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Transcriptional Profiling in Experimental Visceral Leishmaniasis Reveals a Broad Splenic Inflammatory Environment that Conditions Macrophages toward a Disease-Promoting Phenotype.

ABSTRACT: Visceral Leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is characterized by relentlessly increasing visceral parasite replication, cachexia, massive splenomegaly, pancytopenia and ultimately death. Progressive disease is considered to be due to impaired effector T cell function and/or failure of macrophages to be activated to kill the intracellular parasite. In previous studies, we used the Syrian hamster (Mesocricetus auratus) as a model because it mimics the progressive nature of active human VL. We demonstrated previously that mixed expression of macrophage-activating (IFN-?) and regulatory (IL-4, IL-10, IL-21) cytokines, parasite-induced expression of macrophage arginase 1 (Arg1), and decreased production of nitric oxide are key immunopathologic factors. Here we examined global changes in gene expression to define the splenic environment and phenotype of splenic macrophages during progressive VL. We used RNA sequencing coupled with de novo transcriptome assembly, because the Syrian hamster does not have a fully sequenced and annotated reference genome. Differentially expressed transcripts identified a highly inflammatory spleen environment with abundant expression of type I and type II interferon response genes. However, high IFN-? 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. Paradoxically, IFN-? enhanced parasite growth and induced the counter-regulatory molecules Arg1, Ido1 and Irg1 in splenic macrophages. This was mediated, at least in part, through IFN-?-induced activation of STAT3 and expression of IL-10, which suggests that splenic macrophages in VL are conditioned to respond to macrophage activation signals with a counter-regulatory response that is ineffective and even disease-promoting. Accordingly, inhibition of STAT3 activation led to a reduced parasite load in infected macrophages. Thus, the STAT3 pathway offers a rational target for adjunctive host-directed therapy to interrupt the pathogenesis of VL.

SUBMITTER: Kong F

PROVIDER: S-EPMC5283737 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Transcriptional Profiling in Experimental Visceral Leishmaniasis Reveals a Broad Splenic Inflammatory Environment that Conditions Macrophages toward a Disease-Promoting Phenotype.

Kong Fanping F Saldarriaga Omar A OA Spratt Heidi H Osorio E Yaneth EY Travi Bruno L BL Luxon Bruce A BA Melby Peter C PC

PLoS pathogens 20170131 1

Visceral Leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is characterized by relentlessly increasing visceral parasite replication, cachexia, massive splenomegaly, pancytopenia and ultimately death. Progressive disease is considered to be due to impaired effector T cell function and/or failure of macrophages to be activated to kill the intracellular parasite. In previous studies, we used the Syrian hamster (Mesocricetus auratus) as a model because it mimics the pro ...[more]

PMID: 28141856

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Project description:Visceral leishmaniasis (VL), caused by infection with the intracellular protozoan Leishmania donovani, is a chronic progressive disease with a relentlessly increasing parasite burden in the spleen, liver and bone marrow. The disease is characterized by fever, splenomegaly, cachexia, and pancytopenia, and progresses to death if not treated. Control of Leishmania infection is mediated by Th1 (IFNγ-producing) CD4+ T cells, which activate macrophages to produce nitric oxide and kill intracellular parasites. However, despite expansion of CD4+ T cells and increased IFNγ expression in the spleen, humans with active VL do not control the infection. We used an experimental model of chronic progressive VL in hamsters, which mimics clinical and pathological features seen in humans, to better understand the mechanisms that lead to progressive disease. Transcriptional profiling of the spleen during chronic infection revealed expression of markers of both T cell activation and inhibition. CD4+ T cells isolated from the spleen during chronic progressive VL showed mixed expression of Th1 and Th2 cytokines and chemokines, and were marginally effective in controlling infection in an ex vivo T cell-macrophage co-culture system. Splenic CD4+ T cells and macrophages from hamsters with VL showed increased expression of inhibitory receptors and their ligands, respectively. Blockade of the inhibitory receptor PD-L2 led to a significant decrease in parasite burden, revealing a pathogenic role for the PD-1 pathway in chronic VL. PD-L2 blockade was associated with a dramatic reduction in expression of host arginase 1, but no change in IFNγ and inducible nitric oxide synthase. Thus, the expression of counter-regulatory molecules on splenic CD4+ T cells and macrophages promotes a more permissive macrophage phenotype and attenuates intracellular parasite control in chronic progressive VL. Host-directed adjunctive therapy targeting the PD-1 regulatory pathway may be efficacious for VL.

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.

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.

Dataset Information

Transcriptional Profiling in Experimental Visceral Leishmaniasis Reveals a Broad Splenic Inflammatory Environment that Conditions Macrophages toward a Disease-Promoting Phenotype.

Publications

Transcriptional Profiling in Experimental Visceral Leishmaniasis Reveals a Broad Splenic Inflammatory Environment that Conditions Macrophages toward a Disease-Promoting Phenotype.

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