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People are infected with Leishmania donovani when the parasite is deposited in the dermis during the blood meal of the sand fly vector. Most infected people develop a subclinical latent infection, but some develop progressive visceral leishmaniasis. Malnutrition is a risk factor for the development of active VL. We previously demonstrated increased parasite dissemination from the skin to visceral organs in a murine model of malnutrition. Here we investigated the mechanism of early parasite dissemination. After delivery of L. donovani to the skin, we found enhanced capture of parasites by inflammatory monocytes and neutrophils in the skin of malnourished mice. However, parasite dissemination in malnourished mice was driven primarily by infected inflammatory monocytes, which showed increased CCR7 expression, greater intrinsic migratory capacity, and increased trafficking from skin to spleen. PGE2 production, which was increased at the site of skin infection, increased monocyte CCR7 expression and promoted CCR7-related monocyte-mediated early parasite dissemination in malnourished mice. Parasite dissemination in monocytes was reduced by inhibition of PGE2, knockdown or silencing of CCR7 in monocytes, and depletion of inflammatory monocytes through administration of diphtheria toxin to CSFR1-DTR transgenic mice that have monocyte-specific DT receptor expression. CCR7-driven trafficking of infected inflammatory monocytes through the lymph node was accompanied by increased expression of its ligands CCL19 and CCL21. These results show that the CCR7/PGE2 axis is responsible for the increased trafficking of L. donovani-infected inflammatory monocytes from the skin to the spleen in the malnourished host. Undernutrition and production of PGE2 are potential targets to reduce the risk of people developing VL. Nutritional interventions that target improved immune function and reduced PGE2 synthesis should be studied in people at risk of developing VL. Author summary: This study established that inflammatory monocytes contribute to the visceralization of L. donovani parasites in the malnourished host. We found that the prostaglandin E2 produced during the first 72 hours of infection amplified the parasite burden at the site of skin infection and induced a high expression of CCR7 receptor in inflammatory monocytes. CCR7-bearing inflammatory monocytes carried L. donovani parasites via enhanced migration from the skin infection site to the viscera of malnourished mice. This dissemination of Leishmania parasites was abrogated in CCR7 deficient inflammatory monocytes and in mice treated with a prostaglandin E synthase inhibitor. We conclude that PGE2 contributes to monocyte-mediated parasite dissemination through the expression of CCR7 and its ligands in malnourished mice. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is characterized by expansion of myeloid cells in the liver and spleen, which leads to a severe splenomegaly associated with higher risk of mortality. This increased cellularity is thought to be a consequence of recruitment of cells to the viscera. We studied whether the local proliferation of splenic myeloid cells contributes to increased splenic cellularity. We found that a monocyte-like population of adherent splenic cells from Leishmania donovani-infected hamsters had enhanced replicative capacity ex vivo and in vivo (BrdU incorporation, p<0.0001). In vitro assays demonstrated that proliferation was more pronounced in the proinflammatory M1 environment and that intracellular infection prevented proliferation. Secondary analysis of the published splenic transcriptome in the hamster model of progressive VL revealed a gene expression signature that included division of tumoral cells (Z = 2.0), cell cycle progression (Z = 2.3), hematopoiesis (Z = 2.8), proliferation of stem cells (Z = 2.5) and overexpression of proto-oncogenes. Regulators of myeloid cell proliferation were predicted in-silico (CSF2, TLR4, IFNG, IL-6, IL-4, RTK signaling, and STAT3). The in-silico prediction was confirmed with chemical inhibitors of PI3K/AKT, MAPK and STAT3 which decreased splenic myeloid cell division ex vivo. Hamsters infected with L. donovani treated with a STAT3 inhibitor had reduced in situ splenic myeloid proliferation (p = 0.03) and parasite burden. We conclude that monocyte-like myeloid cells have increased STAT3-dependent proliferation in the spleen of hamsters with visceral leishmaniasis and that inhibition of STAT3 reduces myeloid cell proliferation and parasite burden. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The Syrian golden hamster (Mesocricetus aureus) has been used as a model to study infections caused by a number of human pathogens. Studies of immunopathogenesis in hamster infection models are challenging because of the limited availability of reagents needed to define cellular and molecular determinants. Results: We sequenced a hamster cDNA library and developed a first-generation custom cDNA microarray that included 5131 unique cDNAs enriched for immune response genes. We used this microarray to interrogate the hamster spleen response to Leishmania donovani, an intracellular protozoan that causes visceral leishmaniasis. The hamster model of visceral leishmaniasis is of particular interest because it recapitulates clinical and immunopathological features of human disease, including cachexia, massive splenomegaly, pancytopenia, immunosuppression, and ultimately death. In the microarray a differentially expressed transcript was identified as having at least a 2-fold change in expression between uninfected and infected groups and a False Discovery Rate of <5%. Following a relatively silent early phase of infection (at 7 and 14 days post-infection only 8 and 24 genes, respectively, were differentially expressed), there was dramatic upregulation of inflammatory and immune-related genes in the spleen (708 differentially expressed genes were evident at 28 days post-infection). The differentially expressed transcripts included genes involved in inflammation, immunity, and immune cell trafficking. Of particular interest there was concomitant upregulation of the IFN-γ and interleukin (IL)-4 signaling pathways, with increased expression of a battery of IFN-γ- and IL-4-responsive genes. The latter included genes characteristic of alternatively activated macrophages. Conclusions: Transcriptional profiling was accomplished in the Syrian golden hamster, for which a fully annotated genome is not available. In the hamster model of visceral leishmaniasis, a robust and functional IFN-γ response did not restrain parasite load and progression of disease. This supports the accumulating evidence that macrophages are ineffectively activated to kill the parasite. The concomitant expression of IL-4/IL-13 and their downstream target genes, some of which were characteristic of alternative macrophage activation, are likely to contribute to this. Further dissection of mechanisms that lead to polarization of macrophages toward a permissive state is needed to fully understand the pathogenesis of visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In a murine model of moderate childhood malnutrition we found that polynutrient deficiency led to a 4–5-fold increase in early visceralization of L. donovani (3 days post-infection) following cutaneous infection and a 16-fold decrease in lymph node barrier function (p<0.04 for all). To begin to understand the mechanistic basis for this malnutrition-related parasite dissemination we analyzed the cellularity, architecture, and function of the skin-draining lymph node. There was no difference in the localization of multiple cell populations in the lymph node of polynutrient deficient (PND) mice, but there was reduced cellularity with fewer CD11c+dendritic cells (DCs), fibroblastic reticular cells (FRCs), MOMA-2+ macrophages, and CD169+ subcapsular sinus macrophage (p<0.05 for all) compared to the well-nourished (WN) mice. The parasites were equally co-localized with DCs associated with the lymph node conduit network in the WN and PND mice, and were found in the high endothelial venule into which the conduits drain. When a fluorescent low molecular weight (10 kD) dextran was delivered in the skin, there was greater efflux of the marker from the lymph node conduit system to the spleens of PND mice (p<0.04), indicating that flow through the conduit system was altered. There was no evidence of disruption of the conduit or subcapsular sinus architecture, indicating that the movement of parasites into the subcortical conduit region was due to an active process and not from passive movement through a leaking barrier. These results indicate that the impaired capacity of the lymph node to act as a barrier to dissemination of L. donovani infection is associated with a reduced number of lymph node phagocytes, which most likely leads to reduced capture of parasites as they transit through the sinuses and conduit system. Author Summary: The impact of malnutrition in the world is staggering. Malnutrition is thought to directly or indirectly contribute to more than half of all childhood deaths, most of them related to heightened susceptibility to infection. 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. Most people who get infected with this parasite have only an asymptomatic latent infection, however, people who are malnourished have a greatly increased risk of developing severe VL. We initiated these studies of an experimental model that mimics human childhood malnutrition to better understand how malnutrition increases the susceptibility to VL at the molecular and cellular level. In this model we found that malnutrition led to failure of the skin-draining lymph node to act as a barrier to dissemination. This loss of lymph node barrier function was associated with a significant reduction in the numbers of dendritic cells and macrophages, phagocytic cells that capture and kill invading pathogens, and alteration of the flow of lymph through the lymph node. [ABSTRACT FROM AUTHOR]

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,0.001) and increased polyamine synthesis (p,0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p,0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p,0.001) and fibroblasts (p,0.01), and in a hamster fibroblast cell line (p,0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p,0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p,0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection. [ABSTRACT FROM AUTHOR]