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Inflammasome 2014; 1: 76–80 Mini Review Open Access Takato Takenouchi*, Mitsutoshi Tsukimoto, Makoto Hashimoto, Hiroshi Kitani* Inflammasome activation by danger signals: extracellular ATP and pH Abstract: Extracellular ATP has been recognized as a danger signal that alerts the innate immune system. High extracellular concentrations of ATP can trigger the maturation and secretion of pro-inflammatory cytokines (e.g., interleukin-1β and interleukin-18) through the inflammasome-dependent activation of caspase-1. The P2X7 receptor, an ATP-gated cation channel, plays a pivotal role in ATP-induced NLRP3 inflammasome assembly. Recently, intriguing evidence has emerged that acidic extracellular pH acts as a danger signal that activates inflammasomes. Extracellular acidification frequently occurs at sites of inflammation, infection, or injury. In addition, large amounts of ATP are readily released into the extracellular space from damaged cells at such sites. Thus, it is assumed that the ATP/P2X7 receptor pathway regulates the inflammatory response under acidic extracellular conditions. Here, we briefly discuss the mutual effects of extracellular ATP and pH on inflammasome activation and consider their roles in the regulation of inflammation. Keywords: inflammasome; interleukin-1β; extracellular acidosis; P2X7 receptor ATP; DOI 10.2478/infl-2014-0008 Received September 2, 2014; accepted September 22, 2014 1 Introduction Inflammasomes are large intracellular multiprotein complexes that are basically composed of a cytosolic *Corresponding authors: Takato Takenouchi, Hiroshi Kitani: Animal Immune and Cell Biology Research Unit, Division of Animal Sciences, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan Tel&Fax: +81-29-838-6034; E-mail: [email protected], [email protected] Mitsutoshi Tsukimoto: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan Makoto Hashimoto: Division of Sensory and Motor Systems, Tokyo Metropolitan Institute of Medical Science, Tokyo, 156-0057, Japan pattern recognition receptor (PRR) [e.g., nucleotidebinding domain and leucine-rich repeat containing receptor (NLR) protein 1 (NLRP1), NLRP3, NLR family CARD (caspase recruitment domain) domain-containing protein 4 (NLRC4; also known as IPAF), or AIM2 (absent in melanoma 2)] and an inactive procaspase [1]. Some PRR (e.g., NLRP3 and AIM2) require the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) in order to form inflammasomes [2]. Upon PRR stimulation by “danger signals” that are released or elaborated by pathogens or damaged/stressed host cells [3], procaspase-1 is canonically recruited to inflammasomes and converted into its catalytically active form, caspase-1 [2]. Then, active caspase-1 proteolytically cleaves the precursor forms of interleukin (IL)-1β (proIL-1β) and IL-18 (pro-IL-18) into their mature biologically active forms, which are subsequently released into the extracellular milieu through unconventional secretory pathways [4]. Both of these cytokines are initially synthesized as inactive precursor forms in response to inflammatory stimuli, e.g., lipopolysaccharides (LPS), and require post-translational processing by active caspase-1 to exert their biological functions [5]. Since the mature forms of IL-1β and IL-18 are potent proinflammatory cytokines of the innate immune system, inflammasomes are considered to play an important role in the homeostasis of the innate immune system. Moreover, the uncontrolled production and release of these cytokines is linked to various inflammatory conditions [6], suggesting that inflammasomes are potential therapeutic targets for the treatment of inflammation-related disorders [7]. A variety of endogenous and exogenous danger signals have been identified as inducers of inflammasome assembly [8]. Among them, extracellular ATP has been widely recognized as an endogenous danger signal that activates inflammasomes. The P2X7 receptor (P2X7R), an extracellular ATP-gated cation channel, plays a key role in ATP-induced inflammasome activation [9]. Inflammasomes can also sense cellular stress caused by microenvironmental changes such as a reduction in © 2014 T. Takenouchi et al., licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Unauthenticated Download Date | 6/16/17 1:50 PM Inflammasome activation by danger signals: extracellular ATP and pH extracellular osmolarity [10]. In this regard, emerging evidence suggests that extracellular acidosis represents a danger signal that alerts the innate immune system [11]. It is conceivable that extracellular acidosis modulates the innate immune system because its development is a hallmark of inflammatory processes [12]. In this commentary, we summarize the recent advances in our knowledge regarding the involvement of extracellular ATP and pH in the activation of inflammasomes and consider their putative roles in the regulation of inflammation. 2 Extracellular ATP activates inflammasomes via the P2X7R The biological actions of extracellular ATP are generally mediated by cell surface P2R. To date, two types of P2R, ligand-gated P2X ion channels and G protein-coupled P2Y receptors, have been identified [13]. The P2X7R is a member of the P2X subfamily, and its activation by ATP opens cation channels that are permeable to several cations such as K+, Na+, Ca2+, and Mg2+ [14]. P2X7R are abundantly expressed by cells involved in the inflammatory and immune systems, such as monocytes, macrophages, dendritic cells, and T cells. In contrast to other P2R, higher concentrations of extracellular ATP (in the mM range) are required to elicit P2X7R-dependent cellular responses in vitro [15]. Accumulating evidence suggests that the P2X7R plays a critical role in ATP-induced inflammasome activation [9]. NLRP3 inflammasomes are known to sense P2X7R-mediated cytoplasmic signals in monocytes and macrophages [4,16]. It has also been reported that the P2X7R mediates the activation of novel NLRP2 inflammasomes by ATP in human astrocytes [17]. Similar to other poreforming toxins, such as maitotoxin and nigericin, the depletion of intracellular K+ following K+ efflux through P2X7R channels is a key event in the ATP-induced activation of NLRP3 inflammasomes (Fig. 1, left side) [14]. Based on the close association between reactive oxygen species (ROS) and NLRP3 inflammasome activation, it is also suggested that P2X7R-mediated ROS production leads to the activation of NLRP3 inflammasomes [18]. 2.1 Acidic extracellular pH acts as a danger signal via inflammasome activation Local extracellular acidosis is often observed at sites of ischemia and inflammation as well in injured or malignant tissues, probably due to the stimulation of anaerobic 77 glycolysis [19]. Thus, it is plausible that extracellular acidosis plays a role in the regulation of inflammatory processes by modulating the functions of innate immune cells [12]. In this context, acidic extracellular pH (6.5) selectively stimulated the secretion of mature IL-1β from human monocytes without affecting the production of other pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and IL-6 [20,21]. The finding that mature IL-1β secretion by monocytes that were cultured at pH 6.5 was dependent on caspase-1 activity would suggest that inflammasome activation is involved in the abovementioned process [20]. It is worth noting that the latter researchers detected increased pro-IL-1β mRNA expression in the monocytes cultured at pH 6.5, suggesting that the acidic extracellular pH-induced rise in mature IL-1β secretion was partly due to the enhanced synthesis of pro-IL-1β [20]. Recent reports have described the role of acidic extracellular pH in the activation of inflammasomes. Rajamaki and coll. demonstrated that acidic media (pH 6.0-7.0) can trigger the pH-dependent secretion of mature IL-1β via activation of NLRP3 inflammasomes in LPS-primed human macrophages [11]. Since it was demonstrated that alkaline extracellular pH (pH 8.08.5) inversely inhibit the IL-1β secretion induced by several known NLRP3 activators, it has been suggested that the extracellular pH has the bipartite regulatory potential on the activation of NLRP3 inflammasomes [11]. Rajamaki and coll. did not observe any significant change in pro-IL-1β mRNA expression under acidic extracellular conditions [11], supporting the notion that acidic extracellular pH-induced secretion of mature IL-1β results from inflammasome activation rather than the enhanced synthesis of its precursor. Consequently, this study proposes the intriguing concept that extracellular acidosis acts as a novel endogenous danger signal that alerts the innate immune system via NLRP3 inflammasomes [11]. Rapid intracellular acidification caused by extracellular acidosis seems to be important for acidic pH-dependent inflammasome activation (Fig. 1, right side) [11,20]. As for the ATP/P2X7R pathway, it has also been reported that ATP induced a reduction in intracellular pH in rat pancreatic ducts via P2X7R activation [22]. We further observed that the activation of the P2X7R by ATP induced an increase in lysosomal pH in microglial cells [23], which might have caused a reduction in cytosolic pH by impairing the intracellular proton balance. These studies imply that P2X7R-mediated intracellular acidification is implicated in the ATP-induced activation of inflammasomes (Fig. 1). Unauthenticated Download Date | 6/16/17 1:50 PM 78 T. Takenouchi et al. Figure 1: Schematic representation of the potential effects of extracellular ATP and acidic pH on inflammasome activation, and IL-1β processing and secretion in monocyte/macrophage-lineage cells. ATP-induced P2X7R activation triggers the NLRP3 inflammasome-dependent production of active caspase-1 (casp1) followed by the unconventional secretion of mature IL-1β (mIL-1β) (left side). Acidic extracellular pH-induced reductions in intracellular pH also trigger the NLRP3 inflammasome-dependent production and secretion of mIL-1β (right side). At lower extracellular pH, P2X7R activation by ATP preferentially produces and secretes 20-kDa IL-1β rather than mIL-1β (central part). The lysosomal enzyme cathepsin D seems to be responsible for the production of 20-kDa IL-1β. If cytoplasmic pro-IL1b translocates to secretory lysosomes, as was suggested by previous reports [32,33], 20-kDa IL-1β might be produced within lysosomes and secreted into the extracellular space via Ca2+-dependent exocytosis (pathway 1). If cathepsin D is released from lysosomes into the cytoplasm due to P2X7R-dependent lysosomal membrane damage, 20-kDa IL-1β might be produced within the cytoplasm and secreted via an unconventional secretory pathway (pathway 2). We have proposed an alternative pathway in which pro-IL-1β leaks from cells after P2X7R stimulation and is then converted to the 20-kDa form by exocytosed cathepsin D in the acidic extracellular space (pathway 3). 2.2 Acidic extracellular pH inhibits P2X7R ion channel functions Extracellular protons represent an important regulatory factor for voltage and ligand-gated ion channels, and the P2X7R is subject to proton modulation [24]. Indeed, a previous study showed that extracellular protons potently inhibited P2X7R-dependent ion currents and the induction of membrane pore formation through the allosteric modulation of P2X7R channels [25]. The charges of several amino acid residues in the extracellular domain of the rat P2X7R, including Lys, Asp, and His, are likely to be involved in the functional inhibition of this receptor by acidic pH [26,27]. In addition, a reduction in the concentration of the genuine P2X7R agonist ATP4by the protonation of ATP was reported to contribute to Unauthenticated Download Date | 6/16/17 1:50 PM Inflammasome activation by danger signals: extracellular ATP and pH the inhibitory effect of acidic pH on the P2X7R [28]. In any case, P2X7R ion channel functions are negatively regulated under acidic extracellular conditions (Fig. 1). 2.3 Acidic extracellular pH modulates ATPinduced IL-1β processing Despite the inhibitory effect of acidic pH on P2X7R channel activity, the production and secretion of significant amounts of mature IL-1β have been observed after treatment with high concentrations of extracellular ATP, even under acidic extracellular conditions. We found that P2X7R activation by ATP triggered the caspase-1dependent production and secretion of mature IL-1β at pH 6.2-6.7 in LPS-primed mouse microglial cells [29]. This finding is supported by another study in which it was found that extracellular ATP triggered mature IL-1β secretion at pH 6.2 in LPS-primed mouse mixed glia [30]. In these experimental systems, acidic pH alone failed to induce the production and secretion of mature IL-1β [29,30]. Considering that acidic extracellular pH perturbs the functions of P2X7R channels, acidic pH-induced reductions in intracellular pH might be involved in the facilitation of P2X7R-mediated inflammasome assembly. It is worth noting that acidic extracellular pH promoted the production and secretion of an unconventional 20-kDa form of IL-1β instead of the mature 17-kDa form upon stimulation with ATP in LPS-primed mouse microglial cells in a low pH-dependent manner (Fig. 1, central part) [29,30]. Interestingly, lactic acid, which causes a reduction in tissue pH in vivo, induced the preferential production of 20-kDa IL-1β in LPS-primed mouse mixed glia [30]. The production and secretion of 20-kDa IL-1β was also observed in LPS-primed human THP-1 macrophages after 6 h of incubation at lower pH (6.0), even without ATP stimulation [11]. The lysosomal protease cathepsin D was shown to be responsible for the production of 20-kDa IL-1β (Fig. 1, central part), whereas active caspase-1 is not involved in this process [29,30]. Considering that proIL-1β is used as a substrate for the production of both the 20-kDa and mature forms of IL-1β, it is likely that the enhanced production of 20-kDa IL-1β induced by lower pH results in a relative reduction in the amount of bioactive mature IL-1β during acidosis, which might be involved in the physiological regulation of inflammatory responses. 3 Conclusion and perspectives Recent studies have demonstrated that NLRP3 inflammasomes are able to sense a diverse range of danger 79 signals and then initiate the innate immune response [18]. Given the local accumulation of these danger signals in affected tissues, their cooperative effects need to be considered when attempting to understand the regulation of inflammatory process in vivo. In particular, since extracellular acidosis is a common feature of inflammatory loci it is likely that some danger signals exert their functions under acidic extracellular conditions. From this point of view, we discussed the possible mutual effects of extracellular ATP and acidic pH on inflammasome activation and IL-1β-dependent innate immunity. Extracellular ATP or acidic pH alone can stimulate the NLRP3 inflammasome-dependent production and secretion of mature IL-1β in monocyte/macrophagelineage cells (Fig. 1, left and right sides). However, when they co-exist pro-IL-1β seems to be preferentially converted into the unconventional 20-kDa form of IL-1β rather than the 17-kDa mature form (Fig. 1, central part) [29,30]. Since 20-kDa IL-1β is suggested to be ~5-fold less active at the IL-1 receptor than the mature form [31], it is speculated that extracellular acidosis suppresses ATPinduced IL-1β-dependent innate immune responses. This system might contribute to the physiological control of inflammation by preventing excessive inflammatory responses. If so, it is possible that the dysregulation of 20-kDa IL-1β production exacerbates IL-1β-driven inflammation. 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