Regulation Of Immunopathology In Bacterial And Parasitic Disease

National Institute Of Allergy And Infectious Diseases

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Abstract

Necrotic cell death during Mycobacterium tuberculosis (Mtb) infection is considered host detrimental since it facilitates mycobacterial spread. As introduced in previous reports, we have obtained evidence for the role in tuberculosis of ferroptosis, a type of iron-mediated necrosis characterized by the accumulation of toxic lipid peroxides on biological membranes. Our in vitro and in vivo findings implicated ferroptosis as a potential target for host directed therapy of Mtb infection and disease. In subsequent work we sought genetic confirmation of the role of the ferroptotic pathway in Mtb induced cell death and necrosis. In these experiments Dr. Amaral targeted GPX4, the major anti-oxidant enzyme protecting the host against ferroptotic lipid peroxidation in steady state. He did so by generating different conditional-knockout mice for Gpx4 that delete expression of the enzyme in all cells, hemapoetic cells only and myeloid cells only. By infecting these conditional-knockout mice, Dr. Amaral observed that absence or low expression of Gpx4 is detrimental to host defense against Mtb in all of the KO animal models as indicated by worsening disease correlates such as lung tissue necrosis and high bacterial burden in the lung and spleens and lowered survival rates. Moreover, he found that Gpx4 overexpression augmented mouse resistance in high dose Mtb-infected mice. Further work on this project was interrupted by the pandemic as we had to stop breeding the above animals. Nevertheless, in 2021 when we were able to resume our research Dr Amaral was able to perform In vitro experiments revealing that Gpx4-deficient macrophages obtained from the different conditional knockout mice display elevated susceptibility to Mtb infection as evidenced by enhanced necrosis induction as well as increased intracellular bacterial loads. Importantly, the Mtb-induced necrotic cell death seen in the Gpx4-deficient cultures was suppressed by treating cells with the lipid peroxidation inhibitor ferostatin-1. Together these findings presented in a recently accepted publication in the Journal of Experimental Medicine, provide major genetic support for the importance of ferroptosis in promoting Mtb infection and disease as well as its role as a necrotic cell death mechanism in TB infected macrophages. In related studies introduced last year, Dr. Amaral examined the importance of glutathione metabolism to host defense against Mtb infection as well as the impact of this pathway on Mtb-induced cell death and tissue damage. The transcription factor Bach1 represses the expression of genes encoding antioxidant proteins, mainly through its blockade of Nrf2-DNA binding. Dr. Amaral observed that macrophages deficient in Bach1 exhibit upregulated Gpx4 expression as well as increased levels of glutathione, a cofactor required for Gpx4 enzyme activity. Importantly he found that these Bach1-deficient macrophages are more resistant to Mtb-induced necrosis in vitro. Moreover, when infected at high dose with Mtb Bach1 deficient mice displayed a significant reduction in bacterial burden as well as a pronounced decrease in lung necrosis and lipid peroxide levels relative to control WT animals. This decreased disease was reflected in enhanced survival of the infected Bach1 deficient animals. To further assess the association of the Bach1/Gpx4 axis and lipid peroxidation with necrotic pathology, we utilized a well-established B6Sst1s murine model which generates human-like hypernecrotic granulomatous pathology in response to Mtb. To do so we aerosol infected B6Sst1s and B6Sst1sBach1-/- mice and compared their lung histology and bacterial loads 35 days later. Interestingly, B6Sst1s mice deficient in Bach1 displayed enhanced protection against Mtb infection as evidenced by a striking reduction in lung tissue destruction and bacterial burden compared to Mtb-infected Bach sufficient B6Sst1s mice. The reduction in pathology seen in the infected B6Sst1sBach1-/- mice was reflected in their enhanced survival. Supporting the relevance of these findings to human TB, our clinical collaborators observed elevated Bach 1 gene expression in peripheral blood and lung tissue in patients with more severe active TB. These new observations reveal Bach1 as a potential host-directed therapeutic target for preventing the exacerbated necrotic tissue damage and bacterial dissemination seen in active TB. Based on our experience studying the role of oxidative stress and its regulation in infectious disease, as a pandemic irelated project we entered into a collaboration with Dr. Irini Sereti and her colleagues studying the role of this pathway in the systemic hyperinflammation seen in patients suffering from COVID 19. Although inflammasome-derived cytokines and oxidative stress have independently been found to play a role in COVID-19, it has been unclear whether these two important host responses cooperatively contribute to COVID-19 severity. We observed a striking enrichment of CD14highCD16- monocytes displaying inflammasome activation evidenced by caspase-1/ASC-speck formation in COVID-19 patients compared to healthy controls (HCs). Interestingly, CD14highCD16- monocytes from COVID-19 patients also showed aberrant levels of mitochondrial superoxide (MitoSOX) and lipid peroxidation, both hallmarks of the oxidative stress response. MitoSOX and lipid peroxidation strongly correlated with caspase-1 activity in CD14highCD16- monocytes from COVID-19 patients, which was more pronounced in patients displaying severe forms of the disease. Moreover, lower levels of the potent antioxidant glutathione were found in PBMC lysates from COVID-19 patients compared to HCs. We further found that IL-1 beta secretion by SARS-CoV-2-exposed monocytes in vitro depends on both NLRP3 inflammasome activation and lipid peroxidation. Importantly, elevated levels of inflammasome and oxidative stress responses were observed even in mild disease and persisted after short-term patient recovery. Together these findings which were published this year (Lage et al. Frontiers in Immunology) suggest this myeloid-oxidative stress/NLRP3 signaling pathway as a potential target for host-directed therapies aimed at mitigating early COVID-19 hyperinflammation as well as its long-term outcomes. Previous studies from our group in both animal models and patients have characterized the host anti-oxidant enzyme heme oxygenase-1 (HO-1) as an important biomarker of active tuberculosis and work by a former postdoc Diego Costa showed that administration of tin protoporphyrin IX (SnPPIX), a well-characterized HO-1 enzymatic inhibitor, to mice during acute Mtb infection induces substantial reductions in pulmonary bacterial loads and when combined with conventional TB antibiotics results in accelerated clearance of infection. These results led us to explore SnPPIX and related HO-1 inhibitors as potential host directed therapies (HDT) for TB. In work performed in 2021 in collaboration with both the TB Drug Alliance and Dr. Costa (now working in his own lab in Brazil), we confirmed the anti-Mtb activity of Snppx, studied its pharmokinetics and compared its efficacy with that of Stannsoporfin, a clinical grade tin based HO-1 inhibitor manufactured by Mallinkrodt Pharmaceuticals for treatment of infant jaundice. This drug was found to have similar if not greater activity than SnPPIX supporting its potential use as a host directed therapy, Current studies performed in collaboration with the TB Alliance and Petros C. Karakousis (Johns Hopkins) are evaluating the efficacy of Stannsoporfin to enhance bacterial control and prevent relapse when used adjunctively with TB antibiotics.