NLRP3 ablation enhances heat tolerance in endotoxemic mice by inhibiting IL-1β-mediated neuroinammation

Background: Patients with pre-existing illness are more vulnerable to heat stroke-induced injury, but the underlying mechanism is unknown. Recent studies suggested that NLRP3 inammasome played an important role in the pathophysiology of heat stroke. Methods: In this study, we exposed primary murine peritoneal macrophages to different temperature and time length combinations with and without lipopolysaccharide (LPS: 20ng/mL). Cell viability, lactate dehydrogenase (LDH) and pro-inammatory cytokine were measured. We also established heat stroke mice model with preexisting endotoxemia (LPS: 1 mg/kg). Mice survival analysis curve and core temperature (T C ) elevation curve were produced. In vitro, NLRP3 inammasome activation was measured in vitro by using Real-time PCR and Western blot. In vivo, mice hypothalamus was dissected and NLRP3 inammasome activation was also explored. To further demonstrate the participation of NLRP3 inammasome, Nlrp3 knock out mice were used. In addition, IL-1β neutralizing antibody was injected to test whether its effect on heat stroke induce injury. Results: LPS administration was found to exacerbate macrophage damage and heat stroke-induced injury in mice. LPS administration also intensively activated NLRP3 inammasome and increased the production of IL-1β and Caspase-1 p10 in heat stroke group both in vitro and in vivo. Nlrp3 knockout protected peritoneal macrophage from LPS/heat treatment and reduced IL-1β release in primary mice macrophages in vitro. The use of Nlrp3 knock out mice further enhanced heat tolerance and alleviated heat stroke-induced death by reducing mice hypothalamus IL-1β production in given heat stroke condition. Furthermore, IL-1β neutralizing antibody injection signicantly extended endotoxemic mice survival under heat stroke. Conclusions: Based on the above results, we concluded that the NLRP3 inammasome mediates the exacerbation of heat stroke-induced injury in preexisting endotoxemia, and antagonizing NLRP3 inammasome activity might be a potential


Introduction
Heatstroke is characterized clinically by severe hyperthermia, de ned as a core temperature of > 40.5 °C.
The manifestations included headache, sweating, tachycardia, lightheadedness and might soon progress to muscle cramps, oliguria, hypotension, syncope, confusion and coma if the core temperature isn't immediately reduced [1]. Many studies implicated systemic in ammatory response syndromes (SIRS) and central nervous system (CNS) collapse in this disorder [2,3].
Body's tolerance to heat varies widely among individuals. A mild illness develops in response to heat in some people, whereas in others the condition progresses to heat stroke. Widely accepted predisposing factors include prolonged or intense exercise, lack of heat acclimatization, sleep deprivation, dehydration, alcohol abuse, drug abuse, chronic in ammation, febrile illness and older age [4]. Among all the factors, preexisting illness such as endotoxemia from elevated plasma lipopolysaccharide (LPS) was believed to play a crucial role in heat tolerance impairment and increased susceptibility to heat stroke [5]. What's more, latent infection was a common accompanying factor during heat stroke. Heat stroke was commonly reported to compromise the epithelial junction integrity of intestinal mucosa and induce LPS to leak into the circulating blood stream, which made heat stroke resembled sepsis in many aspects [6].
During the 1995 heat wave in Chicago, a high percentage (57%) of classic heat stroke patients had evidence of infection on admission and the mortality rate was as high as 21 percent among these patients during acute hospitalization [7]. Multiple ideas have been put forth as to how pre-existing endotoxemia compromises heat tolerance and increases the risk of heatstroke during heat exposure, the underlying mechanisms are still controversial.
CNS maintains body temperature during environmental temperature challenges and alters body temperature during the in ammatory response and behavioral states and in response to declining energy homeostasis [8,9]. Thermoregulatory circuitry in the hypothalamus exerts heat defense mechanisms by serious effectors to increase heat loss, such as cardiac output increase, vasodilation, peripheral blood ow increase and sweating [10]. Therefore, CNS dysfunction, in particular hypothalamus thermoregulatory network dysfunction, was identi ed as the initial and driving factor during heat stroke [11].
In the past decades, a variety of studies indicated that the pathophysiologic responses to heat stroke are associated with the systemic in ammatory response syndrome that follows thermal injury, which gutderived endotoxin might be implicated [12,13]. In ammatory cytokines such as interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) play an essential role in the pathogenesis of heat stroke. Elevated concentrations of these cytokines have been found in experimental animals and patients with heat stroke [14][15][16]. NACHT, LRR and PYD domains-containing protein 3 (NALP3) is a cytosolic pattern recognition receptor and could form a caspase-1 activating complex (NLRP3 in ammasome) with the adaptor ASC protein PYCARD, which then regulates the cleavage and maturation of IL-1β and IL-18 [17].
However, to our knowledge, the role of NLRP3 in ammasome in the CNS during heat stroke has also not been fully investigated before. This study aimed to investigate the effect of CNS NLRP3 in ammasome in heat stroke in endotoxemic mice. It was hypothesized that pre-existing LPS compromised heat tolerance through the activation of NLRP3 in ammasome, and Nlrp3 knockout or against IL-1β therapy during heat exposure could increase heat tolerance.

Animals
Wild type (WT) C57BL/6 male mice, 5-7 weeks age, were purchased from Super-B&K Laboratory Animal Corp. Ltd, Shanghai, China. Nlrp3 knockout mice were obtained from the Model Animal Research Centre of Nanjing University (AAALAC accredited) as previously reported [18]. The animals were housed in Speci c Pathogen Free (SPF) animal facility for at least 1 week under a 12-h light/dark cycles (8:00-20:00 light and 20:00-8:00 dark). The mice were acclimatized to room temperature at 22±2°C under relative humidity of 50±5%, with free access to water and standard laboratory chow ad libitum.

Cells isolation and culture
The isolation and culture of murine peritoneal macrophage was performed as Davies et al. described [19].
Mice were given 2ml of 3% thioglycollate. Three days later, they were euthanized using CO 2 and 10 ml of DMEM was used to ush the peritoneal cavity. The ush medium was centrifuged and the cell was collected. The remaining cells were seeded in plates and incubated in complete DMEM medium with 10% FBS and penicillin-streptomycin.

Heat stroke protocol and treatment
In vivo, the methods to induce heat stroke in mice were carried out as previously described with small modi cation because of different animal strain [2]. The mice were moved to the Climatic & Environmental Simulation Center in the Navy Medical University where whole-body heating (WBH) with a condition of 41.2°C, relative humidity 50±5% was used in an environment-controlled cabin. The moment at which core temperature reached 42°C was set as the time point for the onset of heat stroke [20]. The pre-existing LPS groups were intraperitoneally injected with 1 mg/kg LPS 4 hours before heat stroke experiment. Normal saline was administered as control. The experiment started at 09:30 AM, core temperature (T C ) was monitored every 15 min using a digital thermometer (ALC-ET06, Shanghai Alcott Biotech Co., China) which was inserted 2 cm into the rectum. The mice underwent the same experimental procedure as the heat stroke mice with a cabin temperature of 22±2 o C and relative humidity of 50±5% throughout the experiment were used as no heat group. Mice with the IL-1β neutralizing antibody (Cat: AF-401-NA, R&D Systems, Minneapolis, MN) were injected intravenously 30 minutes before heat stroke in two dosages which were 0.04 μg/g and 0.2 μg/g body weight. All animal experiments used in this study were approved by the Institutional Animal Care and Use Committee of Navy Medical University (No. 20120025, Shanghai, China) and all procedures were performed in compliance with the Guide line for Care and Use of Laboratory Animals published by the National Institutes of Health, USA. For in vitro assay, peritoneal macrophage was exposed to different temperatures and different time lengths to attain the desired condition. The temperature was set as 38°C, 40°C and 42°C and time length was 1 hour, 2 hours and 4 hours. LPS treatment condition was 20ng/mL. Cell viability and cytokine levels were measured under different temperature and time length combinations. The following four groups were created both in vivo and in vitro studies: control/no heat, control/heat, LPS/no heat and LPS/heat groups.

Cell viability assay
Thirty thousand cells per well were seeded in to 96-well plates in 100μl of DMEM supplemented with 10% FBS, 0.1 mg/ml Penicillin/Streptomycin (P/S) and incubated as described before. The LPS group cells were treated with 100 ng/mL LPS 6h before. Then, the cells were exposed to 38 o C, 40 o C, and 42 o C for 1h, 2h, and 4h. The number of surviving cells was measured by Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan). Data acquisition was performed on SpectraMax M2e Microplate Spectrophotometer (Molecular Devices, CA). Cell viability was calculated according to the formula: cell viability (%) = [(As−Ab)/(Ac−Ab) × 100%, where As, Ac and Ab represent the A450 in treated, untreated and blank groups, respectively.

Serum collection
The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) at heat stroke time point. Blood samples were harvested from abdominal aorta, and the serum was separated by centrifugation at 3000 rpm for 30 min at 4 o C (Eppendorf 5801R centrifuge, Germany), and then stored at -80 o C for biochemical and enzyme-linked immunosorbent assay (ELISA) analysis.

Biochemical and cytokines measurement
The levels of LDH were determined by HITACHI 7080 automated analyzer (Japan). Commercial ELISA kits were used for the measurement of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and IL-1β (R&D system) using a SpectraMax M2e Microplate Spectrophotometer (Molecular Devices, CA, USA) according to the manufacturer's instruction.
Real-time PCR analysis of NLRP3 and pro-IL-1β mRNA Total RNA was isolated by using Trizol reagent (Life Technologies, USA). Reverse transcription was performed with 1ug total RNA using Transcriptor First Strand cDNA Synthesis Kit (Roche Ltd, Swiss). Total of 2ul first-strand cDNA solution was used for real time RT-PCR in combination with Fast Start Universal Probe Master (ROX) in a nal volume of 20μl. All experiments were run in triplicate and underwent 40 ampli cation cycles by using Applied Biosystems 7500 system (Life Technologies Corporation., USA). The primers used for RT-PCR were listed in Table 1. The threshold cycle (CT) of target product was normalized to internal standard GADPH and calculated by using the comparative cycle threshold (ΔΔCt) method.

Western blot analysis
For Western Blot analysis, Cells were lysed and the protein concentrations were measured as we described previously [15]. Cleared lysates were separated by 10% SDS-PAGE, transferred onto PVDF membranes and then blocked for 2 h at room temperature with 5% nonfat dried milk and incubated with anti-NLRP3 Ab (AdipoGen Corp., San Diego, CA, USA), anti-Caspase-1 (p45) Ab, anti-pro-IL-1β (Abcam, Cambridge, UK) and anti-cleaved-Caspase-1 (p10) Ab (Santa Cruz Biotechnology, lnc., Dallas, TX), anti-IL-1β Ab (Cell Signaling Technology, Beverly, MA, USA) and exposed with an Amersham Imager 600 (GE Healthcare Bio-Sciences, Amersham Biosciences, Piscataway, NJ). The band intensity values of the target proteins were normalized to that of β-actin.
Immunofluorescence staining assay Immuno uorescent staining was performed using mouse brain tissues fixed in a fresh solution of 4% paraformaldehyde (pH 7.4) at 4°C. Coronal sections (30 um) containing hypothalamus were prepared.
The slides were incubated with primary antibodies against NLRP3 and Caspase-1 p10 at 4˚C overnight. DAPI (4,6-diamidino-2-phenylindole) was used for nuclear staining. Then, these slides were incubated with corresponding uorescent dye-conjugated secondary antibodies which were Alexa Fluor 488 and Alexa Fluor 594. Finally, these slides were subjected to uorescent microscopy examination at Guge Biotechnologies Co., Ltd. (Wuhan, China) by using Olympus Research Inverted System Microscope IX71 (Olympus, Japan).

Immunohistochemistry analysis of IL-1β
Formalin-buffered tissue samples were embedded in para n wax and subsequently cut into 2-μm slices, using a rotary microtome. The sections were subjected to an antigen retrieval procedure and then rinsed in distilled water, washed in 0.1 M PB for 10 min and incubated in a blocking buffer (0.5% goat serum S-1000, Vector Labs, in 0.1 M PB) at room temperature for 1 h. They were then incubated overnight at 4 °C in a humidi ed chamber with primary anti-IL-1β ab (Abcam, Cambridge, UK) with blocking buffer. The rest procedures were conducted according to manufacturer's protocols (Vectastain ABC kit elite DK-6100 standard, Vector Labs). Finally, sections were washed, dehydrated and mounted with non-aqueous mounting medium (Permount, Fisher) to perform cell counts. As a negative control, sections were incubated without primary antibodies. The sections were examined using an Olympus AX70 microscope.
For the intensity, the quantitation and score were conducted as previously reported [21].

Statistical analysis
The results are measured and expressed as the mean±SEM. One-way ANOVA was used to detected statistical signi cance among group means and Dunnett's test analysis was used to compare speci c two groups when ANOVA showed signi cant difference. The comparisons made between two groups were evaluated using Student's independent t test. In survival curve, the comparison was conducted by using log-rank test method. All statistical analyses were performed using SPSS 21 software. P<0.05 was considered to be statistically signi cant.

Results
Murine peritoneal macrophages cell death results under variant temperature and time length conditions Cultured murine peritoneal macrophages were exposed to 38 o C, 40 o C, and 42 o C 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 Combined with cell death and pro-in ammatory cytokines results, we chose the 40 o C (4h) as the desired condition in following vitro study.
Heat stroke promoted NLRP3 in ammasome activation in murine peritoneal macrophage In order to test whether NLRP3 in ammasome 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 in ammasome [22]. Therefore, both NLRP3 transcription and expression levels were investigated. LPS administration signi cantly induced NLRP3 gene transcription in LPS/no heat mice compared with control/no heat (p<0.01, gure 3a). The LPS/heat mice exhibited signi cantly increased NLRP3 and pro-IL-1β mRNA levels when compared with that of the LPS/no heat mice (both p<0.01, gure 3a and 3b).
In the protein expression analysis, our result showed LPS alone greatly increased NLRP3 production, LPS/heat treatment signi cantly augment this effect (p<0.01, gure 3d). IL-1β protein was the main product for NLRP3 in ammasome 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, gure 3e). Concerning the caspase-1 p10 which was the major effector protein of NLRP3 in ammasome 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 in ammasome. 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 in ammasome assembly. However, LPS/heat prompted not only NLRP3 in ammasome 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 in ammasome 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 ( gure 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, gure 4b). Furthermore, ELISA results indicated that the production of IL-1β induced by heat stroke was signi cantly 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, gure 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 gure 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 signi cant difference between these two groups (p<0.01).
The mean time duration for T C to increase from resting to 42°C were measured every 15 minutes in four groups. The T C 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, gure 5b).

Heat stroke promoted NLRP3 in ammasome activation in murine hypothalamus
We further test whether NLRP3 in ammasome was activated in vivo under heat stroke condition. It has been suggested that hypothalamus was the central nerve region in temperature regulation [23]. Its damage plays a crucial role in temperature disturbances, such as fever or hypothermia [24]. 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 gure 6a and 6b, control/heat mice failed to demonstrate signi cant increase compared with control/no heat mice. Nevertheless, LPS/no heat mice showed signi cant increased level of both NLRP3 and pro-IL-1β in comparison with control/no heat mice. LPS/heat mice exhibited signi cantly 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 gure 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 signi cant caspase-1 p10 and IL-1β increase in comparison with LPS/no heat mice which provided a strong evidence of NLRP3 in ammasome assembly (p=0.03 for caspase-1 p10, gure 6e and p=0.02 for caspase-1p10, gure 6f). To further detect NLRP3 in ammasome activation, immuno uorescence was used and similar result was obtained. Co-localized of NLRP3 (red) and caspase-1 p10 (green) protein was also signi cantly increased in the LPS/Heat mice compared with any other groups ( gure 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 signi cantly 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, gure 7a, log-rank p<0.01). The mean time duration taken for T C to increase from resting to 42°C was signi cantly longer in Nlrp3 -/mice (83±6.15 min versus 134±6.60 min, p<0.01, gure 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 signi cant reduced IL-1β in Nlrp3 -/mice compared with WT mice under LPS/Heat condition ( gure 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 gure 7f, after quanti cation of multiple histological sections we found signi cantly decreased IL-1β production in Nlrp3 -/mice hypothalamus.

IL-1β neutralizing antibody in ated 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 signi cantly 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 ( gure 6a). Furthermore, the mean duration taken for T C 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.

Discussion
Heat stroke is a severe condition clinically diagnosed as a body temperature elevation with CNS disorder including delirium, seizures an coma. Because of further climate change, heat waves are expected to be longer and more intense in the future and heat stroke-related diseases have drawn increasing attention from public health policies [25]. The risk of heat stroke increases dramatically and even to be lifethreatening in immunocompromised individuals with pre-existing illness, cardiovascular disease, drug use, and poor tness level [26]. Besides from heat stroke itself, patients with latent in ammatory condition were more prone to heat stroke-induced injuries, and the injuries in these patients were often lethal [27]. These facts triggered our interest to explore the underlining mechanism of heat stroke in susceptible individuals. Despite above facts, during heat stroke, the blood is redistributed throughout the body during heat stress, leading to intestinal ischemic damage and increased permeability, which in turn causes numerous intestinal gram-negative bacilli to enter the blood and produce endotoxemia, thus initiating systemic in ammatory response syndrome or multiple organ dysfunction including CNS [28]. Lipopolysaccharide (LPS) treatment was commonly used to mimic bacterial infection and preexisting in ammatory condition in heat stroke [12]. In this study, we established a heat stroke animal model with preexisting endotoxemia, which we believe is a better simulation of clinical scenario in susceptible population.
In the survival analysis and T C elevation experiments based on animal model, we deliberately chose a low dose of LPS (1 mg/kg) to mimic non-symptomatic endotoxemia compared with previous publication [12]. The results con rmed that low dose of LPS used here (1 mg/kg) itself made no difference in the survival analysis and T C change in mice (Fig. 5). However, the preexisting condition could largely amplify the effect of heat stroke as shown in the comparison between control/heat and LPS/heat groups (Fig. 5,  Fig. 6). The above results suggested a promoting effect of in ammatory state on heat stroke injuries which are consistent with what was observed in clinical practice.
The NLRP3 in ammasome is one of the most important components of the innate immunity of the body. NLRP3 can bind to ASC and Caspase-1 to form a complex that promotes the maturation of in ammatory factors such as IL-1β and IL-18 [29]. IL-1β is a single-chain polypeptide glycoprotein that is mainly secreted by macrophages or monocytes. Studies have shown that the mortality of heat stroke was closely related to the level of IL-1β [14]. The injection of IL-1β in rats causes symptoms such as low blood pressure, visceral vasoconstriction, and decreased cardiac output, which are similar to the vital signs in rats with heatstroke [30]. In vivo this study found that short-term heat stoke alone failed to activate the NLRP3 in ammasome, whereas heat stroke in combination with even minor LPS pretreatment greatly activated NLRP3 in ammasome in mice hypothalamus which was further con rmed by immuno uorescence staining assay (Fig. 6). To our knowledge this is the rst study demonstrated CNS NLRP3 in ammasome activation in heat stroke under preexisting illness condition.
In recent years, NLRP3 in ammasome upregulation and activation has been found to be associated in the development of many major diseases such as gout, type 2 diabetes, obesity-induced insulin resistance and depression [31,32]. In particular, the NLRP3 in ammasome is activated in response to cellular stresses through a two-component pathway, involving Toll-like receptor 4-ligand interaction (priming) followed by a second signal, such as ATP-dependent P2X purinoreceptor 7 receptor activation [33]. LPS was the most widely used as the priming agent in the rst process. The rst signal leads to the production of IL-1β and IL-18 precursors and the second signal refers to in ammasome assembly and cleaves of pro-Caspase-1 to its active form Caspase-1 p10, which further cleaves pro-IL-1β to IL-1β. Our team has been interested in the potential role of physical stimuli in NLRP3 in ammasome activation [18,34]. Based on that, we then hypothesized that heat was likely served as the second signal for the activation and maturation of the in ammasome. Therefore, different combination of temperature and time length combination was designed and pro-in ammatory cytokines were measured under each condition. The combination of 40 o C (4 h) was chosen as the desired condition based on the consideration of maximum pro-in ammatory cytokines production and adequate cell survival. Then the hypothesis was con rmed by showing that either LPS or heat stroke alone failed to induce signi cant Caspase-1 p10 or IL-1β maturation. Interestingly, when LPS administration preceded heat, NLRP3 in ammasome was strongly activated and IL-1β was secreted (Fig. 3). This result provided evidence that heat stroke was capable to induce NLRP3 in ammasome assembly and was functionally similar with the second signal.
To further demonstrate the role of NLRP3 in ammasome activation in heat stroke, Nlrp3 knockout mice were used. In vitro study, Nlrp3 knockout reduced macrophages death with low production of LDH and IL-1β (Fig. 4). In vivo, Nlrp3 knockout enhanced mice heat tolerance and extended survival time by decreasing both systemic and hypothalamus IL-1β production, whereas the IL-6 and TNF-α levels in these mice were not different from those of the wild-type mice. This result assured the central pathological role of IL-1β in heat stroke (Fig. 7). Furthermore, we found that the administration of IL-1β receptor neutralizing antibodies signi cantly slowed down T C elevation and prolonged mice survival time under heat stroke. These results, together with other study reporting the participation of IL-1β in heat stroke, indicated that importance of NLRP3/IL-1β pathway and relevant inhibition maybe an alteration for the therapy.
In summary, this study demonstrated CNS NLRP3 in ammasome activation in heat stroke mediated pathophysiological mechanism both in macrophages and in mice. This study rst revealed that knockout of the NLRP3 gene alleviated the exacerbation of heat stroke induced hypothalamic neuronal damage.
This nding not only facilitates the mechanism of heat stroke injuries, but also provides a therapeutic target for the prevention and treatment of heat stroke in immune-compromised individuals. Moreover, this is also the rst study reporting the capability of physical stimulation acting as the second signal in NLRP3 in ammasome activation which greatly deepened our understanding of innate immune system.

Declarations Competing interests
The authors declare that they have no competing interests.
Availability of data and materials The data generated during our study cannot be publicly available due to the data safety concern. But the data are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Band intensities were quanti ed by ImageJ software and the values of the target proteins were normalized to that of β-actin. (e) DAPI (blue), NLRP3 (red) and Caspase-1 p10 (green) were stained by immuno uorescent in hypothalamus sections. The merged image showed the co-localization of the NLRP3 and Caspase-1 p10 proteins, scale bar: 20 μm. The threshold cycle (CT) of target product was normalized to internal standard GADPH and calculated by using the comparative cycle threshold (ΔΔCt) method. Band intensities were quanti ed by ImageJ software and the values of the target proteins were normalized to that of β-actin. The results were expressed as the mean ± SEM. (n=5 to 10). **p<0.01, *p<0.05.

Figure 7
Nlrp3 knock out protected mice from heat stroke-induced mice death by inhibiting IL-1β releasing. The experiment was designed into four groups: control/no heat, control/heat, LPS/no heat and LPS/heat groups. LPS treatment condition was 1mg/kg and whole-body heating (WBH) temperature was 41.2°C.
Systemic pro-in ammatory cytokines and hypothalamus IL-1β were measured by ELISA and immunohistochemistry, respectively. The comparison was conducted between Nlrp3 knock out and wildtype mice. (a) Survival curve was monitored. The statistical analysis was conducted by Log-rank test