Fig 2: HIV-1 coinfection and degree of immunosuppression relates to heme oxygenase-1 (HO-1) concentrations in plasma from tuberculosis (TB) patients prior to antitubercular treatment. (A) Spearman correlation analysis of HO-1 levels in plasma by CD4+ T-cell counts in HIV-infected patients before initiation of antibubercular therapy. (B) HIV-infected patients were further classified based on CD4+ T-cell counts and HO-1 levels. (C) HO-1 levels were also compared between antiretroviral therapy (ART)-naïve patients and those who were on ART before TB treatment. In panels (B,C), data were compared using the Mann–Whitney U test. (D) Spearman correlation analysis of HO-1 levels in plasma with HIV viral load at pretreatment in HIV-infected patients.

Fig 3: ESAT6-mediated heme oxygenase-1 (HO-1) induction in Mycobacterium tuberculosis (Mtb)-infected macrophages is associated with reactive oxygen species (ROS)-dependent release of NRF-2 to the nucleus. (A) Human monocyte-differentiated macrophages were infected with Mtb H37Rv or an H37Rv mutant strain lacking ESAT-6 expression (both at multiplicity of infection: 3) for 24 h. Cells infected with the ESAT-6-deficient Mtb strain were treated with 20 µg/mL of the fusion protein Lfn-ESAT-6 with the anthrax-protective antigen cytosolic delivery system. Generation of superoxide anions was quantified in cell supernatants using a colorimetric assay, while intracellular ROS production was assessed by flow cytometry. Results are plotted as histograms where the mean fluorescence intensity (MFI) was compared between the experimental groups. (B) Lipid peroxidation and oxidative DNA damage were assessed as described in Section “Materials and Methods.” (C) Activation of NRF-2 in nuclear extracts (12 h postinfection) and of HO-1 levels in whole cell lysates (24 h postinfection) were determined in cultures of infected macrophages in the presence or absence of a TAT-conjugated NRF-2 sequence peptide that interacts with the Keap-1/NRF-2 complex (TAT-14, 50 µM). Data are from at least three independent experiments using cells from a total of up to six healthy donors. Data were compared using the Wilcoxon matched-pairs test (*p < 0.05, **p < 0.01, ***p < 0.0001). MDA, malondialdehyde; NRF-2, nuclear factor erythroid-derived 2-like 2; 8-OH-dG, 8-hydroxy-2′-deoxyguanosine.

Fig 4: Illustration of the cholesteric liquid crystal (CLC) biological sensor chip in the presence of heme oxygenase (HO)-1 biomolecules in the DMOAP-coated cell. Different color arrows indicate the different light penetration intensities.

Fig 5: Experimental Mycobacterium tuberculosis (Mtb) infection in vivo induces heme oxygenase-1 (HO-1) expression in lungs and blood, which is reversed during antitubercular therapy. (A) HO-1 protein expression was quantitatively assessed in lung homogenates and in plasma samples from rabbits bronchoscopically infected with Mtb by ELISA. (B) HO-1 levels in lung homogenates and serum samples were measured in a tuberculosis treatment model using mice infected with a low dose of Mtb. Graph shows representative data from different experiments using 4 mice in each time point per experimental group (total animal number = 8–12 mice per group). (C) Prospective changes in HO-1 concentrations in plasma were evaluated in non-human primates experimentally infected with Mtb and treated with rifampin or isoniazid. Data were analyzed using the Mann–Whitney U test (two unmatched groups), the Kruskal–Wallis test with Dunn’s multiple comparisons or linear trend ad hoc tests (more than two groups) or the Wilcoxon matched-pairs test (*p < 0.05, **p < 0.01, ***p < 0.0001).