Defining the transcriptional signature of healing and non-healing lesions during murine cutaneous leishmaniasis
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ABSTRACT: Leishmaniasis is a group of diseases caused by parasites of the genus Leishmania that affects millions of people worldwide. The disease outcome is determined by both the parasite species and the host's immune response. Leishmania major infection causes a localized cutaneous lesion in patients and has been widely used to study the development of T cell responses in mice. L. major infected C57BL/6 mice are resistant to infection due to the development of Th1 responses, whereas BALB/c mice develop a Th2 response resulting in disease susceptibility and failure to control parasite replication. However, these disparate host phenotypes are not observed with all Leishmania species. For example, during L. braziliensis infection both BALB/c and C57BL/6 mice are resistant. In order to better understand the host genetic basis underlying disease susceptibility in vivo, we performed a whole genome transcriptional analysis from skin lesions of BALB/c and C57BL/6 mice infected intradermally for 4 weeks with either L. braziliensis or L. major.
Project description:Skin from Balb/c mice after Leishmania major infection. Promastigote forms of the protozoon were inoculated in mice footpads. The skin biopsies were analyzed after 60 days of infection.
Project description:Gene expression profiles of in vitro-infected (Leishmania major) and uninfected bone-marrow-derived macrophages from BALB/c, C57BL/6, C.B6-(lmr1, lmr2) and B6.c-(lmr1, lmr2) mouse strains. We utilised microarrays to investigate a number of issues. Firstly, we determined which genes were differentially regulated in macrophages in response to infection with Leishmania major. Secondly, we hoped to gain some insight into the differences between C-B6-(lmr1, lmr2) congenic mice and their parental control BALB/c, as well as differences between B6-c-(lmr1, lmr2) and their parental control C57BL/6. This would aid us in the search for the genes underlying our loci, since the only genomic regions differing between the two strains are those of the two congenic intervals on chromosomes 9 and 17. Any genes differentially regulated would be contributing to the experimental infection differences observed between the congenic mice and their parental controls. Experiment Overall Design: L. major-infected and uninfected bone marrow-derived macrophages from C57BL/6, BALB/c, B6.c-(lmr1, lmr2) and C.B6-(lmr1, lmr2) mice were analyzed. Macrophages from 6 mice were pooled for each sample.
Project description:Gene expression profiles of in vitro-infected (Leishmania major) and uninfected bone-marrow-derived macrophages from BALB/c, C57BL/6, C.B6-(lmr1, lmr2) and B6.c-(lmr1, lmr2) mouse strains. We utilised microarrays to investigate a number of issues. Firstly, we determined which genes were differentially regulated in macrophages in response to infection with Leishmania major. Secondly, we hoped to gain some insight into the differences between C-B6-(lmr1, lmr2) congenic mice and their parental control BALB/c, as well as differences between B6-c-(lmr1, lmr2) and their parental control C57BL/6. This would aid us in the search for the genes underlying our loci, since the only genomic regions differing between the two strains are those of the two congenic intervals on chromosomes 9 and 17. Any genes differentially regulated would be contributing to the experimental infection differences observed between the congenic mice and their parental controls.
Project description:The mechanisms that mediate immunopathologic responses in infectious diseases are often less well understood than how the pathogens are controlled. Here, we have investigated what causes increased pathology following infection with the protozoan parasite, Leishmania braziliensis. We focused on CD8 T cells since their presence in leishmanial lesions has been correlated with increased disease. By adoptively transferring CD8 T cells to L. braziliensis infected RAG mice, we found that unregulated CD8 T cells promote severe pathology at the infection site, as well as the development of metastatic lesions in other skin sites. In mice with severe pathology, we visualized CD8 T cells degranulating and lysing L. braziliensis infected cells, and in parallel studies with L. braziliensis patients we confirmed that CD8 T cells within lesions exhibit a cytolytic phenotype. We found that perforin deficient CD8 T cells failed to induce disease, indicating that the increased disease induced by CD8 T cells was due perforin-dependent cytotoxicity. In contrast, although we found that CD8 T cells made both IFN-γ and IL-17, neither of these cytokines is required for the development of pathology. Thus, we show for the first time that immunopathology in leishmaniasis can be mediated by cytolytic CD8 T cells. Twelve skin biopsy samples were analyzed, including 2 normal skin biopsies obtained from patients in N. America, and 10 skin biosies obtained from Leishmania brazilensis infected patients presenting at the Corte de Pedra Health Post in Corte de Pedra, Bahia, Brazil
Project description:We evaluated the trancriptome of primary cutaneous leisions caused by infection with Leishmania braziliensis. mRNA-seq technique was used to study the trancriptome of both host and parasite. A total of 10 samples was obtained from primary skin ulcers of two extreme clinical forms of American tegumentary leishmaniasis: (i) individuals that after antimonial treatment cured completely (localized cutaneous leishmaniasis - LCL, n=5) and (ii) individuals that developed mucosal lesions in naso and oropharynx areas long after initial healing of the cutaneous lesion (mucosal leishmaniasis - ML, n=5). The sequencing generated an average of 13+ 5 million reads per samples. The reads were aligned to Homo sapiens (USCS - hg19) and to Leishmania braziliensis (Wellcome Trust Sanger Institute - V2_29072008) genomes. Approximately, 15,000 human genes could be detected in the samples. Low amount of L. braziliensis reads did not allow the evaluation of parasite gene expression. LCL and ML samples showed different patterns of gene expression, indicating a more robust immune response in LCL individuals. In summary, this study demonstrated that next-generation sequencing can be used for identification of potentially important biological pathways and drug targets in the host-response to L. braziliensis infection and for characterization of a gene expression signature that could be used to predict the disease outcome. Moreover, we also showed the ability of this technique in, simultaneously, sequence host and pathogen mRNA. Examination of 10 fragments of cutaneous lesions: 5 from localized cutaneous leishmaniasis patients and 5 from mucosal leishmaniasis patients.
Project description:We evaluated the trancriptome of primary cutaneous leisions caused by infection with Leishmania braziliensis. mRNA-seq technique was used to study the trancriptome of both host and parasite. A total of 10 samples was obtained from primary skin ulcers of two extreme clinical forms of American tegumentary leishmaniasis: (i) individuals that after antimonial treatment cured completely (localized cutaneous leishmaniasis - LCL, n=5) and (ii) individuals that developed mucosal lesions in naso and oropharynx areas long after initial healing of the cutaneous lesion (mucosal leishmaniasis - ML, n=5). The sequencing generated an average of 13+ 5 million reads per samples. The reads were aligned to Homo sapiens (USCS - hg19) and to Leishmania braziliensis (Wellcome Trust Sanger Institute - V2_29072008) genomes. Approximately, 15,000 human genes could be detected in the samples. Low amount of L. braziliensis reads did not allow the evaluation of parasite gene expression. LCL and ML samples showed different patterns of gene expression, indicating a more robust immune response in LCL individuals. In summary, this study demonstrated that next-generation sequencing can be used for identification of potentially important biological pathways and drug targets in the host-response to L. braziliensis infection and for characterization of a gene expression signature that could be used to predict the disease outcome. Moreover, we also showed the ability of this technique in, simultaneously, sequence host and pathogen mRNA.
Project description:The main objective of this study is to identify the list of genes differentially expressed between infected with Leishmania braziliensis and non-infected macrophage cultures based on gene expression microarray profiling The dataset is comprised by the expression profile of 6 samples from three independent experiments and each experiment had three technical replicates. 3 of the 6 samples were U937 derived macrophages infected by Leishmania braziliensis and the other 3 were U937 derived macrophages without infection with Leishmania braziliensis. A total of 18 microarrays analysis were performed.
Project description:Skin from Balb/c mice after Leishmania amazonensis infection. Mice were infected through their footpads with the promastigote form of the protozoon
Project description:Genome sequencing of species of the kinetoplastid parasite, Leishmania, that give rise to a range of disease phenotypes in the host has revealed highly conserved gene content and synteny across the genus. Only a small number of genes are differentially distributed between the three species studied to date, L. major, L. infantum and L. braziliensis. Here, we focus on RNA expression in the disease-promoting intracellular amastigotes and use customised oligonucleotide microarrays to confirm that all of these differentially-distributed genes are expressed in this critical stage of the parasite life cycle, with only a few regulated between species.
Project description:The mechanisms that mediate immunopathologic responses in infectious diseases are often less well understood than how the pathogens are controlled. Here, we have investigated what causes increased pathology following infection with the protozoan parasite, Leishmania braziliensis. We focused on CD8 T cells since their presence in leishmanial lesions has been correlated with increased disease. By adoptively transferring CD8 T cells to L. braziliensis infected RAG mice, we found that unregulated CD8 T cells promote severe pathology at the infection site, as well as the development of metastatic lesions in other skin sites. In mice with severe pathology, we visualized CD8 T cells degranulating and lysing L. braziliensis infected cells, and in parallel studies with L. braziliensis patients we confirmed that CD8 T cells within lesions exhibit a cytolytic phenotype. We found that perforin deficient CD8 T cells failed to induce disease, indicating that the increased disease induced by CD8 T cells was due perforin-dependent cytotoxicity. In contrast, although we found that CD8 T cells made both IFN-γ and IL-17, neither of these cytokines is required for the development of pathology. Thus, we show for the first time that immunopathology in leishmaniasis can be mediated by cytolytic CD8 T cells.