Modulation of protective immunity in human tuberculosis

This thesis presents four studies aimed at elucidating immune responses to Mycobacterium tuberculosis (Mtb) infection, with a focus on the associations with clinical features, such as tuberculosis (TB) disease severity and anemia, and the role of histone deacetylase inhibitors (HDACi) in modulating these responses.

In Study I, we developed a protocol for differentiation and polarization of monocyte-derived macrophages into M1 and M2 cells. Setting up a macrophage infection model with the virulent Mtb-H37Rv strain expressing green fluorescent protein (GFP), allowed assessment of the phenotype and function of Mtb-infected macrophages using flow cytometry. Results from this study indicated that Mtb modulates the polarization of macrophages and is efficiently internalized inside M2 polarized macrophages, which have lower capabilities of clearing the infection effectively. M1 macrophages, known for their robust response to bacterial infections, showed an overall lower Mtb-GFP signal potentially indicating an enhanced capacity for bacterial clearance, although limitations in bacterial containment were also noted in the M1 subset.

In Study II, we investigated how peripheral inflammatory mediator and cytokine levels associated with distinct features in patients with pulmonary TB, including clinical disease severity and anemia. This TB patient cohort represented a sub- group obtained from a randomized clinical trial previously conducted in Ethiopia. Multiplex Magpix data from plasma was examined with machine learning and manual analyses which identified key inflammatory markers that differentiated between severe and mild TB, anemic and non-anemic cases, and patients with extensive and limited lung involvement. These analyses revealed effective ranking of immune markers that separated the immune responses at baseline and after the start of anti-TB treatment in the different patient groups and compared to the healthy controls. Notably, while certain markers correlated with immune protection, other markers decreased with treatment, highlighting their potential as biomarkers for disease progression and response to therapy.

Previous work on the HDACi, phenylbutyrate (PBA), in the presence or absence of vitamin D, have demonstrated enhanced immune functions in Mtb-infected macrophages in vitro and clinical improvement of pulmonary TB upon administration to patients in vivo. However, PBA is a broad-spectrum HDACi with relatively weak HDACi activity in the millimolar-range. Therefore, Study III was designed to test a panel of commercially available HDACi compounds for their efficacy to reduce intracellular Mtb growth in immune cells. For this purpose, we used the macrophage infection model of Study I and IncuCyte live-cell imaging to enable medium-throughput screening of different compounds at different doses in cells obtained from healthy blood donors. The results demonstrated that four different SIRT1 and SIRT2 inhibitors, particularly tenovin, effectively enhanced restriction of Mtb growth in both Mtb-infected macrophages and bulk peripheral blood mononuclear cells (PBMCs). Specific SIRT2 inhibitors emerged as promising compounds useful for boosting intracellular bacterial clearance, although the efficacy of these molecules seemed dependent on the concentration and kinetics used. This study contributes to the growing body of research on HDACi as potential host-directed therapies for TB.

To continue the exploration of immune responses in the clinical trial cohort used in Study II, the work in Study IV was focused on phenotypical and transcriptional profiling of peripheral immune cells in TB patients using bulk RNA-sequencing (seq) on FACS-sorted lymphocytes and myeloid cells in combination with flow cytometry. Bulk RNA-seq data obtained from patients with mild, moderate or severe TB disease as compared to the controls, revealed that several genes where differentially regulated in the CD4+, CD8+, and monocyte cell subsets. Moreover, markers such as CD25, CD38, and HLA-DR were elevated in TB patients, particularly in those with severe disease and anemia, and were linked to disease severity and immune activation. Activated T cells expressing Ox40 as well as CD25+FoxP3+ and CD38+HLA-DR+ T cells were associated with disease severity and of potential relevance in chronic TB. These markers and T cell subsets decreased with effective treatment, suggesting potential use as prognostic markers of treatment response.

Together, these studies highlight significant insights into protective and pathological immune responses involved in TB, the impact of clinical features on immune responses, and the potential of HDAC inhibitors as adjunct host-directed therapies for modulation of TB immunity and to improve Mtb control.

List of scientific papers

I. Mily A, Kalsum S, Loreti MG, Rekha RS, Muvva JR, Lourda M, Brighenti S. Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon M. tuberculosis Infection. Journal of Visualized Experiments, 2020 Sep 18;(163). https://doi.org/10.3791/61807

II. Ashenafi At, Loreti MG+, Bekele A, Aseffa G, Amogne W, Kassa E, Aderaye G, Brighenti S (+ Equal contribution) Inflammatory immune profiles associated with disease severity in pulmonary TB patients with moderate to severe clinical TB or anemia Frontiers in Immunology, 2023 Dec 12 (Vol 14 - 2023). https://doi.org/10.3389/fimmu.2023.1296501

III. Kalsum S, Akber A, Loreti MG, Andersson B, Danielson E, Lerm M, Brighenti Sirtuin inhibitors reduce intracellular growth of M. tuberculosis in human macrophages via modulation of host cell immunity. [Submitted]

IV. Loreti MG, Mily A, Akber M, Ashenafi S, Bekele A, Bekele A, Aseffa G, Amogne W, Kassa E, Aderaye G, Lourda M, Brighenti S Immune profiling of peripheral T cells and myeloid cells in patients with pulmonary TB exhibiting diverse disease severity traits. [Manuscript]