Migraine therapeutics differentially modulate the CGRP pathway.

BACKGROUND: The clinical efficacy of migraine therapeutic agents directed towards the calcitonin-gene related peptide (CGRP) pathway has confirmed the key role of this axis in migraine pathogenesis. Three antibodies against CGRP - fremanezumab, galcanezumab and eptinezumab - and one antibody against the CGRP receptor, erenumab, are clinically approved therapeutics for the prevention of migraine. In addition, two small molecule CGRP receptor antagonists, ubrogepant and rimegepant, are approved for acute migraine treatment. Targeting either the CGRP ligand or receptor is efficacious for migraine treatment; however, a comparison of the mechanism of action of these therapeutic agents is lacking in the literature. METHODS: To gain insights into the potential differences between these CGRP pathway therapeutics, we compared the effect of a CGRP ligand antibody (fremanezumab), a CGRP receptor antibody (erenumab) and a CGRP receptor small molecule antagonist (telcagepant) using a combination of binding, functional and imaging assays. RESULTS: Erenumab and telcagepant antagonized CGRP, adrenomedullin and intermedin cAMP signaling at the canonical human CGRP receptor. In contrast, fremanezumab only antagonized CGRP-induced cAMP signaling at the human CGRP receptor. In addition, erenumab, but not fremanezumab, bound and internalized at the canonical human CGRP receptor. Interestingly, erenumab also bound and internalized at the human AMY1 receptor, a CGRP receptor family member. Both erenumab and telcagepant antagonized amylin-induced cAMP signaling at the AMY1 receptor while fremanezumab did not affect amylin responses. CONCLUSION: The therapeutic effect of agents targeting the CGRP ligand versus receptor for migraine prevention (antibodies) or acute treatment (gepants) may involve distinct mechanisms of action. These findings suggest that differing mechanisms could affect efficacy, safety, and/or tolerability in migraine patients.

Senso-Immunologic Prospects for Complex Regional Pain Syndrome Treatment.

Complex regional pain syndrome (CRPS) is a chronic pain syndrome that occurs in tissue injuries as the result of surgery, trauma, or ischemia. The clinical features of this severely painful condition include redness and swelling of the affected skin. Intriguingly, it was recently suggested that transient receptor potential ankyrin 1 (TRPA1) is involved in chronic post-ischemia pain, a CRPS model. TRPA1 is a non-selective cation channel expressed in calcitonin gene-related peptide (CGRP)-positive primary nociceptors that becomes highly activated in ischemic conditions, leading to the generation of pain. In this review, we summarize the history of TRPA1 and its involvement in pain sensation, inflammation, and CRPS. Furthermore, bone atrophy is also thought to be a characteristic clinical sign of CRPS. The altered bone microstructure of CRPS patients is thought to be caused by aggravated bone resorption via enhanced osteoclast differentiation and activation. Although TRPA1 could be a target for pain treatment in CRPS patients, we also discuss the paradoxical situation in this review. Nociceptor activation decreases the risk of bone destruction via CGRP secretion from free nerve endings. Thus, TRPA1 inhibition could cause severe bone atrophy. However, the suitable therapeutic strategy is controversial because the pathologic mechanisms of bone atrophy in CRPS are unclear. Therefore, we propose focusing on the remission of abnormal bone turnover observed in CRPS using a recently developed concept: senso-immunology.

Galcanezumab for the prevention of cluster headache.

INTRODUCTION: Cluster headache (CH) is among the worst painful conditions. The available therapies are scarce and not specific, leaving many patients unsatisfied because of poor efficacy and/or tolerability. Patients not responding to common treatments are offered semi-invasive and invasive procedures with uncertain results. Based on the current understanding of CH pathophysiology, new possible therapeutic approaches come from drugs interfering with Calcitonin Gene Related Peptide (CGRP). AREAS COVERED: After summarizing the evidence for CGRP involvement in CH pathophysiology, we review the published literature (PubMed) and information (clinicaltrials.gov, EudraCT, EMA and FDA websites) regarding a novel anti-CGRP monoclonal antibody, Galcanezumab, its pharmacological properties, development, and evidence for the treatment of CH. Publications regarding other indications (migraine) are considered for completeness and safety/tolerability profile. EXPERT OPINION: In one randomized clinical trial, Galcanezumab has proven to be effective and safe as a preventive treatment in episodic CH, with a favorable tolerability profile offering a potential new option in the therapeutic arsenal. Inefficacy of galcanezumab in chronic CH as well as the inefficacy of another monoclonal antibody against CGRP (fremanezumab) in both episodic and chronic CH question the scalability of the drug in CH management. Further, studies comparing galcanezumab to the current standard treatments are highly desirable.

A novel immunocompetent model of metastatic prostate cancer-induced bone pain.

BACKGROUND: Over 70% to 85% of men with advanced prostate cancer (PCa) develop bone metastases characterized by severe bone pain and increased likelihood of bone fracture. These clinical features result in decreased quality of life and act as a predictor of higher mortality. Mechanistically, the skeletal pathologies such as osteolytic lesions and abnormal osteoblastic activity drive these symptoms. The role of immune cells in bone cancer pain remains understudied, here we sought to recapitulate this symptomology in a murine model. METHODS: The prostate cancer bone metastasis-induced pain model (CIBP) was established by transplanting a mouse prostate cancer cell line into the femur of immunocompetent mice. Pain development, gait dynamics, and the changes in emotional activities like depression and anxiety were evaluated. Animal tissues including femurs, dorsal root ganglion (DRG), and spinal cord were collected at killing and microcomputed tomography (µCT), histology/immunohistochemistry, and quantitative immunofluorescent analysis were performed. RESULTS: Mice receiving prostate cancer cells showed a significantly lower threshold for paw withdrawal responses induced by mechanical stimulation compared with their control counterparts. Zero maze and DigiGait analyses indicated reduced and aberrant movement associated emotional activity compared with sham control at 8-weeks postinjection. The µCT analysis showed osteolytic and osteoblastic changes and a 50% reduction of the trabecular volumes within the prostate cancer group. Neurologically we demonstrated, increased calcitonin gene-related peptide (CGRP) and neuronal p75NTR immune-reactivities in both the projected terminals of the superficial dorsal horn and partial afferent neurons in DRG at L2 to L4 level in tumor-bearing mice. Furthermore, our data show elevated nerve growth factor (NGF) and TrkA immunoreactivities in the same segment of the superficial dorsal horn that were, however, not colocalized with CGRP and p75NTR . CONCLUSIONS: This study describes a novel immunocompetent model of CIBP and demonstrates the contribution of NGF and p75NTR to chronic pain in bone metastasis.

CGRP pathway monoclonal antibodies for cluster headache.

INTRODUCTION: The involvement of the calcitonin gene-related peptide (CGRP) pathway in primary headache disorders, especially migraine, had led to recent success in the development of new migraine therapies. The CGRP pathway also plays a role in the pathophysiology of cluster headache, so CGRP pathway monoclonal antibodies have been studied in the prevention of cluster headache attacks. AREAS COVERED: This review will outline the trials of fremanezumab and galcanezumab, the two CGRP pathway monoclonal antibodies that have undergone trials in cluster headache prevention. This review will highlight key efficacy and safety outcomes from the trials. EXPERT OPINION: Galcanezumab was shown to be efficacious, reducing the frequency of attacks in episodic cluster headache, while fremanezumab failed its primary endpoint in episodic cluster headache. Both fremanezumab and galcanezumab trials in chronic cluster headache were terminated after futility analysis predicting the failure of both trials to fulfil their primary endpoint. The role of CGRP in cluster headache supports ongoing trials of the remaining CGRP pathway monoclonal antibodies and gepants for preventive and acute treatment. A broad view would include targeting neuropeptides involved in parasympathetic signaling in cluster headache, such as pituitary adenylate cyclase-activating peptide (PACAP); such targets warrant exploration in the search of new treatments.

Peptides, MAbs, Molecules, Mechanisms, and More: Taking a Stab at Cluster Headache.

BACKGROUND: Cluster headache is a highly disabling neurological disorder. PURPOSE: The purpose of this review is to highlight recent therapeutic advances in the treatment of cluster headache such as monoclonal antibodies as well as non-invasive vagus nerve stimulation, and examine future potential therapeutic targets. DISCUSSION: Several therapeutic agents currently in use may have underlying mechanisms important to cluster headache pathophysiology and have yet to be completely elucidated. The psychobiological aspects of cluster headache have a significant impact on patients, as well as pose limitations for treatment. Neuropeptides may play a role in underlying mechanisms in why cluster headache patients are frequent tobacco smokers. Alterations in the hypothalamic-pituitary-adrenal axis and neuroinflammation may play a role in suicidality. The circadian nature of cluster headache may generate the development of future treatment options. New understanding of mechanisms underlying post-traumatic headache may also provide insights into cluster headache pathophysiology. CONCLUSION: Molecular targets and neuromodulation advances have paved the way for a new generation of therapeutic agents in cluster headache. There are several other potential targets.

CGRP-CRLR/RAMP1 signal is important for stress-induced hematopoiesis.

Ecotropic viral integration site-1 (EVI1) has a critical role in normal and malignant hematopoiesis. Since we previously identified high expression of calcitonin receptor like receptor (CRLR) in acute myeloid leukemia (AML) with high EVI1 expression, we here characterized the function of CRLR in hematopoiesis. Since higher expression of CRLR and receptor activity modifying protein 1 (RAMP1) was identified in immature hematopoietic bone marrow (BM) cells, we focused on calcitonin gene-related peptide (CGRP), a specific ligand for the CRLR/RAMP1 complex. To elucidate the role of CGRP in hematopoiesis, Ramp1-deficient (Ramp1-/-) mice were used. The steady-state hematopoiesis was almost maintained in Ramp1-/- mice; however, the BM repopulation capacity of Ramp1-/- mice was significantly decreased, and the transplanted Ramp1-/- BM mononuclear cells had low proliferation capacity with enhanced reactive oxygen species (ROS) production and cell apoptosis. Thus, CGRP is important for maintaining hematopoiesis during temporal exposures with proliferative stress. Moreover, continuous CGRP exposure to mice for two weeks induced a reduction in the number of BM immature hematopoietic cells along with differentiated myeloid cells. Since CGRP is known to be increased under inflammatory conditions to regulate immune responses, hematopoietic exhaustion by continuous CGRP secretion under chronic inflammatory conditions is probably one of the important mechanisms of anti-inflammatory responses.

The Therapeutic Impact of New Migraine Discoveries.

BACKGROUND: Migraine is one of the most disabling neurological conditions and associated with high socio-economic costs. Though certain aspects of the pathomechanism of migraine are still incompletely understood, the leading hypothesis implicates the role of the activation of the trigeminovascular system. Triptans are considered to be the current gold standard therapy for migraine attacks; however, their use in clinical practice is limited. Prophylactic treatment includes non-specific approaches for migraine prevention. All these support the need for future studies in order to develop innovative anti-migraine drugs. OBJECTIVE: The present study is a review of the current literature regarding new therapeutic lines in migraine research. METHODS: A systematic literature search in the database of PUBMED was conducted concerning therapeutic strategies in a migraine published until July 2017. RESULTS: Ongoing clinical trials with 5-HT1F receptor agonists and glutamate receptor antagonists offer promising new aspects for acute migraine treatment. Monoclonal antibodies against CGRP and the CGRP receptor are revolutionary in preventive treatment; however, further long-term studies are needed to test their tolerability. Preclinical studies show positive results with PACAP- and kynurenic acid-related treatments. Other promising therapeutic strategies (such as those targeting TRPV1, substance P, NOS, or orexin) have failed to show efficacy in clinical trials. CONCLUSION: Due to their side-effects, current therapeutic approaches are not suitable for all migraine patients. Especially frequent episodic and chronic migraine represents a therapeutic challenge for researchers. Clinical and preclinical studies are needed to untangle the pathophysiology of migraine in order to develop new and migraine-specific therapies.

CGRP/CGRP Receptor Antibodies: Potential Adverse Effects Due to Blockade of Neovascularization?

Migraine is a severe neurological disorder in which calcitonin gene-related peptide (CGRP) is a key molecule in pathophysiology. Neuronal system-derived CGRP enhances neovascularization in several important pathological conditions and sends a cue to the vascular system. In 2018, the FDA approved erenumab and fremanezumab, antibodies against CGRP receptor and CGRP, as the first new class of drugs for migraine. Treatment of migraine with these antibodies requires great care because neovascularization-related adverse effects may be induced in some patients. Here, we focus on enhancement of neovascularization by CGRP and discuss possible adverse effects resulting from blocking neovascularization. We also suggest that CGRP antibodies may also be used as novel antitumor agents by suppressing tumor-associated angiogenesis.

Calcitonin Gene-Related Peptide Regulates the Potential Antigen Uptake Ability of Human Bronchial Epithelial Cells.

In this study we tried to explore whether calcitonin gene-related peptide (CGRP) regulates the potential antigen uptaking ability of human bronchial epithelial cells (HBECs) and promoting the differentiation of Th1/Th2. We found that CGRP increased the uptake of fluorescein isothiocyanate labeled ovalbumin (FITC-OVA) by HBECs using fluorescence microscopy and flow cytometry analysis. MTT assay showed that T cells proliferated in a dose-dependent manner in the presence of OVA-pretreated HBECs and CGRP inhibited the proliferation of T cells. CGRP decreased secretion of IFN-γ, while it had no influence on secretion of IL-4 by ELISA. Our data suggest that CGRP enhanced HBECs antigen uptake ability and inhibits HBECs induced T cells proliferation.

Calcitonin gene-related peptide exerts anti-inflammatory property through regulating murine macrophages polarization in vitro.

Acute lung injury (ALI) is a condition resulting from direct or indirect lung injury associated with high mortality and morbidity. The phenotype of macrophages in lung contributes to the pathological progress of ALI. Calcitonin gene-related peptide (CGRP) is one of the most abundant neuropeptides in lung, and attenuates lipopolysaccharide (LPS)-induced ALI in rats. However, the exact effect of CGRP on the activation of macrophages remains unknown. Here we investigate the effect of CGRP on the macrophages activation and inflammation in murine macrophages in vitro. We found that LPS increased the expression of CGRP in a LPS-induced ALI murine model and LPS-stimulated murine macrophages. Although CGRP didn't alter the expression of tumor necrosis factor-α (a marker of pro-inflammatory phenotype of macrophages, M1 macrophages) or Arginase 1 (Arg1, a marker of M2 macrophages) in non-differentiated macrophages, CGRP significantly reduced the NLRP3 and pro-IL-1ß mRNA expression induced by LPS, as well as NLRP3 protein and IL-1ß secretion induced by LPS+ATP in macrophages in vitro. On the other hand, CGRP dramatically enhanced the Arg1 expression and activity induced by IL-4 in the time- and dose-dependent manners. CGRP also promoted the expression of markers of M2 macrophages (IL-10, Fizz1 and Mrc1) induced by IL-4 in murine macrophages. These effects of CGRP were also observed in primary murine peritoneal macrophages. In addition, we found that CGRP regulated macrophages polarization partially through calmodulin, PKC and PKA pathways. Specifically, CGRP could inhibit the degradation of I-κB induced by LPS, and enhance the phosphorylation of STAT6 induced by IL-4 in macrophages. In conclusion, our results indicate that CGRP regulates macrophage polarization and inhibits inflammation in murine macrophages.

Migraine, Neurogenic Inflammation, Drug Development - Pharmacochemical Aspects.

BACKGROUND: Migraine is a primary headache disorder. Despite numerous studies conducted with the aim to understand the pathophysiology of migraine, several aspects are still unclear. The trigeminovascular system plays a key role. Neurogenic inflammation is presumed to be an important factor in migraine pathophysiology, mediated by the activation of primary neurons, leading to the release of various pro-inflammatory neuropeptides and neurotransmitters such as Calcitonin Gene-Related Peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP). Nitric oxide (NO), Pituitary adenylate cyclase-activating polypeptide (PACAP) and Glutamate (Glu) also play an important role in the modulation of inflammatory mechanisms. OBJECTIVE: To review the literature focusing on novel therapeutic targets in migraine, related to neurogenic inflammation. METHOD: A systematic literature search in the database of PUBMED was conducted regarding therapeutic strategies in migraine, focusing on substances and cytokines released during neurogenic inflammation, published until January 2017. RESULTS: Ongoing phase III clinical studies with monoclonal antibodies against CGRP and CGRP receptors offer promising novel aspects for migraine treatment. Preclinical and clinical studies targeting SP and nitric oxide synthase (NOS) were all terminated with no significant results compared to placebo. New promising therapeutic goal could be PACAP and its receptor (PAC1), and kynurenic acid (KYNA) analogues. CONCLUSION: Current migraine treatment offers pain relief only for a small proportion of migraine patients and might not be adequate for patients with cardiovascular comorbidity due to side effects. Better understanding of migraine pathophysiology might, therefore, lead to novel therapeutic lines both in migraine attack treatment and prophylaxis.

Chemical Coding of Sensory Neurons Supplying the Hip Joint Capsule in the Sheep.

Immunohistochemical properties of nerve fibres supplying the joint capsule were previously described in many mammalian species, but the localization of sensory neurons supplying this structure was studied only in laboratory animals, the rat and rabbit. However, there is no comprehensive data on the chemical coding of sensory neurons projecting to the hip joint capsule (HJC). The aim of this study was to establish immunohistochemical properties of sensory neurons supplying HJC in the sheep. The study was carried out on 10 sheep, weighing about 30-40 kg. The animals were injected with a retrograde neural tracer Fast Blue (FB) into HJC. Sections of the spinal ganglia (SpG) with FB-positive (FB+) neurons were stained using antibodies against calcitonin gene-related peptide (CGRP) substance P (SP), pituitary adenylate cyclase-activating peptide (PACAP), nitric oxide synthase (n-NOS), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), Leu-5-enkephalin (Leu-Enk), galanin (GAL) and vesicular acetylcholine transporter (VACHT). The vast majority of FB+ neurons supplying HJC was found in the ganglia from the 5th lumbar to the 2nd sacral. Immunohistochemistry revealed that most of these neurons were immunoreactive to CGRP or SP (80.7 ± 8.0% or 56.4 ± 4.8%, respectively) and many of them stained for PACAP or GAL (52.9 ± 2.9% or 50.6 ± 19.7%, respectively). Other populations of FB+ neurons were those immunoreactive to n-NOS (37.8 ± 9.7%), NPY (34.6 ± 6.7%), VIP (28.7 ± 4.8%), Leu-Enk (27.1 ± 14.6) and VACHT (16.7 ± 9.6).

Calcitonin Peptide Family Members Are Differentially Regulated by LPS and Inhibit Functions of Rat Alveolar NR8383 Macrophages.

Members of the calcitonin peptide family-calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin2/intermedin (IMD)-exert modulatory effects upon monocytes and macrophages of various extrapulmonary origins. Utilizing the rat alveolar macrophage (AMφ) cell line NR8383, we here set out to determine to which extent these three peptides and their receptors are differentially regulated in AMφ and what specific effects they have on AMφ key functions. LPS treatment differentially up-regulated expression of the peptides and receptors. Among the three peptides, IMD mRNA content was lowest both in primary rat AMφ and NR8383 cells, whereas IMD peptide dominated in basal and LPS-stimulated secretion from NR8383 cells. Fcγ receptor-mediated phagocytosis and TNF-α production were inhibited by AM, IMD, and CGRP, whereas pro-IL-1ß mRNA was slightly down-regulated exclusively by CGRP. Neither of these peptides affected IL-6 or IL-10 production. None increased intracellular calcium concentration, but AM significantly inhibited store-operated calcium entry. In conclusion, the rat AMφ cell line NR8383 is both a source and a target of the calcitonin peptide family members AM, IMD, and CGRP. Despite sharing proteins of the receptor complexes, AM, IMD, and CGRP each showed a characteristic pattern of effects and regulation, suggesting that these closely related peptides are not just redundant members of one common signaling pathway but act in concert by addressing parallel signaling cascades. Since peptide and receptor expression are up-regulated by LPS, these signaling pathways might act as inhibitory feedback mechanisms in pulmonary bacterial infection.

Exogenous α-calcitonin gene-related peptide attenuates lipopolysaccharide-induced acute lung injury in rats.

α-Calcitonin gene-related peptide (α-CGRP) is a 37 amino-acid neuropeptide that is primarily released from C-type sensory neurons. α-CGRP exerts multiple modulatory effects on immune responses and visceral organ function, but the role of exogenous α-CGRP in lipopolysaccharide (LPS)-induced acute lung injury (ALI) has remained to be elucidated. Forty-eight rats were randomized to receive continuous intraperitoneal infusion of α-CGRP (0.4 µg/kg/min) or normal saline for 30 min, followed by intratracheal injection of 0.5 mg/kg LPS or saline. There were four groups of animals: The saline-saline (S-S) group; the saline-α-CGRP (S-C) group; the LPS-saline (L-S) group and the LPS-α-CGRP (L-C) group. Mean arterial pressure and arterial blood gases were assessed prior to α-CGRP and LPS administration and every hour following LPS treatment. After 4 h, bronchoalveolar lavage was performed and used to assess total cell count and levels of tumor necrosis factor-α, interleukin-1ß, intracellular cell adhesion molecule 1 and macrophage inflammatory protein 2. Lung tissue was also collected for assessing wet-to-dry (W/D) ratio, histology and Evans blue (EB) dye extravasation. Pulmonary α-CGRP concentration and α-CGRP receptor expression were also examined, and inducible cyclic adenosine monophosphate early repressor (ICER) and TNF-α mRNA expression levels were measured. Treatment with exogenous α-CGRP improved oxygenation during LPS-induced ALI. Correspondingly, histological injury, total cell count, inflammatory cytokine levels, W/D ratio and EB dye extravasation were also significantly reduced. α-CGRP receptor 1 expression was noted in pulmonary endothelial cells and alveolar macrophages and α-CGRP receptor expression levels were decreased during ALI, whereas pulmonary α-CGRP expression was continuously increased. Furthermore, exogenous α-CGRP induced upregulation of ICER during LPS-induced ALI. In conclusion, exogenous α-CGRP improved oxygenation and ameliorated lung damage in LPS-induced ALI, and these effects were associated with the upregulation of ICER.

Endogenous prostaglandins and afferent sensory nerves in gastroprotective effect of hydrogen sulfide against stress-induced gastric lesions.

Hydrogen sulfide (H2S) plays an important role in human physiology, exerting vasodilatory, neuromodulatory and anti-inflammatory effects. H2S has been implicated in the mechanism of gastrointestinal integrity but whether this gaseous mediator can affect hemorrhagic lesions induced by stress has been little elucidated. We studied the effect of the H2S precursor L-cysteine, H2S-donor NaHS, the H2S synthesizing enzyme (CSE) activity inhibitor- D,L-propargylglycine (PAG) and the gastric H2S production by CSE/CBS/3-MST activity in water immersion and restraint stress (WRS) ulcerogenesis and the accompanying changes in gastric blood flow (GBF). The role of endogenous prostaglandins (PGs) and sensory afferent nerves releasing calcitonin gene-related peptide (CGRP) in the mechanism of gastroprotection induced by H2S was examined in capsaicin-denervated rats and those pretreated with capsazepine to inhibit activity of vanilloid receptors (VR-1). Rats were pretreated with vehicle, NaHS, the donor of H2S and or L-cysteine, the H2S precursor, with or without the concurrent treatment with 1) nonselective (indomethacin) and selective cyclooxygenase (COX)-1 (SC-560) or COX-2 (rofecoxib) inhibitors. The expression of mRNA and protein for COX-1 and COX-2 were analyzed in gastric mucosa pretreated with NaHS with or without PAG. Both NaHS and L-cysteine dose-dependently attenuated severity of WRS-induced gastric lesions and significantly increased GBF. These effects were significantly reduced by pretreatment with PAG and capsaicin denervation. NaHS increased gastric H2S production via CSE/CBS but not 3-MST activity. Inhibition of COX-1 and COX-2 activity significantly diminished NaHS- and L-cysteine-induced protection and hyperemia. NaHS increased expression of COX-1, COX-2 mRNAs and proteins and raised CGRP mRNA expression. These effects of NaHS on COX-1 and COX-2 protein contents were reversed by PAG and capsaicin denervation. We conclude that H2S exerts gastroprotection against WRS-induced gastric lesions by the mechanism involving enhancement in gastric microcirculation mediated by endogenous PGs, sensory afferent nerves releasing CGRP and the activation of VR-1 receptors.

TRPM8 on mucosal sensory nerves regulates colitogenic responses by innate immune cells via CGRP.

TRPM8 is the molecular sensor for cold; however, the physiological role of TRPM8+ neurons at mucosal surfaces is unclear. Here we evaluated the distribution and peptidergic properties of TRPM8+ fibers in naive and inflamed colons, as well as their role in mucosal inflammation. We found that Trpm8(-/-) mice were hypersusceptible to dextran sodium sulfate (DSS)-induced colitis, and that Trpm8(-/-) CD11c+ DCs (dendritic cells) showed hyperinflammatory responses to toll-like receptor (TLR) stimulation. This was phenocopied in calcitonin gene-related peptide (CGRP) receptor-deficient mice, but not in substance P receptor-deficient mice, suggesting a functional link between TRPM8 and CGRP. The DSS phenotype of CGRP receptor-deficient mice could be adoptively transferred to wild-type (WT) mice, suggesting that CGRP suppresses the colitogenic activity of bone marrow-derived cells. TRPM8+ mucosal fibers expressed CGRP in human and mouse colon. Furthermore, neuronal CGRP contents were increased in colons from naive and DSS-treated Trpm8(-/-) mice, suggesting deficient CGRP release in the absence of TRPM8 triggering. Finally, treatment of Trpm8(-/-) mice with CGRP reversed their hyperinflammatory phenotype. These results suggest that TRPM8 signaling in mucosal sensory neurons is indispensable for the regulation of innate inflammatory responses via the neuropeptide CGRP.

Neuroimmunological communication via CGRP promotes the development of a regulatory phenotype in TLR4-stimulated macrophages.

Environmental signals shape the phenotype and function of activated macrophages. Here, we show that the neuropeptide calcitonin gene-related peptide (CGRP), which is released from sensory nerves, modulates the phenotype of TLR4-activated murine macrophages by enhancing expression of the regulatory macrophage markers IL-10, sphingosine kinase 1 (SPHK1), and LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes). In contrast, CGRP inhibits production of cytokines characteristic of inflammatory macrophages and does not affect expression of wound-healing macrophage markers upon TLR4 engagement. In IL-4-stimulated macrophages, CGRP increased LIGHT expression, but failed to induce IL-10 and SPHK1. The stimulatory effect of CGRP on IL-10 production required activation of protein kinase A and was linked to prolonged phosphorylation of CREB and sustained nuclear accumulation of CRTC2 and CRTC3 (where CRTC is CREB-regulated transcriptional cofactor). CGRP enhanced expression of regulatory macrophage markers during the early, but not late, phase of LPS-stimulation and this effect was independent of autocrine type-I IFN activity. In contrast, autocrine type-I IFN activity and treatment of macrophages with IFN-ß promoted late-phase IL-10 production, but had only minor influence on LIGHT and SPHK1 expression. Together, the results identify neuroimmunological communication through CGRP as a novel costimulatory pathway promoting the development of a regulatory phenotype of TLR4-stimulated macrophages. CGRP appears to act through a mechanism that involves sustained activation of CREB-dependent gene transcription.

CGRP, a neurotransmitter of enteric sensory neurons, contributes to the development of food allergy due to the augmentation of microtubule reorganization in mucosal mast cells.

Neuro-immune interaction in the gut is substantially involved in the maintenance of intestinal immune homeostasis and the pathology of intestinal immune diseases. We have previously demonstrated that mucosal mast cells and nerve fibers containing CGRP, a neurotransmitter of intrinsic enteric sensory neurons, are markedly increased and exist in close proximity to each other in the colon of food allergy (FA) mice. In the present study, a CGRP-receptor antagonist BIBN4096BS significantly alleviated allergic symptoms in the murine FA model. In addition, the elevated numbers of mucosal mast cells in the proximal colon of FA mice were significantly decreased in that of BIBN4096BS-treated FA mice. Thus, we investigated the effects of CGRP on calcium-independent process in degranulation of mucosal mast cells since CGRP increases intracellular cAMP levels, but not Ca(2+) concentration. CGRP did not alter a calcium ionophore A23187-increased cytosolic Ca(2+) concentration in mucosal-type bone marrow-derived mast cells (mBMMCs), but did augment microtubule reorganization in resting and A23187-activated mBMMCs. Furthermore, CGRP alone failed to cause the degranulation of mBMMCs, but CGRP significantly enhanced the degranulation of mBMMCs induced by A23187. Together, these data indicate that CGRP- enhanced microtubule reorganization augments IgE-independent/non-antigenic stimuli-induced mucosal mast cell degranulation, thereby contributing to the development of FA.