Inflammatory immune profiles associated with disease severity in pulmonary tuberculosis patients with moderate to severe clinical TB or anemia.

Background: Immune control of Mycobacterium tuberculosis (Mtb) infection is largely influenced by the extensive disease heterogeneity that is typical for tuberculosis (TB). In this study, the peripheral inflammatory immune profile of different sub-groups of pulmonary TB patients was explored based on clinical disease severity, anemia of chronic disease, or the radiological extent of lung disease. Methods: Plasma samples were obtained from n=107 patients with active pulmonary TB at the time of diagnosis and after start of standard chemotherapy. A composite clinical TB symptoms score, blood hemoglobin status and chest X-ray imaging were used to sub-group TB patients into 1.) mild and moderate-severe clinical TB, 2.) anemic and non-anemic TB, or 3.) limited and extensive lung involvement. Plasma levels of biomarkers associated with inflammation pathways were assessed using a Bio-Plex Magpix 37-multiplex assay. In parallel, Th1/Th2 cytokines were quantified with a 27-multiplex in matched plasma and cell culture supernatants from whole blood stimulated with M. tuberculosis-antigens using the QuantiFERON-TB Gold assay. Results: Clinical TB disease severity correlated with low blood hemoglobin levels and anemia but not with radiological findings in this study cohort. Multiplex protein analyses revealed that distinct clusters of inflammation markers and cytokines separated the different TB disease sub-groups with variable efficacy. Several top-ranked markers overlapped, while other markers were unique with regards to their importance to differentiate the TB disease severity groups. A distinct immune response profile defined by elevated levels of BAFF, LIGHT, sTNF-R1 and 2, IP-10, osteopontin, chitinase-3-like protein 1, and IFNα2 and IL-8, were most effective in separating TB patients with different clinical disease severity and were also promising candidates for treatment monitoring. TB patients with mild disease displayed immune polarization towards mixed Th1/Th2 responses, while pro-inflammatory and B cell stimulating cytokines as well as immunomodulatory mediators predominated in moderate-severe TB disease and anemia of TB. Conclusions: Our data demonstrated that clinical disease severity in TB is associated with anemia and distinct inflammatory immune profiles. These results contribute to the understanding of immunopathology in pulmonary TB and define top-ranked inflammatory mediators as biomarkers of disease severity and treatment prognosis.

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection.

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of tuberculosis (TB). We have established an experimental protocol to follow immune polarization of myeloid-derived cells into M1 (classically activated) or M2 (alternatively activated) macrophage-like cells through assessment with a 10-color flow cytometry panel that allows visualization and deep-characterization of green-fluorescent-protein (GFP)-labeled Mtb in diverse macrophages subsets. Monocytes obtained from healthy blood donors were polarized into M1 or M2 cells using differentiation with granulocyte macrophage-colony-stimulating factor (GM-CSF) or macrophage-colony stimulating factor (M-CSF) followed by polarization with IFN-γ and lipopolysaccharide (LPS) or IL-4, respectively. Fully polarized M1 and M2 cells were infected with Mtb-GFP for 4 hours before detached Mtb-infected macrophages were stained with flow cytometry at 4- or 24-hours post-infection. Sample acquisition was performed with flow cytometry and the data was analyzed using a flow cytometry analysis software. Manual gating as well as dimensionality reduction with Uniform Manifold Approximation and Projection (UMAP) and phenograph analysis was performed. This protocol resulted in effective M1/M2 polarization characterized by elevated levels of CD64, CD86, TLR2, HLA-DR and CCR7 on uninfected M1 cells, while uninfected M2 cells exhibited a strong up-regulation of the M2 phenotype markers CD163, CD200R, CD206 and CD80. M1-polarized cells typically contained fewer bacteria compared to M2-polarized cells. Several M1/M2 markers were downregulated after Mtb infection, which suggests that Mtb can modulate macrophage polarization. In addition, 24 different cell clusters of different sizes were found to be uniquely distributed among the M1 and M2 uninfected and Mtb-infected cells at 24-hours post-infection. This M1/M2 flow cytometry protocol could be used as a backbone in Mtb-macrophage research and be adopted for special needs in different areas of research.