Background The innate immune mediators are likely to influence the clinical phenotype of leishmaniasis by primary responses which limit or facilitate the spread of the parasite, as well as by modulating adaptive immunity. This study investigated the response of key innate immune cells in a focus which regularly reports localised cutaneous leishmaniasis (LCL) caused by Leishmania donovani, a species which typically causes visceral disease. Methods Peripheral blood mononuclear cell (PBMC) derived macrophages and dendritic cells from patients with LCL and healthy controls from endemic and non-endemic areas, were stimulated with soluble Leishmania antigen (SLA). Inflammatory mediators produced by macrophages (TNF-α/TGF-β/IL-10, ELISA; NO, Griess method) and dendritic cells (IL-12p70, IL-10, flowcytometry) and macrophage expression of surface markers of polarization, activation and maturation (flowcytometry) were determined at 24h, 48h and 72h and compared. Study was conducted prospectively from 2015–2019. Results Patient derived macrophages and dendritic cells produced higher levels of both pro and anti-inflammatory mediators compared to controls (p<0.05) with the best discrimination for active disease observed at 72h. Data demonstrated an early activation of macrophages and a subsequent pro-inflammatory bias, as indicated by temporal profiles of TNF-α/TGF-β and TNF-α/IL-10 ratios and higher proportions of classical (M1) macrophages. Higher TGF-β levels were observed in cells from patients with ulcerated or persistent lesions. Immune responses by cells derived from controls in endemic and non-endemic regions did not differ significantly from each other. Conclusions The overall immunophenotypic profile suggests that LCL observed in the country is the result of a balancing immune response between pro-inflammatory and regulatory mediators. The mediators which showed distinct profiles in patients warrant further investigation as potential candidates for immunotherapeutic approaches. A comparison with visceral leishmaniasis caused by the same species, would provide further evidence on the differential role of these mediators in the resulting clinical phenotype.

Background: The innate immune mediators are likely to influence the clinical phenotype of leishmaniasis by primary responses which limit or facilitate the spread of the parasite, as well as by modulating adaptive immunity. This study investigated the response of key innate immune cells in a focus which regularly reports localised cutaneous leishmaniasis (LCL) caused by Leishmania donovani, a species which typically causes visceral disease. Methods: Peripheral blood mononuclear cell (PBMC) derived macrophage and dendritic cell responses to soluble Leishmania antigen (SLA) were compared between patients with LCL and healthy controls from endemic and non-endemic areas. Inflammatory mediators produced by macrophages (TNF-α, NO, TGF-β and IL-10) and dendritic cells (IL-12p70, IL-10) and cell surface markers of macrophage polarization, activation and maturation were determined at 24h, 48h and 72h by Enzyme-linked immunosorbent assay (ELISA) and flowcytometry. Results: Patient derived macrophages and dendritic cells produced higher levels of both pro and anti-inflammatory mediators compared to controls (p<0.05) with the best discrimination for active disease observed at 72h. Data demonstrated an early activation of macrophages and a subsequent pro-inflammatory bias, as indicated by temporal profiles of TNF-α/TGF-β and TNF-α/IL-10 ratios and higher proportions of classical (M1) macrophages. Higher TGF-β levels were observed in cells from patients with ulcerated or persistent lesions. Immune responses by cells derived from controls in endemic and non-endemic regions did not differ significantly from each other. Conclusions: The overall immunophenotypic profile suggests that LCL observed in the country is the result of a balancing immune response between pro-inflammatory and regulatory mediators. The mediators which showed distinct profiles in patients warrant further investigation as potential candidates for immunotherapeutic approaches. A comparison with visceral leishmaniasis caused by the same species, would provide further evidence on the differential role of these mediators in the resulting clinical phenotype.

Background: Leishmaniasis is a neglected tropical disease caused by parasitic protozoa of the genus Leishmania. It has a spectrum of manifestations including cutaneous, mucosal and visceral disease. The clinical outcome of infection in humans is determined primarily by the infecting species and the immune response by the host. Sri Lanka is endemic for localised cutaneous leishmaniasis (LCL) caused by Leishmania donovani; a species which usually causes visceral disease. The aim of this study was to characterize the immune response in LCL by macrophages; the cells responsible for survival as well as eventual elimination of the parasite. Methods & Materials: Peripheral blood mono nuclear cell (PBMC) derived macrophages from newly diagnosed LCL patients (n = 8) and healthy non endemic controls (n = 8) were stimulated with L. donavani antigen (50 g/ml) in vitro. The production of IL-10, TNF␣ and Nitric Oxide (NO) were measured by ELISA and Griess reaction at predetermined time intervals. The differences between experimental groups were analysed using the Student's t-test for parametric data and Mann-Whitney test for non-parametric data. Results: Macrophages from patients produced more cytokines and NO at all time points. IL-10 production by patient macrophages was significantly higher (105.68 ± 26.05 vs 19.81 ± 28.24 pg/mL; p<0.01) at 72 hours but did not vary markedly at 24 and 48 hours. TNF␣ production by patient macrophages was significantly higher at both 24 hours (23.05 ± 13.97 vs 4.01 ± 2.26 pg/mL; p<0.01) and 48 hours (311.33 ± 206.29 vs 17.61 ± 21.09 pg/mL; p<0.01). Levels of production of NO remained similar at 24 and 48 hours but showed increased levels by patient macrophages at 72 hours (5.40 ± 1.15 vs 2.36 ± 1.21 pg/mL; p<0.01). Conclusion: These data suggest that IL-10, TNF␣ and NO play a role in determining disease outcome in LCL due to L. donovani. In contrast to TNF␣, the contribution of IL-10 and NO appear to be later in the infection. The findings should be interpreted in the context of changes in other imflammatory mediators, to better understand the underlying pathogenic mechanisms where a visceralizing Leishmania species is localized to the skin.

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