We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL‐1β, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X7, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP‐induced shedding and IL‐1β release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL‐1β release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL‐1β release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

Microvesicles (MVs) released into the brain microenvironment are emerging as a novel way of cell‐to‐cell communication. We have recently shown that microglia, the immune cells of the brain, shed MVs upon activation but their possible role in microglia‐to‐neuron communication has never been explored. To investigate whether MVs affect neurotransmission, we analysed spontaneous release of glutamate in neurons exposed to MVs and found a dose‐dependent increase in miniature excitatory postsynaptic current (mEPSC) frequency without changes in mEPSC amplitude. Paired‐pulse recording analysis of evoked neurotransmission showed that MVs mainly act at the presynaptic site, by increasing release probability. In line with the enhancement of excitatory transmission in vitro , injection of MVs into the rat visual cortex caused an acute increase in the amplitude of field potentials evoked by visual stimuli. Stimulation of synaptic activity occurred via enhanced sphingolipid metabolism. Indeed, MVs promoted ceramide and sphingosine production in neurons, while the increase of excitatory transmission induced by MVs was prevented by pharmacological or genetic inhibition of sphingosine synthesis. These data identify microglia‐derived MVs as a new mechanism by which microglia influence synaptic activity and highlight the involvement of neuronal sphingosine in this microglia‐to‐neuron signalling pathway.

Duchenne muscular dystrophy is a relatively common disease that affects skeletal muscle, leading to progressive paralysis and death. There is currently no resolutive therapy. We have developed a treatment in which we combined the effects of nitric oxide with nonsteroidal antiinflammatory activity by using HCT 1026, a nitric oxide-releasing derivative of flurbiprofen. Here, we report the results of long-term (1-year) oral treatment with HCT 1026 of two murine models for limb girdle and Duchenne muscular dystrophies (α-sarcoglycan-null and mdx mice). In both models, HCT 1026 significantly ameliorated the morphological, biochemical, and functional phenotype in the absence of secondary effects, efficiently slowing down disease progression. HCT 1026 acted by reducing inflammation, preventing muscle damage, and preserving the number and function of satellite cells. HCT 1026 was significantly more effective than the corticosteroid prednisolone, which was analyzed in parallel. As an additional beneficial effect, HCT 1026 enhanced the therapeutic efficacy of arterially delivered donor stem cells, by increasing 4-fold their ability to migrate and reconstitute muscle fibers. The therapeutic strategy we propose is not selective for a subset of mutations; it provides ground for immediate clinical experimentation with HCT 1026 alone, which is approved for use in humans; and it sets the stage for combined therapies with donor or autologous, genetically corrected stem cells.

Objective— Tumor necrosis factor α (TNF-α), a key proinflammatory cytokine acting on the endothelium, activates endothelial nitric oxide synthase (eNOS). We have examined the signaling pathway leading to this activation and its biological role in endothelium, which are still unknown. Methods and Results— In human endothelial cells, we found that eNOS activation by TNF-α is time dependent and requires activation of Akt, a known eNOS activator. eNOS activation was preceded by sequential activation of neutral-sphingomyelinase-2 (N-SMase2) and sphingosine-kinase-1 (SK1) and generation of sphingosine-1-phosphate (Sph1P). Inhibition of N-SMase2 inhibited Sph1P formation, whereas inhibition of SK1 did not affect N-SMase2 activation by TNF-α. Blockade of N-SMase2, SK1, or the Sph1P receptors S1P ₁ and S1P ₃ , either by silencing or pharmacological inhibitors, prevented eNOS activation. Thus, eNOS is activated by TNF-α via S1P receptors, activated by Sph1P generated through N-SMase2 and SK1 activation. We found that nitric oxide generated through this pathway has a biological role, because it inhibits the expression of E-selectin and the adhesion of dendritic cells to the endothelium stimulated by TNF-α. Conclusions— This study establishes a previously undescribed link among TNF-α, Sph1P, and eNOS in a same signaling pathway of biological relevance in the process of endothelial cell activation by TNF-α.

The endocrine system participates in regulating macrophage maturation, although little is known about the modulating role of the thyroid hormones. In vitro results demonstrate a negative role of one such hormone, triiodothyronine (T3), in triggering the differentiation of bone marrow-derived monocytes into unpolarized macrophages. T3-induced macrophages displayed a classically activated (M1) signature. A T3-induced M1-priming effect was also observed on polarized macrophages because T3 reverses alternatively activated (M2) activation, whereas it enhances that of M1 cells. In vivo, circulating T3 increased the content of the resident macrophages in the peritoneal cavity, whereas it reduced the content of the recruited monocyte-derived cells. Of interest, T3 significantly protected mice against endotoxemia induced by lipopolysaccharide i.p. injection; in these damaged animals, decreased T3 levels increased the recruited (potentially damaging) cells, whereas restoring T3 levels decreased recruited and increased resident (potentially beneficial) cells. These data suggest that the anti-inflammatory effect of T3 is coupled to the modulation of peritoneal macrophage content, in a context not fully explained by the M1/M2 framework. Thyroid hormone receptor expression analysis and the use of different thyroid hormone receptor antagonists suggest thyroid hormone receptor β1 as the major player mediating T3 effects on macrophages. The novel homeostatic link between thyroid hormones and the pathophysiological role of macrophages opens new perspectives on the interactions between the endocrine and immune systems.

Tumor microenvironment is fundamental for cancer progression and chemoresistance. Among stromal cells tumor-associated macrophages (TAMs) represent the largest population of infiltrating inflammatory cells in malignant tumors, promoting their growth, invasion, and immune evasion. M2-polarized TAMs are endowed with the nitric oxide (NO)-generating enzyme inducible nitric oxide synthase (iNOS). NO has divergent effects on tumors, since it can either stimulate tumor cells growth or promote their death depending on the source of it; likewise the role of iNOS in cancer differs depending on the cell type. The role of NO generated by TAMs has not been investigated. Using different tumor models in vitro and in vivo we found that NO generated by iNOS of M2-polarized TAMs is able to protect tumor cells from apoptosis induced by the chemotherapeutic agent cisplatin (CDDP). Here, we demonstrate that the protective effect of NO depends on the inhibition of acid sphingomyelinase (A-SMase), which is activated by CDDP in a pathway involving the death receptor CD95. Mechanistic insights indicate that NO actions occur via generation of cyclic GMP and activation of protein kinase G (PKG), inducing phosphorylation of syntaxin 4 (synt4), a SNARE protein responsible for A-SMase trafficking and activation. Noteworthy, phosphorylation of synt4 at serine 78 by PKG is responsible for the proteasome-dependent degradation of synt4, which limits the CDDP-induced exposure of A-SMase to the plasma membrane of tumor cells. This inhibits the cytotoxic mechanism of CDDP reducing A-SMase-triggered apoptosis. This is the first demonstration that endogenous NO system is a key mechanism through which TAMs protect tumor cells from chemotherapeutic drug-induced apoptosis. The identification of the pathway responsible for A-SMase activity downregulation in tumors leading to chemoresistance warrants further investigations as a means to identify new anti-cancer molecules capable of specifically inhibiting synt4 degradation.