Fig 1.
Generation of a Syrian hamster de novo assembled transcriptome.
(A) Summary of the RNA sequencing and transcriptome assembly workflow. (B) E-value of BLAST results of transcripts generated by the Trinity and BRANCH software. (C) Distribution of BLAST alignment scores generated by the Trinity and BRANCH software. (D) Lengths of transcripts generated by BRANCH that passed or failed a BLAST E-value threshold of <1e-3. (E) Distribution of number of transcripts generated by the Trinity and BRANCH software according to length of transcript. (F) Percent BLAST alignment of the assembled transcriptome with the published NCBI Mesocricetus auratus genome, and conversely, the percent BLAST alignment of the NCBI genome with our de novo assembled transcriptome.
Fig 2.
Characterization of a highly enriched macrophage population from the hamster spleen.
(A) Percent of macrophages, neutrophils, eosinophils and lymphocytes determined by microscopy of Hematoxylin and Eosin stained cytospin preparations of spleen cells from uninfected control (C) and 28-day L. donovani infected (Inf) hamsters (n = 4 per group). *p<0.05 (B) Immunostaining of the enriched splenic macrophage population for intracellular CD68. An isotype-matched antibody was used as a control, and cells were counterstained with Mayer’s hematoxylline. (C) Expression of specific cell lineage markers in the enriched splenic macrophage population. Data are shown as the fold-enrichment of sequence counts in the splenic macrophages relative to the total spleen tissue.
Fig 3.
Identification of differentially expressed transcripts in spleen tissue and splenic macrophages in hamsters with VL.
The number of differentially expressed transcripts in spleen (A) and splenic macrophages (B) from hamsters with VL determined by exact tests (FET) and generalized linear models with the likelihood ratio test (LRT) using the BioConductor R package EdgeR, and the Wald test in DESeq. A false discovery rate (FDR) <0.01 was used as the cutoff. 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. The lower panels show volcano plots and heat maps of the differentially expressed transcripts (DETs).
Fig 4.
Functional characterization of differentially expressed transcripts in the spleen and splenic macrophages from hamsters with VL.
(A) Top 10 canonical pathways (CPs) in the spleen (blue bars) compared to splenic macrophages (gray bars). The pathways were generated by loading all transcripts into IPA. (B) Top 10 canonical pathways (CPs) in splenic macrophages (gray bars) compared to spleen tissue (blue bars). (C) Top 10 gene sets identified in spleen and splenic macrophages determined by Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO) analysis. The Normalized Enrichment Score (NES), nominal p value, and False Discovery Rate (FDR) are shown for each gene set in the table. A network representation of the inflammatory response, cytokines and chemokines, and collagen and extracellular matrix gene sets is shown.
Fig 5.
Differential expression of cytokine and chemokine transcripts in spleens and splenic macrophages of hamsters with VL.
Heat maps showing the differential expression of selected transcripts in spleen tissue (A) and splenic macrophages (B) in uninfected (left side) and 28-day L. donovani infected (right side) hamsters (n = 4 per group). Transcripts down-regulated during infection are shown in green and upregulated transcripts are in red. The source or general function of the cytokine or chemokine is annotated to the left.
Fig 6.
Expression of M1-associated transcripts in spleen and splenic macrophages in VL.
Heat maps showing the differential expression of selected M1-associated transcripts in spleen tissue (A) and splenic macrophages (B) in uninfected (left side) and 28-day L. donovani infected (right side) hamsters (n = 4 per group). Transcripts down-regulated during infection are shown in green and upregulated transcripts are in red. The expression of selected M1-associated transcripts were confirmed by real-time RT-PCR in spleen tissue (C) and splenic macrophages (D) from hamsters with VL (n = 6–8 per group). Data are shown as the mean and SEM of the fold-change relative to the uninfected group. *p<0.05; **p<0.01; ***p<0.001.
Fig 7.
Expression of M2-associated transcripts in spleen and splenic macrophages in VL.
Heat maps showing the differential expression of selected M2-associated transcripts in spleen tissue (A) and splenic macrophages (B) in uninfected (left side) and 28-day L. donovani infected (right side) hamsters (n = 4 per group). Transcripts down-regulated during infection are shown in green and upregulated transcripts are in red. The expression of selected M2-associated transcripts was confirmed by real-time PCR in spleen tissue (C) and splenic macrophages (D) from hamsters with VL (n = 6–8 per group). Data are shown as the mean and SEM of the fold-change relative to the uninfected group. *p<0.05; **p<0.01; ***p<0.001.
Fig 8.
Single-cell expression of Cxcl9, Arg1, and Ido1 RNAs in splenic macrophages from uninfected and L. donovani infected hamsters.
The single-cell expression of M1-associated (Ido1 and Cxcl9) and M2-associated (Arg1) mRNA transcripts and CD68 in splenic macrophages from uninfected (row A) and L. donovani infected hamsters (rows B, C, and D) was determined by in situ amplification and fluorescence hybridization. To determine co-expression, cells were hybridized with fluorescent probes specific to CD68, CXCL9, and Arginase 1 (upper panel) or CD68, IDO1, and Arginase 1 (lower panel) and imaged using confocal microscopy. Nuclei were detected by DAPI staining and cellular morphology by Differential interference contrast (DIC) microscopy. In some cases, parasite nuclei could be seen adjacent to macrophage nuclei (indicated by white arrowheads in the DAPI stained cells). The percent of single-positive and double-positive cells was calculated by counting 100 CD68-positive cells. Uninfected cells (row A) were negative for CXCL9, IDO1, and Arginase 1.
Fig 9.
Predicted transcription factor regulation of M1- and M2-associated gene expression in splenic macrophages from hamsters with VL.
The set of differentially expressed genes in splenic macrophages was loaded into IPA software to predict the transcription factors likely to be activated. Shown is the overlapping network analysis of these transcription factors and the differentially expressed transcripts.
Fig 10.
IFN-γ signaling leads to altered gene expression and increased parasite load in infected splenic macrophages.
Differentially expressed transcripts from spleen tissue (A) and splenic macrophages (B) were loaded into IPA and the canonical IFN-γ signaling pathway generated. Transcripts upregulated in infection are shaded in red. (C) Expression of Cxcl9 and Cxcl10 in bone marrow derived macrophages (BMDMs) that were uninfected (Un) or infected in vitro with L. donovani (Ld), and left unstimulated (C) or stimulated with IFN-α, IFN-γ, or a combination of both (IFNα/γ) for 24 hrs. Data are shown as the mean and SEM of the fold-change relative to the uninfected, unstimulated group. (D) Parasite burden in bone marrow derived macrophages infected in vitro with L. donovani and left unstimulated (Con) or stimulated by IFN-α, IFN-γ, or a combination of both (IFNα/γ) for 24 hrs. (E) Relative parasite burden in splenic macrophages isolated from hamsters at 7, 14, 21, and 42 days after L. donovani infection, cultured and stimulated ex vivo for 24 hrs with hamster IFNγ (+) or mock supernatant (-). Parasite load was determined by expression of Leishmania 18S gene and fold-increase calculated against uninfected cells. (F) Expression of Arg1, Ido1, and Irg1 in uninfected (Un) and infected (Ld) BMDMs treated for 24 hrs with recombinant mouse IFN-α, recombinant hamster IFN-γ, or a combination of both (IFNα/γ). (G) Expression of Arg1 in uninfected BMDMs treated for 24 hrs with recombinant hamster IL-4, IFN-γ, or a combination of both (IL4-IFNγ). *p<0.05; **p<0.01; ***p<0.001.
Fig 11.
IFN-γ-mediated counter-regulatory response and increased parasite load in macrophages is dependent on STAT3 activation.
(A) Representative immunoblots showing phosphorylation of STAT1 and STAT3 in splenic macrophages from uninfected (day 0) and L. donovani infected (day 7, 14 and 28) hamsters. The relative band intensities are graphed from data from 3 experiments with samples pooled from 4 hamsters per time point. (B) Representative immunoblot of phospho-STAT3 (p-STAT3) expression in infected splenic macrophages after 20 min of IFN-γ exposure with and without treatment with 100 μM STAT3 inhibitor (STAT-3i) before IFN-γ stimulation. The relative intensities of the p-STAT3 bands are graphed, representative of 3 experiments. (C) Relative parasite burden (left panel) and arginase-1 (Arg-1) expression (right panel) in hamster BMDM infected in vitro with L. donovani and treated or not for 24 hrs with IFN-γ, with or without pre-treatment with the STAT3 inhibitor (STAT-3i). Fold change compared to mock treated macrophages. (D) Relative parasite burden (left panel) and arginase-1 (Arg-1) expression (right panel) in splenic macrophages from L. donovani infected hamsters (21 days p.i.), cultured and stimulated ex vivo for 24h with or without IFN-γ, with or without pre-treatment with the STAT3 inhibitor (STAT3i). Fold change compared to mock treated macrophages. (E) STAT3 Luciferase reporter assay in BHK cells transduced with STAT3 lentiviral reporter (Cignal Lenti, Qiagen) exposed or not for 4h or 48h to L. donovani with or without IFN-γ, with or without or pre-treatment with 2 μg/mL of anti-mouse/rat IL-10 neutralizing antibody (AF519, R&D). (F) Interleukin-10 (IL-10) mRNA expression in hamster BMDM uninfected or infected in vitro with L. donovani and stimulated or not for 24 hours with IFN-γ, with or without pre-treatment with the STAT3i. Fold change compared to mock treated macrophages. (G) Interleukin-10 (IL-10) expression in splenic macrophages from hamsters isolated 7, 14, or 21 days after infection with L. donovani, and cultured and stimulated ex vivo with IFN-γ for 24h, with or without pre-treatment with the STAT3 inhibitor (STAT3i). Fold change compared to mock treated macrophages. *p<0.05, **p<0.01, ***p<0.001.