Caspase-11 promotes allergic airway inflammation

Abstract

Activated caspase-1 and caspase-11 induce inflammatory cell death in a process termed pyroptosis. Here we show that Prostaglandin E₂ (PGE₂) inhibits caspase-11-dependent pyroptosis in murine and human macrophages. PGE₂ suppreses caspase-11 expression in murine and human macrophages and in the airways of mice with allergic inflammation. Remarkably, caspase-11-deficient mice are strongly resistant to developing experimental allergic airway inflammation, where PGE₂ is known to be protective. Expression of caspase-11 is elevated in the lung of wild type mice with allergic airway inflammation. Blocking PGE₂ production with indomethacin enhances, whereas the prostaglandin E₁ analog misoprostol inhibits lung caspase-11 expression. Finally, alveolar macrophages from asthma patients exhibit increased expression of caspase-4, a human homologue of caspase-11. Our findings identify PGE₂ as a negative regulator of caspase-11-driven pyroptosis and implicate caspase-4/11 as a critical contributor to allergic airway inflammation, with implications for pathophysiology of asthma.

Fig. 1. Prostaglandin E2 protects against pyroptosis.

a Murine BMDMs were treated with 1 μM PGE₂ or 500 μM 6-Bnz-cAMP (PKA ag) for 30 min followed by priming with 100 ng/ml of LPS for 4 h and 2 μg of LPS transfection using FuGENE 0.25% v/v liposomes o/n. Supernatants were collected and analyzed for cell death using an LDH assay. The results shown are from three independent experiments (with 2–4 mice per group in each experiment. Individual data points on the graph represent means from two to three technical replicates). Ordinary one-way ANOVA with Tukey’s multiple comparison test has been used, **P < 0.01, ***P < 0.001, error bars represent mean ± SD. b BMDMs were treated with 1 μM PGE₂ or DMSO for 30 min followed by 100 ng/ml LPS for 4 h, and subjected to qPCR analysis of caspase-11 expression. The results shown are from a single experiment, with three mice in each group, and are representative of three independent experiments; individual data points on the graph are means from technical duplicates. *P < 0.01, two-tailed Student’s t test, error bars represent mean ± SD. c BMDMs were pretreated with PGE₂ for 30 min and then treated with LPS for 4 h. Lysates were assessed with caspase-11 expression by western blotting. A representative western blot from three independent experiments is shown. d BMDMs from wild-type or EP2-deficient mice were assessed for caspase-11 expression by western blotting. The results shown are representative of BMDMs prepared from three mice of each genotype in a single experiment. Densitometry values are shown above each blot.

Fig. 2. Prostaglandin E₂ inhibits caspase-11 expression by inhibition of IFN-β production.

a BMDMs were treated with 1 μM PGE₂ or DMSO for 30 min followed by 100 ng/ml LPS for 4 h, and subjected to qPCR analysis for IFN-β. Data from BMDMs from three mice are shown, and are representative of three independent experiments, each involving three mice, with individual data points on the graph, which are means from technical duplicates. ***P < 0.0001, two-tailed Student’s t test, error bars represent mean ± SD. b BMDMs were treated with 1 μM PGE₂ for 30 min followed by 100 ng/ml of LPS or IFN-β for 4 h, and assessed for caspase-11 by western blotting. The results shown are representative of three independent experiments. c BMDMs were treated with 1 μM PGE₂ for 30 min followed by 100 ng/ml of LPS for 2 h, and assessed for phosphor-STAT-1 by western blotting. The results shown are representative of three independent experiments. d BMDMs were treated with 1 μM PGE₂ or DMSO for 30 min followed by 100 ng/ml of LPS for 2 h. An oligonucleotide pulldown using the caspase-11 promoter was then carried out with samples and then immunoblotted for STAT-1. The results shown are from a single experiment. Densitometry values are shown above each blot. e Schematic representation of PGE₂-mediated inhibition of caspase-11 transcription.

Fig. 3. Prostaglandin E2 inhibits capase-11-driven pyroptosis in LPS-primed cells.

a Murine BMDMs were primed with 100 ng/ml of LPS for 4 h followed by stimulation with 1 μM PGE₂ for 30 min; media was removed and followed by 2 μg of LPS transfected using FuGENE 0.25% v/v liposomes overnight. Cell death was assessed by measuring LDH activity. The results shown are from a single experiment, with three mice in each group, and are representative of three independent experiments. Individual data points on the graph are means from technical duplicates. Ordinary one-way ANOVA with Tukey’s multiple comparison test has been used, *P < 0.01, error bars represent mean ± SD. b BMDMs were primed with 100 ng/ml of LPS for 4 h followed by stimulation with 1 μM PGE₂ for 30 min, and lysates were assessed for caspase-11 expression by western blotting. The results shown are representative of three separate experiments. c Human monocyte-derived macrophages were treated with 1 μM PGE₂ or DMSO for 30 min followed by 100 ng/ml LPS for 4 h. Lysates were assessed for caspase-4 expression by western blotting. The results shown are representative of three independent experiments, each utilizing one or two donors. Densitometry from all four donors is provided (*P < 0.01, two-tailed paired Student’s t test, error bars represent mean ± SEM). d Human monocyte-derived macrophages were treated with 1 μM PGE₂ for 30 min before priming with 100 ng/ml of LPS for 4 h or for 30 min before transfection with 2 μg/mL of LPS using FuGENE 0.25% v/v liposomes o/n. Supernatants were collected and analyzed for cell death using LDH assay. Data from three independent experiments are shown, each experiment utilizing cells from one donor. Individual data points on the graph are means from technical duplicates (*P < 0.01, two-tailed paired Student’s t test, error bars represent mean ± SEM). e Streptavidin pull-down assay. BMDMs were treated with 100 ng/ml of LPS for 4 h, lysed, precleared with strepatvidin beads and incubated with 2 μg of biotinylated LPS, PGE₂ and arachidonic acid, and streptavidin beads for 1 h, and blotted for caspase-11. The results shown are representative of two separate experiments.

Fig. 4. Indomethacin induces whereas misoprostol decreases caspase-11 expression in allergic airway inflammation.

a–e Allergic airway inflammation (AAI) was induced by intraperitoneal injection of 20 μg of OVA mixed with 2 mg of alum, followed 7 days later by two airway challenges with 1% OVA and lung lysate collection 24 h after the final airway challenge. In all, 2 mg/kg of misoprostol was given 2 h before sensitization and 2 h before each airway challenge. Lungs were homogenized, and cellular infiltration as well as cytokine production was analyzed using flow cytometry. To discriminate blood-borne circulating cells from lung-localized cells, we used CD45 i.v. administration 10 min before the mouse was killed and lungs were harvested. Data are from one experiment using five mice per group (*P < 0.01, **P < 0.01, ***P < 0.001, two-tailed paired Student’s t test, error bars represent mean ± SD). f The total lung lysates, obtained by homogenization of whole-lung lobes with tissue grinder, were probed for caspase-11 and IL-1β expression. There were four mice in each group, and analysis of samples from each mouse is shown. g Densitometry from the expression of caspase-11 presented in panel F (*P < 0.01, two-tailed paired Student’s t test, error bars represent mean ± SD). h In total, 2 mg/kg of indomethacin was given 2 h before sensitization and 2 h before each airway challenge. Total lungs lysates were probed for IL-1β and caspase-11 expression, with six mice in each group. Analysis of samples from each mouse are shown. Data are from two independent experiments, each with three mice per group. i Densitometry from the expression of caspase-11 presented in panel h is shown (*P < 0.01, two-tailed paired Student’s t test, error bars represent mean ± SD).

Fig. 5. Caspase-11 drives allergic airway inflammation.

WT (white bars) and caspase-11-deficient (black bars) mice were subjected to allergic airway inflammation protocol as explained below. Briefly, allergic airway inflammation was induced by intraperitoneal injection of 20 μg of OVA mixed with 2 mg of alum, followed 7 days later by two airway challenges with 1% OVA and lung lysate collection 24 h after the final airway challenge. To discriminate blood-borne circulating cells from lung-localized cells, we used CD45 i.v. administration 10 min before the mouse was killed and lungs were harvested. Lung lavage fluid was collected and subjected to (a) differential cell count. b ELISA analysis. c H&E staining of lung sections. Lungs were homogenized, and cellular infiltration as well as cytokine production was analyzed using flow cytometry (d–f). Data for each mouse tested are shown, with 4–5 mice per group, and are represenative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t test, error bars represent mean ± SD). g Blood was collected from control and OVA-treated mice 24 h after the final airway challenge, and serum was isolated and analyzed for IgE levels by ELISA. Two independent experiments were carried out with five mice per group; individual data points on the graph are means from technical duplicates, **P < 0.01 (two-tailed Student’s t test, error bars represent mean ± SD). h Alveolar macrophages from asthmatic or control patients were subjected to qPCR analysis for caspase-4 (eight healthy and seven asthmatic patients, data points on the graph are means from technical duplicates, **P < 0.01, Mann–Whitney test. Error bars represent mean ± SD).

Fig. 6. Schematic representation of the protective role of PGE2 in inhibition of caspase-11 and allergic airway inflammation.

In the lung, extracellular LPS from the lung microbiome or Gram-negative bacterial infections triggers LPS signaling, which induces IFN-β production. IFN-β in turn activates STAT-1, which gets phosphorylated and initiates transcription of caspase-11. Intracellular LPS activates caspase-11, which initiates a highly inflammatory process of cell death termed pyroptosis. Pyroptosis is therefore likely to participate or even initiate allergic airway inflammation. PGE₂ can inhibit IFN-β production and caspase-11 transcription, as well as caspase-11 activation by LPS, thereby limiting pyroptosis.