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Influence of effector molecules released by Th1 and Th17 cellson glial cells

Prajeeth Chittappen Kandiyila, Julius Kornischa, Reza Khorooshib,Henrik Toft-hansenb, Kirsten Löhrc, Trevor Owensb, Jochen Huehnc,Martin Stangela

aClinicalNeuroimmunology andNeurochemistry, Department of Neurology,Hannover Medical School, Hannover, Germany; bDepartment ofNeurobiology, Institute of Molecular Medicine, University of SouthernDenmark, Odense, Denmark; cExperimental Immunology, Helmholtz Centrefor Infection Research, Braunschweig, Germany

In neurodegenerative diseases like multiple sclerosis and exper-imental autoimmune encephalomyelitis, pathogenic CD4+ T helpercell subsets that characteristically secrete IFN-gamma (Th1) or IL-17(Th17) infiltrate from the periphery into the central nervous system(CNS) and cause tissue damage. Their interaction with astrocytes andmicroglia is crucial in the regulation of neuroinflammation. Thepotential effects of distinct effector molecules released by pathogenicTh1 and Th17 cells in this process are poorly understood. In thisstudy, we tested the ability of factors secreted by in vitro generatedTh1 and Th17 cells to induce glial cell activation. Recently, we haveshown that only effector molecules secreted by Th1 cells activate andpolarize microglia to an M1-like phenotype, whereas the effectorsreleased by Th17 cells had no direct influence on microglia. To furthercharacterize the interaction between T cells and glia we investigatedthe effect of Th1- and Th17-derived factors on astrocytes. For thispurpose, we treated astrocytes harvested from mixed glial prepara-tions with Th1- and Th17-derived culture supernatants and assessedthe expression of factors that are known to be involved in mediatingneuroinflammation. Measurement of culture supernatants revealedsignificantly higher amounts of monocyte chemoattractant protein-1(MCP-1/CCL2)when astrocytes were exposed to Th1 and Th17 derivedfactors. RT-PCR analysis of astrocytes revealed that both Th1- and Th17-derived factors significantly upregulated the expression of induciblenitric oxide synthase (NOS2). Interestingly, expression of IL6 and CCL20that are involved in differentiation and recruitment of Th17 cells wasupregulated only in those astrocytes that were treated with Th17-derived factors. Furthermore, transwell migration assays demonstratethat Th17-activated astrocytes efficiently attracted microglia whichreflects the situation in the CNSwherein astrocytes have been shown tobe crucial in recruiting microglia. Taken together, in our attempt todissect the individual contribution of Th1 and Th17 cells in propagatingneuroinflammation we show that Th1-derived factors can directlyinfluence both microglia and astrocytes, whereas the targets of Th17-derived factors in this process seem to be mainly astrocytes.

doi:10.1016/j.jneuroim.2014.08.385

246

The impact of neuroinflammation on tau pathology in healthyaging and neurodegeneration

Alexander W. Collcutt, Jessica L. Teeling, Ayodeji A. Asuni

Centre for Biological Sciences, University of Southampton, Southampton,United Kingdom

Systemic infections in a population accelerate the progression ofneurodegenerative conditions such as Alzheimer’s Disease (AD) but

reasons for this remain unclear. Pathology of AD is characterised byaccumulating misfolded protein aggregates of amyloid-beta and Tauneurofibrillary tangles, as well as neuroinflammation. Although amy-loid and tau pathology are synergistic, therapeutically A(beta) hasreceived the most attention to date and has not as yet yielded a viabletreatment. Neuroinflammation and Tau pathology positively correlatewith cognitive deficits and there is some evidence to suggest the formeraccelerates the latter. To explore this mechanism we injected LPS orsaline unilaterally into the hippocampus of C57BL/6 wild-type mice.Using histological methods we found markers of microglial activationsuch as Fc(gamma)RI and CD11b had increased after 1 day. In addition,therewas an increase in tau phosphorylation (PHF-1, CP13) at the sametime point. Both of these increases had returned to control levelsafter 28 days. In comparison, a pronounced immune activation wasalso observed in tau transgenic mice (Tg4510) at 1 year of age, withsignificant tau phosphorylation and neurodegeneration seen previouslyin this model. We have shown that an acute inflammatory insult cancause tau phosphorylation in the brain, which is resolved quickly, butthe immune cells may subsequently remain primed. We hypothesisethat many of these acute insults or a (systemic) low-grade chronicinflammatory state may increase an individual’s risk to a neurodegen-erative disease or worsen a current one, through modulation of tauphosphorylation. Components of this mechanismmay act as targets fornovel therapeutics against neurodegenerative diseases like AD.

doi:10.1016/j.jneuroim.2014.08.386

367

Fingolimod may support neuroprotection via blockade ofastrocyte S1P and cytokine signaling cascades inMultiple Sclerosis

Emanuela Colomboa, Marco Di Darioa, Eleonora Capitoloa,Linda Chaabanea, Jia Newcombeb, Gianvito Martinoa, Cinthia Farinaa

aInstitute of Experimental Neurology (INSpe), San Raffaele ScientificInstitute, Milan, Italy; bNeuroResource, UCL Institute of Neurology,London, United Kingdom

Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipidthat regulates different cellular processes such as growth, survivaland differentiation. S1P signaling supports growth factor signaltransduction and triggers activation of nuclear transcription factorNFκB and of nitric oxide (NO) synthase. S1P acts via binding tospecific G protein-coupled receptors (S1P1-5), which may beexpressed by resident cells in the central nervous system (CNS),including astrocytes. Upon CNS injury astrocytes actively participatein glial scar formation and tissue repair, and dysregulation of theiractivity can have detrimental effects on CNS function. Recent reportssuggest that activation of pathways leading to NFκB activation andNO production in astrocytes contributes to multiple sclerosis (MS)pathology. Published studies have shown that the proinflammatorycytokines IL1 and IL17 are produced in the CNS during neuroinflamma-tion and that cytokines may induce inside-out S1P signaling. Neverthe-less, the possibility and relevance of the crosstalk between the signalingcascades of S1P and of IL1 and IL17 in MS remain largely unaddressed.

Here we show that, while absent in normal appearing and controlwhite matter, IL1R, IL17RA and S1P receptors S1P1 and S1P3 are allupregulated on astrocytes in MS lesions and in the spinal cord ofexperimental autoimmune encephalomyelitis (EAE) mice. In vitroassays demonstrate that IL1, IL17 and S1P induce NFκB translocationand NO production in astrocytes, and that the astrocyte conditionedmedia trigger neuronal death.

Abstracts144

Importantly, in vitro targeting of S1P signaling by Fingolimodblocks NFκB translocation and NO production in astrocytes evokedby either S1P or inflammatory cytokines and this effect results in theinhibition of astrocyte-mediated neurodegeneration. Finally, thera-peutic administration of Fingolimod to EAE mice hampers astrocyteactivation and nitrosative stress in vivo.

In conclusion these data indicate that neurotoxic mediatorsreleased by astrocytes following activation of S1P and cytokine signallingcascades may contribute to neuronal damage in MS via NFκB pathwayactivation and NO production, and that the neuroprotective effect ofFingolimod in vivo may be the indirect result of its action on astrocyteactivation.

This study was supported by Fondazione Italiana Sclerosi Multipla(FISM) (2012/R/7), Amici Centro Sclerosi Multipla (Acesm) andItalian Ministry of Health.

doi:10.1016/j.jneuroim.2014.08.387

102

Shedding microvesicles released by a human microglial cell line(CHME-5): A good platform for studying the biology of the system

Federico Colomboa, Annamaria Finardib, Gabriella Racchettib,Roberto Furlanb

aSan Raffaele Scientific Institute, Università Vita-Salute San Raffaele,Milano, Italy; bSan Raffaele Scientific Institute, Ospedale San Raffaele,Milano, Italy

Cell-to-cell communication is a field of growing interest in thecurrent cell biology since the recent discovery of multiple andunexpected mechanisms by which cells can propagate informationamong them through active biomolecules. One of these mechanismsis based on the release of membrane particles of different size,generally called microvesicles (MVs), filled by either proteins ornucleic acids whose composition seems to precisely reflect that ofthe cell of origin.

Until now two main microvesicle populations have been isolatedand characterized: exosomes and shedding vesicles (SVs). Theformer are nanoparticles (diameter: 30–100 nm) of endocyticorigin constitutively released by cells upon the fusion of multi-vesicular bodies (MVBs) with the plasma membrane whereas thelatter are larger vesicles (diameter: 100–1500 nm) formed by theoutward bending of the plasma membrane (called budding orectocytosis) triggered by few specific signals as ATP. The mecha-nisms by which the deformation of membranes occurs are knownonly in part and the biology of this process is still very far from befully understood.

For a long time MVs were considered as mere cell artifacts untilrecent evidences demonstrating the importance of their ability incarrying and protecting molecular cargoes in the extracellular spaceto be crucial importance either in physiological or in pathologicalconditions ranging from tumor cell migration and metastasisdissemination, to coagulation and inflammation. Recently our grouphas reported a massive increase of shedding vesicles of myeloidorigin in the cerebrospinal fluid of multiple sclerosis patients respectto the healthy controls and that their number correlates with theseverity of the disease. In order to elucidate some aspects of SVsbiology in the contest of human physiology and pathology we choseas a model a microglia cell line of human embryonal origin, namelyCHME-5. Here I report the detailed characterization of the ability of thiscell line to release detectable amounts of SVs upon ATP or cytokine

stimulation (conditions mimic the inflamed tissues) by using multipleapproaches ranging from western blotting to immunofluorescence,flowcytometry and electronmicroscopy. Thus this cell linewill offer thecrucial advantage of producing enough microvesicles to performOMICS analyses in order to reveal their content and possibly also theirfunctions.

doi:10.1016/j.jneuroim.2014.08.388

420

Neutrophils induce Alzheimer's-like disease via LFA-1-integrinand neutrophil extracellular traps

Elena Zenaroa, Enrica Pietronigroa, Vittorina Della Biancaa,Gennj Piacentinoa, Alessio Montresora, Ermanna Turanob,Bruno Bonettib, Gabriela Constantina

aDepartment of Pathology and Diagnostics, University of Verona,Verona, Italy; bDepartment of Neurological Sciences, University ofVerona, Verona, Italy

Inflammation has been correlated to Alzheimer's disease (AD) and abetter understanding of inflammation mechanisms may potentiallyhelp to develop new approaches to treat this disorder.

The aim of our study was to investigate the role of vascularinflammation and immune cell trafficking in animal models of AD.We detected enhanced expression of vascular adhesion moleculesand increased accumulation of neutrophils in 5XFAD and 3xTg-ADtransgenicmicewith cognitive impairment during early stage of AD-likedisease compared to age-matched wild-type controls. Using two-photon laser-scanning microscopy we observed that neutrophils crawlin blood vessels and transmigrate in high numbers in areaswith amyloidplaques in both AD-like models. Interestingly, neutrophils releasedintravascular and intraparenchymal neutrophil extracellular traps(NETs), suggesting that NETosis may represent a potential neutrophil-dependent disease mechanism in AD. Amyloid beta peptide 1-42induced high affinity state of LFA-1 integrin and LFA-1-dependentadhesion of neutrophils. Moreover, two-photon microscopy exper-iments showed that LFA-1 integrin blockade prevented neutrophilextravasation, and inhibited intraparenchymal motility. Notably,neutrophil depletion or blockade of neutrophil trafficking by ananti-LFA-1 integrin antibody inhibited amyloid deposition and tauphosphorylation, and rescued cognitive deficits in behavioral testsin 5XFAD and 3xTg mice, suggesting that neutrophils play a keyrole in AD-like disease. Importantly, restoration of cognitivefunction in mice with temporary inhibition of neutrophil functionduring early disease was maintained also at later time points inaged animals. To understand the relevance of our data in humans,we analyzed human cortical brain samples from subjects with AD.Our results showed that neutrophils adhered and spread insidebrain venules or migrated into the parenchyma in high numbersand release NETs in human AD brains compared to control subjects.Taken together our results demonstrate that neutrophils inducecognitive impairment in AD-like disease and suggest that inhibitionof neutrophil trafficking may represent a new therapeutic strategyin AD.

This work was supported by the European Research Council(ERC) (261079) grant Neurotrafficking (to G.C.).

doi:10.1016/j.jneuroim.2014.08.389

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