Project description:Leishmania spp. is a protozoan parasite that affects millions of people around the world. At present, there is no effective vaccine to prevent leishmaniases in humans. A major limitation in vaccine development is the lack of precise understanding of the particular immunological mechanisms that allow parasite survival in the host. The parasite-host cell interaction induces dramatic changes in transcriptome patterns in both organisms, therefore, a detailed analysis of gene expression in infected tissues will contribute to the evaluation of drug and vaccine candidates, the identification of potential biomarkers, and the understanding of the immunological pathways that lead to protection or progression of disease. High-throughput real-time quantitative PCR is a highly sensitive, and specific technique that allows analysis of gene expression in multiple genes at the same time. Hence, it is an interesting approach that may bring light to this complex topic. In this large-scale analysis, forty-seven BALB/c mice were divided in two groups: control mice and mice infected with L. infantum promastigotes. After infection animals were sacrificed at different timepoints, RNA was purified from spleens and expression of 112 genes related with immune system was determined by high-throughput qPCR. These extensive gene-expression analyses reveal a selective recruitment of lymphocytes to the spleen and an immunosuppressive response, which favors parasite persistence, in early stages of the infection. In later stages of infection, gene-expression patterns suggest an inflammatory process that should resolve infection. Nevertheless, a detailed analysis reveals that L. infantum infection induces a regulatory process that counteracts the Th1/M1 response. This large pool of data was also used to identify a group of potential biomarkers of infection and parasitic burden in spleen, on the bases of two different statistical models, a logistic regression analysis and a linear regression analysis. Given the results, it seems clear that gene expression signature analysis is a useful tool to identify the mechanisms involved in disease outcome and to establish a rational approach for the identification of potential biomarkers useful for monitoring disease progression, new therapies or vaccine development in Leishmaniases.

Project description:Leishmania spp. is a protozoan parasite that affects millions of people around the world. At present, there is no effective vaccine to prevent leishmaniases in humans. A major limitation in vaccine development is the lack of precise understanding of the particular immunological mechanisms that allow parasite survival in the host. The parasite-host cell interaction induces dramatic changes in transcriptome patterns in both organisms, therefore, a detailed analysis of gene expression in infected tissues will contribute to the evaluation of drug and vaccine candidates, the identification of potential biomarkers, and the understanding of the immunological pathways that lead to protection or progression of disease. In this large-scale analysis, differential expression of 112 immune-related genes has been analyzed using high-throughput qPCR in spleens of infected and naïve Balb/c mice at four different time points. This analysis revealed that early response against Leishmania infection is characterized by the upregulation of Th1 markers and M1-macrophage activation molecules such as Ifng, Stat1, Cxcl9, Cxcl10, Ccr5, Cxcr3, Xcl1, and Ccl3. This activation doesn't protect spleen from infection, since parasitic burden rises along time. This marked difference in gene expression between infected and control mice disappears during intermediate stages of infection, probably related to the strong anti-inflammatory and immunosuppresory signals that are activated early upon infection (Ctla4) or remain activated throughout the experiment (Il18bp). The overexpression of these Th1/M1 markers is restored later in the chronic phase (8 wpi), suggesting the generation of a classical "protective response" against leishmaniasis. Nonetheless, the parasitic burden rockets at this timepoint. This apparent contradiction can be explained by the generation of a regulatory immune response characterized by overexpression of Ifng, Tnfa, Il10, and downregulation Il4 that counteracts the Th1/M1 response. This large pool of data was also used to identify potential biomarkers of infection and parasitic burden in spleen, on the bases of two different regression models. Given the results, gene expression signature analysis appears as a useful tool to identify mechanisms involved in disease outcome and to establish a rational approach for the identification of potential biomarkers useful for monitoring disease progression, new therapies or vaccine development.

Project description:Leishmania spp. is a protozoan parasite that affects millions of people around the world. At present, there is no effective vaccine to prevent leishmaniases in humans. A major limitation in vaccine development is the lack of precise understanding of the particular immunological mechanisms that allow parasite survival in the host. The parasite-host cell interaction induces dramatic changes in transcriptome patterns in both organisms, therefore, a detailed analysis of gene expression in infected tissues will contribute to the evaluation of drug and vaccine candidates, the identification of potential biomarkers, and the understanding of the immunological pathways that lead to protection or progression of disease. High-throughput real-time quantitative PCR is a highly sensitive, and specific technique that allows analysis of gene expression in multiple genes at the same time. Hence, it is an interesting approach that may bring light to this complex topic. In this large-scale analysis, forty-seven BALB/c mice were divided in two groups: control mice and mice infected with L. infantum promastigotes. After infection animals were sacrificed at different timepoints, RNA was purified from spleens and expression of 112 genes related with immune system was determined by high-throughput qPCR. These extensive gene-expression analyses reveal a selective recruitment of lymphocytes to the spleen and an immunosuppressive response, which favors parasite persistence, in early stages of the infection. In later stages of infection, gene-expression patterns suggest an inflammatory process that should resolve infection. Nevertheless, a detailed analysis reveals that L. infantum infection induces a regulatory process that counteracts the Th1/M1 response. Given the results, it seems clear that gene expression signature analysis is a useful tool to identify the mechanisms involved in disease outcome and to establish a rational approach for the identification of potential biomarkers useful for monitoring disease progression, new therapies or vaccine development in Leishmaniases.

Project description:BackgroundIn the Mediterranean basin, three Leishmania species have been identified: L. infantum, L. major and L. tropica, causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L. infantum, L. major, and L. tropica, as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L. infantum) and parasites involved in CL (i.e., L. major, L. tropica).Methodology/principal findingsU937 cells differentiated into macrophage-like cells were infected with L. infantum, L. major and L. tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L. infantum-infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization.ConclusionsThe identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy.

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Transcriptional profiling of immune-related genes in Leishmania infantum-infected mice: identification of potential biomarkers of infection and progression of disease

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