Date Published: January 31, 2017
Publisher: Public Library of Science
Author(s): Fanping Kong, Omar A. Saldarriaga, Heidi Spratt, E. Yaneth Osorio, Bruno L. Travi, Bruce A. Luxon, Peter C. Melby, Christian R. Engwerda.
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.
Visceral leishmaniasis (VL), caused by the intracellular protozoa Leishmania donovani and L. infantum (syn L. chagasi), affects nearly a half-million people each year . It occurs in tropical and subtropical regions of the world and is commonly associated with poverty. Infection is initiated when parasites are deposited in the skin by the sand fly vector. In most infected people, the infection is controlled by a type 1 cellular immune response and there are no signs of disease. However, some infected individuals develop a chronic progressive illness characterized by fever, splenomegaly, cachexia, pancytopenia and a relentlessly increasing parasite burden in the spleen, liver and bone marrow. Susceptibility is associated with decreased antigen-induced IFN-γ and IL-12 responses in peripheral blood mononuclear cells [2,3], CD8 T cell exhaustion , reduced T cell-mediated macrophage activation and parasite killing , and increased IL-10 production [6–8]. In contrast to the in vitro finding of decreased antigen-induced IFN-γ, there is a high level of plasma and splenic IFN-γ production [6,9–11] and evidence of antigen-induced IFN-γ production in ex vivo whole blood assays  in patients with VL. The disconnect between what should be a protective IFN-γ response and the relentless parasite replication and disease progression in VL remains an enigma. In vitro models of L. donovani infection identified several pathways of impaired macrophage function , but macrophage function in vivo has not been investigated.
Genome-wide expression analysis revealed evidence of a broad inflammatory signature that included an extensive array of upregulated interferon response genes in the spleen during progressive VL. This type of gene expression would be expected to drive macrophages toward a M1 phenotype and protect against Leishmania . However, M1 polarization was not dominant and IFN-γ paradoxically enhanced parasite growth in splenic macrophages. Importantly, the parasite-promoting effect of IFN-γ was more pronounced in splenic macrophages isolated later in the course of infection. This suggests that as VL progresses, splenic macrophages in VL are conditioned by the chronic inflammatory environment to respond to macrophage activation signals in an aberrant, pathological way that contributes to the progressive infection. Several mechanisms could account for this. First, the finding of fewer upregulated IFN-response genes in splenic macrophages relative to the whole spleen, including transcripts known to be induced in macrophages, suggests relative macrophage unresponsiveness to IFN-γ. The absence of NOS2 upregulation is likely a central determinant of ineffective parasite killing. Impaired IFN-γ signaling, which has been well-described in in vitro infected macrophages [120–124], is likely to contribute to impaired NOS2 expression during VL. Second, the co-expression of M1- and M2-associated transcripts, and the finding that IFN-γ induces Arg1, are suggestive of a misdirected signaling in splenic macrophages during VL. There is a growing body of evidence that Arg1 has a significant pathological role in Leishmania infection, including human VL [19,20,80–82,125,126]. Our data indicate that expression of Arg1 is not part of a conventional Th2-driven M2 macrophage phenotype, but identify a previously unrecognized mechanism of IFN-induced Arg1. This has significant bearing on the pathogenesis of VL so the mechanisms of IFN regulation of Arg1 in VL need further investigation. Third, parasite-derived signals and anti-inflammatory/regulatory cues (e.g. IL-10 and IL-21) [19,20] may impair macrophage effector function and lead to disease-promoting gene expression. Fourth, the massive interferon response in the spleen appears to have a counter-protective effect through initiation of an exuberant counter-regulatory response mediated via STAT3 and IL-10. The STAT3-dependent IFN-γ-induced Arg1 expression may paradoxically lead to impaired macrophage or T cell responses, as we and others have described previously in VL [19,20,80–82,125,126]. Lastly, the high expression of a broad array of chemokines in the spleen is likely to lead to accumulation of immature myeloid cell populations, which have some features consistent with myeloid-derived suppressor cells, that are less responsive to classical activation signals. Collectively, these data identify a number of molecules, pathways and transcription factors that contribute to the pathogenesis of VL. The STAT3 pathway in particular is an attractive target for adjunctive host-directed therapy for VL.