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The emergence of drug-resistant Mycobacterium tuberculosis (M.tb) has led to the development of novel anti-tuberculosis (anti-TB) drugs. Common methods for testing the efficacy of new drugs, including two-dimensional cell culture models or animal models, have several limitations. Therefore, an appropriate model representative of the human organism is required. Here, we developed an M.tb infection model using human lung organoids (hLOs) and demonstrated that M.tb H37Rv can infect lung epithelial cells and human macrophages (hMφs) in hLOs. This novel M.tb infection model can be cultured long-term and split several times while maintaining a similar number of M.tb H37Rv inside the hLOs. Anti-TB drugs reduced the intracellular survival of M.tb in hLOs. Notably, M.tb growth in hLOs was effectively suppressed at each passage by rifampicin and bedaquiline. Furthermore, a reduction in inflammatory cytokine production and intracellular survival of M.tb were observed upon knockdown of MFN2 and HERPUD1 (host-directed therapeutic targets for TB) in our M.tb H37Rv-infected hLO model. Thus, the incorporation of hMφs and M.tb into hLOs provides a powerful strategy for generating an M.tb infection model. This model can effectively reflect host-pathogen interactions and be utilized to test the efficacy of anti-TB drugs and host-directed therapies. Author summary: Establishment of M.tb infection model is imperative to develop new anti-TB drugs based on the pathogenesis of TB. Various animal models, including mice, rats, guinea pigs, non-human primates, rabbits, cattle, and zebrafish, are commonly used in TB research to mimic TB symptoms and study immune responses to M.tb infection. In vitro models, such as agent-based models allow examination of host-pathogen interactions, early granuloma formation and drug screening, providing cellular-level insights. However, these models may not fully represent human immunopathology owing to differences in immune cell distributions. Lung organoids mimic human lung dynamics and functions, providing crucial insights into immune responses to TB. In this study, an M.tb infection model developed using hLOs demonstrated infection of lung epithelial cells and human macrophages, reflecting host-pathogen interactions. This model is attractive for evaluating the efficacy of anti-TB drugs and host-directed therapies. [ABSTRACT FROM AUTHOR]

Tumor suppressor p53 is not only affects immune responses but also contributes to antibacterial activity. However, its bactericidal function during mycobacterial infection remains unclear. In this study, we found that the p53-deficient macrophages failed to control Mycobacterium tuberculosis (Mtb), manifested as a lower apoptotic cell death rate and enhanced intracellular survival. The expression levels of p53 during Mtb infection were stronger in M1 macrophages than in M2 macrophages. The TLR2/JNK signaling pathway plays an essential role in the modulation of M1 macrophage polarization upon Mtb infection. It facilitates p53-mediated apoptosis through the production of reactive oxygen species, nitric oxide and inflammatory cytokines in Mtb-infected M1 macrophages. In addition, nutlin-3 effectively abrogated the intracellular survival of mycobacteria in both TB patients and healthy controls after H37Ra infection for 24 h, indicating that the enhancement of p53 production effectively suppressed the intracellular survival of Mtb in hosts. These results suggest that p53 can be a new therapeutic target for TB therapy. [ABSTRACT FROM AUTHOR]

Background: Apoptosis is thought to play a role in host defenses against intracellular pathogens, including Mycobacterium tuberculosis (Mtb), by preventing the release of intracellular components and the spread of mycobacterial infection. This study aims to investigate the role of endoplasmic reticulum (ER) stress mediated apoptosis in mycobacteria infected macrophages. Methodology/Principal Findings: Here, we demonstrate that ER stress-induced apoptosis is associated with Mtb H37Rv-induced cell death of Raw264.7 murine macrophages. We have shown that Mtb H37Rv induced apoptosis are involved in activation of caspase-12, which resides on the cytoplasmic district of the ER. Mtb infection increase levels of other ER stress indicators in a time-dependent manner. Phosphorylation of eIF2α was decreased gradually after Mtb H37Rv infection signifying that Mtb H37Rv infection may affect eIF2α phosphorylation in an attempt to survive within macrophages. Interestingly, the survival of mycobacteria in macrophages was enhanced by silencing CHOP expression. In contrast, survival rate of mycobacteria was reduced by phosphorylation of the eIF2α. Futhermore, the levels of ROS, NO or CHOP expression were significantly increased by live Mtb H37Rv compared to heat-killed Mtb H37Rv indicating that live Mtb H37Rv could induce ER stress response. Conclusion/Significance: These findings indicate that eIF2α/CHOP pathway may influence intracellular survival of Mtb H37Rv in macrophages and only live Mtb H37Rv can induce ER stress response. The data support the ER stress pathway plays an important role in the pathogenesis and persistence of mycobacteria. [ABSTRACT FROM AUTHOR]

It has been known that infection plays a role in the development of hypertension. However, the role of hypertension in the progression of infectious diseases remain unknown. Many countries with high rates of hypertension show geographical overlaps with those showing high incidence rates of tuberculosis (TB). To explore the role of hypertension in tuberculosis, we compared the effects of hypertension during mycobacterial infection, we infected both hypertensive Angiotensin II (Ang II) and control mice with Mycobacterium tuberculosis (Mtb) strain H37Ra by intratracheal injection. Ang II-induced hypertension promotes cell death through both apoptosis and necrosis in Mtb H37Ra infected mouse lungs. Interestingly, we found that lipid accumulation in pulmonary tissues was significantly increased in the hypertension group compared to the normal controls. Ang II-induced hypertension increases the formation of foamy macrophages during Mtb infection and it leads to cell death. Moreover, the hypertension group showed more severe granuloma formation and fibrotic lesions in comparison with the control group. Finally, we observed that the total number of mycobacteria was increased in the lungs in the hypertension group compared to the normal controls. Taken together, these results suggest that hypertension increases intracellular survival of Mtb through formation of foamy macrophages, resulting in severe pathogenesis of TB. [ABSTRACT FROM AUTHOR]

Apoptosis is an important host defense mechanism against mycobacterial infection. However, the molecular mechanisms regulating apoptosis during mycobacterial infection are not well known. Recent reports suggest that bacterial infection regulates mitochondrial fusion and fission in various ways. Here, we investigated the role of mitochondria in Mycobacterium tuberculosis (Mtb)-infected macrophages. Mtb H37Rv (Rv) infection induced mitofusin 2 (MFN2) degradation, leading to mitochondrial fission. Interestingly, Mtb H37Ra (Ra) infection induced significantly greater mitochondrial fragmentation than Rv infection. Mtb-mediated Parkin, an E3 ubiquitin ligase, contributed to the degradation of MFN2. To evaluate the role of endoplasmic reticulum stress in the production of Parkin during Mtb infection, we analyzed Parkin production in 4-phenylbutyric acid (4-PBA)-pretreated macrophages. Pretreatment with 4-PBA reduced Parkin production in Mtb-infected macrophages. In contrast, the level of MFN2 production recovered to a level similar to that of the unstimulated control. In addition, Ra-infected macrophages had reduced mitochondrial membrane potential (MMP) compared to those infected with Rv. Interestingly, intracellular survival of mycobacteria was decreased in siMFN2-transfected macrophages; in contrast, overexpression of MFN2 in macrophages increased Mtb growth compared with the control. [ABSTRACT FROM AUTHOR]