Splenic Transcriptional Responses in Severe Visceral Leishmaniasis: Impaired Leukocyte Chemotaxis and Cell Cycle Arrest.
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ABSTRACT:
SUBMITTER: de Melo CVB
PROVIDER: S-EPMC8602831 | biostudies-literature | 2021
REPOSITORIES: biostudies-literature
Publications
Splenic Transcriptional Responses in Severe Visceral Leishmaniasis: Impaired Leukocyte Chemotaxis and Cell Cycle Arrest.
de Melo Caroline Vilas Boas CVB Guimarães Torres Felipe F Hermida Micely D'El-Rei MD Fontes Jonathan L M JLM Mesquita Bianca Ramos BR Brito Reginaldo R Ramos Pablo Ivan P PIP Fernandes Gabriel R GR Freitas Luiz Antônio Rodrigues LAR Khouri Ricardo R Costa Carlos Henrique Nery CHN Dos-Santos Washington L C WLC
Frontiers in immunology 20211105
Structural changes in the spleen have been reported in several infectious diseases. In visceral leishmaniasis (VL), a severe parasitic disease caused by <i>Leishmania</i> spp., the loss of white pulp accompanies a severe clinical presentation. Hamster model reproduces aspects of human VL progression. In the early stages, a transcriptomic signature of leukocyte recruitment was associated with white pulp hyperplasia. Subsequently, impaired leukocyte chemotaxis with loss of T lymphocytes in the per ...[more]
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Project description:Therapeutic vaccines, when used alone or in combination therapy with antileishmanial drugs, may have an important place in the control of a variety of forms of human leishmaniasis. Here, we describe the development of an adenovirus-based vaccine (Ad5-KH) comprising a synthetic haspb gene linked to a kmp11 gene via a viral 2A sequence. In nonvaccinated Leishmania donovani-infected BALB/c mice, HASPB- and KMP11-specific CD8(+) T cell responses were undetectable, although IgG1 and IgG2a antibodies were evident. After therapeutic vaccination, antibody responses were boosted, and IFNγ(+)CD8(+) T cell responses, particularly to HASPB, became apparent. A single vaccination with Ad5-KH inhibited splenic parasite growth by ∼66%, a level of efficacy comparable to that observed in early stage testing of clinically approved antileishmanial drugs in this model. These studies indicate the usefulness of adenoviral vectors to deliver leishmanial antigens in a potent and host protective manner to animals with existing L. donovani infection.
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2021-07-12 | GSE159195 | GEO
Project description:BackgroundNew drugs are needed to treat visceral leishmaniasis (VL) because the current therapies are toxic, expensive, and parasite resistance may weaken drug efficacy. We established a novel ex vivo splenic explant culture system from hamsters infected with luciferase-transfected Leishmania donovani to screen chemical compounds for anti-leishmanial activity.Methodology/principal findingsTHIS MODEL HAS ADVANTAGES OVER IN VITRO SYSTEMS IN THAT IT: 1) includes the whole cellular population involved in the host-parasite interaction; 2) is initiated at a stage of infection when the immunosuppressive mechanisms that lead to progressive VL are evident; 3) involves the intracellular form of Leishmania; 4) supports parasite replication that can be easily quantified by detection of parasite-expressed luciferase; 5) is adaptable to a high-throughput screening format; and 6) can be used to identify compounds that have both direct and indirect anti-parasitic activity. The assay showed excellent discrimination between positive (amphotericin B) and negative (vehicle) controls with a Z' Factor >0.8. A duplicate screen of 4 chemical libraries containing 4,035 compounds identified 202 hits (5.0%) with a Z score of <-1.96 (p<0.05). Eighty-four (2.1%) of the hits were classified as lead compounds based on the in vitro therapeutic index (ratio of the compound concentration causing 50% cytotoxicity in the HepG(2) cell line to the concentration that caused 50% reduction in the parasite load). Sixty-nine (82%) of the lead compounds were previously unknown to have anti-leishmanial activity. The most frequently identified lead compounds were classified as quinoline-containing compounds (14%), alkaloids (10%), aromatics (11%), terpenes (8%), phenothiazines (7%) and furans (5%).Conclusions/significanceThe ex vivo splenic explant model provides a powerful approach to identify new compounds active against L. donovani within the pathophysiologic environment of the infected spleen. Further in vivo evaluation and chemical optimization of these lead compounds may generate new candidates for preclinical studies of treatment for VL.
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Project description:We used next generation sequencing to examine global changes in gene expression in the spleen and splenic macrophages in a hamster model of progressive visceral leishmaniasis. We used RNA sequencing coupled with de novo transcriptome assembly, because the Syrian hamster does not have a fully sequenced and annotated reference genome. Libraries for deep sequencing were constructed from poly-A RNA isolated from the spleens or splenic macrophages of uninfected and 28-day L. donovani-infected hamsters (n=4 per group) using standard Illumina protocols. The library quality was confirmed by Agilent Bioanalyzer. Paired-end 50-base sequencing was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1000.The quality of raw sequencing reads was checked using FastQC (v0.10.1). To avoid the contamination of pathogen sequences, we filtered out reads (using the FASTX Toolkit v0.0.13) that aligned to the Leishmania donovani BPK282A1 genome (NCBI BioProject PRJEA61817) using Bowtie 2 (v2.0.0-beta5) under default options. To reduce the effect of low quality reads, we further filtered out artifacts and reads having a phred score <28 in more than 10% of nucleotides using FASTX Toolkit (v0.0.13). Both forward and reverse reads were removed if any of them failed to pass the filters. To obtain a complete transcriptome, we used two steps. First, the cleaned sequencing reads from different spleen samples were pooled together and de novo assembled using Trinity software. Second, the resulting transcriptome was combined with all cleaned reads from hamster spleen and splenic macrophages and the CHO-K1 RefSeq genome to perform a second de novo assembly using BRANCH. We first created a customized reference library using Rattus norvegicus (Rnor_5.0.73) and Mus musculus (GRCm38.73) genomes. We then used BLAST (v 2.2.28+) to align each transcript generated from BRANCH against the customized library to assign it a gene name based upon sequence similarity. An E-value cutoff of <1e-3 was used. Additionally, we compared the Trinity transcripts with the CHO-K1 Ref Seq genome. All the non-Leishmania-like raw sequencing reads were first mapped to our de novo reference genome using Bowtie2 (v2.1.0) with default options, but allowing one read to map to as many as 500 different transcripts. We then measured the expression abundance, count of reads mapped to each transcript, using the software eXpress. To identify differentially expressed transcripts in each experiment, 3 different approaches were applied: exact tests of Robinson and Smyth and generalized linear models with the likelihood ratio test using the BioConductor R package EdgeR, and the Wald test using DESeq2. Only transcripts with at least 1 count per million in at least 3 out of 4 samples in the control or experimental group were included in the analysis. A transcript was considered differentially expressed only when it was identified by all three different approaches. Analysis of differentially expressed transcripts identified a highly inflammatory spleen environment with abundant expression of type I and type II interferon response genes. However, high IFN-γ expression was ineffective in directing exclusive M1 macrophage polarization, suppressing M2-associated gene expression, and restraining parasite replication and disease. While many IFN-inducible transcripts were upregulated in the infected spleen, fewer were induced in splenic macrophages in VL suggesting suppression of macrophage responsiveness to activating cytokines. The transcription factor STAT3 was predicted and confirmed to be activated, and found to orchestrate an exuberant counter-regulatory response that favors parasite replication.
2017-01-11 | GSE91187 | GEO
Project description:Background: Human visceral leishmaniasis, caused by infection with Leishmania donovani or L. infantum, is a potentially fatal disease affecting 50,000-90,000 people yearly in 75 disease endemic countries, with more than 20,000 deaths reported. Experimental models of infection play a major role in understanding parasite biology, host-pathogen interaction, disease pathogenesis, and parasite transmission. In addition, they have an essential role in the identification and pre-clinical evaluation of new drugs and vaccines. However, our understanding of these models remains fragmentary. Although the immune response to Leishmania donovani infection in mice has been extensively characterized, transcriptomic analysis capturing the tissue-specific evolution of disease has yet to be reported. Methods: We provide an analysis of the transcriptome of spleen, liver and peripheral blood of BALB/c mice infected with L. donovani. Where possible, we compare our data in murine experimental visceral leishmaniasis with transcriptomic data in the public domain obtained from the study of L. donovani-infected hamsters and patients with human visceral leishmaniasis. Digitised whole slide images showing the histopathology in spleen and liver are made available via a dedicated website, www.leishpathnet.org. Results: Our analysis confirms marked tissue-specific alterations in the transcriptome of infected mice over time and identifies previously unrecognized parallels and differences between murine, hamster and human responses to infection. We show commonality of interferon-regulated genes whilst confirming a greater activation of type 2 immune pathways in infected hamsters compared to mice. Cytokine genes and genes encoding immune checkpoints were markedly tissue specific and dynamic in their expression, and pathways focused on non-immune cells reflected tissue specific immunopathology. Our data also addresses the value of measuring peripheral blood transcriptomics as a potential window into underlying systemic disease. Conclusions: Our transcriptomic data, coupled with histopathologic analysis of the tissue response, provide an additional resource to underpin future mechanistic studies and to guide clinical research.
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