Fig 2: Myeloid Specific HO-1 Knockout Mice (HO-1LysM−/−) Are Susceptible to Mtb Infection(A) Kaplan-Meier survival analysis of Mtb-infected HO-1LysM+/+ and HO-1LysM (n = 10 mice per group).(B and C) Bacterial burden in lungs (B) and spleens (C) of Mtb-infected HO-1LysM+/+ and HO-1LysM−/− mice at 6, 12, and 24 weeks post-infection.(D) Flow cytometry gating strategy for identification of myeloid cells.(E and F) Absolute numbers in cells per gram of tissue (E) and proportion of CD45+ cells for lung neutrophils (F) from uninfected or Mtb-infected HO-1LysM+/+ and HO-1LysM−/− mice at 12 and 24 weeks post-infection.(G and H) Absolute numbers in cells per gram of tissue (G) and proportion of CD45+ cells for lung macrophages (H) from uninfected or Mtb-infected HO-1LysM+/+ and HO-1LysM−/− mice at 12 and 24 weeks post-infection.(I) The sequential flow cytometry gating strategy for identification of IFN-γ-producing lymphocytes and T cells.(J) Representative gated population of CD45+IFN-γ+ T cells.(K and L) Differences in proportions of CD45+ cells for IFN-γ+ T cells (K) and IFN-γ MFI (L) isolated from the lungs of Mtb-infected HO-1LysM+/+ and HO-1LysM−/− mice at 24 weeks post-infection.(M) Representative gated population of CD45+CD4+IFN-γ+ T cells.(N) Differences in proportions of CD45+ cells for CD4+ T cells in the lungs of Mtb-infected HO-1LysM+/+ and HO-1LysM−/− mice at 24 weeks post-infection.(O and P) Differences in proportions of CD45+ cells (O) and IFN-γ MFI (P) for CD45+CD4+IFN-γ+ T cells isolated from the lungs of Mtb-infected HO-1LysM+/+ and HO-1LysM−/− mice at 24 weeks post-infection.(Q) Representative gated population of CD45+CD4+CD69+IFN-γ+ T cells.(R) Differences in proportions of CD45+ cells for CD45+CD4+CD69+ T cells in the lungs of Mtb-infected HO-1LysM+/+ and HO-1LysM−/− mice at 24 weeks post-infection.(S and T) Differences in proportions of CD45+ cells (S) and IFN-γ MFI (T) for CD45+CD4+CD69+IFN-γ+ T cells isolated from the lungs of Mtb-infected HO-1LysM+/+ and HO-1LysM−/− mice at 24 weeks post-infection.n = 4 for each time point. Statistical testing was performed using the unpaired Student’s t test. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 3: Cytokine Response in HO-1 LysM Mice and Proposed Model for HO-1-Mediated Protection against Mtb Infection(A–K) Cytokine analysis (A), IL-2 (B), IL-3 (C), IL-5 (D), IL-6 (E), IL-10 (F), IL-17A (G), MIP-1α (H), MIP-1β (I), G-CSF (J), KC (K) in the BALF of uninfected and Mtb-infected HO-1LysM+/+ and HO-1LysM−/− mice at 6, 12, and 18 weeks post-infection. n = 4 for each time point. Statistical testing was performed using the unpaired Student’s t test. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.(L) Proposed model for HO-1-mediated protection during Mtb infection. Mtb infection induces HO-1 expression and its downstream enzymatic cascade. Together with its enzymatic products and ferritin, which is induced by HO-1, HO-1 protects against TB immunopathology and thereby contributes to overall TB disease tolerance via its anti-oxidant, anti-inflammatory, and anti-proliferative properties. Therefore, maintaining the physiological levels of HO-1 is important to limit TB disease pathology (green shaded area). Contrarily, in HO-1-deficient conditions, the levels of key pro-inflammatory cytokines are significantly elevated. This results in rapid and dysregulated myeloid cell infiltration to the infected sites, which results in significantly elevated levels of ROS and RNS as well as rapid karyorrhexis and NETosis. ROS and RNS also generate increased localized concentrations of peroxynitrite (ONOO−) and subsequent suppression of T cell responses. In addition, Mtb infection also causes endothelial injury and intravascular coagulation, resulting in significant accumulation of heme, a potent proinflammatory and pro-oxidant molecule. Excess heme overwhelms the cytoprotective activity of HO-1, thereby contributing pro-oxidant-mediated inflammatory responses. Together, these dysregulated inflammatory responses result in chronic TB immunopathology and rapid disease progression (red shaded area).

Fig 4: Nuclear-directed expression of HO-1 in HEK293 cells. HO-1 and HO-1/NLS were constructed into pEGFP-N3 vector. Resultant constructs, (A) pEGFP-HO-1 and (B) pEGFP-HO-1/NLS, were transfected in HEK293 cells. After 24 h, cells were fixed and stained with F-actin phalloidin and DAPI. Images were taken by using Zeiss LSM 510 Meta confocal microscope. Bar, 50 μm.

Fig 5: Tolvaptan induces Nrf2 nuclear translocation and HO-1 expression in mpkCCD cells. (a) Tolvaptan promotes Nrf2 nuclear translocation. (Left panel) Western blotting of Nrf2 in nuclear extract. mpkCCD cells were treated with 200 μM tolvaptan or 5 μM sulforaphane on filter for 12 h, following which nuclear fraction was separated using commercially available reagents for nuclear extraction. Arrow indicates the band of Nrf2. (Right panel) Densitometric analysis of Nrf2. Error bars are mean values ± S.E. from three experiments. Tukey’s test, *P < 0.05. C: control (DMSO), Tol: 200 μM tolvaptan, Sul: 5 μM sulforaphane. P.C.: positive control. (b) Tolvaptan increases HO-1 mRNA expression in a dose-dependent manner. mpkCCD cells were treated with 10–200 μM tolvaptan for 4 h, following which HO-1 mRNA expression was examined using qPCR. Error bars are mean values ± S.E. from three experiments. Tukey’s test, *P < 0.05, **P < 0.01. (c) Tolvaptan induces HO-1 protein expression in a dose-dependent manner. (Top panel) mpkCCD cells were treated with 10–200 μM tolvaptan for 12 h. (Bottom panel) Densitometric analysis of HO-1 is presented. Error bars are mean values ± S.E. from three experiments. Tukey’s test, *P < 0.05, **P < 0.01. (d) An Nrf2 inhibitor, ML385, inhibits the HO-1 induction by tolvaptan. (Top panel) Following the pre-treatment of mpkCCD cells using DMSO (C, T, and S) or 50 μM ML385 (T + M and S + M) for 1 h, mpkCCD cells were treated with 200 μM tolvaptan or 5 μM sulforaphane in the presence or absence of 50 μM ML385 for 12 h. C: control (DMSO), T: 200 μM tolvaptan, M: 50 μM ML385, S: 5 μM sulforaphane. (Bottom panel) Densitometric analysis of HO-1 is presented. Error bars are mean values ± S.E. from three experiments. Tukey’s test, *P < 0.05, **P < 0.01.