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Jagged-1 is required for the expansion of CD4+ CD25+ FoxP3+ regulatory T cells and tolerogenic dendritic cells by murine mesenchymal stromal cells.

Cahill EF, Tobin LM, Carty F, Mahon BP, English K - Stem Cell Res Ther (2015)

Bottom Line: MSC, but not Jagged-1 knock down MSC, reduced pathology in a mouse model of allergic airway inflammation.Protection mediated by MSC was associated with enhanced Treg in the lung and significantly increased production of interleukin (IL)-10 in splenocytes re-stimulated with allergen.The current study suggests that MSC-mediated immune modulation involves the education and expansion of regulatory immune cells in a Jagged-1 dependent manner and provides the first report of the importance of Jagged-1 signalling in MSC protection against inflammation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Institute of Immunology, Maynooth University, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland. emerfc@gmail.com.

ABSTRACT

Introduction: Mesenchymal stromal cells (MSC) have well defined immunomodulatory properties including the suppression of lymphocyte proliferation and inhibition of dendritic cell (DC) maturation involving both cell contact and soluble factors. These properties have made MSC attractive candidates for cellular therapy. However, the mechanism underlying these characteristics remains unclear. This study sought to investigate the mechanisms by which MSC induce a regulatory environment.

Method: Allogeneic bone marrow mesenchymal stromal cells were cultured with T cells or dendritic cells in the presence or absence of gamma secretase inhibitor to block Notch receptor signalling. T cells and dendritic cells were examined by flow cytometry for changes in phenotype marker expression. Stable knock down MSC were generated to examine the influence of Jagged 1 signalling by MSC. Both wildtype and knockdown MSC were subsequently used in vivo in an animal model of allergic airway inflammation.

Results: The Notch ligand Jagged-1 was demonstrated to be involved in MSC expansion of regulatory T cells (Treg). Additionally, MSC-induced a functional semi-mature DC phenotype, which further required Notch signalling for the expansion of Treg. MSC, but not Jagged-1 knock down MSC, reduced pathology in a mouse model of allergic airway inflammation. Protection mediated by MSC was associated with enhanced Treg in the lung and significantly increased production of interleukin (IL)-10 in splenocytes re-stimulated with allergen. Significantly less Treg and IL-10 was observed in mice treated with Jagged-1 knock down MSC.

Conclusions: The current study suggests that MSC-mediated immune modulation involves the education and expansion of regulatory immune cells in a Jagged-1 dependent manner and provides the first report of the importance of Jagged-1 signalling in MSC protection against inflammation in vivo.

No MeSH data available.


Related in: MedlinePlus

MSC educated tolerogenic DC induce regulatory T cells. C57BL/6 DC were cultured in the absence (immature DC) or presence (mature DC) of LPS without or with MSC (tolerogenic DC), and harvested from adherent MSC via gentle aspiration after 48 hours. These DC cells were washed and subsequently cultured with sorted CD4+CD25−FoxP3− T cells at a ratio of 1:4 DC to T cell, for 72 hours. T cells were gated on CD3 to discriminate them from the DC. Expression of CD25 and FoxP3-GFP was examined by flow cytometry following co-culture with immature DC (A), mature DC (B) or tolerogenic DC (C). MHCIIlow CD86low tolerogenic DC significantly increased the induction of Tregs compared to immature DC or mature DC (**, P <0.01) (D). Data in figures A, B and C are representative flow cytometry plots while figure D shows statistics from three studies. DC, dendritic cells; LPS, lipopolysaccharide; MHC, major histocompatibility complex; MSC, mesenchymal stromal cells.
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Fig3: MSC educated tolerogenic DC induce regulatory T cells. C57BL/6 DC were cultured in the absence (immature DC) or presence (mature DC) of LPS without or with MSC (tolerogenic DC), and harvested from adherent MSC via gentle aspiration after 48 hours. These DC cells were washed and subsequently cultured with sorted CD4+CD25−FoxP3− T cells at a ratio of 1:4 DC to T cell, for 72 hours. T cells were gated on CD3 to discriminate them from the DC. Expression of CD25 and FoxP3-GFP was examined by flow cytometry following co-culture with immature DC (A), mature DC (B) or tolerogenic DC (C). MHCIIlow CD86low tolerogenic DC significantly increased the induction of Tregs compared to immature DC or mature DC (**, P <0.01) (D). Data in figures A, B and C are representative flow cytometry plots while figure D shows statistics from three studies. DC, dendritic cells; LPS, lipopolysaccharide; MHC, major histocompatibility complex; MSC, mesenchymal stromal cells.

Mentions: Induction of Treg could account for the inhibition observed in the proliferation assay. Therefore, the influence of MSC on DC driven induction of Treg was studied. C57BL/6 DC were co-cultured with allogeneic MSC in the presence or absence of LPS. TLR stimulation of MSC has been shown to alter both the expression of immune modulatory factors and the immune suppressive function of MSC [40-42]. However, these findings are conflicting, with some studies showing that TLR activation reduces, or others that it enhances, MSC immune suppressive effects. In addition, these studies examining LPS stimulation of MSC focus on MSC effects on T cell proliferation. After 48 hours, DC were recovered by gentle aspiration from adherent MSC and subsequently cultured with sorted FoxP3-eGFP− CD25− T cells. DC matured with LPS (mDC) induced two fold more FoxP3+ Tregs than immature DC (iDC). In line with this, studies have shown that LPS-matured DC secrete anti-inflammatory cytokines (IL-10) and promote the induction of Treg [43-45]. Interestingly, DC that had previously been co-cultured with MSC and expressed MHC ClassIIlow and CD86low cells markers (tDC) induced greater numbers of FoxP3+ Treg (approximately seven fold more than iDC). These data provided strong evidence to suggest that MSC educated DC promote a regulatory environment (Figure 3).Figure 3


Jagged-1 is required for the expansion of CD4+ CD25+ FoxP3+ regulatory T cells and tolerogenic dendritic cells by murine mesenchymal stromal cells.

Cahill EF, Tobin LM, Carty F, Mahon BP, English K - Stem Cell Res Ther (2015)

MSC educated tolerogenic DC induce regulatory T cells. C57BL/6 DC were cultured in the absence (immature DC) or presence (mature DC) of LPS without or with MSC (tolerogenic DC), and harvested from adherent MSC via gentle aspiration after 48 hours. These DC cells were washed and subsequently cultured with sorted CD4+CD25−FoxP3− T cells at a ratio of 1:4 DC to T cell, for 72 hours. T cells were gated on CD3 to discriminate them from the DC. Expression of CD25 and FoxP3-GFP was examined by flow cytometry following co-culture with immature DC (A), mature DC (B) or tolerogenic DC (C). MHCIIlow CD86low tolerogenic DC significantly increased the induction of Tregs compared to immature DC or mature DC (**, P <0.01) (D). Data in figures A, B and C are representative flow cytometry plots while figure D shows statistics from three studies. DC, dendritic cells; LPS, lipopolysaccharide; MHC, major histocompatibility complex; MSC, mesenchymal stromal cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4414370&req=5

Fig3: MSC educated tolerogenic DC induce regulatory T cells. C57BL/6 DC were cultured in the absence (immature DC) or presence (mature DC) of LPS without or with MSC (tolerogenic DC), and harvested from adherent MSC via gentle aspiration after 48 hours. These DC cells were washed and subsequently cultured with sorted CD4+CD25−FoxP3− T cells at a ratio of 1:4 DC to T cell, for 72 hours. T cells were gated on CD3 to discriminate them from the DC. Expression of CD25 and FoxP3-GFP was examined by flow cytometry following co-culture with immature DC (A), mature DC (B) or tolerogenic DC (C). MHCIIlow CD86low tolerogenic DC significantly increased the induction of Tregs compared to immature DC or mature DC (**, P <0.01) (D). Data in figures A, B and C are representative flow cytometry plots while figure D shows statistics from three studies. DC, dendritic cells; LPS, lipopolysaccharide; MHC, major histocompatibility complex; MSC, mesenchymal stromal cells.
Mentions: Induction of Treg could account for the inhibition observed in the proliferation assay. Therefore, the influence of MSC on DC driven induction of Treg was studied. C57BL/6 DC were co-cultured with allogeneic MSC in the presence or absence of LPS. TLR stimulation of MSC has been shown to alter both the expression of immune modulatory factors and the immune suppressive function of MSC [40-42]. However, these findings are conflicting, with some studies showing that TLR activation reduces, or others that it enhances, MSC immune suppressive effects. In addition, these studies examining LPS stimulation of MSC focus on MSC effects on T cell proliferation. After 48 hours, DC were recovered by gentle aspiration from adherent MSC and subsequently cultured with sorted FoxP3-eGFP− CD25− T cells. DC matured with LPS (mDC) induced two fold more FoxP3+ Tregs than immature DC (iDC). In line with this, studies have shown that LPS-matured DC secrete anti-inflammatory cytokines (IL-10) and promote the induction of Treg [43-45]. Interestingly, DC that had previously been co-cultured with MSC and expressed MHC ClassIIlow and CD86low cells markers (tDC) induced greater numbers of FoxP3+ Treg (approximately seven fold more than iDC). These data provided strong evidence to suggest that MSC educated DC promote a regulatory environment (Figure 3).Figure 3

Bottom Line: MSC, but not Jagged-1 knock down MSC, reduced pathology in a mouse model of allergic airway inflammation.Protection mediated by MSC was associated with enhanced Treg in the lung and significantly increased production of interleukin (IL)-10 in splenocytes re-stimulated with allergen.The current study suggests that MSC-mediated immune modulation involves the education and expansion of regulatory immune cells in a Jagged-1 dependent manner and provides the first report of the importance of Jagged-1 signalling in MSC protection against inflammation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Institute of Immunology, Maynooth University, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland. emerfc@gmail.com.

ABSTRACT

Introduction: Mesenchymal stromal cells (MSC) have well defined immunomodulatory properties including the suppression of lymphocyte proliferation and inhibition of dendritic cell (DC) maturation involving both cell contact and soluble factors. These properties have made MSC attractive candidates for cellular therapy. However, the mechanism underlying these characteristics remains unclear. This study sought to investigate the mechanisms by which MSC induce a regulatory environment.

Method: Allogeneic bone marrow mesenchymal stromal cells were cultured with T cells or dendritic cells in the presence or absence of gamma secretase inhibitor to block Notch receptor signalling. T cells and dendritic cells were examined by flow cytometry for changes in phenotype marker expression. Stable knock down MSC were generated to examine the influence of Jagged 1 signalling by MSC. Both wildtype and knockdown MSC were subsequently used in vivo in an animal model of allergic airway inflammation.

Results: The Notch ligand Jagged-1 was demonstrated to be involved in MSC expansion of regulatory T cells (Treg). Additionally, MSC-induced a functional semi-mature DC phenotype, which further required Notch signalling for the expansion of Treg. MSC, but not Jagged-1 knock down MSC, reduced pathology in a mouse model of allergic airway inflammation. Protection mediated by MSC was associated with enhanced Treg in the lung and significantly increased production of interleukin (IL)-10 in splenocytes re-stimulated with allergen. Significantly less Treg and IL-10 was observed in mice treated with Jagged-1 knock down MSC.

Conclusions: The current study suggests that MSC-mediated immune modulation involves the education and expansion of regulatory immune cells in a Jagged-1 dependent manner and provides the first report of the importance of Jagged-1 signalling in MSC protection against inflammation in vivo.

No MeSH data available.


Related in: MedlinePlus