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Congenic mesenchymal stem cell therapy reverses hyperglycemia in experimental type 1 diabetes.

Jurewicz M, Yang S, Augello A, Godwin JG, Moore RF, Azzi J, Fiorina P, Atkinson M, Sayegh MH, Abdi R - Diabetes (2010)

Bottom Line: NOR MSCs were evaluated with regard to their in vitro immunomodulatory function in the context of autoreactive T-cell proliferation and dendritic cell (DC) generation.NOR MSCs were shown to suppress diabetogenic T-cell proliferation via PD-L1 and to suppress generation of myeloid/inflammatory DCs predominantly through an IL-6-dependent mechanism.NOR MSC treatment of experimental type 1 diabetes resulted in long-term reversal of hyperglycemia, and therapy was shown to alter diabetogenic cytokine profile, to diminish T-cell effector frequency in the pancreatic lymph nodes, to alter antigen-presenting cell frequencies, and to augment the frequency of the plasmacytoid subset of DCs.

View Article: PubMed Central - PubMed

Affiliation: Transplantation Research Center, Children's Hospital and Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Objective: A number of clinical trials are underway to test whether mesenchymal stem cells (MSCs) are effective in treating various diseases, including type 1 diabetes. Although this cell therapy holds great promise, the optimal source of MSCs has yet to be determined with respect to major histocompatibility complex matching. Here, we examine this question by testing the ability of congenic MSCs, obtained from the NOR mouse strain, to reverse recent-onset type 1 diabetes in NOD mice, as well as determine the immunomodulatory effects of NOR MSCs in vivo.

Research design and methods: NOR MSCs were evaluated with regard to their in vitro immunomodulatory function in the context of autoreactive T-cell proliferation and dendritic cell (DC) generation. The in vivo effect of NOR MSC therapy on reversal of recent-onset hyperglycemia and on immunogenic cell subsets in NOD mice was also examined.

Results: NOR MSCs were shown to suppress diabetogenic T-cell proliferation via PD-L1 and to suppress generation of myeloid/inflammatory DCs predominantly through an IL-6-dependent mechanism. NOR MSC treatment of experimental type 1 diabetes resulted in long-term reversal of hyperglycemia, and therapy was shown to alter diabetogenic cytokine profile, to diminish T-cell effector frequency in the pancreatic lymph nodes, to alter antigen-presenting cell frequencies, and to augment the frequency of the plasmacytoid subset of DCs.

Conclusions: These studies demonstrate the inimitable benefit of congenic MSC therapy in reversing experimental type 1 diabetes. These data should benefit future clinical trials using MSCs as treatment for type 1 diabetes.

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Related in: MedlinePlus

MSC suppression of DC differentiation. A: Using an established model of DC generation from NOD bone marrow mononuclear cells, coculture with NOR MSCs was shown to markedly reduce the CD11c+CD11b+ population, so that the predominant cell phenotype was CD11clowCD11blow (CD11c+CD11b+ cells = 40.7 ± 2.6% and 22.1 ± 2.4% for control and NOR MSC-treated, respectively, n = 5, P = 0.0007), whereas treatment with anti-IL-6 in large part abrogated this effect (CD11c+CD11b+ cells = 30.9 ± 4.7%, not significant in comparison to control [−/−] and MSCs alone [+/−]). Analysis of expression of Ly-6c in the CD11b+ fraction demonstrated that coculture with NOR MSCs resulted in a decrease in both the CD11b+Ly-6chigh and CD11b+Ly-6cint populations (n = 4, P = 0.0053 and P = 0.02, respectively), which was fully abrogated by blockade of IL-6 (p = not significant). B: The population of lineage-negative cells was evaluated in DC culture as a function of progenitor frequency; coculture with MSCs increased the percentage of Lin− cells (n = 4, Lin− cells = 8.94 ± 0.87% and 13.73 ± 1.08% for control and NOR MSC-treated, respectively, P = 0.004), which was in part rescued by addition of anti-IL-6 (p = not significant). Similarly, Sca-1 expression within the lineage-negative population was markedly increased in the presence of MSCs (n = 4, Lin− Sca-1+ cells = 6.98 ± 1.27% and 30.53 ± 6% for control and NOR MSC-treated, respectively, P = 0.0085). Although treatment with anti-IL-6 resulted in loss of significance of this effect, IL-6 blockade appeared to be incompletely effective in reducing Sca-1 expression in response to MSCs. C: Cytokine analysis of cocultures of DCs and NOR MSCs demonstrated marked IL-6 production in the presence of MSCs (n = 4, P = 0.0074) as well as efficient blockade of IL-6 in response to treatment with anti-IL-6. Both Flt3L and M-CSF levels were substantially increased in response to MSC coculture (P = 0.03 and P = 0.04, respectively), and IL-6 blockade had no effect on these growth factors (P = 0.013 and P = 0.018, respectively, in comparison to DCs alone). Conversely, TNF-α production was reduced in the presence of MSCs (P = 0.0056), and anti-IL-6 treatment resulted in abrogation of this effect (p = not significant). D: Giemsa staining of DC culture cytospins demonstrated a lower nuclear/cytoplasmic ratio in response to coculture with MSCs, and IL-6 blockade appeared to in large part abrogate this effect. Experiments were performed between 3 and 5 times, and data are displayed with means and SEM. (A high-quality color representation of this figure is available in the online issue.)
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Figure 3: MSC suppression of DC differentiation. A: Using an established model of DC generation from NOD bone marrow mononuclear cells, coculture with NOR MSCs was shown to markedly reduce the CD11c+CD11b+ population, so that the predominant cell phenotype was CD11clowCD11blow (CD11c+CD11b+ cells = 40.7 ± 2.6% and 22.1 ± 2.4% for control and NOR MSC-treated, respectively, n = 5, P = 0.0007), whereas treatment with anti-IL-6 in large part abrogated this effect (CD11c+CD11b+ cells = 30.9 ± 4.7%, not significant in comparison to control [−/−] and MSCs alone [+/−]). Analysis of expression of Ly-6c in the CD11b+ fraction demonstrated that coculture with NOR MSCs resulted in a decrease in both the CD11b+Ly-6chigh and CD11b+Ly-6cint populations (n = 4, P = 0.0053 and P = 0.02, respectively), which was fully abrogated by blockade of IL-6 (p = not significant). B: The population of lineage-negative cells was evaluated in DC culture as a function of progenitor frequency; coculture with MSCs increased the percentage of Lin− cells (n = 4, Lin− cells = 8.94 ± 0.87% and 13.73 ± 1.08% for control and NOR MSC-treated, respectively, P = 0.004), which was in part rescued by addition of anti-IL-6 (p = not significant). Similarly, Sca-1 expression within the lineage-negative population was markedly increased in the presence of MSCs (n = 4, Lin− Sca-1+ cells = 6.98 ± 1.27% and 30.53 ± 6% for control and NOR MSC-treated, respectively, P = 0.0085). Although treatment with anti-IL-6 resulted in loss of significance of this effect, IL-6 blockade appeared to be incompletely effective in reducing Sca-1 expression in response to MSCs. C: Cytokine analysis of cocultures of DCs and NOR MSCs demonstrated marked IL-6 production in the presence of MSCs (n = 4, P = 0.0074) as well as efficient blockade of IL-6 in response to treatment with anti-IL-6. Both Flt3L and M-CSF levels were substantially increased in response to MSC coculture (P = 0.03 and P = 0.04, respectively), and IL-6 blockade had no effect on these growth factors (P = 0.013 and P = 0.018, respectively, in comparison to DCs alone). Conversely, TNF-α production was reduced in the presence of MSCs (P = 0.0056), and anti-IL-6 treatment resulted in abrogation of this effect (p = not significant). D: Giemsa staining of DC culture cytospins demonstrated a lower nuclear/cytoplasmic ratio in response to coculture with MSCs, and IL-6 blockade appeared to in large part abrogate this effect. Experiments were performed between 3 and 5 times, and data are displayed with means and SEM. (A high-quality color representation of this figure is available in the online issue.)

Mentions: Aberrant DC development and imbalance in antigen-presenting cell (APC) subsets have been reported to be responsible for the lack of tolerance mechanisms in NOD mice (22,23). To examine the effect of NOR MSCs on DC generation, we performed in vitro studies of NOR MSCs and NOD DCs using an established method of DC culture (16,24). Because of the substantial production of IL-6 in our NOR MSC cultures (Fig. 2A) as well as the fact that IL-6 has been demonstrated to both suppress and alter DC differentiation (2,7,25,26), we performed IL-6 blocking studies in conjunction with coculture of MSCs and DCs. The presence of MSCs strikingly reduced CD11c and CD11b expression of DCs, so that the predominant population induced by MSCs was CD11clowCD11blow (Fig. 3A, CD11c+CD11b+ cells = 40.7 ± 2.6% and 22.1 ± 2.4% for control and NOR MSC-treated, respectively, n = 5, P = 0.0007). Addition of anti-IL-6 somewhat abrogated the change in phenotype observed in DC coculture with NOR MSCs (Fig. 3A, n = 4, not significant in comparison to control [−/−] and in comparison to MSCs alone [−/+]). Of note, costimulatory molecule expression in the CD11c+ population was not found to be significantly different in the presence or absence of MSCs (data not shown). The CD11b+ population was also evaluated with respect to Ly-6c expression, as both CD11b+Ly-6chigh and CD11b+Ly-6cint cells have been demonstrated to be inflammatory monocytes recruited to sites of inflammation (22,27). Coculture with NOR MSCs resulted in downregulation of both the CD11b+Ly-6chigh and CD11b+Ly-6cint populations (Fig. 3A, n = 4, P = 0.0053 and P = 0.02, respectively), and this difference was again abrogated by blockade of IL-6. We then assessed the number of lineage-negative cells as a function of progenitor frequency or lack of differentiation, and NOR MSC treatment was shown to increase lineage-negative cells as well as increase the expression of Sca-1 within the lineage-negative population (Fig. 3B, n = 4, P = 0.004 and P = 0.0085, respectively). Treatment with anti-IL-6 was somewhat efficacious in abrogating the suppression of differentiation observed in response to MSC coculture, suggesting that other factors may be involved in the effect of MSCs on DC differentiation. We therefore examined the supernatants of DC and MSC cocultures at day 8 for cytokine production. As shown in Fig. 3C, coculture with MSCs significantly enhanced IL-6 levels (n = 4, P = 0.0074), and addition of IL-6 blocking antibody efficiently suppressed IL-6 production. Moreover, Flt3L and M-CSF production was increased in response to MSCs (n = 4, P = 0.03 and P = 0.04, respectively), and IL-6 blockade had no effect on increased levels of these cytokines (P = 0.013 and P = 0.018, respectively, in comparison to DCs alone, Fig. 3C). Conversely, production of TNF-α, a growth factor involved in the maturation of DCs as well as a cytokine secreted by mature DCs (16,28), was reduced in the presence of MSCs (Fig. 3C, n = 4, P = 0.0056), and blocking of IL-6 resulted in abrogation of this effect. To examine the morphology of DCs in response to MSCs, we performed Giemsa staining of day 8 DC cultures and found that the nuclear-to-cytoplasmic ratio appeared to decrease after coculture with MSCs, a feature commonly associated with earlier stages of differentiation (29), and IL-6 blockade appeared to partially reverse this effect (Fig. 3D). Examination of side scatter of DCs by flow cytometric analysis revealed that coculture with MSCs resulted in a dramatic reduction in the degree of granularity (data not shown), again demonstrating a lack of differentiation in response to MSCs (30). Addition of MSCs to plasmacytoid DC (pDC) cultures resulted in enhanced pDC frequency, and this effect was fully reversed by IL-6 blockade (supplementary Fig. 1 in the online appendix available at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0542/DC1), as IL-6 has been previously demonstrated to be important for pDC generation (31). Taken together, these data demonstrate a marked effect of NOR MSCs on DC phenotype, differentiation, and cytokine production, which is in large part mediated by IL-6.


Congenic mesenchymal stem cell therapy reverses hyperglycemia in experimental type 1 diabetes.

Jurewicz M, Yang S, Augello A, Godwin JG, Moore RF, Azzi J, Fiorina P, Atkinson M, Sayegh MH, Abdi R - Diabetes (2010)

MSC suppression of DC differentiation. A: Using an established model of DC generation from NOD bone marrow mononuclear cells, coculture with NOR MSCs was shown to markedly reduce the CD11c+CD11b+ population, so that the predominant cell phenotype was CD11clowCD11blow (CD11c+CD11b+ cells = 40.7 ± 2.6% and 22.1 ± 2.4% for control and NOR MSC-treated, respectively, n = 5, P = 0.0007), whereas treatment with anti-IL-6 in large part abrogated this effect (CD11c+CD11b+ cells = 30.9 ± 4.7%, not significant in comparison to control [−/−] and MSCs alone [+/−]). Analysis of expression of Ly-6c in the CD11b+ fraction demonstrated that coculture with NOR MSCs resulted in a decrease in both the CD11b+Ly-6chigh and CD11b+Ly-6cint populations (n = 4, P = 0.0053 and P = 0.02, respectively), which was fully abrogated by blockade of IL-6 (p = not significant). B: The population of lineage-negative cells was evaluated in DC culture as a function of progenitor frequency; coculture with MSCs increased the percentage of Lin− cells (n = 4, Lin− cells = 8.94 ± 0.87% and 13.73 ± 1.08% for control and NOR MSC-treated, respectively, P = 0.004), which was in part rescued by addition of anti-IL-6 (p = not significant). Similarly, Sca-1 expression within the lineage-negative population was markedly increased in the presence of MSCs (n = 4, Lin− Sca-1+ cells = 6.98 ± 1.27% and 30.53 ± 6% for control and NOR MSC-treated, respectively, P = 0.0085). Although treatment with anti-IL-6 resulted in loss of significance of this effect, IL-6 blockade appeared to be incompletely effective in reducing Sca-1 expression in response to MSCs. C: Cytokine analysis of cocultures of DCs and NOR MSCs demonstrated marked IL-6 production in the presence of MSCs (n = 4, P = 0.0074) as well as efficient blockade of IL-6 in response to treatment with anti-IL-6. Both Flt3L and M-CSF levels were substantially increased in response to MSC coculture (P = 0.03 and P = 0.04, respectively), and IL-6 blockade had no effect on these growth factors (P = 0.013 and P = 0.018, respectively, in comparison to DCs alone). Conversely, TNF-α production was reduced in the presence of MSCs (P = 0.0056), and anti-IL-6 treatment resulted in abrogation of this effect (p = not significant). D: Giemsa staining of DC culture cytospins demonstrated a lower nuclear/cytoplasmic ratio in response to coculture with MSCs, and IL-6 blockade appeared to in large part abrogate this effect. Experiments were performed between 3 and 5 times, and data are displayed with means and SEM. (A high-quality color representation of this figure is available in the online issue.)
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Figure 3: MSC suppression of DC differentiation. A: Using an established model of DC generation from NOD bone marrow mononuclear cells, coculture with NOR MSCs was shown to markedly reduce the CD11c+CD11b+ population, so that the predominant cell phenotype was CD11clowCD11blow (CD11c+CD11b+ cells = 40.7 ± 2.6% and 22.1 ± 2.4% for control and NOR MSC-treated, respectively, n = 5, P = 0.0007), whereas treatment with anti-IL-6 in large part abrogated this effect (CD11c+CD11b+ cells = 30.9 ± 4.7%, not significant in comparison to control [−/−] and MSCs alone [+/−]). Analysis of expression of Ly-6c in the CD11b+ fraction demonstrated that coculture with NOR MSCs resulted in a decrease in both the CD11b+Ly-6chigh and CD11b+Ly-6cint populations (n = 4, P = 0.0053 and P = 0.02, respectively), which was fully abrogated by blockade of IL-6 (p = not significant). B: The population of lineage-negative cells was evaluated in DC culture as a function of progenitor frequency; coculture with MSCs increased the percentage of Lin− cells (n = 4, Lin− cells = 8.94 ± 0.87% and 13.73 ± 1.08% for control and NOR MSC-treated, respectively, P = 0.004), which was in part rescued by addition of anti-IL-6 (p = not significant). Similarly, Sca-1 expression within the lineage-negative population was markedly increased in the presence of MSCs (n = 4, Lin− Sca-1+ cells = 6.98 ± 1.27% and 30.53 ± 6% for control and NOR MSC-treated, respectively, P = 0.0085). Although treatment with anti-IL-6 resulted in loss of significance of this effect, IL-6 blockade appeared to be incompletely effective in reducing Sca-1 expression in response to MSCs. C: Cytokine analysis of cocultures of DCs and NOR MSCs demonstrated marked IL-6 production in the presence of MSCs (n = 4, P = 0.0074) as well as efficient blockade of IL-6 in response to treatment with anti-IL-6. Both Flt3L and M-CSF levels were substantially increased in response to MSC coculture (P = 0.03 and P = 0.04, respectively), and IL-6 blockade had no effect on these growth factors (P = 0.013 and P = 0.018, respectively, in comparison to DCs alone). Conversely, TNF-α production was reduced in the presence of MSCs (P = 0.0056), and anti-IL-6 treatment resulted in abrogation of this effect (p = not significant). D: Giemsa staining of DC culture cytospins demonstrated a lower nuclear/cytoplasmic ratio in response to coculture with MSCs, and IL-6 blockade appeared to in large part abrogate this effect. Experiments were performed between 3 and 5 times, and data are displayed with means and SEM. (A high-quality color representation of this figure is available in the online issue.)
Mentions: Aberrant DC development and imbalance in antigen-presenting cell (APC) subsets have been reported to be responsible for the lack of tolerance mechanisms in NOD mice (22,23). To examine the effect of NOR MSCs on DC generation, we performed in vitro studies of NOR MSCs and NOD DCs using an established method of DC culture (16,24). Because of the substantial production of IL-6 in our NOR MSC cultures (Fig. 2A) as well as the fact that IL-6 has been demonstrated to both suppress and alter DC differentiation (2,7,25,26), we performed IL-6 blocking studies in conjunction with coculture of MSCs and DCs. The presence of MSCs strikingly reduced CD11c and CD11b expression of DCs, so that the predominant population induced by MSCs was CD11clowCD11blow (Fig. 3A, CD11c+CD11b+ cells = 40.7 ± 2.6% and 22.1 ± 2.4% for control and NOR MSC-treated, respectively, n = 5, P = 0.0007). Addition of anti-IL-6 somewhat abrogated the change in phenotype observed in DC coculture with NOR MSCs (Fig. 3A, n = 4, not significant in comparison to control [−/−] and in comparison to MSCs alone [−/+]). Of note, costimulatory molecule expression in the CD11c+ population was not found to be significantly different in the presence or absence of MSCs (data not shown). The CD11b+ population was also evaluated with respect to Ly-6c expression, as both CD11b+Ly-6chigh and CD11b+Ly-6cint cells have been demonstrated to be inflammatory monocytes recruited to sites of inflammation (22,27). Coculture with NOR MSCs resulted in downregulation of both the CD11b+Ly-6chigh and CD11b+Ly-6cint populations (Fig. 3A, n = 4, P = 0.0053 and P = 0.02, respectively), and this difference was again abrogated by blockade of IL-6. We then assessed the number of lineage-negative cells as a function of progenitor frequency or lack of differentiation, and NOR MSC treatment was shown to increase lineage-negative cells as well as increase the expression of Sca-1 within the lineage-negative population (Fig. 3B, n = 4, P = 0.004 and P = 0.0085, respectively). Treatment with anti-IL-6 was somewhat efficacious in abrogating the suppression of differentiation observed in response to MSC coculture, suggesting that other factors may be involved in the effect of MSCs on DC differentiation. We therefore examined the supernatants of DC and MSC cocultures at day 8 for cytokine production. As shown in Fig. 3C, coculture with MSCs significantly enhanced IL-6 levels (n = 4, P = 0.0074), and addition of IL-6 blocking antibody efficiently suppressed IL-6 production. Moreover, Flt3L and M-CSF production was increased in response to MSCs (n = 4, P = 0.03 and P = 0.04, respectively), and IL-6 blockade had no effect on increased levels of these cytokines (P = 0.013 and P = 0.018, respectively, in comparison to DCs alone, Fig. 3C). Conversely, production of TNF-α, a growth factor involved in the maturation of DCs as well as a cytokine secreted by mature DCs (16,28), was reduced in the presence of MSCs (Fig. 3C, n = 4, P = 0.0056), and blocking of IL-6 resulted in abrogation of this effect. To examine the morphology of DCs in response to MSCs, we performed Giemsa staining of day 8 DC cultures and found that the nuclear-to-cytoplasmic ratio appeared to decrease after coculture with MSCs, a feature commonly associated with earlier stages of differentiation (29), and IL-6 blockade appeared to partially reverse this effect (Fig. 3D). Examination of side scatter of DCs by flow cytometric analysis revealed that coculture with MSCs resulted in a dramatic reduction in the degree of granularity (data not shown), again demonstrating a lack of differentiation in response to MSCs (30). Addition of MSCs to plasmacytoid DC (pDC) cultures resulted in enhanced pDC frequency, and this effect was fully reversed by IL-6 blockade (supplementary Fig. 1 in the online appendix available at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0542/DC1), as IL-6 has been previously demonstrated to be important for pDC generation (31). Taken together, these data demonstrate a marked effect of NOR MSCs on DC phenotype, differentiation, and cytokine production, which is in large part mediated by IL-6.

Bottom Line: NOR MSCs were evaluated with regard to their in vitro immunomodulatory function in the context of autoreactive T-cell proliferation and dendritic cell (DC) generation.NOR MSCs were shown to suppress diabetogenic T-cell proliferation via PD-L1 and to suppress generation of myeloid/inflammatory DCs predominantly through an IL-6-dependent mechanism.NOR MSC treatment of experimental type 1 diabetes resulted in long-term reversal of hyperglycemia, and therapy was shown to alter diabetogenic cytokine profile, to diminish T-cell effector frequency in the pancreatic lymph nodes, to alter antigen-presenting cell frequencies, and to augment the frequency of the plasmacytoid subset of DCs.

View Article: PubMed Central - PubMed

Affiliation: Transplantation Research Center, Children's Hospital and Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Objective: A number of clinical trials are underway to test whether mesenchymal stem cells (MSCs) are effective in treating various diseases, including type 1 diabetes. Although this cell therapy holds great promise, the optimal source of MSCs has yet to be determined with respect to major histocompatibility complex matching. Here, we examine this question by testing the ability of congenic MSCs, obtained from the NOR mouse strain, to reverse recent-onset type 1 diabetes in NOD mice, as well as determine the immunomodulatory effects of NOR MSCs in vivo.

Research design and methods: NOR MSCs were evaluated with regard to their in vitro immunomodulatory function in the context of autoreactive T-cell proliferation and dendritic cell (DC) generation. The in vivo effect of NOR MSC therapy on reversal of recent-onset hyperglycemia and on immunogenic cell subsets in NOD mice was also examined.

Results: NOR MSCs were shown to suppress diabetogenic T-cell proliferation via PD-L1 and to suppress generation of myeloid/inflammatory DCs predominantly through an IL-6-dependent mechanism. NOR MSC treatment of experimental type 1 diabetes resulted in long-term reversal of hyperglycemia, and therapy was shown to alter diabetogenic cytokine profile, to diminish T-cell effector frequency in the pancreatic lymph nodes, to alter antigen-presenting cell frequencies, and to augment the frequency of the plasmacytoid subset of DCs.

Conclusions: These studies demonstrate the inimitable benefit of congenic MSC therapy in reversing experimental type 1 diabetes. These data should benefit future clinical trials using MSCs as treatment for type 1 diabetes.

Show MeSH
Related in: MedlinePlus