A Preliminary Study of Mild Heat Stress on Inflammasome Activation in Murine Macrophages

Abstract

Inflammation and mitochondrial-dependent oxidative stress are interrelated processes implicated in multiple neuroinflammatory disorders, including Alzheimer's disease (AD) and depression. Exposure to elevated temperature (hyperthermia) is proposed as a non-pharmacological, anti-inflammatory treatment for these disorders; however, the underlying mechanisms are not fully understood. Here we asked if the inflammasome, a protein complex essential for orchestrating the inflammatory response and linked to mitochondrial stress, might be modulated by elevated temperatures. To test this, in preliminary studies, immortalized bone-marrow-derived murine macrophages (iBMM) were primed with inflammatory stimuli, exposed to a range of temperatures (37-41.5 °C), and examined for markers of inflammasome and mitochondrial activity. We found that exposure to mild heat stress (39 °C for 15 min) rapidly inhibited iBMM inflammasome activity. Furthermore, heat exposure led to decreased ASC speck formation and increased numbers of polarized mitochondria. These results suggest that mild hyperthermia inhibits inflammasome activity in the iBMM, limiting potentially harmful inflammation and mitigating mitochondrial stress. Our findings suggest an additional potential mechanism by which hyperthermia may exert its beneficial effects on inflammatory diseases.

Keywords: IL-1β; caspase 1; cytokine; hyperthermia; inflammasome; mild heat stress; mitochondria; pyrexia.

Figure 1

Mild and extreme heat shock differentially inhibit proinflammatory cytokines. (A) Timing of heat exposure and LPS/ATP stimulation in iBMMs. In each model, macrophages were treated with LPS (1 μg/mL) for 4 h, then ATP (5 mM) was added for 30 min to induce the release of IL-1β. Supernatants and cell lysate were analyzed by ELISA for cytokines and assayed for LDH release. One set of samples was incubated at 37 °C for 4 h as a control (black bars). Co-exposure of the “no priming” model: cells were stimulated with LPS and incubated at 41.5 °C or 39 °C for 1 h, followed by incubation at 37 °C for 3 h (hatched bars). “Priming” model: cells were stimulated with LPS and incubated at 37 °C for 3 h, followed by incubation at 41.5 °C or 39 °C for one hour (checked bars). (B–D) The co-exposure model at 41.5 °C shows inhibition of TNFα, IL-6, and intracellular IL-1β (hatched bars, 41.5 °C→37 °C). The primed model at 41.5 °C shows some inhibition of TNFα and IL-6 and complete inhibition of IL-1β (checked bars, 37 °C→41.5 °C). (E–G) 39 °C co-exposure model shows inhibition of IL-1β, TNFα, and IL-6 (hatched bars). The primed model at 39 °C shows inhibition of IL-1β, but not TNFα and IL-6 (checked bars). (H,I) The LDH-release assay shows cell death in the co-exposure model at both temperatures (hatched bars), but not in the primed model (checked bars). Results are the combined results of 2–3 independent experiments (IL-1β and LDH) or representative of 2–3 independent experiments (TNFα and IL-6). **** p < 0.0001 as determined by a two-way ANOVA with Tukey’s multicorrection comparison. *** p < 0.001, ** p < 0.01, ns—not significant.

Figure 2

Mild heat shock inhibits membrane permeability. (A) unstimulated controls show no increased permeability: iBMMs were incubated at 37 °C (black circles) or exposed to 39 °C for one hour (red squares) followed by addition of PI (5 μM) to assay membrane permeability over time. (B) LPS controls show no increased permeability: iBMMs were primed with LPS and incubated at 37 °C for the entire 4 hours (black circles) or switched to 39 °C for the last hour (red squares), followed by addition of PI. (C) LPS+ATP stimulation shows that exposure to 39 °C inhibits ATP-induced pore formation in the membrane in LPS-primed cells: iBMMs were primed with LPS and incubated at 37 °C for the entire 4 hours (black circles) or switched to 39 °C for the last hour (red squares), followed by addition of ATP + PI The results are representative of two independent experiments. **** p < 0.0001 as determined by a two-way ANOVA with Tukey’s multicorrection comparison.

Figure 3

Mild heat shock rapidly inhibits caspase 1 and IL-1β processing. (A) Western blot shows presence of IL-1β processed fragment p17 and caspase 1 processed fragment p20 in LPS primed iBMMs treated with ATP incubated at 37 °C (Left panels), and decreased processing in heat-exposed cells (Right panels). (B) Time course: cells were primed for 3 h with LPS at 37 °C, then exposed to 39 °C for the indicated times, followed by addition of ATP to activate the processing of IL-1β. One of two independent experiments.

Figure 4

Mild heat shock inhibits the formation of ASC specks but does not inhibit mitochondrial membrane potential. (A) Representative images of iBMMs expressing ASC-citrine stained with Mitotracker CMX ROS and Hoescht nuclear stain show inhibition of ASC specks (white arrows) and increased numbers of cells with polarized mitochondria in heat-exposed cells. Live cells were imaged using the IXM live cell microscopy system. Images are from a representative experiment. (B) Quantification of ASC specks per DAPI nuclei. Results are a representative experiment of 3 independent experiments, counted manually with >5 (40×) fields/conditions. (C) Quantification of the number of cells/well with respiring mitochondria, normalized to the unstimulated 37 °C control condition, greater than 16 wells/condition/experiment combined over three independent experiments. **** p < 0.0001 as determined by a two-way ANOVA with Tukey’s multicorrection comparison; ns, nonsignificant.

Figure 5

Exposure to a mildly elevated temperature of 39 °C leads to rapid inhibition of inflammasome activation, associated with decreased caspase 1 and IL-1β processing, decreased plasma membrane permeability, decreased ASC speck formation, and increased numbers of polarized mitochondria.