Caratterizzazione del ruolo fisiopatologico e funzionale del canali Transient Receptor Potential (TRP) nelle patologie dolorose di origine infiammatoria e neuropatica

Summary The TRPA1 receptor has been identified as a pivotal molecular entity in sensory biology, especially as a sensor of chemical species/irritants present in foods, atmospheric pollutants and toxicants. Rapid progress is being made in characterizing the function of TRPA1 in various tissues and organs under normal and pathophysiological conditions. TRPA1 receptor is an excitatory ion channel expressed by a subpopulation of primary afferent somatosensory neurons that contain SP and CGRP. Environmental irritants (mustard oil, allicin, acrolein) can activate TRPA1, causing acute pain, neuropeptide release, and neurogenic inflammation. Genetic studies indicate that TRPA1 is also activated downstream of one or more proalgesic agents that stimulate phospholipase C signaling pathways, thereby implicating this channel in peripheral mechanisms controlling pain hypersensitivity. The commonly accepted paradigm is that TRPA1 expressed in primary sensory neurons is directly activated by ROS/RNS/RCS, thus signalling pain from the PNS to the CNS. While direct stimulation of nociceptor TRPA1 mediates acute spontaneous pain, recent findings from our laboratory also, have shifted the focus to the TRPA1 expressed in non-neuronal cells as the critical factor to sustain allodynia and chronic pain. In our study we aim at identifying the role of TRPA1 in different model of pain ranging from inflammatory to neuropathic pain. The first aim of the present study was to identify the role of TRPA1 in a mouse model of trigeminal neuropathic pain produced by the constriction of the infraorbital nerve (CION) and to explore the molecular and cellular pathways that, from the initial nerve injury, result in channel engagement. The monocyte chemoattractant protein 1 (MCP-1), also known as chemoattractant chemokine (C-C motif) ligand 2 (CCL2), by binding to the chemotactic cytokine receptor 2 (CCR- 2), promotes monocyte transendothelial migration to the site of nerve injury. In various paradigms of peripheral nerve injury, CCL2 inhibition and CCR-2 genetic ablation abrogates mechanical allodynia. In addition, antioxidants have been reported to attenuate neural hypersensitivity in various models of neuropathic pain, such as sciatic chronic constriction injury and spinal nerve ligation. Thus, the contribution of monocyte/macrophage infiltration and the ensuing oxidative stress in the TRPA1- mediated pain-like behaviors was investigated in the CION model. Results propose that CCL2-driven monocyte/macrophage accumulation within the injured nerve and the neighboring tissue generates oxidative burst that, by TRPA1 targeting, promotes and maintains CION-evoked pain-like behaviors. However, while exploring a similar model (partial sciatic nerve ligation, pSNL), we surprisingly observed that mice with genetic deletion or pharmacological blockade of TRPA1 not only showed the expected reduced mechanical allodynia, but also exhibited a marked reduction in 2 macrophages infiltration and H2O2 generation in the injured nerve trunk. Thus, by a series of genetic and pharmacological interventions and, more importantly, by generating mice with conditional TRPA1 deletion in different cells, we identified the expression of TRPA1 in Schwann cells and showed a role of the receptor in molecular events that sustain allodynia after a damaged of the nerve trunk. Our results reveal distinct kinetics of monocyte/macrophage accumulation by CCL2 and the TRPA1/oxidative stress pathways. The most parsimonious explanation of the present results is that oxidative stress generated by Schwann cell TRPA1/NOX1 has bidirectional effects. The inwardly released H2O2 targets TRPA1 on adjacent nociceptor nerve fibers in a paracrine fashion to sustain allodynia. The outwardly released H2O2 promotes the final part (about 200 μm) of the journey of macrophages, which, deriving from the blood stream, slowly accumulate into the perineural space following the CCL2 gradient. Thereafter, following the Schwann cell derived oxidative stress gradient, macrophages rapidly pass across the perineurium to enter the damaged nerve trunk. TRPA1 has been identified in oligodendrocytes, with possible detrimental roles in ischemia and neurodegeneration (Hamilton et al., 2016). Herein, we extend this observation to Schwann cells, the peripheral analogues of oligodendrocytes, which, via TRPA1, orchestrate neuroinflammation and ensuing neuropathic pain. Amelioration of neuropathic pain by currently developed TRPA1 antagonists may derive from their ability to attenuate macrophage-dependent neuroinflammation. Since TRPA1 is involved in multiple painful condition, we also investigate the role of TRPA1 in pain symptoms associated with the treatment of the third-generation AIs, which include the steroidal agent exemestane (Aromasin) the triazoles anastrozole (Arimidex) and letrozole (Femara), currently recommended for adjuvant endocrine treatment as primary, sequential, or extended therapy with tamoxifen, for postmenopausal women diagnosed with estrogen receptor-positive breast cancer. Among these, the AI-associated musculoskeletal symptoms (AIMSS) are characterized by morning stiffness and pain of the hands, knees, hips, lower back, and shoulders. In addition to musculoskeletal pain, pain symptoms associated with AIs have recently been more accurately described with the inclusion of neuropathic, diffused, and mixed pain (Laroche et al., 2014). The chemical structure of exemestane includes a system of highly electrophilic conjugated Michael acceptor groups, which might react with the thiol groups of reactive cysteine residues. Michael addition reaction with specific cysteine residues is a major mechanism that results in TRPA1 activation by a large variety of electrophilic compounds. In addition, aliphatic and aromatic nitriles can react with cysteine to form thiazoline derivatives and accordingly the tear gas 2-chlorobenzylidene malononitrile (CS) has been identified as a TRPA1 agonist. We noticed that both letrozole and anastrozole possess nitrile moieties. Thus, we hypothesized that exemestane, letrozole and anastrozole may produce neurogenic inflammation, nociception and hyperalgesia by targeting TRPA1. The ability to gate TRPA1 in vitro 3 was confirmed in vivo by the observation that the pain-like behaviors evoked by AIs in mice are abrogated by genetic deletion or pharmacological blockade of the channel. However, AI concentrations required for TRPA1 gating in vitro are 1-2 order of magnitude higher than those found in patient plasma. In addition, an important proportion (30-40%), but not all, of treated patients develop the painful condition. These observations suggest that exposure to AIs is necessary, but not sufficient, to produce AIMSS, and that additional factors should cooperate with AIs to promote pain symptoms. Aromatase inhibition, while reducing downstream production of estrogens, moderately increases upstream plasma concentrations of androgens, including androstenedione (ASD) (Gallicchio et al., 2011). Exemestane, a false aromatase substrate, blocks enzymatic activity by accommodating in the binding pocket that snugly encloses ASD. We showed that ASD, which retains some of the reactive chemical features of exemestane, such as the α,β-carbonyl moiety of the A ring and the ketone group at the 17 position, targets TRPA1, thus evoking pain-like responses and neurogenic inflammation, supported the hypothesis that channel activation in peptidergic nociceptors promotes AIMSS. Another painful condition is represented by migraine pain. Occupational exposure to, or treatment with, organic nitrates has long been known to provoke headaches. These observations have led to the clinical use of glyceryl trinitrate (GTN) as a reliable provocation test for migraine attacks (Iversen et al., 1989; Olesen, 2008; Sicuteri et al., 1987). In most subjects, including healthy controls, GTN administration causes a mild headache that develops rapidly and is short-lived. However, after a remarkable time lag (hours) from GTN exposure, migraineurs develop severe headaches that fulfill the criteria of a typical migraine attack (Iversen et al., 1989; Olesen, 2008; Sicuteri et al., 1987). GTN administration to rodents and humans produces a delayed and prolonged (hours) hyperalgesia that temporally correlates with GTN-induced migraine-like attacks in humans (Ferrari et al., 2016; Tassorelli et al., 2003). Several mechanisms have been proposed to explain the mechanism, including degranulation of meningeal mast cells (Ferrari et al., 2016), delayed meningeal inflammation sustained by induction of NO synthase and prolonged NO generation, and the release of CGRP (Ramachandran et al., 2014), a primary migraine neuropeptide (Edvinsson, 2015), but the precise mechanisms was not identified. In our study we identified a pivotal role of TRPA1 expressed in trigeminal ganglion neurons in sustaining the delayed mechanical allodynia induced by GTN administration, which recapitulate the migraine attach. Our data clearly showed that TRPA1 receptor can be considered as a novel and major general pain mechanism and TRPA1 antagonists may represent novel analgesic for a long-waited advancement for the treatment of various types of pain.