Immune mechanisms controlling tuberculosis-diabetes co-morbidity
Authors: Hanxiong Li 
Published: November 2024

<p dir="ltr">Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), remains a leading global health concern, responsible for millions of infections annually. Despite extensive scientific efforts, the biological and immunological mechanisms that predispose certain individuals to develop active TB, rather than containing the infection in a latent state, are not fully understood. Notably, the incidence of active TB is significantly higher among individuals with diabetes mellitus (DM), which suggests a profound interaction between metabolic disorders and immune responses in the progression of M. tuberculosis infection.</p><p dir="ltr">The immune defense against M. tuberculosis is mainly mediated by macrophages, a major target of infection. Macrophages are activated by CD4+ Th1 cells, which respond to mycobacterial antigens by secreting cytokines such as interferon- gamma (IFN-y), thereby enhancing the macrophage ability to contain or kill the intracellular bacteria.</p><p dir="ltr">This thesis presents an in-depth study of the molecular factors that modulate the immune responses of macrophages and T cells to M. tuberculosis infection. Through a series of controlled experiments using M. tuberculosis-infected in vitro and in vivo models, we investigate how these cells orchestrate a defense against the bacterium and how their efficacy is compromised under diabetic conditions.</p><p dir="ltr">In DM, the high glucose levels cause alterations in the carbohydrate metabolism. These result in elevated levels of methylglyoxal (MGO), a reactive compound that is a byproduct of glycolysis. MGO glycates various cellular macromolecules altering their function, in turn accounting for DM pathogenesis. In paper I, we found that MGO irreversibly binds to thioredoxin reductase 1 (TXNRD1), a mammalian selenoprotein, transforming it into a NADPH oxidase. This modification is crucial for the activation of NRF2, a typically cytoprotective transcription factor. We showed that NRF2 activation suppressed the secretion of IL-1B and nitric oxide (NO). Deficiency of the secretion of these molecules accounts for the enhanced intracellular proliferation of M. tuberculosis. The overexpression of NRF2 by genetic deficiency of its inhibitor KEAP1, was also sufficient to reduce the inflammatory responses in bone marrow-derived macrophages (BMMs) and promoted the intracellular growth of M. tuberculosis. Using various specific inhibitors of TXNRD1, including the FDA-approved auranofin, we replicated the effects of MGO on M. tuberculosis-infected BMMs. Treatment of M. tuberculosis- infected mice with MGO and auranofin increased the expression of antioxidant genes, hampered the production of protective molecules by macrophages and increased the bacterial levels in lungs. Altogether we propose that alterations of carbohydrate metabolism in DM account for the increased risk for TB in DM patients by targeting a TXNRD1-NRF2 redox pathway.</p><p dir="ltr">In paper II, we extend the exploration of host defense against M. tuberculosis under hyperglycemic conditions and attempt to define a role for hypoxia- inducible factor-1 (HIF-1), a key regulator of responses to hypoxia. We found that M. tuberculosis infection significantly stabilized the expression of HIF-la protein and the downstream responses of HIF-1 in BMMs and mice, enhancing immune and metabolic functions. Treatment with deferoxamine (DFO), a hypoxia mimetic, further amplified these HIF-1-dependent responses, reducing bacterial loads both in vitro and in vivo. Conversely, exposure to high glucose or MGO attenuated these responses and increased M. tuberculosis burden in BMMs. Similar trends were observed in hyperglycemic Leprdb/db mice, which exhibited higher bacterial loads and reduced expression of HIF-1-targeted genes in the lungs compared to controls. Importantly, the negative effects of high glucose and MGO were reversible with DFO treatment. Additionally, BMMs lacking Hif1a showed diminished protective immune and glycolytic responses after mycobacterial infection under both high glucose treatment and normal conditions. Our findings suggest targeting HIF-1 as a novel strategy to mitigate TB progression in diabetic patients.</p><p dir="ltr">In paper III, we investigated the role of HIF-1 in T cells during M. tuberculosis infection. We found that mice with HIF-1 stabilization in T cells, due to genetic deficiency of its inhibitor VHL (Vhl cKO), showed a higher susceptibility to M. tuberculosis, with increased bacterial loads and exacerbated lung pathology. The heightened vulnerability was associated with fewer M. tuberculosis-specific T cells in the lungs, which exhibited diminished proliferation, altered transcriptional profiles, and increased expression of inhibitory receptors. By contrast, mice with HIF-1 deficiency in T cells exhibited similar responses to those of wild-type controls during M. tuberculosis infection. In addition to these findings, the study revealed that Vhl cKO mice presented blunted T-cell responses following immunization with the BCG, the only available vaccine against TB, suggesting a broader defect in T-cell functionality beyond natural infection scenarios. Moreover, our results underscore the critical role of VHL in regulating MYC activity, which is pivotal for T-cell activation, growth, expansion, and survival upon TCR signaling. The absence of VHL led to significant alterations in these processes, highlighting the intricate link between cellular oxygen-sensing mechanisms and T- cell-mediated immunity.</p><p dir="ltr">In conclusion, we found that:</p><p dir="ltr">1. MGO binds and inhibits TXNRD1 reductase and converts it to a NADPH- oxidase that mediates NRF2 activation, impairing the macrophage control of M. tuberculosis.</p><p dir="ltr">2. While HIF-1 is protective in macrophages against M. tuberculosis, its function and expression are diminished under conditions of hyperglycemia and carbonyl stress.</p><p dir="ltr">3. Stabilization of HIF-1 in T cells disrupts the control of M. tuberculosis infection in mice by impairing T cell activation.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>Hanxiong Li</b>, Ruining Liu*, Gokul Raj Kathamuthu*, Radosveta Gencheva, Axel Tobias Scholz, Mohammad Alzrigat, Lucia Coppo, Elias S.J. Arnér, Martin E. Rottenberg. The inhibition of TXNRD1 by methylglyoxal impairs the intracellular control of Mycobacterium tuberculosis. [Manuscript]</p><p dir="ltr">II. Terán G*, Li H*, Catrina SB, Liu R, Brighenti S, Zheng X, Grünler J, Nylén S, Carow B, Rottenberg ME. High Glucose and Carbonyl Stress Impair HIF-1-Regulated Responses and the Control of Mycobacterium tuberculosis in Macrophages. Mbio. 2022 Sep 19:e01086-22. <a href="https://doi.org/10.1128/mbio.01086-22" rel="noreferrer" target="_blank">https://doi.org/10.1128/mbio.01086-22</a></p><p dir="ltr">III. Liu R, Muliadi V, Mou W, Li H, Yuan J, Holmberg J, Chambers BJ, Ullah N, Wurth J, Alzrigat M, Schlisio S, Carow B, Larsson LG, Rottenberg ME. HIF-1 stabilization in T cells hampers the control of Mycobacterium tuberculosis infection. Nature communications. 2022 Sep 5;13(1):1-20. <a href="https://doi.org/10.1038/s41467-022-32639-9" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41467-022-32639-9</a></p><p dir="ltr">* Equal contribution</p>