Mechanisms of the alternative macrophage activation in TB - Pulmonary tuberculosis (TB) remains a leading cause of death from a single infectious agent, Mycobacterium tuberculosis (Mtb). Mtb is a specialized human pathogen whose evolutionary success is dependent on the ability to cause extensive lung damage that enables the pathogen transmission via aerosols. Lung vulnerability to TB represents a major obstacle for the development of efficacious vaccines and therapies. However, mechanisms of the particular lung vulnerability to TB remain unknown. We have developed a genetic mouse model of pulmonary TB, where the human-like organized necrotic granulomas develop in immunocompetent inbred mouse strain B6.Sst1S after aerosol, intravenous or subcutaneous infection. The necrotic lesion development is associated with the accumulation of Arginase 1 (Arg1)-positive myeloid cells within the pulmonary TB lesions. Previous studies also described the appearance of Arg1+ cells in TB lesions of susceptible mice, NHP and human and predicted their roles in TB progression. We propose to use the B6.Sst1S mouse model of chronic pulmonary TB to study the phenotypes and genesis of the Arg1+ myeloid cells in pulmonary TB. Our preliminary data demonstrate that iNOS+ and Arg1+ macrophages in pulmonary TB lesions represent non-overlapping populations and that the coordinated expression of Arg1 and other M2 polarization markers increases in the peripheral blood and bone marrow myeloid cells in parallel with pulmonary TB progression. We hypothesize that the lung damage-associated distant signals induce the M2-committed myeloid progenitors that upregulate M2 polarization markers in bone marrow, prior to their recruitment to the lung lesions, and subsequently become unresponsive to M1-polarizing signals. We propose to address the following questions: What specific cell populations in peripheral blood express Arg1+ mRNA at various stages of TB? Do these cells co-express specific surface markers and receptors? Would depletion of these cells in the blood prevent their accumulation in pulmonary TB lesions? Our specific aims are: Aim 1: To define populations of Arg1+ cells in peripheral blood during TB progression. We propose to characterize these populations using single cell RNAseq and multiplexed spectral flow cytometry, and to develop a multiplexed gene expression panel for monitoring myeloid cell polarization by sampling peripheral blood in longitudinal studies. Aim 2. To determine the roles of IL-4 in driving the Arg1+ cell genesis during chronic TB. We propose to treat Mtb-infected mice with IL-4 neutralizing antibodies and monitor the M2 polarization in peripheral blood using the macrophage gene expression panel and detect their presence and phenotypes in pulmonary TB lesions using multiplexed immunochemistry panels. We anticipate that the proposed studies will identify unique biomarkers of the M2 polarized myeloid cells in peripheral blood associated with TB and allow us to enrich them for functional studies in vitro and to deplete these cells in vivo, and, thus, establish their roles in pulmonary TB.
