As introduced in last years report we have been investigating the use of heme oxygenase-1 and related molecules produced in response to tissue damage as disease biomarkers in human tuberculosis. M. tuberculosis triggers free radical production in infected macrophages which leads to oxidative burst and lipid peroxidation. Excessive oxidative stress is seen in patients with pulmonary TB and is associated with tissue necrosis and cavitary disease. The host produces a variety of anti-oxidant mediators to circumvent the detrimental effects of oxidative stress. In the lungs, the transcription factor NRF-2 is highly expressed and is thought to be a major regulator of the anti-oxidant responses. One of the most important NRF-2 products is the enzyme heme oxygenase-1 (HO-1) that induces cytoprotection by metabolizing free heme, a highly toxic molecule released as a consequence of tissue damage. As introduced previously and published this year (see Scientific Advance below), we measured the enzyme in plasma samples in Indian TB patients and found that concentrations of HO-1 are increased during pulmonary TB and are highest in those patients with bilateral lung lesions and smear positive M. tuberculosis. Moreover, statistical analysis demonstrated that HO-1 levels can be used to discriminate active from latent TB and importantly that the elevations in plasma HO-1 levels seen in TB patients were completely reversed after successful anti-TB therapy. The association of HO-1 levels and active TB was later confirmed in a second study population in Salvador Brazil by our collaborator Theolis Barbosa Bessa. In work performed during this report period we compared HO-1 levels with those of other systemically expressed products of oxidative stress and tissue damage and asked whether combined measurement of these mediators with HO-1 can increase the accuracy and sensitivity of the HO-1 based assay. We found that plasma concentrations of several metalloproteinases (MMP), such as MMP-1, MMP-8 and MMP-9, are increased in active TB patients compared to those with latent infection. Intriguingly, systemic concentrations of these three MMPs are inversely correlated with HO-1 values, such that patients exhibiting high levels of HO-1 display relatively low levels of MMPs and vice versa. While testing combinations of different MMPs and HO-1 to increase accuracy in detecting active disease in our Indian cohort, we observed that the diagnosis performance achieves maximum levels when values of HO-1 and MMP-1 are considered, rather than each biomarker alone. The intriguing observation of a dichotomy in active TB patients with regard to plasma concentrations of HO-1 and MMPs led us to examine the cellular basis of this phenomenon . To do so we employed in vitro models using M. tuberculosis infection of human monocyte-differentiated macrophages and verified that macrophages upregulate production of HO-1 and preferentially MMP-1 (amongst other MMPs). Moreover, MMP-1 induction is reduced in conditions in which HO-1 is overexpressed as a result of pharmacological treatment. We have also demonstrated that both production of reactive oxygen species (ROS) and viable mycobacteria are required for proper induction of HO-1, but not MMP-1, upon M. tuberculosis infection. These findings argue that in macrophages M. tuberculosis induces HO-1 and MMP-1 expression via distinct signals and pathways that may cross regulate each other. In another study, based on results generated in the mouse model of tuberculosis, we investigated the relationship of IL-1 family members, lipid mediators and interferons in TB patients. In two separate study sites, India and China, respectively, we found that the IL-1 response was dampened with pulmonary TB disease status compared to healthy controls or latently infected individuals. Moreover, profiling lipid mediators, such as prostaglandins and lipoxins in combination with IL-1 and interferons, allowed for faithful discrimination of and stratification of disease severity among active TB patients, suggesting that these pathways are uniquely associated with the spectra of pulmonary pathology observed in TB patients.
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