Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases. The relationship between these distinct processes and the implications for TB diagnosis and clinical disease staging are poorly understood. Our previously published studies in this area have focused on the anti-oxidant enzyme heme oxygenase-1 (HO-1 in both human and experimental TB infection. Our collaborative studies in India, Brazil and the NIH clinical center identified HO-1 as a biomarker that distinguishes patients with active TB disease from those with latent or successfully treated Mtb infection. Currently our work on human TB is focused on a collaboration with colleagues at the University of Cape Town (funded in part by a joint US-South Africa UO-1 grant). An important component of this project is to extend our analysis of host inflammatory markers to a larger, better characterized treatment cohort. After an extensive delay because of problems in obtaining approval for our clinical protocol, patient recruitment for the project commenced in March. At the time of this report, 225 healthy controls have been screened of which 89 have been enrolled as either TB- (Quantiferon negative) or TB + (Quantiferon +). In addition, 8 patients with active pulmonary TB have been recruited and enrolled In the meantime as described in last years report and published earlier this year (Rockwood et al, 2017). Bruno Andrade (a former fellow now participating as a collaborator from Brazil) teaming up with Cape Town colleagues was able to utilize archived serum and plasma samples to perform an extensive analysis of over 30 inflammation associated biomarkers in TB and HIV/TBs patients prior to and following anti-TB treatment. Levels of HO-1 were found to decrease following chemotherapy but HO-1 did not on its own predict successful treatment outcome. The data also revealed that HO-1 levels discriminate between HIV/TB patients with low vs high CD4+ T cell counts. These preliminary associations will be re-evaluated in the more extensive UO-1 sponsored post-treatment study which also incorporates cellular immune response studies using cells from different disease tissue sites. HO-1 expression is upregulated in both experimental and human Mycobacterium tuberculosis (Mtb) infection and as noted above is a biomarker of active disease in patients. In experiments described in last years report and published during review period (see Scientific Advance), we showed that administration of tin protoporphyrin IX (SnPPIX), a well-characterized HO-1 enzymatic inhibitor, to mice during acute Mtb infection results in substantial reductions in pulmonary bacterial loads comparable to that achieved following conventional antibiotic therapy. We additionally showed that both the pulmonary induction of HO-1 expression and the efficacy of SnPPIX treatment in reducing bacterial burden are dependent on the presence of host T lymphocytes. Together, these findings revealed mammalian HO-1 as a target for host directed therapy of Mtb infection and identified the immune response as a critical regulator of this function. Our current follow-up experiments suggest that the effects of HO-1 inhibition on Mtb infection may be due to reduction in the generation from heme of free iron needed for intracellular bacterial growth. Consistent with this hypothesis we have shown in a related line of experiments that M. tuberculosis infection induces an increase in serum levels of hepcidin, a peptide hormone that regulates the expression of ferroportin, a protein that exports iron from the cytoplasm to the extracellular compartment. Indeed, this infection induced increase in serum hepcidin was accompanied by a reduction in the expression of ferroportin in pulmonary myeloid cells obtained from infected mice. Thus, the observed hepcidin response by promoting iron accumulation in M. tuberculosis containing cells in the lungs may favor pathogen growth and fitness and be host detrimental. In additional experiments published during the year (Rockwood et al 2017) we explored the pathway responsible for HO-1 induction in Mtb infected macrophages in vitro. We found that this response is dependent on macrophage ROS production induced by the Mtb virulence factor ESAT-6 which then triggers the release of the transcription factor NRF2 from its complex with the protein KEAP1 in the cytosol of infected cells. Free NRF2 then translocates to the nucleus, where it binds to the promoter region of the HO-1 gene inducing its transcription. In related work, we were able to identify a myeloid cell population in the lungs of infected mice that serves as source of the HO-1 in vivo. Finally, a new project was initiated by Eduardo Amaral examining the role of ferroptosis, a form of iron dependent programmed necrosis in experimental TB infection. Using the inhibitor ferrostatin, which blocks the final lipid peroxidation step in ferroptosis, he obtained evidence linking Mtb induced death of infected macrophages both in vitro and in vivo. In infected mice ferrostatin treatment resulted in substantially decreased bacterial loads thus revealing ferroptosis as a potential target for host directed therapy and revealing another host process involving iron which influences resistance to tuberculosis.
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