The Cag pathogenicity island and interaction between TLR2/NOD2 and NLRP3 regulate IL-1β production in Helicobacter pylori infected dendritic cells

Abstract

Helicobacter pylori colonization of the stomach affects about half of the world population and is associated with the development of gastritis, ulcers, and cancer. Polymorphisms in the IL1B gene are linked to an increased risk of H. pylori associated cancer, but the bacterial and host factors that regulate interleukin (IL)-1β production in response to H. pylori infection remain unknown. Using murine BM-derived DCs, we show that the bacterial virulence factors cytotoxin-associated genes pathogenicity island and CagL, but not vacuolating cytotoxin A or CagA, regulate the induction of pro-IL-1β and the production of mature IL-1β in response to H. pylori infection. We further show that the host receptors, Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain 2 (NOD2), but not NOD1, are required for induction of pro-IL-1β and NOD-like receptor pyrin domain containing 3 (NLRP3) in H. pylori infected DCs. In contrast, NLRP3 and the adaptor ASC were essential for the activation of caspase-1, processing of pro-IL-1β into IL-1β, and IL-1β secretion. Finally, we show that mice deficient in caspase-1, IL-1β, and IL-1 receptor, but not NLRP3, are impaired in the clearance of CagA-positive H. pylori from the stomach when compared with WT mice. These studies identify bacterial cag pathogenicity island and the cooperative interaction among host innate receptors TLR2, NOD2, and NLRP3 as important regulators of IL-1β production in H. pylori infected DCs.

Keywords: Helicobacter pylori; IL-1β; Inflammasome; NLRP3; NOD2.

Figure 1. H. pylori cagPAI and CagL, but not VacA, enhances IL-1 β induction in DCs

A, DCs were infected with wild-type P12 H. pylori and isogenic mutants deficient in VacA, cagPAI, or CagL at a multiplicity of infection (MOI) of 1/20 overnight. IL-1β production was determined by ELISA. B, uptake of wild-type P12 H. pylori and isogenic mutants by DCs. C–E, DCs were infected with indicated wild-type H. pylori and isogenic mutants at a multiplicity of infection (MOI) of 1/20 overnight. F–G, DCs were infected with wild-type H. pylori and indicated isogenic mutant strains with or without LPS priming (6 hr). IL-1β production was determined by ELISA. The mRNA expression of Il1b was evaluated by real-time PCR at 6 hr after infection and fold increase (arbitrary unit) was obtained by comparison to the level of uninfected DCs. Data shown are means ± SD of triplicate samples of one experiment representative of three independent experiments. ** p < 0.01

Figure 2. TLR2 and NOD2 induce pro-IL-1β expression and IL-1β release upon H. pylori infection in DCs

A–D, DCs from wild-type, Tlr2^−/−, Nod1^−/−, Nod2^−/− and Nod2^−/−Tlr2^−/− mice were infected with H. pylori at a MOI of 1/20 overnight (A,B) or for 6 hr (C,D). IL-1β secretion was determined by ELISA (A–B) and induction of pro-IL-1β was analyzed by immunoblotting (C). mRNA expression of Il1b was evaluated by real-time PCR and fold increase (arbitrary units) was obtained by comparison to the level of uninfected DCs (D). Immunoblotting for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. For A, B, and D, data shown are means ± SD of triplicate samples of one experiment representative of three independent experiments. * p < 0.05, ** p < 0.01.

Figure 3. Processing of IL-1β upon H. pylori infection depends on Caspase-1

A–F, DCs from wild-type and Casp1^−/− mice were infected with H. pylori (HP 29965 or HP SPM326) at the indicated MOI overnight (A,B,D,E) or at a MOI of 1/100 for 6 hr (C,F). IL-1β and TNF-α production was determined by ELISA (A,B,D,E) and induction of pro-IL-1β and p17 was analyzed by immunoblotting (C,F). For A, B, D, and E, data shown are means ± SD of triplicate samples of one experiment representative of three independent experiments. * p < 0.05, ** p < 0.01.

Figure 4. TLR2 and NOD2 induce Nlrp3 expression and pro-IL-1β processing upon H. pylori infection

A, DCs from WT and Nod2^−/−Tlr2^−/− mice were infected with H. pylori at a MOI of 1/100 for 6 hr. The production of the p20 subunit of Caspase-1 and processed IL-1β (p17) were analyzed by immunoblotting. B, DCs from WT and indicated WT and mutant DCs were infected with H. pylori at a MOI of 1/20 for 6 hr. mRNA expression of Nlrp3 was evaluated by real-time PCR and fold increase (arbitrary units) was obtained by comparison to the level of uninfected DCs. C, DCs from wild-type and indicated mutant DCs were infected with H. pylori overnight with or without LPS priming (6 hr). IL-1β production was determined by ELISA. For B and C, data shown are means ± SD of triplicate samples of one experiment representative of three independent experiments. * p < 0.05, ** p < 0.01.

Figure 5. H. pylori infection activates Caspase-1 via the NLRP3 inflammasome in DCs

A–C, DCs from wild-type and Nlrp3^−/−, Nlrp4^−/−, Asc^−/− and Nlrc4^−/− mice were infected with H. pylori at the indicated MOI overnight (A–B) or at a MOI of 1/100 for 6 hr (C). IL-1β and TNF-α production was determined by ELISA (A–B) and activated Caspase-1 (p20) and processed IL-1β (p17) was analyzed by immunoblotting (C). For AB, data shown are means ± SD of triplicate samples of one experiment representative of three independent experiments. * p < 0.05, ** p < 0.01.

Figure 6. IL-1β signaling regulates H. pylori colonization in vivo

A–D, H. pylori colonization was determined in the stomach of wild-type, Il1b^−/−, l1r^−/−, Casp1^−/− and Nlrp3^−/− mice 4 weeks post-infection by quantitative culture. Values are expressed as colony forming units (CFUs) per gram stomach tissue. Each dot represents one animal. N. S., not significant.