RESUMEN
Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania, which is endemic in certain areas of Europe, such as southern Spain. The disease manifests in various clinical phenotypes, including visceral, cutaneous, mucosal, or asymptomatic leishmaniasis. This diversity in clinical outcomes may be influenced by the host immune response, with human leukocyte antigen (HLA) molecules playing a crucial role in determining susceptibility and progression of the infection. This study explores the association between specific HLA variants and Leishmania infantum infection. We recruited four cohorts: a control group, asymptomatic individuals, patients with symptomatic disease, and cohabitants of infected individuals. HLA typing was performed for all participants, followed by an association analysis with infection status and disease progression. Our findings indicate that the HLA-B*38 and HLA-C*03 alleles are associated with protection against L. infantum infection. These results contribute to a better understanding of the disease's progression, offer potential for new therapeutic approaches such as vaccines, and expand the existing knowledge in the literature.
Asunto(s)
Alelos , Leishmania infantum , Humanos , Leishmania infantum/genética , España/epidemiología , Masculino , Femenino , Adulto , Predisposición Genética a la Enfermedad , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/epidemiología , Estudios de Cohortes , Persona de Mediana Edad , Antígenos HLA/genética , Frecuencia de los GenesRESUMEN
BACKGROUND: Currently, treatment regimens for visceral leishmaniasis (VL) are limited because of the presence of numerous adverse effects. Nicotinamide, a readily available and cost-effective vitamin, has been widely acknowledged for its safety profile. Several studies have demonstrated the anti-leishmanial effects of nicotinamide in vitro. However, the potential role of nicotinamide in Leishmania infection in vivo remains elusive. METHODS: In this study, we assessed the efficacy of nicotinamide as a therapeutic intervention for VL caused by Leishmania infantum in an experimental mouse model and investigated its underlying molecular mechanisms. The potential molecular mechanism was explored through cytokine analysis, examination of spleen lymphocyte subsets, liver RNA-seq analysis, and pathway validation. RESULTS: Compared to the infection group, the group treated with nicotinamide demonstrated significant amelioration of hepatosplenomegaly and recovery from liver pathological damage. The NAM group exhibited parasite reduction rates of 79.7% in the liver and 86.7% in the spleen, respectively. Nicotinamide treatment significantly reduced the activation of excessive immune response in infected mice, thereby mitigating hepatosplenomegaly and injury. Furthermore, nicotinamide treatment enhanced fatty acid ß-oxidation by upregulating key enzymes to maintain lipid homeostasis. CONCLUSIONS: Our findings provide initial evidence supporting the safety and therapeutic efficacy of nicotinamide in the treatment of Leishmania infection in BALB/c mice, suggesting its potential as a viable drug for VL.
Asunto(s)
Leishmania infantum , Leishmaniasis Visceral , Metabolismo de los Lípidos , Hígado , Ratones Endogámicos BALB C , Niacinamida , Bazo , Animales , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/inmunología , Niacinamida/farmacología , Niacinamida/uso terapéutico , Ratones , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/parasitología , Hígado/efectos de los fármacos , Hígado/patología , Leishmania infantum/efectos de los fármacos , Bazo/parasitología , Bazo/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Inflamación/tratamiento farmacológico , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéuticoRESUMEN
Repurposing drugs and adjuvants is an attractive choice of present therapy that reduces the substantial costs, chances of failure, and systemic toxicity. Mycobacterium indicus pranii was originally developed as a leprosy vaccine but later has been found effective against Leishmania donovani infection. To extend our earlier study, here we reported the immunotherapeutic modulation of the splenic and circulatory neutrophils in favour of hosts as neutrophils actually serve as the pro-parasitic portable shelter to extend the Leishmania infection specifically during the early entry into the hosts' circulation. We targeted to disrupt this early pro-parasitic incidence by the therapeutic combination of M. indicus pranii and heat-induced promastigotes against antimony-resistant L. donovani infection. The combination therapy induced the functional expansion of CD11b+Ly6CintLy6Ghi neutrophils both in the post-infected spleen, and also in the circulation of post-treated animals followed by the immediate Leishmania infection. More importantly, the enhanced expression of MHC-II, phagocytic uptake of the parasites by the circulatory neutrophils as well as the oxidative burst were induced that limited the chances of the very early establishment of the infection. The enhanced expression of pro-inflammatory cytokines, like IL-1α and TNF-α indicated resistance to the parasite-mediated takeover of the neutrophils, as these cytokines are critical for the activation of T cell-mediated immunity and host-protective responses. Additionally, the induction of essential transcription factors and cytokines for early granulocytic lineage commitment suggests that the strategy not only contributed to the peripheral activation of the neutrophils but also promoted granulopoiesis in the bone marrow.
Asunto(s)
Antimonio , Leishmania donovani , Leishmaniasis Visceral , Neutrófilos , Leishmania donovani/inmunología , Animales , Neutrófilos/inmunología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/tratamiento farmacológico , Ratones , Antimonio/farmacología , Mycobacterium/inmunología , Activación Neutrófila/inmunología , Bazo/inmunología , Calor , Citocinas/metabolismo , Ratones Endogámicos BALB C , Resistencia a MedicamentosRESUMEN
In the Americas, L. infantum (syn. chagasi) is the main cause of human visceral leishmaniasis. The role of neutrophils as part of the innate response to Leishmania spp. infection is dubious and varies according to the species causing the infection. Global expression of coding RNAs, microRNAs and long non-coding RNAs changes as part of the immune response against pathogens. Changes in mRNA and non-coding RNA expression resulting from infection by Leishmania spp. are widely studied in macrophages, but scarce in neutrophils, the first cell to encounter the trypanosomatid, especially following infection by L. infantum. Herein, we aimed to understand the expression patterns of coding and non-coding transcripts during acute in vitro infection of human neutrophils by L. infantum. We isolated neutrophils from whole blood of healthy male donors (n = 5) and split into groups: 1) infected with L. infantum (MOI = 5:1), and 2) uninfected controls. After 3 hours of exposure of infected group to promastigotes of L. infantum, followed by 17 hours of incubation, total RNA was extracted and total RNA-Seq and miRNA microarray were performed. A total of 212 genes were differentially expressed in neutrophils following RNA-Seq analysis (log2(FC)±0.58, FDR≤0.05). In vitro infection with L. infantum upregulated the expression of 197 and reduced the expression of 92 miRNAs in human neutrophils (FC±2, FDR≤0.01). Lastly, 5 downregulated genes were classified as lncRNA, and of the 10 upregulated genes, there was only 1 lncRNA. Further bioinformatic analysis indicated that changes in the transcriptome and microtranscriptome of neutrophils, following in vitro infection with L. infantum, may impair phagocytosis, apoptosis and decrease nitric oxide production. Our work sheds light on several mechanisms used by L. infantum to control neutrophil-mediated immune response and identifies several targets for future functional studies, aiming at the development of preventive or curative treatments for this prevalent zoonosis.
Asunto(s)
Leishmania infantum , MicroARNs , Neutrófilos , ARN Largo no Codificante , ARN Mensajero , Humanos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Leishmania infantum/genética , Leishmania infantum/inmunología , ARN Largo no Codificante/genética , MicroARNs/genética , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/genética , Adulto , Perfilación de la Expresión GénicaRESUMEN
Surveillance and sustained control of visceral leishmaniasis (VL) require reliable serodiagnostic tools. rK39, the gold standard antigen for VL diagnosis, is limited by its documented poor sensitivity in certain endemic regions, such as East Africa, and by the longevity of its antibodies, making it difficult to distinguish active from cured infections. In a recent publication in mBio, Roberts et al. (A. J. Roberts, H.B. Ong, S. Clare, C. Brandt, et al., mBio 15:e00859-24, 2024, https://doi.org/10.1128/mbio.00859-24) identified new immunogenic Leishmania candidates in dogs and humans. In dogs, combined antigens LdBPK_290790.1 + LdBPK_362700.1 (D4 +D46) distinguished symptomatic from asymptomatic infections. For humans, LdBPK_323600.1 (D36) antigen produced short-lived antibodies and performed well in patient cohorts from Bangladesh and Ethiopia, but not Kenya. This study adds promising new candidates to our serodiagnostic toolbox but highlights the need for more antigen discovery studies that may have to be focused on regional performance.
Asunto(s)
Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Enfermedades de los Perros , Leishmaniasis Visceral , Pruebas Serológicas , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/veterinaria , Leishmaniasis Visceral/inmunología , Perros , Animales , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/genética , Pruebas Serológicas/métodos , Humanos , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitología , Enfermedades de los Perros/inmunología , Anticuerpos Antiprotozoarios/sangre , Sensibilidad y Especificidad , EtiopíaRESUMEN
BACKGROUND: In endemic areas, Leishmania infantum and feline immunodeficiency virus (FIV) co-infection occurs in cats, and may favour a progressive course of feline leishmaniosis. Abnormalities in serum protein fractions have been reported, but inflammation markers have scarcely been studied. Erythrocyte sediment rate (ESR) is a marker of inflammation that is poorly used in veterinary medicine, but it has been evaluated in EDTA blood using a recently introduced automatic device. We studied ESR and a pool of feline markers of inflammation (MoI) in cats L. infantum (Li+) and/or FIV antibody-positive (Li+FIV+/FIV+) with the aims (a) to evaluate ESR as MoI in cats with the infectious and clinical conditions considered and (b) to provide data about a pool of MoI never investigated in the feline infections studied and in other cat diseases before. METHODS: This prospective controlled study included 35 study group cats (Li+, n = 20; FIV +, n = 8; Li+FIV+, n = 7) and ten healthy antibody-negative control cats. Clinical findings at physical examination and selected clinical pathological abnormalities related to inflammation were statistically analysed in relation to the infectious status and ESR values. RESULTS: ESR values were higher in Li+, FIV+, and Li+FIV+ cats compared with control cats, and 40% of the study group cats had ESR values above the reference interval (RI). ESR positively correlated with some positive MoI and negatively with some negative MoI studied. Additionally, a higher prevalence of ESR values above the RI has been detected in cats with hypoalbuminemia or hypergammaglobulinemia and higher ESR values were measured in cats with serum protein electrophoresis (SPE) fraction abnormalities. Correlations were also found with erythrocytes, hemoglobin, hematocrit and some erythrocyte indices. FIV+ and Li+FIV+ cats had a higher prevalence of increased ESR values, and almost all had SPE abnormalities and more severe clinical presentations compared with Li+ cats. CONCLUSIONS: Abnormal levels of MoI were found in almost all parameters studied, particularly in FIV+ and Li+FIV+ cats. Also, ESR can be used as a marker of inflammation in cats with L. infantum and/or FIV infection.
Asunto(s)
Biomarcadores , Sedimentación Sanguínea , Enfermedades de los Gatos , Virus de la Inmunodeficiencia Felina , Inflamación , Leishmania infantum , Leishmaniasis Visceral , Gatos , Animales , Leishmania infantum/inmunología , Virus de la Inmunodeficiencia Felina/inmunología , Enfermedades de los Gatos/sangre , Enfermedades de los Gatos/parasitología , Enfermedades de los Gatos/inmunología , Inflamación/veterinaria , Inflamación/sangre , Biomarcadores/sangre , Leishmaniasis Visceral/veterinaria , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Masculino , Estudios Prospectivos , Anticuerpos Antivirales/sangre , Femenino , Síndrome de Inmunodeficiencia Adquirida del Felino/sangre , Síndrome de Inmunodeficiencia Adquirida del Felino/inmunología , Coinfección/veterinaria , Coinfección/parasitología , Coinfección/virología , Anticuerpos Antiprotozoarios/sangreRESUMEN
Post Kala-azar dermal leishmaniasis (PKDL) arises as a significant dermal sequel following Visceral leishmaniasis (VL) caused by protozoan parasite Leishmania donovani (LD). PKDL acts as a significant constrain for VL elimination serving as a crucial reservoir for LD. PKDL patients exhibit depigmented macular and papular lesions on their skin, which results in social discrimination due to loss of natural skin color. Inflammatory reactions, prevalent in both VL and PKDL, potentially lead to tissue damage in areas harboring the parasite. Disruption of the immune-inflammasomal network not only facilitates LD persistence but also leads to the skin hypopigmentation seen in PKDL, impacting social well-being. Activation of inflammasomal markers like STAT1, NLRP1, NLRP3, AIM2, CASP11, and NLRP12 have been identified as a common host-defense mechanism across various Leishmania infections. Conversely, Leishmania modulates inflammasome activation to sustain its presence within the host. Nevertheless, in specific instances of Leishmania infection, inflammasome activation can worsen disease pathology by promoting parasite proliferation and persistence. This study encompasses recent transcriptomic analyses conducted between 2016 and 2023 on human and murine subjects afflicted with VL/PKDL, elucidating significant alterations in inflammasomal markers in both conditions. It offers a comprehensive understanding how these markers contribute in disease progression, drawing upon available literature for logical analysis. Furthermore, our analysis identifies validated miRNA network that could potentially disrupt this crucial immune-inflammasomal network, thereby offering a plausible explanation on how secreted LD-factors could enable membrane-bound LD, isolated from the host cytoplasm, to modulate cytoplasmic inflammasomal markers. Insights from this study could guide the development of host-directed therapeutics to impede transmission and address hypopigmentation, thereby mitigating the social stigma associated with PKDL.
Asunto(s)
Inflamasomas , Leishmania donovani , Leishmaniasis Cutánea , Leishmaniasis Visceral , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Humanos , Inflamasomas/metabolismo , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Leishmania donovani/inmunología , AnimalesRESUMEN
Leishmania donovani surface glycoprotein 63 (GP63) is a major virulence factor involved in parasite escape and immune evasion. In this study, we generated virus-like particles (VLPs) expressing L. donovani GP63 using the baculovirus expression system. Mice were intramuscularly immunized with GP63-VLPs and challenged with L. donovani promastigotes. GP63-VLP immunization elicited higher levels of L. donovani antigen-specific serum antibodies and enhanced splenic B cell, germinal center B cell, CD4+, and CD8+ T cell responses compared to unimmunized controls. GP63-VLPs inhibited the influx of pro-inflammatory cytokines IFN-γ and IL-6 in the livers, as well as thwarting the development of splenomegaly in immunized mice. Upon L. donovani challenge infection, a drastic reduction in splenic parasite burden was observed in VLP-immunized mice. These results indicate that GP63-VLPs immunization conferred protection against L. donovani challenge infection by inducing humoral and cellular immunity in mice.
Asunto(s)
Leishmania donovani , Leishmaniasis Visceral , Ratones Endogámicos BALB C , Vacunas de Partículas Similares a Virus , Animales , Leishmania donovani/inmunología , Ratones , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Femenino , Leishmaniasis Visceral/prevención & control , Leishmaniasis Visceral/inmunología , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Vacunas contra la Leishmaniasis/inmunología , Vacunas contra la Leishmaniasis/administración & dosificación , Eficacia de las Vacunas , Inmunidad Celular , Bazo/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos B/inmunología , Inmunidad Humoral , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/genética , Citocinas/inmunología , MetaloendopeptidasasRESUMEN
Centrin1 gene deleted Leishmania donovani parasite (LdCen1-/-) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1-/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1-/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1-/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1-/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1-/-, there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1-/- manufactured under cGMP complaint conditions can be suitable for future clinical trials.
Asunto(s)
Eliminación de Gen , Leishmania donovani , Leishmaniasis Visceral , Vacunas Atenuadas , Leishmania donovani/inmunología , Leishmania donovani/genética , Animales , Humanos , Perros , Vacunas Atenuadas/inmunología , Leishmaniasis Visceral/prevención & control , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Cricetinae , Vacunas contra la Leishmaniasis/inmunología , Vacunas contra la Leishmaniasis/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Leucocitos Mononucleares/inmunología , FemeninoRESUMEN
Visceral leishmaniasis is a potentially devastating neglected tropical disease caused by the protozoan parasites Leishmania donovani and L. infantum (chagasi). These parasites reside in tissue macrophages and survive by deploying a number of mechanisms aimed at subverting the host immune response. CD4+ T cells play an important role in controlling Leishmania parasites by providing help in the form of pro-inflammatory cytokines to activate microbiocidal pathways in infected macrophages. However, because these cytokines can also cause tissue damage if over-produced, regulatory immune responses develop, and the balance between pro-inflammatory and regulatory CD4+ T cells responses determines the outcomes of infection. Past studies have identified important roles for pro-inflammatory cytokines such as IFNγ and TNF, as well as regulatory co-inhibitory receptors and the potent anti-inflammatory cytokine IL-10. More recently, other immunoregulatory molecules have been identified that play important roles in CD4+ T cell responses during VL. In this review, we will discuss recent findings about two of these molecules; the NK cell granule protein Nkg7 and the anti-inflammatory cytokine TGFß, and describe how they impact CD4+ T cell functions and immune responses during visceral leishmaniasis.
Asunto(s)
Linfocitos T CD4-Positivos , Leishmania donovani , Leishmaniasis Visceral , Factor de Crecimiento Transformador beta , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Humanos , Linfocitos T CD4-Positivos/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/inmunología , Leishmania donovani/inmunología , Animales , Macrófagos/inmunología , Leishmania infantum/inmunología , Citocinas/metabolismoRESUMEN
A large proportion of HIV-coinfected visceral leishmaniasis (VL-HIV) patients exhibit chronic disease with frequent VL recurrence. However, knowledge on immunological determinants underlying the disease course is scarce. We longitudinally profiled the circulatory cellular immunity of an Ethiopian HIV cohort that included VL developers. We show that chronic VL-HIV patients exhibit high and persistent levels of TIGIT and PD-1 on CD8+/CD8- T cells, in addition to a lower frequency of IFN-γ+ TIGIT- CD8+/CD8- T cells, suggestive of impaired T cell functionality. At single T cell transcriptome and clonal resolution, the patients show CD4+ T cell anergy, characterised by a lack of T cell activation and lymphoproliferative response. These findings suggest that PD-1 and TIGIT play a pivotal role in VL-HIV chronicity, and may be further explored for patient risk stratification. Our findings provide a strong rationale for adjunctive immunotherapy for the treatment of chronic VL-HIV patients to break the recurrent disease cycle.
Asunto(s)
Coinfección , Infecciones por VIH , Leishmaniasis Visceral , Humanos , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/complicaciones , Leishmaniasis Visceral/parasitología , Infecciones por VIH/inmunología , Infecciones por VIH/complicaciones , Coinfección/inmunología , Masculino , Adulto , Femenino , Linfocitos T CD8-positivos/inmunología , Persona de Mediana Edad , Enfermedad Crónica , Linfocitos T CD4-Positivos/inmunología , EtiopíaRESUMEN
Berardinelli-Seip congenital lipodystrophy (CGL), a rare autosomal recessive disorder, is characterized by a lack of adipose tissue. Infections are one of the major causes of CGL individuals' premature death. The mechanisms that predispose to infections are poorly understood. We used Leishmania infantum as an in vitro model of intracellular infection to explore mechanisms underlying the CGL infection processes, and to understand the impact of host mutations on Leishmania survival, since this pathogen enters macrophages through specialized membrane lipid domains. The transcriptomic profiles of both uninfected and infected monocyte-derived macrophages (MDMs) from CGL (types 1 and 2) and controls were studied. MDMs infected with L. infantum showed significantly downregulated expression of genes associated with infection-response pathways (MHC-I, TCR-CD3, and granzymes). There was a transcriptomic signature in CGL cells associated with impaired membrane trafficking and signaling in response to infection, with concomitant changes in the expression of membrane-associated genes in parasites (e.g. δ-amastins). We identified pathways suggesting the lipid storage dysfunction led to changes in phospholipids expression and impaired responses to infection, including immune synapse (antigen presentation, IFN-γ signaling, JAK/STAT); endocytosis; NF-kappaB signaling; and phosphatidylinositol biosynthesis. In summary, lipid metabolism of the host plays an important role in determining antigen presentation pathways.
Asunto(s)
Leishmania infantum , Lipodistrofia Generalizada Congénita , Macrófagos , Transducción de Señal , Humanos , Macrófagos/metabolismo , Macrófagos/parasitología , Macrófagos/inmunología , Lipodistrofia Generalizada Congénita/genética , Lipodistrofia Generalizada Congénita/metabolismo , Leishmania infantum/genética , Transcriptoma , Masculino , Femenino , Perfilación de la Expresión Génica , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/metabolismoRESUMEN
The treatment for visceral leishmaniasis (VL) causes toxicity in patients, entails high cost and/or leads to the emergence of resistant strains. No human vaccine exists, and diagnosis presents problems related to the sensitivity or specificity of the tests. Here, we tested two phage clones, B1 and D11, which were shown to be protective against Leishmania infantum infection in a murine model as immunotherapeutics to treat mice infected with this parasite species. The phages were used alone or with amphotericin B (AmpB), while other mice received saline, AmpB, a wild-type phage (WTP) or WTP/AmpB. Results showed that the B1/AmpB and D11/AmpB combinations induced polarised Th1-type cellular and humoral responses, which were primed by high levels of parasite-specific IFN-γ, IL-12, TNF-α, nitrite and IgG2a antibodies, which reflected in significant reductions in the parasite load in distinct organs of the animals when analyses were performed 1 and 30 days after the treatments. Reduced organic toxicity was also found in these animals, as compared with the controls. In conclusion, preliminary data suggest the potential of the B1/AmpB and D11/AmpB combinations as immunotherapeutics against L. infantum infection.
Asunto(s)
Anfotericina B , Anticuerpos Antiprotozoarios , Inmunoterapia , Leishmania infantum , Leishmaniasis Visceral , Ratones Endogámicos BALB C , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/tratamiento farmacológico , Animales , Anfotericina B/uso terapéutico , Anfotericina B/administración & dosificación , Anticuerpos Antiprotozoarios/sangre , Leishmania infantum/inmunología , Leishmania infantum/efectos de los fármacos , Ratones , Inmunoterapia/métodos , Femenino , Antiprotozoarios/uso terapéutico , Antiprotozoarios/administración & dosificación , Inmunoglobulina G/sangre , Carga de Parásitos , Modelos Animales de Enfermedad , Técnicas de Visualización de Superficie Celular , Citocinas/metabolismo , Células TH1/inmunologíaRESUMEN
Host anti-inflammatory responses are critical for the progression of visceral leishmaniasis, and the pleiotropic cytokine interleukin (IL)-33 was found to be upregulated in infection. Here, we documented that IL-33 induction is a consequence of elevated cAMP-mediated exchange protein activated by cAMP (EPAC)/calcineurin-dependent signaling and essential for the sustenance of infection. Leishmania donovani-infected macrophages showed upregulation of IL-33 and its neutralization resulted in decreased parasite survival and increased inflammatory responses. Infection-induced cAMP was involved in IL-33 production and of its downstream effectors PKA and EPAC, only the latter was responsible for elevated IL-33 level. EPAC initiated Rap-dependent phospholipase C activation, which triggered the release of intracellular calcium followed by calcium/calmodulin complex formation. Screening of calmodulin-dependent enzymes affirmed involvement of the phosphatase calcineurin in cAMP/EPAC/calcium/calmodulin signaling-induced IL-33 production and parasite survival. Activated calcineurin ensured nuclear localization of the transcription factors, nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha required for IL-33 transcription, and we further confirmed this by chromatin immunoprecipitation assay. Administering specific inhibitors of nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha in BALB/c mouse model of visceral leishmaniasis decreased liver and spleen parasite burden along with reduction in IL-33 level. Splenocyte supernatants of inhibitor-treated infected mice further documented an increase in tumor necrosis factor alpha and IL-12 level with simultaneous decrease of IL-10, thereby indicating an overall disease-escalating effect of IL-33. Thus, this study demonstrates that cAMP/EPAC/calcineurin signaling is crucial for the activation of IL-33 and in effect creates anti-inflammatory responses, essential for infection.
Asunto(s)
Calcineurina , AMP Cíclico , Interleucina-33 , Leishmania donovani , Leishmaniasis Visceral , Ratones Endogámicos BALB C , Transducción de Señal , Animales , Ratones , Calcineurina/metabolismo , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Interleucina-33/metabolismo , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/metabolismo , Leishmaniasis Visceral/parasitología , Macrófagos/metabolismo , Macrófagos/parasitologíaRESUMEN
Apolipoprotein E (ApoE) has been associated with several diseases including Parkinson's disease, Alzheimer's and multiple sclerosis. ApoE also has documented immunomodulatory functions. We investigated gene expression in circulating monocytes and in bone marrows of patients with visceral leishmaniasis (VL) living in an endemic area in Bihar, India, and contrasted these with control healthy subjects or other diagnostic bone marrows from individuals in the same region. Samples from VL patients were obtained prior to initiating treatment. Our study revealed significant upregulated expression of the apoE transcript in patients with VL. Furthermore, the levels of ApoE protein were elevated in serum samples of subjects with VL compared with healthy endemic controls. These observations may provide clues regarding the complex interactions between lipid metabolism and immunoregulation of infectious and inflammatory diseases.
Asunto(s)
Apolipoproteínas E , Leishmaniasis Visceral , Monocitos , Regulación hacia Arriba , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Apolipoproteínas E/genética , Médula Ósea , India/epidemiología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/parasitología , Monocitos/inmunologíaRESUMEN
Leishmaniasis, a major globally re-emerging neglected tropical disease, has a restricted repertoire of chemotherapeutic options due to a narrow therapeutic index, drug resistance, or patient non-compliance due to toxicity. The disease is caused by the parasite Leishmania that resides in two different forms in two different environments: as sessile intracellular amastigotes within mammalian macrophages and as motile promastigotes in sandfly gut. As mitogen-activated protein kinases (MAPKs) play important roles in cellular differentiation and survival, we studied the expression of Leishmania donovani MAPKs (LdMAPKs). The homology studies by multiple sequence alignment show that excepting LdMAPK1 and LdMAPK2, all thirteen other LdMAPKs share homology with human ERK and p38 isoforms. Expression of LdMAPK4 and LdMAPK5 is less in avirulent promastigotes and amastigotes. Compared to miltefosine-sensitive L. donovani parasites, miltefosine-resistant parasites have higher LdMAPK1, LdMAPK3-5, LdMAPK7-11, LdMAPK13, and LdMAPK14 expression. IL-4-treatment of macrophages down-regulated LdMAPK11, in virulent amastigotes whereas up-regulated LdMAPK5, but down-regulated LdMAPK6, LdMAPK12-15, expression in avirulent amastigotes. IL-4 up-regulated LdMAPK1 expression in both virulent and avirulent amastigotes. IFN-γ-treatment down-regulated LdMAPK6, LdMAPK13, and LdMAPK15 in avirulent amastigotes but up-regulated in virulent amastigotes. This complex profile of LdMAPKs expression among virulent and avirulent parasites, drug-resistant parasites, and in amastigotes within IL-4 or IFN-γ-treated macrophages suggests that LdMAPKs are differentially controlled at the host-parasite interface regulating parasite survival and differentiation, and in the course of IL-4 or IFN-γ dominated immune response.
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Interacciones Huésped-Parásitos , Leishmania donovani , Macrófagos , Proteínas Quinasas Activadas por Mitógenos , Leishmania donovani/enzimología , Animales , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ratones , Macrófagos/parasitología , Macrófagos/metabolismo , Humanos , Ratones Endogámicos BALB C , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/inmunología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Interferón gamma/metabolismo , Resistencia a MedicamentosRESUMEN
Visceral leishmaniasis (VL) is a potentially fatal parasitic infection caused by Leishmania donovani in India. L. donovani is an obligate intracellular protozoan residing mostly in macrophages of the reticuloendothelial system throughout chronic infection. Monocytic phagocytes are critical in the pathogenesis of different forms of leishmaniasis. Subsets of monocytes are distinguished by their surface markers into CD14+CD16- classical monocytes, CD14+CD16+ intermediate monocytes, and CD16++CD14low non-classical monocyte subsets. During cutaneous leishmaniasis (CL), intermediate monocyte are reported to be a source of inflammatory cytokines IL-1ß and TNF, and they express CCR2 attracting them to sites of inflammatory pathology. We examined monocyte subsets in the blood and bone marrow of patients with VL from an endemic site in Bihar, India, and found these contrasted with the roles of monocytes in CL. During VL, intermediate and non-classical CD16+ monocyte subsets expressed instead a non-inflammatory phenotype with low CCR2, high CX3CR1 and low microbicidal oxidant generation, making them more similar to patrolling monocytes than inflammatory cells. Bone marrow CD16+ monocyte subsets expressed a phenotype that might be more similar to the inflammatory subsets of CL, although our inability to obtain bone marrow from healthy donors in the endemic region hampered this interpretation Overall the data suggest that CD16+ intermediate monocyte subsets in VL patients express a phenotypes that contributes to an immunosuppressed pathologic immune state, but in contrast to CL, these do not mediate localized inflammatory responses.
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Médula Ósea , Leishmaniasis Visceral , Monocitos , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Humanos , Monocitos/inmunología , India , Adulto , Masculino , Médula Ósea/parasitología , Femenino , Receptores de IgG/análisis , Receptores de IgG/metabolismo , Leishmania donovani/inmunología , Leishmania donovani/fisiología , Adulto Joven , Adolescente , Receptores CCR2/metabolismo , Persona de Mediana Edad , Niño , Receptores de Quimiocina/metabolismo , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Citocinas/metabolismoRESUMEN
Visceral leishmaniasis (VL) is an infectious disease caused by Leishmania infantum. Clinically, VL evolves with systemic impairment, immunosuppression and hyperactivation with hypergammaglobulinemia. Although renal involvement has been recognized, a dearth of understanding about the underlying mechanisms driving acute kidney injury (AKI) in VL remains. We aimed to evaluate the involvement of immunoglobulins (Igs) and immune complexes (CIC) in the occurrence of AKI in VL patients. Fourteen VL patients were evaluated between early treatment and 12 months post-treatment (mpt). Anti-Leishmania Igs, CIC, cystatin C, C3a and C5a were assessed and correlated with AKI markers. Interestingly, high levels of CIC were observed in VL patients up to 6 mpt. Concomitantly, twelve patients met the criteria for AKI, while high levels of cystatin C were observed up to 6 mpt. Plasmatic cystatin C was positively correlated with CIC and Igs. Moreover, C5a was correlated with cystatin C, CIC and Igs. We did not identify any correlation between amphotericin B use and kidney function markers in VL patients, although this association needs to be further explored in subsequent studies. Our data reinforce the presence of an important renal function impairment during VL, suggesting the involvement of Igs, CIC, and C5a in this clinical condition.
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Lesión Renal Aguda , Complejo Antígeno-Anticuerpo , Leishmaniasis Visceral , Humanos , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/sangre , Lesión Renal Aguda/sangre , Lesión Renal Aguda/inmunología , Lesión Renal Aguda/parasitología , Masculino , Femenino , Complejo Antígeno-Anticuerpo/sangre , Adulto , Biomarcadores/sangre , Persona de Mediana Edad , Cistatina C/sangre , Adolescente , Adulto Joven , Anfotericina B/uso terapéutico , Leishmania infantum/inmunologíaRESUMEN
Visceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE: Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.
Asunto(s)
Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Leishmania donovani , Leishmaniasis Visceral , Proteínas Protozoarias , Pruebas Serológicas , Leishmania donovani/genética , Leishmania donovani/inmunología , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Animales , Humanos , Ratones , Perros , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Pruebas Serológicas/métodos , Biomarcadores/sangre , Femenino , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Ratones Endogámicos BALB C , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Sensibilidad y Especificidad , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitologíaRESUMEN
Our developed cell division-specific 'centrin' gene deleted Leishmania donovani (LdCen1-/-) the causative parasite of the fatal visceral-leishmaniasis (VL), exhibits a selective growth arrest at the intracellular stage and is anticipated as a live attenuated vaccine candidate against VL. LdCen1-/- immunization in animals has shown increased IFN-γ secreting CD4+ and CD8+ T cells along with protection conferred by a protective proinflammatory immune response. A label-free proteomics approach has been employed to understand the physiology of infection and predict disease interceptors during Leishmania-host interactions. Proteomic modulation after infection of human macrophage cell lines suggested elevated annexin A6, implying involvement in various biological processes such as membrane repair, transport, actin dynamics, cell proliferation, survival, differentiation, and inflammation, thereby potentiating its immunological protective capacity. Additionally, S100A8 and S100A9 proteins, known for maintaining homeostatic balance in regulating the inflammatory response, have been upregulated after infection. The inhibitory clade of serpins, known to inhibit cysteine proteases (CPs), was upregulated in host cells after 48 h of infection. This is reflected in the diminished expression of CPs in the parasites during infection. Such proteome analysis confirms LdCen1-/- efficacy as a vaccine candidate and predicts potential markers in future vaccine development strategies against infectious diseases.