Spatiotemporal interrogation of interactions of pneumolysin, pulmonary epithelium, and PMNs that result in age-associated susceptibility to Streptococcus pneumoniae disease

/ABSTRACT Streptococcus pneumoniae (Sp) is the most common cause of community-acquired pneumonia and the elderly are at increased risk of disseminated infection. A hallmark of Sp infection is robust polymorphonuclear leucocyte (PMN/neutrophil) influx, driven by upregulation of the 12/15-lipoxygenase (12/15-LOX) pathway, which produces the chemoattractant hepoxilin A3 (HXA3). HXA3 also triggers tissue-destructive neutrophil elastase (NE) release by PMNs, and NE inhibition diminishes Sp bacteremia. Thus, while PMNs are essential for Sp infection control, excessive PMN influx also drives tissue damage. Age-associated increases in inflammation and decreases in apical junctional complex (AJC) components critical for barrier integrity contribute to susceptibility of the elderly to Sp disease; reduction of Sp bacteremia levels in aged mice to that of young mice requires both PMN NE inhibition and epithelial barrier fortification. Notably, the Sp virulence factor pneumolysin (PLY) drives both direct AJC disruption and 12/15-LOX expression. PLY- and age-mediated AJC weakening may also diminish PMN function, as PMN mechanosensing during transendothelial migration enhances their killing activity. The level of PLY production is a key determinant to inflammation and barrier disruption, but is heterogeneous. Thus, with PLY and PMNs as marked examples, a detailed understanding of Sp lung infection pathogenesis requires investigation of the relationship between anatomic location, tissue microenvironment, and heterogeneity of microbial and host cell phenotypes. In contrast, studies of Sp pulmonary infection to date have examined only global readouts of the behavior of populations of bacteria, epithelial cells, and PMNs. We posit that during Sp lung infection, ply expression results in localized AJC disruption and epithelial 12/15-LOX expression, driving focal PMN migration and epithelial damage. This is exacerbated by intrinsic diminished AJC integrity and enhanced PMN NE secretion in aged hosts. To test this model, Aim 1 utilizes confocal microscopy analysis of Sp infection (wild type, ∆ply) of young and aged mice to quantify Sp ply expression at the single bacterium level in situ, to elucidate the spatiotemporal relationships of PLY-producing Sp, 12/15-LOX expression, and PMN recruitment. We will also identify the cells that express 12/15-LOX in response to Sp and determine their proximity to transmigrating PMNs, and the effect of aging on these parameters. In Aim 2, analysis of intravascular and transmigrated airway PMNs by flow cytometry or thick tissue 3D confocal imaging of optically cleared tissue will be used to determine how local PLY- and age-mediated weakening of epithelial AJCs may alter PMN phenotype and bactericidal activity. Together, these studies will establish both a new framework understanding of how Sp-host epithelial interaction heterogeneity affects local PMN response and the effect of aging on these interactions, as well as tractable methods to explore mechanistic details of this framework in situ. Project Number: 1R21AI193724-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: JOHN LEONG (+1 co-PI) | Institution: TUFTS UNIVERSITY BOSTON, BOSTON, MA | Award Amount: $450,630 | Activity Code: R21 | Study Section: Bacterial-Host Interactions Study Section [BHI] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19372401