title: Expression of genes involved in immune response and in vitro immunosuppressive effect of equine MSCs.
abstract: The immunomodulatory capacities of mesenchymal stem cells (MSCs) have made them the subject of increased clinical interest for tissue regeneration and repair. We have studied the immunomodulatory capacity of equine MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) in cocultures with allogeneic peripheral blood mononuclear cells (PBMCs). Different isoforms and concentrations of phytohaemaglutinin (PHA) were tested to determine the best stimulation conditions for PBMC proliferation and a proliferation assay was performed for 7 days to determine the optimal day of stimulation of PBMCs. The effect of the dose and source of MSCs was evaluated in cocultures of 10(5) PBMCs with different ratios of AT- and BM-MSCs (1:1, 1:10, 1:20 and 1:50). Proliferation rates of the PBMCs were evaluated using BrdU ELISA colorimetric assay. PHA stimulated equine PBMCs reached their peak of growth after 3 days of culture. The immunoassay showed a decrease of the PBMCs growth at high ratio cocultures (1:1 and 1:10). Equine BM-MSCs and AT-MSCs demonstrated an ability to suppress the proliferation of stimulated PBMCs. Although MSCs derived from both sources displayed immunosuppressive effects, AT-MSCs were slightly more potent than BM-MSCs. In addition, the expression of 26 genes coding for different molecules implicated in the immune response was analyzed in cocultures of BM-MSCs and PHA stimulated PBMSCs by reverse transcriptase real time quantitative PCR (RT-qPCR). An upregulation in genes associated with the production of interleukins and cytokines such as TNF-α and TGF-β1 was observed except for IFN-γ whose expression significantly decreased. The variations of interleukins and cytokine receptors showed no clear patterns. COX-1 and COX-2 showed similar expression patterns while INOs expression significantly decreased in the two cell types present in the coculture. Cyclin D2 and IDO-1 showed an increased expression and CD90, ITG-β1 and CD44 expression decreased significantly in BM-MSCs cocultured with PHA stimulated PBMCs. On the contrary, CD6 and VCAM1 expression increased in these cells. With regard to the expression of the five genes involved in antigen presentation, an upregulation was observed in both cocultured MSCs and stimulated PBMCs. This study contributes to the knowledge of the immunoregulatory properties of equine MSCs, which are notably important for the treatment of inflammation processes, such as tendinitis and osteoarthritis.
authors.affiliation: Laboratorio de Genética Bioquímica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain.
authors.forename: Ana Rosa
authors.initials: AR
authors.lastname: Remacha
authors.affiliation: Laboratorio de Genética Bioquímica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Barrachina
authors.affiliation: Laboratorio de Genética Bioquímica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain.
authors.forename: Samuel
authors.initials: S
authors.lastname: Álvarez-Arguedas
authors.affiliation: Laboratorio de Genética Bioquímica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Ranera
authors.affiliation: Laboratorio de Genética Bioquímica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain; Hospital Veterinario, Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Romero
authors.affiliation: Laboratorio de Genética Bioquímica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain; Hospital Veterinario, Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain.
authors.forename: Francisco José
authors.initials: FJ
authors.lastname: Vázquez
authors.affiliation: Laboratorio de Genética Bioquímica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Zaragoza
authors.affiliation: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER/FJD, Madrid, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Yañez
authors.affiliation: Laboratorio de Genética Bioquímica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain.
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Martín-Burriel
authors.affiliation: Laboratorio de Genética Bioquímica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain. Electronic address: rodellar@unizar.es.
authors.forename: Clementina
authors.initials: C
authors.lastname: Rodellar
mesh_terms: D000818:Animals; D054360:Chemokine CXCL1 / Q000502:physiology; D005802:Genes, MHC Class II / Q000235:genetics; D006736:Horses / Q000235:genetics / Q000276:immunology*; D007165:Immunosuppression Therapy / Q000379:methods / Q000662:veterinary*; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000502:physiology; D016753:Interleukin-10 / Q000502:physiology; D015850:Interleukin-6 / Q000502:physiology; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000502:physiology; D059630:Mesenchymal Stem Cells / Q000276:immunology* / Q000502:physiology; D054367:Receptors, CXCR3 / Q000502:physiology; D019718:Receptors, CXCR4 / Q000502:physiology; D059467:Transcriptome / Q000276:immunology; D053773:Transforming Growth Factor beta1 / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology
chemical_list: D054360:Chemokine CXCL1; D015850:Interleukin-6; D054367:Receptors, CXCR3; D019718:Receptors, CXCR4; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D007371:Interferon-gamma
keywords: Adipose tissue; Bone marrow; Horse; Immunomodulation; Mesenchymal stem cells