The Helicobacter pylori type IV secretion system promotes IL-8 synthesis in a model of pediatric airway epithelium via p38 MAP kinase

Abstract

Epidemiologic studies have reported an inverse relationship between childhood Helicobacter pylori infection and development of allergic asthma. Because lung epithelium plays an important role in allergic asthma pathogenesis, we hypothesized that H. pylori may directly influence airway epithelial cell innate immune function, particularly in early childhood. To test our hypothesis, we established an in vitro H. pylori infection model using primary tracheobronchial epithelial cell cultures derived from infant, juvenile and adult rhesus monkeys. Airway epithelial cell cultures were infected with wild-type or cag pathogenicity island mutant H. pylori strains, followed by evaluation of IL-8 and IL-6 protein synthesis. We found that H. pylori primarily increased IL-8 synthesis in a MOI and age-dependent fashion, with a greater than 4-fold induction in infant versus adult cultures. H. pylori-induced IL-8 synthesis in infant and juvenile cultures was significantly reduced by cag pathogenicity island mutants, indicating a requirement for the type IV secretion system. Although peptidoglycan recognition of nucleotide binding oligomerization domain-containing protein 1 (NOD1) and NF-kappaB have been implicated as key cytokine signaling molecules for H. pylori infection in gastric epithelium, NOD1 (ML130) or NF-kappaB (JSH-23) inhibitors minimally affected IL-8 synthesis in airway epithelial cell cultures following H. pylori infection. In contrast, inhibition of the p38 MAP kinase pathway (SB203580) resulted in almost complete suppression of H. pylori-induced IL-8 synthesis. Collectively, these results indicate that H. pylori can preferentially elicit IL-8 synthesis in a model of pediatric airway epithelium using the type IV secretion system via p38 MAP kinase.

Fig 1. H. pylori infection can induce IL-8 and IL-6 synthesis in the 16-HBE human bronchial epithelial cell line.

Supernatants from 16-HBE cell cultures were collected following infection with either H. pylori strain PMSS1 or J166. (A and C) Effect of 24 hr incubation with PMSS1 on IL-8 (n = 9) and IL-6 (n = 9) protein concentration. (B and D) Effect of 24 hr incubation with J166 on IL-8 (n = 10) and IL-6 (= 7) protein concentration. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. Error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001

Fig 2. Age-dependent IL-8 synthesis in primary airway epithelial cell cultures following H. pylori infection.

Supernatants from infant, juvenile or adult monkey primary airway epithelial cell cultures were collected following infection with H. pylori strain PMSS1. (A and D) Effect of 24 hr incubation with H. pylori PMSS1 on IL-8 and IL6 protein concentration in infant cultures (n = 10). (B and E) Effect of 24 hr incubation with H. pylori PMSS1 on IL-8 and IL-6 protein concentration in juvenile cultures (n = 3). (C and F) Effect of 24 hr incubation with H. pylori PMSS1 on IL-8 and IL-6 protein concentration in adult cultures (n = 4). Statistical significance was determined using one-way ANOVA followed by Bonferroni’s multiple comparison test. Error bars represent SEM. ** p <0.01, **** p< 0.0001

Fig 3. H. pylori viability is essential for optimal IL-8 synthesis in primary airway epithelial cell cultures.

Supernatants from juvenile primary airway epithelial cell cultures were collected following infection with (A) H. pylori PMSS1 (MOI = 2.5–15) for 24 hr or treatment with (B) 10⁸ cells/ml heat-killed H. pylori for 24 hr. Statistical significance was determined using unpaired t-test *p<0.05. Error bars represent SEM.

Fig 4. Effect of cagPAI mutation on H. pylori-induced proinflammatory cytokines in primary airway epithelial cell cultures.

Primary airway epithelial cell cultures were infected with PMSS1 WT, cagY knockout or cagA knockout strains for 24 hr, followed by collection of supernatant for ELISA. (A and D) Effect of WT or mutant stains on IL-8 and IL-6 protein concentration in cultures of infant epithelial cells (n = 3). (B and E) Effect of WT or mutant stains on IL-8 and IL-6 protein concentration in cultures of juvenile airway epithelial cells (n = 3). (C and F) Effect of WT or mutant strains on IL-8 and IL-6 protein concentration in cultures of adult airway epithelial cells (n = 3). Statistical significance was determined by two-way ANOVA followed by Bonferroni’s multiple comparison test. Error bars represent SEM. **p < 0.01 (G) Western blot analysis for PMSS1 CagA phosphorylation following infection of the 16-HBE cell line. 16-HBE cell cultures were assessed at 0, 1, 6, 12 and 18 hrs post-infection with PMSS1 (MOI = 10). Arrows point to detected anti-phosphotyrosine labelled bands at 150 kDa for 6 and 12 hrs.

Fig 5. Activation of NOD1 promotes IL-8 synthesis in both infant and adult airway epithelial cell cultures.

(A) NOD1 real time PCR analysis for both infant (n = 4) and adult (n = 9) primary cultures. *p<0.05 as determined by unpaired t-test. (B) Fold change of IL-8 protein in infant (n = 5) or adult (n = 3) primary cultures following treatment with iE-DAP. *p<0.05 by two-way ANOVA followed by Bonferroni’s multiple comparison test. (C) IL-8 protein synthesis in infant (n = 8) or adult (n = 9) primary cultures following treatment with iE-DAP only or iE-DAP in the presence of ML130. *p<0.05 by two-way ANOVA followed by Bonferroni’s multiple comparison test, infant iE-DAP vs infant ML130, infant iE-DAP vs adult ML130. Error bars represent SEM.

Fig 6. H. pylori induced IL-8 synthesis is inhibited by the p38 MAP kinase inhibitor SB203580.

Juvenile primary cultures were infected with H. pylori (PMSS1) in the presence of inhibitors for NOD1, NF-kappaB and p38 kinase. Supernatant were harvested after 24hr. (A) H. pylori-induced IL-8 synthesis following treatment with ML130 (n = 6). *p<0.05 (H. pylori vs media). (B) H. pylori-induced IL-8 synthesis following treatment with JSH-23 (n = 4). **p<0.01 (H. pylori vs media). (C) H. pylori-induced IL-8 synthesis following treatment with SB203580 (n = 6). *p<0.05 (H. pylori vs media), ***p<0.001 (H. pylori vs. H. pylori + SB203584). Statistical significance was determined by repeated measures one-way ANOVA.