Barbara Viviani

Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis.

Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)–expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R–expressing inflammatory cells.

Cytokines and neuronal ion channels in health and disease.

The biophysical properties and the spatial distribution of ion channels define the signaling characteristics of individual neurons. Function, number localization, and ratio of receptor and ion channels are dynamically modulated in response to diverse stimuli and undergo dynamic changes in both physiological and pathological conditions. Increasing evidence indicates that cytokines may specifically interact with receptor and ion channels regulating neuronal excitability, synaptic plasticity, and injury. Interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha, two proinflammatory cytokines implicated in various pathophysiological conditions of the CNS, have been particularly studied. Literature data indicate that these cytokines (1) directly and promptly modulate ion channel activity, (2) exert different (and often opposite) effects on the same channels, and (3) act on ion channels both at physiological and pathological concentrations. Consequently, cytokines are now regarded as novel neuromodulators, opening important perspectives in the current view of brain behavior.

Interleukin-1β Released by gp120 Drives Neural Death through Tyrosine Phosphorylation and Trafficking of NMDA Receptors

Interleukin-1β is a proinflammatory cytokine implicated under pathological conditions involving NMDA receptor activation, including the AIDS dementia complex (HAD). No information is available on the molecular mechanisms recruited by native interleukin-1β produced under this type of condition. Using a sandwich co-culture of primary hippocampal neurons and glia, we investigated whether native interleukin-1β released by HIV-gp120-activated glia (i) affects NMDAR functions and (ii) the relevance on neuronal spine density and survival, two specific traits of HAD. Increased phosphorylation of NR2B Tyr-1472 was observed after 24 h of exposure of neurons to 600 pm gp120. This effect occurred only when neurons were treated in the presence of glial cells and was abolished by the interleukin-1 receptor antagonist (IL-1ra). Gp120-induced phosphorylation of NR2B resulted in a sustained elevation of intracellular Ca2+ in neurons and in a significant increase of NR2B binding to PSD95. Increased intracellular Ca2+ was prevented by 10 μm ifenprodil, that selectively inhibits receptors containing the NR2B, by interleukin-1ra and by Ca-pYEEIE, a Src family SH2 inhibitor peptide. These last two inhibitors, prevented also NR2B binding to PSD95. Finally, gp120 reduced by 35% of the total PSD95 positive spine density after 48 h of treatment and induced by 30% of the neuronal death. Again, both of these effects were blocked by Ca-pYEEIE. Altogether, our data show that gp120 releasing interleukin-1β from glia increases tyrosine phosphorylation of NMDAR. Thus, tyrosine phosphorylation may contribute to the sensitization of the receptor increasing its function and synaptic localization. Both of these effects are relevant for neurodegeneration.

In vitro characterization of the immunotoxic potential of several perfluorinated compounds (PFCs)

We have previously shown that PFOA and PFOS directly suppress cytokine secretion in immune cells, with different mechanisms of action. In particular, we have demonstrated a role for PPAR-α in PFOA-induced immunotoxicity, and that PFOS has an inhibitory effect on LPS-induced I-κB degradation. These studies investigate the immunomodulatory effects of four other PFCs, namely PFBS, PFOSA, PFDA, and fluorotelomer using in vitro assays. The release of the pro-inflammatory cytokines IL-6 and TNF-α was evaluated in lipolysaccharide (LPS)-stimulated human peripheral blood leukocytes (hPBL) and in the human promyelocytic cell line THP-1, while the release of IL-10 and IFN-γ was evaluated in phytohemagglutinin (PHA)-stimulated hPBL. All PFCs suppressed LPS-induced TNF-α production in hPBL and THP-1 cells, while IL-6 production was suppressed by PFOSA, PFOS, PFDA and fluorotelomer. PFBS, PFOSA, PFOS, PFDA and fluorotelomer inhibited PHA-induced IL-10 release, while IFN-γ secretion was affected by PFOSA, PFOS, PFDA and fluorotelomer. Leukocytes obtained from female donors appear to be more sensitive to the in vitro immunotoxic effects of PFCs when their responses are compared to the results obtained using leukocytes from male donors. Mechanistic investigations demonstrated that inhibition of TNF-α release in THP-1 cells occurred at the transcriptional level. All PFCs, including PFOA and PFOS, decreased LPS-induced NF-κB activation. With the exception of PFOA, none of the PFCs tested was able to activate PPARα driven transcription in transiently transfected THP-1 cells, excluding a role for PPARα in the immunomodulation observed. PFBS and PFDA prevented LPS-induced I-κB degradation. Overall, these studies suggest that PFCs affect NF-κB activation, which directly suppresses cytokine secretion by immune cells. Our results indicate that PFOA is the least active of the PFCs examined followed by PFBS, PFDA, PFOS, PFOSA and fluorotelomer.

Reactive oxygen species generated by glia are responsible for neuron death induced by human immunodeficiency virus-glycoprotein 120 in vitro

Human immunodeficiency virus infection is often followed by neurodegeneration, the cause of motor and cognitive impairment in some patients affected by acquired immunodeficiency. Several in vitro data indicate glycoprotein (gp) 120 as one of the substances responsible for the neurodegenerative event that takes place only if non-neuronal cells (glial cells) are present. Our purpose was to investigate the molecular mechanisms through which glial cells could affect neuron viability after exposure to gp120 protein. We used a sandwich co-culture of primary hippocampal neurons and primary glial cells, where the two cell populations face each other but are separable. Exposure of 1-week-old rat hippocampal neurons in co-culture with glia to 600 pM gp120 protein resulted in the death of 30% of neurons after 6 days of treatment. A significant increase of intracellular calcium ([Ca2+]i), evident 72 h after gp120 exposure (control 45.8+/-7.6 nM, gp120 176.5+/-43.6 nM), preceded neuron death. The gp120 protein affected neither the viability nor the morphology or [Ca2+]i of glial cells. However, a significant amount of reactive oxygen species as well as of interleukin-1beta was produced. Treatment of the co-culture with an antibody against interleukin-1beta prevented neuron increase of [Ca2+]i and cell death but not glial production of reactive oxygen species, whereas prior incubation of glial cells with Trolox, an antioxidant analog of vitamin E, down-regulated interleukin-1beta expression and completely prevented neuron cell death. Our results indicate that reactive oxygen species produced in glial cells by gp120 exposure cause neurodegeneration by inducing the synthesis of interleukin-1beta.

Effects of central and peripheral inflammation on hippocampal synaptic plasticity.

The central nervous system (CNS) and the immune system are known to be engaged in an intense bidirectional crosstalk. In particular, the immune system has the potential to influence the induction of brain plastic phenomena and neuronal networks functioning. During direct CNS inflammation, as well as during systemic, peripheral, inflammation, the modulation exerted by neuroinflammatory mediators on synaptic plasticity might negatively influence brain neuronal networks functioning. The aim of the present study was to investigate, by using electrophysiological techniques, the ability of hippocampal excitatory synapses to undergo synaptic plasticity during the initial clinical phase of an experimental model of CNS (experimental autoimmune encephalomyelitis, EAE) as well as following a systemic inflammatory trigger. Moreover, we compared the morphologic, synaptic and molecular consequences of central neuroinflammation with those accompanying peripheral inflammation. Hippocampal long-term potentiation (LTP) has been studied by extracellular field potential recordings in the CA1 region. Immunohistochemistry was performed to investigate microglia activation. Western blot and ELISA assays have been performed to assess changes in the subunit composition of the synaptic glutamate NMDA receptor and the concentration of pro-inflammatory cytokines in the hippocampus. Significant microglial activation together with an impairment of CA1 LTP was present in the hippocampus of mice with central as well as peripheral inflammation. Interestingly, exclusively during EAE but not during systemic inflammation, the impairment of hippocampal LTP was paralleled by a selective reduction of the NMDA receptor NR2B subunit levels and a selective increase of interleukin-1β (IL1β) levels. Both central and peripheral inflammation-triggered mechanisms can activate CNS microglia and influence the function of CNS synapses. During direct CNS inflammation these events are accompanied by detectable changes in synaptic glutamate receptors subunit composition and in the levels of the pro-inflammatory cytokine IL1β.

Levels and actions of progesterone and its metabolites in the nervous system during physiological and pathological conditions

Progesterone is synthesized and actively metabolized in the central and peripheral nervous system, into neuroactive steroid metabolites, such as dihydroprogesterone, allopregnanolone and isopregnanolone. Progesterone and/or its metabolites exert a variety of effects acting as physiological regulators of neuronal and glial development and plasticity, controlling reproduction, neuroendocrine events, mood and affection. In addition, these neuroactive steroids maintain neural homeostasis and exert neuroprotective actions. In agreement, metabolic pathways of progesterone are affected by modifications in the level of gonadal hormones and by pathology or injury with a regional specificity and in a sex-dimorphic way. Therefore, observations here summarized may provide a background to design sex-specific therapies based on progesterone metabolites. On this point of view, considering that one of the major limits of a therapy based on neuroactive steroids could be modifications in their plasma levels and their consequent peripheral effects, pharmacological treatments aimed to increase their levels in the nervous system could provide an interesting therapeutic option.

CysLT1 receptor-induced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation

BackgroundCysteine-containing leukotrienes (cysteinyl-LTs) are pivotal inflammatory mediators that play important roles in the pathophysiology of asthma, allergic rhinitis, and other inflammatory conditions. In particular, cysteinyl-LTs exert a variety of effects with relevance to the aetiology of asthma such as smooth muscle contraction, eosinophil recruitment, increased microvascular permeability, enhanced mucus secretion and decreased mucus transport and, finally, airway smooth muscle cells (ASMC) proliferation. We used human ASMC (HASMC) to identify the signal transduction pathway(s) of the leukotriene D4 (LTD4)-induced DNA synthesis.MethodsProliferation of primary HASMC was measured by [3H]thymidine incorporation. Phosphorylation of EGF receptor (EGF-R) and ERK1/2 was assessed with a polyclonal anti-EGF-R or anti-phosphoERKl/2 monoclonal antibody. A Ras pull-down assay kit was used to evaluate Ras activation. The production of reactive oxygen species (ROS) was estimated by measuring dichlorodihydrofluorescein (DCF) oxidation.ResultsWe demonstrate that in HASMC LTD4-stimulated thymidine incorporation and potentiation of EGF-induced mitogenic signaling mostly depends upon EGF-R transactivation through the stimulation of CysLT1-R. Accordingly, we found that LTD4 stimulation was able to trigger the increase of Ras-GTP and, in turn, to activate ERK1/2. We show here that EGF-R transactivation was sensitive to pertussis toxin (PTX) and phosphoinositide 3-kinase (PI3K) inhibitors and that it occurred independently from Src activity, despite the observation of a strong impairment of LTD4-induced DNA synthesis following Src inhibition. More interestingly, CysLT1-R stimulation increased the production of ROS and N-acetylcysteine (NAC) abolished LTD4-induced EGF-R phosphorylation and thymidine incorporation.ConclusionCollectively, our data demonstrate that in HASMC LTD4 stimulation of a Gi/o coupled CysLT1-R triggers the transactivation of the EGF-R through the intervention of PI3K and ROS. While PI3K and ROS involvement is an early event, the activation of Src occurs downstream of EGF-R activation and is followed by the classical Ras-ERK1/2 signaling pathway to control G1 progression and cell proliferation.

Distribution of interleukin-1 receptor complex at the synaptic membrane driven by interleukin-1β and NMDA stimulation

Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that contributes to neuronal injury in various degenerative diseases, and is therefore a potential therapeutic target. It exerts its biological effect by activating the interleukin-1 receptor type I (IL-1RI) and recruiting a signalling core complex consisting of the myeloid differentiation primary response protein 88 (MyD88) and the IL-1R accessory protein (IL-1RAcP). This pathway has been clearly described in the peripheral immune system, but only scattered information is available concerning the molecular composition and distribution of its members in neuronal cells. The findings of this study show that IL-1RI and its accessory proteins MyD88 and IL-1RAcP are differently distributed in the hippocampus and in the subcellular compartments of primary hippocampal neurons. In particular, only IL-1RI is enriched at synaptic sites, where it co-localises with, and binds to the GluN2B subunit of NMDA receptors. Furthermore, treatment with NMDA increases IL-1RI interaction with NMDA receptors, as well as the surface expression and localization of IL-1RI at synaptic membranes. IL-1β also increases IL-1RI levels at synaptic sites, without affecting the total amount of the receptor in the plasma membrane. Our results reveal for the first time the existence of a dynamic and functional interaction between NMDA receptor and IL-1RI systems that could provide a molecular basis for IL-1β as a neuromodulator in physiological and pathological events relying on NMDA receptor activation.

Further development of the NCTC 2544 IL-18 assay to identify in vitro contact allergens

Several European Union legislations request the use of in vitro methods for toxicological evaluations, including sensitization, in order to increase consumer safety but also to reduce the use of animals. The EU project SENS-IT-IV addresses the need of developing predictive in vitro tests to assess contact and respiratory hypersensitivity reactions. In this context, we have recently reported the possibility to use IL-18 production in the human keratinocyte cell line NCTC 2544 to discriminate contact sensitizer from irritants and low molecular weight respiratory allergens. The aims of the present study were to further develop this assay in order to optimize experimental conditions; to develop a 96-well plate format to establish a high throughput assay; to test the performance of other available keratinocyte cell lines, and to understand the signal transduction pathway involved in p-phenylenediamine (PPD)-induced IL-18 production. If cells reach confluence at the moment of treatment, the ability to identify contact allergens is lost; therefore a careful check for the optimal cell density using PPD as reference contact allergen is critical. In our hands, a cell density of 1-2.5 × 10(5)cells/ml gave optimal stimulation. In order to develop a high throughput test, cells seeded in 96-well plate were exposed to contact allergens (2,4-dinitrochlorobenzene, p-phenylenediamine, isoeugenol, cinnamaldehyde, tetramethylthiuram disulfite, resorcinol, cinnamic alcohol and eugenol), irritants (phenol, sodium laurel sulphate, lactic acid and salicylic acid) and respiratory allergens (hexachloroplatinate, diphenylmethane diisocyanate, trimellitic anhydride). A selective increase in total (intracellular plus released) IL-18 was observed 24h later in cells treated with contact allergens, whereas no changes were observed following treatment with respiratory allergens and irritants, confirming previous results obtained in a 24-well format assay. A selective induction of IL-18 was also obtained testing with PPD other keratinocyte cell lines, namely HPKII and HaCaT, with the HPKII showing the highest stimulation index. Regarding the signal transduction pathway, we could demonstrate using selective inhibitors a role for oxidative stress, NF-κB and p38 MAPK activation in PPD-induced IL-18 production. In conclusion, results obtained suggest that the production of IL-18 represents a promising endpoint for the screening of potential contact allergens. The assay can be performed in a 96-well plate format, different keratinocyte cell lines can be used, and a role for oxidative stress in contact allergen-induced IL-18 was demonstrated.