Immunopathogenesis in pulmonary tuberculosis : impact of immunomodulation and diabetes co-morbidity

Abstract: Even in the 21st century, tuberculosis (TB) remains a major global health threat, primarily due to the emergence of antibiotic resistance. Presence of co-morbidities such as diabetes mellitus (DM) has worsened the current situation and made it more difficult to treat this deadly disease, especially in resource-poor settings. It is well-known that Mtb (Mycobacterium tuberculosis) bacilli can manipulate both innate and adaptive arms of the human immune system, but how Mtb evade host antimicrobial mechanism is not fully understood. Therefore, a deeper understanding of the immunomodulation caused by Mtb, with and without co-existing illnesses, is essential to develop more effective treatment strategies. The work in this thesis was intended to uncover Mtb-mediated immune alterations, particularly in TB-DM disease, and to examine the feasibility of novel host-directed therapy (HDT). In Paper I, we set out to study the efficacy of HDT using phenylbutyrate (PBA) and vitamin D (vitD) to strengthen host immune defenses upon administration to pulmonary TB patients. In a randomized controlled trial conducted in Bangladesh, we previously reported positive effects on clinical as well as microbiological TB outcomes upon daily PBA and vitD treatment together with standard chemotherapy for 8 weeks. Stored samples obtained from the clinical trial subjects were now used to assess secondary outcomes including cytokine/chemokine secretion by peripheral blood mononuclear cell (PBMC) cultures (Luminex assay), endoplasmic reticulum (ER) stress markers expressed in monocyte-derived-macrophages (MDMs) (quantitative real-time PCR), and activation of LC3-dependent autophagy in Mtb-infected MDMs (confocal microscopy). We observed a marked reduction in the concentration of inflammatory mediators including tumor necrosis factor (TNF)-α, CC motif chemokine ligand (CCL)-11 and CCL5 after 8 weeks of PBA treatment compared to the placebo group. Similarly, vitD treatment effectively reduced CCL11, C-X-C motif chemokine ligand (CXCL)-10 and PDGF concentrations after 8 weeks of treatment. Both PBA- and vitD-treatment contributed to reduced mRNA levels of the ER stress marker, x-box binding protein1spliced (XBP1sp)-l. Autophagy was enhanced in MDMs obtained from all intervention groups after 8 weeks of treatment as compared to placebo. These findings suggested that the improvement of primary outcomes observed in the clinical trial, were associated with reduced inflammation and ER stress and instead enhanced autophagy in Mtb-infected patient cells. In paper II, we aimed to explore DM-associated immune alterations of clinical, radiological, and immunological outcomes in TB disease using TB and TB-DM study cohorts collected in Bangladesh. Clinical samples from peripheral blood and sputum from patients and controls were analyzed (blood chemistry, Luminex, quantitative real-time PCR) along with clinical data (composite clinical TB score and demographics) and chest radiography (chest X-ray score) before and after 1, 2 and 6 months of standard anti-TB treatment. TB-DM patients were significantly older, had higher body mass index (BMI), were less anemic and from a better socio-economic background compared to TB patients. Intriguingly, clinical TB symptoms and time to bacterial clearance in sputum were similar comparing TB and TB-DM patients. Even so, TB-DM patients had poorly managed glycemic control throughout the study period and glycemic status was positively associated with BMI. Importantly, the TB-DM cohort showed reduced resolution of inflammation in the middle and lower lung zones compared with TB patients, which was correlated to plasma leptin concentrations at all time points. These changes were associated with upregulated mRNA expression of inflammatory TNF-α and IL-1β in PBMCs as well as higher CD8 mRNA levels but downregulated CD4 and IL-10 transcripts in sputum cells after standard treatment in TB-DM compared to TB patients. Additionally, glycemic status in TB-DM patients was inversely correlated to sputum IL-10 transcript levels observed after start of anti-TB treatment. These results indicate that TB-DM disease is characterized by low-grade inflammation that persist even after completion of successful anti-TB chemotherapy. In Paper III, we developed a protocol for assessment of M1/M2 polarization of human myeloid-derived cells using 10-color flow cytometry of adherent macrophages infected with green fluorescent protein (GFP)-expressing Mtb. The experimental protocol involved in vitro polarization of MDMs into classically activated (M1) or alternatively activated (M2) macrophages and assessment of phenotype and function before, and 4 to 24 hours after Mtb infection. M1 or M2 cells were successfully differentiated with granulocyte monocyte colony stimulating factor (GM-CSF) or monocyte colony stimulating factor (M-CSF), followed by polarization with interferon (IFN)-γ and lipopolysaccharide (LPS), or interleukin (IL)-4, respectively. This protocol allowed us to polarize and define M1 cells by elevated levels of CD64 and CD86 co-expression, while M2 cells were characterized by a high CD163 and CD200R co-expression. The level of Mtb infection was generally higher in M2 as compared to M1 cells, although the relative increase in infected cells from 4 to 24 hours was higher in M1- compared with M2-polarized cells. Manual gating as well as unsupervised analysis using dimensionality reduction with Uniform Manifold Approximation and Projection (UMAP) and phenograph clustering, showed that Mtb infection altered the expression of M1 and M2 markers after 24 hours and generated clearly separated cell clusters of different sizes. This M1/M2 flow cytometry protocol could be used as a backbone in Mtb-macrophage research and be adopted for special needs including assessment of cells cultured in vitro or obtained ex vivo from clinical patient samples.

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