Cortistatin as a Novel Multimodal Therapy for the Treatment of Parkinson's Disease.

Parkinson's disease (PD) is a complex disorder characterized by the impairment of the dopaminergic nigrostriatal system. PD has duplicated its global burden in the last few years, becoming the leading neurological disability worldwide. Therefore, there is an urgent need to develop innovative approaches that target multifactorial underlying causes to potentially prevent or limit disease progression. Accumulating evidence suggests that neuroinflammatory responses may play a pivotal role in the neurodegenerative processes that occur during the development of PD. Cortistatin is a neuropeptide that has shown potent anti-inflammatory and immunoregulatory effects in preclinical models of autoimmune and neuroinflammatory disorders. The goal of this study was to explore the therapeutic potential of cortistatin in a well-established preclinical mouse model of PD induced by acute exposure to the neurotoxin 1-methil-4-phenyl1-1,2,3,6-tetrahydropyridine (MPTP). We observed that treatment with cortistatin mitigated the MPTP-induced loss of dopaminergic neurons in the substantia nigra and their connections to the striatum. Consequently, cortistatin administration improved the locomotor activity of animals intoxicated with MPTP. In addition, cortistatin diminished the presence and activation of glial cells in the affected brain regions of MPTP-treated mice, reduced the production of immune mediators, and promoted the expression of neurotrophic factors in the striatum. In an in vitro model of PD, treatment with cortistatin also demonstrated a reduction in the cell death of dopaminergic neurons that were exposed to the neurotoxin. Taken together, these findings suggest that cortistatin could emerge as a promising new therapeutic agent that combines anti-inflammatory and neuroprotective properties to regulate the progression of PD at multiple levels.

Cortistatin deficiency reveals a dysfunctional brain endothelium with impaired gene pathways, exacerbated immune activation, and disrupted barrier integrity.

BACKGROUND: Brain activity governing cognition and behaviour depends on the fine-tuned microenvironment provided by a tightly controlled blood-brain barrier (BBB). Brain endothelium dysfunction is a hallmark of BBB breakdown in most neurodegenerative/neuroinflammatory disorders. Therefore, the identification of new endogenous molecules involved in endothelial cell disruption is essential to better understand BBB dynamics. Cortistatin is a neuroimmune mediator with anti-inflammatory and neuroprotective properties that exerts beneficial effects on the peripheral endothelium. However, its role in the healthy and injured brain endothelium remains to be evaluated. Herein, this study aimed to investigate the potential function of endogenous and therapeutic cortistatin in regulating brain endothelium dysfunction in a neuroinflammatory/neurodegenerative environment. METHODS: Wild-type and cortistatin-deficient murine brain endothelium and human cells were used for an in vitro barrier model, where a simulated ischemia-like environment was mimicked. Endothelial permeability, junction integrity, and immune response in the presence and absence of cortistatin were evaluated using different size tracers, immunofluorescence labelling, qPCR, and ELISA. Cortistatin molecular mechanisms underlying brain endothelium dynamics were assessed by RNA-sequencing analysis. Cortistatin role in BBB leakage was evaluated in adult mice injected with LPS. RESULTS: The endogenous lack of cortistatin predisposes endothelium weakening with increased permeability, tight-junctions breakdown, and dysregulated immune activity. We demonstrated that both damaged and uninjured brain endothelial cells isolated from cortistatin-deficient mice, present a dysregulated and/or deactivated genetic programming. These pathways, related to basic physiology but also crucial for the repair after damage (e.g., extracellular matrix remodelling, angiogenesis, response to oxygen, signalling, and metabolites transport), are dysfunctional and make brain endothelial barrier lacking cortistatin non-responsive to any further injury. Treatment with cortistatin reversed in vitro hyperpermeability, tight-junctions disruption, inflammatory response, and reduced in vivo BBB leakage. CONCLUSIONS: The neuropeptide cortistatin has a key role in the physiology of the cerebral microvasculature and its presence is crucial to develop a canonical balanced response to damage. The reparative effects of cortistatin in the brain endothelium were accompanied by the modulation of the immune function and the rescue of barrier integrity. Cortistatin-based therapies could emerge as a novel pleiotropic strategy to ameliorate neuroinflammatory/neurodegenerative disorders with disrupted BBB.

Neuropeptide Cortistatin Regulates Dermal and Pulmonary Fibrosis in an Experimental Model of Systemic Sclerosis.

INTRODUCTION: Scleroderma, or systemic sclerosis, is a complex connective tissue disorder characterized by autoimmunity, vasculopathy, and progressive fibrosis of the skin and internal organs. Because its aetiology is unknown, the identification of genes/factors involved in disease severity, differential clinical forms, and associated complications is critical for understanding its pathogenesis and designing novel treatments. Neuroendocrine mediators in the skin emerge as potential candidates. We investigated the role played by the neuropeptide cortistatin in a preclinical model of scleroderma. METHODS: Dermal fibrosis was induced by repetitive intradermal injections of bleomycin in wild-type and cortistatin-deficient mice. The histopathological signs and expression of fibrotic markers were evaluated in the skin and lungs. RESULTS: An inverse correlation between cortistatin levels and fibrogenic activation exists in the damaged skin and dermal fibroblasts. Bleomycin-challenged skin lesions of mice that are partially and totally deficient in cortistatin showed exacerbated histopathological signs of scleroderma, characterized by thicker and more fibrotic dermal layer, enlarged epidermis, and increased inflammatory infiltration in comparison to those of wild-type mice. Cortistatin deficiency enhanced dermal collagen deposits, connective tissue growth factor expression, loss of microvessels, and predisposition to suffer severe complications that co-occur with dermal exposition to bleomycin, including pulmonary fibrotic disease and increased mortality. Treatment with cortistatin mitigated these pathological processes. DISCUSSION/CONCLUSION: We identify cortistatin as an endogenous break of skin inflammation and fibrosis. Deficiency in cortistatin could be a marker of poor prognosis of scleroderma and associated complications. Cortistatin-based therapies emerge as attractive candidates to treat severe forms of systemic sclerosis and to manage fibrosis-related side effects of bleomycin chemotherapy in oncologic patients.

Cortistatin regulates fibrosis and myofibroblast activation in experimental hepatotoxic- and cholestatic-induced liver injury.

BACKGROUND AND PURPOSE: Liver fibrosis induced by chronic hepatic injury remains a major cause of morbidity and mortality worldwide. Identification of susceptibility/prognosis factors and new therapeutic tools for treating hepatic fibrotic disorders are urgent medical needs. Cortistatin is a neuropeptide with potent anti-inflammatory and anti-fibrotic activities in lung that binds to receptors that are expressed in liver fibroblasts and hepatic stellate cells. We evaluated the capacity of cortistatin to regulate liver fibrosis. EXPERIMENTAL APPROACH: We experimentally induced liver fibrosis in mice by chronic CCl4 exposure and bile duct ligation and evaluated the histopathological signs and fibrotic markers. KEY RESULTS: Hepatic expression of cortistatin inversely correlated with liver fibrosis grade in mice and humans with hepatic disorders. Cortistatin-deficient mice showed exacerbated signs of liver damage and fibrosis and increased mortality rates when challenged by hepatotoxic and cholestatic injury. Compared with wild-type mice, non-parenchymal liver cells isolated from cortistatin-deficient mice showed increased presence of cells with activated myofibroblast phenotypes and a differential genetic signature that is indicative of activated hepatic stellate cells and periportal fibroblasts and of myofibroblasts with active contractile apparatus. Cortistatin treatment reversed in vivo and in vitro these exaggerated fibrogenic phenotypes and protected from progression to severe liver fibrosis in response to hepatic injury. CONCLUSION AND IMPLICATIONS: We identify cortistatin as an endogenous molecular brake on liver fibrosis and its deficiency as a potential poor-prognosis marker for chronic hepatic disorders that link with fibrosis. Cortistatin-based therapies emerge as attractive strategies for ameliorating severe hepatic fibrosis of various aetiologies.

The Neuropeptide Cortistatin Alleviates Neuropathic Pain in Experimental Models of Peripheral Nerve Injury.

Neuropathic pain is one of the most severe forms of chronic pain caused by the direct injury of the somatosensory system. The current drugs for treating neuropathies have limited efficacies or show important side effects, and the development of analgesics with novel modes of action is critical. The identification of endogenous anti-nociceptive factors has emerged as an attractive strategy for designing new pharmacological approaches to treat neuropathic pain. Cortistatin is a neuropeptide with potent anti-inflammatory activity, recently identified as a natural analgesic peptide in several models of pain evoked by inflammatory conditions. Here, we investigated the potential analgesic effect of cortistatin in neuropathic pain using a variety of experimental models of peripheral nerve injury caused by chronic constriction or partial transection of the sciatic nerve or by diabetic neuropathy. We found that the peripheral and central injection of cortistatin ameliorated hyperalgesia and allodynia, two of the dominant clinical manifestations of chronic neuropathic pain. Cortistatin-induced analgesia was multitargeted, as it regulated the nerve damage-induced hypersensitization of primary nociceptors, inhibited neuroinflammatory responses, and enhanced the production of neurotrophic factors both at the peripheral and central levels. We also demonstrated the neuroregenerative/protective capacity of cortistatin in a model of severe peripheral nerve transection. Interestingly, the nociceptive system responded to nerve injury by secreting cortistatin, and a deficiency in cortistatin exacerbated the neuropathic pain responses and peripheral nerve dysfunction. Therefore, cortistatin-based therapies emerge as attractive alternatives for treating chronic neuropathic pain of different etiologies.

Protective role of cortistatin in pulmonary inflammation and fibrosis.

BACKGROUND AND PURPOSE: Acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and pulmonary fibrosis remain major causes of morbidity, mortality and a healthcare burden in critically ill patient. There is an urgent need to identify factors causing susceptibility and for the design of new therapeutic agents. Here, we evaluate the effectiveness of the immunomodulatory neuropeptide cortistatin to regulate pulmonary inflammation and fibrosis in vivo. EXPERIMENTAL APPROACH: ALI/ARDS and pulmonary fibrosis were induced experimentally in wild-type and cortistatin-deficient mice by pulmonary infusion of the bacterial endotoxin LPS or the chemotherapeutic drug bleomycin, and the histopathological signs, pulmonary leukocyte infiltration and cytokines, and fibrotic markers were evaluated. KEY RESULTS: Partially deficient mice in cortistatin showed exacerbated pulmonary damage, pulmonary inflammation, alveolar oedema and fibrosis, and subsequent increased respiratory failure and mortality when challenged to LPS or bleomycin, even at low doses. Treatment with cortistatin reversed these aggravated phenotypes and protected from progression to severe ARDS and fibrosis, after high exposure to both injury agents. Moreover, cortistatin-deficient pulmonary macrophages and fibroblasts showed exaggerated ex vivo inflammatory and fibrotic responses, and treatment with cortistatin impaired their activation. Finally, the protective effects of cortistatin in ALI and pulmonary fibrosis were partially inhibited by specific antagonists for somatostatin and ghrelin receptors. CONCLUSION AND IMPLICATIONS: We identified cortistatin as an endogenous inhibitor of pulmonary inflammation and fibrosis. Deficiency in cortistatin could be a marker of poor prognosis in inflammatory/fibrotic pulmonary disorders. Cortistatin-based therapies could emerge as attractive candidates to treat severe ALI/ARDS, including SARS-CoV-2-associated ARDS.

Correction to: The atypical RhoGTPase RhoE/Rnd3 is a key molecule to acquire a neuroprotective phenotype in microglia.

In the version of this article that was originally published [1]; some information in the "Author's contributions" section was omitted.

The atypical RhoGTPase RhoE/Rnd3 is a key molecule to acquire a neuroprotective phenotype in microglia.

BACKGROUND: Over-activated microglia play a central role during neuroinflammation, leading to neuronal cell death and neurodegeneration. Reversion of over-activated to neuroprotective microglia phenotype could regenerate a healthy CNS-supporting microglia environment. Our aim was to identify a dataset of intracellular molecules in primary microglia that play a role in the transition of microglia to a ramified, neuroprotective phenotype. METHODS: We exploited the anti-inflammatory and neuroprotective properties of conditioned medium of adipose-derived mesenchymal stem cells (CM) as a tool to generate the neuroprotective phenotype of microglia in vitro, and we set up a microscopy-based siRNA screen to identify its hits by cell morphology. RESULTS: We initially assayed an array of 157 siRNAs against genes that codify proteins and factors of cytoskeleton and activation/inflammatory pathways in microglia. From them, 45 siRNAs significantly inhibited the CM-induced transition from a neurotoxic to a neuroprotective phenotype of microglia, and 50 siRNAs had the opposite effect. As a proof-of-concept, ten of these targets were validated with individual siRNAs and by downregulation of protein expression. This validation step resulted essential, because three of the potential targets were false positives. The seven validated targets were assayed in a functional screen that revealed that the atypical RhoGTPase RhoE/Rnd3 is necessary for BDNF expression and plays an essential role in controlling microglial migration. CONCLUSIONS: Besides the identification of RhoE/Rnd3 as a novel inducer of a potential neuroprotective phenotype in microglia, we propose a list of potential targets to be further confirmed with selective activators or inhibitors.

Vasoactive Intestinal Peptide Ameliorates Acute Myocarditis and Atherosclerosis by Regulating Inflammatory and Autoimmune Responses.

Vasoactive intestinal peptide (VIP) is a neuropeptide that exerts various vascular and cardioprotective functions and regulates immune function and inflammatory response at multiple levels. However, its role in inflammatory cardiovascular disorders is largely unknown. Myocarditis and atherosclerosis are two inflammatory and autoimmune cardiovascular diseases that cause important adverse circulatory events. In this study, we investigate the therapeutic effects of VIP in various well-established preclinical models of experimental autoimmune myocarditis and atherosclerosis. Intraperitoneal injection of VIP during the effector phase of experimental autoimmune myocarditis in susceptible BALB/c mice significantly reduced its prevalence, ameliorated signs of heart hypertrophy and injury, attenuated myocardial inflammatory infiltration, and avoided subsequent profibrotic cardiac remodeling. This effect was accompanied by a reduction of Th17-driven cardiomyogenic responses in peripheral lymphoid organs and in the levels of myocardial autoantibodies. In contrast, acute and chronic atherosclerosis was induced in apolipoprotein E-deficient mice fed a hyperlipidemic diet and subjected to partial carotid ligation. Systemic VIP treatment reduced the number and size of atherosclerotic plaques in carotid, aorta, and sinus in hypercholesterolemic mice. VIP reduced Th1-driven inflammatory responses and increased regulatory T cells in atherosclerotic arteries and their draining lymph nodes. VIP also regulated cholesterol efflux in macrophages and reduced the formation of foam cells and their presence in atherosclerotic plaques. Finally, VIP inhibited proliferation and migration of smooth muscle cells and neointima formation in a mouse model of complete carotid ligation. These findings encourage further studies aimed to assess whether VIP can be used as a pharmaceutical agent to treat heart inflammation and atherosclerosis.

Alkylated resveratrol prodrugs and metabolites as potential therapeutics for neurodegenerative diseases.

Resveratrol is a naturally occurring stilbene which has shown promising results as treatment for several neurodegenerative diseases. However, its application is limited due to its low efficacy and bioavailability. Here, we have designed and synthesized alkylated resveratrol prodrugs combining structural modification to improve antioxidant and anti-inflammatory properties and the preparation of prodrugs to extend drug bioavailability. For comparison we also studied resveratrol prodrugs and alkylated resveratrol derivatives. Methylated and butylated resveratrol derivatives showed the best in vitro neuroprotective and anti-inflammatory activity. The glucosyl- and glucosyl-acyl- prodrugs of these derivatives showed lower toxicity on zebra fish embryo. When neuroprotection was examined on pentylenetetrazole challenged zebra fish, they were capable of reverting neuronal damage but to a lower extent than resveratrol. Nevertheless, 3-O-(6'-O-octanoyl)-ß-d-glucopyranoside resveratrol (compound 8) recovered AChE activity over 100% whereas resveratrol only up to 92%. In a 3-nitropropionic acid mice model of Huntington's disease, resveratrol derivative 8 delayed the onset and reduced the severity of HD-like symptoms, by improving locomotor activity and protecting against weight loss. Its effects involved an equal antioxidant but better anti-inflammatory profile than resveratrol as shown by SOD2 expression in brain tissue and circulating levels of IL-6 (11 vs 18 pg/mL), respectively. Finally, the octanoyl chain in compound 8 could be playing a role in inflammation and neuronal development indicating it could be acting as a double-drug, instead of as a prodrug.

Human amnion favours tissue repair by inducing the M1-to-M2 switch and enhancing M2 macrophage features.

Human amniotic mesenchymal cells (hAMTCs) possess interesting immunomodulatory properties, making them attractive candidates for regenerative medicine applications. Recent in vivo reports argue in favour of an important role for macrophages as targets of hAMTC-mediated suppression of inflammation and the enhancement of tissue repair. However, a comprehensive study of the effects of hAMTCs and their conditioned medium (CM) on human macrophage differentiation and function is unavailable. In the present study we found that hAMTCs and CM induce the differentiation of myeloid cells (U937 and monocytes) towards macrophages. We then investigated their effects on monocytes differentiated toward pro-inflammatory M1 and anti-inflammatory M2 macrophages. Monocytes treated under M1 conditions in the presence of hAMTCs or CMs shifted towards M2-like macrophages, which expressed CD14, CD209, CD23, CD163 and PM-2 K, possessed higher phagocytic activity and produced higher IL-10 and lower pro-inflammatory cytokines. They were also poor T cell stimulators and Th1 inducers, while they were able to increase activated and naïve suppressive Treg subsets. We show that prostaglandins, and not IL-6, play a role in determining the M2 activation status. Instead, monocytes treated under M2 conditions in the presence of hAMTCs or CM retained M2-like features, but with an enhanced anti-inflammatory profile, having a reduced expression of the co-stimulatory molecule CD80, reduced phagocytosis activity and decreased the secretion of inflammatory chemokines. Importantly, we provide evidence that macrophages re-educated by CM improve tissue regeneration/repair in wound-healing models. In conclusion, we identified new cell targets of hAMTCs and their bioactive factors and here provide insight into the beneficial effects observed when these cells are used in therapeutic approaches in vivo. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Mesenchymal stem cells induce the ramification of microglia via the small RhoGTPases Cdc42 and Rac1.

Activated microglia play a central role in the course of neurodegenerative diseases as they secrete cytotoxic substances which lead to neuronal cell death. Understanding the mechanisms that drive activation of microglia is essential to reverse this phenotype and to protect from neurodegeneration. With some exceptions, evidence indicates that changes in cell morphology from a star shape to a round and flat shape accompany the process of activation in microglia. In this study, we investigated the effect of adipose-tissue-derived mesenchymal stem cells (ASCs), which exert important anti-inflammatory actions, in microglia morphology. Microglia exposed to ASCs or their secreted factors (conditioned medium) underwent a cell shape change into a ramifying morphology in basal and inflammatory conditions, similar to that observed in microglia found in healthy brain. Colony-stimulating factor-1 secreted by ASCs played a critical role in the induction of this phenotype. Importantly, ASCs reversed the activated round phenotype induced in microglia by bacterial endotoxins. The ramifying morphology of microglia induced by ASCs was associated with a decrease of the proinflammatory cytokines tumor necrosis factor-α and interleukin-6, an increase in phagocytic activity, and the upregulation of neurotrophic factors and of Arginase-1, a marker for M2-like regulatory microglia. In addition, activation of the phosphoinositide-3-kinase/Akt pathway and the RhoGTPases Rac1 and Cdc42 played a major role in the acquisition of this phenotype. Therefore, these RhoGTPases emerge as key players in the ramification of microglia by anti-inflammatory agents like ASCs, being fundamental to maintain the tissue-surveying, central nervous system supporting state of microglia in healthy conditions.

Therapeutic efficacy of stable analogues of vasoactive intestinal peptide against pathogens.

Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide recently identified as a potential antimicrobial peptide. To overcome the metabolic limitations of VIP, we modified the native peptide sequence and generated two stable synthetic analogues (VIP51 and VIP51(6-30)) with better antimicrobial profiles. Herein we investigate the effects of both VIP analogues on cell viability, membrane integrity, and ultrastructure of various bacterial strains and Leishmania species. We found that the two VIP derivatives kill various non-pathogenic and pathogenic Gram-positive and Gram-negative bacteria as well as the parasite Leishmania major through a mechanism that depends on the interaction with certain components of the microbial surface, the formation of pores, and the disruption of the surface membrane. The cytotoxicity of the VIP derivatives is specific for pathogens, because they do not affect the viability of mammalian cells. Docking simulations indicate that the chemical changes made in the analogues are critical to increase their antimicrobial activities. Consequently, we found that the native VIP is less potent as an antibacterial and fails as a leishmanicidal. Noteworthy from a therapeutic point of view is that treatment with both derivatives increases the survival and reduces bacterial load and inflammation in mice with polymicrobial sepsis. Moreover, treatment with VIP51(6-30) is very effective at reducing lesion size and parasite burden in a model of cutaneous leishmaniasis. These results indicate that the VIP analogues emerge as attractive alternatives for treating drug-resistant infectious diseases and provide key insights into a rational design of novel agents against these pathogens.

Adrenomedullin protects from experimental autoimmune encephalomyelitis at multiple levels.

Adrenomedullin is a neuropeptide known for its cardiovascular activities and anti-inflammatory effects. Here, we investigated the effect of adrenomedullin in a model of experimental autoimmune encephalomyelitis (EAE) that mirrors chronic progressive multiple sclerosis. A short-term systemic treatment with adrenomedullin reduced clinical severity and incidence of EAE, the appearance of inflammatory infiltrates in spinal cord and the subsequent demyelination and axonal damage. This effect was exerted at multiple levels affecting both early and late events of the disease. Adrenomedullin decreased the presence/activation of encephalitogenic Th1 and Th17 cells and down-regulated several inflammatory mediators in peripheral lymphoid organs and central nervous system. Noteworthy, adrenomedullin inhibited the production by encephalitogenic cells of osteopontin and of Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), two critical cytokines in the development of EAE. At the same time, adrenomedullin increased the number of IL-10-producing regulatory T cells with suppressive effects on the progression of EAE. Furthermore, adrenomedullin generated dendritic cells with a semi-mature phenotype that impaired encephalitogenic responses in vitro and in vivo. Finally, adrenomedullin regulated glial activity and favored an active program of neuroprotection/regeneration. Therefore, the use of adrenomedullin emerges as a novel multimodal therapeutic approach to treat chronic progressive multiple sclerosis.

Protective role of the neuropeptide urocortin II against experimental sepsis and leishmaniasis by direct killing of pathogens.

We currently face an alarming resurgence in infectious diseases characterized by antimicrobial resistance and therapeutic failure. This has generated the urgent need of developing new therapeutic approaches that include agents with nontraditional modes of action. A recent interest focused on approaches based on our natural immune defenses, especially on peptides that combine innate antimicrobial activity against diverse pathogens and immunoregulatory functions. In this study, to our knowledge, we describe for the first time the antimicrobial activity of the neuropeptide urocortin II (UCNII) against a panel of Gram-positive and Gram-negative bacteria and tropical parasites of the genus Leishmania. Importantly, this cytotoxicity was selective for pathogens, because UCNII did not affect mammalian cell viability. Structurally, UCNII has a cationic and amphipathic design that resembles antimicrobial peptides. Using mutants and UCNII fragments, we determined the structural requirements for the interaction between the peptide and the surface of pathogen. Following its binding to pathogen, UCNII caused cell death through different membrane-disrupting mechanisms that involve aggregation and membrane depolarization in bacteria and pore formation in Leishmania. Noteworthily, UCNII killed the infective form of Leishmania major even inside the infected macrophages. Consequently, UCNII prevented mortality caused by polymicrobial sepsis and ameliorated pathological signs of cutaneous leishmaniasis. Besides its presence in body physical and mucosal barriers, we found that innate immune cells produce UCNII in response to infections. Therefore, UCNII could be considered as an ancient highly-conserved host peptide involved in the natural antimicrobial defense and emerge as an attractive alternative to current treatments for microbial disorders with associated drug resistances.

Paradoxical effect of cortistatin treatment and its deficiency on experimental autoimmune encephalomyelitis.

Cortistatin is a cyclic-neuropeptide produced by brain cortex and immune cells that shows potent anti-inflammatory activity. In this article, we investigated the effect of cortistatin in two models of experimental autoimmune encephalomyelitis (EAE) that mirror chronic and relapsing-remitting multiple sclerosis. A short-term systemic treatment with cortistatin reduced clinical severity and incidence of EAE, the appearance of inflammatory infiltrates in spinal cord, and the subsequent demyelination and axonal damage. This effect was associated with a reduction of the two deleterious components of the disease, namely, the autoimmune and inflammatory response. Cortistatin decreased the presence/activation of encephalitogenic Th1 and Th17 cells in periphery and nervous system, and downregulated various inflammatory mediators, whereas it increased the number of regulatory T cells with suppressive effects on the encephalitogenic response. Moreover, cortistatin regulated glial activity and favored an active program of neuroprotection/regeneration. We further used cortistatin-deficient mice to investigate the role of endogenous cortistatin in the control of immune responses. Surprisingly, cortistatin-deficient mice were partially resistant to EAE and other inflammatory disorders, despite showing competent inflammatory/autoreactive responses. This unexpected phenotype was associated with elevated circulating glucocorticoids and an anxiety-like behavior. Our findings provide a powerful rationale for the assessment of the efficacy of cortistatin as a novel multimodal therapeutic approach to treat multiple sclerosis and identify cortistatin as a key endogenous component of neuroimmune system.

Analgesic effect of the neuropeptide cortistatin in murine models of arthritic inflammatory pain.

OBJECTIVE: To investigate the role of the antiinflammatory neuropeptide cortistatin in chronic pain evoked by joint inflammation. METHODS: Thermal and mechanical hyperalgesia was evoked in mouse knee joints by intraplantar injection of tumor necrosis factor α and intraarticular infusion of Freund's complete adjuvant, and the analgesic effects of cortistatin, administered centrally, peripherally, and systemically, were assessed. In addition, the effects of cortistatin on the production of nociceptive peptides and the activation of pain signaling were assayed in dorsal root ganglion cultures and in inflammatory pain models. The role of endogenous cortistatin in pain sensitization and perpetuation of chronic inflammatory states was evaluated in cortistatin-deficient mice. Finally, the effect of noxious/inflammatory stimuli in the production of cortistatin by the peripheral nociceptive system was assayed in vitro and in vivo. RESULTS: Expression of cortistatin was observed in peptidergic nociceptors of the peripheral nociceptive system, and endogenous cortistatin was found to participate in the tuning of pain sensitization, especially in pathologic inflammatory conditions. Results showed that cortistatin acted both peripherally and centrally to reduce the tactile allodynia and heat hyperalgesia evoked by arthritis and peripheral tissue inflammation in mice, via mechanisms that were independent of its antiinflammatory action. These mechanisms involved direct action on nociceptive neurons and regulation of central sensitization. The analgesic effects of cortistatin in murine arthritic pain were linked to binding of the neuropeptide to somatostatin and ghrelin receptors, activation of the G protein subunit Gαi , impairment of ERK signaling, and decreased production of calcitonin gene-related peptide in primary nociceptors. CONCLUSION: These findings indicate that cortistatin is an antiinflammatory factor with potent analgesic effects that may offer a new approach to pain therapy in pathologic inflammatory states, including osteoarthritis and rheumatoid arthritis.

Adipose-derived mesenchymal stromal cells induce immunomodulatory macrophages which protect from experimental colitis and sepsis.

OBJECTIVE: To investigate the effect of adipose-derived mesenchymal stromal cells (ASCs) on the activation state of macrophages (MΦ) in vitro, and the potential therapeutic effect of these cells in experimental colitis and sepsis. DESIGN: Murine bone marrow-derived macrophages were cultured with ASCs or with ASC conditioned media (ASC-MΦ) and characterised for the expression of several regulatory macrophage markers, including enzymes and cytokines, and for their immunomodulatory capacity in vitro. The therapeutic effect was investigated of ASC-MΦ in two models of experimental inflammatory colitis induced by trinitrobenzene sulphonic acid and dextran sodium sulphate, and in polymicrobial sepsis induced by caecal ligation and puncture. RESULTS: ASC-MΦ showed a phenotype that clearly differed from the classically activated macrophages or the alternatively activated macrophages induced by interleukin (IL)-4, characterised by high arginase activity, increased production of IL-10 upon restimulation and potent immunosuppressive activity on T cells and macrophages. Activation of cyclo-oxygenase-2 on ASCs seems to be critically involved in inducing this phenotype. Systemic infusion of ASC-MΦ inhibited colitis in mice, reducing mortality and weight loss while lowering the colonic and systemic levels of inflammatory cytokines. Importantly, therapeutic injection of ASC-MΦ in established chronic colitis alleviated its progression and avoided disease recurrence. Moreover, ASC-MΦ protected from severe sepsis by reducing the infiltration of inflammatory cells into various organs and by downregulating the production of several inflammatory mediators, where ASC-MΦ-derived IL-10 played a critical role. CONCLUSION: ASCs induce a distinct regulatory activation state of macrophages which possess potent immunomodulatory ability and therapeutic potential in inflammatory bowel diseases and sepsis.

Neuropeptides as pleiotropic modulators of the immune response.

Although necessary to eliminate pathogens, inflammation can lead to serious deleterious effects in the host if left unchecked. During the inflammatory response, further damage may arise from potential autoimmune responses occurring when the immune cells and molecules that respond to pathogen-derived antigens also react to self-antigens. In this sense, the identification of endogenous factors that control exacerbated immune responses is a key goal for the development of new therapeutic approaches for inflammatory and autoimmune diseases. Some neuropeptides that are produced during the ongoing inflammatory response have emerged as endogenous anti-inflammatory agents that could collaborate in tuning the balanced steady state of the immune system. These neuropeptides participate in maintaining immune tolerance through two distinct mechanisms: by regulating the balance between pro-inflammatory and anti-inflammatory factors, and by inducing the emergence of regulatory T cells with suppressive activity against autoreactive T cell effectors. Indeed, a functioning neuropeptide system contributes to general health, and alterations in the levels of these neuropeptides and/or their receptors lead to changes in susceptibility to inflammatory and autoimmune diseases. Recently, we found that some neuropeptides also have antimicrobial and antiparasitic actions, suggesting that they could act as primary mediators of innate defense, even in the most primitive organisms. In this review, we use the vasoactive intestinal peptide as example of an immunomodulatory neuropeptide to summarize the most relevant data found for other neuropeptides with similar characteristics, including adrenomedullin, urocortin, cortistatin and ghrelin.

Glial innate immunity generated by non-aggregated alpha-synuclein in mouse: differences between wild-type and Parkinson's disease-linked mutants.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the presence in the brain of intracellular protein inclusions highly enriched in aggregated alpha-synuclein (α-Syn). Although it has been established that progression of the disease is accompanied by sustained activation of microglia, the underlying molecules and factors involved in these immune-triggered mechanisms remain largely unexplored. Lately, accumulating evidence has shown the presence of extracellular α-Syn both in its aggregated and monomeric forms in cerebrospinal fluid and blood plasma. However, the effect of extracellular α-Syn on cellular activation and immune mediators, as well as the impact of familial PD-linked α-Syn mutants on this stimulation, are still largely unknown. METHODS AND FINDINGS: In this work, we have compared the activation profiles of non-aggregated, extracellular wild-type and PD-linked mutant α-Syn variants on primary glial and microglial cell cultures. After stimulation of cells with α-Syn, we measured the release of Th1- and Th2- type cytokines as well as IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2 and MIP-1α/CCL3 chemokines. Contrary to what had been observed using cell lines or for the case of aggregated α-Syn, we found strong differences in the immune response generated by wild-type α-Syn and the familial PD mutants (A30P, E46K and A53T). CONCLUSIONS: These findings might contribute to explain the differences in the onset and progression of this highly debilitating disease, which could be of value in the development of rational approaches towards effective control of immune responses that are associated with PD.