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Transcriptional profiling of the spleen in progressive visceral leishmaniasis reveals mixed expression of type 1 and type 2 cytokine-responsive genes.

ABSTRACT: BACKGROUND:The Syrian golden hamster (Mesocricetus aureus) has been used as a model to study infections caused by a number of human pathogens. Studies of immunopathogenesis in hamster infection models are challenging because of the limited availability of reagents needed to define cellular and molecular determinants. RESULTS:We sequenced a hamster cDNA library and developed a first-generation custom cDNA microarray that included 5131 unique cDNAs enriched for immune response genes. We used this microarray to interrogate the hamster spleen response to Leishmania donovani, an intracellular protozoan that causes visceral leishmaniasis. The hamster model of visceral leishmaniasis is of particular interest because it recapitulates clinical and immunopathological features of human disease, including cachexia, massive splenomegaly, pancytopenia, immunosuppression, and ultimately death. In the microarray a differentially expressed transcript was identified as having at least a 2-fold change in expression between uninfected and infected groups and a False Discovery Rate of <5%. Following a relatively silent early phase of infection (at 7 and 14 days post-infection only 8 and 24 genes, respectively, were differentially expressed), there was dramatic upregulation of inflammatory and immune-related genes in the spleen (708 differentially expressed genes were evident at 28 days post-infection). The differentially expressed transcripts included genes involved in inflammation, immunity, and immune cell trafficking. Of particular interest there was concomitant upregulation of the IFN-? and interleukin (IL)-4 signaling pathways, with increased expression of a battery of IFN-?- and IL-4-responsive genes. The latter included genes characteristic of alternatively activated macrophages. CONCLUSIONS:Transcriptional profiling was accomplished in the Syrian golden hamster, for which a fully annotated genome is not available. In the hamster model of visceral leishmaniasis, a robust and functional IFN-? response did not restrain parasite load and progression of disease. This supports the accumulating evidence that macrophages are ineffectively activated to kill the parasite. The concomitant expression of IL-4/IL-13 and their downstream target genes, some of which were characteristic of alternative macrophage activation, are likely to contribute to this. Further dissection of mechanisms that lead to polarization of macrophages toward a permissive state is needed to fully understand the pathogenesis of visceral leishmaniasis.

SUBMITTER: Espitia CM

PROVIDER: S-EPMC4253007 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Transcriptional profiling of the spleen in progressive visceral leishmaniasis reveals mixed expression of type 1 and type 2 cytokine-responsive genes.

Espitia Claudia M CM Saldarriaga Omar A OA Travi Bruno L BL Osorio E Yaneth EY Hernandez Alvaro A Band Mark M Patel Mandakini J MJ Medina Audrie A AA Cappello Michael M Pekosz Andrew A Melby Peter C PC

BMC immunology 20141126

<h4>Background</h4>The Syrian golden hamster (Mesocricetus aureus) has been used as a model to study infections caused by a number of human pathogens. Studies of immunopathogenesis in hamster infection models are challenging because of the limited availability of reagents needed to define cellular and molecular determinants.<h4>Results</h4>We sequenced a hamster cDNA library and developed a first-generation custom cDNA microarray that included 5131 unique cDNAs enriched for immune response genes ...[more]

PMID: 25424735

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Project description:Background: Visceral leishmaniasis (VL) is caused by Leishmania infantum or L. donovani infection. One of the main problems related to this disease is the emergence of severe clinical forms with a lethality of 5-20%, even while under specific treatment. In humans and other species susceptible to fatal VL, such as dogs and hamsters, the disruption of splenic white pulp (WP) is accompanied by disease progression. Control of VL progression is seen in BALB/c mice, as evidenced by a mild clinical presentation and controlled parasite replication in the liver and spleen. In this study, we investigated the features involved in the morphological remodeling of splenic compartments associated with the control of VL progression to death. Methods: We evaluated cohorts of BALB/c mice after 30, 60, and 90 days of infection by L. infantum. Spleen morphology, cell population subsets and cytokine production were studied in the spleen using flow- and histo-cytometry. Results: Intraperitoneal infection with 108 promastigotes of L. infantum led to progressive increases in spleen size at 60 and 90 days after infection. Splenomegaly was the only clinical sign of disease observed. At 30 days after infection, hyperplasia in the WP and decreased numbers of plasmacytoid dendritic cells were observed. The WP hyperplasia subsided at 60 days post-infection. However, the splenomegaly remained in association with increased numbers of macrophages, B and T lymphocytes and plasma cells. An increased number of lymphoid tissue inducer (LTi) cells was observed; these were distributed around the periarteriolar lymphoid sheath in control mice and scattered throughout the red pulp in the Leishmania-infected mice. After 90 days of infection, increased IL-6 and IFN-? production was seen in the spleen, as well as higher frequencies of follicular and plasmacytoid dendritic cells. Conclusion: The data presented herein emphasizes the potential role of spleen remodeling in the control of severe forms of VL and highlights features potentially involved in this process.

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: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: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.

Project description:The Syrian golden hamster (Mesocricetus auratus) is uniquely susceptible to a variety of intracellular pathogens and is an excellent model for a number of human infectious diseases. The molecular basis for this high level of susceptibility is unknown, and immunological studies related to this model have been limited by the lack of available reagents. In this report we describe the cloning and sequence analysis of portions of the Syrian hamster interleukin 2 (IL-2), IL-4, gamma interferon (IFN-gamma), tumor necrosis factor alpha, IL-10, IL-12p40, and transforming growth factor beta cDNAs. In addition, we examined the cytokine response to infection with the intracellular protozoan Leishmania donovani in this animal model. Sequence analysis of the hamster cytokines revealed 69 to 93% homology with the corresponding mouse, rat, and human nucleotide sequences and 48 to 100% homology with the deduced amino acid sequences. The hamster IFN-gamma, compared with the mouse and rat homologs, had an additional 17 amino acids at the C terminus that could decrease the biological activity of this molecule and thus contribute to the extreme susceptibility of this animal to intracellular pathogens. The splenic expression of these genes in response to infection with L. donovani, the cause of visceral leishmaniasis (VL), was determined by Northern blotting. VL in the hamster is a progressive, lethal disease which very closely mimics active human disease. In this model there was pronounced expression of the Th1 cytokine mRNAs, with transcripts being detected as early as 1 week postinfection. Basal expression of IL-4 in uninfected hamsters was prominent but did not increase in response to infection with L. donovani. IL-12 transcript expression was detected at low levels in infected animals and paralleled the expression of IFN-gamma. Expression of IL-10, a potent macrophage deactivator, increased throughout the course of infection and could contribute to the progressive nature of this infection. These initial studies are the first to examine the molecular immunopathogenesis of a hamster model of VL infection and indicate that progressive disease in this model of VL is not associated with early polarization of the splenic cellular immune response toward a Th2 phenotype and away from a Th1 phenotype.

Dataset Information

Transcriptional profiling of the spleen in progressive visceral leishmaniasis reveals mixed expression of type 1 and type 2 cytokine-responsive genes.

Publications

Transcriptional profiling of the spleen in progressive visceral leishmaniasis reveals mixed expression of type 1 and type 2 cytokine-responsive genes.

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