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Role of gamma-secretase in human umbilical-cord derived mesenchymal stem cell mediated suppression of NK cell cytotoxicity.

Chatterjee D, Marquardt N, Tufa DM, Beauclair G, Low HZ, Hatlapatka T, Hass R, Kasper C, von Kaisenberg C, Schmidt RE, Jacobs R - Cell Commun. Signal (2014)

Bottom Line: The main soluble immunosuppressant was identified as prostaglandin (PG)-E2.IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity.Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Mesenchymal stem cells (MSCs) are increasingly considered to be used as biological immunosuppressants in hematopoietic stem cell transplantation (HSCT). In the early reconstitution phase following HSCT, natural killer (NK) cells represent the major lymphocyte population in peripheral blood and display graft-vs-leukemia (GvL) effects. The functional interactions between NK cells and MSCs have the potential to influence the leukemia relapse rate after HSCT. Until date, MSC-NK cell interaction studies are largely focussed on bone marrow derived (BM)-MSCs. Umbilical cord derived (UC)-MSCs might be an alternative source of therapeutic MSCs. Thus, we studied the interaction of UC-MSCs with unstimulated allogeneic NK cells.

Results: UC-MSCs could potently suppress NK cell cytotoxicity in overnight cultures via soluble factors. The main soluble immunosuppressant was identified as prostaglandin (PG)-E2. Maximal PGE2 release involved IL-1β priming of MSCs after close contact between the NK cells and UC-MSCs. Interestingly, blocking gamma-secretase activation alleviated the immunosuppression by controlling PGE2 production. IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity.

Conclusion: Although the role of PGE2 in NK cell-MSC has been reported, the requirement of cell-cell contact for PGE2 induced immunosuppression remained unexplained. Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.

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Related in: MedlinePlus

Effect of gamma-secretase on IL-1 signalling and PGE2 content. A: UC-MSCs were cultured in presence of different doses of IL-1β stimulation with or without DAPT (gamma-secretase inhibitor). UC-MSCs were detached using accutase and stained to analyse intracellular COX-2 expression (n = 5). B: Gamma-secretase activity in UC-MSCs was silenced using siRNA targeting a catalytic subunit of gamma-secretase (PSEN-1). Scrambled siRNA (SCR) was also introduced into UC-MSCs as control. After 24 hours, PSEN-1 or SCR siRNA treated MSCs were stimulated with IL-1β overnight. UC-MSCs were detached using accutase and stained to compare intracellular COX-2 expression with or without IL-1β stimulation (n = 3). C: UC-MSCs were stimulated with IL-1β for 40 minutes, with or without 2.5 hours pre-treatment with DAPT. MSCs were detached using accutase, immediately fixed, permeabilised and stained to analyse intracellular pJNK levels (n = 4). D: NK cells were cultured with or without MSCs or co-cultured in presence of 6 μM DAPT, 10 μM DAPT or DMSO. CD107a degranulation assay was performed with K562 target cells. The bars represent the effect of gamma-secretase inhibition on degranulative capacity of NK cells (n = 5). E: NK cells were cultured in NK cell conditioned media (NK cm) or in NK-MSC conditioned media (NK-MSC cm) or NK-MSC conditioned media where they were co-cultured in presence of 10 μM DAPT or DMSO (NK-MSC-10 μM DAPT cm or NK-MSC-DMSO cm respectively). Chromium release assay was performed with K562 target cells (n = 3). F: PGE2 concentration in MSC cm, NK-MSC cm or NK-MSC-10 μM DAPT cm as determined by competitive ELISA (n = 3).
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Fig4: Effect of gamma-secretase on IL-1 signalling and PGE2 content. A: UC-MSCs were cultured in presence of different doses of IL-1β stimulation with or without DAPT (gamma-secretase inhibitor). UC-MSCs were detached using accutase and stained to analyse intracellular COX-2 expression (n = 5). B: Gamma-secretase activity in UC-MSCs was silenced using siRNA targeting a catalytic subunit of gamma-secretase (PSEN-1). Scrambled siRNA (SCR) was also introduced into UC-MSCs as control. After 24 hours, PSEN-1 or SCR siRNA treated MSCs were stimulated with IL-1β overnight. UC-MSCs were detached using accutase and stained to compare intracellular COX-2 expression with or without IL-1β stimulation (n = 3). C: UC-MSCs were stimulated with IL-1β for 40 minutes, with or without 2.5 hours pre-treatment with DAPT. MSCs were detached using accutase, immediately fixed, permeabilised and stained to analyse intracellular pJNK levels (n = 4). D: NK cells were cultured with or without MSCs or co-cultured in presence of 6 μM DAPT, 10 μM DAPT or DMSO. CD107a degranulation assay was performed with K562 target cells. The bars represent the effect of gamma-secretase inhibition on degranulative capacity of NK cells (n = 5). E: NK cells were cultured in NK cell conditioned media (NK cm) or in NK-MSC conditioned media (NK-MSC cm) or NK-MSC conditioned media where they were co-cultured in presence of 10 μM DAPT or DMSO (NK-MSC-10 μM DAPT cm or NK-MSC-DMSO cm respectively). Chromium release assay was performed with K562 target cells (n = 3). F: PGE2 concentration in MSC cm, NK-MSC cm or NK-MSC-10 μM DAPT cm as determined by competitive ELISA (n = 3).

Mentions: IL-1β is known to act via IL-1 receptor present on target cells. IL-1 receptor (IL-1R) activity has been recently described to be gamma-secretase proteolysis dependent in HEK293T cells as well as mouse embryonic fibroblasts (MEFS) [17]. We examined if IL-1R signaling in human UC-MSCs depended on gamma-secretase activity. We cultured UC-MSCs with varying concentrations of IL-1β overnight in presence or absence of gamma-secretase inhibitor, DAPT. IL-1β was very efficient in upregulating COX-2 levels. However, the presence of DAPT significantly reduced COX-2 upregulation (Figure 4A). Pharmacological inhibitors can have a wide range of targets. To eliminate this possibility, we performed small interfering RNA (siRNA) mediated silencing of gamma-secretase activity in MSCs. We chose to knock-down Presenilin (PSEN)-1 because it is one of the catalytic subunits of gamma-secretase complex [20,21]. siRNA targeting PSEN-1 or control scrambled (SCR) siRNAs were introduced into the MSCs by electroporation. Knock-down of PSEN-1 mRNA was analysed after 24 hours by real-time PCR. PSEN-1 siRNA caused a 75% decrease in PSEN-1 mRNA compared to SCR siRNA (Additional file 8: Figure S8). In parallel with the assessment of mRNA knock-down, siRNA-treated MSCs were cultured for 16 hours in presence or absence of IL-1β. Similar to DAPT-mediated inhibition, PSEN-1 silencing significantly reduced COX-2 upregulation in response to IL-1β (Figure 4B). Introduction of PSEN-1 siRNA did not affect the basal COX-2 expression in MSCs. This clearly indicates the involvement of gamma-secretase in IL-1R signalling. Next, we tested if this effect of gamma-secretase blocking stemmed from impairment of IL-1R downstream signalling. We pre-treated MSCs with DAPT for 2.5 hours followed by IL-1β stimulation for 40 minutes. The DAPT pre-treated MSCs showed significant impairment in their ability to phosphorylate c-Jun N-terminal kinases (JNK) as mirrored in reduced phosphorylation of JNK following stimulation by IL-1β (Figure 4C). We further investigated if gamma-secretase influenced IL-1R signalling in our culture-setting. Pharmacological inhibition of gamma-secretase activity in NK-MSC co-cultures resulted in a restoration of NK cell degranulative capacity (Figure 4D). To establish that gamma-secretase blocking indeed affected the release of soluble suppressants, we performed the following experiment. Cell-free supernatants were collected from the NK-MSC co-cultures conducted in presence of DAPT or DMSO. They have been referred to as MSC-NK-DAPT conditioned medium (NK-MSC-DAPT cm) and MSC-NK-DMSO conditioned medium (NK-MSC-DMSO cm), respectively. NK cm and NK-MSC cm were also collected as controls. NK cells were cultured in these conditioned media followed by chromium release assay. NK-MSC cm caused a significant decline in lytic potential of NK cells. The cell-free co-culture supernatant generated in presence of gamma-secretase blocker, DAPT, had non-significant suppressive capacity. This indicated that NK-MSC-DAPT cm contained much lower quantity of the suppressant (Figure 4E). To directly test if the release of PGE2 was gamma-secretase dependent, we performed ELISA to analyse the concentration of PGE2 in MSC cm, NK-MSC cm, and NK-MSC-10 μM DAPT cm. We found that the amount of PGE2 increased significantly upon NK-MSC co-culture, but there was no increase in PGE2 if gamma-secretase activity was blocked (Figure 4F).Figure 4


Role of gamma-secretase in human umbilical-cord derived mesenchymal stem cell mediated suppression of NK cell cytotoxicity.

Chatterjee D, Marquardt N, Tufa DM, Beauclair G, Low HZ, Hatlapatka T, Hass R, Kasper C, von Kaisenberg C, Schmidt RE, Jacobs R - Cell Commun. Signal (2014)

Effect of gamma-secretase on IL-1 signalling and PGE2 content. A: UC-MSCs were cultured in presence of different doses of IL-1β stimulation with or without DAPT (gamma-secretase inhibitor). UC-MSCs were detached using accutase and stained to analyse intracellular COX-2 expression (n = 5). B: Gamma-secretase activity in UC-MSCs was silenced using siRNA targeting a catalytic subunit of gamma-secretase (PSEN-1). Scrambled siRNA (SCR) was also introduced into UC-MSCs as control. After 24 hours, PSEN-1 or SCR siRNA treated MSCs were stimulated with IL-1β overnight. UC-MSCs were detached using accutase and stained to compare intracellular COX-2 expression with or without IL-1β stimulation (n = 3). C: UC-MSCs were stimulated with IL-1β for 40 minutes, with or without 2.5 hours pre-treatment with DAPT. MSCs were detached using accutase, immediately fixed, permeabilised and stained to analyse intracellular pJNK levels (n = 4). D: NK cells were cultured with or without MSCs or co-cultured in presence of 6 μM DAPT, 10 μM DAPT or DMSO. CD107a degranulation assay was performed with K562 target cells. The bars represent the effect of gamma-secretase inhibition on degranulative capacity of NK cells (n = 5). E: NK cells were cultured in NK cell conditioned media (NK cm) or in NK-MSC conditioned media (NK-MSC cm) or NK-MSC conditioned media where they were co-cultured in presence of 10 μM DAPT or DMSO (NK-MSC-10 μM DAPT cm or NK-MSC-DMSO cm respectively). Chromium release assay was performed with K562 target cells (n = 3). F: PGE2 concentration in MSC cm, NK-MSC cm or NK-MSC-10 μM DAPT cm as determined by competitive ELISA (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Fig4: Effect of gamma-secretase on IL-1 signalling and PGE2 content. A: UC-MSCs were cultured in presence of different doses of IL-1β stimulation with or without DAPT (gamma-secretase inhibitor). UC-MSCs were detached using accutase and stained to analyse intracellular COX-2 expression (n = 5). B: Gamma-secretase activity in UC-MSCs was silenced using siRNA targeting a catalytic subunit of gamma-secretase (PSEN-1). Scrambled siRNA (SCR) was also introduced into UC-MSCs as control. After 24 hours, PSEN-1 or SCR siRNA treated MSCs were stimulated with IL-1β overnight. UC-MSCs were detached using accutase and stained to compare intracellular COX-2 expression with or without IL-1β stimulation (n = 3). C: UC-MSCs were stimulated with IL-1β for 40 minutes, with or without 2.5 hours pre-treatment with DAPT. MSCs were detached using accutase, immediately fixed, permeabilised and stained to analyse intracellular pJNK levels (n = 4). D: NK cells were cultured with or without MSCs or co-cultured in presence of 6 μM DAPT, 10 μM DAPT or DMSO. CD107a degranulation assay was performed with K562 target cells. The bars represent the effect of gamma-secretase inhibition on degranulative capacity of NK cells (n = 5). E: NK cells were cultured in NK cell conditioned media (NK cm) or in NK-MSC conditioned media (NK-MSC cm) or NK-MSC conditioned media where they were co-cultured in presence of 10 μM DAPT or DMSO (NK-MSC-10 μM DAPT cm or NK-MSC-DMSO cm respectively). Chromium release assay was performed with K562 target cells (n = 3). F: PGE2 concentration in MSC cm, NK-MSC cm or NK-MSC-10 μM DAPT cm as determined by competitive ELISA (n = 3).
Mentions: IL-1β is known to act via IL-1 receptor present on target cells. IL-1 receptor (IL-1R) activity has been recently described to be gamma-secretase proteolysis dependent in HEK293T cells as well as mouse embryonic fibroblasts (MEFS) [17]. We examined if IL-1R signaling in human UC-MSCs depended on gamma-secretase activity. We cultured UC-MSCs with varying concentrations of IL-1β overnight in presence or absence of gamma-secretase inhibitor, DAPT. IL-1β was very efficient in upregulating COX-2 levels. However, the presence of DAPT significantly reduced COX-2 upregulation (Figure 4A). Pharmacological inhibitors can have a wide range of targets. To eliminate this possibility, we performed small interfering RNA (siRNA) mediated silencing of gamma-secretase activity in MSCs. We chose to knock-down Presenilin (PSEN)-1 because it is one of the catalytic subunits of gamma-secretase complex [20,21]. siRNA targeting PSEN-1 or control scrambled (SCR) siRNAs were introduced into the MSCs by electroporation. Knock-down of PSEN-1 mRNA was analysed after 24 hours by real-time PCR. PSEN-1 siRNA caused a 75% decrease in PSEN-1 mRNA compared to SCR siRNA (Additional file 8: Figure S8). In parallel with the assessment of mRNA knock-down, siRNA-treated MSCs were cultured for 16 hours in presence or absence of IL-1β. Similar to DAPT-mediated inhibition, PSEN-1 silencing significantly reduced COX-2 upregulation in response to IL-1β (Figure 4B). Introduction of PSEN-1 siRNA did not affect the basal COX-2 expression in MSCs. This clearly indicates the involvement of gamma-secretase in IL-1R signalling. Next, we tested if this effect of gamma-secretase blocking stemmed from impairment of IL-1R downstream signalling. We pre-treated MSCs with DAPT for 2.5 hours followed by IL-1β stimulation for 40 minutes. The DAPT pre-treated MSCs showed significant impairment in their ability to phosphorylate c-Jun N-terminal kinases (JNK) as mirrored in reduced phosphorylation of JNK following stimulation by IL-1β (Figure 4C). We further investigated if gamma-secretase influenced IL-1R signalling in our culture-setting. Pharmacological inhibition of gamma-secretase activity in NK-MSC co-cultures resulted in a restoration of NK cell degranulative capacity (Figure 4D). To establish that gamma-secretase blocking indeed affected the release of soluble suppressants, we performed the following experiment. Cell-free supernatants were collected from the NK-MSC co-cultures conducted in presence of DAPT or DMSO. They have been referred to as MSC-NK-DAPT conditioned medium (NK-MSC-DAPT cm) and MSC-NK-DMSO conditioned medium (NK-MSC-DMSO cm), respectively. NK cm and NK-MSC cm were also collected as controls. NK cells were cultured in these conditioned media followed by chromium release assay. NK-MSC cm caused a significant decline in lytic potential of NK cells. The cell-free co-culture supernatant generated in presence of gamma-secretase blocker, DAPT, had non-significant suppressive capacity. This indicated that NK-MSC-DAPT cm contained much lower quantity of the suppressant (Figure 4E). To directly test if the release of PGE2 was gamma-secretase dependent, we performed ELISA to analyse the concentration of PGE2 in MSC cm, NK-MSC cm, and NK-MSC-10 μM DAPT cm. We found that the amount of PGE2 increased significantly upon NK-MSC co-culture, but there was no increase in PGE2 if gamma-secretase activity was blocked (Figure 4F).Figure 4

Bottom Line: The main soluble immunosuppressant was identified as prostaglandin (PG)-E2.IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity.Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Mesenchymal stem cells (MSCs) are increasingly considered to be used as biological immunosuppressants in hematopoietic stem cell transplantation (HSCT). In the early reconstitution phase following HSCT, natural killer (NK) cells represent the major lymphocyte population in peripheral blood and display graft-vs-leukemia (GvL) effects. The functional interactions between NK cells and MSCs have the potential to influence the leukemia relapse rate after HSCT. Until date, MSC-NK cell interaction studies are largely focussed on bone marrow derived (BM)-MSCs. Umbilical cord derived (UC)-MSCs might be an alternative source of therapeutic MSCs. Thus, we studied the interaction of UC-MSCs with unstimulated allogeneic NK cells.

Results: UC-MSCs could potently suppress NK cell cytotoxicity in overnight cultures via soluble factors. The main soluble immunosuppressant was identified as prostaglandin (PG)-E2. Maximal PGE2 release involved IL-1β priming of MSCs after close contact between the NK cells and UC-MSCs. Interestingly, blocking gamma-secretase activation alleviated the immunosuppression by controlling PGE2 production. IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity.

Conclusion: Although the role of PGE2 in NK cell-MSC has been reported, the requirement of cell-cell contact for PGE2 induced immunosuppression remained unexplained. Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.

Show MeSH
Related in: MedlinePlus