Impact of cagPAI and T4SS on the inflammatory response of human neutrophils to Helicobacter pylori infection

Abstract

Helicobacter pylori contains a pathogenicity island, cagPAI, with genes homologous to components of the type IV secretion system (T4SS) of Agrobacterium tumefaciens. The T4SS components assemble a structure that transfers CagA protein and peptidoglycan into host epithelial cells, causing the increased release of interleukin 8 (IL8) from the cells. The Toll-like receptors on neutrophils recognize H. pylori, initiating signaling pathways that enhance the activation of NF-κB. However, the roles of cagPAI and T4SS in the inflammatory response of neutrophils are unknown. We evaluated the participation of cagPAI and T4SS in the response of human neutrophils to H. pylori infection. Neutrophils were isolated from the blood of healthy donors and infected with H. pylori cagPAI(+), cagPAI(-), and cagPAI mutant strains virB4 (-) and virD4 (-). Whereas cagPAI(+) strain 26695 induced the greatest IL8 production, a proinflammatory response, cagPAI(-) strain 8822 induced the greatest IL10 production, an anti-inflammatory response. In contrast, the virB4 (-) and virD4 (-) mutant strains produced significantly more of the two proinflammatory cytokines IL1β and tumor necrosis factor αthan the cagPAI(+) strain 26695. We observed that H. pylori downregulated the expression of TLRs 2 and 5 but upregulated TLR9 expression in a cagPAI and T4SS-independent manner. These results show for the first time that the response of human neutrophils to H. pylori may vary from a pro-inflammatory to an anti-inflammatory response, depending on cagPAI and the integrity of T4SS.

Figure 1. Helicobacter pylori induced an increase in cytokine expression in human neutrophils, regardless of the status of its cagPAI.

A) Culture supernatants 24 h after infection with cagPAI⁺ or cagPAI^– H. pylori strains were analyzed for IL8 secretion with an ELISA. cagPAI⁺ strain 26695 induced more IL8 than cagPAI^– strain 8822, and the lack of functional OipA protein in the cagPAI⁺256 strain was associated with a reduction in the amount of IL8 induced. B) Clinically isolated cagPAI⁺ strain 261 and cagPAI^– strain 8822 induced the highest levels of IL1β. C) The expression of TNFα did not differ in the cells infected with these two strains. D) cagPAI⁺ strain 261, isolated from a gastric ulcer, and cagPAI^– strain 8822 induced the greatest amounts of IL10. The data shown are from five independent experiments performed in duplicate. The data from these five experiments were pooled and analyzed by ANOVA to assess their statistical significance. *P<0.05 for H. pylori-infected neutrophils versus uninfected neutrophils. Differences between the strains were analyzed with the Student–Newman–Keuls test and were considered statistically significant when *P<0.05. MC = mock control.

Figure 2. Effect of H. pylori T4SS integrity on cytokine induction in human neutrophils.

A) Strains lacking the genes that encode virB4 or virD4 protein in the cagPAI did not modify neutrophil IL8 production compared with the cagPAI⁺26695 reference strain. B) The virB4^– strain induced high levels of IL1β compared with the other mutant virD4^– strain or the 26695 cagPAI⁺ strain. C) High TNFα expression was induced in neutrophils by both mutant strains compared with the cagPAI⁺ strain 26695. D) The virB4^– and virD4^– strains induced the expression of similar amounts of IL10, but these did not differ significantly from that induced by cagPAI⁺ strain 26695. The data shown are from five independent experiments performed in duplicate. The data from these five experiments were pooled and assessed with ANOVA to determine their statistical significance. *P<0.05 for H. pylori-infected neutrophils versus uninfected neutrophils. Differences between the strains were analyzed with the Student–Newman–Keuls test, and differences were considered statistically significant when *P<0.05. MC = mock control.

Figure 3. Helicobacter pylori cagPAI⁺ strain, T4SS mutants, and a cagPAI^– strain downregulated neutrophil TLR2 expression.

Mean fluorescence intensity (MFI) of TLR2 on neutrophils 3, 6, and 24 h after infection (A, B, and C, respectively) with cagPAI⁺ strain 26695, T4SS mutants (virB4^– and virD4^–), or cagPAI^– strain 8822. The data shown are from three independent experiments and line represent median. Flow cytometry results were obtained as medians ± intervals of percentiles and analyzed with the Kruskal–Wallis one-way test. Differences were considered statistically significant when *P<0.05. MC = mock control.

Figure 4. Helicobacter pylori cagPAI⁺ strains and a cagPAI^– strain did not induce changes in TLR4 expression.

Mean fluorescence intensity (MIF) of TLR4 on neutrophils 3, 6, and 24 h after infection (A, B, and C, respectively) with cagPAI⁺ strain 26695, T4SS mutants (virB4^– and virD4^–), or cagPAI^– strain 8822. The data shown are from three independent experiments and line represent median. Flow cytometry results were obtained as medians ± intervals of percentiles and analyzed with the Kruskal–Wallis one-way test. Differences were considered statistically significant when*P<0.05. MC = mock control.

Figure 5. Helicobacter pylori cagPAI⁺ strain, T4SS mutants, and a cagPAI^– strain downregulated neutrophil TLR5 expression.

Mean fluorescence intensity (MFI) of TLR5 on neutrophils 3, 6, and 24 h after infection (A, B, and C, respectively) with cagPAI⁺ strain 26695, T4SS mutants (virB4^– and virD4^–), or cagPAI^– strain 8822. The data shown are from three independent experiments and line represent median. The results were obtained as medians ± intervals of percentiles and analyzed using the Kruskal–Wallis one-way test. Differences were considered statistically significant when *P<0.05. MC = mock control.

Figure 6. Helicobacter pylori cagPAI⁺ strain, T4SS mutants, and a cagPAI^– strain upregulated TLR9 expression.

The percentage of neutrophils expressing TLR9 3, 6, and 24 h after infection (A, B, and C, respectively) with cagPAI⁺ strain 26695, T4SS mutants (virB4^– and virD4^–), and cagPAI^– strain 8822. The data shown are from three independent experiments and line represent median. The results were obtained as medians ± intervals of percentiles and analyzed with the Kruskal–Wallis one-way test. Differences were considered statistically significant when *P<0.05. MC = mock control.