RESUMO
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.
Assuntos
Alelos , Leishmania infantum , Humanos , Leishmania infantum/genética , Espanha/epidemiologia , Masculino , Feminino , Adulto , Predisposição Genética para Doença , Leishmaniose Visceral/genética , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/epidemiologia , Estudos de Coortes , Pessoa de Meia-Idade , Antígenos HLA/genética , Frequência do GeneRESUMO
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.
Assuntos
Leishmania infantum , MicroRNAs , Neutrófilos , RNA Longo não Codificante , RNA Mensageiro , Humanos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Leishmania infantum/genética , Leishmania infantum/imunologia , RNA Longo não Codificante/genética , MicroRNAs/genética , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/genética , Adulto , Perfilação da Expressão GênicaRESUMO
The spleen plays a pivotal role in the pathogenesis of visceral leishmaniasis. In severe forms of the disease, the spleen undergoes changes that can compromise its function in surveilling blood-circulating pathogens. In this study, we present an integrated analysis of the structural and gene expression alterations in the spleens of three patients with relapsing visceral leishmaniasis, two of whom were coinfected with HIV. Our findings reveal that the IL6 signaling pathway plays a significant role in the disorganization of the white pulp, while BCL10 and ICOSLG are associated with spleen organization. Patients coinfected with HIV and visceral leishmaniasis exhibited lower splenic CD4+ cell density and reduced expression of genes such as IL15. These effects may contribute to a compromised immune response against L. infantum in coinfected individuals, further impacting the structural organization of the spleen.
Assuntos
Coinfecção , Infecções por HIV , Leishmaniose Visceral , Baço , Humanos , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/genética , Baço/patologia , Infecções por HIV/complicações , Coinfecção/virologia , Masculino , Adulto , Feminino , Linfócitos T CD4-Positivos/imunologia , Leishmania infantum/genética , Expressão GênicaRESUMO
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.
Assuntos
Leishmania infantum , Lipodistrofia Generalizada Congênita , Macrófagos , Transdução de Sinais , Humanos , Macrófagos/metabolismo , Macrófagos/parasitologia , Macrófagos/imunologia , Lipodistrofia Generalizada Congênita/genética , Lipodistrofia Generalizada Congênita/metabolismo , Leishmania infantum/genética , Transcriptoma , Masculino , Feminino , Perfilação da Expressão Gênica , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/metabolismoRESUMO
Canine leishmaniosis (CanL), caused by Leishmania infantum, is a complex disease of growing importance in Europe. Clinical manifestations result from the down-modulation of the host immune response through multiple host-parasite interactions. Although several factors might influence CanL progression, this is the first known study evaluating risk factors for its different clinical stages in a large referral hospital population (n = 35.669) from an endemic area, over a 20 year period. Genome-wide scans for selection signatures were also conducted to explore the genomic component of clinical susceptibility to L. infantum infection. The prevalence of CanL was 3.2% (16.7% stage I; 43.6% stage II; 32.1% stage III; 7.6% stage IV). Dog breed (crossbreed), bodyweight (<10 kg), living conditions (indoors), regular deworming treatment, and being vaccinated against Leishmania significantly decreased the transmission risk and the risk for developing severe clinical forms. Conversely, the detection of comorbidities was associated with advanced clinical forms, particularly chronic kidney disease, neoplasia, cryptorchidism, infectious tracheobronchitis and urate urolithiasis, although those did not impact the clinical outcome. Significant associations between an increased risk of severe clinical stages and findings in the anamnesis (renal or skin-related manifestations) and physical examination (ocular findings) were also detected, highlighting their diagnostic value in referred cases of CanL. Sixteen breeds were found to be significantly more susceptible to developing severe stages of leishmaniosis (e.g. Great Dane, Rottweiler, English Springer Spaniel, Boxer, American Staffordshire Terrier, Golden Retriever), while 20 breeds displayed a clinical resistantance phenotype and, thus, are more likely to mount an efficient immune response against L. infantum (e.g. Pointer, Samoyed, Spanish Mastiff, Spanish Greyhound, English Setter, Siberian Husky). Genomic analyses of these breeds retrieved 12 regions under selection, 63 candidate genes and pinpointed multiple biological pathways such as the IRE1 branch of the unfolded protein response, which could play a critical role in clinical susceptibility to L. infantum infection.
Assuntos
Doenças do Cão , Predisposição Genética para Doença , Leishmania infantum , Cães , Animais , Doenças do Cão/parasitologia , Doenças do Cão/epidemiologia , Doenças do Cão/genética , Leishmania infantum/genética , Masculino , Fatores de Risco , Leishmaniose Visceral/veterinária , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/genética , Comorbidade , Feminino , Progressão da Doença , Leishmaniose/veterinária , Leishmaniose/epidemiologia , Leishmaniose/parasitologia , Prevalência , Estudo de Associação Genômica AmplaRESUMO
Introduction: Single nucleotide variations (SNVs) are specific genetic variations that commonly occur in a population and often do not manifest phenotypically. However, depending on their location and the type of nucleotide exchanged, an SNV can alter or inhibit the function of the gene in which it occurs. Immunoglobulin G (IgG) receptor genes have exhibited several polymorphisms, including rs1801274, which is found in the FcgRIIa gene. The replacement of A with T results in a Histidine (H) to Arginine (R) substitution, altering the affinity of the IgG receptor for IgG subtypes and C-reactive protein (CRP). In this study, we analyzed rs1801274 and its functional implications concerning L. Infantum uptake and cytokine production. Methods: We genotyped 201 individuals from an endemic area for visceral leishmaniasis to assess the presence of rs1801274 using Taqman probes for a candidate gene study. Additionally, we included seventy individuals from a non-endemic area for a functional study. Subsequently, we isolated and cultivated one-week adherent mononuclear cells (AMCs) derived from the peripheral blood of participants residing in the non-endemic region in the presence of L. infantum promastigotes, with and without antigen-specific IgG and/or CRP. We analyzed the rate of phagocytosis and the production of nitric oxide (NO), tumor necrosis factor (TNF)-a, interleukin (IL)-10, IL-12 p70, IL-1b, IL- 6, and IL-8 in the culture supernatants. Results and discussion: In participants from the endemic region, the A/G (H/R isoform) heterozygous genotype was significantly associated with susceptibility to the disease. Furthermore, SNVs induced a change in the phagocytosis rate in an opsonin-dependent manner. Opsonization with IgG increased the production of IL-10, TNF-a, and IL-6 in AMCs with the H/R isoform, followed by a decrease in NO production. The results presented here suggest that the rs1801274 polymorphism is linked to a higher susceptibility to visceral leishmaniasis.
Assuntos
Leishmania infantum , Leishmaniose Visceral , Humanos , Leishmaniose Visceral/genética , Leishmania infantum/genética , Receptores de IgG/genética , Interleucina-12 , Fator de Necrose Tumoral alfa , Nucleotídeos , Isoformas de Proteínas , Variação Genética , Imunoglobulina GRESUMO
Domestic dogs are the primary urban reservoirs of Leishmania infantum, the causative agent of visceral leishmaniasis. In Canine Leishmaniasis (CanL), modulation of the host's immune response may be associated with the expression of small non-coding RNAs called microRNA (miR). miR-194 expression increases in peripheral blood mononuclear cells (PBMCs) of dogs with leishmaniasis with a positive correlation with the parasite load and in silico analysis demonstrated that the TRAF6 gene is the target of miR-194 in PBMCs from diseased dogs. Here, we isolated PBMCs from 5 healthy dogs and 28 dogs with leishmaniasis, naturally infected with L. infantum. To confirm changes in miR-194 and TRAF6 expression, basal expression of miR-194 and gene expression of TRAF6 was measured using qPCR. PBMCs from healthy dogs and dogs with leishmaniasis were transfected with miR-194 scramble, mimic, and inhibitor and cultured at 37° C, 5% CO2 for 48 hours. The expression of possible targets was measured: iNOS, NO, T-bet, GATA3, and FoxP3 were measured using flow cytometry; the production of cytokines IL-1ß, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and TGF-ß in cell culture supernatants was measured using capture enzyme-linked immunosorbent assays (ELISA). Parasite load was measured using cytometry and qPCR. Functional assays followed by miR-194 inhibitor and IL-1ß blockade and assessment of NO production were also performed. Basal miR-194 expression was increased in PBMC from dogs with Leishmaniasis and was negatively correlated with TRAF6 expression. The mimic of miR-194 promoted an increase in parasite load. There were no significant changes in T-bet, GATA3, or FoxP3 expression with miR-194 enhancement or inhibition. Inhibition of miR-194 increased IL-1ß and NO in PBMCs from diseased dogs, and blockade of IL-1ß following miR-194 inhibition decreased NO levels. These findings suggest that miR-194 is upregulated in PBMCs from dogs with leishmaniasis and increases parasite load, possibly decreasing NO production via IL-1ß. These results increase our understanding of the mechanisms of evasion of the immune response by the parasite and the identification of possible therapeutic targets.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , MicroRNAs , Animais , Cães , Citocinas/genética , Doenças do Cão/parasitologia , Fatores de Transcrição Forkhead , Leishmania infantum/fisiologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/veterinária , Leucócitos Mononucleares , MicroRNAs/genética , Óxido Nítrico/metabolismo , Carga Parasitária , Fator 6 Associado a Receptor de TNF , Leishmaniose/veterinária , Interleucina-1beta/metabolismoRESUMO
Leishmaniasis is a zoonotic parasitic disease, and the main reservoir of the parasite is the dog, although recent years have seen an increase in other mammalian species. In the Mediterranean region, where it is an endemic disease, it is caused by the species Leishmania infantum. The Ibizan hound, an autochthonous breed of this region, appears to have a genetic resistance to parasitic infection, whereas other canine breeds, such as the Boxer, are susceptible to infection. These differences are related to the differentiated activation of the immune response, with the Ibizan hound activating the Th1 immune response, whereas the Boxer breed triggers the Th2 immune response. Cytokine levels and genomic haplotypes of several genes involved in the immune response were analysed in twenty-eight Ibizan hound (resistant canine breed model) and twenty-four Boxer (susceptible canine breed) without clinical signs in the Mediterranean region. Cytokine levels were analysed by ELISA commercial kits and haplotypes were studied using CanineHD DNA Analysis BeadChip including 165,480 mapped positions. The results show 126 haplotypes associated with differential immune response in dogs. Specifically, haplotypes in IL12RB1, IL6R, CIITA, THEMIS, NOXA1, HEY2, RAB38, SLC35D2, SLC28A3, RASEF and DAPK1 genes are associated with serum levels of IFN-γ, IL-2, IL-8, and IL-18. These results suggest that the resistance or susceptibility to Leishmania infantum infection could be a consequence of haplotypes in several genes related to immune response. Future studies are needed to elucidate the relationship of these haplotypes with immune response and gene expression regulation.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Animais , Cães , Leishmania infantum/genética , Haplótipos , Leishmaniose/veterinária , Citocinas , Zoonoses , Imunidade , Doenças do Cão/diagnóstico , Leishmaniose Visceral/genética , Leishmaniose Visceral/veterinária , MamíferosRESUMO
BACKGROUND: The Ibizan Hound is a canine breed native to the Mediterranean region, where leishmaniasis is an endemic zoonosis. Several studies indicate a low prevalence of this disease in Ibizan Hound dogs, whereas other canine breeds present a high prevalence. However, the underlying molecular mechanisms still remain unknown. The aim of this work is to analyse the relationship between serum levels of cytokines and the genomic profiles in two canine breeds, Ibizan Hound (resistant canine breed model) and Boxer (susceptible canine breed model). METHODS: In this study, we analyse the haplotypes of genes encoding cytokines related to immune response of Leishmania infantum infection in twenty-four Boxers and twenty-eight Ibizan Hounds apparently healthy using CanineHD DNA Analysis BeadChip including 165,480 mapped positions. The haplo.glm extension of haplo.score was used to perform a General Linear Model (GLM) regression to estimate the magnitude of individual haplotype effects within each cytokine. RESULTS: Mean levels of interferon gamma (IFN-γ), interleukin 2 (IL-2) and IL-18 in Boxer dogs were 0.19 ± 0.05 ng/ml, 46.70 ± 4.54 ng/ml, and 36.37 ± 30.59 pg/ml, whereas Ibizan Hound dogs present 0.49 ± 0.05 ng/ml, 64.55 ± 4.54 ng/ml, and 492.10 ± 31.18 pg/ml, respectively. The GLM regression shows fifteen haplotypes with statistically significant effect on the cytokine serum levels (P < 0.05). The more relevant are IL6-CGAAG and IFNG-GCA haplotypes, which increase and decrease the IL-2, IL-8 and IFN-γ serum levels, respectively. CONCLUSIONS: Haplotypes in the IFNG and IL6 genes have been correlated to serum levels of IFN-γ, IL-2 and IL-18, and a moderate effect has been found on IL8 haplotype correlated to IL-8 and IL-18 serum levels. The results indicate that the resistance to L. infantum infection could be a consequence of certain haplotypes with a high frequency in the Ibizan Hound dog breed, while susceptibility to the disease would be related to other specific haplotypes, with high frequency in Boxer. Future studies are needed to elucidate whether these differences and haplotypes are related to different phenotypes in immune response and expression gene regulation to L. infantum infections in dogs and their possible application in new treatments and vaccines.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Animais , Cães , Citocinas/genética , Interleucina-6 , Interferon gama/genética , Interleucina-2/genética , Interleucina-18/genética , Leishmania infantum/genética , Haplótipos , Interleucina-8/genética , Doenças do Cão/epidemiologia , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/veterináriaRESUMO
Leishmaniases, a group of vector-borne diseases, are caused by the protozoan intracellular parasite Leishmania (L.) and are transmitted by the phlebotomine sandflies. A wide range of clinical manifestations in L- infection is observed. The clinical outcome ranges from asymptomatic, cutaneous leishmaniasis (CL) to severe mucosal leishmaniasis (ML) or visceral leishmaniasis (VL), depending on the L. species. Interestingly, only a fraction of L.-infected individuals progress to disease development, suggesting a key role of host genetics in the clinical outcome. NOD2 plays a critical role in the control of host defense and inflammation. The NOD2-RIK2 pathway is involved in developing a Th1- type response in patients with VL and C57BL/6 mice infected with L. infantum. We investigated whether variants in the NOD2 gene (R702W rs2066844, G908R rs2066845, and L1007fsinsC rs2066847) are associated with susceptibility to CL caused by L. guyanensis (Lg) in 837 patients with Lg-Cl and 797 healthy controls (HC) with no history of leishmaniasis. Both patients and HC are from the same endemic area of the Amazonas state of Brazil. The variants R702W and G908R were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and L1007fsinsC was by direct nucleotide sequencing. The minor allele frequency (MAF) of L1007fsinsC was 0.5% among the patients with Lg-CL and 0.6% in the healthy controls group. R702W genotypes frequencies were similar in both groups. Only 1% and 1.6% were heterozygous for G908R among the patients with Lg-CL and HC, respectively. None of the variants revealed any association with susceptibility to the development of Lg-CL. Correlations of genotypes with the level of plasma cytokines revealed that individuals with the mutant alleles of R702W tend to have low levels of IFN-γ. G908R heterozygotes also tend to have low IFN-γ, TNF-α, IL-17, and IL-8. Variants of NOD2 are not involved in the pathogenesis of Lg-CL.
Assuntos
Leishmaniose Cutânea , Leishmaniose Visceral , Proteína Adaptadora de Sinalização NOD2 , Animais , Camundongos , Citocinas/genética , Genótipo , Leishmania guyanensis , Leishmaniose Cutânea/genética , Leishmaniose Visceral/genética , Camundongos Endogâmicos C57BL , Humanos , Proteína Adaptadora de Sinalização NOD2/genéticaRESUMO
Gain-of-function mutations in the STAT1 gene have been initially associated with chronic mucocutaneous candidiasis. However, further research has shown that STAT1 GOF variants may increase susceptibility to infection by other intracellular pathogens. This report describes the first case of disseminated leishmaniasis associated with a STAT1 GOF mutation in a pediatric patient who did not have chronic mucocutaneous candidiasis. The patient was a four-year-old boy presenting with fever, severe asthenia, hepatosplenomegaly, pancytopenia, and liver failure. Bone marrow aspirate revealed hemophagocytosis and Leishmania parasites. Treatment consisted primarily of liposomal amphotericin B, as per the Hemophagocytic Lymphohistiocytosis 2004 protocol. After eight weeks of treatment, the patient did not improve and was submitted to diagnostic splenectomy. Activated macrophages and nodular spleen necrosis secondary to the visceral leishmaniasis were detected. Unfortunately, the patient died in the second week after splenectomy due to overwhelming systemic infection. DNA sequencing revealed a pathogenic (p. R274Q) GOF mutation in STAT1.
Assuntos
Candidíase Mucocutânea Crônica , Leishmaniose Visceral , Candidíase Mucocutânea Crônica/complicações , Candidíase Mucocutânea Crônica/genética , Criança , Pré-Escolar , Mutação com Ganho de Função , Humanos , Leishmaniose Visceral/complicações , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/genética , Masculino , Mutação , Fator de Transcrição STAT1/genéticaRESUMO
BACKGROUND: Leishmaniases are a group of anthropo-zoonotic parasitic diseases caused by a protozoan of the Leishmania genus, affecting both humans and other vertebrates, including dogs. L. infantum is responsible for the visceral and occasionally cutaneous form of the disease in humans and canine leishmaniasis. Previously, we have shown that L. infantum induces a mild but significant increase in endoplasmic reticulum (ER) stress expression markers to promote parasites survival in human and murine infected macrophages. Moreover, we demonstrated that the miRNA hsa-miR-346, induced by the UPR-activated transcription factor sXBP1, was significantly upregulated in human macrophages infected with different L. infantum strains. However, the ER stress response in infected dogs, which represent an important reservoir for Leishmania parasite, was described once recently, whereas the miR-346 expression was not reported before. Therefore, this study aimed to investigate these pathways in the canine macrophage-like cell line DH82 infected by Leishmania spp. and to evaluate the presence of cfa-miR-346 in plasma of non-infected and infected dogs. The DH82 cells were infected with L. infantum and L. braziliensis parasites and the expression of cfa-mir-346 and several ER stress markers was evaluated by quantitative PCR (qPCR) at different time points. Furthermore, the cfa-miR-346 was monitored in plasma collected from non-infected dogs (n = 11) and dogs naturally infected by L. infantum (n = 18). RESULTS: The results in DH82 cells showed that cfa-mir-346 was induced at both 24 h and 48 h post-infection with all Leishmania strains but not with tunicamycin, accounting for a mechanism of induction independent from sXBP1, unlike what was previously observed in human cell lines. Moreover, the cfa-miR-346 expression analysis on plasma revealed a significant increase in infected dogs compared to non-infected dogs. CONCLUSIONS: Here for the first time, we report the upregulation of cfa-miR-346 induced by Leishmania infection in canine macrophage-like cells and plasma samples of naturally infected dogs. According to our results, the cfa-miR-346 appears to be linked to infection, and understanding its role and identifying its target genes could contribute to elucidate the mechanisms underlying the host-pathogen interaction in leishmaniasis.
Assuntos
Doenças do Cão , Leishmaniose Visceral , MicroRNAs , Animais , Doenças do Cão/genética , Doenças do Cão/parasitologia , Cães , Leishmania infantum , Leishmaniose Visceral/genética , Leishmaniose Visceral/veterinária , MicroRNAs/genéticaRESUMO
Visceral leishmaniasis (VL; also known as kala-azar), caused by the protozoan parasite Leishmania donovani, is characterized by the inability of the host to generate an effective immune response. The manifestations of the disease depend on the involvement of various immune components such as activation of macrophages, cell mediated immunity, secretion of cytokines and chemokines, etc. Macrophages are the final host cells for Leishmania parasites to multiply, and they are the key to a controlled or aggravated response that leads to clinical symptoms. The two most common macrophage phenotypes are M1 and M2. The pro-inflammatory microenvironment (mainly by IL-1ß, IL-6, IL-12, IL-23, and TNF-α cytokines) and tissue injury driven by classically activated macrophages (M1-like) and wound healing driven by alternatively activated macrophages (M2-like) in an anti-inflammatory environment (mainly by IL-10, TGF-ß, chemokine ligand (CCL)1, CCL2, CCL17, CCL18, and CCL22). Moreover, on polarized Th cells, chemokine receptors are expressed differently. Typically, CXCR3 and CCR5 are preferentially expressed on polarized Th1 cells, whereas CCR3, CCR4, and CCR8 have been associated with the Th2 phenotype. Further, the ability of the host to produce a cell-mediated immune response capable of regulating and/or eliminating the parasite is critical in the fight against the disease. Here, we review the interactions between parasites and chemokines and chemokine receptors in the pathogenesis of VL.
Assuntos
Leishmania donovani , Leishmaniose Visceral , Quimiocinas , Citocinas/genética , Humanos , Leishmaniose Visceral/genética , Leishmaniose Visceral/parasitologia , Células Th1RESUMO
Visceral leishmaniasis (VL) is a vector-borne infectious disease that can be potentially fatal if left untreated. In Brazil, it is caused by Leishmania infantum parasites. Blood transcriptomics allows us to assess the molecular mechanisms involved in the immunopathological processes of several clinical conditions, namely, parasitic diseases. Here, we performed mRNA sequencing of peripheral blood from patients with visceral leishmaniasis during the active phase of the disease and six months after successful treatment, when the patients were considered clinically cured. To strengthen the study, the RNA-seq data analysis included two other non-diseased groups composed of healthy uninfected volunteers and asymptomatic individuals. We identified thousands of differentially expressed genes between VL patients and non-diseased groups. Overall, pathway analysis corroborated the importance of signaling involving interferons, chemokines, Toll-like receptors and the neutrophil response. Cellular deconvolution of gene expression profiles was able to discriminate cellular subtypes, highlighting the contribution of plasma cells and NK cells in the course of the disease. Beyond the biological processes involved in the immunopathology of VL revealed by the expression of protein coding genes (PCGs), we observed a significant participation of long noncoding RNAs (lncRNAs) in our blood transcriptome dataset. Genome-wide analysis of lncRNAs expression in VL has never been performed. lncRNAs have been considered key regulators of disease progression, mainly in cancers; however, their pattern regulation may also help to understand the complexity and heterogeneity of host immune responses elicited by L. infantum infections in humans. Among our findings, we identified lncRNAs such as IL21-AS1, MIR4435-2HG and LINC01501 and coexpressed lncRNA/mRNA pairs such as CA3-AS1/CA1, GASAL1/IFNG and LINC01127/IL1R1-IL1R2. Thus, for the first time, we present an integrated analysis of PCGs and lncRNAs by exploring the lncRNA-mRNA coexpression profile of VL to provide insights into the regulatory gene network involved in the development of this inflammatory and infectious disease.
Assuntos
Leishmania infantum , Leishmaniose Visceral , Leishmaniose , RNA Longo não Codificante , Humanos , Leishmania infantum/genética , Leishmaniose Visceral/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , TranscriptomaRESUMO
Proinflammatory cytokine secretion determines the infection course in leishmaniasis. The immunopathology of canine leishmaniasis (CanL) caused by Leishmania infantum is characterized by low Leishmania-specific IFN-γ and IL-17 production. Mutations in the human IL-17 gene promoter alter cytokine expression and may increase the susceptibility of humans to some infectious diseases. In this study, we correlated canine IL-17 single nucleotide polymorphisms (SNPs) with anti-Leishmania IgG levels, parasite load and external clinical signs in dogs naturally exposed to L. infantum in Brazil. A higher frequency (Chi-square test: X2= 5.378, df= 1, P= 0.020) of major alleles was observed among dogs showing no external clinical signs attributable to Leishmania infection. A high proportion of A allele carriers (mutant) were observed among dogs with high antibody levels, although differences were not statistically significant (Chi-square test: X2= 4.410, df= 4, P= 0.353), as compared to dogs with low antibody levels. In general, the association of canine IL-17 SNPs with disease expression or disease exasperation did not reach enough statistical power to allow the use of these mutations as prognostic markers. This knowledge may pave the way for further investigations on the genetic aspects of CanL and its immunotherapy.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Animais , Citocinas/metabolismo , Doenças do Cão/parasitologia , Cães , Interleucina-17/genética , Leishmaniose/genética , Leishmaniose/veterinária , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/genética , Leishmaniose Visceral/veterináriaRESUMO
Visceral leishmaniasis in humans is a chronic and fatal disease if left untreated. Canine leishmaniasis (CanL) is a severe public health problem because infected animals are powerful transmitters of the parasite to humans via phlebotomine vectors. Therefore, dogs are an essential target for control measures. Progression of canine infection is accompanied by failure of cellular immunity with reduction of circulating lymphocytes and increased cytokines that suppress macrophage function. Studies showed that the regulation of the effector function of macrophages and T cells appears to depend on miRNAs; miRNA-21 (miR-21) shows increased expression in splenic leukocytes of dogs with CanL and targets genes related to the immune response. Mimics and inhibitors of miR-21 were used in vitro to transfect splenic leukocytes from dogs with CanL. After transfection, expression levels of the proteins FAS, FASL, CD69, CCR7, TNF-α, IL-17, IFN-γ, and IL-10 were measured. FAS, FASL, CD69, and CCR7 expression levels decreased in splenic leukocytes from dogs with CanL. The miR-21 mimic decreased CD69 expression in splenic leukocytes from CanL and healthy groups. The miR-21 inhibitor decreased IL-10 levels in culture supernatants from splenic leukocytes in the CanL group. These findings suggest that miR-21 alters the immune response in CanL; therefore, miR-21 could be used as a possible therapeutic target for CanL.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , MicroRNAs , Animais , Doenças do Cão/parasitologia , Cães , Interleucina-10/genética , Interleucina-10/uso terapêutico , Leishmaniose/veterinária , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/genética , Leishmaniose Visceral/veterinária , MicroRNAs/genética , MicroRNAs/uso terapêutico , Receptores CCR7RESUMO
Zoonotic visceral leishmaniosis caused by Leishmania infantum is an endemic disease in the Mediterranean Basin affecting mainly humans and dogs, the main reservoir. The leishmaniosis outbreak declared in the Community of Madrid (Spain) led to a significant increase in human disease incidence without enhancing canine leishmaniosis prevalence, suggesting a better adaptation of the outbreak's isolates by other host species. One of the isolates obtained in the focus, IPER/ES/2012/BOS1FL1 (BOS1FL1), has previously demonstrated a different phenotype than the reference strain MCAN/ES/1996/BCN150 (BCN150), characterized by a lower infectivity when interacting with canine macrophages. Nevertheless, not enough changes in the cell defensive response were found to support their different behavior. Thus, we decided to investigate the molecular mechanisms involved in the interaction of both parasites with DH82 canine macrophages by studying their transcriptomic profiles developed after infection using RNA sequencing. The results showed a common regulation induced by both parasites in the phosphoinositide-3-kinase-protein kinase B/Akt and NOD-like receptor signaling pathways. However, other pathways, such as phagocytosis and signal transduction, including tumor necrosis factor, mitogen-activated kinases and nuclear factor-κB, were only regulated after infection with BOS1FL1. These differences could contribute to the reduced infection ability of the outbreak isolates in canine cells. Our results open a new avenue to investigate the true role of adaptation of L. infantum isolates in their interaction with their different hosts.
Assuntos
Cães/genética , Cães/parasitologia , Leishmania infantum/patogenicidade , Leishmaniose Visceral/genética , Leishmaniose Visceral/veterinária , Estágios do Ciclo de Vida/fisiologia , Macrófagos/parasitologia , Transcriptoma/genética , Animais , Linhagem Celular , Regulação da Expressão Gênica , Ontologia Genética , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Visceral/parasitologia , Macrófagos/metabolismo , Proteínas NLR/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , VirulênciaRESUMO
The implementation of prospective drug resistance (DR) studies in the research-and-development (R&D) pipeline is a common practice for many infectious diseases but not for neglected tropical diseases (NTDs). Here, we explored and demonstrated the importance of this approach using as paradigms Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GlaxoSmithKline (GSK) "Leishbox" to treat VL. We experimentally selected resistance to TCMDC-143345 in vitro and characterized resistant parasites at the genomic and phenotypic levels. We found that it took more time to develop resistance to TCMDC-143345 than to other drugs in clinical use and that there was no cross-resistance to these drugs, suggesting a new and unique mechanism. By whole-genome sequencing, we found two mutations in the gene encoding the L. donovani dynamin-1-like protein (LdoDLP1) that were fixed at the highest drug pressure. Through phylogenetic analysis, we identified LdoDLP1 as a family member of the dynamin-related proteins, a group of proteins that impacts the shapes of biological membranes by mediating fusion and fission events, with a putative role in mitochondrial fission. We found that L. donovani lines genetically engineered to harbor the two identified LdoDLP1 mutations were resistant to TCMDC-143345 and displayed altered mitochondrial properties. By homology modeling, we showed how the two LdoDLP1 mutations may influence protein structure and function. Taken together, our data reveal a clear involvement of LdoDLP1 in the adaptation/reduced susceptibility of L. donovani to TCMDC-143345. IMPORTANCE Humans and their pathogens are continuously locked in a molecular arms race during which the eventual emergence of pathogen drug resistance (DR) seems inevitable. For neglected tropical diseases (NTDs), DR is generally studied retrospectively once it has already been established in clinical settings. We previously recommended to keep one step ahead in the host-pathogen arms race and implement prospective DR studies in the R&D pipeline, a common practice for many infectious diseases but not for NTDs. Here, using Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK Leishbox to treat VL, as paradigms, we experimentally selected resistance to the compound and proceeded to genomic and phenotypic characterization of DR parasites. The results gathered in the present study suggest a new DR mechanism involving the L. donovani dynamin-1-like protein (LdoDLP1) and demonstrate the practical relevance of prospective DR studies.
Assuntos
Antiprotozoários , Resistência a Medicamentos , Dinamina I , Leishmania donovani , Leishmaniose Visceral , Humanos , Antiprotozoários/imunologia , Dinamina I/genética , Dinamina I/imunologia , Genômica , Leishmania donovani/genética , Leishmania donovani/imunologia , Leishmania donovani/parasitologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Filogenia , Estudos Retrospectivos , Resistência a Medicamentos/genética , Resistência a Medicamentos/imunologiaRESUMO
Leishmaniasis is a disease of the subtropical and tropical spheres of the earth and has various clinical manifestations. The different form of leishmaniasis includes cutaneous leishmaniasis, mucocutaneous leishmaniasis, most lethal visceral leishmaniasis and PKDL form. These different forms depend on many factors such as parasite and vector species, geographical, environmental conditions and population ethnicity. Host genetic factors have been widely investigated for their role in developing the disease in various infections. There are several reports on associations or resistance between candidate gene polymorphisms and the risk and outcome of Leishmania infection. Polymorphism in genes involved in both innate and adaptive immune systems, as well as genes of metabolic processes contributes to disease manifestation. The wide availability and advancement of molecular techniques permits to exploration of hereditary factors related to leishmaniasis. Many candidate gene studies were conducted on family-based and population to identify novel biomarkers for understanding disease pathogenesis pathways and possible drug targets. This comprehensive review presents an update on various human genes polymorphism that influence the outcome of different forms of Leishmania infection in endemic regions of the world. Various electronic databases were searched systematically for relevant publications and thoroughly analyzed. Most of the candidate gene studies were found with discrepancies in findings. Genetic and functional studies with adequate power are needed to validate the contribution of host genes in susceptibility or resistance towards Leishmania infection and understanding pathogenesis.
Assuntos
Leishmaniose Cutânea/genética , Leishmaniose Mucocutânea/genética , Leishmaniose Visceral/genética , Polimorfismo Genético , HumanosRESUMO
RecQ helicases are superfamily 2 (SF2) DNA helicases that unwind a wide spectrum of complex DNA structures in a 3' to 5' direction and are involved in maintaining genome stability. RecQ helicases from protozoan parasites have gained significant interest in recent times because of their involvement in cellular DNA repair pathways, making them important targets for drug development. In this study, we report biophysical and biochemical characterization of the catalytic core of a RecQ helicase from hemoflagellate protozoan parasite Leishmania donovani. Among the two putative RecQ helicases identified in L. donovani, we cloned, overexpressed and purified the catalytic core of LdRECQb. The catalytic core was found to be very efficient in unwinding a wide variety of DNA substrates like forked duplex, 3' tailed duplex and Holliday junction DNA. Interestingly, the helicase core also unwound blunt duplex with slightly less efficiency. The enzyme exhibited high level of DNA-stimulated ATPase activity with preferential stimulation by forked duplex, Holliday junction and 3' tailed duplex. Walker A motif lysine mutation severely affected the ATPase activity and significantly affected unwinding activity. Like many other RecQ helicases, L. donovani RECQb also possesses strand annealing activity. Unwinding of longer DNA substrates by LdRECQb catalytic core was found to be stimulated in the presence of replication protein A (LdRPA-1) from L. donovani. Detailed biochemical characterization and comparison of kinetic parameters indicate that L. donovani RECQb shares considerable functional similarity with human Bloom syndrome helicase.