Alexandre H. Lopes

Spinal cord oligodendrocyte-derived alarmin IL-33 mediates neuropathic pain

Neuropathic pain from injury to the peripheral and CNS represents a major health care issue. We have investigated the role of IL‐33/IL‐33 receptor (ST2) signaling in experimental models of neuropathic pain in mice. Chronic constriction injury (CCI) of the sciatic nerve induced IL‐33 production in the spinal cord. IL‐33/citrine reporter mice revealed that oligodendrocytes are the main cells expressing IL‐33 within the spinal cord together with a minor expression by neurons, microglia, and astrocytes. CCI‐induced mechanical hyperalgesia was reduced in IL‐33R (ST2)‐/‐ mice compared with wild‐type (WT) mice. Intrathecal treatment of WT mice with soluble IL‐33 receptor (IL‐33 decoy receptor) markedly reduced CCI‐induced hyperalgesia. Consistent with these observations, intrathecal injection of IL‐33 enhanced CCI hyperalgesia and induced hyperalgesia in naive mice. IL‐33‐mediated hyperalgesia during CCI was dependent on a reciprocal relationship with TNF‐α and IL‐1β. IL‐33‐induced hyperalgesia was markedly attenuated by inhibitors of PI3K, mammalian target of rapamycin, MAPKs (p38, ERK, and JNK), NF‐κB, and also by the inhibitors of glial cells (microglia and astrocytes). Furthermore, targeting these signaling pathways and cells inhibited IL‐33‐induced TNF‐α and IL‐1β production in the spinal cord. Our study, therefore, reveals an important role of oligodendrocyte‐derived IL‐33 in neuropathic pain.— Zarpelon, A. C., Rodrigues, F. C., Lopes, A. H., Souza, G. R., Carvalho, T. T., Pinto, L. G., Xu, D., Ferreira, S. H., Alves‐Filho, J. C., McInnes, I. B., Ryffel, B., Quesniaux, V. F. J., Reverchon, F., Mortaud, S., Menuet, A., Liew, F. Y., Cunha, F. Q., Cunha, T. M., Verri, Jr., W. A. Spinal cord oligodendrocyte‐derived alarmin IL‐33 mediates neuropathic pain. FASEB J. 30, 54‐65 (2016). www.fasebj.org

Targeting the minor pocket of C5aR for the rational design of an oral allosteric inhibitor for inflammatory and neuropathic pain relief

Significance Persistent pain in inflammatory and neuropathic conditions is often refractory to conventional analgesic therapy, with most patients suffering with unrelieved pain and serious treatment-related side effects. There is still a tremendous need to identify novel therapeutics for pain control with innovative biological mechanisms and minimal side effects. In this paper we challenge the hypothesis that a conserved structural motif across the G protein-coupled receptor family plays a regulatory role in the negative modulation of receptor activation and use a multidisciplinary approach to the rational drug design and characterization of a novel potent allosteric inhibitor of the C5a anaphylatoxin receptor (C5aR), thus providing a new promising avenue for the improvement of pharmacotherapy of chronic pain. Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. Pharmacological therapies currently available for certain types of pain are only partially effective and may cause severe adverse side effects. The C5a anaphylatoxin acting on its cognate G protein-coupled receptor (GPCR), C5aR, is a potent pronociceptive mediator in several models of inflammatory and neuropathic pain. Although there has long been interest in the identification of C5aR inhibitors, their development has been complicated, as for many peptidomimetic drugs, mostly by poor drug-like properties. Herein, we report the de novo design of a potent and selective C5aR noncompetitive allosteric inhibitor, DF2593A, guided by the hypothesis that an allosteric site, the “minor pocket,” previously characterized in CXC chemokine receptors-1 and -2, is functionally conserved in the GPCR class. In vitro, DF2593A potently inhibited C5a-induced migration of human and rodent neutrophils. In vivo, oral administration of DF2593A effectively reduced mechanical hyperalgesia in several models of acute and chronic inflammatory and neuropathic pain, without any apparent side effects. Mechanical hyperalgesia after spared nerve injury was also reduced in C5aR−/− mice compared with WT mice. Furthermore, treatment of C5aR−/− mice with DF2593A did not produce any further antinociceptive effect compared with C5aR−/− mice treated with vehicle. The successful medicinal chemistry strategy confirms that a conserved minor pocket is amenable for the rational design of selective inhibitors and the pharmacological results support that the allosteric blockade of the C5aR represents a highly promising therapeutic approach to control chronic inflammatory and neuropathic pain.

The Quassinoid Isobrucein B Reduces Inflammatory Hyperalgesia and Cytokine Production by Post-transcriptional Modulation

Isobrucein B (1) is a quassinoid isolated from the Amazonian medicinal plant Picrolemma sprucei. Herein we investigate the anti-inflammatory and antihyperalgesic effects of this quassinoid. Isobrucein B (1) (0.5-5 mg/kg) inhibited carrageenan-induced inflammatory hyperalgesia in mice in a dose-dependent manner. Reduced hyperalgesia was associated with reduction in both neutrophil migration and pronociceptive cytokine production. Pretreatment with 1 inhibited in vitro production/release of cytokines TNF, IL-1β, and KC/CXCL1 by lipopolysaccharide-stimulated macrophages. To investigate its molecular mechanism, RAW 264.7 macrophages with a luciferase reporter gene controlled by the NF-κB promoter were used (RAW 264.7-Luc). Quassinoid 1 reduced the luminescence emission by RAW 264.7-Luc stimulated by different compounds. Unexpectedly, NF-κB translocation to macrophage nuclei was not inhibited by 1 when evaluated by Western blotting and immunofluorescence. Furthermore, quassinoid 1 did not change the levels of TNF mRNA transcription in stimulated macrophages, suggesting post-transcriptional modulation. In addition, constitutive expression of luciferase in RAW 264.7 cells transiently transfected with a plasmid containing a universal promoter was inhibited by 1. Thus, isobrucein B (1) displays anti-inflammatory and antihyperalgesic activities by nonselective post-transcriptional modulation, resulting in decreased production/release of pro-inflammatory cytokines and neutrophil migration.

CXCL1/CXCR2 signaling in pathological pain: Role in peripheral and central sensitization

Pathological pain conditions can be triggered after peripheral nerve injury and/or inflammation. It is associated with plasticity of nociceptive pathway in which pain is prolonged even after healing of the injured tissue. Generally combinations of analgesic drugs are not sufficient to achieve selective palliation from chronic pain, besides causing a greater number of side effects. In order to identify novel alternatives for more effective treatments, it is necessary to clarify the underlying mechanisms of pathological pain. It is well established that there are two main components in pathological pain development and maintenance: (i) primary sensory neuron sensitization (peripheral sensitization), and (ii) central sensitization. In both components cytokines and chemokines act as key mediators in pain modulation. CXCL1 is a chemokine that promote both nociceptor and central sensitization via its main receptor CXCR2, which is a promising target for novel analgesic drugs. Here, we reviewed and discussed the role of the CXCL1/CXCR2 signaling axis in pathological pain conditions triggered by either peripheral inflammation or nerve injury.

Peripheral NLCR4 inflammasome participates in the genesis of acute inflammatory pain

Abstract Inflammatory hyperalgesia is a complex process that depends on the sensitization of primary nociceptive neurons triggered by proinflammatory mediators, such as interleukin 1&bgr; (IL-1&bgr;). Recently, the peripheral activation of caspase-1 (previously known as IL-1&bgr;-converting enzyme) was implicated in the induction of acute inflammatory pain by promoting the processing of IL-1&bgr; from its precursor form, pro-IL-1&bgr;. Caspase-1 activation in several systems requires the assembly of an intracellular molecular platform called an inflammasome. Inflammasomes consist of 1 nucleotide-binding oligomerization domain–like receptor (NLR), the adapter molecule apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), and caspase-1. NLRP3 and NLRC4 inflammasomes are well described. However, the identity of the inflammasome that is involved in the peripheral activation of caspase-1 that accounts for acute inflammatory hyperalgesia has not been described. The present findings demonstrated that mice deficient in NLRC4 or ASC, but not in NLRP3, present reduced mechanical and thermal acute inflammatory hyperalgesia induced by carrageenan. The reduced hyperalgesia was accompanied by significant impairments in the levels of mature forms of IL-1&bgr; (p17) and caspase-1 (p20) compared to wild-type mice at the inflammatory site. Therefore, these results identified the inflammasome components NLRC4 and ASC as the molecular platform involved in the peripheral activation of caspase-1 and IL-1&bgr; maturation, which are responsible for the induction of acute inflammatory pain. In conclusion, our study provides new therapeutic targets for the control of acute inflammatory pain.

DF2755A, a novel non-competitive allosteric inhibitor of CXCR1/2, reduces inflammatory and post-operative pain

The activation of CXCR1/2 has been implicated in the genesis of inflammatory and postoperative pain. Here, we investigated a novel orally acting allosteric inhibitor of CXCR1/2 (DF2755A) and evaluated its antinociceptive effect in several models of inflammatory and post-operatory pain. DF2755A was tested in vitro for efficacy in the chemotaxis assay, selectivity and toxicity. In vivo, C57Bl/6 mice were treated orally with DF2755A and the following experiments were performed: pharmacokinetic profile; inflammatory hyperalgesia models using electronic pressure meter test; neutrophil migration assay assessed by myeloperoxidase assay. DF2755A selectively inhibited neutrophil chemotaxis induced by CXCR1/2 ligands without effect on CXCL8 binding to neutrophils. A single mutation of the allosteric site at CXCR1 abrogated the inhibitory effect of DF2755A on CXCL8-induced chemotaxis. DF2755A given orally was well absorbed (88.2%), and it was able to reduce, in a dose (3-30mg/kg)-dependent manner, inflammatory hyperalgesia induced by carrageenan, LPS and CXCL1/KC as well as neutrophil recruitment and IL-1β production. In addition, DF2755A was able to reduce post-incisional nociception. Therapeutic treatment with DF2755A reduced CFA-induced inflammatory hyperalgesia even when injected intrathecally. The present results indicate that DF2755A is a novel selective allosteric inhibitor of CXCR1/2 with a favorable oral pharmacokinetic profile. Furthermore, the results might suggest that DF2755A might be a candidate of a novel therapeutic option to control inflammatory and post-operative pain.

Neuroimmune–Glia Interactions in the Sensory Ganglia Account for the Development of Acute Herpetic Neuralgia

Herpetic neuralgia is the most important symptom of herpes zoster disease, which is caused by Varicella zoster. Nevertheless, the pathophysiological mechanisms involved in herpetic neuralgia are not totally elucidated. Here, we examined the neuroimmune interactions at the sensory ganglia that account for the genesis of herpetic neuralgia using a murine model of Herpes Simplex Virus Type-1 (HSV-1) infection. The cutaneous HSV-1 infection of mice results in the development of a zosteriform-like skin lesion followed by a time-dependent increase in pain-like responses (mechanical allodynia). Leukocytes composed mainly of macrophages and neutrophils infiltrate infected DRGs and account for the development of herpetic neuralgia. Infiltrating leukocytes are responsible for driving the production of TNF, which in turn mediates the development of herpetic neuralgia through downregulation of the inwardly rectifying K+ channel Kir4.1 in satellite glial cells. These results revealed that neuroimmune–glia interactions at the sensory ganglia play a critical role in the genesis of herpetic neuralgia. In conclusion, the present study elucidates novel mechanisms involved in the genesis of acute herpetic pain and open new avenues for its control. SIGNIFICANCE STATEMENT Acute herpetic neuralgia is the most important symptom of herpes zoster disease and it is very difficult to treat. Using a model of peripheral infection of mice with HSV-1, we have characterized for the first time the neuroimmune–glia interactions in the sensory ganglia that account for the development of acute herpetic neuralgia. Among these mechanisms, leukocytes composed mainly of macrophages and neutrophils infiltrate infected sensory ganglia and are responsible for driving the production of TNF. TNF, via TNFR1, mediates herpetic neuralgia development through downregulation of the inwardly rectifying K+ channel Kir4.1 in satellite glial cells. This study elucidates novel mechanisms involved in the genesis of acute herpetic neuralgia and open new avenues for its control.

Chronic restraint stress increases angiotensin II potency in the rat carotid: role of cyclooxygenases and reactive oxygen species

To investigate the mechanisms underlying the effects of chronic restraint stress on the vascular contractile response induced by angiotensin (Ang) II in rat carotid.

Carotid sinus nerve electrical stimulation in conscious rats attenuates systemic inflammation via chemoreceptor activation

Recent studies demonstrated a critical functional connection between the autonomic (sympathetic and parasympathetic) nervous and the immune systems. The carotid sinus nerve (CSN) conveys electrical signals from the chemoreceptors of the carotid bifurcation to the central nervous system where the stimuli are processed to activate sympathetic and parasympathetic efferent signals. Here, we reported that chemoreflex activation via electrical CSN stimulation, in conscious rats, controls the innate immune response to lipopolysaccharide attenuating the plasma levels of inflammatory cytokines such as tumor necrosis factor (TNF), interleukin 1β (IL-1β) and interleukin 6 (IL-6). By contrast, the chemoreflex stimulation increases the plasma levels of anti-inflammatory cytokine interleukin 10 (IL-10). This chemoreflex anti-inflammatory network was abrogated by carotid chemoreceptor denervation and by pharmacological blockade of either sympathetic - propranolol - or parasympathetic - methylatropine – signals. The chemoreflex stimulation as well as the surgical and pharmacological procedures were confirmed by real-time recording of hemodynamic parameters [pulsatile arterial pressure (PAP) and heart rate (HR)]. These results reveal, in conscious animals, a novel mechanism of neuromodulation mediated by the carotid chemoreceptors and involving both the sympathetic and parasympathetic systems.

The NOD2 signaling in peripheral macrophages contributes to neuropathic pain development

Abstract Neuropathic pain is one of the most important types of chronic pain. It is caused by neuronal damage. Clinical and experimental studies suggest a critical role for neuroimmune interactions in the development of neuropathic pain. In this article, we have shown that the cytoplasmic receptor Nod-like receptor-2, NOD2, and its adaptor-signaling molecule RIPK2 participate in the development of neuropathic pain after peripheral nerve injury (spared nerve injury model). The activation of NOD2 signaling in peripheral macrophage mediates the development of neuropathic pain through the production of pronociceptive cytokines (tumor necrosis factor and IL-1&bgr;). This study found that peripheral nerve injury promoted a systemic increase in the NOD2 ligand. These results highlight a previously undetermined role for NOD2 signaling in the development of neuropathic pain, suggesting a new potential target for preventing neuropathic pain.