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Host arginase 1 (arg1) expression is a significant contributor to the pathogenesis of progressive visceral leishmaniasis (VL), a neglected tropical disease caused by the intracellular protozoan Leishmania donovani. Previously we found that parasite-induced arg1 expression in macrophages was dependent on STAT6 activation. Arg1 expression was amplified by, but did not require, IL-4, and required de novo synthesis of unknown protein(s). To further explore the mechanisms involved in arg1 regulation in VL, we screened a panel of kinase inhibitors and found that inhibitors of growth factor signaling reduced arg1 expression in splenic macrophages from hamsters with VL. Analysis of growth factors and their signaling pathways revealed that the Fibroblast Growth Factor Receptor 1 (FGFR-1) and Insulin-like Growth Factor 1 Receptor (IGF-1R) and a number of downstream signaling proteins were activated in splenic macrophages isolated from hamsters infected with L. donovani. Recombinant FGF-2 and IGF-1 increased the expression of arg1 in L. donovani infected hamster macrophages, and this induction was augmented by IL-4. Inhibition of FGFR-1 and IGF-1R decreased arg1 expression and restricted L. donovani replication in both in vitro and ex vivo models of infection. Inhibition of the downstream signaling molecules JAK and AKT also reduced the expression of arg1 in infected macrophages. STAT6 was activated in infected macrophages exposed to either FGF-2 or IGF-1, and STAT6 was critical to the FGFR-1- and IGF-1R-mediated expression of arg1. The converse was also true as inhibition of FGFR-1 and IGF-1R reduced the activation of STAT6 in infected macrophages. Collectively, these data indicate that the FGFR/IGF-1R and IL-4 signaling pathways converge at STAT6 to promote pathologic arg1 expression and intracellular parasite survival in VL. Targeted interruption of these pathological processes offers an approach to restrain this relentlessly progressive disease., Author Summary Visceral leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is a progressive infection that is particularly common in impoverished populations of the world. People die from this disease unless it is treated. We used an experimental infection model that mimics the clinical and pathological features of human VL to study how the parasite causes this severe disease. We found that host macrophages infected with Leishmania donovani are activated in a way that leads to the expression of arginase, an enzyme that counteracts the cell's mechanisms that control the infection. This disease-promoting activation pathway was driven by the convergence of growth factor and cytokine signaling pathways and activation of the transcription factor STAT6. Chemical inhibition of signaling through the fibroblast growth factor receptor-1 (FGFR-1) or insulin-like growth factor-1 receptor (IGF-IR), or genetic knockdown of STAT6 led to reduced expression of arginase and enhanced control of the infection by macrophages. This indicates that the growth factor signaling pathways together with the cytokine pathways promote this disease. Interventions designed to disrupt this signaling could help in the treatment of VL.

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., Author Summary Visceral leishmaniasis (VL) is a neglected parasitic disease that is caused by the intracellular protozoan Leishmania donovani. Patients with this disease suffer from muscle wasting, enlargement of the spleen, reduced blood counts and ultimately will die without treatment. Progressive disease is considered to be due to impaired cellular immunity, with T cell or macrophage dysfunction, or both. We studied the Syrian hamster as an infection model because it mimics the progressive nature of human disease. We examined global changes in gene expression in the spleen and splenic macrophages during experimental VL and identified a highly inflammatory spleen environment with abundant expression of interferon and interferon-response genes that would be expected to control the infection. However, the high level of IFN-γ expression was ineffective in mediating a protective macrophage response, restraining parasite replication and halting progression of disease. We found that IFN-γ itself stimulated parasite growth in splenic macrophages and induced expression of counter-regulatory molecules, which may paradoxically make the host more susceptible. These data give insights into the nature of the immune response that promotes the infection, and identifies potential targets for therapeutic intervention.

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p, Author Summary Visceral leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is a progressive, potentially fatal infection found in many resource-poor regions of the world. We initiated these studies of an experimental model of VL to better understand the molecular and cellular determinants underlying this disease. We found that host macrophages or fibroblasts, when infected with Leishmania donovani or exposed to products secreted by the parasite, are permissive to infection because they fail to metabolize arginine to generate nitric oxide, the effector molecule needed to kill the intracellular parasites. Instead, the infected host cells are activated in a way that leads to the expression of arginase, an enzyme that metabolizes arginine to produce polyamines, which support parasite growth. This detrimental activation pathway was dependent on the parasite-induced activation of the transcription factor STAT6, but contrary to the previously accepted paradigm, did not require (but was amplified by) the presence of polarized Th2 cells or type 2 cytokines. Knockdown of host arginase or STAT6 enhanced control of the infection, indicating that this activation pathway has a critical role in the pathogenesis of the disease. Interventions designed to inhibit the STAT6-arginase-polyamine pathway could help in the treatment or prevention of VL.