GPX4 and BACH1 regulate necrotic cell death and host resistance in Mycobacterium tuberculosis infection in vivo
Eduardo P. Amaral1, Sivaranjani Namasivayam1, Kate Aberman1, Diego L. Costa1, Logan Fisher1, Olena Kamenyeva2, Caio Cesar B. Bomfim1, Katrin Mayer-Barber3, Bruno B. Andrade4 and Alan Sher1
1 Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD firstname.lastname@example.org.
2 Research Technology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.
3 Inflammation and Innate Immunity Unit, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health Bethesda, MD.
4 Multinational Organization Network Sponsoring Translational and Epidemiological Research Initiative, José Silveira Foundation, Salvador, Brazil.
Ferroptosis is an iron-mediated form of regulated necrotic cell death characterized by the accumulation of lipid peroxides on biological membranes. We have previously described a major role for ferroptosis in Mtb-induced macrophage necrosis based on its biochemical requirements and its blockade both in vitro and in vivo by a lipid peroxidation inhibitor (ferrostatin-1). To validate these findings and further delineate the mechanism involved we have analyzed Mtb-induced cellular necrosis and host resistance in mice genetically deficient in or overexpressing glutathione peroxidase (Gpx4), an essential regulator of ferroptotic lipid peroxidation. To do so we generated conditional-knockout mice for Gpx4 by using CD45cre and LysMcre systems to target hematopoietic and myeloid lineage cells, respectively. After aerosol low dose Mtb infection these conditional-knockout mice showed both increased lung necrosis and substantially elevated pulmonary and splenic bacterial burdens in lung and spleen. Intriguingly, mice overexpressing Gpx4 were found to display decreased bacterial burdens as well as reduced pulmonary necrosis when infected at high dose, a model that reveals Mtb-induced necrotic pathology. In the opposite direction, genetic ablation of BACH1, a transcription factor known to repress a group of antioxidant genes including Gpx4, increased the levels of intracellular glutathione as well as enhanced the expression of Gpx4 in Mtb-infected macrophage in vitro and in vivo. Consistent with these observations, macrophages deficient in BACH1 were more resistant to both iron-mediated cell death as well as Mtb-induced necrosis. Moreover, Bach1-deficient mice infected at high dose with Mtb displayed a stricking reduction in bacterial loads as well as pulmonary necrosis and lipid peroxidation. This was reflected in the enhanced survival of the infected Bach1-/- animals. Together, these findings provide important genetic evidence supporting and further delineating the role of ferroptosis in host cell death and tissue necrosis in Mtb infection and implicate both Gpx4 and Bach1 as potential targets for host-direct therapy of tuberculosis.