Dataset Information

Inflammatory chemokines and their receptors in human visceral leishmaniasis: Gene expression profile in peripheral blood, splenic cellular sources and their impact on trafficking of inflammatory cells.

ABSTRACT: Chemokines play an important role in determining cellular composition at inflammatory sites, and as such, influence disease outcome. In this study, we investigated the expression profile and splenic cellular source of various inflammatory chemokines and their receptors in human visceral leishmaniasis (VL). The expression of chemokines or their receptors was measured at the gene and protein level by employing real time qPCR and a cytometric bead array assay, respectively. In addition, the cellular source of chemokines and their receptors in the spleen was identified employing gene expression analyses in sequentially selected cell subsets. We identified elevated expression of CXCL10, CXCL9, CXCL8, and decreased CCL2 from VL patients. Further, we found reduced expression of the chemokine receptors CXCR1, CXCR2, CXCR3 and CCR2, but increased expression of CCR7 on VL PBMC, compared to endemic healthy controls. Additionally, splenic monocytes were found to be the major source of CXCL10, CXCL9 and CCR2, whereas T cells were the main source of CXCR3 and CCR7. We also report a strong association between plasma IFN-? and CXCL-10, CXCL-9 levels. Enhanced parasite burden positively correlates with increased expression of CXCL10, CXCL9, IFN-? and IL-10. Overall our result indicates that VL patients have an elevated inflammatory chemokine milieu which correlated with disease severity. However, expression of their chemokine receptors was significantly impaired, which may have contributed to reduced frequencies of blood monocytes and neutrophils in peripheral blood. In contrast, enhanced expression of CCR7 was associated with increased numbers of activated T cells in circulation. These findings highlight the importance of chemokines for recruitment of various cell populations in VL, and the knowledge gained may help in global understandings of the complex interaction between chemokines and pathological processes, and therefore will contribute towards the design of novel chemokine based immunological therapies against VL.

SUBMITTER: Singh N

PROVIDER: S-EPMC6359895 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Inflammatory chemokines and their receptors in human visceral leishmaniasis: Gene expression profile in peripheral blood, splenic cellular sources and their impact on trafficking of inflammatory cells.

Singh Neetu N Sundar Shyam S

Molecular immunology 20170220

Chemokines play an important role in determining cellular composition at inflammatory sites, and as such, influence disease outcome. In this study, we investigated the expression profile and splenic cellular source of various inflammatory chemokines and their receptors in human visceral leishmaniasis (VL). The expression of chemokines or their receptors was measured at the gene and protein level by employing real time qPCR and a cytometric bead array assay, respectively. In addition, the cellula ...[more]

PMID: 28222329

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Project description: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.

Project description:BackgroundNew drugs are needed to treat visceral leishmaniasis (VL) because the current therapies are toxic, expensive, and parasite resistance may weaken drug efficacy. We established a novel ex vivo splenic explant culture system from hamsters infected with luciferase-transfected Leishmania donovani to screen chemical compounds for anti-leishmanial activity.Methodology/principal findingsTHIS MODEL HAS ADVANTAGES OVER IN VITRO SYSTEMS IN THAT IT: 1) includes the whole cellular population involved in the host-parasite interaction; 2) is initiated at a stage of infection when the immunosuppressive mechanisms that lead to progressive VL are evident; 3) involves the intracellular form of Leishmania; 4) supports parasite replication that can be easily quantified by detection of parasite-expressed luciferase; 5) is adaptable to a high-throughput screening format; and 6) can be used to identify compounds that have both direct and indirect anti-parasitic activity. The assay showed excellent discrimination between positive (amphotericin B) and negative (vehicle) controls with a Z' Factor >0.8. A duplicate screen of 4 chemical libraries containing 4,035 compounds identified 202 hits (5.0%) with a Z score of <-1.96 (p<0.05). Eighty-four (2.1%) of the hits were classified as lead compounds based on the in vitro therapeutic index (ratio of the compound concentration causing 50% cytotoxicity in the HepG(2) cell line to the concentration that caused 50% reduction in the parasite load). Sixty-nine (82%) of the lead compounds were previously unknown to have anti-leishmanial activity. The most frequently identified lead compounds were classified as quinoline-containing compounds (14%), alkaloids (10%), aromatics (11%), terpenes (8%), phenothiazines (7%) and furans (5%).Conclusions/significanceThe ex vivo splenic explant model provides a powerful approach to identify new compounds active against L. donovani within the pathophysiologic environment of the infected spleen. Further in vivo evaluation and chemical optimization of these lead compounds may generate new candidates for preclinical studies of treatment for VL.

Dataset Information

Inflammatory chemokines and their receptors in human visceral leishmaniasis: Gene expression profile in peripheral blood, splenic cellular sources and their impact on trafficking of inflammatory cells.

Publications

Inflammatory chemokines and their receptors in human visceral leishmaniasis: Gene expression profile in peripheral blood, splenic cellular sources and their impact on trafficking of inflammatory cells.

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