Knockdown of the AIM2 molecule attenuates ischemia-reperfusion-induced spinal neuronal pyroptosis by inhibiting AIM2 inflammasome activation and subsequent release of cleaved caspase-1 and IL-1β
Introduction
Spinal cord ischemia-reperfusion (IR) injury is a destructive and unpredictable complication with great risk of permanent neurological deficits (Liu Y et al., 2018; Nazli Y et al., 2015). The underlying molecular mechanisms provoking IR-induced neuronal damage are complicated and multifactorial and involve oxidative stress, sterile inflammation and excitotoxicity (Fu J et al., 2018). Inflammatory processes are clearly designed to limit cell damage and occur via responses from residual glial cells to recruit circulatory leukocytes throughout the duration of spinal cord IR injury, thus contributing most to the progression and exacerbation of neurological dysfunction (Anwar MA et al., 2016; Chen J et al., 2017). Given the complex crosstalk during the inflammatory cascade, emerging evidence has indicated that multiprotein complexes termed inflammasomes are indirectly responsible for neuronal death during central nervous system (CNS) injury (de Rivero Vaccari JP et al., 2014; Fann DY et al., 2013; Mortezaee K et al., 2018). Inflammasomes typically consist of three important components: the initiator receptor, the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and precursor caspase-1 (Fann DY et al., 2013; Adamczak SE et al., 2014). When stimulated, the inflammasomes assemble in the cytoplasm and recruit procaspase-1 via ASC (to be converted into cleaved caspase-1) and the mature cytokines interleukin (IL)-1β and IL-18 (de Rivero Vaccari JP et al., 2014; Mortezaee K et al., 2018). Regarding CNS pathology, NOD-like receptor protein (NLRP)1 and NLRP3 are well-known initiator receptors (Fann DY et al., 2013; Fann DY et al., 2018; Zhang B et al., 2017, Supplementary Fig. 1). Blocking the function of NLRP1 and NLRP3 with immunoglobulin has been demonstrated to protect neurons in an experimental stroke model by reducing the release of IL-1β and IL-18 (Fann DY et al., 2013). Moreover, silencing NLRP3 with siRNA and a pharmacological inhibitor, MCC950, can alleviate brain damage by decreasing cleaved caspase-1 and IL-1β and the phosphorylation of nuclear factor (NF)-κB p65 in the penumbral region (Ismael S et al., 2018; Ma Q et al., 2014).
Additionally, cytosolic double-stranded DNA (dsDNA) released from damaged or dying cells can provoke IR-induced sterile inflammation (Kim HY et al., 2015; Sun Q et al., 2017). The absent in melanoma 2 (AIM2) protein is a canonical receptor for cytosolic DNA (Adamczak SE et al., 2014). Recently, there has been increasing focus on the role of AIM2 in the formation of an inflammasome after exposure to an exogenous pathogen or host ectopic nucleic acids (de Zoete MR et al., 2014; Hornung V et al., 2009; Sagulenko V et al., 2013). After binding of the host ectopic dsDNA to AIM2's HIN domain, AIM2 recruits ASC and procaspase-1 around the DNA molecule, thus completing the maturation of caspase-1 and proinflammatory cytokines (de Zoete MR et al., 2014). Moreover, some studies have suggested that AIM2 is highly expressed in rodent neurons after traumatic injury (Adamczak SE et al., 2014; Denes A et al., 2015; de Rivero Vaccari JP et al., 2014). Coculture of mouse embryonic neurons with cerebrospinal fluid (CSF) collected from patients with brain injury has been associated with significant increases in AIM2 protein levels and the induction of cleaved caspase-1 (Adamczak SE et al., 2014). The significance of cleaved caspase-1 in provoking pyroptosis has been well described in several types of cells and is characterized by a loss of membrane integrity, followed by pore formation and an inflammatory response (Adamczak SE et al., 2014; Kim HY et al., 2015; Sagulenko et al., 2018, Supplementary Fig. 1). However, inflammasomes are expressed in a tissue-specific manner and have played contradictory roles in different models (de Rivero Vaccari JP et al., 2014; de Zoete MR et al., 2014); thus, it is unknown whether the AIM2 inflammasome is activated in spinal neurons following IR injury. Inflammasome activation can be regulated by regulating its specific components (de Rivero Vaccari JP et al., 2014; Sagulenko et al., 2013; Sagulenko et al., 2018). Compared with wild-type mice, AIM2, ASC and caspase-1 knockout mice are all resistant to ischemic brain injury because these alterations prevented inflammasome formation (Denes et al., 2015). Thus, in this study, we used siRNA and a specific inhibitor to investigate whether the AIM2 molecule acts as an initiator to trigger AIM2 inflammasome activation and regulate neuronal pyroptosis in both in vivo and in vitro models.
Section snippets
Animals
The protocols were approved by the Ethics Committee of China Medical University. Twelve-to 15-week-old C57BL/6 mice were supplied by the Animal Center of China Medical University (Shenyang, China). Mice had free access to food and water at least 1 week for pre-acclimation. The cages were in well-controlled conditions with a temperature of 22–24 °C, relative humidity of 50–60% and 12/12-h light/dark cycle.
Mouse model of spinal cord IR injury and experimental protocol
The mouse IR injury model was established as previously reported (Li XQ et al., 2017).
Temporal motor dysfunction and spinal AIM2 molecule dysregulation following IR injury
All mice displayed normal hindlimb motor function after 3 d of continuous intrathecal injection. As shown in Fig. 1A and B, both the average and the individual BMS scores were greatly decreased during the 24-h reperfusion period, beginning at 4 h and maintaining a stable low level at 12 h post-IR (P < 0.05), indicating that IR induced motor dysfunction.
Given that the AIM2 inflammasome can be activated during IR-induced sterile inflammation (Kim HY et al., 2015), we also evaluated the changes in
Discussion
Increasing evidence has implicated the AIM2 inflammasome and its major components and released products (e.g., ASC, cleaved caspase-1, and mature IL-1β) as factors that drive IR-induced sterile inflammation and tissue damage (Asmussen A et al., 2016; Fernandes-Alnemri T et al., 2009; Man SM et al., 2016; Kim HY et al., 2015). Thus, attempts at targeting and regulating peripheral factors or common nodes might provide reliable clinical treatments. Previous studies have suggested that blocking the
Conclusions
This study highlighted the functional roles of the AIM2 molecule in initiating AIM2 inflammasome activation in both in vivo and in vitro IR models. Downregulation of the AIM2 molecule partially prevented motor dysfunction and neuronal pyroptosis by inhibiting AIM2 inflammasome activation and subsequently decreasing caspase-1 and IL-1β cleavage.
Conflicts of interest
All authors declare no conflicts of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 81601053 and 81771342), the Scientific Research Project of Liaoning Province Department of Education (No. LK201636) and the Combined Project of the Liaoning Natural Science Foundation (No. 20180530087).
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