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-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 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-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.

Tissue and host species-specific transcriptional changes in models of experimental visceral leishmaniasis

“…In contrast, M2-associated genes were less commonly regulated in mouse compared to hamster spleen (23% of genes listed in Figure 7 of 41, including Ccl2 , Cd209a , Cebpb , Chi3l1 , and Il1rn ). These data are in accord with the above analysis indicating an enhanced Th-2 type inflammation in hamster compared to murine EVL.…”

“…Finally, given its importance to the host response to infection, we examined the status of macrophage activation, and observed a good degree of concordance for M1-associated transcripts (as described in Figure 6 of 41) between hamster and mouse (55% of genes, including Ccl2 , Cxcl9 , Cxcl10 , Ifng , Irf1 , Irf7 , Irg1 , Socs3 , Stat1 and Ccr7 ). In contrast, M2-associated genes were less commonly regulated in mouse compared to hamster spleen (23% of genes listed in Figure 7 of 41, including Ccl2 , Cd209a , Cebpb , Chi3l1 , and Il1rn ).…”

“…In contrast to the murine EVL model, data generated by Melby and colleagues suggests that the immune response in hamster EVL favours a more prominent Th2 like response 40, 41 . In addition, their analysis also indicates that macrophages in hamster EVL are conditioned to respond to activation with the induction of a program of gene expression that favours amastigote growth, including Arg1 , Ido-1 and Irg1 and associated with STAT3 activation 41 .…”

“…However, a detailed comparison of the pathophysiology in infected hamsters and mice has not been conducted. We made use of recently published RNA-Seq data derived from the spleens of hamsters infected with L. donovani infection 41 to compare these two infection models, albeit with the caveat that different technologies were used for measuring mRNA abundance. Kong et al observed 4360 DE transcripts at d28 post infection, of which 2340 (53.7%) were up-regulated and 2020 (46.3%) were down-regulated compared to spleens from naïve hamsters.…”

“…Venn diagrams were generated using Venny 2.1 47 . Hamster transcriptomic datasets were from 41. Human transcriptomic data sets were from 39.…”