Serum amyloid A primes microglia for ATP-dependent interleukin-1\u3b2 release

Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves production of acute-phase proteins, including serum amyloid A (SAA). Interleukin-1\u3b2 (IL-1\u3b2), a master regulator of neuroinflammation produced by activated inflammatory cells of the myeloid lineage, in particular microglia, plays a key role in the pathogenesis of acute and chronic diseases of the peripheral nervous system and CNS. IL-1\u3b2 release is promoted by ATP acting at the purinergic P2X7 receptor (P2X7R) in cells primed with toll-like receptor (TLR) ligands

In Vitro and In Vivo Human Herpesvirus 8 Infection of Placenta

Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8) infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women

Astrocyte-microglia interaction in the expression of a pro-inflammatory or pain-related phenotype: molecular and cellular aspects

In the central nervous system (CNS), glial cells not only serve supportive and nutritive roles for neurons, but also respond to protracted stress and insults by up-regulating inflammatory processes. Reactive astrocytes and microglia have been detected in animal models of neuronal injury such as ischemia, axotomy, and neurotoxic insult, and in the human brain in neurodegenerative diseases. Reactive glial cells produce a wide array of pro-inflammatory molecules, including nitric oxide, cytokines and chemokines. The complexity of studying glial activation in vivo has led to the widespread adoption of in vitro approaches, for example the use of the bacterial toxin lipopolysaccharide (LPS, a ligand for toll-like receptor 4 (TLR4)) as an experimental model of glial activation. In the latter, however, the contribution of microglia, if any, to the response by astrocytes remains an open question, as such astrocyte cultures frequently contain minor numbers of microglia. In the present study, we set up a in vitro model to evaluate the interaction between astrocytes and microglia in the CNS. At first we used mixed glial cultures from neonatal rat cortex and spinal cord, and we control for the presence of other cell types (endothelium, oligodendrocytes, and neurons). Then we purified microglia and enriched astrocyte cultures in order to evaluate the response to inflammatory (LPS), or pain stimuli (substance P, vasoactive intestinal peptide, calcitonin gene related peptide). Under our experimental conditions, we demonstrated that enriched astrocyte cultures respond to LPS for longer times than purified microglia, but glial cultures lack response to peptides involved in neuronal pain transmission. Subsequently, enriched (<5% microglia) astrocytes cultured from neonatal rat cortex and spinal cord were treated with the lysosomotropic agent L-leucine methyl ester to eliminate residual microglia, as confirmed by loss of microglia-specific marker genes. L-Leucine methyl ester treatment lend to a loss of LPS responsiveness, in terms of nitric oxide and cytokine gene up-regulation and mediator output into the culture medium. Astrocyte responsiveness could be reconstituted by re-addition of increasing numbers of purified microglia which, by themselves, yielded little or no signal upon LPS exposure. Given that astrocytes greatly outnumber microglia in the CNS, these data suggest that a similar ‘cross-talk’ between microglia and astrocytes in vivo may be an important element in the evolution of an inflammatory pathology. In the absence of pathogens, TLR signaling can be activated by molecules called damage associated molecular patterns, released by the injured tissue. The astrocyte/microglia co-culture paradigm described here may provide a useful starting point to elucidate the molecular mechanisms underlying astrocyte- and microglia-specific responses pertaining to, although not limited to, CNS inflammation, especially where TLR activation plays a role.Nel sistema nervosa centrale (SNC), le cellule gliali non svolgono solamente una funzione di supporto ai neuroni, ma rispondono “attivandosi” a rilevanti stress e insulti chimici. Astrociti e microglia attivati sono stati individuati in modelli animali in cui è stata indotta una lesione del SNC, ischemia, assotomia, neurotossicità da tossine neurotossiche, e, nel caso dell’uomo, in malattie neurodegenerative. Cellule gliali attivate possono rilasciare diversi fattori pro-infiammatori, come l’ossido nitrico, citochine e varie chemochine, agenti, questi, tutti coinvolti nei processi infiammatori. Tuttavia, poiché questo fenomeno risulta piuttosto complesso in vivo, numerosi studi hanno cercato di allestire dei modelli in vitro in grado di comprendere il contributo delle varie cellule gliali nell’infiammazione, nel dolore cronico e in quello neuropatico. Classici modelli di infiammazione a livello del SNC, coinvolgono i recettori dell’immunità innata della famiglia Toll-like (TLR), in particolare il recettore TLR4. In vitro e in vivo, trattando con lipopolisaccaridi (LPS) batterici colture cellulari da tessuto nervoso o animali, si induce uno stato di attivazione della microglia e degli astrociti simile a quello presente nell’infiammazione. Tuttavia, nonostante in numerosi studi sia stato dimostrato il coinvolgimento della microglia nel fenomeno infiammatorio ed algico, resta ancora poco chiaro il preciso ruolo degli astrociti, poiché le colture di quest’ultimi contengono spesso delle contaminanti di microglia. In questo studio, abbiamo allestito un modello in vitro utile per valutare le interazioni tra astrociti e microglia nei fenomeni infiammatori ed algici. Inizialmente abbiamo utilizzato colture gliali miste ottenute da corteccia e midollo spinale di ratto neonato di due giorni. Abbiamo valutato la presenza nella coltura cellule oligodendrocitarie, endoteliali e neuronali, evidenziando come sostanzialmente la coltura fosse composta quasi esclusivamente da cellule microgliali e astrocitarie. Successivamente, separando la microglia dagli astrociti, abbiamo ottenuto colture arricchite in astrociti, le quali evidenziano una contaminante di microglia inferiore al 5%. Queste colture sono state quindi stimolate con LPS, o con peptidi coinvolti nella genesi o nel mantenimento dello stimolo algico a livello neuronale (quali sostanza P, peptide intestinale vasoattivo, peptide correlato al gene della calcitonina, ecc.). Abbiamo quindi dimostrato che, nelle nostre condizioni sperimentali, le colture arricchite in astrociti rispondono, al trattamento con LPS, per periodi più prolungati rispetto alla microglia purificata. Tuttavia, entrambe le colture gliali non evidenziano nessuna risposta pro-infiammatoria quando trattate con i tre neuro-peptidi testè elencati. Successivamente, le colture arricchite in astrociti sono state trattate con l’agente lisosomotropico L-Leucina metil estere (L-LME), un composto che, come dimostrato nelle nostre colture, è in grado di causare la morte della microglia e la conseguente scomparsa dei suoi marcatori genici specifici. Dopo trattamento con L-LME, la coltura pura di astrociti evidenzia una perdita di risposta all’LPS sia in termini di assenza di induzione trascrizionale dei geni che codificano l’enzima inducibile nitrico sintasi e alcune citochine infiammatorie, come pure di un mancato rilascio di questi fattori nel mezzo di coltura. La risposta degli astrociti all’LPS può essere ripristinata mediante riaggiunta alle colture astrocitarie di quantità crescenti di cellule microgliali. Tuttavia se le stesse quantità di cellule di microglia utilizzate nella riaggiunta, vengono coltivate in assenza di astrociti, esse evidenziano una risposta all’LPS significativamente minore. Poiché nel SNC gli astrociti sono presenti in numero molto superiore alla microglia, questi dati suggeriscono che come in vitro, anche in vivo, potrebbe esistere una cooperazione tra astrociti e microglia e ciò potrebbe risultare di vitale importante nella genesi e nel mantenimento dell’infiammazione e del dolore patologico. Infatti, anche in assenza di patogeni nel SNC, i recettori TLR dell’immunità innata, possono essere attivati da fattori della famiglia delle proteine associate a danni molecolari (damage associated molecular patterns, DAMP) che vengono attivati da danni cellulari che non implicano una risposta infiammatoria da tossine batteriche. La co-coltura di astrociti e microglia descritta nel presente studio può rappresentare, quindi, un punto di partenza per chiarire il meccanismo molecolare che sottintende alla specifica risposta infiammatoria mediata da astrociti e microglia, causata dall’attivazione dei recettori della famiglia TLR

Culture of rat mesencephalic dopaminergic neurons and application to neurotoxic and neuroprotective agents

Dopaminergic neuronal cell degeneration is the principal characteristic feature of the neuropathology of Parkinson disease. Cultures of mesencephalic neurons are widely used as a source of dopaminergic neurons for the study of mechanisms implicated in dopaminergic cell death and for the evaluation of potential dopaminergic neuroprotective agents, including neurotrophic factors. This chapter presents a detailed protocol for the preparation of rat mesencephalic cell cultures and their application to evaluating the effect of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium and the neuroprotective action of brain-derived neurotrophic facto

Toll-Like Receptors 2, -3 and -4 Prime Microglia but not Astrocytes Across Central Nervous System Regions for ATP-Dependent Interleukin-1\u3b2 Release

Interleukin-1 beta (IL-1 beta) is a crucial mediator in the pathogenesis of inflammatory diseases at the periphery and in the central nervous system (CNS). Produced as an unprocessed and inactive pro-form which accumulates intracellularly, release of the processed cytokine is strongly promoted by ATP acting at the purinergic P2X(7) receptor (P2X(7)R) in cells primed with lipopolysaccharide (LPS), a Toll-like receptor (TLR) 4 ligand. Microglia are central to the inflammatory process and a major source of IL-1 beta when activated. Here we show that purified (> 99%) microglia cultured from rat cortex, spinal cord and cerebellum respond robustly to ATP-dependent IL-1 beta release, upon priming with a number of TLR isoform ligands (zymosan and Pam3CSK4 for TLR2, poly(I:C) for TLR3). Cytokine release was prevented by a P2X(7)R antagonist and inhibitors of stress-activated protein kinases. Enriched astrocytes (<= 5% microglia) from these CNS regions displayed responses qualitatively similar to microglia but became unresponsive upon eradication of residual microglia with the lysosomotropic agent Leu-Leu-OMe. Activation of multiple TLR isoforms in nervous system pathology, coupled with elevated extracellular ATP levels and subsequent P2X(7)R activation may represent an important route for microglia-derived IL-1 beta. This phenomenon may have important consequences for neuroinflammation and its position to the common pathology of CNS diseases

Co-ultramicronized palmitoylethanolamide/luteolin promotes the maturation of oligodendrocyte precursor cells

Oligodendrocytes have limited ability to repair the damage to themselves or to other nerve cells, as seen in demyelinating diseases like multiple sclerosis. An important strategy may be to replace the lost oligodendrocytes and/or promote the maturation of undifferentiated oligodendrocyte precursor cells (OPCs). Recent studies show that a composite of co-ultramicronized N-palmitoylethanolamine (PEA) and luteolin (co-ultramicronized PEA/luteolin, 10:1 by mass) is efficacious in improving outcome in experimental models of spinal cord and traumatic brain injuries. Here, we examined the ability of co-ultramicronized PEA/luteolin to promote progression of OPCs into a more differentiated phenotype. OPCs derived from newborn rat cortex were placed in culture and treated the following day with 10 \u3bcM co-ultramicronized PEA/luteolin. Cells were collected 1, 4 and 8 days later and analyzed for expression of myelin basic protein (MBP). qPCR and Western blot analyses revealed a time-dependent increase in expression of both mRNA for MBP and MBP content, along with an increased expression of genes involved in lipid biogenesis. Ultramicronized PEA or luteolin, either singly or in simple combination, were ineffective. Further, co-ultramicronized PEA/luteolin promoted morphological development of OPCs and total protein content without affecting proliferation. Co-ultramicronized PEA/luteolin may represent a novel pharmacological strategy to promote OPC maturation

Co-Ultramicronized Palmitoylethanolamide/Luteolin Facilitates the Development of Differentiating and Undifferentiated Rat Oligodendrocyte Progenitor Cells

Oligodendrocytes, the myelin-producing cells of the central nervous system (CNS), have limited capability to bring about repair in chronic CNS neuroinflammatory demyelinating disorders such as multiple sclerosis (MS). MS lesions are characterized by a compromised pool of undifferentiated oligodendrocyte progenitor cells (OPCs) unable to mature into myelin-producing oligodendrocytes. An attractive strategy may be to replace lost OLs and/or promote their maturation. N-palmitoylethanolamine (PEA) is an endogenous fatty acid amide signaling molecule with anti-inflammatory and neuroprotective actions. Recent studies show a co-ultramicronized composite of PEA and the flavonoid luteolin (co-ultraPEALut) to be more efficacious than PEA in improving outcome in CNS injury models. Here, we examined the effects of co-ultraPEALut on development of OPCs from newborn rat cortex cultured under conditions favoring either differentiation (Sato medium) or proliferation (fibroblast growth factor-2 and platelet-derived growth factor (PDGF)-AA-supplemented serum-free medium (\u201cSFM\u201d)). OPCs in SFM displayed high expression of PDGF receptor alpha gene and the proliferation marker Ki-67. In Sato medium, in contrast, OPCs showed rapid decreases in PDGF receptor alpha and Ki-67 expression with a concomitant rise in myelin basic protein (MBP) expression. In these conditions, co-ultraPEALut (10 \u3bcM) enhanced OPC morphological complexity and expression of MBP and the transcription factor TCF7l2. Surprisingly, co-ultraPEALut also up-regulated MBP mRNA expression in OPCs in SFM. MBP expression in all cases was sensitive to inhibition of mammalian target of rapamycin. Within the context of strategies to promote endogenous remyelination in MS which focus on enhancing long-term survival of OPCs and stimulating their differentiation into remyelinating oligodendrocytes, co-ultraPEALut may represent a novel pharmacological approach