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Phylogenetic distinction of iNOS and IDO function in mesenchymal stem cell-mediated immunosuppression in mammalian species.

Su J, Chen X, Huang Y, Li W, Li J, Cao K, Cao G, Zhang L, Li F, Roberts AI, Kang H, Yu P, Ren G, Ji W, Wang Y, Shi Y - Cell Death Differ. (2013)

Bottom Line: On the basis of the inducibility of IDO and iNOS by inflammatory cytokines in MSCs, the tested mammalian species fall into two distinct groups: IDO utilizers and iNOS utilizers.Interestingly, based on the limited number of species tested, the iNOS-utilizing species all belong to the phylogenetic clade, Glires.Although the evolutionary significance of this divergence is not known, we believe that this study provides critical guidance for choosing appropriate animal models for preclinical studies of MSCs.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai Jiao Tong University School of Medicine, 225 South Chongqing Road, Shanghai 200025, China.

ABSTRACT
Mammalian mesenchymal stem cells (MSCs) have been shown to be strongly immunosuppressive in both animal disease models and human clinical trials. We have reported that the key molecule mediating immunosuppression by MSCs is species dependent: indoleamine 2,3-dioxygenase (IDO) in human and inducible nitric oxide synthase (iNOS) in mouse. In the present study, we isolated MSCs from several mammalian species, each of a different genus, and investigated the involvement of IDO and iNOS during MSC-mediated immunosuppression. The characterization of MSCs from different species was by adherence to tissue culture plastic, morphology, specific marker expression, and differentiation potential. On the basis of the inducibility of IDO and iNOS by inflammatory cytokines in MSCs, the tested mammalian species fall into two distinct groups: IDO utilizers and iNOS utilizers. MSCs from monkey, pig, and human employ IDO to suppress immune responses, whereas MSCs from mouse, rat, rabbit, and hamster utilize iNOS. Interestingly, based on the limited number of species tested, the iNOS-utilizing species all belong to the phylogenetic clade, Glires. Although the evolutionary significance of this divergence is not known, we believe that this study provides critical guidance for choosing appropriate animal models for preclinical studies of MSCs.

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BM-MSCs isolated from different species have different immunosuppression mechanism. (a) The immunosuppressive abilities of MSCs derived from different species. CFSE-stained PBMCs or Spls isolated from rat, rabbit, hamster, or pig were stimulated with conconavalin A (Con A) (1 μg/ml), or from human, monkey, or mouse were stimulated with anti-CD3 (1 μg/ml) in the presence of corresponding BM-MSCs (2.0 × 104 cells per well in 96-well plate) at a ratio of 1 : 10 (MSC: T cell) for 96 h. iNOS inhibitor L-NMMA (1 mM) or IDO inhibitor 1-MT (0.5 mM) were added into the coculture system. CFSE fluorescence intensity reduction of PBMCs or Spls was detected by flow cytometry. Data are representative of three independent experiments. (b) NO production in the MSC and activated Spls/PBMCs cocultures of different species was determined by Griess reagent
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fig5: BM-MSCs isolated from different species have different immunosuppression mechanism. (a) The immunosuppressive abilities of MSCs derived from different species. CFSE-stained PBMCs or Spls isolated from rat, rabbit, hamster, or pig were stimulated with conconavalin A (Con A) (1 μg/ml), or from human, monkey, or mouse were stimulated with anti-CD3 (1 μg/ml) in the presence of corresponding BM-MSCs (2.0 × 104 cells per well in 96-well plate) at a ratio of 1 : 10 (MSC: T cell) for 96 h. iNOS inhibitor L-NMMA (1 mM) or IDO inhibitor 1-MT (0.5 mM) were added into the coculture system. CFSE fluorescence intensity reduction of PBMCs or Spls was detected by flow cytometry. Data are representative of three independent experiments. (b) NO production in the MSC and activated Spls/PBMCs cocultures of different species was determined by Griess reagent

Mentions: In our previous work, we have demonstrated the mechanisms underlying MSC-mediated immunosuppression. They are not innately immunosuppressive, but become so after exposure to pro-inflammatory cytokines. With sufficient cytokine stimulation, MSCs can produce large amounts of chemokines, which recruit immune cells into their proximity.11 The recruited immune cells are then subject to the effects of locally high concentrations of immunosuppressive factors whose production is also induced by pro-inflammatory cytokines. Remarkably, the key molecule mediating this immunosuppression differs according to species; in mouse, it is NO produced by iNOS, whereas in human it is tryptophan depletion resulting from IDO upregulation.12 Yet, most researches to date have employed mouse models to verify the therapeutic effect of MSCs. As the mechanisms of MSC-mediated immunosuppression differ between mouse and human, we propose that other in vivo animal models, more suitable than mouse, should be used to mimic the immunosuppressive function of MSCs in human. To this end, we used specific inhibitors of iNOS and IDO to test whether MSCs from other species also employ either of these two molecules for immunosuppression. Accordingly, NG-monomethyl-L-arginine acetate salt (L-NMMA) or 1-methyl-tryptophan (1-MT), specific inhibitors of iNOS and IDO, respectively, were added to the MSC-Spl (or PBMC) coculture system. Interestingly, we found that immunosuppression by MSCs from rat, hamster, and rabbit was reversible by L-NMMA, but not 1-MT, indicating that they share a similar mechanism with mouse MSCs. Conversely, monkey and pig MSCs were dependent on IDO, making them similar to human MSCs (Figure 5a). The similar results were obtained when we measured the NO production in the supernatant of the MSC-Spl (or PBMC) cocultures. High level of NO concentration was detected in mouse, rat, hamster, and rabbit coculture system, whereas little NO was detected in human, monkey, and pig cocultures (Figure 5b). Therefore, our findings strongly suggest that animal models using monkey or pig would be more appropriate for preclinical studies of the effect of MSC-based therapy in human patients.


Phylogenetic distinction of iNOS and IDO function in mesenchymal stem cell-mediated immunosuppression in mammalian species.

Su J, Chen X, Huang Y, Li W, Li J, Cao K, Cao G, Zhang L, Li F, Roberts AI, Kang H, Yu P, Ren G, Ji W, Wang Y, Shi Y - Cell Death Differ. (2013)

BM-MSCs isolated from different species have different immunosuppression mechanism. (a) The immunosuppressive abilities of MSCs derived from different species. CFSE-stained PBMCs or Spls isolated from rat, rabbit, hamster, or pig were stimulated with conconavalin A (Con A) (1 μg/ml), or from human, monkey, or mouse were stimulated with anti-CD3 (1 μg/ml) in the presence of corresponding BM-MSCs (2.0 × 104 cells per well in 96-well plate) at a ratio of 1 : 10 (MSC: T cell) for 96 h. iNOS inhibitor L-NMMA (1 mM) or IDO inhibitor 1-MT (0.5 mM) were added into the coculture system. CFSE fluorescence intensity reduction of PBMCs or Spls was detected by flow cytometry. Data are representative of three independent experiments. (b) NO production in the MSC and activated Spls/PBMCs cocultures of different species was determined by Griess reagent
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921585&req=5

fig5: BM-MSCs isolated from different species have different immunosuppression mechanism. (a) The immunosuppressive abilities of MSCs derived from different species. CFSE-stained PBMCs or Spls isolated from rat, rabbit, hamster, or pig were stimulated with conconavalin A (Con A) (1 μg/ml), or from human, monkey, or mouse were stimulated with anti-CD3 (1 μg/ml) in the presence of corresponding BM-MSCs (2.0 × 104 cells per well in 96-well plate) at a ratio of 1 : 10 (MSC: T cell) for 96 h. iNOS inhibitor L-NMMA (1 mM) or IDO inhibitor 1-MT (0.5 mM) were added into the coculture system. CFSE fluorescence intensity reduction of PBMCs or Spls was detected by flow cytometry. Data are representative of three independent experiments. (b) NO production in the MSC and activated Spls/PBMCs cocultures of different species was determined by Griess reagent
Mentions: In our previous work, we have demonstrated the mechanisms underlying MSC-mediated immunosuppression. They are not innately immunosuppressive, but become so after exposure to pro-inflammatory cytokines. With sufficient cytokine stimulation, MSCs can produce large amounts of chemokines, which recruit immune cells into their proximity.11 The recruited immune cells are then subject to the effects of locally high concentrations of immunosuppressive factors whose production is also induced by pro-inflammatory cytokines. Remarkably, the key molecule mediating this immunosuppression differs according to species; in mouse, it is NO produced by iNOS, whereas in human it is tryptophan depletion resulting from IDO upregulation.12 Yet, most researches to date have employed mouse models to verify the therapeutic effect of MSCs. As the mechanisms of MSC-mediated immunosuppression differ between mouse and human, we propose that other in vivo animal models, more suitable than mouse, should be used to mimic the immunosuppressive function of MSCs in human. To this end, we used specific inhibitors of iNOS and IDO to test whether MSCs from other species also employ either of these two molecules for immunosuppression. Accordingly, NG-monomethyl-L-arginine acetate salt (L-NMMA) or 1-methyl-tryptophan (1-MT), specific inhibitors of iNOS and IDO, respectively, were added to the MSC-Spl (or PBMC) coculture system. Interestingly, we found that immunosuppression by MSCs from rat, hamster, and rabbit was reversible by L-NMMA, but not 1-MT, indicating that they share a similar mechanism with mouse MSCs. Conversely, monkey and pig MSCs were dependent on IDO, making them similar to human MSCs (Figure 5a). The similar results were obtained when we measured the NO production in the supernatant of the MSC-Spl (or PBMC) cocultures. High level of NO concentration was detected in mouse, rat, hamster, and rabbit coculture system, whereas little NO was detected in human, monkey, and pig cocultures (Figure 5b). Therefore, our findings strongly suggest that animal models using monkey or pig would be more appropriate for preclinical studies of the effect of MSC-based therapy in human patients.

Bottom Line: On the basis of the inducibility of IDO and iNOS by inflammatory cytokines in MSCs, the tested mammalian species fall into two distinct groups: IDO utilizers and iNOS utilizers.Interestingly, based on the limited number of species tested, the iNOS-utilizing species all belong to the phylogenetic clade, Glires.Although the evolutionary significance of this divergence is not known, we believe that this study provides critical guidance for choosing appropriate animal models for preclinical studies of MSCs.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai Jiao Tong University School of Medicine, 225 South Chongqing Road, Shanghai 200025, China.

ABSTRACT
Mammalian mesenchymal stem cells (MSCs) have been shown to be strongly immunosuppressive in both animal disease models and human clinical trials. We have reported that the key molecule mediating immunosuppression by MSCs is species dependent: indoleamine 2,3-dioxygenase (IDO) in human and inducible nitric oxide synthase (iNOS) in mouse. In the present study, we isolated MSCs from several mammalian species, each of a different genus, and investigated the involvement of IDO and iNOS during MSC-mediated immunosuppression. The characterization of MSCs from different species was by adherence to tissue culture plastic, morphology, specific marker expression, and differentiation potential. On the basis of the inducibility of IDO and iNOS by inflammatory cytokines in MSCs, the tested mammalian species fall into two distinct groups: IDO utilizers and iNOS utilizers. MSCs from monkey, pig, and human employ IDO to suppress immune responses, whereas MSCs from mouse, rat, rabbit, and hamster utilize iNOS. Interestingly, based on the limited number of species tested, the iNOS-utilizing species all belong to the phylogenetic clade, Glires. Although the evolutionary significance of this divergence is not known, we believe that this study provides critical guidance for choosing appropriate animal models for preclinical studies of MSCs.

Show MeSH
Related in: MedlinePlus