The hallmark of neuroinflammation is the activation of microglia, the immunocompetent cells of the CNS, releasing a number of pro-inflammatory mediators implicated in the pathogenesis of several neurological diseases such as AD, PD, MS and ischemic stroke. The innate immune TLR4, localized on the surface of microglia, is a first-line host defense receptor against invading microorganisms and LPS, a component of the cell wall of gram-negative bacteria, was first identified as the TLR ligand. ARs interacting with TLR4 influence on many immune properties of microglia and are implicated in numerous physiological and pathophysiological states in CNS. Indeed, Ado, a ubiquitous nucleoside with important immunomodulatory functions, has been found to take part in the principal microglial activation processes spanning from proliferation, process extension, retraction, migration and cytokine production. On this background, the aim of this study was to investigate the adenosinergic contribution to glial cell mediated-neuroinflammation by analyzing the expression of ARs and the signalling pathways, transcription factors and cytokines activated by them in different pathophysiological conditions linked to hypoxic and inflammatory conditions. The aim of studies reported in chapter 1 was to investigate whether Ado may affect microglia functions by acting on HIF-1α modulation, the main transcription factor of hypoxia-inducible genes, involved in the immune response, being regulated in normoxia by inflammatory mediators. Primary murine microglia were activated with LPS with or without Ado, Ado receptor agonists and antagonists and HIF-1α accumulation and downstream genes regulation were determined. Ado increased LPS-induced HIF-1α accumulation leading to an increase in HIF-1α target genes involved in cell metabolism and pathogens killing but did not induce HIF-1α dependent genes related to angiogenesis and inflammation. The stimulatory effect of Ado on HIF-1α and its target genes was essentially exerted by activation of A2A through ERK1/2 (or p44/42) and A2B subtypes via p38 MAPKs and Akt phosphorylation. Furthermore, the nucleoside raised VEGF and decreased TNF-α levels, by activating A2B subtypes. Our results show that Ado increases GLUT-1 and iNOS gene expression in a HIF-1α-dependent way, through A2A and A2B ARs, suggesting their role in the regulation of microglial cells function following injury. However, inhibition of TNF-α adds an important anti-inflammatory effect only for the A2B subtype. The aim of studies reported in chapter 2 was to indagate the potential role of ARs in the modulation of IL-6 secretion and cell proliferation in primary microglial cells. The A2B AR agonist BAY 60-6583 stimulated IL-6 increase under normoxia and hypoxia, in a dose- and time-dependent way. In cells incubated with the blockers of PLC, PKC-ε and PKC-delta the IL-6 increase due to A2B AR activation was strongly reduced, whilst it was not affected by the inhibitor of AC. Investigation of cellular signalling involved in the A2B AR effect revealed that only the inhibitor of p38 MAPK was able to block the agonist effect on IL-6 secretion, whilst inhibitors of ERK1/2, JNK1/2 MAPKs and Akt were not. Stimulation of p38 by BAY 60-6583 was A2B AR dependent, through PLC, PKC-ɛ and PKC-delta but not AC pathway, in both normoxia and hypoxia. Finally, BAY 60-6583 increased microglial cell proliferation involving A2B AR, PLC, PKC-ε PKC-delta and p38 signalling. Our results show for the first time that Ado by activating A2B ARs increases IL-6 protein levels and cell proliferation through a pathway dependent on PLC, PKC-ε, PKC-delta, and p38 signalling. These findings add new molecular pathways activated by Ado in microglia to give a reduction of genes and cytokines involved in inflammation and hypoxic injury that may cohexist in stroke, ischemia and other CNS disorders.

Il segno distintivo del processo neuroinfiammatorio è l'attivazione delle microglia, le cellule immunocompetenti del sistema nervoso centrale (SNC), che sono responsabili del rilascio di numerosi mediatori pro-infiammatori implicati nella patogenesi di diverse malattie neurologiche come la malattia di Alzheimer, il morbo di Parkinson, la sclerosi multipla e l’ictus ischemico. Il recettore dell’immunità innata Toll-like (TLR)4, presente sulla superficie della microglia, gioca un ruolo chiave nella difesa dell'organismo e può essere attivato dal lipopolisaccaride (LPS), uno dei componenti della membrana cellulare esterna dei batteri gram-negativi. I recettori dell'adenosina (AR) interagendo con il recettore TLR4 influenzano numerose proprietà immunologiche delle cellule microgliali e sono implicati in molteplici stati fisiologici e patofisiologici nel SNC. Infatti, è stato dimostrato che l'adenosina (Ado), nucleoside ubiquitario con importanti funzioni immunomodulatorie e ligando endogeno degli AR, prende parte ai principali processi di attivazione della microglia come: proliferazione, estensione e retrazione dei processi cellulari, migrazione e produzione di citochine. Sulla base di queste considerazioni, lo scopo di questa tesi è stato quello di investigare il contributo adenosinergico sulla neuroinfiammazione mediata dalle cellule gliali attraverso l’analisi dell'espressione degli AR e le vie di trasduzione del segnale, i fattori di trascrizione e le citochine attivate da questi recettori in diverse condizioni patofisiologiche legate ad ipossia e infiammazione. Lo scopo dello studio riportato nel capitolo 1 è stato quello di investigare se Ado poteva influenzare le funzioni della microglia agendo sulla modulazione del fattore indotto dall’ipossia-1α (HIF-1α), il principale fattore di trascrizione dei geni ipossia-inducibili, coinvolti nella risposta immunitaria e regolati in normossia da mediatori dell'infiammazione. I dati ottenuti hanno messo in evidenza che Ado è in grado di aumentare l'accumulo di HIF-1α indotto dall’LPS portando ad un aumento dei geni bersaglio di HIF-1α coinvolti nel metabolismo cellulare e nell’eliminazione dei patogeni ma non è in grado di indurre quelli dipendenti da HIF-1α correlati all'angiogenesi e all'infiammazione. L'effetto stimolatorio di Ado su HIF-1α e dei suoi geni bersaglio è stato essenzialmente esercitato dall'attivazione degli A2A AR attraverso le proteine chinasi attivate da mitogeni (MAPK) chinasi regolate da segnali extra cellulari (ERK)1/2 e degli A2B AR tramite la MAPK p38 e la fosforilazione di Akt. Inoltre, Ado attraverso gli A2B AR ha aumentato i livelli del fattore di crescita dell'endotelio vascolare (VEGF) e diminuito quelli del fattore di necrosi tumorale (TNF)-α. Lo scopo dello studio riportato nel capitolo 2 è stato quello di indagare il potenziale ruolo degli AR nella modulazione della secrezione dell'interleuchina(IL)-6 e nella proliferazione cellulare in cellule microgliali murine primarie. I dati ottenuti hanno messo in evidenza che l'agonista A2B (BAY 60-6583) è in grado di stimolare l'aumento di IL-6, in modo dose e tempo-dipendente, sia in normossia che in ipossia e che tale aumento è fortemente ridotto dall’incubazione delle cellule con gli inibitori della fosfolipasi C (PLC), della proteina chinasi C (PKC)-ε e PKC-delta. Lo studio sulle vie di trasduzione del segnale cellulare ha rivelato che solo l'inibitore di p38 è in grado di bloccare l'effetto dell’agonista A2B sulla secrezione di IL-6, mentre gli inibitori di ERK1/2, JNK1/2 e Akt non hanno dato alcun effetto. La stimolazione di p38 da parte di BAY 60-6583 è risultata dipendente dagli A2B AR, tramite PLC, PKC-ɛ e PKC-delta ma non tramite l’adenilato ciclasi, sia in normossia che in ipossia. Infine, BAY 60-6583 ha aumentato la proliferazione delle cellule microgliali attraverso la via di segnale che vede coinvolti A2B AR, PLC, PKC-ε PKC-delta e p38.

Glial cells and neuroinflammation: the adenosinergic contribution

BENCIVENNI, SERENA
2018

Abstract

The hallmark of neuroinflammation is the activation of microglia, the immunocompetent cells of the CNS, releasing a number of pro-inflammatory mediators implicated in the pathogenesis of several neurological diseases such as AD, PD, MS and ischemic stroke. The innate immune TLR4, localized on the surface of microglia, is a first-line host defense receptor against invading microorganisms and LPS, a component of the cell wall of gram-negative bacteria, was first identified as the TLR ligand. ARs interacting with TLR4 influence on many immune properties of microglia and are implicated in numerous physiological and pathophysiological states in CNS. Indeed, Ado, a ubiquitous nucleoside with important immunomodulatory functions, has been found to take part in the principal microglial activation processes spanning from proliferation, process extension, retraction, migration and cytokine production. On this background, the aim of this study was to investigate the adenosinergic contribution to glial cell mediated-neuroinflammation by analyzing the expression of ARs and the signalling pathways, transcription factors and cytokines activated by them in different pathophysiological conditions linked to hypoxic and inflammatory conditions. The aim of studies reported in chapter 1 was to investigate whether Ado may affect microglia functions by acting on HIF-1α modulation, the main transcription factor of hypoxia-inducible genes, involved in the immune response, being regulated in normoxia by inflammatory mediators. Primary murine microglia were activated with LPS with or without Ado, Ado receptor agonists and antagonists and HIF-1α accumulation and downstream genes regulation were determined. Ado increased LPS-induced HIF-1α accumulation leading to an increase in HIF-1α target genes involved in cell metabolism and pathogens killing but did not induce HIF-1α dependent genes related to angiogenesis and inflammation. The stimulatory effect of Ado on HIF-1α and its target genes was essentially exerted by activation of A2A through ERK1/2 (or p44/42) and A2B subtypes via p38 MAPKs and Akt phosphorylation. Furthermore, the nucleoside raised VEGF and decreased TNF-α levels, by activating A2B subtypes. Our results show that Ado increases GLUT-1 and iNOS gene expression in a HIF-1α-dependent way, through A2A and A2B ARs, suggesting their role in the regulation of microglial cells function following injury. However, inhibition of TNF-α adds an important anti-inflammatory effect only for the A2B subtype. The aim of studies reported in chapter 2 was to indagate the potential role of ARs in the modulation of IL-6 secretion and cell proliferation in primary microglial cells. The A2B AR agonist BAY 60-6583 stimulated IL-6 increase under normoxia and hypoxia, in a dose- and time-dependent way. In cells incubated with the blockers of PLC, PKC-ε and PKC-delta the IL-6 increase due to A2B AR activation was strongly reduced, whilst it was not affected by the inhibitor of AC. Investigation of cellular signalling involved in the A2B AR effect revealed that only the inhibitor of p38 MAPK was able to block the agonist effect on IL-6 secretion, whilst inhibitors of ERK1/2, JNK1/2 MAPKs and Akt were not. Stimulation of p38 by BAY 60-6583 was A2B AR dependent, through PLC, PKC-ɛ and PKC-delta but not AC pathway, in both normoxia and hypoxia. Finally, BAY 60-6583 increased microglial cell proliferation involving A2B AR, PLC, PKC-ε PKC-delta and p38 signalling. Our results show for the first time that Ado by activating A2B ARs increases IL-6 protein levels and cell proliferation through a pathway dependent on PLC, PKC-ε, PKC-delta, and p38 signalling. These findings add new molecular pathways activated by Ado in microglia to give a reduction of genes and cytokines involved in inflammation and hypoxic injury that may cohexist in stroke, ischemia and other CNS disorders.
GESSI, Stefania
DI VIRGILIO, Francesco
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2478763
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