Final Report Summary - IMMUNO BALANCE MS (Study of mechanisms underlying the balance between inflammatory and regulatory immune response in multiple sclerosis)
The main objective of the project was to characterise the involvement of antigen presenting cells (APC) in the thelper (Th17)/tregulatory (Treg) balance during multiple sclerosis (MS) disease. The study was performed to elucidate early mechanisms that modulate the establishment of the equilibrium between the inflammatory and immune-suppressive response in MS.
We divided the work in three parts:
1. ex vivo analysis of APC response to toll like receptor (TLR) agonists in MS patients through multi-parameter cytofluorimetric analysis.
2. ex vivo analysis of APC from MS patients through molecular approaches.
3. analysis of T cell activation by APC in MS.
In the first part of the project we studied whether purified APCs from MS patients show an alteration in their response to TLR agonist. TLR signals induce innate immune cell differentiation and influence the immunological outcome of their interactions with T cells.
In order to select the specific APC population to use in our study, we studied the frequency and the response to TLR agonists of each APC subset in the blood from MS patients and healthy donors (HDs).
The use of specific APC markers and multiparameter cytofluorimetric analysis enable us to quantify the cell number of each APC subset (myeloid and plasmacytoid dendritic cells, classical and non classical monocytes). We found that the frequency of myeloid dendritic cells (mDC) was significantly increased, while the number of plasmacytoid dendritic cells (pDC) was significantly decreased in MS patients compared to HDs. Similar number of classical and non classical monocytes was found in MS patients and HDs.
Then, we analysed the response of mDC and pDC, whose frequency was modulated in MS, to TLR7, TLR8, TLR2 and TLR4 agonists (R848, R837 and LPS) in terms of cytokine production (IL-12/23p40, TNF-a and IFN-a). Among those TLR agonists, we selected R848 as the strongest signal able to simultaneously induce high production of cytokines by all DC subsets. This approach did not reveal differences in the percentage of cytokine-producing cells by DCs from MS and HDs. We confirm these results by a different approach: purified pDC and mDC stimulated with R848 released the same amount of IL-1b, IL-6, TNF-a and IFN-a in MS and HDs.
Then, in the second part of the project, we use molecular approaches to investigate whether pDC and mDC from MS patients could have a differential transcriptional profile.
We purified pDC and mDC from the blood of MS patients and HDs. We studied the complete transcriptional profile, covering the whole human transcriptional genome. So far, our study focussed on seven patients and eight HDs. We performed a global gene expression profiling in order to compare pDC and mDC from MS patients and HDs.
The global analysis revealed that differences between pDC and mDC are more significant than differences between HDs and MS patients. We explored all the genes differentially expressed in MS patients versus HDs: 60% and 40% of the genes were upregulated and downregulated, respectively, in mDC from MS compared to HDs; 57% and 43% of the genes were upregulated and downregulated, respectively, in pDC from MS compared to HDs.
Initially we focussed on genes encoding co-stimulatory molecules on DC surface that are known to activate T cells: OX40-L, CD40, CD86, ICOS-L, CD70, 4IBB-L. We found that pDC from MS patients express higher levels of OX40-L, CD86, ICOS-L and 4IBB-L compared to pDC from HDs; mDC from MS patients express higher levels of ICOS-L and 4IBB-L compared to mDC from HDs. These results suggest a potential differential induction of T cell immune response by DCs in MS patients compared to HDs.
Thus, in the third part of the project we explored the functionality of DC-Tcell interaction in MS patients and HDs.
The results show the two DC subsets activate different Th responses: activated pDCs induce IL-10 production (related to Tregulatory function), while activated mDC drive Th17 cell polarisation (related to inflammation). Interestingly, although the tendency is also observed in HDs, DCs from MS patients seem to be more potent polarisers towards Th17 polarisation and less potent polarisers towards IL-10 producing cells. Moreover, we found that in MS patients and not in HDs, pDC cocultured with naïve T cells significantly induce the secretion of IL-9, a cytokine produced by a new Th profile called Th9. The immune function of Th9 cells and its role in MS is still controversial. Although most part of the studies demonstrate an inflammatory role in the mouse model of MS, it has been reported a role of IL-9 either in amplify the inflammatory response than in amplify the regulatory response. Interestingly, in a mouse model it has been demonstrated that OX40-L, a costimulatory molecule that we found highly expressed in pDC from MS patients contributes to IL-9 polarisation. Since OX40-L is also involved in the down-regulation of IL-10 production, we hypothesised that OX40-OX40-L interaction between pDC and T cells in MS patients could be responsible of the simultaneous increase of IL-9 and decrease of IL-10 observed in MS patients compared to HDs. This hypothesis is still under investigation in our lab. We expect that neutralisation of OX40-OX40L in pDC-T cell interaction may modulate the balance between IL-10 and IL-9 favouring the induction of IL-10 and the regulatory response, which should suppress the inflammation. The achievement of the expected results could have important socio-economic impact in the MS field, because the mechanisms identified as regulators of the balance between inflammation and immune regulation could be therapeutically modulated in MS patients towards a protective immune response.
We divided the work in three parts:
1. ex vivo analysis of APC response to toll like receptor (TLR) agonists in MS patients through multi-parameter cytofluorimetric analysis.
2. ex vivo analysis of APC from MS patients through molecular approaches.
3. analysis of T cell activation by APC in MS.
In the first part of the project we studied whether purified APCs from MS patients show an alteration in their response to TLR agonist. TLR signals induce innate immune cell differentiation and influence the immunological outcome of their interactions with T cells.
In order to select the specific APC population to use in our study, we studied the frequency and the response to TLR agonists of each APC subset in the blood from MS patients and healthy donors (HDs).
The use of specific APC markers and multiparameter cytofluorimetric analysis enable us to quantify the cell number of each APC subset (myeloid and plasmacytoid dendritic cells, classical and non classical monocytes). We found that the frequency of myeloid dendritic cells (mDC) was significantly increased, while the number of plasmacytoid dendritic cells (pDC) was significantly decreased in MS patients compared to HDs. Similar number of classical and non classical monocytes was found in MS patients and HDs.
Then, we analysed the response of mDC and pDC, whose frequency was modulated in MS, to TLR7, TLR8, TLR2 and TLR4 agonists (R848, R837 and LPS) in terms of cytokine production (IL-12/23p40, TNF-a and IFN-a). Among those TLR agonists, we selected R848 as the strongest signal able to simultaneously induce high production of cytokines by all DC subsets. This approach did not reveal differences in the percentage of cytokine-producing cells by DCs from MS and HDs. We confirm these results by a different approach: purified pDC and mDC stimulated with R848 released the same amount of IL-1b, IL-6, TNF-a and IFN-a in MS and HDs.
Then, in the second part of the project, we use molecular approaches to investigate whether pDC and mDC from MS patients could have a differential transcriptional profile.
We purified pDC and mDC from the blood of MS patients and HDs. We studied the complete transcriptional profile, covering the whole human transcriptional genome. So far, our study focussed on seven patients and eight HDs. We performed a global gene expression profiling in order to compare pDC and mDC from MS patients and HDs.
The global analysis revealed that differences between pDC and mDC are more significant than differences between HDs and MS patients. We explored all the genes differentially expressed in MS patients versus HDs: 60% and 40% of the genes were upregulated and downregulated, respectively, in mDC from MS compared to HDs; 57% and 43% of the genes were upregulated and downregulated, respectively, in pDC from MS compared to HDs.
Initially we focussed on genes encoding co-stimulatory molecules on DC surface that are known to activate T cells: OX40-L, CD40, CD86, ICOS-L, CD70, 4IBB-L. We found that pDC from MS patients express higher levels of OX40-L, CD86, ICOS-L and 4IBB-L compared to pDC from HDs; mDC from MS patients express higher levels of ICOS-L and 4IBB-L compared to mDC from HDs. These results suggest a potential differential induction of T cell immune response by DCs in MS patients compared to HDs.
Thus, in the third part of the project we explored the functionality of DC-Tcell interaction in MS patients and HDs.
The results show the two DC subsets activate different Th responses: activated pDCs induce IL-10 production (related to Tregulatory function), while activated mDC drive Th17 cell polarisation (related to inflammation). Interestingly, although the tendency is also observed in HDs, DCs from MS patients seem to be more potent polarisers towards Th17 polarisation and less potent polarisers towards IL-10 producing cells. Moreover, we found that in MS patients and not in HDs, pDC cocultured with naïve T cells significantly induce the secretion of IL-9, a cytokine produced by a new Th profile called Th9. The immune function of Th9 cells and its role in MS is still controversial. Although most part of the studies demonstrate an inflammatory role in the mouse model of MS, it has been reported a role of IL-9 either in amplify the inflammatory response than in amplify the regulatory response. Interestingly, in a mouse model it has been demonstrated that OX40-L, a costimulatory molecule that we found highly expressed in pDC from MS patients contributes to IL-9 polarisation. Since OX40-L is also involved in the down-regulation of IL-10 production, we hypothesised that OX40-OX40-L interaction between pDC and T cells in MS patients could be responsible of the simultaneous increase of IL-9 and decrease of IL-10 observed in MS patients compared to HDs. This hypothesis is still under investigation in our lab. We expect that neutralisation of OX40-OX40L in pDC-T cell interaction may modulate the balance between IL-10 and IL-9 favouring the induction of IL-10 and the regulatory response, which should suppress the inflammation. The achievement of the expected results could have important socio-economic impact in the MS field, because the mechanisms identified as regulators of the balance between inflammation and immune regulation could be therapeutically modulated in MS patients towards a protective immune response.