View Abstract

A3849 - Interferon-G Protects Macrophages from Pneumococcus-Induced Necroptosis
Author Block: E. Arafa1, G. A. Wasserman2, A. K. Wooten1, F. T. Coleman3, I. Martin2, M. R. Jones1, L. J. Quinton1, J. P. Mizgerd1; 1Pulmonary Center /Molecular Translational Medicine, Boston University School of Medicine, Boston, MA, United States, 2Pulmonary Center, Boston University School of Medicine, Boston, MA, United States, 3Microbiology, Boston University School of Medicine, Boston, MA, United States.
Rationale: Streptococcus pneumoniae (pneumococcus) is a leading cause of community acquired pneumonia in both children and adults. Virulent pneumococcal isolates cause severe pneumonia by evading immune responses, including diminished NF-κB activation and increased necroptosis in alveolar macrophages (J Infect Dis, PMID:28368460, 2017). However, in mice previously challenged with non-lethal pneumococcal serotypes, heterotypic immunity leads to the development of lung resident memory T (TRM) cells that enhance protection against even the most virulent of pneumococcal isolates (Mucosal Immunol, PMID:28513594, 2017). Here, we examine whether select elements of heterotypic immunity can confer resistance against pneumococcus-induced macrophage necroptosis.
Methods: Macrophage-pneumococcal interactions were investigated using Raw264.7 cells, a mouse myeloid cell line, infected with the highly virulent ATCC 6303 isolate. Cell death was measured using a lactate dehydrogenase assay, whereas cellular oncosis and membrane permeabilization were detected using confocal microscopy, NF-κB activity was quantified using a κB-luciferase reporter cell line, and STAT1 proteins were examined using immunoblot.
Results: Pneumococcus-infected macrophages displayed cellular oncosis and membrane permeabilization, features of programmed cell death. Macrophages pre-treated with Necrostatin-1 showed a significant reduction in membrane permeability, oncosis, and cell death, implicating necroptosis as the cause of cell death. Next, we tested whether recombinant cytokines that were elevated in the air spaces of mice with heterotypic immunity were sufficient to rescue macrophages from pneumococcus-induced necroptosis. Recombinant GM-CSF had inconsistent effects on cell death, cellular oncosis, and membrane permeabilization, and IL-17A had no effect on any of these outcomes. In contrast, IFN-γ pre-treatment caused consistent, strong, and significant reductions in cell death, cellular oncosis, and membrane permeabilization. Since NF-κB activation and necroptosis of macrophages are inversely correlated during pneumococcal infection (J Infect Dis. PMID:28368460, 2017), we tested whether IFN-γ pre-treatment activated the NF-κB signaling pathway, but it did not. Instead, IFN-γ pre-treatment activated STAT1, resulting in increased content and tyrosine-phosphorylation of STAT1 protein in the protected macrophages.
Conclusion: Taken together, our data suggest that heterotypic immunity and CD4+ TRM cells may protect macrophages against pneumococcus-induced necroptosis, and delineate IFN-γ as a promising potential effector mechanism for this protection.