Journal
JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY
Volume 67, Issue 12, Pages 2892-2902Publisher
OXFORD UNIV PRESS
DOI: 10.1093/jac/dks315
Keywords
amphotericin B; cytokines; nitric oxide; arginase1; macrophages
Funding
- Department of Biotechnology (DBT), Government of India
- Council of Scientific and Industrial Research, Government of India, New Delhi
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The aim of this study was to characterize the antileishmanial activity of heat-killed Mycobacterium indicus pranii (Mw) alone or in combination with a subtoxic dose of amphotericin B [AMB(st)]. Mw- and MwAMB(st)-mediated antileishmanial activity was evaluated by microscopic counting of intracellular amastigotes in Giemsa-stained macrophages and real-time PCR analysis of inducible nitric oxide synthase (iNOS) expression and measurement of nitric oxide generation by Griess reagent. The relationship between Mw and Toll-like receptor 4 (TLR4) signalling was studied by fluorescence-activated cell sorting, western blot and confocal microscopy. The effect of Mw alone or in combination with AMB(st) on the expression and production of interleukin (IL)-12, tumour necrosis factor-, IL-10 and transforming growth factor- was analysed by real-time PCR and ELISA, respectively. Mw treatment alone or with AMB(st) caused a significant increase in TLR4 expression of L. donovani-infected macrophages along with the activation of TLR4 downstream signalling, facilitating active nuclear translocation of nuclear factor B (NF-B). These events culminated in the up-regulation of the proinflammatory response, which was abrogated by treatment with TLR4-specific small-interfering RNA. In addition, this study demonstrates that this chemoimmunotherapeutic strategy confers protection against leishmanial pathogenesis via TLR4-dependent counter-regulation of inducible nitric oxide synthase (iNOS) and arginase1 activity. These results provide a mechanistic understanding of Mw- or MwAMB(st)-mediated protection against leishmanial parasites within host macrophages.
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