Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases. The interplay between these distinct processes and the implications for TB diagnosis and clinical disease staging are poorly understood. In the work performed during this report period we have expanded our studies assessing the interplay between the anti-oxidant enzyme heme oxygenase-1 (HO-1) and matrix metalloproteinsases (MMPs) in human tuberculosis. As introduced in last years report, patients with TB, but not healthy controls or individuals with latent TB infection, expressed either very high levels of HO-1 or of MMP-1, revealing a dichotomy within this clinical group. We have detected this dichotomous expression of HO-1 and MMP-1 in plasma samples from patients with pulmonary TB from South India, Brazil and North America. In addition, the levels of both HO-1 and MMP-1 were dramatically reduced with successful anti-TB chemotherapy but not in patients with treatment failure. The inverse expression of HO-1 and MMP-1 was not observed in plasma after successful treatment. These findings led us to hypothesize that the dichotomous expression of HO-1 and MMPs may be a unique biosignature of active TB disease. We tested this hypothesis by comparing the plasma expression profile of HO-1 and MMP-1 in North American patients with TB or other lung diseases, such as asthma, non-TB mycobacteria, lung cancer, idiopathic pulmonary fibrosis and pulmonary sarcoidosis in a multi-institute collaboration with laboratories from the NHLBI (Drs. Joel Moss, Vincent Manganiello, Joseph Fontana and Stewart Levine), NIAID (Drs. Steven Holland, Kenneth Olivier and Irini Sereti) and Bernadette Gochuico (NHGRI). Strikingly, combined assessment of HO-1 and MMP-1 in plasma distinguished TB patients from those with other lung pathologies with a low rate of misclassification using a canonical correlation approach. In summary, these results argue that combined measurement of HO-1 and MMP-1 offers a strategy for discrimination of active TB from other lung diseases and for clinical staging of TB patients. As described in last years report, we have employed in vitro models using M. tuberculosis infection of human monocyte-differentiated macrophages to better understand the potential cross regulation between HO-1 and MMP-1 at a cellular level. We had found that MMP-1 expression is reduced in conditions in which HO-1 is overexpressed as a result of pharmacological treatment. During this report period, we expanded these experimental studies and discovered that carbon monoxide, a product of HO-1 activity, dramatically reduces MMP-1 expression via inhibition of the transcription regulator AP-1 and to a lesser extent NF-kB. Carbon monoxide induced increased IL-10 and reduced TNF-alpha production in infected macrophages. In addition, we found that pharmacological inhibition of MMP-1 enhanced HO-1 expression by nuclear export of the NRF-2 suppressor Bach1. Together, these findings indicate that cross-regulation of HO-1 and MMP-1 is a likely explanation of the dichotomous expression of these markers in TB patients. In a separate study we examined the use of HO-1, MMPs and other host inflammatory molecules as biomarkers for TB type-2 diabetes mellitus (T2DM) comorbidity. Compared with patients with TB without diabetes, those with coincident T2DM exhibited increased Mycobacterium tuberculosis bacillary loads in sputum. Plasma levels of HO-1 but not of other acute phase proteins were higher in patients with TB and T2DM than in patients without diabetes, independent of bacillary sputum loads. Moreover, patients with coincident TB and T2DM exhibited increased plasma levels of TIMP-4 and elevated peripheral blood neutrophil counts, which, when considered together with HO-1, resulted in increased power to discriminate individuals with TB only from those also suffering from T2DM. These findings reveal elevated plasma levels of HO-1 and TIMP-4 and peripheral blood neutrophil counts as potential single and combined markers of pathogenesis in TB and T2DM. Lysosomal cathepsin B is thought to play a major role in the induction of acute inflammation. Cathepsin B plays several functions inside the cell, regulating exocytosis of cytokines, proteolytic activation of mediators as well as the induction of cell death. Cathepsin B levels are increased in the lungs of TB infected mice and rabbits and in plasma of patients withactive tuberculosis. In a new project, we are studying how cathepsin B might contribute to immunopathology and in initial experiments studied the mechanism by which this molecule is released from lysosomes in Mtb infected bone marrow derived macrophages. We observed MTb induced lysosomal leakage of mature cathepsin B in murine bone marrow derived macrophages. Importantly this was dependent on expression of the MTB secretory protein ESAT-6. Addition of the Cathepsin B inhibitor CA074-Me, resulted in a dramatic reduction in levels of mature IL-1beta in infected macrophages and this was associated with inhibition of the interaction of NLRP3 and ASC as measured by co-immunoprecipitation arguing for a critical role of the enzyme in NLRP3-inflammasome activation. In vivo, Mtb infected mice deficient in cathepsin B (CSTB KO) displayed reduced bacterial CFU burdens in lungs and spleens, reduced IFN-gamma production but increased levels of IL-17 at 32 days post-infection. Consistent with these findings infected C57BL/6 mice treated with the cathepsin B inhibitor, CA074, displayed reduced CFU burdens as well as pulmonary pathology. These preliminary observations suggest that cathepsin B plays major roles in host resistance to MTB as well as in bacterial induced inflammasome activation and IL-1beta maturation.
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