Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). One of the pathological hallmarks of MS is the T cells-mediated destruction of myelin sheath, which result in axonal damage and subsequent neurological dysfunction. Current MS therapies are focused on immunosuppression as they are aimed at limiting the entry of immune cells into CNS, thereby preventing neuroinflammation. Although these therapies have been shown to be potent disease-modifying agents they fail to prevent or reverse disease progression. Astrocytes, among CNS resident cells, has been recently suggested as alternative highly promising therapeutic targets because of the key role played by these cells in driving disease progression in MS. Indeed, thanks to a close contact between astrocyte end-feet and blood vessels, the crosstalk of astrocytes with encephalitogenic T cells is centrally implicated in MS pathogenesis (Ponath et al., 2018). In this view, we have recently demonstrated that ShcC/Rai is as a novel astrocytic adaptor whose loss in mice accounts for a reduced demyelination and a milder experimental autoimmune encephalomyelitis (EAE), notwithstanding a higher frequency and pathogenicity of autoreactive T cells infiltrated within CNS highlighting the key role played by astrocytes in T cell modulation during EAE (Ulivieri et al., 2016). In the first part of this project we have investigated the molecular mechanism underlying the ability of ShcC/Rai-deficient astrocytes to generate an efficient T cell suppressive microenvironment in the pathological setting of EAE. At the beginning, we focused to study the ability of astrocytes to control the balance between extracellular ATP and adenosine in response to encephalitogenic T cells. We found that astrocytes respond to autoreactive T cells injury by enhancing the expression and activity of CD39 ectonucleotidase, responsible for the enzymatic hydrolysis of extracellular ATP into the immunosuppressive mediator adenosine, and that ShcC/Rai couples CD39 to its negative regulator RanBPM thereby limiting its activity. Accordingly, we measured high adenosine concentration in conditioned medium of Rai-/- astrocytes. As a result, T cells in the presence of microenvironment shaped by Rai knock-out astrocytes showed reduced proliferation and an up-regulation of inhibitory receptor CTLA-4, indicating that higher levels of adenosine are responsible to immunosuppression. We further characterized the impact of Rai on the protein composition of astrocytes-derived extracellular vesicles (EVs) in response to T cell-derived cytokines. Data obtained using a proteomic approach revealed that several proteins are differentially present in EVs released from Rai deficient or control astrocytes. Interestingly, enrichment analysis showed that these proteins participate in glutamate metabolism, in the control of protein folding and in the protection from oxidative stress suggesting that Rai controls pathways involved in brain homeostasis. Additionally, we examined functional polarization of astrocytes towards a neuroprotective (A2) or a neurotoxic (A1) phenotype. We show that Rai-/- astrocytes skew towards the A2 neuroprotective phenotype in response to encephalitogenic T cells both in vitro and in the EAE mouse model of MS by enhancing the activation of STAT3 transcription factor. In the second part of the project we have analyzed the role of ShcC/Rai in adenosine signaling in T cells. We identify a novel mechanism by which Rai in T cells dampens immunosuppressive effect of adenosine by inhibiting A2A receptor signalling interfering with the activation of transcription factor CREB. Characterization of molecular mechanism in a Jurkat T cell lines overexpressing Rai shows that Rai forms a complex with CREB upon A2AR triggering. In this respect, the phosphorylation/activation of CREB was significantly higher in Rai knock-out T cells compared with control following A2AR stimulation. Collectively, these data identify Rai/ShcC adaptor protein as critical regulator of astrocytes responses to T cells mediated neuroinflammation and highlight a new molecular mechanism to which Rai prevents establishment of an immunosuppressive program in T cells by limiting the transcriptional activity of nuclear factor CREB.

DE TOMMASO, D. (2020). Astrocytes contribute to neuroinflammation during EAE by shaping the CNS microenvironment via Rai signalling.

Astrocytes contribute to neuroinflammation during EAE by shaping the CNS microenvironment via Rai signalling

De Tommaso Domiziana
2020-01-01

Abstract

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). One of the pathological hallmarks of MS is the T cells-mediated destruction of myelin sheath, which result in axonal damage and subsequent neurological dysfunction. Current MS therapies are focused on immunosuppression as they are aimed at limiting the entry of immune cells into CNS, thereby preventing neuroinflammation. Although these therapies have been shown to be potent disease-modifying agents they fail to prevent or reverse disease progression. Astrocytes, among CNS resident cells, has been recently suggested as alternative highly promising therapeutic targets because of the key role played by these cells in driving disease progression in MS. Indeed, thanks to a close contact between astrocyte end-feet and blood vessels, the crosstalk of astrocytes with encephalitogenic T cells is centrally implicated in MS pathogenesis (Ponath et al., 2018). In this view, we have recently demonstrated that ShcC/Rai is as a novel astrocytic adaptor whose loss in mice accounts for a reduced demyelination and a milder experimental autoimmune encephalomyelitis (EAE), notwithstanding a higher frequency and pathogenicity of autoreactive T cells infiltrated within CNS highlighting the key role played by astrocytes in T cell modulation during EAE (Ulivieri et al., 2016). In the first part of this project we have investigated the molecular mechanism underlying the ability of ShcC/Rai-deficient astrocytes to generate an efficient T cell suppressive microenvironment in the pathological setting of EAE. At the beginning, we focused to study the ability of astrocytes to control the balance between extracellular ATP and adenosine in response to encephalitogenic T cells. We found that astrocytes respond to autoreactive T cells injury by enhancing the expression and activity of CD39 ectonucleotidase, responsible for the enzymatic hydrolysis of extracellular ATP into the immunosuppressive mediator adenosine, and that ShcC/Rai couples CD39 to its negative regulator RanBPM thereby limiting its activity. Accordingly, we measured high adenosine concentration in conditioned medium of Rai-/- astrocytes. As a result, T cells in the presence of microenvironment shaped by Rai knock-out astrocytes showed reduced proliferation and an up-regulation of inhibitory receptor CTLA-4, indicating that higher levels of adenosine are responsible to immunosuppression. We further characterized the impact of Rai on the protein composition of astrocytes-derived extracellular vesicles (EVs) in response to T cell-derived cytokines. Data obtained using a proteomic approach revealed that several proteins are differentially present in EVs released from Rai deficient or control astrocytes. Interestingly, enrichment analysis showed that these proteins participate in glutamate metabolism, in the control of protein folding and in the protection from oxidative stress suggesting that Rai controls pathways involved in brain homeostasis. Additionally, we examined functional polarization of astrocytes towards a neuroprotective (A2) or a neurotoxic (A1) phenotype. We show that Rai-/- astrocytes skew towards the A2 neuroprotective phenotype in response to encephalitogenic T cells both in vitro and in the EAE mouse model of MS by enhancing the activation of STAT3 transcription factor. In the second part of the project we have analyzed the role of ShcC/Rai in adenosine signaling in T cells. We identify a novel mechanism by which Rai in T cells dampens immunosuppressive effect of adenosine by inhibiting A2A receptor signalling interfering with the activation of transcription factor CREB. Characterization of molecular mechanism in a Jurkat T cell lines overexpressing Rai shows that Rai forms a complex with CREB upon A2AR triggering. In this respect, the phosphorylation/activation of CREB was significantly higher in Rai knock-out T cells compared with control following A2AR stimulation. Collectively, these data identify Rai/ShcC adaptor protein as critical regulator of astrocytes responses to T cells mediated neuroinflammation and highlight a new molecular mechanism to which Rai prevents establishment of an immunosuppressive program in T cells by limiting the transcriptional activity of nuclear factor CREB.
2020
DE TOMMASO, D. (2020). Astrocytes contribute to neuroinflammation during EAE by shaping the CNS microenvironment via Rai signalling.
DE TOMMASO, Domiziana
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/1105117
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo