Systemic Disease during Streptococcus pneumoniae Acute Lung Infection Requires 12-Lipoxygenase–Dependent Inflammation

Abstract

Acute pulmonary infection by Streptococcus pneumoniae is characterized by high bacterial numbers in the lung, a robust alveolar influx of polymorphonuclear cells (PMNs) and a risk of systemic spread of the bacterium. We investigated host-mediators of S. pneumoniae-induced PMN migration and the role of inflammation in septicemia following pneumococcal lung infection. Hepoxilin A3 (HXA3) is a PMN chemoattractant and a metabolite of the 12-lipoxygenase (12-LOX) pathway. We observed that S. pneumoniae infection induced the production of 12-lipoxygenase in cultured pulmonary epithelium and in the lungs of infected mice. Inhibition of the 12- LOX pathway prevented pathogen-induced PMN transepithelial migration in vitro and dramatically reduced lung inflammation upon high-dose pulmonary challenge with S. pneumoniae in vivo, thus implicating HXA3 in pneumococcus-induced pulmonary inflammation. PMN basolateral-to-apical transmigration in vitro significantly increased apical-to-basolateral transepithelial migration of bacteria. Mice suppressed in the expression of 12-lipoxygenase exhibited little or no bacteremia and survived an otherwise lethal pulmonary challenge. Our data suggest that pneumococcal pulmonary inflammation is required for high level bacteremia and systemic infection, partly by disrupting lung epithelium through 12-LOX-dependent HXA3 production and subsequent PMN transepithelial migration.

Cytosolic Phospholipase A2α Promotes Pulmonary Inflammation and Systemic Disease during Streptococcus pneumoniae Infection

“…Robust recruitment of PMNs into alveolar spaces is a hallmark of pneumococcal pneumonia, and we previously showed that blocking the 12-LOX pathway abrogated PMN migration response across pulmonary epithelia (27). As arachidonic acid (AA) is the substrate for 12-LOX activity, we sought to determine whether pneumococcal infection leads to AA release from pulmonary epithelial cells.…”

“…Infection with B. subtilis resulted in significantly less release of AA than with the S. pneumoniae strains (2.5-fold lower than with strain TIGR4 and 2-fold lower than with strains D39 and G54; at 5 h postinfection, P Ͻ 0.005, P Ͻ 0.05, P Ͻ 0.05, respectively) and was statistically indistinguishable from treatment with HBSS. Strain TIGR4 (55) was previously used for monolayer and murine infection experiments (27,56), including in investigations that revealed 12-LOX-dependent inflammation, and thus was utilized for the studies described below. cPLA 2 has a high specificity for membrane phospholipids with AA at the sn-2 position, and its activation has been implicated in AA release and inflammation (54,57).…”

“…Arachidonic acid (AA), the precursor of eicosanoids, is acted upon by cyclooxygenases (COX) to produce prostaglandins and thromboxanes or by lipoxygenases (LOX) to produce leukotrienes, lipoxins, and hepoxilins (24)(25)(26). We previously showed that the 12-LOX pathway and its products are important for PMN transepithelial migration during S. pneumoniae infection (27). This observation is consistent with other studies that have shown the 12-LOX pathway to be critical for PMN transepithelial migration across pulmonary epithelia during Pseudomonas aeruginosa infection and across in vitro intestinal epithelia during infection by Salmonella enterica serovar Typhimurium, Shigella flexneri, or enteroaggregative Escherichia coli (28)(29)(30)(31)(32)(33).…”

<em>In vitro</em> Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

“…Several studies have demonstrated that pathogen-infected epithelial layers facilitate neutrophil trans-epithelial migration 3,8,19,[24][25][26][27][28]31,32 . This occurs via a pathogen-specific induction of an epithelial cell-derived neutrophil chemotactic gradient 3,23 .…”

“…Furthermore, several additional strains of P. aeruginosa including clinical isolates from cystic fibrosis patients have been validated as inducers in this assay 25 . Streptococcus pneumoniae and Klebsiella pneumoniae, Gram-positive and Gram-negative bacterial pathogens, respectively, are commonly associated with pneumonia and have been shown to be capable of inducing neutrophil trans-epithelial migration using the in vitro coculture model 26,32 . Thus, this model system offers a robust in vitro approach using human derived cells to explore inflammatory processes instigated by mucosal pathogens.…”