Studies on the role of HIF-1 in the control of infection with Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis (TB), is responsible for most deaths caused by a bacterium globally. Two main reasons for this are that (Bacille Calmette–Guérin) BCG, the only available vaccine against TB, cannot provide a full protection against TB in adults and that antibiotic treatment is hampered by the increase of drug resistances to M. tuberculosis. In addition, it is still not completely understood why some people are more prone to developing active TB after infection with M. tuberculosis, while others remain asymptomatic. As the co-infection with the human immunodeficiency virus or the comorbidity with diabetes mellitus (DM) increases the risk of TB reactivation, this indicates that interactions between host immune factors and M. tuberculosis determine the outcome of infection. The interaction would lead to the development of granulomas composed of macrophages, granulocytes, lymphocytes and fibroblasts. M. tuberculosis control is primarily mediated by macrophages, the main host cell of intracellular M. tuberculosis, that are activated by mycobacteria-specific CD4 TH1 cells.

The studies presented in this thesis analyze molecular factors that may control the response of macrophages and T cells to M. tuberculosis infection. For increased understanding of generated T cell responses upon vaccination, we studied the phenotype of T cells and their localization after mucosal and distal BCG immunization. Studies were evaluated by using M. tuberculosis-infected mice and were further complemented with mechanistical in vitro studies.

Studied molecules in paper I are the hypoxia-inducible factors (HIFs), transcription factors regulating the adaptation to hypoxia of different cell populations, which are negatively regulated by von Hippel-Lindau factor (VHL). One of the transcription factors, HIF-1, has been broadly studied in immune cells. It can mediate metabolic and immunoregulatory responses of immune cells. Therefore, we investigated the role of HIF-1 overexpression in CD4 T cells during infection with M. tuberculosis and found that mice with VHL deficiency in T cells (Vhl cKO) were more susceptible to M. tuberculosis infection. In contrast, mice with HIF-1 deficiency in T cells behave similar to wild type control during M. tuberculosis infection, which suggests that HIF-1 deficiency plays a redundant role in T cells in M. tuberculosis-infected mice. The increased susceptibility of Vhl cKO mice is attributed to a less M. tuberculosis-specific T cells accumulated in the lungs that show an impaired cell proliferation, a perturbed differentiation and an amplified expression of inhibitory receptors. We also observe blunt T cell responses in Vhl cKO mice immunized with BCG. Moreover, VHL plays an essential role in MYC activity, cell activation, growth, expansion and survival of CD4 T cells activation via TCR signaling. The impaired responses of VHL deficient T cell are reversed by HIF-1 deletion, indicating that the increased susceptibility of M. tuberculosis infection and impaired T cell responses in Vhl cKO mice is attributed to HIF-1 stabilization in T cells.

In paper II, we analyzed the localization and differentiation of mycobacteria-specific CD4 and CD8 T cells after subcutaneous (s.c) and intratracheal (i.t.) immunization with BCG and aerosol infection with M. tuberculosis in mice. We find mycobacteria-specific and proliferating CD4 and CD8 T cells accumulating in the lung but not in the lung draining lymph node (dLN) at several time points after infection or immunization. After M. tuberculosis infection CD4 and CD8 T cells that secrete IFN-γ after stimulation with different mycobacterial peptides or PPD (a mycobacterial protein extract) are found in lungs, but not in dLN. Resident memory CD4 and CD8 T cells (TRM) expressing PD-1 that are mycobacteria-specific accumulate in the lung parenchyma after aerosol infection and i.t. -rather than s.c.- immunization. Fingolimod treatment inhibiting recirculation indicates that TRM are generated in the lung parenchyma after i.t. BCG booster. Collectively, our data suggests mycobacteria-specific T cells during mucosal BCG immunization and M. tuberculosis infection may be generated and/ or expanded in the lung.

DM is positively associated with active TB development, but the mechanisms remain unclear. In paper III, we investigated the role of HIF-1 in bone marrow-derived macrophages (BMM) during M. tuberculosis or BCG infection. We observe expression of HIF-1, HIF-1 regulated genes involved in metabolic transcripts and immune molecules are significantly induced in BMM and lung from mice with mycobacteria infection. Deferoxamine (DFO) treatment that mimics hypoxia further enhances HIF-1-regulated responses and restricted bacterial growth both in vitro and in vivo. HIF-1-regulated responses are diminished and the intracellular bacterial load in BMM is increased by the treatment with high glucose levels or methylglyoxal (MGO), a highly reactive dicarbonyl metabolite enriched in DM. In accordance with this, more levels of M. tuberculosis loads, and lower levels of HIF-1-regulated genes were shown in lungs from hyperglycemic Leprdb/db mice infected with M. tuberculosis. The effects of high glucose or MGO on intracellular M. tuberculosis-growth and HIF-1-regulated transcripts in BMM were reverted by addition of DFO. Besides, loss of Hif1a in BMM diminishes expression of HIF-1-regulated inflammatory and metabolic transcripts after mycobacterial infection under normal or high glucose conditions. In summary, our data suggests that HIF-1 could be a potential target for improved control of M. tuberculosis during DM.

In conclusion, we found that: 1. Stabilized HIF-1 in T cells hinders M. tuberculosis infection control by impairing T cell activation; 2. Mycobacteria-specific T cells accumulate in the lung but not in the dLN during M. tuberculosis infection or mucosal BCG immunization; 3. Although HIF-1 in macrophages plays a protective role against M. tuberculosis, its function and levels are reduced under hyperglycemia and carbonyl stress.

List of scientific papers

I. Liu R, Muliadi V, Mou W, Li H, Yuan J, Holmberg J, Chambers BJ, Ullah N, Wurth J, Alzrigat M, Schlisio S. HIF-1 stabilization in T cells hampers the control of Mycobacterium tuberculosis infection. Nature communications. 2022 Sep 5;13(1):1-20.
https://doi.org/10.1038/s41467-022-32639-9

II. Basile JI*, Liu R*, Mou W, Gao Y, Carow B, Rottenberg ME. Mycobacteria-specific T cells are generated in the lung during mucosal BCG immunization or infection with mycobacterium tuberculosis. Frontiers in immunology. 2020 Oct 22;11:566319. * Shared first authors.
https://doi.org/10.3389/fimmu.2020.566319

III. 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.
https://doi.org/10.1128/mbio.01086-22