For several years, pharmacological management of IBD was limited to aminosalicylates, thiopurines, and steroids, which, despite providing symptomatic relief, affected scarcely the disease course . Subsequently, advances in understanding the molecular mechanisms underlying the pathogenesis of IBD allowed us to identify novel therapies based on biological entities. These therapies aimed at curbing the activity of pro-inflammatory cytokines pivotally involved in the onset and progression of IBDs . Even though biological drugs undoubtedly represent an evolution in the therapeutic approach to IBD, these are often characterized by a high degree of therapeutic failure and in many cases by heavy adverse effects . For these reasons, the identification of new therapeutic targets with immunomodulating/anti-inflammatory properties represents a field of extreme interest for the scientific community.
In this regard, increasing attention has been paid to the design and synthesis of novel pharmacological entities acting on the purinergic system as viable way to regulate the development and course of several immuno-mediated inflammatory diseases (IMIDs) . In particular, the active role of P2X4 receptor subtype has been well defined in driving the immunostimulating effects of extracellular ATP, eliciting pro-inflammatory responses in both myeloid or lymphoid cells . Therefore, this receptor subtype represents an ideal target for pharmacological management several immune/inflammatory diseases [8, 9, 11, 12].
The aims of our study were: a) to evaluate the putative anti-inflammatory effects of NC-2600 and NP-1815-PX, two novel and selective P2X4 receptor antagonists, and b) to characterize the molecular mechanisms underlying their immunomodulatory effect in a murine model of DNBS-induced colitis. DEX was employed as a comparative agent with known anti-inflammatory activity to evaluate the relative anti-inflammatory effects of both P2X4 ligands.
The DNBS-induced colitis closely mimics the pathology of Crohn's disease as it is characterized by body weight loss, diarrhea, colon ulceration and bleeding, the depletion of goblet cells, and the formation of granulomas within the gut wall [24, 25]. This represents a predictive model to assess the efficacy of novel anti-inflammatory agents. In this regard, the suitability of this model was corroborated by the efficacy of DEX to ameliorate the macroscopic and histological features of colitis as well as to reduce tissue TNF and IL-1β levels, in line with previous reports showing similar beneficial effects of this glucocorticoid drugs [16, 26].
In the first set of experiments, we observed that the pharmacological blockade of P2X4 receptor via NC-2600 and NP-1815-PX elicited a significant improvement in several inflammatory parameters, including body weight, spleen weight, macroscopic and microscopic tissue injury score, as well as down-regulated IL-1β levels in DNBS-rats, thus providing evidence that the pharmacological modulation of this receptor is a viable option to manage enteric inflammatory processes. This is in line with a number of recent studies describing the critical role of P2X4 receptor in protecting against infections, inflammation, and organ injury . Indeed, this receptor has been shown to regulate a variety of pathophysiological processes characterized by an excessive immune cell activity such as autoimmune and neuroinflammatory diseases . In particular, P2X4, widely expressed on cells of both the innate and adaptive immune cells, is emerging as a pivotal pathway in mediating the immunostimulating/pro-inflammatory effect of ATP , thus representing an attractive therapeutic target in multiple diseases, especially in chronic inflammation and neuropathic pain [27–29].
Interestingly, in our experimental model, both P2X4 antagonists failed to reduce tissue TNF levels. By contrast, NC-2600 and NP-1815-PX were able to counteract the increase of IL-1β concentration in colonic tissues from rats with DNBS-induced colitis. IL-1β is a potent inflammatory cytokine centrally involved in the development of colitis and TH17-associated immune responses in the gut . On the same line, patients with Crohn's disease showed a positive correlation between the severity of mucosal inflammation and the levels of IL-1β, thus corroborating a relevant role for this cytokine in the pathophysiology of IBDs .
Despite the mechanism of IL-1β release has proven to be complex a set of molecular and biochemical investigations allowed to demonstrate that the bioactive form of this cytokine is produced by the inflammasome NLRP3, a caspase-1 activating molecular platform, in response to selected danger-associated molecular patterns and pathogen-associated molecular patterns . In particular, the NLRP3 inflammasome complex, including NLRP3, ASC and pro-caspase-1, through the processing and release of IL-1β and IL-18, acts as a key player both in shaping the central and/or peripheral immune/inflammatory responses in several inflammatory diseases, including IBDs . Such pathway is currently designated as “canonical NLRP3 inflammasome activation.” In addition, a “non-canonical NLRP3 inflammasome activation,” which depends on caspase-11 in mice (caspase-4 and caspase-5 in human) and caspase-8, has been reported to play a pivotal role in the pathophysiological events underlying bowel inflammation .
Based on these findings we designed several experiments to evaluate the putative effect of NC-2600 and NP-1815-PX in counteracting inflammasome NLRP3 activation, especially focusing on the effect of tested drugs on the expression and activity of caspase-1, the canonical activation pathway of NLRP3 complex. Our findings showed that treatment with NC-2600 and NP-1815-PX, despite not reducing the caspase-1 expression, was able to decrease its activity in colonic tissues from rats with colitis. This evidenced the anti-inflammatory activity of this novel P2X4 antagonists by inhibiting the canonical caspase-1-dependent NLRP3 inflammasome signaling pathway. This is in line with previous studies, performed in murine models of diabetic nephropathy, bladder inflammation and acute kidney injury, showing that the pharmacological blockade of P2X4 receptors blunted the IL-1β release through the inhibition of NLRP3 inflammasome pathway [29, 35, 36].
In order to corroborate the results obtained from in vivo experiments, we designed a set of in vitro experiments to evaluate the effect of the P2X4 antagonists on modulating the canonical and non-canonical pathways involved in NLRP3 activation. Therefore, we performed experiments in LPS-primed and PMA-differentiated THP-1 cells treated with ATP, an established model to investigate the functions of purinergic receptors in monocyte/macrophage cells [37–39]. THP-1 cells were incubated with LPS, a well-recognized activator of the first step of NLRP3 signaling as well as a hallmark of altered intestinal permeability, a condition typically observed in the murine model of colitis and IBD patients [40, 41]. We observed that co-treatment THP-1 cell line with LPS and ATP determined an increase in the expression of inflammasome components, including NLRP3, ASC and caspase-1, followed by marked IL-1β release. Under this condition, the incubation with NC-2600 and NP-1815-PX decreased all of the above mentioned parameters, indicating an inhibitory effect of test drugs on the canonical caspase-1-dependent NLRP3 inflammasome pathway. This result is in line with a previous report displaying an inhibitory effect on canonical NLRP3 activation following the pharmacological blockade of P2X4 receptors in rat urothelial cells, neuronal dorsal horn cells and renal tubule epithelial cells incubated with ATP [29, 35, 36, 42].
In parallel with the canonical NLRP3 inflammasome activation, a “non-canonical NLRP3 inflammasome activation,” depending on caspase-11, has been described to be pivotal in the maintenance of intestinal immune homeostasis [43, 44]. Recent studies have also reported the involvement of caspase-8 in regulating the expression and release of IL-1β by NLRP3 inflammasome activation, highlighting a novel non-apoptotic role of caspase-8 in the context of inflammation [45, 46]. In the present study, sets of experiments were devoted to evaluate the modulatory effect of NC-2600 and NP-1815-PX also on the non-canonical NLRP3 inflammasome pathway. The pharmacological blockade of P2X4 receptors affects also the non-canonical NLRP3 inflammasome pathways, reducing the caspase-5 and caspase-8 expression in THP-1 cells treated with LPS and ATP. Of note, no effect for both tested drugs was observed on caspase-4 expression. However, since uncleaved caspase-4 can promote the release of IL-1β through activation of the canonical NLRP3-ASC-caspase-1 signaling [47, 48], we could rule out that the inhibition of IL-1β release resulting from the blockade of P2X4 receptors would depend, at least in part, in an uncleaved caspase-4 manner.
Taken together, these results provide convincing evidence that the blockade of P2X4 receptors exerts an inhibitory effect on both the canonical and non-canonical pathways involved in NLRP3 activation. This represents the novelty of the present study, and further studies aimed at better characterizing the molecular mechanisms underlying the P2X4 receptors/NLRP3 inflammasome interplay represent the logical continuation of this research topic.
IL-1β, beyond being involved in the initiation and amplification of inflammatory responses, plays a critical role in the apoptosis of epithelial cells causing tissue damage and barrier dysfunction. This, in turn, leads to increased intestinal permeability typically associated with IBDs . Consistently with this evidence, our experimental model showed a decreased expression levels of colonic tight junctions. Interestingly, treatment with NC-2600 and NP-1815-PX counteracted the reduction of tight junction expression associated with experimental colitis, suggesting that the pharmacological blockade of P2X4 receptors can exert beneficial effect in the maintenance of intestinal epithelial barrier integrity. Despite the fact that no data are available about the molecular mechanisms underlying the beneficial effects of this novel P2X4 antagonists in the maintenance of intestinal epithelial barrier, it is conceivable that these likely depend, at least in part, on the action of these drugs on the NLRP3/caspase-1/IL-1β axis.
In conclusion, the present study expands current knowledge about the beneficial effects of the P2X4 receptor blockade in modulating immune/inflammatory responses. Our results demonstrated here for the first time that the direct and selective inhibition of P2X4 receptors represent a viable approach for the management of bowel inflammation via the inhibition of the canonical and non-canonical NLRP3 inflammasome signaling pathways. According to the present results, the blockade of P2X4 receptors allows an effective control of experimental intestinal inflammation, and novel and selective P2X4 antagonists can provide a basis for development of anti-inflammatory drugs suitable for treatment of IBD.