Antibiotics in mycobacteria-macrophage system and its consequences at both microorganism and host cell level

Dos Santos Olivera, Renato Antonio (2010). Antibiotics in mycobacteria-macrophage system and its consequences at both microorganism and host cell level. PhD thesis The Open University.



To select potentially therapeutic anti-mycobacterial agents, 9 compounds were screened on the basis of their capacity to arrest M. avium growth in liquid broth. Out of the 8 compounds that proved suitable, 2 were singled out upon evaluation of their apoptotic/necrotic effects on THP-1 macrophage-like cells. One of them controlled poorly M. avium intra-macrophage growth and showed deleterious effects on some function parameters of resting and activated host cells that led to its elimination as a potential antibiotic. The remaining compound was the substituted hydroxy-naphthoquinone lapachol, a natural product from a South American tree bark. Lapachol was bacteriostatic toward M. avium, did not induce apoptosis or necrosis of THP-1 macrophages at ≤ 32 μg/mL (132 pM) and arrested the intra-macrophage growth of M. avium at the lower dose of 16 μg/mL (66 pM). The study of the effects of lapachol on the expression of function parameters and cytokine secretion by THP-1 macrophages showed that it immunomodulated resting and TLR2-agonised human macrophage functions, some of which can lead to improved host cell responses to infection. Favourable effects were increases in IFNɣR1 and MHCII surface expression and a marked inhibition of IL-10 secretion. The up-regulation of IFNɣRl and MHCII can potentially improve the capacity to respond to IFNɣ and present antigens, respectively. The inhibition of IL-10 secretion would prevent macrophage de-activation, improving the capacity of macrophages to control the intracellular growth of M. avium. Lapachol did not affect important aspects of host cell function such as ER or oxidative stress and TLR2 agonism-induced capacity to produce oxygen and nitrogen metabolites and secrete TNF-α. The drug resulted in a reduction of IL-1β and TNF-α secretion only from resting and IFN-ɣ-treated cells, and in a decrease in latex bead and M. avium internalization. A proteomic approach to study the influence of lapachol on the expression of proteins modulated by IFN-ɣ or TLR2 agonism showed important reductions in the expression levels of three proteases, the ER protein DnaJ homolog that forms part of complexes together with grp78 regulates grp78 activity and two cytoskeletal proteins, L-plastin and fascin. The down-regulation of these cytoskeletal proteins could hypothetically affect cell migration and phagocytosis. Lapachol treatment resulted in important increases in cytosolic protein disulfide isomerase A3 (ERp57), thought to be involved in Stat3 complexing and in disulfide bond cleavages coupled to protein degradation, and glucose-6-P-dehydrogenase, involved in NADPH generation and, as a consequence, in the protection against damage by hydrogen peroxide. The results obtained during this work suggest the convenience of in vivo experiments to further test lapachol regarding its potential as a new anti-mycobacterial agent and immunomodulator.

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