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A p38 MAPK-mediated alteration of COX-2/PGE2 regulates immunomodulatory properties in human mesenchymal stem cell aging.

Yu KR, Lee JY, Kim HS, Hong IS, Choi SW, Seo Y, Kang I, Kim JJ, Lee BC, Lee S, Kurtz A, Seo KW, Kang KS - PLoS ONE (2014)

Bottom Line: In this study, we evaluated the effect of replicative senescence on the immunomodulatory ability of hMSCs in vitro and in vivo.Among the anti-inflammatory cytokines, the production of prostaglandin E2 (PGE2) and the expression of its primary enzyme, cyclooxygenase-2 (COX-2), were profoundly increased by pre-stimulation with interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and this response was significantly decreased with consecutive passages.In conclusion, our data indicate that the immunomodulatory ability of hMSCs gradually declines with consecutive passages via a p38-mediated alteration of COX-2 and PGE2 levels.

View Article: PubMed Central - PubMed

Affiliation: Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, South Korea; Research Institute for Veterinary Medicine, College of Veterinary Medicine, Seoul National University, Seoul, Korea.

ABSTRACT
Because human mesenchymal stem cells (hMSC) have profound immunomodulatory effects, many attempts have been made to use hMSCs in preclinical and clinical trials. For hMSCs to be used in therapy, a large population of hMSCs must be generated by in vitro expansion. However, the immunomodulatory changes following the in vitro expansion of hMSCs have not been elucidated. In this study, we evaluated the effect of replicative senescence on the immunomodulatory ability of hMSCs in vitro and in vivo. Late-passage hMSCs showed impaired suppressive effect on mitogen-induced mononuclear cell proliferation. Strikingly, late-passage hMSCs had a significantly compromised protective effect against mouse experimental colitis, which was confirmed by gross and histologic examination. Among the anti-inflammatory cytokines, the production of prostaglandin E2 (PGE2) and the expression of its primary enzyme, cyclooxygenase-2 (COX-2), were profoundly increased by pre-stimulation with interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and this response was significantly decreased with consecutive passages. We demonstrated that the impaired phosphorylation activity of p38 MAP kinase (p38 MAPK) in late-passage hMSCs led to a compromised immunomodulatory ability through the regulation of COX-2. In conclusion, our data indicate that the immunomodulatory ability of hMSCs gradually declines with consecutive passages via a p38-mediated alteration of COX-2 and PGE2 levels.

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Declined immune-inhibitory effect of late-passage hMSCs is regulated by PGE2 and COX-2.(A–E) Early- and late-passage hMSCs were treated with or without IFN-γ and TNF-α for 24 hours. (A, D) The PGE2 concentration was measured from the culture supernatant by an ELISA after IFN-γ and TNF-α treatment for 24 hours. n = 3. (**P<0.01) (B, C, E) COX-2 expression was investigated using Western blot and immunocytochemistry after exposure to IFN-γ and TNF-α for 24 hours. GAPDH was used for normalization. n = 3. (**P<0.01) (F) Concanavalin A stimulated hMNCs were cultured alone or co-cultured with early-, late- passage and celecoxib-treated hMSCs at a 1∶10 ratio (MSC:MNC). The proliferation of hMNCs was measured by a BrdU assay after 72 hours. n = 3. (**P<0.01).
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pone-0102426-g004: Declined immune-inhibitory effect of late-passage hMSCs is regulated by PGE2 and COX-2.(A–E) Early- and late-passage hMSCs were treated with or without IFN-γ and TNF-α for 24 hours. (A, D) The PGE2 concentration was measured from the culture supernatant by an ELISA after IFN-γ and TNF-α treatment for 24 hours. n = 3. (**P<0.01) (B, C, E) COX-2 expression was investigated using Western blot and immunocytochemistry after exposure to IFN-γ and TNF-α for 24 hours. GAPDH was used for normalization. n = 3. (**P<0.01) (F) Concanavalin A stimulated hMNCs were cultured alone or co-cultured with early-, late- passage and celecoxib-treated hMSCs at a 1∶10 ratio (MSC:MNC). The proliferation of hMNCs was measured by a BrdU assay after 72 hours. n = 3. (**P<0.01).

Mentions: NO, PGE2, IDO and TGF-β1 have previously been identified as relevant mediators in the regulation of the immunomodulatory properties of MSCs [25]. To determine whether compromised immunosuppressive functions in hMSC aging are affected by these mediators, we measured the expression levels of each molecule from the culture media of early- and late-passage hMSCs. Because previous reports have shown evidence that the immunosuppressive functions of MSCs are elicited by proinflammatory cytokines [6], early- and late-passage hMSCs were primed with IFN-γ and TNF-α for 24 hours. There was no significant difference in the NO and TGF-β1 levels in the presence or absence of proinflammatory cytokines in both early- and late-passage hMSCs (Figure S2A and B). However, the PGE2 concentration was dramatically increased in the presence of proinflammatory cytokines. Interestingly, the PGE2 concentration in early-passage hMSCs was higher than in late-passage hMSCs (Figure 4A). We next investigated the expression levels of immunomodulatory molecules after proinflammatory cytokine activation using Western blot analysis. Consistent with the results from an ELISA assay, only the expression of COX-2, a key enzyme in production of PGE2, was significantly decreased, while the expression of p16INK4A, a senescence marker, was increased in late-passage hMSCs (Figure 4B and S2C). Furthermore, COX-2 expression was significantly down-regulated in late-passage hMSCs compared to early-passage hMSCs, whereas Fibronectin expression was not changed, which was confirmed by immunocytochemistry (Figure 4C). To identify whether the expression of PGE2 and COX-2 decreased following hMSC passages, we investigated the secretion level of PGE2 and the expression of the COX-2 protein in consecutive passages of hMSCs. The PGE2 concentration and COX-2 expression decreased gradually with the increasing hMSC passage number (Figure 4D and E). To determine the effect of COX-2/PGE2 on hMSC immunosuppressive functions, we observed the inhibitory effect of hMSCs on hMNC proliferation in the presence or absence of celecoxib, a selective COX-2 inhibitor. Celecoxib-treated, early-passage hMSCs exhibited significantly compromised immunosuppressive abilities (Figure 4F). Taken together, these results indicate that the immunosuppressive properties of hMSCs gradually decrease upon consecutive passages via down-regulation of COX-2/PGE2 expression.


A p38 MAPK-mediated alteration of COX-2/PGE2 regulates immunomodulatory properties in human mesenchymal stem cell aging.

Yu KR, Lee JY, Kim HS, Hong IS, Choi SW, Seo Y, Kang I, Kim JJ, Lee BC, Lee S, Kurtz A, Seo KW, Kang KS - PLoS ONE (2014)

Declined immune-inhibitory effect of late-passage hMSCs is regulated by PGE2 and COX-2.(A–E) Early- and late-passage hMSCs were treated with or without IFN-γ and TNF-α for 24 hours. (A, D) The PGE2 concentration was measured from the culture supernatant by an ELISA after IFN-γ and TNF-α treatment for 24 hours. n = 3. (**P<0.01) (B, C, E) COX-2 expression was investigated using Western blot and immunocytochemistry after exposure to IFN-γ and TNF-α for 24 hours. GAPDH was used for normalization. n = 3. (**P<0.01) (F) Concanavalin A stimulated hMNCs were cultured alone or co-cultured with early-, late- passage and celecoxib-treated hMSCs at a 1∶10 ratio (MSC:MNC). The proliferation of hMNCs was measured by a BrdU assay after 72 hours. n = 3. (**P<0.01).
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pone-0102426-g004: Declined immune-inhibitory effect of late-passage hMSCs is regulated by PGE2 and COX-2.(A–E) Early- and late-passage hMSCs were treated with or without IFN-γ and TNF-α for 24 hours. (A, D) The PGE2 concentration was measured from the culture supernatant by an ELISA after IFN-γ and TNF-α treatment for 24 hours. n = 3. (**P<0.01) (B, C, E) COX-2 expression was investigated using Western blot and immunocytochemistry after exposure to IFN-γ and TNF-α for 24 hours. GAPDH was used for normalization. n = 3. (**P<0.01) (F) Concanavalin A stimulated hMNCs were cultured alone or co-cultured with early-, late- passage and celecoxib-treated hMSCs at a 1∶10 ratio (MSC:MNC). The proliferation of hMNCs was measured by a BrdU assay after 72 hours. n = 3. (**P<0.01).
Mentions: NO, PGE2, IDO and TGF-β1 have previously been identified as relevant mediators in the regulation of the immunomodulatory properties of MSCs [25]. To determine whether compromised immunosuppressive functions in hMSC aging are affected by these mediators, we measured the expression levels of each molecule from the culture media of early- and late-passage hMSCs. Because previous reports have shown evidence that the immunosuppressive functions of MSCs are elicited by proinflammatory cytokines [6], early- and late-passage hMSCs were primed with IFN-γ and TNF-α for 24 hours. There was no significant difference in the NO and TGF-β1 levels in the presence or absence of proinflammatory cytokines in both early- and late-passage hMSCs (Figure S2A and B). However, the PGE2 concentration was dramatically increased in the presence of proinflammatory cytokines. Interestingly, the PGE2 concentration in early-passage hMSCs was higher than in late-passage hMSCs (Figure 4A). We next investigated the expression levels of immunomodulatory molecules after proinflammatory cytokine activation using Western blot analysis. Consistent with the results from an ELISA assay, only the expression of COX-2, a key enzyme in production of PGE2, was significantly decreased, while the expression of p16INK4A, a senescence marker, was increased in late-passage hMSCs (Figure 4B and S2C). Furthermore, COX-2 expression was significantly down-regulated in late-passage hMSCs compared to early-passage hMSCs, whereas Fibronectin expression was not changed, which was confirmed by immunocytochemistry (Figure 4C). To identify whether the expression of PGE2 and COX-2 decreased following hMSC passages, we investigated the secretion level of PGE2 and the expression of the COX-2 protein in consecutive passages of hMSCs. The PGE2 concentration and COX-2 expression decreased gradually with the increasing hMSC passage number (Figure 4D and E). To determine the effect of COX-2/PGE2 on hMSC immunosuppressive functions, we observed the inhibitory effect of hMSCs on hMNC proliferation in the presence or absence of celecoxib, a selective COX-2 inhibitor. Celecoxib-treated, early-passage hMSCs exhibited significantly compromised immunosuppressive abilities (Figure 4F). Taken together, these results indicate that the immunosuppressive properties of hMSCs gradually decrease upon consecutive passages via down-regulation of COX-2/PGE2 expression.

Bottom Line: In this study, we evaluated the effect of replicative senescence on the immunomodulatory ability of hMSCs in vitro and in vivo.Among the anti-inflammatory cytokines, the production of prostaglandin E2 (PGE2) and the expression of its primary enzyme, cyclooxygenase-2 (COX-2), were profoundly increased by pre-stimulation with interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and this response was significantly decreased with consecutive passages.In conclusion, our data indicate that the immunomodulatory ability of hMSCs gradually declines with consecutive passages via a p38-mediated alteration of COX-2 and PGE2 levels.

View Article: PubMed Central - PubMed

Affiliation: Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, South Korea; Research Institute for Veterinary Medicine, College of Veterinary Medicine, Seoul National University, Seoul, Korea.

ABSTRACT
Because human mesenchymal stem cells (hMSC) have profound immunomodulatory effects, many attempts have been made to use hMSCs in preclinical and clinical trials. For hMSCs to be used in therapy, a large population of hMSCs must be generated by in vitro expansion. However, the immunomodulatory changes following the in vitro expansion of hMSCs have not been elucidated. In this study, we evaluated the effect of replicative senescence on the immunomodulatory ability of hMSCs in vitro and in vivo. Late-passage hMSCs showed impaired suppressive effect on mitogen-induced mononuclear cell proliferation. Strikingly, late-passage hMSCs had a significantly compromised protective effect against mouse experimental colitis, which was confirmed by gross and histologic examination. Among the anti-inflammatory cytokines, the production of prostaglandin E2 (PGE2) and the expression of its primary enzyme, cyclooxygenase-2 (COX-2), were profoundly increased by pre-stimulation with interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and this response was significantly decreased with consecutive passages. We demonstrated that the impaired phosphorylation activity of p38 MAP kinase (p38 MAPK) in late-passage hMSCs led to a compromised immunomodulatory ability through the regulation of COX-2. In conclusion, our data indicate that the immunomodulatory ability of hMSCs gradually declines with consecutive passages via a p38-mediated alteration of COX-2 and PGE2 levels.

Show MeSH
Related in: MedlinePlus