Dr Michelangelo Certo | University of Birmingham (original) (raw)

Papers by Dr Michelangelo Certo

Science Translational Medicine, 2018

Silencing key neurons with botulinum toxin conjugates exerts long-lasting pain relief in mouse mo... more Silencing key neurons with botulinum toxin conjugates exerts long-lasting pain relief in mouse models of chronic pain.

ASSAY and Drug Development Technologies, 2016

Repurposing the macrolide antibiotic azithromycin has recently been suggested as a promising neur... more Repurposing the macrolide antibiotic azithromycin has recently been suggested as a promising neuroprotective strategy for the acute treatment of ischemic stroke. Here, we aim at further characterizing the immunomodulatory properties of intraperitoneal (i.p.) administration of this drug and, more importantly, at assessing whether neuroprotection can also be achieved by the more clinically relevant intravenous (i.v.) route of administration in a mouse model of focal cerebral ischemia induced by transient (30-min) middle cerebral artery occlusion (MCAo). A single i.p. injection of azithromycin (150 mg/kg) upon reperfusion prevented ischemia-induced spleen contraction and increased the number of MAC-1-immunopositive microglia/macrophages in the ischemic hemisphere 48 h after the insult. This was paralleled by an elevation of alternatively activated phenotypes (i.e., Ym1immunopositive M2-polarized cells) and by a reduced expression of the pro-inflammatory marker myeloperoxidase. More importantly, i.v. administration of azithromycin upon reperfusion reduced MCAoinduced infarct volume and cerebral edema to an extent comparable to that obtained via the i.p. route. Although the i.p. route is often used for research purposes, it is impractical in the clinical setting; however, i.v. administration can easily be used in ischemic stroke patients who usually have i.v. access already established on hospital admission. The neuroprotective efficacy of the clinically relevant i.v. administration of azithromycin, together with its beneficial immunomodulatory properties reported in mice subjected to transient MCAo, suggests that this macrolide antibiotic can be effectively repurposed for the acute treatment of ischemic stroke. To this end, further work is needed to validate the efficacy of azithromycin in the clinical setting.

Mini-Reviews in Medicinal Chemistry, 2016

Ischemic stroke is a devastating condition primarily caused by reduced blood supply to the brain.... more Ischemic stroke is a devastating condition primarily caused by reduced blood supply to the brain. Interleukin (IL)-1β is a pro-inflammatory cytokine that plays a pivotal role in the detrimental inflammatory processes that participate to cerebral ischemic damage. After injury, it is produced by distinct cells of the neurovascular unit as an inactive precursor, pro-IL-1β. Although previous studies have suggested that caspase-1 is the main enzyme implicated in the cleavage of pro-IL-1β into the biologically active cytokine, recent work has demonstrated that, under ischemia-reperfusion conditions, other mechanisms may be involved in cytokine maturation. Indeed, we have shown that in rats subjected to transient middle cerebral artery occlusion (MCAo), elevation of IL-1β levels is paralleled by an elevation of gelatinolytic, but not caspase-1 activity in the injured hemisphere and pharmacological inhibition of gelatinases, i.e. matrix metalloproteases (MMP)-2 and MMP-9 prevents cytokine maturation. These findings further support the hypothesis that, under ischemia-reperfusion injury, cerebral elevation of IL-1β occurs via mechanisms other than caspase-1, likely involving gelatinases.

Pharmacological Research, 2015

The identification of novel drug targets for the treatment of ischemic stroke is currently an urg... more The identification of novel drug targets for the treatment of ischemic stroke is currently an urgent challenge. Recent experimental findings have highlighted the neuroprotective potential of immunomodulatory strategies, based on polarization of myeloid cells toward non-inflammatory, beneficial phenotypes. Given the role of retinoid X receptors (RXR) in myeloid cells differentiation and polarization, here we have explored the neuroprotective potential of the RXR agonist bexarotene in mice subjected to focal cerebral ischemia. Acute administration of bexarotene significantly reduced blood brain barrier leakage, brain infarct damage and neurological deficit produced by transient middle cerebral artery occlusion in mice, without affecting cerebral blood flow. The rexinoid exerted neuroprotection with a wide time-window, being effective when administered up to 4.5h after the insult. The amelioration of histological outcome, as well as the ability of bexarotene to revert middle cerebral artery occlusion (MCAo)-induced spleen atrophy, was antagonised by BR1211, a pan-RXR antagonist, or by the selective peroxisome proliferator-activated receptor (PPAR)γ antagonist bisphenol A diglycidyl ether (BADGE), highlighting the involvement of the RXR/PPARγ heterodimer in the beneficial effects exerted by the drug. Immunofluorescence analysis revealed that bexarotene elevates Ym1-immunopositive N2 neutrophils both in the ipsilateral hemisphere and in the spleen of mice subjected to transient middle cerebral artery occlusion, pointing to a major role for peripheral neutrophil polarization in neuroprotection. Thus, our findings suggest that the RXR agonist bexarotene exerts peripheral immunomodulatory effects under ischemic conditions to be effectively repurposed for the acute therapy of ischemic stroke.

Experimental Neurology, 2016

To develop novel and effective treatments for ischemic stroke, we investigated the neuroprotectiv... more To develop novel and effective treatments for ischemic stroke, we investigated the neuroprotective effects of the macrolide antibiotic azithromycin in a mouse model system of transient middle cerebral artery occlusion. Intraperitoneal administration of azithromycin significantly reduced blood-brain barrier damage and cerebral infiltration of myeloid cells, including neutrophils and inflammatory macrophages. These effects resulted in a dose-dependent reduction of cerebral ischemic damage, and in a remarkable amelioration of neurological deficits up to 7days after the insult. Neuroprotection was associated with increased arginase activity in peritoneal exudate cells, which was followed by the detection of Ym1- and arginase I-immunopositive M2 macrophages in the ischemic area at 24-48h of reperfusion. Pharmacological inhibition of peritoneal arginase activity counteracted azithromycin-induced neuroprotection, pointing to a major role for drug-induced polarization of migratory macrophages towards a protective, non-inflammatory M2 phenotype.

Frontiers in Neuroscience, 2015

Introduction Recent microarray studies have revealed that the majority of the genes acutely modul... more Introduction Recent microarray studies have revealed that the majority of the genes acutely modulated in the blood of ischemic stroke patients regulate the immune system and are expressed in circulating neutrophils and macrophages (Tang et al, J Cereb Blood Flow Metab 2006;26:1089-102; Barr et al., Neurology 2010;75:1009-1014; Oh et al, J. Neuroimmunol. 2012;249:60-65). Accordingly, systemic inflammatory reactions contribute to ischemic stroke injury, involving activation and brain recruitment of macrophages, neutrophils, dendritic cells and lymphocytes. Cerebral infiltration of blood-borne macrophages and neutrophils exerts a dualistic role on the evolution of brain damage depending on their ability to acquire specific phenotypes (Amantea et al, Curr Med Chem 2014;21:2098-117; Cuartero et al Stroke 2013, 44:3498-508; Fumagalli et al Glia 2013, 61:827-42; Perego et al J Neuroinflamm 2011, 8:174). Thus, targeting the peripheral immune system, by inhibiting detrimental inflammatory reactions, while promoting polarization of circulating leukocytes towards beneficial non-inflammatory phenotypes, represents a promising innovative strategy for stroke treatment. Accordingly, reducing cerebral infiltration of neutrophils and detrimental M1 macrophages, while elevating the number of Ym1- and arginase I-immunopositive M2 macrophages in the ischemic hemisphere, the macrolide antibiotic azithromycin provides neuroprotection in a mouse model of transient middle cerebral artery occlusion (tMCAo) (Amantea et al., 2012, BiS12). Methods Focal brain ischemia was induced by transient (30-min) MCAo with an intraluminal filament (Amantea et al Neuroscience 2008,152:8-17) in adult C57Bl/6 male mice receiving azithromycin (0.15-150 mg/kg) or vehicle (0.9% NaCl) i.p. upon reperfusion. Sham animals were subjected to same surgical procedure without filament insertion. The peripheral activation and brain infiltration of systemic immune cells was characterised by immunofluorescence labelling of specific cell markers (i.e., CD11b and Iba1 for microglia/macrophages, Ly6B for neutrophils, Ym1 and arginase for M2-macrophages) combined with flow cytometry analysis (Pallotta et al Nat Immunol. 2011;12:870-8) of brain and peritoneal samples from mice undergone tMCAo. Arginase activity was assessed in peritoneal exudate cells by measuring the concentration of urea generated by the arginase-dependent hydrolysis of L-arginine (Arranza et al PNAS USA 2012;109:9517-22). Results and conclusions Here, we report that neuroprotection afforded by azithromycin is associated to significant elevation of the percentage of F4/80+/Ym1+ M2 macrophages - coincident with increased arginase activity - in peritoneal exudate from mice subjected to tMCAo. Pharmacological inhibition of peritoneal arginase activity abolishes azithromycin-induced neuroprotection, underscoring the crucial role of drug-induced polarization of migratory macrophages towards a protective, non-inflammatory M2 phenotype. This, together with our recent evidence that induction of N2-like phenotypes in infiltrating neutrophils contributes to neuroprotection by PPAR /RXR receptor activation, underlies the importance of peripheral immune system polarization for the amelioration of ischemic stroke outcome.

Cell death & disease, 2011

Clinical Pharmacognosy, 2011

ABSTRACT Leptin is an adipose hormone whose neurotrophic effects have recently been described. He... more ABSTRACT Leptin is an adipose hormone whose neurotrophic effects have recently been described. Here, we investigate its neuroprotective potential in an experimental model of focal brain ischemia induced in rats by permanent occlusion of the middle cerebral artery (pMCAo). A single, subcutaneous (s.c.) administration of leptin (1 mg/kg, 3 h before pMCAo) significantly reduces brain infarct volume and neurological deficit measured up to 7 days after pMCAo. Western blot analysis combined with immunohistofluorescence show that the ischemic insult significantly increases STAT3 phosphorylation in both the cytosolic and nuclear fractions of the ipsilateral, ischemic cortex 3 h after pMCAo and this further increases in the nuclear but not in the cytosolic fraction of the ischemic cortex 24 h after occlusion. In comparison to vehicle-treated animals, 3h after pMCAo, leptin further increases STAT3 phosphorylation in the nuclear fraction of the astrocytes populating the penumbra. By contrast, after 24 h of ischemia, leptin further increases STAT3 phosphorylation in the cytosolic fraction of neurons and this is associated with increased expression of TIMP-1. Thus, the early activation of astrocytes, together with the late elevation of TIMP-1 in neurons, may represent crucial mechanisms by which STAT3 mediates neuroprotection afforded by leptin in focal ischemia.

ABSTRACT Activation of RAGE (receptor for advanced glycation endproducts) plays a role in neurona... more ABSTRACT Activation of RAGE (receptor for advanced glycation endproducts) plays a role in neuronal damage and neuroinflammation associated with brain ischemia, though the underlying mechanisms remain unclear. Poly(ADP-ribose) polymerase-1 (PARP-1) is a coactivator of the transcription factor NF-kB, that mediates inflammatory responses in cerebral ischemia. The PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34) prevents IkB-alpha degradation, thus blocking NF-kB activation, and reduces brain damage following cerebral ischemia. In this study, we have investigated whether inhibition of PARP modulates RAGE expression in the cerebral cortex and soluble RAGE form (sRAGE) in male Wistar rats subjected to transient (1h) middle cerebral artery occlusion (tMCAo). After 24h of reperfusion, cortical RAGE expression was evaluated by western blotting, while circulating RAGE was quantified by ELISA. RAGE expression in the ischemic cortex and circulating sRAGE were significantly decreased after tMCAo as compared to sham-operated animals. This effect was abolished by systemic administration of PJ34. Our data demonstrate that neuroprotection by PJ34 is associated with modulation of RAGE expression in cerebral ischemia and that sRAGE levels may represent a useful peripheral marker for stroke.

ABSTRACT Neuropathic pain is a form of chronic pain consequent to an injury delivered to single o... more ABSTRACT Neuropathic pain is a form of chronic pain consequent to an injury delivered to single or multiple nerves in the Peripheral Nervous System (PNS) or Central Nervous System (CNS). Almost 20% of the European population is affected by chronic or intermittent pain and this causes a reduction of the patient’s life quality. Gabapentin is a widely used drug in the treatment of neuropathic pain likely acting via the voltage-dependent calcium channel subunit alpha2-delta1. It has been shown that the anti-allodynic effect of gabapentin depends on the expression of this target molecule and that parameters like circadian oscillation can affect a2δ-1 expression and consequently gabapentin’s efficacy. Aim of this study was to verify the anti-allodynic effect of a chronic treatment with gabapentin and to correlate it with 2δ-1 expression in an experimental model of neuropathic pain. Furthermore, 2δ-1 expression was characterized in mice of different age. The study was conducted using male mice (22-25g) that underwent Spinal Nerve Ligation (SNL), according to the model described by Kim & Chung (1992). Both sham (n=2) and SNL animals (n=8) were subjected to behavioural tests to assess their mechanical (Von Frey’s test) and thermal (Hargreaves’ test) sensitivity. Western blot experiments were conducted on sham and SNL animals to assess the expression of alpha2-delta1 subunit of voltage gated Ca2+ channels in C57Bl/6 mice of different age (2-6-11 months). In SNL, but not sham animals, severe mechanical allodynia is fully developed at day 1-3 after ligature and peaks at day seven after surgery, lasting for up to thirty days. Western blotting results demonstrate that behavioural effects are paralleled by 2δ-1 overexpression in the spinal cord sections ipsilateral to the nerve injury and this is detectable on day 3 and 7 after surgery; the latter observation is conserved in SNL animals of 6 and 11 months of age. No sensitization to thermal sensitivity develops in SNL animals. Under these experimental conditions, administration of gabapentin (100 mg/Kg given i.p. once daily 1 h before behavioural tests), starting from day seven after surgery, minimizes allodynia assessed for up to two weeks. By contrast, this treatment schedule does not affect allodynia measured in later phases of neuropathic pain. In conclusion, our data provide further evidence to the rational use of gabapentin in the early treatment of neuropathic pain in view of its action on 2δ-1 subunit, which is overexpressed in the initial stages of neuropathic state development. Also, these results open new horizons to further investigations on the role of 2δ-1 subunit in the mechanisms underlying neuropathic pain development in aged animals. The experimental protocols were in accordance to the guidelines of the Italian Ministry of Health for animal care (D.M. 116/1992). This work was supported by the Italian Ministry of Health (Ricerca Finalizzata 2005, ex 56/05/15).

The development of novel effective therapies aimed at reducing post-ischemic brain damage is curr... more The development of novel effective therapies aimed at reducing post-ischemic brain damage is currently a urgent challenge. Research should be focused on rescuing the peripheral territories of the penumbra, perfused by collateral blood vessels, where the evolution of tissue damage is strongly affected by inflammatory events that involve soluble mediators and specialised cells activated locally or recruited from the periphery (see Amantea et al., FEBS J 2009; 276:13-26; Iadecola & Anrather, Nat Medicine 2011; 17:796-808). Indeed, brain infiltration of dendritic cells, lymphocytes, macrophages and neutrophils does occur during the early stages after the insult (Gelderblom et al., Stroke 2009; 40:1849-57), and may significantly affect the evolution of brain damage as emphasised by the evidence that severe combined immunodeficient mice are protected from focal ischemic injury (Hurn et al., J Cereb Blood Flow Metab 2007; 27:1798-1805) and brain accumulation of neutrophils after stroke correlates with the severity of brain tissue damage and poor neurological outcome in humans (Akopov et al., Stroke 1996; 27:1739-43). More interestingly, the genes acutely induced in the blood of ischemic stroke patients are expressed mainly by polymorphonuclear leukocytes and to a lesser degree by monocytes and most of these genes regulate the inflammatory response (Tang et al J Cereb Blood Flow Metab 2006; 26:1089-1102; Oh et al J Neuroimmunol 2012; 249:60-65). Thus, immunity and inflammation are involved in all stages of the ischemic cascade, from the early damaging events triggered by arterial occlusion to the late regenerative processes underlying post-ischemic tissue repair. We have observed that a macrolide compound, azithromycin, administered upon reperfusion, improves the beneficial early activation of astrocytes, thus promoting glial scar formation, while reducing brain infiltration of pro-inflammatory M1-macrophages few hours after reperfusion in rodents subjected to transient middle cerebral artery occlusion (MCAo). At later stages after the insult, azithromycin promotes brain activation/infiltration of macrophages, likely resembling “anti-inflammatory” M2-phenotype cells (2.3 fold increase of F480+/Ym1+ cells after 24h of reperfusion) and reduces by 2-fold CD11b+/Gr1+ neutrophil infiltration in the ischemic hemisphere. These effects result in a significant reduction of brain infarct volume and neurological deficit assessed up to 7 days after reperfusion in rodents subjected to either mild (30 min) or robust (2h) MCAo. Thus, modulation of the immune response by a multitarget therapy aimed at improving the beneficial while reducing the detrimental components of the immune/inflammatory response may represent a promising approach for the development of novel drugs for stroke.

Stroke is one of the most common causes of disability and mortality worldwide [1]. Although consi... more Stroke is one of the most common causes of disability and mortality worldwide [1]. Although considerable effort has been made to limit stroke incidence and improve outcome, strategies aimed at protecting injured neurons in the brain have all failed and the only pharmacological treatment currently available is thrombolysis. Stroke triggers a complex cascade of events contributing to the progression of brain damage, also including the activation of inflammatory responses that play a crucial role in neuronal injury, highlighting the therapeutic potential of anti-inflammatory drugs [2]. Nevertheless, recent evidence suggests that inflammatory reactions can also be neuroprotective. Thus, in addition to the prevention of the detrimental inflammatory processes, drug therapies should be aimed at the promotion of the reparative mechanisms orchestrated by beneficial inflammatory cells, such as M2 macrophages, which are able to scavenge debris and promote angiogenesis, tissue remodelling and repair [2, 3]. Retinoid X receptor (RXR) and/or peroxisome proliferator-activated receptor-γ (PPAR-γ) have been suggested to control macrophages and neutrophils polarization [4]. Bexarotene, a selective RXR agonist, is currently used to treat cutaneous T-cell lymphoma and it has been recently shown to be neuroprotective in animal models of Alzheimer and Parkinson diseases [5,6]. Here, we demonstrate that bexarotene has neuroprotective and immunomodulatory effects in a mouse model of cerebral ischemia. Acute treatment with bexarotene (0.5-25 mg/kg, i.p., upon reperfusion) dose-dependently reduces cerebral infarct volume, edema and neurological impairments induced by transient (30 min) middle cerebral artery occlusion (MCAo) in mice. The most effective dose of bexarotene (25 mg/kg, i.p.) induces a 75% reduction of the infarct volume caused by transient MCAo and this effect is accompanied by a reduction of the blood brain barrier disruption assessed 2 hours after reperfusion. By immunofluorescence analysis, we observe that bexarotene reduces the activation of microglia in the ischemic hemisphere after 48h of reperfusion and, paradoxically, it increases brain recruitment of macrophages and neutrophils, with a peak of infiltration 48 hours after the insult. In the mice treated with bexarotene, the percentage of polarization of these cells toward the M2-like (CD11b+/Ym1+) and N2 (LY-6B+/Ym1+) phenotype is significantly elevated as compared to vehicle-injected animals. The relevant effect of bexarotene on systemic inflammation is confirmed by the inhibition of the spleen size reduction induced by the ischemic insult. More importantly, the neuroprotective effects of bexarotene are mediated by the activation of RXR and their partner PPAR-γ as demonstrated by the evidence that the pan-RXR antagonist BR1211 and the PPAR-γ antagonist bisphenol A diglycidyl ether (BADGE) significantly revert neuroprotection.

European Journal of Pharmacology, 2014

The receptor for advanced glycation end products (RAGE) has a potential role as a damage-sensing ... more The receptor for advanced glycation end products (RAGE) has a potential role as a damage-sensing molecule; however, to date, its involvement in the pathophysiology of stroke and its modulation following neuroprotective treatment are not completely understood. We have previously demonstrated that expression of distinct RAGE isoforms, recognized by different antibodies, is differentially modulated in the brain of rats subjected to focal cerebral ischemia. Here, we focus on the full-length membrane-bound RAGE isoform, showing that its expression is significantly elevated in the striatum, whereas it is reduced in the cortex of rats subjected to transient middle cerebral artery occlusion (MCAo). Notably, the reduction of cortical levels of full-length RAGE detected 24 h after reperfusion is abolished by systemic administration of a neuroprotective dose of the poly(ADP-ribose) polymerase (PARP) inhibitor, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34). More interestingly, a significant reduction of plasma soluble RAGE (sRAGE) occurs 24 h after reperfusion and this effect is reverted by a neuroprotective dose of PJ34. Soluble forms of RAGE, generated either by alternative splicing or by proteolysis of the full-length form, effectively bind advanced glycation end products, thereby competing with the cell surface full-length RAGE, thus providing a 'decoy' function that may counteract the adverse effects of receptor signaling in neurons and may possibly exert cytoprotective effects. Thus, our data confirm the important role of RAGE in ischemic cerebral damage and, more interestingly, suggest the potential use of sRAGE as a blood biomarker of stroke severity and of neuroprotective treatment efficacy.

Frontiers in Physiology, 2020

Several studies have highlighted the interplay between metabolism, immunity and inflammation. Bot... more Several studies have highlighted the interplay between metabolism, immunity and inflammation. Both tissue resident and infiltrating immune cells play a major role in the inflammatory process of rheumatoid arthritis (RA) via the production of cytokines, adipocytokines and metabolic intermediates. These functions are metabolically demanding and require the most efficient use of bioenergetic pathways. The synovial membrane is the primary site of inflammation in RA and exhibits distinctive histological patterns characterized by different metabolism, prognosis and response to treatment. In the RA synovium, the high energy demand by stromal and infiltrating immune cells, causes the accumulation of metabolites, and adipo-cytokines, which carry out signaling functions, as well as activating transcription factors which act as metabolic sensors. These events drive immune and joint-resident cells to acquire pro-inflammatory effector functions which in turn perpetuate chronic inflammation. Whether metabolic changes are a consequence of the disease or one of the causes of RA pathogenesis is still under investigation. This review covers our current knowledge of cell metabolism in RA. Understanding the intricate interactions between metabolic pathways and the inflammatory and immune responses will provide more awareness of the mechanisms underlying RA pathogenesis and will identify novel therapeutic options to treat this disease.

Current Medicinal Chemistry, 2014

The evolution of ischemic brain damage is strongly affected by an inflammatory reaction that invo... more The evolution of ischemic brain damage is strongly affected by an inflammatory reaction that involves soluble mediators, such as cytokines and chemokines, and specialized cells activated locally or recruited from the periphery. The immune system affects all phases of the ischemic cascade, from the acute intravascular reaction due to blood flow disruption, to the development of brain tissue damage, repair and regeneration. Increased endothelial expression of adhesion molecules and blood-brain barrier breakdown promotes extravasation and brain recruitment of blood-borne cells, including macrophages, neutrophils, dendritic cells and T lymphocytes, as demonstrated both in animal models and in human stroke. Nevertheless, most anti-inflammatory approaches showing promising results in experimental stroke models failed in the clinical setting. The lack of translation may reside in the redundancy of most inflammatory mediators, exerting both detrimental and beneficial functions. Thus, this review is aimed at providing a better understanding of the dualistic role played by each component of the inflammatory/immune response in relation to the spatio-temporal evolution of ischemic stroke injury.

The Journal of Steroid Biochemistry and Molecular Biology, 2021

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of immune fun... more The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of immune function, promoting anti-inflammatory, tolerogenic T cell responses by modulating antigen presentation by dendritic cells (DC). Transcriptomic analyses indicate that DC responses to 1,25D involve changes in glycolysis, oxidative phosphorylation, electron transport and the TCA cycle. To determine the functional impact of 1,25D-mediated metabolic remodelling, human monocyte-derived DC were differentiated to immature (+vehicle, iDC), mature (+LPS, mDC), and immature tolerogenic DC (+1,25D, itolDC) and characterised for metabolic function. In contrast to mDC which showed no change in respiration, itolDC showed increased basal and ATP-linked respiration relative to iDC. Tracer metabolite analyses using 13C -labeled glucose showed increased lactate and TCA cycle metabolites. Analysis of lipophilic metabolites of 13C-glucose revealed significant incorporation of label in palmitate and palmitoleate, indicating that 1,25D promotes metabolic fatty acid synthesis in itolDC. Inhibition of fatty acid synthesis in itolDC altered itolDC morphology and suppressed expression of CD14 and IL-10 by these cells. These data indicate that the ability of 1,25D to induce tolerogenic DC involves metabolic remodelling leading to synthesis of fatty acids.

Nature reviews, Immunology, 2021

The microenvironment in cancerous tissues is immunosuppressive and pro-tumorigenic, whereas the m... more The microenvironment in cancerous tissues is immunosuppressive and pro-tumorigenic, whereas the microenvironment of tissues affected by chronic inflammatory disease is pro-inflammatory and anti-resolution. Despite these opposing immunological states, the metabolic states in the tissue microenvironments of cancer and inflammatory diseases are similar: both are hypoxic, show elevated levels of lactate and other metabolic by-products and have low levels of nutrients. In this Review, we describe how the bioavailability of lactate differs in the microenvironments of tumours and inflammatory diseases compared with normal tissues, thus contributing to the establishment of specific immunological states in disease. A clear understanding of the metabolic signature of tumours and inflammatory diseases will enable therapeutic intervention aimed at resetting the bioavailability of metabolites and correcting the dysregulated immunological state, triggering beneficial cytotoxic, inflammatory responses in tumours and immunosuppressive responses in chronic inflammation.

Science Translational Medicine, 2018

ASSAY and Drug Development Technologies, 2016

Mini-Reviews in Medicinal Chemistry, 2016

Pharmacological Research, 2015

Experimental Neurology, 2016

Frontiers in Neuroscience, 2015

Cell death & disease, 2011

Clinical Pharmacognosy, 2011

European Journal of Pharmacology, 2014

Frontiers in Physiology, 2020

Current Medicinal Chemistry, 2014

The Journal of Steroid Biochemistry and Molecular Biology, 2021

Nature reviews, Immunology, 2021

Cell death & disease, 2011

The majority of the genes acutely modulated in the blood of ischemic stroke patients regulate the... more The majority of the genes acutely modulated in the blood of ischemic stroke patients regulate the immune system and are expressed in circulating neutrophils and macrophages. Accordingly, systemic inflammatory reactions contribute to ischemic stroke injury, prompting activation and brain recruitment of macrophages, neutrophils, dendritic cells and lymphocytes. Cerebral infiltration ofblood-borne macrophages and neutrophils exerts a dualistic role on the evolution of brain damage depending on their ability to acquire specific phenotypes. Accordingly, we have recently observed that by reducing cerebral infiltration of neutrophils and detrimental M1 macrophages, while elevating the number of Ym1-and arginase I-immunopositive M2 macrophages in the ischemic hemisphere, the macrolide antibiotic azithromycin provides neuroprotection in a mouse model of transient middle cerebral artery occlusion (MCAo). Here, we aim at validating whether repurposing existing drugs that target the peripheral immune system, promoting polarization of circulating leukocytes towards non-inflammatory phenotypes, represents a promising strategy for stroke treatment. Focal brain ischemia was induced by transient (30-min) MCAo with an intraluminal filament in adult C57Bl/6 male mice receiving azithromycin (0.15-150 mg/kg) or bexarotene (0.5-25 mg/kg) i.p. up to 4.5 hours after reperfusion. The peripheral activation and brain infiltration of systemic immune cells was characterised by immunofluorescence labelling of specific cell markers (i.e., CD11b and Iba1 for microglia/macrophages, Ly6B for neutrophils, Ym1 and arginase for M2-macrophages) combined with flow cytometry analysis of brain and peritoneal samples from mice undergone transient MCAo. Arginase activity was assessed in peritoneal exudate cells by measuring the concentration of urea generated by the arginase-dependent hydrolysis of L-arginine. Neuroprotection afforded by azithromycin is associated to significant elevation of the percentage of F4/80+/Ym1+ M2 macrophages-coincident with increased arginase activity-in peritoneal exudate from mice subjected to transient MCAo. Pharmacological inhibition of peritoneal arginase activity abolishes azithromycin induced neuroprotection, underscoring the crucial role of drug-induced polarization of migratory macrophages towards a protective, non-inflammatory M2 phenotype. Conversely, by activating PPAR /RXR receptor, bexarotene promotes peripheral polarization and brain infiltration of N2 neutrophils, thus reducing MCAo-induced brain infarct damage and neurological deficit. Repurposing existing drugs with immunomodulatory effects represents an innovative strategy for the acute treatment of ischemic stroke.

With an incidence of 400 new cases each year per 100000 inhabitants and a mortality rate of 20%, ... more With an incidence of 400 new cases each year per 100000 inhabitants and a mortality rate of 20%, ischemic brain injury (stroke, trauma, cardiac arrest) is the second leading cause of death and the first cause of permanent disability in industrialized countries. Yet, most stroke cases are not medically treated and most of the therapies now available are either not effective or can be applied to only a small percentage of patients reaching the stroke unit. Discoveries of the past three decades have led to the unifying " excitotoxic " glutamate-mediated pathogenesis of post-ischemic neuronal death that, unfortunately, failed to translate into effective therapeutics. In fact, clinical trials testing NMDA antagonists, calcium channel blockers, spin traps for free radical scavenging, etc., in stroke, have all failed. The causes for these disappointing results should be looked for in the complex, often dual, role (both deleterious and beneficial) of biological mediators identified as important players of the excitotoxic cascade. Rescuing the peripheral tissue of the penumbra, where active death mechanisms are recruited and proceed more slowly than in the core, might widen the therapeutic window for intervention. In the penumbra, tissue damage evolution is strongly affected by neuroinflammatory events that involve soluble mediators (e.g. IL-1β), enzymes (e.g. MMPs) and specialised cells activated locally (e.g. astrocytes and microglia) or recruited from the periphery (e.g. neutrophils, dendritic cells, lymphocytes). An integrated modulation of these responses may represent an innovative approach to limit the evolution of ischemic brain damage. We have observed that a macrolide compound, azithromycin, administered upon reperfusion, improves the beneficial early activation of astrocytes, thus promoting glial scar formation, while reducing brain infiltration of pro-inflammatory M1-macrophages few hours after reperfusion in rodents subjected to transient middle cerebral artery occlusion (MCAo). At later stages after the insult, azithromycin promotes brain activation/infiltration of macrophages, likely resembling " anti-inflammatory " M2-phenotype cells (2.3 fold increase of F480+/Ym1+ cells after 24h of reperfusion) and reduces by 2-fold CD11b+/Gr1+ neutrophil infiltration in the ischemic hemisphere. These effects result in a significant reduction of brain infarct volume and neurological deficit assessed up to 7 days after reperfusion in rodents subjected to either mild (30 min) or robust (2h) MCAo. Thus, modulation of the immune response by a multitarget therapy aimed at improving the beneficial while reducing the detrimental components of the immune/inflammatory response may represent a promising approach for the development of novel drugs for stroke.

Activation of RAGE (receptor for advanced glycation endproducts) plays a role in neuronal damage ... more Activation of RAGE (receptor for advanced glycation endproducts) plays a role in neuronal damage and neuroinflammation associated with brain ischemia, though the underlying mechanisms remain unclear. Poly(ADP-ribose) polymerase-1 (PARP-1) is a coactivator of the transcription factor NF-kB, that mediates inflammatory responses in cerebral ischemia. The PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34) prevents IkB-alpha degradation, thus blocking NF-kB activation, and reduces brain damage following cerebral ischemia. In this study, we have investigated whether inhibition of PARP modulates RAGE expression in the cerebral cortex and soluble RAGE form (sRAGE) in male Wistar rats subjected to transient (1h) middle cerebral artery occlusion (tMCAo). After 24h of reperfusion, cortical RAGE expression was evaluated by western blotting, while circulating RAGE was quantified by ELISA. RAGE expression in the ischemic cortex and circulating sRAGE were significantly decreased after tMCAo as compared to sham-operated animals. This effect was abolished by systemic administration of PJ34. Our data demonstrate that neuroprotection by PJ34 is associated with modulation of RAGE expression in cerebral ischemia and that sRAGE levels may represent a useful peripheral marker for stroke.

Leptin is an adipose hormone whose neurotrophic effects have recently been described. Here, we in... more Leptin is an adipose hormone whose neurotrophic effects have recently been described. Here, we investigate its neuroprotective potential in an experimental model of focal brain ischemia induced in rats by permanent occlusion of the middle cerebral artery (pMCAo). A single, subcutaneous (s.c.) administration of leptin (1 mg/kg, 3 h before pMCAo) significantly reduces brain infarct volume and neurological deficit measured up to 7 days after pMCAo. Western blot analysis combined with immunohistofluorescence show that the ischemic insult significantly increases STAT3 phosphorylation in both the cytosolic and nuclear fractions of the ipsilateral, ischemic cortex 3 h after pMCAo and this further increases in the nuclear but not in the cytosolic fraction of the ischemic cortex 24 h after occlusion. In comparison to vehicle-treated animals, 3h after pMCAo, leptin further increases STAT3 phosphorylation in the nuclear fraction of the astrocytes populating the penumbra. By contrast, after 24 h of ischemia, leptin further increases STAT3 phosphorylation in the cytosolic fraction of neurons and this is associated with increased expression of TIMP-1. Thus, the early activation of astrocytes, together with the late elevation of TIMP-1 in neurons, may represent crucial mechanisms by which STAT3 mediates neuroprotection afforded by leptin in focal ischemia.

In addition to its effects in the hypothalamus to control body weight, leptin is involved in the ... more In addition to its effects in the hypothalamus to control body weight, leptin is involved in the regulation of neuronal function, development and survival [1]. Recent findings have highlighted the neuroprotective effects of leptin against ischemic brain injury; however, to date, little is known about the role played by signal transducer and activator of transcription (STAT)-3, a major mediator of ObR transduction pathway in the brain, in the beneficial effects exerted by the hormone. We have investigated the neuroprotective potential of leptin by using an experimental model of focal brain ischemia induced in rats by permanent occlusion of the middle cerebral artery (MCAo) with an intraluminal filament [2]. A single, subcutaneous (s.c.) administration of leptin (1 mg/kg, 3 h before MCAo) significantly reduced brain infarct volume measured 24 hours, 3 days or 7 days after permanent MCAo. Neuroprotection was also associated with reduced neurological deficit as assessed up to 7 days following ischemia. Subcellular fractionation western blotting studies revealed that the ischemic insult significantly increased levels of STAT3 phosphorylation in both the cytosolic and nuclear fractions of the ipsilateral, ischemic brain cortex 3 h after MCAo (P<0.05 vs contralateral) and these further increased in the nuclear (P<0.001 vs 3 h) but not in the cytosolic fraction of the ischemic cortex 24 h after occlusion. In comparison to vehicle-treated animals, 3h after MCAo, leptin further increased STAT3 phosphorylation in the nuclear fraction of the astrocytes populating the penumbra, as confirmed by immunofluorescence. The early enhancement of nuclear phospho-STAT3 induced by leptin in the astrocytes of the ischemic penumbra may contribute to a beneficial effect of these cells on the evolution of tissue damage. By contrast, after 24 h of ischemia leptin further increased STAT3 phosphorylation in the cytosolic fraction. At this stage pSTAT3 immunoreactivity was mainly detected in neurons and other (presumably microglial) cells of the peri-infarct region of the motor cortex. This latter effect was associated with an increased expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1 in the cortex, suggesting its possible involvement to the neuroprotection produced by the adipokine. REFERENCES

Neuropathic pain is a form of chronic pain consequent to an injury delivered to single or multipl... more Neuropathic pain is a form of chronic pain consequent to an injury delivered to single or multiple nerves in the Peripheral Nervous System (PNS) or Central Nervous System (CNS). Almost 20% of the European population is affected by chronic or intermittent pain and this causes a reduction of the patient's life quality. Gabapentin is a widely used drug in the treatment of neuropathic pain likely acting via the voltage-dependent calcium channel subunit alpha2-delta1. It has been shown that the anti-allodynic effect of gabapentin depends on the expression of this target molecule and that parameters like circadian oscillation can affect α2δ-1 expression and consequently gabapentin's efficacy. Aim of this study was to verify the anti-allodynic effect of a chronic treatment with gabapentin and to correlate it with α2δ-1 expression in an experimental model of neuropathic pain. Furthermore, α2δ-1 expression was characterized in mice of different age. The study was conducted using male mice (22-25g) that underwent Spinal Nerve Ligation (SNL), according to the model described by Kim & Chung (1992). Both sham (n=2) and SNL animals (n=8) were subjected to behavioural tests to assess their mechanical (Von Frey's test) and thermal (Hargreaves' test) sensitivity. Western blot experiments were conducted on sham and SNL animals to assess the expression of alpha2-delta1 subunit of voltage gated Ca2+ channels in C57Bl/6 mice of different age (2-6-11 months). In SNL, but not sham animals, severe mechanical allodynia is fully developed at day 1-3 after ligature and peaks at day seven after surgery, lasting for up to thirty days. Western blotting results demonstrate that behavioural effects are paralleled by α2δ-1 overexpression in the spinal cord sections ipsilateral to the nerve injury and this is detectable on day 3 and 7 after surgery; the latter observation is conserved in SNL animals of 6 and 11 months of age. No sensitization to thermal sensitivity develops in SNL animals. Under these experimental conditions, administration of gabapentin (100 mg/Kg given i.p. once daily 1 h before behavioural tests), starting from day seven after surgery, minimizes allodynia assessed for up to two weeks. By contrast, this treatment schedule does not affect allodynia measured in later phases of neuropathic pain. In conclusion, our data provide further evidence to the rational use of gabapentin in the early treatment of neuropathic pain in view of its action on α2δ-1 subunit, which is overexpressed in the initial stages of neuropathic state development. Also, these results open new horizons to further investigations on the role of α2δ-1 subunit in the mechanisms underlying neuropathic pain development in aged animals. The experimental protocols were in accordance to the guidelines of the Italian Ministry of Health for animal care (D.M. 116/1992). This work was supported by the Italian Ministry of Health (Ricerca Finalizzata 2005, ex 56/05/15).

The development of novel effective therapies aimed at reducing post-ischemic brain damage is curr... more The development of novel effective therapies aimed at reducing post-ischemic brain damage is currently a urgent challenge. Research should be focused on rescuing the peripheral territories of the penumbra, perfused by collateral blood vessels, where the evolution of tissue damage is strongly affected by inflammatory events that involve soluble
mediators and specialised cells activated locally or recruited from the periphery (see Amantea et al., FEBS J 2009; 276:13-26; Iadecola & Anrather, Nat Medicine 2011; 17:796-808). Indeed, brain infiltration of dendritic cells, lymphocytes, macrophages and neutrophils does occur during the early stages after the insult (Gelderblom et al., Stroke 2009; 40:1849-57), and may significantly affect the evolution of brain damage as emphasised by the evidence that severe combined immunodeficient mice are protected from focal ischemic injury (Hurn et al., J Cereb Blood
Flow Metab 2007; 27:1798-1805) and brain accumulation of neutrophils after stroke correlates with the severity of brain tissue damage and poor neurological outcome in humans (Akopov et al., Stroke 1996; 27:1739-43). More interestingly, the genes acutely induced in the blood of ischemic stroke patients are expressed mainly by polymorphonuclear leukocytes and to a lesser degree by monocytes and most of these genes regulate the inflammatory response (Tang et al J Cereb Blood Flow Metab 2006; 26:1089-1102; Oh et al J Neuroimmunol 2012;
249:60-65). Thus, immunity and inflammation are involved in all stages of the ischemic cascade, from the early damaging events triggered by arterial occlusion to the late regenerative processes underlying post-ischemic tissue repair.
We have observed that a macrolide compound, azithromycin, administered upon reperfusion, improves the beneficial early activation of astrocytes, thus promoting glial scar formation, while reducing brain infiltration of pro-inflammatory M1-macrophages few hours after reperfusion in rodents subjected to transient middle cerebral artery occlusion (MCAo). At later stages after the insult, azithromycin promotes brain activation/infiltration of macrophages, likely resembling “anti-inflammatory” M2-phenotype cells (2.3 fold increase of F480+/Ym1+ cells after 24h of reperfusion) and reduces by 2-fold CD11b+/Gr1+ neutrophil infiltration in the ischemic hemisphere.
These effects result in a significant reduction of brain infarct volume and neurological deficit assessed up to 7 days after reperfusion in rodents subjected to either mild (30 min) or robust (2h) MCAo.
Thus, modulation of the immune response by a multitarget therapy aimed at improving the beneficial while reducing the detrimental components of the immune/inflammatory response may represent a promising approach for the development of novel drugs for stroke.

Methods
Focal brain ischemia was induced by transient (30-min) MCAo with an intraluminal filament (Amantea et al Neuroscience 2008,152:8-17) in adult C57Bl/6 male mice receiving azithromycin (0.15-150 mg/kg) or
vehicle (0.9% NaCl) i.p. upon reperfusion. Sham animals were subjected to same surgical procedure without filament insertion.
The peripheral activation and brain infiltration of systemic immune cells was characterised by immunofluorescence labelling of specific cell markers (i.e., CD11b and Iba1 for microglia/macrophages, Ly6B for neutrophils, Ym1 and arginase for M2-macrophages) combined with flow cytometry analysis (Pallotta et al Nat Immunol. 2011;12:870-8) of brain and peritoneal samples from mice undergone tMCAo.
Arginase activity was assessed in peritoneal exudate cells by measuring the concentration of urea generated by the arginase-dependent hydrolysis of L-arginine (Arranza et al PNAS USA 2012;109:9517-22).

Results and conclusions
Here, we report that neuroprotection afforded by azithromycin is associated to significant elevation of the percentage of F4/80+/Ym1+
M2 macrophages - coincident with increased arginase activity - in peritoneal exudate from mice subjected to tMCAo. Pharmacological inhibition of peritoneal arginase activity abolishes azithromycin-induced neuroprotection, underscoring the crucial role of drug-induced polarization of migratory macrophages towards a protective, non-inflammatory M2 phenotype. This, together with our recent evidence that induction of N2-like phenotypes in infiltrating neutrophils contributes to
neuroprotection by PPAR /RXR receptor activation, underlies the importance of peripheral immune system polarization for the amelioration of ischemic stroke outcome.

Stroke is a devastating disease associated with high morbidity and mortality worldwide (Mozaffari... more Stroke is a devastating disease associated with high morbidity and mortality worldwide (Mozaffarian et al., 2015). Nevertheless, all the neuroprotective approaches tested to date have failed to translate in the clinical setting and the only pharmacological treatment currently available is thrombolysis, though its use is limited given the strict eligibility criteria and the risk of adverse effects such as haemorrhagic transformation. Recent evidence has highlighted the neuroprotective potential of immunomodulatory strategies, based on polarization of myeloid cells towards non-inflammatory, beneficial phenotypes (Cuartero et al., 2013; Amantea et al., 2015). Given the role of retinoid X receptors (RXR) in myeloid cells differentiation and polarization, here we have explored the neuroprotective potential of the RXR agonist bexarotene in mice subjected to focal cerebral ischemia. A single dose of bexarotene (5 or 25 mg/kg, i.p., upon reperfusion) significantly reduced cerebral infarct volume, edema and neurological impairments produced by transient (30 min) middle cerebral artery occlusion (MCAo) in mice. The most effective dose of the rexinoid (25 mg/kg, i.p.) induced a 75% reduction of the infarct volume evaluated by cresyl violet staining of coronal brain slices 48h after reperfusion. This effect was associated with a significant reduction of the blood brain barrier rupture assessed 2 hours after reperfusion by Evans blue leakage. Bexarotene exerted neuroprotection with a wide time-window, being effective when administered up to 4.5 hours after the insult. Immunofluorescence analysis revealed that bexarotene increases brain recruitment of macrophages and neutrophils, with a peak of infiltration 48 hours after the insult. Interestingly, the percentage of polarization of these cells toward the M2-like (CD11b+/Ym1+) and N2 (LY-6G+/Ym1+) phenotype is significantly elevated by the drug as compared to vehicle-injected animals. This was coincident with a higher density of Ly-6G/Ym1-immunopositive N2 neutrophils in the spleen of ischemic mice treated with the rexinoid. The effect of bexarotene on systemic inflammation is confirmed by its ability to inhibit spleen atrophy induced by the ischemic insult. The improvement of histological outcomes, as well as the ability of bexarotene to revert MCAo-induced spleen atrophy, was antagonised by BR1211, a pan-RXR antagonist, or by the selective PPARγ antagonist BADGE, highlighting the involvement of the RXR/PPARγ heterodimer in the beneficial effects exerted by the drug. In conclusion, our findings demonstrate that due to its peripheral immunomodulatory effects, consisting in the polarization of myeloid cells toward non-inflammatory (M2 and N2) phenotypes, the RXR agonist bexarotene may be effectively repurposed for the acute therapy of ischemic stroke (Certo et al., 2015).

Botulinum neurotoxin/A (BoNT/A) reversibly blocks neuromuscular synaptic transmission by cleavage... more Botulinum neurotoxin/A (BoNT/A) reversibly blocks neuromuscular synaptic transmission by cleavage of the specific target SNAP25 essential for neurotransmitter release. However, there is also growing evidence that BoNT/A will ameliorate chronic pain states although the mechanism remains unclear. Recently, a botulinum A-based molecule (BiTOX) has been developed that maintains neuronal silencing capacity and reduces peripheral neuropathic pain states but without muscle paralysis (Mangione et al.,2016). Here we have extended this approach by conjugating the silencing domain of BoNT/A (BOT) to either substance P or dermorphin to reversibly inhibit key NK1 or mu-opiate receptor-expressing neurons in the pain pathways and ameliorate chronic pain states. SP-BOT or Derm-BOT conjugates were injected intrathecally in mice either 2 weeks prior to surgery or 5 days after surgery for the spared nerve injury model of neuropathic pain (SNI). Von Frey's filaments were used to monitor mechanical sensitivity. The targeting of conjugates was monitored using immunochemistry. Lumbar spinal cord sections were stained for cleaved SNAP25 using a specific antibody and tyramide amplification followed by mu-opiate receptor antibody or NK1-rceptor antibody. SP-BOT (100ng/2μl) treatment before surgery attenuated the increase in mechanical sensitivity seen after SNI (55%). Similarly, when the construct was injected after SNI surgery SP-BOT injected mice showed reduced neuropathic sensitivity compared with the vehicle-injected SNI mice (40%). Derm-BOT conjugates (100ng/3μl) also reduced mechanical hypersensitivity when injected intrathecally in mice after SNI surgery. Immunohistochemistry demonstrated subsets of cleaved SNAP 25-positive neurons in the dorsal horn of conjugate injected mice. Double staining with either NK1 or mu-receptor antibodies indicated that up take of conjugates was largely specific: SP-BOT was found in NK1 positive neurons and Derm-SAP in mu-receptor positive neurons. In conclusion, the data strongly suggests that both BOT constructs have translational potential for the treatment of chronic pain by selectively and reversibly silencing central neurons involved in pain processing.