Helicobacter urease-induced activation of the TLR2/NLRP3/IL-18 axis protects against asthma

Abstract

Inflammasome activation and caspase-1-dependent (CASP1-dependent) processing and secretion of IL-1β and IL-18 are critical events at the interface of the bacterial pathogen Helicobacter pylori with its host. Whereas IL-1β promotes Th1 and Th17 responses and gastric immunopathology, IL-18 is required for Treg differentiation, H. pylori persistence, and protection against allergic asthma, which is a hallmark of H. pylori-infected mice and humans. Here, we show that inflammasome activation in DCs requires the cytoplasmic sensor NLRP3 as well as induction of TLR2 signaling by H. pylori. Screening of an H. pylori transposon mutant library revealed that pro-IL-1β expression is induced by LPS from H. pylori, while the urease B subunit (UreB) is required for NLRP3 inflammasome licensing. UreB activates the TLR2-dependent expression of NLRP3, which represents a rate-limiting step in NLRP3 inflammasome assembly. ureB-deficient H. pylori mutants were defective for CASP1 activation in murine bone marrow-derived DCs, splenic DCs, and human blood-derived DCs. Despite colonizing the murine stomach, ureB mutants failed to induce IL-1β and IL-18 secretion and to promote Treg responses. Unlike WT H. pylori, ureB mutants were incapable of conferring protection against allergen-induced asthma in murine models. Together, these results indicate that the TLR2/NLRP3/CASP1/IL-18 axis is critical to H. pylori-specific immune regulation.

Figure 3. H. pylori urease is required for CASP1 activation, persistence, and asthma protection in neonatally infected mice.

(A–E) Neonatal C57BL/6 mice were infected for 1 month with WT or Δure H. pylori PMSS1 and assessed with respect to (A) gastric colonization, (B) gastric mucosal NLRP3 and CASP1 (asterisk indicates the NLRP3-specific band), (C) Nlrp3 expression, (D) IL-18, and (E) Ifng expression, as analyzed by (B) Western blotting, (D) ELISA, and/or (C and E) qRT-PCR. (F–M) Neonatally infected mice were additionally sensitized and challenged (s.c.) with (F–I) ovalbumin or (J–M) house dust mite (HDM) allergen starting at 4 weeks after infection to induce allergic asthma; αIL-18 mAb was administered weekly starting at the time of infection. Inf, infection. (F and J) Eosinophils in 1 ml of bronchoalveolar lavage fluid (BALF). (G) IL-5 ELISA of ovalbumin-restimulated lung single cell preparations. (H and L) Lung inflammation, as assessed on H&E-stained sections. (I and M) Goblet cell metaplasia, as quantified on PAS-stained sections. BM, basement membrane. (K) House dust mite–specific serum IgE, as determined by ELISA. (N) H. pylori colonization of WT, Tlr2^–/–, and Nlrp3^–/– mice 1 month after infection. Symbols represent individual animals, and horizontal lines indicate the medians. Pooled data from 2 (A–E and N) and 3 (F–M) independent experiments are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, Mann-Whitney U test.

Figure 2. IL-1β secretion upon H. pylori infection requires LPS-induced transcriptional activation of pro–IL-1β and urease- and TLR2-dependent expression of NLRP3.

(A) Genome-wide screening for H. pylori tn mutants incapable of IL-1β secretion identifies the indicated mutant categories. R-M, restriction modification. (B) Relative IL-1β secretion of individual tn clones with insertions in ureA, ureB, and HPG27_146. (C–E and I–M) Murine BMDCs, (F and G) splenic CD11c⁺ DCs, and (H) human blood-derived DCs were infected overnight or for the indicated time points with G27 WT, Δure, or Δ146 strains of H. pylori and analyzed by (C, F, H, K, and M) IL-1β ELISA; (G) IL-18 ELISA; (D, J, and L) CASP1 p10 and p45, pro–IL-1β, and NLRP3 Western blotting; and (E and I) qRT-PCR (normalized to Gapdh and to uninfected controls). BMDCs were prestimulated with E. coli LPS where indicated, and 1 μg/ml recombinant GST-tagged UreA, UreB (native or heat-inactivated [HI] for 10 minutes at 70°C), or GST was added to cocultures as noted. Pooled data from 3 to 4 independent experiments are show in C, E, I, K, and M; a representative of 2 experiments is shown in F–H. *P ≤ 0.05, **P ≤ 0.01, Mann-Whitney U test. Error bars represent mean + SD.

Figure 1. CASP1 activation by H. pylori depends on NLRP3, ASC, and TLR2.

(A–G) BMDCs from mice of the indicated genotypes were infected overnight with H. pylori NSH57, G27, and/or PMSS1. (A, C, and E) Western blot analysis of CASP1 activation (p10) in the cell supernatant compared to full-length CASP1 p45 and GAPDH in the extract. Representative results of 3 independent experiments are shown (n = 3). (B, D, and F) IL-1β ELISA of culture supernatants; cells were prestimulated with E. coli LPS prior to infection. Uninf, uninfected. (G) Il1b transcription, as measured by qRT-PCR (normalized to Gapdh and to uninfected controls). Mean + SD of 3 independent experiments is shown (n = 3). (H and I) CD11c⁺ splenic DCs were infected overnight with G27. (H) IL-1β and (I) IL-18 secretion was measured by ELISA. Representative data of 3 independent experiments are shown (n = 3). (J and K) Mice were infected for 1 month with PMSS1 prior to the quantification of (J) gastric colonization and (K) Ifng expression. Pooled data from 2 studies are shown (n = 2). Horizontal lines indicate medians. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, Mann-Whitney U test. Error bars represent mean + SD.