The number of marked clones observed after intrathoracic inoculation of <i>C. taeniopus</i> mosquitoes.

<p>The mean number of VEEV clones present in the tissues of mosquitoes (legs/wings, saliva) 8 days after intrathoracic infection.</p

The number of marked VEEV clones observed during transmission from <i>C. taeniopus</i> mosquitoes to the vertebrate host.

<p>The number of clones present in transmitting mosquitoes and infected mice. (A) Mosquitoes infected at a high dose bloodmeal (5.7 log₁₀ pfu/ml) and (B) Mosquitoes infected with a low dose bloodmeal (4.9 log₁₀ pfu/ml).</p

The effect of bottlenecks on a population of marked mixed VEEV clones during infection of <i>C. taeniopus</i> with two different starting titers.

<p>The mean number of clones present at various time points and in various tissues (midgut, body, legs/wings, saliva) from <i>C. taeniopus</i> mosquitoes sampled following oral infection. (A) Mosquitoes infected with a high titer bloodmeal 5.7 log₁₀ pfu/ml (n = 4). (B) Mosquitoes exposed to a low titer bloodmeal 4.9 log₁₀ pfu/ml (n = 9).</p

The effect of bottlenecks on a population of marked mixed VEEV clones during experimental infection of <i>C. taeniopus</i>.

<p>The mean number of clones identified in each tissue, [body, legs/wings (representing the hemocoel), saliva] from mosquitoes sampled daily following oral infection (n = 3). Statistical significance between bodies versus legs/wings and bodies versus saliva was determined by a paired t-test.</p

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

<div><p>Visceral Leishmaniasis (VL), caused by the intracellular protozoan <i>Leishmania donovani</i>, 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 (<i>Mesocricetus auratus</i>) as a model because it mimics the progressive nature of active human VL. We demonstrated previously that mixed expression of macrophage-activating (IFN-γ) 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 <i>de novo</i> 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-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. Paradoxically, IFN-γ 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-γ-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.</p></div

IFN-γ-mediated counter-regulatory response and increased parasite load in macrophages is dependent on STAT3 activation.

<p><b>(A)</b> Representative immunoblots showing phosphorylation of STAT1 and STAT3 in splenic macrophages from uninfected (day 0) and <i>L</i>. <i>donovani</i> 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>B)</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. <b>(C)</b> Relative parasite burden (left panel) and arginase-1 (Arg-1) expression (right panel) in hamster BMDM infected <i>in vitro</i> with <i>L</i>. <i>donovani</i> 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. <b>(D)</b> Relative parasite burden (left panel) and arginase-1 (Arg-1) expression (right panel) in splenic macrophages from <i>L</i>. <i>donovani</i> infected hamsters (21 days p.i.), cultured and stimulated <i>ex vivo</i> for 24h with or without IFN-γ, with or without pre-treatment with the STAT3 inhibitor (STAT3i). Fold change compared to mock treated macrophages. (<b>E)</b> STAT3 Luciferase reporter assay in BHK cells transduced with STAT3 lentiviral reporter (Cignal Lenti, Qiagen) exposed or not for 4h or 48h to <i>L</i>. <i>donovani</i> 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). <b>(F)</b> Interleukin-10 (IL-10) mRNA expression in hamster BMDM uninfected or infected <i>in vitro</i> with <i>L</i>. <i>donovani</i> 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. (<b>G)</b> Interleukin-10 (IL-10) expression in splenic macrophages from hamsters isolated 7, 14, or 21 days after infection with <i>L</i>. <i>donovani</i>, and cultured and stimulated <i>ex vivo</i> 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.</p

Predicted transcription factor regulation of M1- and M2-associated gene expression in splenic macrophages from hamsters with VL.

<p>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.</p

Expression of M2-associated transcripts in spleen and splenic macrophages in VL.

<p>Heat maps showing the differential expression of selected M2-associated transcripts in spleen tissue <b>(A)</b> and splenic macrophages <b>(B)</b> in uninfected (left side) and 28-day <i>L</i>. <i>donovani</i> 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 <b>(C)</b> and splenic macrophages <b>(D)</b> 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.</p

IFN-γ signaling leads to altered gene expression and increased parasite load in infected splenic macrophages.

<p>Differentially expressed transcripts from spleen tissue <b>(A)</b> and splenic macrophages <b>(B)</b> were loaded into IPA and the canonical IFN-γ signaling pathway generated. Transcripts upregulated in infection are shaded in red. <b>(C)</b> Expression of Cxcl9 and Cxcl10 in bone marrow derived macrophages (BMDMs) that were uninfected (Un) or infected <i>in vitro</i> with <i>L</i>. <i>donovani</i> (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. <b>(D)</b> Parasite burden in bone marrow derived macrophages infected <i>in vitro</i> with <i>L</i>. <i>donovani</i> and left unstimulated (Con) or stimulated by IFN-α, IFN-γ, or a combination of both (IFNα/γ) for 24 hrs. <b>(E)</b> Relative parasite burden in splenic macrophages isolated from hamsters at 7, 14, 21, and 42 days after <i>L</i>. <i>donovani</i> infection, cultured and stimulated <i>ex vivo</i> 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. <b>(F)</b> 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). <b>(G)</b> Expression of Arg1 in uninfected BMDMs treated for 24 hrs with recombinant hamster IL-4, IFN-γ, or a combination of both (IL4-IFNγ). *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

Identification of differentially expressed transcripts in spleen tissue and splenic macrophages in hamsters with VL.

<p>The number of differentially expressed transcripts in spleen <b>(A)</b> and splenic macrophages <b>(B)</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).</p