Necrotic cell death during Mycobacterium tuberculosis (Mtb) infection is considered host detrimental since it facilitates mycobacterial spread. As introduced in previous reports we have been examining the role in tuberculosis of ferroptosis, a type of necrosis induced by the accumulation of free iron and toxic lipid peroxides. We observed that Mtb-induced macrophage necrosis is associated with reduced levels of glutathione and glutathione peroxidase-4 (Gpx4) along with increased free iron, mitochondrial superoxide and lipid peroxidation, all of which are important hallmarks of ferroptosis. Moreover, necrotic cell death in Mtb-infected macrophage cultures was suppressed by both iron chelation and the addition of ferrostatin-1 (Fer-1), a major ferroptosis inhibitor. Additional in vivo experiments revealed that pulmonary necrosis in acutely infected mice is associated with reduced glutathione and Gpx4 as well as increased lipid peroxidation and is likewise inhibited by Fer-1 treatment. Importantly, Fer-1-treated infected animals also exhibited marked 1 log or greater reductions in bacterial load. Together, these findings which were published this year (Amaral et al, 2019) implicated ferroptosis as a major mechanism of necrosis in Mtb infection and as a potential target for host directed therapy in tuberculosis. We pursued additional studies later in the year to obtain genetic confirmation of the role of the ferroptotic pathway in Mob induced cell death and necrosis. In these experiments Dr. Amaral targeted GPX4 the major enzyme protecting the host against ferroptotic lipid peroxidation in steady state. To do so he infected two different genetically engineered mice in which gpx4 can be conditionally deleted either in the whole animal or specifically in the myeloid (LysM) lineage. In both cases, the mice were more susceptible to infection consistent with a role for GPX4 in limiting necrosis induced spread of the pathogen. In a second approach he tested mice engineered to overexpress GPX4. These animals were more resistant to infection, showed less pulmonary necrosis and in vitro experiments confirmed that their macrophages underwent less Mtb induced necrotic cell death. These ongoing experiments provide important genetic support for the importance of ferroptosis in promoting Mtb infection and disease in the murine animal model. In additional work, we sought evidence for a role of ferroptosis in human tuberculosis. We found (Amaral et al, 2019) that MTb induces necrosis of human monocyte derived macrophages and that this cell death response is prevented by treatment of the cultures with the lipid peroxidation inhibitor Fer1. In addition, with our colleague Bruno Andrade we have initiated an analysis of ferroptosis biomarkers in pulmonary TB patients from Brazil. We found that patients with active disease display increased plasma lipid peroxidation products and decreased PBMC glutathione levels than endemic controls. In addition patients with more severe disease (bilateral vs unilateral lung lesions ; cavitation vs no cavitation) and higher sputum bacteria levels showed significantly lowered blood monocyte gpx4 mRNA levels. These early findings are consistent with a role for ferroptosis in TB disease although they do not directly establish a link with Mtb induced tissue necrosis. Previous studies from our group in both animal models and patients have characterize the host anti-oxidant enzyme heme oxygenase-1 (HO-1) as an important biomarker of active tuberculosis. Amongst its other activities, this enzyme releases free iron from heme thus helping supply Mtb with the iron that it depends on for its growth. Support for this pro-bacterial function of HO-1 came from work from our lab showing 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. Moreover, SnPPIX administration when combined with conventional TB antibiotics resulted in accelerated clearance of infection. These results have led us to explore SnPPIX as a potential host directed therapy (HDT) for TB. However, a major hurdle has been the daily intraperitoneal (IP) injection regimen required for drug efficacy in our murine model which would be unacceptable for use in humans. Dr. Olu Adeleke, a visiting pharmaceutical scientist from South Africa, has developed a potential solution to this problem. In her project she incorporates SnPPIX in a thermoresponsive, biocompatible/biodegradable copolymeric matrix which upon intramuscular injection forms a gel-like depot for extended release of the drug. Using this delivery system she has been able to achieve efficacy (as measured by bacterial load reduction) equivalent to that obtained with daily IP injection but with dosing intervals as long as a fortnight and with no histopathologic evidence of toxicity. Parallel pharmacokinetic measurements confirmed the effects of this formulation in achieving slow release of the drug. These encouraging findings now allow us to reconsider the possible clinical testing of this SnPPX based experimental HDT.
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