IL-1β, IL-18, and eicosanoids promote neutrophil recruitment to pore-induced intracellular traps following pyroptosis

Abstract

Inflammasomes activate caspase-1, initiating a lytic form of programmed cell death termed pyroptosis, which is an important innate immune defense mechanism against intracellular infections. We recently demonstrated in a mouse infection model of pyroptosis that instead of releasing bacteria into the extracellular space, bacteria remain trapped within the pyroptotic cell corpse, termed the pore-induced intracellular trap (PIT). This trapping mediates efferocytosis of the PIT and associated bacteria by neutrophils; bacteria are subsequently killed via neutrophil ROS. Using this pyroptosis model, we now show that the pro-inflammatory cytokines IL-1β and IL-18 and inflammatory lipid mediators termed eicosanoids are required for effective clearance of bacteria downstream of pyroptosis. We further show that IL-1β, IL-18, and eicosanoids affect this in part by mediating neutrophil recruitment to the PIT. This is in addition to our prior findings that complement is also important to attract neutrophils. Thus, the PIT initiates a robust and coordinated innate immune response involving multiple mediators that attract neutrophils to efferocytose the PIT and its entrapped bacteria.

Keywords: Caspase-1; Inflammasome; Neutrophil recruitment; Pore-induced intracellular trap; Pyroptosis.

Figure 1. IL-1β, IL-18, and pyroptosis cooperatively clear FliC^ind S. Typhimurium

(A–E, H, I) Mice (n=5) were infected with (A, B) high dose (10⁵ CFUs) or (C–I) low dose (10³ CFUs) (of 1:1 ratio of FliC^ind Amp^R and WT Kan^R S. Typhimurium IP for 17 hours prior to doxycycline injection; organs were harvested 7 hours later. CIs are expressed as the log FliC^ind/WT (Amp/Kan CFUs). (F–G) Mice were infected with WT or FliC^ind S. Typhimurium and treated with doxycycline at 24 hours pi for 5 hours. Spleens were prepared for single cell suspension, stained and analyzed by flow cytometry. Neutrophils were defined as the CD11b⁺ Ly6G⁺. (F) The gating strategy represents a WT mouse infected with FliC^ind S. Typhimurium. (G) The mean percentage of neutrophils of the total splenocytes population is stated in the figure for each condition. (H–I) For NK-cell depletion, mice were injected with 75 ng anti-NK1.1 PK136 or isotype control C1.18.4 (BioXCell) at −3 and −1 dpi. Depletion of NK1.1-positive cells was confirmed by flow cytometry (left). NK cells were defined as NK1.1⁺ F4/80⁻, and the percentage of each cell type is indicated. All data are shown as mean + SE of n=3 (F, G) and n=5 (A–E, H, I) mice and are from single experiments representative of two (A, H, I) or three (B–G) experiments. * p < 0.05; two-tailed student t-test.

Figure 2. Prostaglandins and leukotrienes are required to clear FliC^ind S. Typhimurium

(A) Schematic overview of eicosanoid synthesis and inhibitor action. (B–D) Mice (n=5) were infected with 10³ CFUs of 1:1 ratio of FliC^ind Amp^R and WT Kan^R S. Typhimurium IP for 17 hours, then treated with doxycycline in the presence of (B, C) DMSO vehicle, (B) indomethacin, zileuton, (C) a combination of indomethacin and zileution, or (D) licofelone and harvested 7 hours later. CIs are expressed as the log FliC^ind/WT (Amp/Kan CFUs). (E) WT BMMs in vitro were pre-treated with PBS, zileuton, or indomethacin for 4 hours, and infected with S. Typhimurium MOI 50 SPI-1 in the presence of inhibitors. Cytotoxicity was measured by % LDH release at 2 hours post infection. (F) Mice (n=3) were infected with WT or FliC^ind S. Typhimurium and both groups of mice were treated with doxycycline and inhibitors as in (A), and splenocytes analyzed by flow cytometry. Neutrophils were defined as CD11b⁺ Ly6G⁺. The mean percentage of neutrophils of the total splenocytes population is stated in the figure for each condition. All data are shown as mean + SE of n=5 (B–D), and n=3 (F) mice. All panels (A–F) are representative of three independent experiments. * p < 0.05; two-tailed student t-test.