccinated counterparts. Following infection, there was also a less prominent inflammatory profile particularly in the vaccinated aged group, with lower production of TNF-α and nitrite compared to the respective unvaccinated group. Interestingly, the LaAg intranasal vaccination promoted increased production of IFN-γ that was observed in both young- and aged vaccinated groups. Additionally, CD4+ and CD8+T cells from both vaccinated groups presented decreased expression of the inhibitory receptors PD-1 and KLRG1 compared to their unvaccinated controls. Interestingly, a strong positive correlation was observed between the expression of both inhibitory receptors PD-1 and KLRG1 and parasitism, which was more conspicuous in the unvaccinated-aged mice than in the others. Overall, this study helps define new strategies to improve vaccine effectiveness and provides a perspective for prophylactic alternatives against leishmaniasis.
Assuntos
Leishmania infantum , Leishmania mexicana , Vacinas contra Leishmaniose , Leishmaniose Visceral , Leishmaniose , Vacinas Protozoárias , Humanos , Animais , Camundongos , Idoso , Leishmaniose Visceral/prevenção & controle , Receptor de Morte Celular Programada 1 , Antígenos de Protozoários , Camundongos Endogâmicos BALB C , CitocinasRESUMO
It is well accepted that the impact of diseases is generally more detrimental in elderly individuals than in younger ones. Changes in the immune system due to ageing can directly affect the ability to respond effectively to infections and may contribute to the higher morbidities and mortalities in the elderly population. Leishmaniasis is a complex of clinically unique diseases caused by obligate intracellular protozoa belonging to genus Leishmania, wherein visceral leishmaniasis (VL) is the most severe form and is fatal if left untreated. In this study, aged mice (72 weeks old) presented increased susceptibility to L. infantum infection compared to younger mice (46-week-old), with notable parasitism in both the spleen and liver, as well as exhibiting hepatosplenomegaly. A pronounced inflammatory profile was observed in the aged-infected mice, with excessive production of TNF-α and nitrite, along with diminished IFN-γ production and reduced proliferative capacity of T cells (assessed by expression of the Ki67 marker). Additionally, both CD4+ and CD8+ T cells from the aged-infected mice presented increased expression of the inhibitory receptors PD-1 and KLRG1 that strongly correlated with the parasitism found in the liver and spleen of this group. Overall, the data reported in this study suggests for the first time that ageing may negatively impact the VL outcome and provides a perspective for new therapeutic strategies involving manipulation of immunosenescence features against Leishmania infection.
Assuntos
Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Idoso , Envelhecimento , Animais , Linfócitos T CD8-Positivos , Humanos , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The severity of lesions that develop in patients infected by Leishmania braziliensis is mainly associated with a highly cytotoxic and inflammatory cutaneous environment. Recently, we demonstrated that senescent T and NK cells play a role in the establishment and maintenance of this tissue inflammation. Here, we extended those findings using transcriptomic analyses that demonstrate a strong co-induction of senescence and pro-inflammatory gene signatures in cutaneous leishmaniasis (CL) lesions. The senescence-associated signature was characterized by marked expression of key genes such as ATM, Sestrin 2, p16, p21 and p38. The cell type identification from deconvolution of bulk sequencing data showed that the senescence signature was linked with CD8+ effector memory and TEMRA subsets and also senescent NK cells. A key observation was that the senescence markers in the skin lesions are age-independent of patients and were correlated with lesion size. Moreover, a striking expression of the senescence-associated secretory phenotype (SASP), pro-inflammatory cytokine and chemokines genes was found within lesions that were most strongly associated with the senescent CD8 TEMRA subset. Collectively, our results confirm that there is a senescence transcriptomic signature in CL lesions and supports the hypothesis that lesional senescent cells have a major role in mediating immunopathology of the disease.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Imunossenescência/genética , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/etiologia , Leishmaniose Cutânea/patologia , Transcriptoma , Biomarcadores , Biópsia , Biologia Computacional/métodos , Citocinas/genética , Citocinas/metabolismo , Bases de Dados Genéticas , Suscetibilidade a Doenças/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Leishmaniose Cutânea/metabolismo , Carga Parasitária , Pele/patologiaRESUMO
BACKGROUND: Regulatory T cells (Tregs) play a critical role during Mycobacterium tuberculosis (Mtb) infection, modulating host responses while neutralizing excessive inflammation. However, their impact on regulating host protective immunity is not completely understood. Here, we demonstrate that Treg cells abrogate the in vitro microbicidal activity against Mtb. METHODS: We evaluated the in vitro microbicidal activity of peripheral blood mononuclear cells (PBMCs) from patients with active tuberculosis (TB), individuals with latent tuberculosis infection (LTBI, TST+/IGRA+) and healthy control (HC, TST-/IGRA-) volunteers. PBMCs, depleted or not of CD4+CD25+ T-cells, were analyzed to determine frequency and influence on microbicidal activity during in vitro Mtb infection with four clinical isolates (S1, S5, R3, and R6) and one reference strain (H37Rv). RESULTS: The frequency of CD4+CD25highFoxP3+ cells were significantly higher in Mtb infected whole blood cultures from both TB patients and LTBI individuals when compared to HC. Data from CD4+CD25+ T-cells depletion demonstrate that increase of CD4+CD25highFoxP3+ is associated with an impairment of Th-1 responses and a diminished in vitro microbicidal activity of LTBI and TB groups. CONCLUSIONS: Tregs restrict host anti-mycobacterial immunity during active disease and latent infection and thereby may contribute to both disease progression and pathogen persistence.
Assuntos
Atividade Bactericida do Sangue , Antígenos CD4/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Tuberculose Latente/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/imunologia , Antígenos CD4/genética , Estudos de Casos e Controles , Fatores de Transcrição Forkhead/genética , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Linfócitos T ReguladoresRESUMO
Patients infected by Leishmania braziliensis develop debilitating skin lesions. The role of inhibitory checkpoint receptors (ICRs) that induce T cell exhaustion during this disease is not known. Transcriptional profiling identified increased expression of ICRs including PD-1, PDL-1, PDL-2, TIM-3, and CTLA-4 in skin lesions of patients that was confirmed by immunohistology where there was increased expression of PD-1, TIM-3, and CTLA-4 in both CD4+ and CD8+ T cell subsets. Moreover, PDL-1/PDL-2 ligands were increased on skin macrophages compared to healthy controls. The proportions PD1+, but not TIM-3 or CTLA-4 expressing T cells in the circulation were positively correlated with those in the lesions of the same patients, suggesting that PD-1 may regulate T cell function equally in both compartments. Blocking PD-1 signaling in circulating T cells enhanced their proliferative capacity and IFN-γ production, but not TNF-α secretion in response to L. braziliensis recall antigen challenge in vitro. While we previously showed a significant correlation between the accumulation of senescent CD8+CD45RA+CD27- T cells in the circulation and skin lesion size in the patients, there was no such correlation between the extent of PD-1 expression by circulating on T cells and the magnitude of skin lesions suggesting that exhausted-like T cells may not contribute to the cutaneous immunopathology. Nevertheless, we identified exhausted-like T cells in both skin lesions and in the blood. Targeting this population by PD-1 blockade may improve T cell function and thus accelerate parasite clearance that would reduce the cutaneous pathology in cutaneous leishmaniasis.
Assuntos
Inibidores de Checkpoint Imunológico/farmacologia , Leishmaniose Cutânea/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Adulto , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Proteínas de Checkpoint Imunológico/metabolismo , Imunossenescência , Inflamação , Interferon gama/imunologia , Leishmania braziliensis/patogenicidade , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Pele/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Cytotoxic activity mediated by CD8+ T cells is the main signature of the immunopathogenesis of cutaneous leishmaniasis (CL). Here, we performed a broad evaluation of natural killer (NK) cell phenotypic and functional features during cutaneous leishmaniasis. We demonstrate for the first time that CL patients present the accumulation of circulating NK cells with multiple features of replicative senescence including low proliferative capacity and shorter telomeres, elevated expression of CD57, KLRG1 but diminished CD27 stimulatory receptor expression. Moreover, they exhibited higher cytotoxic and inflammatory potential than age-matched controls. The accumulation of circulating senescent NK cells (CD56dim CD57bright ) correlated positively with skin lesion size in the same patients, suggesting that they, like circulating senescent CD8+ T cells, may contribute to the immunopathology of CL. However, this senescent population had lower cutaneous lymphocyte antigen expression and so had diminished skin-homing potential compared with total or senescent CD8+ T cells. This was confirmed in CL skin lesions where we found a predominance of CD8+ T cells (both senescent and non-senescent) that correlated with the severity of the disease. Although there was also a correlation between the proportions of senescent NK cells (CD56+ CD57+ ) in the skin and lesion size, this was less evident. Collectively our results demonstrate first-hand that senescent cytotoxic cells may mediate skin pathology during human cutaneous leishmaniasis. However, as senescent cytotoxic CD8+ T cells predominate in the skin lesions, they may have a greater role than NK cells in mediating the non-specific skin damage in CL.
Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/patologia , Leishmania braziliensis/patogenicidade , Leishmaniose Cutânea/patologia , Pele/patologia , Linfócitos T Citotóxicos/patologia , Antígeno CD56/genética , Antígeno CD56/imunologia , Antígenos CD57/genética , Antígenos CD57/imunologia , Estudos de Casos e Controles , Senescência Celular/imunologia , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/parasitologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Masculino , Oligossacarídeos/genética , Oligossacarídeos/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Índice de Gravidade de Doença , Antígeno Sialil Lewis X/análogos & derivados , Antígeno Sialil Lewis X/genética , Antígeno Sialil Lewis X/imunologia , Transdução de Sinais , Pele/imunologia , Pele/parasitologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/parasitologiaRESUMO
The use of adjuvants in vaccine formulations is a well-established practice to improve immunogenicity and protective immunity against diseases. Previously, we have demonstrated the feasibility of intranasal vaccination with the antigen of killed Leishmania amazonensis promastigotes (LaAg) against experimental leishmaniasis. In this work, we sought to optimize the immunogenic effect and protective immunity against murine visceral leishmaniasis conferred by intranasal delivery of LaAg in combination with a synthetic TLR1/TLR2 agonist (Pam3CSK4). Intranasal vaccination with LaAg/PAM did not show toxicity or adverse effects, induced the increase of delayed-type hypersensitivity response and the production of inflammatory cytokines after parasite antigen recall. However, mice vaccinated with LaAg/PAM and challenged with Leishmania infantum presented significant reduction of parasite burden in both liver and spleen, similar to those vaccinated with LaAg. Although LaAg/PAM intranasal vaccination had induced higher frequencies of specific CD4+ and CD8+ T cells and increased levels of IgG2a antibody isotype in serum, both LaAg and LaAg/PAM groups presented similar levels of IL-4 and IFN-y and decreased production of IL-10 when compared to controls. Our results provide the first evidence of the feasibility of intranasal immunization with antigens of killed Leishmania in association with a TLR agonist, which may be explored for developing an effective and alternative strategy for vaccination against visceral leishmaniasis.
Assuntos
Antígenos de Protozoários/imunologia , Leishmania/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Lipopeptídeos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/administração & dosagem , Citocinas/sangue , Feminino , Vacinas contra Leishmaniose/administração & dosagem , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/prevenção & controle , Lipopeptídeos/administração & dosagem , Fígado/metabolismo , Fígado/parasitologia , Linfócitos/imunologia , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Baço/metabolismo , Baço/parasitologia , VacinaçãoRESUMO
An increased number of regulatory T (Treg) cells has been reported in patients with HTLV-1 and Strongyloides stercoralis co-infection, suggesting the contribution of these cells to worm survival. As Strongyloides infections have been found to be highly prevalent in chronic alcoholics, we investigated the effect of abusive ethanol ingestion on the induction of Treg cells in alcoholic patients with Strongyloides infection. Treg cells were assessed by flow cytometry in the peripheral blood of 12 healthy non-alcoholic (control) and 14 alcoholic patients (alcoholic) without Strongyloides infection and five non-alcoholics (controlSs) and five chronic alcoholics (alcoholSs) with Strongyloides infection. The results showed significantly higher frequencies of Treg cells in the alcoholic, controlSs and alcoholSs group patients than in the control group patients. However, the frequencies of Treg cells did not differ between the alcoholSs and controlSs groups. In conclusion, our results demonstrate that ethanol consumption induced an increase in the number of circulating Treg cells in chronic alcoholics in this study but was unable to potentiate the induction of these cells in alcoholics with Strongyloides infection.
Assuntos
Alcoolismo/sangue , Strongyloides stercoralis/fisiologia , Estrongiloidíase/sangue , Linfócitos T Reguladores/citologia , Adulto , Alcoolismo/imunologia , Alcoolismo/parasitologia , Animais , Feminino , Citometria de Fluxo , Humanos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Estrongiloidíase/imunologia , Estrongiloidíase/parasitologia , Linfócitos T Reguladores/imunologiaRESUMO
Obesity is the main causal factor for metabolic syndrome and chronic systemic inflammation, which impacts on immune function and increases susceptibility to pathogens. Here, we investigated the effect of obesity on the outcome of visceral leishmaniasis caused by Leishmaniasis infantum chagasi. C57BL/6 mice fed with high-sugar and butter diet (HSB) showed a significant increase in body weight, adiposity index and morphological changes in adipocyte. To investigate the consequences of obesity on the specific immunity against Leishmania, both control and HSB diet groups were infected with 107 L. infantum chagasi promastigotes in the eighth-week after diet started and euthanized 4 weeks later. HSB-diet fed mice exhibited a significantly higher parasite burden in both liver and spleen compared with control- diet group. Gonadal adipocyte tissue from HSB-diet mice showed increased TNF-α, IL-6 and leptin and diminished IL-10 production compared with control. Cytokines production analysis in the spleen and liver from these animals also demonstrated higher production of IFN-γ, TNF-α, IL-6 and nitric oxide and diminished production of IL-10 and TGF-ß, which correlate with inflammatory foci and the cell hyperplasia observed. Taken together, obesity can interfere with responses to pathogen-derived signals and impair the development of protective anti-Leishmania immunity.