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A7574 - Mutation of Pneumococcal Phosphodiesterase 2 Skews Macrophage Responses Toward Type I Interferon Expression
Author Block: A. K. Wooten1, F. T. Coleman1, E. I. Arafa1, M. R. Jones1, L. J. Quinton1, S. I. Pelton2, G. Bai3, J. P. Mizgerd1; 1Pulmonary Center, Boston University School of Medicine, Boston, MA, United States, 2Pediatric Infectious Diseases, Boston Medical Center, Boston, MA, United States, 3Immunology and Microbial Disease, Albany Medical College, Albany, NY, United States.
Rationale: Streptococcus pneumoniae can cause disparate innate immune responses within the lungs. Some pneumococci drive macrophages towards necroptosis, and these are more virulent cases of pneumonia (J Infect Dis 216:425, 2017). Pneumococcus produces cyclic di-AMP (cdA), a PAMP that can stimulate type I interferon production, and type I interferons promote necroptosis in some settings. Within pneumococci, cdA is degraded by two enzymes, phosphodiesterase 1 (pde1) and phosphodiesterase 2 (pde2). We hypothesized that mutated pneumococcal phosphodiesterases would alter macrophage responses, resulting in increased type I interferons and necroptosis.
Methods: Pneumococci included clinical isolates of varied virulence properties and genetically engineered D39 isolates in which pde1, pde2, or both were deleted. Cyclic di-AMP levels in bacteria were measured by ELISA. Macrophage responses were studied using RAW264.7 cell cultures and alveolar macrophages lavaged from C57BL/6 mice. Morphology was visualized using confocal microscopy. Cytokine transcripts were quantified using RT-qPCR. IRF content and phosphorylation were assessed using immunoblot.
Results: Across 6 pneumococcal isolates previously studied in mouse lungs, the 3 more virulent isolates displaying more severe lung infection and greater macrophage necroptosis had higher cdA content compared to their less virulent counterparts. Mutations of the phosphodiesterases that degrade cdA led D39 pneumococci to induce increased cellular oncosis (characteristic of necroptosis) in RAW264.7 cells, 4-6 fold the D39 WT values in single mutants and 10-fold more in the double mutant. Interestingly, IFNβ expression in RAW264.7 was increased by phosphodiesterase mutation, but only after stimulation by Δpde2 single mutants, and not from the Δpde1 or Δpde1Δpde2 double mutants. None of the phosphodiesterase mutations influenced TNFα or IL-1β induction, suggesting specificity to the IFNβ pathway and pde2 enzyme. Phosphorylation of IRF3 and IRF7 was observed only in the Δpde2 mutants, suggesting direct activation of these IFN-inducing transcription factors. The selective effects of pde2 (but not other) mutants on IFNβ (but not TNFα or IL-1β) expression were confirmed in alveolar macrophages collected from mice infected in vivo.
Conclusion: Cyclic di-AMP and pneumococcal phosphodiesterases clearly impact macrophage innate immunity and survival, but the biology is more complex than originally envisioned. Phosphodiesterase 2 has unique roles in regulating type I interferon pathways, due to changes in bacterial metabolites other than cdA.