Comparative roles of free fatty acids with reactive nitrogen intermediates and reactive oxygen intermediates in expression of the anti-microbial activity of macrophages against Mycobacterium tuberculosis

Abstract

We assessed the role of free fatty acids (FFA) in the expression of the activity of macrophages against Mycobacterium tuberculosis in relation to the roles of two major anti-microbial effectors, reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI). Intracellular growth of M. tuberculosis residing inside macrophages was accelerated by treatments of macrophages with either quinacrine (phospholipase A2 (PLA2) inhibitor), arachidonyl trifuloromethylketone (type IV cytosolic PLA2 inhibitor), NG-monomethyl-L-arginine (nitric oxide synthase inhibitor), and superoxide dismutase plus catalase (ROI scavengers). In addition, M. tuberculosis-infected macrophages produced and/or secreted these effectors sequentially in the order ROI (0-3 h), FFA (0-48 h), and RNI (3 to at least 72 h). Notably, membranous FFA (arachidonic acid) of macrophages translocated to M. tuberculosis residing in the phagosomes of macrophages in phagocytic ability- and PLA2-dependent fashions during cultivation after M. tuberculosis infection. FFA, RNI and H2O2-mediated halogenation system (H2O2-halogenation system) displayed strong activity against M. tuberculosis in cell-free systems, while ROI alone exerted no such effects. Combinations of 'FFA + RNI' and 'RNI + H2O2-halogenation system' exhibited synergistic and additive effects against M. tuberculosis, respectively, while 'FFA + H2O2-halogenation system' had an antagonistic effect. Moreover, a sequential attack of FFA followed by RNI exerted synergistic activity against M. tuberculosis. Since M. tuberculosis-infected macrophages showed simultaneous production of RNI with FFA secretion for relatively long periods (approx. 45 h) and prolonged RNI production was seen thereafter, RNI in combination with FFA appear to play critical roles in the manifestation of the activity of macrophages against M. tuberculosis.

Fig. 1

Effects of some metabolic inhibitors on the growth of Mycobacterium tuberculosis residing in IFN-γ-activated murine peritoneal macrophages. Mycobacterium tuberculosis-infected macrophages were cultured in the presence or absence of either quinacrine (5 μm), N^G-monomethyl-l-arginine (NMMA; 0·5 mm), or ‘superoxide dismutase (SOD; 1000 U/ml) + catalase (900 U/ml)’ for 5 days, and thereafter the number of colony-forming units (CFU) of intracellular organisms were enumerated. Each bar indicates the mean ± s.e.m. (n = 3). *Significantly different from the value of control macrophages (without test metabolic inhibitors) (P < 0·01; Student's t-test).

Fig. 2

Time course of production/secretion of reactive oxygen intermediates (ROI) (○), free fatty acids (FFA) (Δ), and reactive nitrogen intermediates (RNI) (•) by macrophages in response to stimulation due to Mycobacterium tuberculosis infection. ROI and RNI production was measured in terms of chemiluminescence and ${\text{NO}}_2^{\text{−}}$ accumulation into culture medium, respectively. FFA secretion was measured in terms of liberation of radioactive arachidonic acid from macrophages which had been labelled with ³H-arachidonic acid (³H-AA). Each plot indicates the mean ± s.e.m. (n = 3).

Fig. 3

Evidence for the translocation of membranous free fatty acid (FFA) molecules to the intracellular Mycobacterium tuberculosis residing in macrophages. (a) Macrophages loaded with ³H-arachidonic acid (³H-AA) were infected with M. tuberculosis for 2 h and cultivated for up to 12 h (see Materials and methods). ○, Uninfected macrophages; •, M. tuberculosis-infected macrophages. Each plot indicates the mean ± s.e.m. (n = 3). Significantly greater than the values of uninfected macrophages: *P < 0·05; **P < 0·01; Student's t-test. (b) ³H-AA-loaded macrophages were infected with M. tuberculosis in the presence or absence of quinacrine or cytochalasin D for 2 h and then cultivated in the medium containing corresponding metabolic inhibitors for 12 h. □, Uninfected macrophages; hatched bars, M. tuberculosis-infected macrophages cultured in medium alone (the control for quinacrine treatment) or medium containing 1% dimethyl sulfoxide (DMSO) (the solute control for cytochalasin D treatment); ▪, metabolic inhibitor-treated macrophages. Each bar indicates the mean ± s.e.m. (n = 5). Significantly greater than the value of uninfected macrophages: *P < 0·01; significantly smaller than the values of corresponding control macrophages without drug treatment: †P < 0·01.

Fig. 4

The role of type IV cytosolic phospholipase A₂ (cPLA₂) in the expression of anti-microbial activity by IFN-γ-activated murine peritoneal macrophages against Mycobacterium tuberculosis. (a) Mycobacterium tuberculosis-infected macrophages were cultured in the presence or absence of 10 or 50 μm of either manoalide (type IIa secretory PLA₂ (sPLA₂) inhibitor) or arachidonyl trifluoromethylketone (a-TFMK) (type IV cPLA₂ inhibitor) for 5 days and, thereafter, the number of colony-forming units (CFU) of intracellular organisms was enumerated. Each bar indicates the mean ± s.e.m. (n = 4). Significantly different from the value of control macrophages (without test metabolic inhibitors): *P < 0·01 (Student's t-test). (b) Expression of inducible nitric oxide synthase (iNOS), type IIa sPLA₂, and type IV cPLA₂ mRNAs by IFN-γ-activated macrophages during the course of M. tuberculosis infection. Murine peritoneal macrophages were precultivated in medium with or without 500 U/ml IFN-γ for 3 days. The resulting macrophages were infected with M. tuberculosis organisms for 2 h and the infected macrophages were cultured for up to 12 h. At intervals, the macrophages were subjected to reverse transcription-polymerase chain reaction assay. The values in parentheses are iNOS or PLA₂ band/β-actin band ratios.

Fig. 5

Anti-Mycobacterium tuberculosis activities of the combinations of (a) ‘free fatty acids (FFA) + reactive nitrogen intermediates (RNI)’, (b) ‘RNI + H₂O₂-halogenation system’, (c) ‘FFA + H₂O₂-halogenation system’, and (d) ‘RNI + xanthine oxidase-acetaldehyde (XOA) system’. Mycobacterium tuberculosis was treated with either RNI (2·5 or 3·0 mg/ml NaNO₂), FFA (5 or 10 μg/ml arachidonic acid), H₂O₂-halogenation system (10 μm H₂O₂, 10 μm NaI, 1 μm FeSO₄), XOA system (20 μg/ml xanthine oxidase, 10 mm acetaldehyde, 100 μm FeSO₄, 100 μm EDTA), or combinations of them for 2 h (a,b,d) or 1 h (c). Each plot indicates the mean ± s.e.m. (n = 3). Significant synergistic/additive effect: *P < 0·05 (Bonferroni's multiple t-test); significant antagonistic effect: †P < 0·05 (Bonferroni's multiple t-test). Treatment of M. tuberculosis with buffer alone was not toxic to the organisms.