Leonardo Cavone

Histone deacetylase (HDAC) inhibitors reduce the glial inflammatory response in vitro and in vivo.

Histone deacetylase inhibitors (HDACi) are emerging tools for epigenetic modulation of gene expression and suppress the inflammatory response in models of systemic immune activation. Yet, their effects within the brain are still controversial. Also, whether HDACs are expressed in astrocytes or microglia is unclear. Here, we report the identification of transcripts for HDAC 1-11 in cultured mouse glial cells. Two HDACi such as SAHA and ITF2357 induce dramatic increase of histone acetylation without causing cytotoxicity of cultured cells. Of note, the two compounds inhibit expression of pro-inflammatory mediators by LPS-challenged glial cultures, and potentiate immunosuppression triggered by dexamethasone in vitro. The anti-inflammatory effect is not due to HDACi-induced transcription of immunosuppressant proteins, (including SOCS-1/3) or microRNA-146. Rather, it is accompanied by direct alteration of transcription factor DNA binding and ensuing transcriptional activation. Indeed, both HDACi impair NFkappaB-dependent IkappaBalpha resynthesis in glial cells exposed to LPS, and, among various AP1 subunits and NFkappaB p65, affect the DNA binding activity of c-FOS, c-JUN and FRA2. Importantly, ITF2357 reduces the expression of pro-inflammatory mediators in the striatum of mice iontophoretically injected with LPS. Data demonstrate that mouse glial cells have ongoing HDAC activity, and its inhibition suppresses the neuroinflammatory response because of a direct impairment of the transcriptional machinery.

The Therapeutic Potential of HDAC Inhibitors in the Treatment of Multiple Sclerosis

Multiple sclerosis (MS) is a devastating autoimmune disorder of the central nervous system (CNS) for which there is no efficacious cure. Thanks to numerous preclinical and clinical studies, drugs able to mitigate the inexorable course of the disease have been made available recently. Still, there is a terrible need for compounds capable of reducing the severity of the autoimmune attack and of blocking progression of the disorder. Also, besides the classic immunosuppressive strategies, it is now appreciated that compounds directly targeting neuronal death can be of relevance to the treatment of MS patients. Acetylation homeostasis is a key regulator of both immune cell activation and neuronal survival. Of note, potent histone deacetylase inhibitors (HDACi) endowed with antiinflammatory and neuroprotective properties have been identified. Efficacy of HDACi in experimental models of MS has been reported consistently. In this review, we provide an appraisal of the literature on HDACi and MS, also discussing the mechanisms by which HDACi can suppress the autoimmune attack to the CNS.

Functional study in a yeast model of a novel succinate dehydrogenase subunit B gene germline missense mutation (C191Y) diagnosed in a patient affected by a glomus tumor

Mutations of succinate dehydrogenase (SDH) subunits B, C and D are associated to pheochromocytoma/paraganglioma (PGL) development. The mechanisms linking SDH mutations to tumorigenesis are currently unknown. We report a novel germline missense SDHB mutation (C191Y) in a patient affected by a glomus tumor. The missense mutation hits an amino acid residue conserved from mammals to the yeast Saccharomyces cerevisiae. The pathogenic significance of the human mutation was validated in a yeast model. SDH2(C184Y) mutant allele equivalent to human SDHB(C191Y) did not restore the OXPHOS phenotype of the Deltasdh2 null mutant. In the mutant, SDH activity was also abolished along with a reduction in respiration. Sensitivity to oxidative stress was increased in the mutant, as revealed by reduced growth in the presence of menadione. Remarkably, the frequency of petite colony formation was increased in the mutant yeast strain, indicating an increased mtDNA mutability. Histochemistry demonstrates that SDH activity was selectively absent in the patient tumor tissue. Overall, our results demonstrate that the C191Y SDHB mutation suppresses SDH enzyme activity leading to increased ROS formation and mtDNA mutability in our yeast model. These findings further our understanding of the mechanisms underlying PGL development and point to the yeast model as a valid tool to investigate on the possible pathogenic relevance of SDH novel mutations and/or rare polymorphism.

Neurological basis of AMP-dependent thermoregulation and its relevance to central and peripheral hyperthermia

Therapeutic hypothermia is of relevance to treatment of increased body temperature and brain injury, but drugs inducing selective, rapid, and safe cooling in humans are not available. Here, we show that injections of adenosine 5′-monophosphate (AMP), an endogenous nucleotide, promptly triggers hypothermia in mice by directly activating adenosine A1 receptors (A1R) within the preoptic area (POA) of the hypothalamus. Inhibition of constitutive degradation of brain extracellular AMP by targeting ecto 5′-nucleotidase, also suffices to prompt hypothermia in rodents. Accordingly, sensitivity of mice and rats to the hypothermic effect of AMP is inversely related to their hypothalamic 5′-nucleotidase activity. Single-cell electrophysiological recording indicates that AMP reduces spontaneous firing activity of temperature-insensitive neurons of the mouse POA, thereby retuning the hypothalamic thermoregulatory set point towards lower temperatures. Adenosine 5′-monophosphate also suppresses prostaglandin E2-induced fever in mice, having no effects on peripheral hyperthermia triggered by dioxymetamphetamine (ecstasy) overdose. Together, data disclose the role of AMP, 5′-nucleotidase, and A1R in hypothalamic thermoregulation, as well and their therapeutic relevance to treatment of febrile illness.

Ischemic neuroprotection by TRPV1 receptor-induced hypothermia

Although treatment of stroke patients with mild hypothermia is a promising therapeutic approach, chemicals inducing prompt and safe reduction of body temperature are an unmet need. We measured the effects of the transient receptor potential vanilloid-1 (TRPV1) agonist rinvanil on thermoregulation and ischemic brain injury in mice. Intraperitoneal or intracerebroventricular injection of rinvanil induces mild hypothermia that is prevented by the receptor antagonist capsazepine. Both intraischemic and postischemic treatments provide permanent neuroprotection in animals subjected to transient middle cerebral artery occlusion (MCAo), an effect lost in mice artificially kept normothermic. Data indicate that TRPV1 receptor agonists are promising candidates for hypothermic treatment of stroke.

GPR35 Activation Reduces Ca2+ Transients and Contributes to the Kynurenic Acid-Dependent Reduction of Synaptic Activity at CA3-CA1 Synapses

Limited information is available on the brain expression and role of GPR35, a Gi/o coupled receptor activated by kynurenic acid (KYNA). In mouse cultured astrocytes, we detected GPR35 transcript using RT-PCR and we found that KYNA (0.1 to 100 µM) decreased forskolin (FRSK)-induced cAMP production (p<0.05). Both CID2745687 (3 µM, CID), a recently described GPR35 antagonist, and GPR35 gene silencing significantly prevented the action of KYNA on FRSK-induced cAMP production. In these cultures, we then evaluated whether GPR35 activation was able to modulate intracellular Ca2+ concentration ([Ca2+]i ) and [Ca2+]i fluxes. We found that both KYNA and zaprinast, a phosphodiesterase (PDE) inhibitor and GPR35 agonist, did not modify either basal or peaks of [Ca2+]i induced by challenging the cells with ATP (30 µM). However, the [Ca2+]i plateau phase following peak was significantly attenuated by these compounds in a store-operated Ca2+ channel (SOC)-independent manner. The activation of GPR35 by KYNA and zaprinast was also studied at the CA3-CA1 synapse in the rat hippocampus. Evoked excitatory post synaptic currents (eEPSCs) were recorded from CA1 pyramidal neurons in acute brain slices. The action of KYNA on GPR35 was pharmacologically isolated by using NMDA and α7 nicotinic receptor blockers and resulted in a significant reduction of eEPSC amplitude. This effect was prevented in the presence of CID. Moreover, zaprinast reduced eEPSC amplitude in a PDE5- and cGMP-independent mechanism, thus suggesting that glutamatergic transmission in this area is modulated by GPR35. In conclusion, GPR35 is expressed in cultured astrocytes and its activation modulates cAMP production and [Ca2+]i. GPR35 activation may contribute to KYNA effects on the previously reported decrease of brain extracellular glutamate levels and reduction of excitatory transmission.

Induction of CD83+CD14+ Nondendritic Antigen-Presenting Cells by Exposure of Monocytes to IFN-α

IFN-α is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-α effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-α levels during treatment of infections and cancers. We show that in vitro IFN-α exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-α induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-α concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-α-challenged CD83+ cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-α-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-α-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4+ T cells. Notably, autologous memory CD4+ T cells proliferated when exposed to tetanus toxoid-pulsed IFN-α-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-α showed long-lasting STAT-1 phosphorylation. Remarkably, CD83+CD14+ cells were present in varicella skin lesions in close contact with IFN-α-producing cells. The present findings suggest that IFN-α alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-α in vivo.

PARP-1 inhibition prevents CNS migration of dendritic cells during EAE, suppressing the encephalitogenic response and relapse severity.

Background: Pharmacological inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) are currently evaluated in clinical trials for various malignancies but, interestingly, also proved of remarkable efficacy in preclinical models of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE). Objectives: The objectives of the study were to determine molecular mechanisms underlying suppression of the encephalitogenic response by these drugs; likewise, whether clinically-relevant post-treatment paradigms with PARP-1 inhibitors could prevent EAE relapses. Methods: Adopted both in vitro techniques (bone marrow-derived cultured DC) as well as in vivo models of chronic or relapsing–remitting (RR) EAE. Results: We report that two structurally unrelated PARP-1 inhibitors negatively regulated NFκB activation, as well as maturation, cytokine production and APC function of cultured mouse bone marrow-derived dendritic cells (DCs). PARP-1 inhibitors also reduced the number and APC function of DCs migrating in the draining lymph nodes of ovalbumin-immunized mice. In C57Bl mice with chronic EAE or SJL mice with RR EAE, pharmacological inhibition of PARP-1 reduced CNS DC migration and demyelination as well as neurological impairment to an extent similar to that achieved with the potent immunosuppressant cyclosporine A. Remarkably, PARP-1 inhibitors injected after the first phase of disease reduced relapse incidence and severity, as well as the spinal cord number of autoreactive Th17 cells. Under this clinically-relevant treatment paradigm, PARP inhibitors also suppressed epitope spreading of the encephalitogenic response. Conclusions: Overall, data underscore the potential relevance of PARP-1 inhibitors to MS therapy and suppression of autoimmunity.

Dipotassium Glycyrrhizate Inhibits HMGB1-Dependent Inflammation and Ameliorates Colitis in Mice

Background High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. Advances in targeting HMGB1 represent a major challenge to improve the treatment of acute/chronic inflammation. Aim This study is aimed at verifying whether the inhibition of HMGB1 through dipotassium glycyrrhizate (DPG) is a good strategy to reduce intestinal inflammation. Methods Human colon adenocarcinoma cell line, HT29, human epithelial colorectal adenocarcinoma, Caco2, and murine macrophage cell line, RAW 264.7, were cultured to investigate the effect of DPG on the secretion of HMGB1. Acute colitis was induced in C57BL/6 mice through administration of 3% dextran sodium sulphate (DSS); a combined treatment with DSS and 3 or 8 mg/kg/day DPG was used to investigate the effects of DPG on intestinal inflammation. Animals were euthanized at seventh day and colonic samples underwent molecular and histological analyses. Results DPG significantly reduces in vitro the release of HMGB1 in the extracellular matrix as well as expression levels of pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6, by inhibiting HMGB1. Moreover, DPG significantly decreases the severity of DSS-induced colitis in mice. Murine colonic samples show decreased mRNA levels of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6, as well as HMGB1 receptors, RAGE and TLR4. Finally, HMGB1, abundantly present in the feces of mice with DSS-induced colitis, is strongly reduced by DPG. Conclusions HMGB1 is an early pro-inflammatory cytokine and an active protagonist of mucosal gut inflammation. DPG exerts inhibitory effects against HMGB1 activity, significantly reducing intestinal inflammation. Thus, we reason that DPG could represent an innovative tool for the management of human intestinal inflammation.

Dysregulation of sphingosine 1 phosphate receptor-1 (S1P1) signaling and regulatory lymphocyte-dependent immunosuppression in a model of post-fingolimod MS rebound.

Fingolimod affords protection from MS by sequestering lymphocytes in secondary lymphoid organs via down regulation of their sphingosine 1 phosphate receptor (S1P1). Unexpectedly, accumulating evidence indicates that patients who discontinue fingolimod treatment may be at risk of rehearsal of magnetic resonance (MR) and clinical disease activity, sometimes featuring dramatic rebound. We therefore developed in vivo and in vitro models of post-fingolimod MS rebound to unravel its cellular and molecular mechanisms. The impact of fingolimod withdrawal on T regulatory lymphocytes was also investigated by means of cytofluorimetric analysis and antigen-specific lymphocyte proliferation assays. We show that mice with relapsing-remitting experimental autoimmune encephalomyelitis (EAE) undergo extremely severe, chronic disease rebound upon discontinuation of fingolimod. Remarkably, rebound is preceded by a burst of S1P1 overexpression in lymph node-entrapped lymphocytes that correlates with subsequent massive lymphocyte egress and widespread CNS immune infiltration. Also, consistent with the ability of S1P1 to counteract polarization and function of T regulatory lymphocytes their number and suppression of effector T cells is reduced by fingolimod suspension. Data disclose the first pathogenic mechanisms of post-fingolimod rebound that may be targeted for therapeutic intervention.