Increasing evidence suggest that inflammasomes play key roles in regulating neuroinflammatory response following TBI [29]. The current study has shown that targeting NLRP3 inflammasome with our small molecule inhibitor JC124 is neuroprotective for TBI. Specifically, post-TBI treatment with JC124 at the acute stage following injury significantly decreased injury-induced neuronal degeneration and cortical tissue damage. This protective effect is likely mediated through specific targeting of TBI-induced activation of NLRP3 inflammasome and its downstream neuroinflammatory cascade as demonstrated by completely blocking of TBI-enhanced expression of NLRP3 and its adaptor protein ASC, reduction of downstream caspase-1 activation, reactive oxygen species (iNOS) and pro-inflammatory cytokines IL-1β, TNFα protein expressions. Our results suggest that NLRP3 inflammasome is involved in the development of secondary injury following TBI, and targeting NLRP3 inflammasome is a viable strategy for TBI treatment.

Neuroinflammation is an essential player dictating disease progression in many neurological insults including TBI. Injury-induced neuroinflammatory response, activated by the release of host-derived proteins termed danger-associated molecular patterns, significantly contributes to the progression of secondary injury and impact post-injury recovery. Recent evidence indicates a critical role for the inflammasome complex in initiating neuroinflammatory response after brain trauma [29]. Inflammasomes are essential intracellular multiprotein complexes that direct the innate immune responses to pathogenic stimuli, regulate the activation of caspase-1, production of IL-1β and IL-18, and induction of cell death [7]. Among known inflammasomes, the NOD-like receptors (NLRs) family members NLRP1 and NLRP3 are the most widely studied in the brain and capable to activate caspase-1, IL-1β, and IL-18 [7]. Thus far, activation of NLRP1 and NLRP3 inflammasomes has been reported following TBI in both pre-clinical and clinical studies [30, 31]. In a rat fluid percussive injury model, formation of NLPR1 inflammasome complex, upregulation of caspase-1, and increased IL-1β were detected at 4 h following injury [32]. In a rat weight drop injury model, increased level of NLRP3 and its downstream substrates including caspase-1, ASC, IL-1β, and IL-18 were detected in the peri-injury cortex at both mRNA and protein levels from 6 h to 7 days post-injury [13]. In a mouse cortical impact injury model, increased protein expression of NLRP3, caspase-1, and ASC in the peri-injury cortex was also reported at 1 to 7 days post-injury with the peak expression at 3 days [26]. Study has also reported that TBI led to NLRs and AIM2 inflammasome-mediated pyroptosis in brain microvascular endothelial cells in the injured cerebral cortex in a mouse CCI model [33]. In clinic, NLRP1 and caspase-1 proteins are found in cerebrospinal fluid (CSF) in severe adult TBI patients, and the level is correlated with prognosis [34]. In pediatric patients with severe TBI, increased NLRP3 but not NLRP1 was found in the CSF and was associated with poor prognosis [31]. In a weight drop diffuse injury model using transgenic mice lacking NLRP3, reduced brain tissue damage and inflammatory cell response with preserved cognitive function were observed [35]. In contrast, in transgenic mice which lack NLRP1 or ASC genes, although reduced IL-1β was observed, no protective effect was found following TBI [28]. Collectively, these studies suggest that TBI induces activation of NLRs family members of inflammasomes and activation of inflammasomes particularly the NLRP3 is associated with the injury progression.

Activation of the inflammasome complex is an essential step for the development of neuroinflammation in secondary brain damage. Although the activation pathway is not completely understood, many signals that are related to tissue damage including TBI have been suggested to trigger NLRP3 inflammasome activation including extracellular ATP, K+ efflux, damaged mitochondria, elevated reactive oxygen species, influx of Ca2+, endoplasmic reticulum stress and cathepsin release [36,37,38,39,40]. Among these signals K+ efflux is the best-characterized minimal stimulus for NLRP3 inflammasome activation [41]. Once activated, the NLRP3 inflammasome forms a molecular platform for caspase-1 activation which leads to subsequent release of IL-1β and IL-18 and the eventual amplification of inflammatory responses [7]. The brain is particularly sensitive to IL-1β and IL-18 signaling, as both neurons and glial cells express receptors for these cytokines [42]. IL-1β is the conversion product of caspase-1 activation and triggers NF-KΒ signaling that up-regulates transcription of other pro-inflammatory genes [43]. Ample evidence indicates that IL-1β and IL-18 are involved in the onset and development of the inflammatory cascade following TBI [44,45,46,47]. Elevated IL-1β is found in the CSF and brain parenchyma within hours after brain injury in both humans and rodents [44, 48]. It is suggested that the damaging effects of IL-1β is related to its effects on activating other pro-inflammatory cytokines such as TNF-a and IL-6, leading to activation and recruitment of microglia and leukocytes, and disruption of the BBB [42, 48]. Infiltration of macrophages and activation of resident microglial cells further release inflammatory mediators that are cytotoxic to neurons contributing to neurodegeneration and tissue damage [49]. In our study, CCI induces activation of cascape1, leading to upregulated expression of proinflammatory mediators including IL-1β, TNFα, and iNOS, as well as inflammatory cell response in the brain causing eventual neuronal cell degeneration.

Because of the crucial role of NLRP3 inflammasomes in controlling neuroinflammatory response and neural tissue damage following TBI, drug development targeting activation of NLRP3 inflammasome could be a viable therapeutic strategy for TBI. Thus far, studies have reported varying non-specific pharmacological agents with function on NLRP3 inflammasome inhibition having beneficial effect for TBI such as omega-3 fatty acids [50], propofol [51], and resveratrol [52]. Studies also reported that treatment with an anti-ASC neutralizing antibodies can reduce innate immune response and significantly decrease contusion volume in a rat fluid percussive injury model [32], and a NLRP3 inhibitor BAY 11-7-82 post-TBI treatment showing protective effect with reduced brain damage and inflammatory cells was reported [53]. Recently, a small-molecule NLRP3 inhibitor MCC950 has been shown neuroprotective effect in stroke, cerebral hemorrhage, and TBI models [26, 54,55,56]. In TBI, MCC950 treatment given at 1 and 3 h following a CCI injury in mice, reduction of caspase-1, and IL-1β was observed accompanied with improved motor and sensory function at 1 and 3 days post-injury [56]. When MCC 950 was given i.p. daily for the first 3 days followed by every other day until the end of experiments up to 21 days post-injury, it can attenuate microglia-derived NLRP3 inflammasome activation and production of IL-1β, reduce brain edema, lesion volume, inflammatory cell response, and cell death, as well as improve neurological functions [26].

Our laboratories have recently designed and developed a sulfonamide analog JC124 based on the structure of glyburide. Sulfonylurea-containing compounds such as glyburide and CP-456,773 (now named MCC950) potently inhibit ATP- or hypotonicity-induced IL-1β processing via specific inhibition of the NLRP3 inflammasome [15, 57]. These compounds specifically inhibit the triggering step of NLRP3 activation without affecting the NF-κB signaling-related priming step or the activation of other inflammasomes thus is NLRP3 specific [15, 58]. The mechanism by which these compounds inhibit NLRP3 activation is currently not understood. It is likely that sulfonylurea containing compounds act at downstream of K+ depletion as they do not prevent K+ efflux and the inhibition mechanism is not related to K+ channels. Glyburide was shown to inhibit the ATPase activity of NLRP3, whereas MCC950 does not affect the Ca2+ flux in cells treated with ATP thus with different mechanism [15, 58]. Furthermore, several other small molecule compounds have been reported to target the NLRP3 inflammasome pathway [58,59,60]. However, the mechanism of action or biological targets of these compounds either act upstream of the inflammasome complex or remain unknown. Glyburide is used in clinic for diabetic treatment, the dose for its NLRP3 inflammasome inhibition effects is at the risk of inducing hypoglycemia, thus cannot be used directly as NLRP3 inhibitor [15]. JC124 was rationally designed based on the structure of glyburide to remove the potential hypoglycemic effects. Our studies have established that JC124 is an active and selective NLRP3 inhibitor by blocking ASC aggregation, activation of caspase-1, and release of IL-1β in macrophages that constitutively express active NLRP3 [16]. Using photoaffinity-labeling probes, we have found that JC124 directly targets inflammasome complex without affecting its ATPase activity, thus representing a novel mechanism (unpublished data). Our studies have also demonstrated the protective effect of this compound in a mouse acute myocardial infarction model [18] and transgenic Alzheimer’s disease models [18, 19]. In this current study, in a rat focal brain injury model, JC124 has shown neuroprotective effects for TBI when given at the acute stage following TBI, similar to what have been reported with MCC950 treatment [56]. Furthermore, compared to reported MCC950 studies done by Xu et al. [26], MCC950 was given during the entire experimental period up to 21 days post-injury, whereas in our study, JC124 was given only during the first 30 h post-injury when NLRP3 inflammasome activation was trigged by brain injury suggesting the benefits of direct inhibition of the inflammasome complex by our novel compound.

Post-traumatic inflammation is detrimental at the early stage but can be beneficial during the chronic stage as it promotes both clearance of debris and regeneration. The target of anti-inflammatory interventions for TBI is to remove danger signals and clear debris during the acute stage, prevent the development of chronic neuroinflammation and promote regenerative immune phenotype in the chronic stage [20]. Thus far, many anti-inflammatory agents have shown beneficial effects in pre-clinical TBI models; however, these effects have failed to translate into clinic. Cautious must be taken in developing new agents targeting neuroinflammation. More studies are needed to evaluate NLRP3 inflammasome inhibitors including our compound JC124.