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

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.

Figure 1

Morphology of BM-MSCs from different species. BM-MSCs before passage 10 from different species were isolated and maintained in DMEM complete medium as described. Their morphology was monitored under microscope and the scale bar indicates 100 μm

Figure 2

Surface markers of different species-derived BM-MSCs. All BM-MSCs were seeded in six-well plates and collected upon confluence for subsequent RNA extraction, RNA reverse transcription, and PCR assay. A panel of surface markers including CD105, CD73, CD29, CD44, and CD45 was detected for identification of BM-MSCs while GAPDH served as an input control

Figure 3

Adipocyte and osteoblast differentiation of BM-MSCs from different species. BM-MSCs before passage 12 were induced to differentiate into adipocytes and osteoblasts in differential medium and detected as described in Materials and Methods, untreated group used as control. (a) Adipocyte differentiation indicated by oil red O staining; the scale bar indicates 100 μm. (b) Osteoblast differentiation indicated by Alizarin red S staining; the scale bar indicates 500 μm

Figure 4

BM-MSCs isolated from different species have the same immunosuppression effects on Spls or PBMCs. PBMCs or Spls stained with CFSE were cocultured with BM-MSCs (2.0 × 10⁴ cells per well in 96-well plate) at a ratio of 1 : 10 (MSC: T cell) for 96 h, in the presence of conconavalin A (Con A) (1 μg/ml; for rat, rabbit, hamster, and pig), or anti-CD3 (1 μg/ml; for human, monkey, and mouse). CFSE fluorescence intensity reduction of PBMCs or Spls was detected by flow cytometry. Data are representative of three independent experiments

Figure 5

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

Figure 6

iNOS-based phylogenetic tree. The iNOS-coding region of amino-acid sequences of different species were used for evolutionary relationship alignment by neighbor-joining method. Numbers at nodes indicate the bootstrap support values. Domestic pig: Sus scrofa domesticus; bovine: Bos taurus; horse: Equus caballus; domestic dog: Canis lupus; rhesus macaques: Macaca mulatta; human: Homo sapiens; chimpanzee: Pan troglodytes; New Zealand rabbits: Oryctolagus cuniculus; Syrian hamsters: Mesocricetus auratus; BALB/c, C57BL/6 mice: Mus musculus; Sprague–Dawley rats: Rattus norvegicus