HMGB1-induced NLRP3 Inammasome Participating in Platelet Activation and Thrombocytopenia in Heatstroke

Background: Previous studies have suggested that NOD-like receptor family pyrin domain containing 3 (NLRP3) inammasome plays an important role in heat stroke (HS). As a common complication in HS, thrombocytopenia has been widely considered as a good predictor of HS-related mortality. However, little is known about the relationship between inammasome and thrombocytopenia as well as platelet activation in HS. Methods: We established a rat HS model to investigate the roles of NLRP3 inammasome in both platelet activation and thrombocytopenia, platelet activation was reected with Flow cytometry while thrombocytopenia was measured by platelet count. The colocalization of NLRP3 inammasome was detected by confocal uorescence microscopy. Mitochondrial-derived reactive oxygen species (ROS) were detected using the molecular probes. Plasma HMGB1 levels were measured by ELISA. Results: Activation of the inammasome was detected in platelet of rats in HS. Elevated ROS activated NLRP3 inammasome in HS group could signicantly induce platelet activation and thrombocytopenia. The upregulated P-selectin (CD62P ) and decreased platelet count triggered by NLRP3 inammasome were attributed to the high mobility group box protein 1 (HMGB1) in plasma. Moreover, inhibition of HMGB1, caspase-1, NLRP3, or ROS in rats with HS suppressed platelet activation and the decline of platelet count. Similar results were obtained when the receptor toll-like receptor 4 (TLR4) / advanced glycation end product (RAGE) was blocked. Conclusions: This study indicated that platelets were activated by NLRP3 inammasome through TLR4/RAGE/HMGB1 signaling pathway. The NLRP3 inammasome might be the potential target for HS treatment. results indicate that both TLR4 and RAGE receptor-specic antibodies can inhibit activation of the NLRP3 inammasome, platelet activation and thrombocytopenia. Also, anti-TLR4 antibody pretreatment signicantly shortened the heat exposure and time to HS onset in rats. These results suggest that HMGB1 activates the NLRP3 inammasome via TLR4 and RAGE receptors and participates in platelet activation and thrombocytopenia.


Background
Heat stroke (HS), a life-threatening disease with the core body temperature rising above 40.5℃, often develops multiple organ dysfunction syndrome, which is dominated by central nervous system dysfunction and causes high rates of mortality and disability [1,2]. Thrombocytopenia, a common phenomenon that occurs within 24 h after HS onset, could prognose the higher mortality rates [3].
However, little is known about the mechanism of thrombocytopenia in HS.
Platelets, mainly involved in hemostasis and thrombosis, also play important roles as in ammatory cells in innate and adaptive immune responses [4,5]. The NOD-like receptor family pyrin domain containing 3 (NLRP3) in ammasome is involved both in the initiation of many in ammatory responses and thrombosis under many insults [6,7]. A recent in vitro study showed that the NLRP3 in ammasome contributed to platelet activation, aggregation, and consequently thrombus formation [7]. Hottz et al. found that activated platelets, with increased expression of P-selectin (CD62P), could activate NLRP3 in ammasome and release increased interleukin-1β (IL-1β), which is associated with thrombocytopenia in patients with dengue fever [7]. IL-1β, an important in ammatory factor mediated by NLRP3-dependent caspase-1 activation, has been linked to increased endothelial permeability [8,9], thrombosis, and dysregulated hemostasis in dengue fever [10]. Signi cantly increased IL-1β levels had been found in HS [11]. However, whether IL-1β induced by NLRP3 in ammasome is involved in platelet activation and thrombocytopenia in HS is not yet clear.
High mobility group box protein 1 (HMGB1), a typical damage-related molecular pattern molecule, is often transferred from the nucleus to the cytoplasm and can be extracellularly released under many stimuli [12]. We previously found that elevated plasma HMGB1 levels in early HS patients prognosed the severity and mortality of disease [13]. More importantly, Gen et al. [11] found that HS induced liver injury via HMGB1-induced NLRP3 in ammasome as well as the release of IL-1β . It is well known that HMGB1 plays a role in regulating platelet activation, microparticle secretion, adhesion, and thrombus formation [14,15]. Mice lacking HMGB1 in platelets exhibited increasing bleeding time and reduced formation of thrombus, platelet aggregation, in ammation, and organ damage during experimental trauma/hemorrhagic shock [14]. Unfortunately, whether HMGB1-induced NLRP3 in ammasome participates in platelet activation and thrombocytopenia in HS is still unknown.
In this study, using a rat HS model, we demonstrated that HMGB1-induced NLRP3 in ammasome was activated via toll-like receptor 4 (TLR4) and the receptor for advanced glycation end product (RAGE) signaling pathway, and contributed to platelet activation and thrombocytopenia. The ndings of this study suggest that targeting the in ammasome may represent a novel therapeutic strategy to recover from a depletion of platelets in HS.

Experimental animals
Because estrogen has certain effects on HS pathogenesis and organ injury [16], only adult male, pathogen-free Sprague-Dawley (SD) rats (Experimental Animal Center of the General Hospital of Southern Theatre Command, license number for animal experimentation: SCXK, GuangDong 2016-0041), weighing 220 to 250 g, were used in this study. The rats received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23, 1985 revision). We adhered to the guidelines for animal care of the General Hospital of Southern Theatre Command. All procedures were approved by the Institutional Animal Care and Use Committee before the experiments were conducted.

Rat model of HS
Rats were housed for 6 h at ambient temperature (25℃ ± 0.5℃) with a humidity of 35% ± 5% and were randomly divided into 21 groups, with 6 rats per group. To induce HS, rats were placed in a prewarmed incubator maintained at 39.5℃ ± 0.2℃ with a relative humidity of 60.0% ± 5.0%. The rectal temperature (Tr) was monitored at 10-min intervals using a thermocouple (BW-TH1101; Biowill, Shanghai, China), which was inserted 6.5 cm into the rectum of each rat. The time point at which the Tr reached 43℃ was considered a reference point of HS onset [11]. The rats were removed from the incubator after HS onset, transferred to room temperature (22.0℃ ± 0.5℃) with ice-cold saline (0.09%, 20ml) and were fed with adequate food and water. The rats in control group were sham-heated at a temperature of 25℃ ± 0.5℃ and a humidity level of 35% ± 5%. Ethyl pyruvate (EP, 50 mg/kg body weight, MCE, USA) was injected intraperitoneally (i.p.) to inhibit HMGB1 release, or phosphate buffered solution (PBS) were injected as a negative control before rats subjected to heat stress. A neutralizing antibody against HMGB1 (3 mg/kg body weight, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or nonimmune control IgG were immediately injected i.p. into the rats before heat stress. In some rats, TLR4 neutralizing antibody (8 µg/kg body weight, Abcam, Cambridge, MA, USA), RAGE neutralizing antibody (80 µg/kg body weight, R&D, USA), or both were injected i.p. before rats subjected to heat stress. Other rats were injected i.p. with the caspase-1 inhibitor Ac-Tyr-Val-Ala-Asp-chloromethylketone (ac-YVAD-cmk, 0.3 mg/kg body weight, Enzo Biochem Inc., New York, USA) or PBS containing 2.8% dimethyl sulfoxide (DMSO) as a control before rats subjected to heat stress. Some rats were given MCC950 (20 mg/kg body weight, Selleckchem, Houston, TX, USA), a speci c blocker of NLRP3, 1 h before heat stress. Some rats were injected i.p. with the antioxidant N-acetylcysteine (NAC, 300 mg/kg body weight, MCE) immediately before heat stress.

Blood collection and platelet count
Rats were anesthetized by inhalation of iso urane. Blood was collected from the abdominal aorta, and 50 μL of whole blood was collected into a sodium citrate anticoagulation tube to measure the platelet count using a blood cell analyzer (BC-3000, Mairui, Shenzhen, China).

Plasma HMGB1 level measurement
Plasma HMGB1 levels were measured by a commercially available HMGB1 enzyme-linked immunosorbent assay kit according to the manufacturer's instructions (Shino-Test Corporation, Sagamihara, Japan).

Statistical analysis
All data were presented as the mean ± SD unless stated otherwise. Statistical signi cance was determined with the least signi cant difference t test or one-way analysis of variance (ANOVA) using GraphPad Prism 5.0 software (GraphPad, San Diego CA, USA). P < 0.05 was considered statistically signi cant.

HS-induced platelet activation and thrombocytopenia
The platelet count began to decline at 3 h after HS onset (Fig. 1A), whereas the expressions of IL-1β (Fig.  1D) and CD62P (Fig. 1E) in platelets increased progressively during the same period after HS onset. All changes reached peak at 9 h after HS onset. The platelet parameters such as mean platelet volume (MPV) and platelet distribution width (PDW) were assessed. MPV (Fig. 1B) and PDW (Fig. 1C) at 3 h and 6 h after HS were within normal ranges, with no statistical differences comparing to the sham group.
However, both values increased signi cantly compared to those in the control group after 9 h of HS (P < 0.01). Therefore, for the remaining experiments, we observed heat-stressed animals only at the time point of 9 h after HS onset.

Activation of NLRP3 in ammasome in platelets from rats with HS
The association of NLRP3 and ASC as well as caspase-1 represents activation of the in ammasome. The colocalization of NLRP3 (green) and ASC (red) in platelets was observed with a confocal microscopy (Fig. 2C). In addition, ow cytometric analyses showed that NLRP3 ( Fig. 2A) and cleaved caspase-1 (Fig.  2B) increased at 3 h after HS onset and reached peak levels at 9 h after HS onset. All results indicate the HS-inducing NLRP3 in ammasome activation in platelets.

NLRP3 in ammasome mediates platelet activation and thrombocytopenia in HS
To investigate whether HS-activated NLRP3 in ammasome mediates platelet activation and thrombocytopenia, we injected rats i.p. with MCC950, a selective inhibitor of the NLRP3 in ammasome, before the rats being subjected to heat stress. The results showed that inhibited NLRP3 in ammasome signi cantly down-regulated the levels of cleaved caspase-1 (

HMGB1 is involved in platelet NLRP3 in ammasome activation in HS rats
Previous studies found that increased extracellular HMGB1 levels early in HS were negatively correlated with its prognosis [11,19]. HMGB1 activates the NLRP3 in ammasome in liver cells, which in turn promotes hepatocyte pyroptosis [11]. Consistent with previous studies (13), we found that the concentration of HMGB1 in plasma of HS rats gradually increased (Fig. 4A). We also unraveled the role of HMGB1 in inducing NLRP3 in ammasome activation, platelet activation, and thrombocytopenia in HS.
We injected rats i.p. with EP to inhibit HMGB1 release and used anti-HMGB1 neutralizing antibody to block the effect of HMGB1 before submitting rats to HS.
EP inhibiting or neutralizing antibody against HMGB1 signi cantly reduced the expression of NLRP3 (Fig. 4B) and cleaved caspase-1 (Fig. 4C) at 9 h after HS onset. Notably, levels of IL-1β (Fig. 4D), expression of CD62P (Fig. 4E), and thrombocytopenia (Fig. 4F) at 9 h after HS onset were signi cantly alleviated after pretreatment with EP and anti-HMGB1 antibody. These results indicate that HMGB1 plays an important role in the activation of the NLRP3 in ammasome in platelets and participate in platelet activation and thrombocytopenia in HS.
In the early stages of sterile in ammation, HMGB1 activates the NLRP3 in ammasome through TLR4 or RAGE receptors [20]. As shown in Figures 4G-I, expressions of TLR2, TLR4, and RAGE in platelets were gradually increased after HS onset. To de ne the receptors that mediate HMGB1-induced NLRP3 in ammasome activation, we applied anti-TLR4 and anti-RAGE neutralizing antibodies before heat exposure. Either anti-TLR4 or anti-RAGE neutralizing antibody signi cantly inhibited HS-induced NLRP3 (Fig. 4J) and cleaved caspase-1 (Fig. 4K) expression in platelets at 9 h after HS onset. In addition, the combination of anti-TLR4 and anti-RAGE neutralizing antibody induced greater inhibition of NLRP3 and cleavage of caspase-1 expression. Synchronous changes were also observed in HMGB1-induced upregulated expression of IL-1β (Fig. 4L), CD62P (Fig. 4M), and thrombocytopenia (Fig. 4N) at 9 h after HS.
These data indicate that both TLR4 and RAGE receptors are involved in activation of NLRP3 in ammasome in platelet and consequently are associated with platelet activation and thrombocytopenia in HS.
HMGB1 activates platelet NLRP3 in ammasome by upregulating ROS in HS rats ROS mainly include the superoxide anion (O2-), hydrogen peroxide (H2O2), and the hydroxyl radical [HO- ; 21]. Many studies con rmed that ROS can be regulated by HMGB1 [22,23], and is the key factor affecting the activation of NLRP3 in ammasome [24][25][26]. However, the effect of HS-induced ROS on NLRP3 in ammasome is not yet clearly understood yet.
To study the changes of ROS in platelets and the effect of ROS on the HMGB1-induced NLRP3 in ammasome in HS, we used a uorescence probe to detect the expression of mitochondrial O 2 •-, cytoplasmic O 2 •-, and H 2 O 2 in platelets of rats with HS. We found that mitochondrial O 2 •- (Fig. 5A), cytoplasmic O 2 •- (Fig. 5B), and H 2 O 2 (Fig. 5C) increased at 3 h after HS onset. Inhibiting HMGB1 signi cantly reduced the level of ROS, which indicates that HMGB1 is involved in high-level of ROS in HS (Fig. 5D-F).
To explore the relationship between ROS and the NLRP3 in ammasome activation, we pretreated rats with the antioxidant NAC. In platelets, NAC signi cantly inhibited the expression of NLRP3 (Fig. 5G) and cleaved caspase-1 (Fig. 5H) at 9 h after HS onset. Similar results were observed for IL-1β (Fig. 5I), CD62P (Fig. 5J), and thrombocytopenia (Fig. 5K). These results indicate that ROS is responsible for platelet NLRP3 in ammasome-induced activation and thrombocytopenia in HS.

Discussion
Thrombocytopenia is commonly observed and associated with poor clinical outcomes in HS (3). The mechanism by which HS induces thrombocytopenia and platelet activation remains unclear. Here, we uncovered an important role of the NLRP3 in ammasome in mediating platelet activation and thrombocytopenia in a rat HS model. We demonstrated that high levels of extracellular HMGB1 induced by HS activate the NLRP3 in ammasome through TLR4 and RAGE pathway. This activation in turn promotes platelet activation and thrombocytopenia (Fig. 6).
Gader previously demonstrated that thrombocytopenia appeared in patients with mild HS and that it was more pronounced when body temperatures increased above 41°C. Furthermore, when the temperature increased within a certain range, the aggregation rate of platelets were also increased [27]. Platelet aggregation plays a key role in hemostasis and thrombosis, and it may be involved in HS-induced thrombocytopenia as well. On the basis of our results, we speculate that HS-induced thrombocytopenia is possibly caused by platelet activation and depletion.
MPV and PDW are indices of platelet size in circulation, MPV measures the average platelet volume, while PDW describes the variation in platelet size [28], both of which are markers of platelet activation and thrombocytopenia [29,30]. In this experiment, we found that MPV and PDW in rats with HS did not change at 3 h but were increased signi cantly at 9 h after HS onset. Previous study showed that thrombocytopenia in patients with HS without damage to megakaryocytes in bone marrow [27]. Combined with the changes in platelet counts and platelet activation, we speculate that thrombocytopenia in rats with HS may not be related to impaired platelet production in bone marrow but is mainly caused by peripheral platelet consumption.
Zhuang et al. found that heat stress facilitated the procession of Px-caspase-1 (a caspase gene) expression, an important effector molecule of the NLRP3 in ammasome [31]. One team in our institute previously found that HMGB1-induced NLRP3 in ammasome activation in liver cells during HS was associated with liver damage [11]. Hottz et al. [7] also reported NLRP3 in ammasome activation in platelets of patients with dengue fever. Pranav et al. [32] found that NLRP3 in ammasome affects platelet activation, aggregation, and thrombosis. Therefore, it is speculated that the HMGB1-induced NLRP3 in ammasome could mediate platelet activation and thrombocytopenia in HS. We observed that the expressions of NLRP3, ASC, and cleaved caspase-1 in platelets in rats with HS were increased early during HS onset and remained increased thereafter ( Fig. 2A, B, C). By laser confocal microscopy, we con rmed that the NLRP3 in ammasome assembly presented at 9 h after HS onset ( Fig.  2A). All these results re ect the assembly and activation of NLRP3 in ammasome in HS. Consistent with the results of a previous study [32], we found that inhibition of NLRP3 and cleavage of caspase-1 alleviated platelet activation and thrombocytopenia (Fig. 3), indicating that the NLRP3 in ammasome possibly plays an important role in platelet activation and thrombocytopenia. HMGB1, a class of non-histone chromosomal binding protein present in eukaryotic cells, has been extensively studied as a transcription factor and growth-promoting factor [12] and an important molecule that can activate NLRP3 in ammasome [33]. Our previous studies showed that plasma HMGB1 was signi cantly elevated at an early stage in HS, and was associated with more critical disease [13]. Inhibiting HMGB1 release or blocking HMGB1 can alleviate liver sinusoidal endothelial damage and reduce thrombosis in HS [19,34]. In this experiment, we also found that inhibiting HMGB1 can inhibit the activation of the NLRP3 in ammasome, platelet activation, and thrombocytopenia in HS. Previous studies have showed that HMGB1 promotes in ammation via the main receptors, such as TLR2 and TLR4 [35,36]. Because there was no anti-TLR2 neutralizing antibody for rats, we only tested the effects of TLR4 and RAGE receptors. It has been reported that RAGE receptors are involved in mediating biological functions outside the cell and the binding of the receptors to their ligands cannot independently conduct intracellular signaling pathways but requires interactions with other receptors [37,38]. Different to the results of a prior study that visfatin-induced disruption on junction proteins of mouse vascular endothelial cells was conducted through the HMGB1-RAGE, rather than the HMGB1-TLR4 pathway [39], our results indicate that both TLR4 and RAGE receptor-speci c antibodies can inhibit activation of the NLRP3 in ammasome, platelet activation and thrombocytopenia. Also, anti-TLR4 antibody pretreatment signi cantly shortened the heat exposure and time to HS onset in rats. These results suggest that HMGB1 activates the NLRP3 in ammasome via TLR4 and RAGE receptors and participates in platelet activation and thrombocytopenia.
ROS have an important role in the homeostasis of aging and death of platelets in internal and external environments [23], which are involved in platelet activation, adhesion, and aggregation and cause platelet apoptosis [21]. Studies have con rmed that HS can lead to an explosive increase in intracellular ROS [40]. In our experiments, we found that 3 kinds of ROS in the platelets of rats with HS increased progressively with time ( Fig. 5A, B, C). HMGB1 can upregulate intracellular levels of ROS in many diseases including HS [41]. We found that inhibition or blockade of HMGB1 could partially inhibit the production all of 3 kinds of ROS (Fig. 5D,E), suggesting a mechanism of HMGB1-induced ROS in the rat model of HS. ROS induced by HMGB1 is known to be a key mechanism for triggering NLRP3 in ammasome formation and activation in response to many exogenous stimuli [25]. Many researchers in recent years have shown that elevated intracellular ROS promote the activation of the NLRP3 in ammasome [42,43]. We found that NAC pretreatment could inhibit the expression of platelet NLRP3 and cleaved caspase-1 in rats with HS, indicating that ROS can upregulate the activation of the NLRP3 in ammasome in platelets.
Consistent with previous studies, we found that NAC could inhibit platelet activation and thrombocytopenia in rats with HS. Mitochondria is the main source of ROS, and a large amount of ROS can damage mitochondria, causing changes in mitochondrial membrane permeability that in turn triggers autophagy, apoptosis, and necrosis [44,45]. Here, we observed the ultrastructural changes of platelets in HS by transmission electron microscopy and found mitochondria swelling, cristae vague, autophagy (supplementary Fig. A), and platelet necrosis (supplementary Fig. B). However, whether the thrombocytopenia in rats with HS is related to platelet death caused by ROS and which type of death is dominant needs further study.
Pyroptosis, a caspase-1 dependent programmed cell death [11], possibly occurs in platelets because of caspase-1 activation and thrombocytopenia in rats with HS. During the time course of the study, thrombocytopenia and caspase-1 activation showed opposite trends. Typically, both activated caspase-1 and PI positive are both used to de ne pyroptosis in eukaryotic cells; however, because platelets have no nuclei, we cannot use this de nition to describe pyroptosis of platelets. However, the relationship between cleaved caspase-1 and thrombocytopenia implies that a link may exist between caspase-1 and platelet death. It has been reported that HS can cause hepatocyte pyroptosis [11]. In this study, by transmission electron microscopy, we found a catheter expansion, a vacuole as one of the characteristics of pyroptosis, in the platelets of rats at 9 h after HS onset (supplementary Fig. C). However, whether HS can cause platelet pyroptosis is still unknown and needs further research.

Conclusions
The elevated HMGB1 in HS via both of TLR4 and RAGE receptors induced high level of ROS, which activates NLRP3 in ammasome, ultimately aggravating in ammatory response, platelet activation and thrombocytopenia ( Figure 6).      In the model, HS-induced HMGB1 in plasma acting through TLR4 and RAGE signaling induces high level of ROS, which activates NLRP3 in ammasome, which in turn promotes IL-1βrelease, platelet activation and thrombocytopenia.

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