Murine peritoneal macrophages cell death results under variant temperature and time length conditions
Cultured murine peritoneal macrophages were exposed to 38oC, 40oC, and 42oC for 1h, 2h, and 4h respectively. A cell viability assay was performed to evaluate the survival rate of cells under different condition combinations. As shown in Figure 1a, without the presence of LPS, the cell viability of 42oC (4h) was 84.0%±3.0% which was significant increased compared with 40oC (4h), (55.1%±2.0%). In contrast, there was no difference between 42oC (2h) and 40oC (2h). Same null difference was also found between 42oC (1h) and 40oC (1h). Under the temperature of 42oC, LPS treatment caused considerable peritoneal macrophages loss in either 2h or 4h compared with non-LPS treatment.
Cell death was also assessed by measuring the release of LDH in Figure 1b. In the condition of 38oC (4h), LPS treatment induced significant LDH level increase compared with non-LPS treatment. The significant influence of LPS was also observed in the environment of 40oC (4h). On the contrary, LPS usage failed to generate significant change when the heat duration shorten from 38oC (4h), 40oC (4h) to 38oC (2h), 40oC (2h).
Murine peritoneal macrophages pro-inflammatory cytokines production results under variant temperature and time length conditions
ELISA analysis of the pro-inflammatory cytokines of IL-1β, IL-6, and TNF-α in supernatant was shown in Figure 2. In Figure 2a, it is interesting to find that the IL-1β level was not significantly altered regardless of different temperature and time length combinations, this result demonstrated that the presence of LPS was the single most dominant factor on IL-1β production.
In the result of IL-6 (Figure 2b), the result seemed to be the same with IL-1β except for the condition of 42oC (4h). Under LPS condition, when compared 42oC (4h) and 42oC (2h), the result showed unexpectedly decreased IL-6 production in longer tome duration. In Figure 2c, LPS treatment also played principle role in TNF-α production. With LPS treatment, there was no significant difference was found between different time length under the temperature of either 38oC or 40oC. Moreover, with the presence of LPS and a temperature condition of 42oC, 42oC (1h) indicated a significantly high level of TNF-α in comparison with 42oC (2h) and 42oC (4h).
Combined with cell death and pro-inflammatory cytokines results, we chose the 40oC (4h) as the desired condition in following vitro study.
Heat stroke promoted NLRP3 inflammasome activation in murine peritoneal macrophage
In order to test whether NLRP3 inflammasome was activated in vitro under above heat stroke condition, we created four groups: control/no heat, control/heat, LPS/no heat and LPS/heat groups. NLRP3 gene transcription and protein expression were the crucial step for the formation and activation of the NLRP3 inflammasome . Therefore, both NLRP3 transcription and expression levels were investigated. LPS administration significantly induced NLRP3 gene transcription in LPS/no heat mice compared with control/no heat (p<0.01, figure 3a). The LPS/heat mice exhibited significantly increased NLRP3 and pro-IL-1β mRNA levels when compared with that of the LPS/no heat mice (both p<0.01, figure 3a and 3b).
In the protein expression analysis, our result showed LPS alone greatly increased NLRP3 production, LPS/heat treatment significantly augment this effect (p<0.01, figure 3d). IL-1β protein was the main product for NLRP3 inflammasome activation for that reason IL-1β was also measured. Compared with the control/no heat and control/heat groups, LPS/heat group demonstrated considerably increased IL-1β expression (p<0.01, figure 3e). Concerning the caspase-1 p10 which was the major effector protein of NLRP3 inflammasome activation, our result suggested that there was almost no detectable caspase-1 p10 production in control/no heat group. LPS/heat group increased caspase-1 p10 generation when compared with either control/heat or LPS/no heat and suggested an intense activation of NLRP3 inflammasome. After comprehensive consideration of the results, we observed control/heat, LPS/no heat could induce NLRP3 and IL-1β expression in varying degrees but were far from enough to start NLRP3 inflammasome assembly. However, LPS/heat prompted not only NLRP3 inflammasome elements expression but also activation.
Nlrp3 knock out protected mice from heat stroke induced murine peritoneal macrophages death and IL-1β releasing
To determine the role of NLRP3 inflammasome in heat stroke induced cell death, murine peritoneal macrophages were isolated from Nlrp3-/- mice and then exposed to LPS/heat treatment. Using this strategy, we observed that Nlrp3-/- macrophages exhibit resistance to heat stroke induced cell death (figure 4a). Knock out of Nlrp3 rescued heat stroke induced cell death from 80.27±2.09% to 87.39±2.09% (p<0.05). A test of the supernatant LDH showed that knock out of Nlrp3 suppressed heat stroke induced LDH release from 229.30±9.79% to 200.17±8.67% (p<0.05, figure 4b). Furthermore, ELISA results indicated that the production of IL-1β induced by heat stroke was significantly lessened by knockout of Nlrp3 from 278.12±29.44pg/ml to 26.30±4.80pg/ml to in murine peritoneal macrophage from (p<0.01, figure 4c).
LPS compromised survival time and heat tolerance under heat stroke.
In vivo study, animals were also divided into four groups: control/no heat, control/heat, LPS/no heat and LPS/heat groups. The survival study result was drawn into curve in figure 5a. All of the mice in control/no heat and LPS/no heat group that were not exposed to heat survived. The duration of survival was the shortest in the LPS/heat mice (84.24±4.84 min), followed by the control/heat mice (165.42±5.32 min) and the log-rank test found significant difference between these two groups (p<0.01).
The mean time duration for TC to increase from resting to 42°C were measured every 15 minutes in four groups. The TC of control/no heat and LPS/no heat mice that accommodated in normal temperature remained stable in the experiment procedure. To reach 42°C, it took 80±4.3 min to reach 42°C in LPS/heat mice, 120±5.3 min in control/heat mice and these heating durations were signiﬁcantly different (p<0.01, figure 5b).
Heat stroke promoted NLRP3 inflammasome activation in murine hypothalamus
We further test whether NLRP3 inflammasome was activated in vivo under heat stroke condition. It has been suggested that hypothalamus was the central nerve region in temperature regulation. Its damage plays a crucial role in temperature disturbances, such as fever or hypothermia . So mice hypothalamus was dissected and prepared for RT-PCR and Western Blot analysis.
NLRP3 and pro-IL-1β mRNA levels were measured by using RT-PCR. As shown in figure 6a and 6b, control/heat mice failed to demonstrate significant increase compared with control/no heat mice. Nevertheless, LPS/no heat mice showed significant increased level of both NLRP3 and pro-IL-1β in comparison with control/no heat mice. LPS/heat mice exhibited significantly increased NLRP3 and pro-IL-1β mRNA levels in the hypothalamus when compared with that of the control/no heat mice (p<0.01). We then performed western blot to detect the target protein: NLRP3, caspase-1 p10 and IL-1β. The blot result was shown in figure 6c. In NLRP3 expression, LPS/Heat mice showed remarkable increase when compared with control/no heat mice. Once NLRP3 was activated, pro-IL-1β was cleaved by caspase-1 p10 in to IL-1β, so caspase-1 p10 and IL-1β were measured. In caspase-1 p10 and IL-1β, though LPS administration alone could to some extent induce their production. LPS/Heat mice illustrated significant caspase-1 p10 and IL-1β increase in comparison with LPS/no heat mice which provided a strong evidence of NLRP3 inflammasome assembly (p=0.03 for caspase-1 p10, figure 6e and p=0.02 for caspase-1p10, figure 6f). To further detect NLRP3 inflammasome activation, immunofluorescence was used and similar result was obtained. Co-localized of NLRP3 (red) and caspase-1 p10 (green) protein was also significantly increased in the LPS/Heat mice compared with any other groups (figure 6g).
Nlrp3 knock out protected mice from heat stroke-induced mice death by inhibiting IL-1β releasing
To address whether Nlrp3 knock out can overcome the lethal effect of heat stroke, Nlrp3-/- mice was monitored in comparison with WT mice. In the survival study, Nlrp3 knockout was associated with significantly improved survival after LPS/Heat treatment compared with WT LPS/Heat group (average survival time: 96.66±5.14 min versus 149.94±4.94 min respectively, figure 7a, log-rank p<0.01). The mean time duration taken for TC to increase from resting to 42°C was significantly longer in Nlrp3-/- mice (83±6.15 min versus 134±6.60 min, p<0.01, figure 7b). In order to further test the effect of Nlrp3 knock out in heat stroke mice, mice serum was collected and IL-1β, IL-6 and TNF-α were measured. As expected, the result indicated significant reduced IL-1β in Nlrp3-/- mice compared with WT mice under LPS/Heat condition (figure 7c). In contrast to IL-1β, there was no difference observed in IL-6 and TNF-α.
We then further explored the in situ IL-1β production in hypothalamus by using immunohistochemistry assay. The immunohistochemistry result was shown in figure 7f, after quantification of multiple histological sections we found significantly decreased IL-1β production in Nlrp3-/- mice hypothalamus.
IL-1β neutralizing antibody inflated heat intolerance induced by heat stroke
To further investigate the role of the IL-1β in heat stroke induced damage, IL-1β neutralizing antibody was administrate just 30 minutes before heat. IL-1β neutralizing antibody was associated with significantly improved survival time after LPS/Heat treatment with p<0.01 in both 0.04 μg/g and 0.2 μg/g groups compared with saline (figure 6a). Furthermore, the mean duration taken for TC to increase from resting to 42°C was 120±6.3 min for 0.04 μg/g IL-1β neutralizing antibody group, 150±5.2 min for 0.2 μg/g IL-1β neutralizing antibody group (Figure 6b). These heating durations were also signiﬁcantly different when compared with saline group with p<0.01 for each comparison.