Lower Temperatures May Worsen Gout by Increasing Crystal Formation and Triggering Inflammation

Bone marrow-derived macrophages (BMDMs) were differentiated from progenitors isolated from the tibias and femurs of C57BL/6 mice using conditioned media containing L929 cell-cultured media (30%, a source of a macrophage colony-stimulating factor), fetal bovine serum (FBS, 10%) and antibiotics at 37 °C in 5% CO₂ atmosphere. After seven days, the BMDMs were subjected to inflammasome activation. The mice for the BMDMs were approved by the Institutional Animal Care and Use Committee of Kangwon National University (KW-210317-2).

BMDMs (1 × 10⁶ cells/well in a 12-well-plate) were primed with LPS (1 μg/mL) for 3 h [14]. The LPS-primed BMDMs were replaced with RPMI 1640 media containing the following inflammasome triggers: nigericin (NG, 40 μM) for 1 h; MSU crystals (400 μg/mL) for 3 h; aluminum potassium sulfate (alum, 400 μg/mL) and dsDNA (1 μg/mL) with jetPRIME^TM (2 μL/mL) for 1 h; and flagellin (500 ng/mL) with Lipofectamine 2000 (10 μL/mL) for 3 h. The trigger-treated BMDMs were incubated in an incubator set to various temperatures in an atmosphere containing 5% CO₂. The concentration of CO₂ was maintained strictly by monitoring with a CO₂ analyzer to prevent the pH changing, which is another factor contributing to MSU crystallization [2].

After inflammasome activation, the cellular supernatant (Sup) of the BMDMs were collected, and the cells were lysed using a buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tri-base, pH 8.0) by adding a proteinase inhibitor cocktail. The cellular lysate (Lys) was harvested by centrifugation (15,000 rcf, 5 min), and the remaining debris was treated with suberic acid bis (2 mM) for cross-linking. After 1 h, the pellet (Pellet) was suspended in loading dye for immunoblotting [15].

The samples (i.e., Lys, Sup, and Pellet) were separated by SDS-PAGE (10 or 16%) and transferred to a PVDF membrane using a Mini-PROTEAN Tetra cell system. The membrane was incubated with the following antibodies overnight at 4 °C: anti-Casp1 (p20) antibody, anti-ASC antibody, or anti-Actin antibody. The membrane was further probed with a second anti-sera conjugated with horseradish peroxidase and visualized with a chemiluminescence solution using a chemiluminescent system (EZ-Capture II). The density of the image was analyzed using a CS Analyzer.

The secretion of IL-1β was analyzed using an ELISA kit. For the phagocytosis rates, BMDMs (1 × 10⁴ cells/well in a 96-well-black plate) were incubated in RPMI 1640 containing 10% FBS, antibiotics, and fluorescent beads at the indicated temperature. After 1 or 6 h, the remaining beads in the media were removed by washing with PBS, and the fluorescence (Ex/Em at 407/505) was measured. For MSU solubility, the MSU crystals were dissolved in distilled water as indicated, and the optical density (OD) from 280 to 330 nm was measured. In addition, MSU crystals were added to RPMI 1640 and incubated at various temperatures (4, 25, 37, 42, and 65 °C). After 3 h, the RPMI 1640 containing MSU was filtered through a syringe filter (with a pore size of 0.45 μm) while maintaining the incubation temperature. The filtered RPMI 1640 was then subjected to OD analysis. All assays were conducted using a microplate spectrophotometer.

Statistical analyses were conducted using GraphPad Prism6. Two groups were analyzed using a Mann–Whitney test and multiple groups were calculated using a one-way non-parametric ANOVA (Kruskal–Wallis test). The significant differences (p-values) are marked in the figures.

Mouse BMDMs were treated with LPS at 37 °C for 3 h to induce the priming step of inflammasome activation. Subsequently, the cells were incubated separately at different temperatures (25, 33, 37, 39, and 42 °C) for the activation step. The BMDMs were subjected to the NLRP3, NLRC4, or AIM2 triggers. The IL-1β secretion was then measured as an indicator of inflammasome activation. The cells incubated at 37 °C showed the most abundant secretion of IL-1β in response to the NG, flagellin, or dsDNA treatments compared to the other temperatures, as shown in Figure 1A. The level of IL-1β release was reduced if the temperature decreased or increased from 37 °C. On the other hand, the MSU crystal-mediated IL-1β secretion was the highest in the BMDMs at 25 °C and decreased with increasing temperatures. Furthermore, Casp1 (p20) secretion was analyzed by immunoblotting (Figure 1B,C). Similar to the above IL-1β data, Casp1 (p20) secretion from BMDMs treated with the inflammasome triggers (i.e., NG, flagellin or dsDNA) was decreased when the incubation temperature deviated from 37 °C. Nevertheless, the MSU crystals inducing the release of Casp1 (p20) increased with decreasing temperatures from 37 °C and decreased with increasing temperatures. Overall, the incubation at 37 °C was the optimal temperature for the activation of inflammasome activation. In one exception, MSU crystals, an NLRP3 trigger, enhanced inflammasome activation at low temperatures.

NG and MSU crystals are all NLRP3 triggers, while the active modes differ [5]. NG interacting with pannexin-1 acts as a potassium ionophore to induce NLRP3 inflammasome assembly. On the other hand, MSU crystals are consumed by cells via phagocytosis, which induces NLRP3 inflammasome activation through lysosomal rupture and cathepsin B release. This study elucidated the effects of temperatures on inflammasome activation in the BMDMs treated with MSU crystals or alum particles and found that the effects of NLRP3 occur through phagocytosis. As shown in Figure 2A, IL-1β secretion in response to MSU crystals and alum particles was increased significantly in the BMDM incubated at 33 °C and decreased in the cells at 39 °C compared to that of the cells at 37 °C. In contrast, the IL-1β secretion elicited by the dsDNA treatment was relatively unaffected by changes in temperature. Although the release pattern of IL-1β differed depending on the trigger, the incubating temperature of the LPS-primed BMDMs during the activation step was sufficient to regulate the phagocytosis-mediating inflammasome activation. Furthermore, the formation of ASC pyroptosome, the other readout of inflammasome activation, was observed in response to MSU crystals at 33 or 37 °C (Figure 2B). As expected, the MSU-treated BMDMs at 33 °C showed more Casp1 (p20) secretion and the formation of ASC pyroptosome than that at 37 °C. Overall, a lower temperature might augment inflammasome activation in response to MSU crystals.

The effects of a temperature change on the priming step of inflammasome activation were examined further. BMDMs were primed with LPS at different temperatures (25, 33, 37, 39, and 42 °C), and the levels of NLRP3, pro-IL-1β, and pro-Casp1 were observed (Figure 3A,B). As a result, all the proteins were expressed abundantly at 37 and 39 °C. On the other hand, the expression of NLRP3 and pro-IL-1β decreased at 33 °C and was barely detectable at 25 and 42 °C. The pro-Casp1 protein was also lower at 42 °C, similar to the others, but it was not tightly regulated by LPS priming. Hence, it was found that incubation at 37 °C was optimal for the priming step, and inflammasome activation may deteriorate at the highest temperature (42 °C).

Several inhibitors of the NLRP3 inflammasome pathway were adopted to determine if the MSU crystals at lower temperatures activated NLRP3 inflammasome (Figure 4). LPS-primed BMDMs were treated with MSU crystals at 33 or 37 °C in the presence of inhibitors. As a result, Z-VAD-FMK (a pan-Casp inhibitor), a KCl solution (a blocker of potassium efflux), DPI (an inhibitor for ROS production), cytochalasin D (a phagocytosis inhibitor), and CA-074 Me (a cathepsin B inhibitors) inhibited IL-1β secretion in a dose-dependent manner [8,16]. On the other hand, the levels of IL-1β differed significantly between temperatures; the IL-1β released at 33 °C was significantly higher than at 37 °C. Next, the effects of temperature on phagocytosis were examined. BMDMs were incubated in the medium containing two sizes of fluorescent beads for 1 or 6 h, and the beads consumed by BMDMs were measured by fluorescence (Figure 5A). As a result, phagocytosis was not altered by temperature. Thus, a lower temperature may enhance IL-1β secretion only via the NLRP3 inflammasome pathway.

This study focused on the solubility of MSU depending on temperature. According to the literature [17], the optical density between 280 to 320 nm increased depending on the concentration of the MSU solution in DW (Figure 5B). The MSU crystals were mixed with a medium (i.e., RPMI 1640) in order to trigger NLRP3 inflammasome and were incubated at different temperatures (4 to 65 °C). After 3 h, each MSU mixture was filtered to remove the crystals, and the optical density was measured (Figure 5C). The MSU levels increased with increasing temperatures. As such, a lower temperature formed more crystals. Overall, the lower temperature during the activation step might augment NLRP3 inflammasome activation by forming more MSU crystals.