NADPH-derived reactive oxygen species is required for induction of heme oxygenase-1 (HO-1) in macrophages infected with Mycobacterium tuberculosis ( Mtb ) . (A) Human monocyte-derived macrophages were infected with Mtb H37Rv [multiplicity of infection (MOI): 3] in the presence or absence of an antioxidant (Tempol) for 24 h and HO-1 levels were measured in whole cell extracts by ELISA. Right panel shows HO-1 levels in infected macrophages treated with Tempol in the presence of a TAT-conjugated <t>NRF-2</t> sequence peptide that interacts with the <t>Keap-1/NRF-2</t> complex (TAT-14, 50 µM). (B) Generation of superoxide anions was quantified in cell supernatants of cultures shown in panel (A) . H 2 O 2 was used as positive control. (C) Activation of the HO-1 transcription factor NRF-2 in nuclear extracts was quantitatively assessed 12 h after infection using a colorimetric DNA-binding ELISA kit. (D) Bone marrow-differentiated macrophages were prepared from wild type (WT), p47phox −/− , or gp91phox −/− (deficient in two distinct subunits of the NADPH oxidase system) mice and infected in vitro with Mtb (MOI: 3) for 24 h. HO-1 levels were then assessed in whole cell extracts by ELISA. (E,F) HO-1 levels in cell extracts as well as activation of NRF-2 in nuclear extracts were quantified in macrophage cultures in the presence of TAT-14 (50 µM). Data are from at least three experiments using cells from a total of up to six healthy donors. (D–F) Three independent experiments were performed, with samples run in triplicates. Data from different biological groups were analyzed using the Kruskal–Wallis test, with the Dunn's multiple-comparison test, whereas matched analyses were performed using the Wilcoxon matched-pairs test (* p
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