Abstract
Leishmania mexicana causes localized (LCL) or diffuse cutaneous leishmaniasis (DCL). The cause of dissemination in DCL remains unknown, yet NK cells possibly play a role in activating leishmanicidal mechanisms during innate and adaptive immune responses. We had previously shown that Leishmania lipophosphoglycan (LPG) is a ligand for TLR2, activating human NK cells. We have now analyzed NK cells in LCL and DCL patients. NK numbers and effector mechanisms differed drastically between both groups of patients: DCL patients showed reduced NK cell numbers; diminished IFN-γ and TNF-α production; and lower TLR2, TLR1, and TLR6 expression as compared to LCL patients. The altered protein expression found in NK cells of DCL patients correlated with their down-regulation of IFN-γ gene expression in LPG-stimulated and non-stimulated cells as compared to LCL patients. NK cell response was further analyzed according to gender, age, and disease evolution in LCL patients showing that female patients produced higher IFN-γ levels throughout the disease progression, whereas TLR2 expression diminished in both genders with prolonged disease evolution and age. We furthermore show the activation pathway of LPG binding to TLR2 and demonstrated that TLR2 forms immunocomplexes with TLR1 and TLR6. In addition to the reduced NK cell numbers in peripheral blood, DCL patients also showed reduced NK cell numbers in the lesions. They were randomly scattered within the lesions, showing diminished cytokine production, which contrasts with those of LCL lesions, where NK cells produced IFN-γ and TNF-α and were found within organized granulomas. We conclude that in DCL patients the reduced NK-cell numbers and their diminished activity, evidenced by low TLR expression and low cytokine production, are possibly involved in the severity of the disease. Our results provide new information on the contribution of NK cells in Leishmania infections of the human host.
Highlights
Leishmania mexicana causes a wide spectrum of cutaneous diseases, ranging from localized cutaneous leishmaniasis (LCL), characterized by ulcers at sites of parasite inoculation, to diffuse cutaneous leishmaniasis (DCL), where parasites spread throughout the skin forming disfiguring nodules [1]
In order to analyze whether TLR2 binds to TLR1 and TLR6 in NK cells, we immunoprecipitated with anti-TLR2 and Western blotted with a-TLR1 or a-TLR6
We speculate that the reduced binding of TLR1 or TLR6 to TLR2 after incubation with LPG is possibly due to partial obstruction of the docking site after LPG binds to TLR2
Summary
Leishmania mexicana causes a wide spectrum of cutaneous diseases, ranging from localized cutaneous leishmaniasis (LCL), characterized by ulcers at sites of parasite inoculation, to diffuse cutaneous leishmaniasis (DCL), where parasites spread throughout the skin forming disfiguring nodules [1]. Among the first innate cells capable of early IFN-c and TNFa production are NK cells [11] They can be divided into 2 subsets: CD56dim and CD56bright, yet the roles of these subsets have not been clearly characterized in leishmaniasis [12,13]. We had previously shown that Leishmania LPG activates human NK cells through TLR2 stimulation, leading to IFN-c and TNF-a production [3]. These cytokines synergize in the macrophage to induce iNOS leading to NO production, one of the molecules responsible for intracellular Leishmania destruction [14]. Even though NK cells have been shown to play an important protective role in mouse Leishmania infections [11,15], their response has not been analyzed in patients with LCL and DCL
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