Pneumolysin Induces 12-Lipoxygenase-Dependent Neutrophil Migration during Streptococcus pneumoniae Infection

Abstract

Streptococcus pneumoniae is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that S. pneumoniae infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)-dependent lipid inflammatory mediator hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major S. pneumoniae virulence factor that generates ∼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient S. pneumoniae mutant was impaired in triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic Bacillus subtilis with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the α-toxin of Clostridium septicum, which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non-pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX-dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOX-dependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX-deficient mice indicates that 12-LOX-independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indicate that PLY is an important factor in promoting hepoxilin A3-dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion.

Fig. 1.. Pneumolysin is required for PMN transepithelial migration during S. pneumoniae infection.

Apical sides of H292 monolayers on transwells were infected with 1X10⁷ CFU per transwell of wild type, ply-deficient, or complemented S. pneumoniae strains for 2.5 hours. After infection, 1x10⁶ human PMNs were added to the basolateral side post-infection. PMN transepithelial migration was quantified by MPO activity. fMLP and buffer alone were used as positive and negative controls, respectively. For each panel, shown is a representative of two to four independent experiments where each condition was tested in triplicate per experiment. Asterisk(s) indicate migration is significantly (* = p<0.05, **** = p<0.0001) greater than the corresponding S. pneumoniae Δply mutant, as determined by one-way ANOVA, Post-hoc: Tukey.

Fig. 2.. Pneumolysin is sufficient to induce PMN transepithelial migration in a dose dependent manner.

(A) Apical sides of H292 monolayers on transwells were infected with 1x10⁷ wild type B. subtilis or B. subtilis expressing ply. After infection, transepithelial migration of 1x10⁶ human PMNs added to the basolateral side was quantified by MPO assay. fMLP and buffer alone were used as positive and negative controls, respectively. Asterisks indicate migration is significantly (**** = p<0.0001) greater than migration induced by the parental B. subtilis strain, as determined by one-way ANOVA, Post-hoc: Tukey. Shown is a representative of two independent experiments where each condition was tested in triplicate per experiment. (B) Apical sides of H292 monolayers were treated with indicated units of recombinant PLY (PLY). Transepithelial migration of 1x10⁶ PMNs added to the basolateral side post-infection was quantified. fMLP and buffer alone were used as positive and negative controls, respectively. Shown is one representative experiment of four independent experiments where each condition was tested in triplicate per experiment. Asterisks indicate migration is significantly (** = p<0.005, **** = p<0.0001) greater than migration induced by buffer alone, as determined by one-way ANOVA, Post-hoc: Dunnett.

Fig. 3.. Diverse pore-forming agonists induce PMN transmigration.

The apical face of H292 monolayers were treated with the indicated units of intermedilysin (ILY) of S. intermedius, streptolysin O (SLO) of S. pyogenes, perfringolysin O (PFO) of C. perfringens, or alpha toxin of C. septicum. Transepithelial migration of 1 x 10⁶ human PMNs added to the basolateral side was quantified by MPO assay. Buffer alone was used as negative control. Each panel depicts one representative of between two and four independent experiments where each condition was tested in triplicate per experiment. Asterisks indicate migration is significantly (* = p<0.05, ** = p<0.005, *** = p<0.0005, **** = p<0.0001) greater than migration induced by buffer alone, as determined by one-way ANOVA. Post-hoc: Dunnett.

Fig. 4.. PLY requires pore-forming activity to induce PMN migration.

(A) Apical sides of H292 monolayers were treated for one hour with equimolar concentrations of functional PLY or a PLY cholesterol binding site mutant (PLY_CBS). (B) Apical sides of H292 monolayers were treated for 1 hour with equimolar concentrations of PLY, a PLY mutant locked in an early prepore conformation (PLY_EP), or a PLY mutant locked in a late prepore conformation (PLY_LP). 1x10⁶ human PMNs were added to the basolateral side and PMN transepithelial migration was quantified by MPO assay. Buffer alone and fMLP were used as negative and positive controls respectively. Shown is one representative experiment of two independent experiments where each condition was tested in triplicate per experiment. Asterisks indicate migration induced by wild type PLY is significantly greater (* = p<0.05, ** = p<0.005, **** = p<0.0001) than equimolar concentrations of toxoid as determined by (A) Welch’s t-test or (B) one-way ANOVA, Post-hoc: Tukey.

Fig. 5.. 12-LOX is required for PLY induced PMN transmigration.

H292 monolayers were incubated for 3 hours with buffer alone, the 12-LOX inhibitor Ci-Di-Cy, or the 12-LOX inhibitor Baicalein. Following this incubation, apical sides of H292 monolayers were treated for one hour with 2.5 units of PLY. 1x10⁶ human PMNs were added to the basolateral side and PMN transepithelial migration was quantified by MPO assay. Buffer alone and fMLP were used as negative and positive controls respectively. Shown is a representative of six independent experiments where each condition was tested in triplicate per experiment. Treatment with inhibitors reached statistical significance in five out of six independent experiments. Asterisks indicate that PMN migration is significantly greater (**** = p<0.0001) than migration induced by the negative control of buffer without PLY, 2.5 units PLY with Ci-Di-Cy, and 2.5 units PLY with Baicalein as determined by one-way ANOVA, Post-hoc: Tukey.

Fig. 6.. PLY requires pore formation and the 12-LOX pathway to induce robust PMN migration in vivo.

Female WT mice were challenged intratracheally (i.t.) with PLY, PLY_CBS, PLY_EP, or PLY_LP. Female Alox15^−/− mice were challenged i.t. with PLY. Control mice received buffer alone. (A-F) Mice were sacrificed and H&E-stained lung sections were prepared. Sections were examined by light microscopy (at original magnification x 20). Histology images shown are representative data from one of three independent experiments (using three to five mice per condition). (G) BALF samples were collected and the number of PMNs was measured by flow cytometry. The data presented is the average value for each treatment condition across three independent experiments (using three to five mice per condition). # indicates that PMN number in the BALF was significantly greater in WT mice challenged with PLY than in all other groups (# = p<0.05) except Alox15^−/− mice challenged with PLY (p = 0.1012) as determined by one-way ANOVA, Post-hoc: Tukey. Alox15^−/− mice challenged with PLY were not significantly different than WT mice that received buffer alone (p = 0.7135) as determined by one-way ANOVA, Post-hoc: Tukey.