Characterization of Inflammasomes and Their Regulation in the Red Fox

Abstract

Background: Inflammasomes recognize endogenous and exogenous danger signals, and subsequently induce the secretion of IL-1β. Studying inflammasomes in the red fox (Vulpes vulpes) is crucial for wildlife veterinary medicine, as it can help control inflammatory diseases in foxes.

Methods: We investigated the activation and intracellular mechanisms of three inflammasomes (NLRP3, AIM2, and NLRC4) in fox peripheral blood mononuclear cells (PBMCs), using established triggers and inhibitors derived from humans and mice.

Results: Fox PBMCs exhibited normal activation and induction of IL-1β secretion in response to representative inflammasome triggers (ATP and nigericin for NLRP3, dsDNA for AIM2, flagellin for NLRC4). Additionally, PBMCs showed normal IL-1β secretion when inoculated with inflammasome-activating bacteria. In inhibitors of the inflammasome signaling pathway, fox inflammasome activation was compared with mouse inflammasomes. MCC950, a selective NLRP3 inhibitor, suppressed the secretion of dsDNA- and flagellin-mediated IL-1β in foxes, unlike mice.

Conclusions: These findings suggest that NLRP3 may have a common role in dsDNA- and flagellin-mediated inflammasome activation in the red fox. It implies that this fox inflammasome biology can be applied to the treatment of inflammasome-mediated diseases in the red fox.

Keywords: Vulpes vulpes; cytokine; inflammasome; interleukin-1beta; red foxes.

Figure 1

Comparison of inflammasome activation between dog and fox PBMCs. (A) Schematic diagram representing the priming and activation steps of inflammasomes. (B) LPS-primed PBMCs were treated with NG (40 μM) and ATP (5 mM) for 1 h, and the expression of pro-IL-1β in the cellular lysate (Lys) and the secretion of IL-1β (p17) in the cellular supernatant (Sup) were detected by immunoblotting. Lys: cellular lysate; Sup: cellular supernatant.

Figure 2

Optimization of the NLRP3 inflammasome in red foxes. (A) Fox PBMCs were primed with LPS (1 to 1000 ng/mL) and further treated with ATP (5 mM) or NG (40 μM). (B) Fox PBMCs primed with LPS (1 ng/mL) were treated with ATP, NG, and CaCl₂ at indicated concentrations. The secreted IL-1β was analyzed by immunoblotting. Images are representative of two independent experiments. Lys: cellular lysate; Sup: cellular supernatant.

Figure 3

Optimization of AIM2 and NLRC4 inflammasomes in red foxes. (A) Fox PBMCs were primed with LPS (1 to 1000 ng/mL) and further transfected with dsDNA (4 μg/mL). (B) LPS (1 ng/mL)-primed fox PBMCs were transfected with dsDNA at indicated concentrations. (C) Fox PBMCs were treated with LPS (1 to 1000 ng/mL), followed by the introduction of flagellin (500 ng/mL). (D) LPS-primed PBMCs of red foxes were treated with flagellin as presented. IL-1β secretion was measured using immunoblotting or ELISA. Images are representative of two independent experiments, and bar graphs present the mean ± standard deviation (SD) of at least three independent experiments. Lys: cellular lysate; Sup: cellular supernatant.

Figure 4

Effect of bacteria on inflammasome activation in fox PBMCs. Fox PBMCs were primed with LPS (1 ng/mL) and then inoculated with bacteria at the indicated multiplicity of infection (MOI) for the presented times. The release of IL-1β was measured using ELISA. The bar graphs present the mean ± SD of at least three independent experiments.

Figure 5

Effect of inhibitors on the activation of fox and mouse inflammasomes. LPS-primed fox PBMCs (A) and mouse BMDMs (B) were treated with a ROS scavenger (diphenyleneiodonium (DPI)), a potassium efflux inhibitor (KCl and glibenclamide (Glib)), and an NLRP3 selective inhibitor (MCC950 (MCC)) in the presence of inflammasome triggers such as NG for NLRP3, dsDNA for AIM2, and flagellin for NLRC4. The secretion of IL-1β was analyzed by ELISA. Bar graphs present the mean ± SD of at least three independent experiments.