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Utility of a Mouse Model of Osteoarthritis to Demonstrate Cartilage Protection by IFN γ -Primed Equine Mesenchymal Stem Cells

View Article: PubMed Central - PubMed

ABSTRACT

Objective: Mesenchymal stem cells isolated from adipose tissue (ASC) have been shown to influence the course of osteoarthritis (OA) in different animal models and are promising in veterinary medicine for horses involved in competitive sport. The aim of this study was to characterize equine ASCs (eASCs) and investigate the role of interferon-gamma (IFNγ)-priming on their therapeutic effect in a murine model of OA, which could be relevant to equine OA.

Methods: ASC were isolated from subcutaneous fat. Expression of specific markers was tested by cytometry and RT-qPCR. Differentiation potential was evaluated by histology and RT-qPCR. For functional assays, naïve or IFNγ-primed eASCs were cocultured with peripheral blood mononuclear cells or articular cartilage explants. Finally, the therapeutic effect of eASCs was tested in the model of collagenase-induced OA (CIOA) in mice.

Results: The immunosuppressive function of eASCs on equine T cell proliferation and their chondroprotective effect on equine cartilage explants were demonstrated in vitro. Both cartilage degradation and T cell activation were reduced by naïve and IFNγ-primed eASCs, but IFNγ-priming enhanced these functions. In CIOA, intra-articular injection of eASCs prevented articular cartilage from degradation and IFNγ-primed eASCs were more potent than naïve cells. This effect was related to the modulation of eASC secretome by IFNγ-priming.

Conclusion: IFNγ-priming of eASCs potentiated their antiproliferative and chondroprotective functions. We demonstrated that the immunocompetent mouse model of CIOA was relevant to test the therapeutic efficacy of xenogeneic eASCs for OA and confirmed that IFNγ-primed eASCs may have a therapeutic value for musculoskeletal diseases in veterinary medicine.

No MeSH data available.


Related in: MedlinePlus

IFNγ-priming improves the immunosuppressive and chondroprotective properties of eASCs in vitro. (A) Proliferation of equine PBMCs in presence of naïve eASCs or IFNγ-primed eASCs at different ratios. Results are expressed as the percentage of PHA-induced proliferation of equine PBMCs, which was assigned the value of 100% and represented as mean ± SEM for three independent biological replicates. (B) Gene expression level of TGFβ1, COX-2, and IL-6 in eASCs. Gene expression in IFNγ-primed eASCs was normalized to that obtained in naïve eASCs. (C) PGE2 concentration in supernatants of naïve and IFNγ-primed eASCs. (D) GAG release quantification from cartilage explants incubated with IL1β (1 ng/mL) and Oncostatin M (10 ng/mL) during 7, 9, or 12 days and cocultured with or without 25,000 or 100,000 naïve and IFNγ-primed eASCs (100 ng/mL, 24 h before coculture). Results are expressed as the percentage of GAG release normalized to control condition (explant alone) at each time points and represented as mean ± SEM for 10–14 independent biological replicates. Data were analyzed using the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons for (A, D) and using the Mann–Whitney test for (B,C) *p < 0.05 in samples versus PBMC alone (A) or control (D); #p < 0.05 between primed and unprimed samples.
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Figure 2: IFNγ-priming improves the immunosuppressive and chondroprotective properties of eASCs in vitro. (A) Proliferation of equine PBMCs in presence of naïve eASCs or IFNγ-primed eASCs at different ratios. Results are expressed as the percentage of PHA-induced proliferation of equine PBMCs, which was assigned the value of 100% and represented as mean ± SEM for three independent biological replicates. (B) Gene expression level of TGFβ1, COX-2, and IL-6 in eASCs. Gene expression in IFNγ-primed eASCs was normalized to that obtained in naïve eASCs. (C) PGE2 concentration in supernatants of naïve and IFNγ-primed eASCs. (D) GAG release quantification from cartilage explants incubated with IL1β (1 ng/mL) and Oncostatin M (10 ng/mL) during 7, 9, or 12 days and cocultured with or without 25,000 or 100,000 naïve and IFNγ-primed eASCs (100 ng/mL, 24 h before coculture). Results are expressed as the percentage of GAG release normalized to control condition (explant alone) at each time points and represented as mean ± SEM for 10–14 independent biological replicates. Data were analyzed using the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons for (A, D) and using the Mann–Whitney test for (B,C) *p < 0.05 in samples versus PBMC alone (A) or control (D); #p < 0.05 between primed and unprimed samples.

Mentions: With the rationale in mind that priming eASCs will improve their immunosuppressive properties, we evaluated their capacities to inhibit the proliferation of ePBMCs. eASCs were pretreated or not with IFNγ for 24 h and then cultured with PHA-activated ePBMCs at different ratios for 3 days. When compared with ePBMCs alone, both eASCs- and IFNγ-primed eASCs were able to inhibit T cell proliferation in a dose-dependent manner (Figure 2A). The antiproliferative effect of IFNγ-primed eASCs was, however, significantly higher, whatever the eASC/ePBMC ratio. In order to determine whether this higher suppressive effect of IFNγ-primed eASCs was related to higher paracrine functions, expression of several known immunosuppressive mediators was quantified by RT-qPCR. We noticed that TGFβ1 expression levels were decreased in IFNγ-primed eASCs, while IL6 expression was increased (Figure 2B). No change in expression of COX-2 transcripts or PGE2 secretion was observed (Figures 2B,C).


Utility of a Mouse Model of Osteoarthritis to Demonstrate Cartilage Protection by IFN γ -Primed Equine Mesenchymal Stem Cells
IFNγ-priming improves the immunosuppressive and chondroprotective properties of eASCs in vitro. (A) Proliferation of equine PBMCs in presence of naïve eASCs or IFNγ-primed eASCs at different ratios. Results are expressed as the percentage of PHA-induced proliferation of equine PBMCs, which was assigned the value of 100% and represented as mean ± SEM for three independent biological replicates. (B) Gene expression level of TGFβ1, COX-2, and IL-6 in eASCs. Gene expression in IFNγ-primed eASCs was normalized to that obtained in naïve eASCs. (C) PGE2 concentration in supernatants of naïve and IFNγ-primed eASCs. (D) GAG release quantification from cartilage explants incubated with IL1β (1 ng/mL) and Oncostatin M (10 ng/mL) during 7, 9, or 12 days and cocultured with or without 25,000 or 100,000 naïve and IFNγ-primed eASCs (100 ng/mL, 24 h before coculture). Results are expressed as the percentage of GAG release normalized to control condition (explant alone) at each time points and represented as mean ± SEM for 10–14 independent biological replicates. Data were analyzed using the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons for (A, D) and using the Mann–Whitney test for (B,C) *p < 0.05 in samples versus PBMC alone (A) or control (D); #p < 0.05 between primed and unprimed samples.
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Figure 2: IFNγ-priming improves the immunosuppressive and chondroprotective properties of eASCs in vitro. (A) Proliferation of equine PBMCs in presence of naïve eASCs or IFNγ-primed eASCs at different ratios. Results are expressed as the percentage of PHA-induced proliferation of equine PBMCs, which was assigned the value of 100% and represented as mean ± SEM for three independent biological replicates. (B) Gene expression level of TGFβ1, COX-2, and IL-6 in eASCs. Gene expression in IFNγ-primed eASCs was normalized to that obtained in naïve eASCs. (C) PGE2 concentration in supernatants of naïve and IFNγ-primed eASCs. (D) GAG release quantification from cartilage explants incubated with IL1β (1 ng/mL) and Oncostatin M (10 ng/mL) during 7, 9, or 12 days and cocultured with or without 25,000 or 100,000 naïve and IFNγ-primed eASCs (100 ng/mL, 24 h before coculture). Results are expressed as the percentage of GAG release normalized to control condition (explant alone) at each time points and represented as mean ± SEM for 10–14 independent biological replicates. Data were analyzed using the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons for (A, D) and using the Mann–Whitney test for (B,C) *p < 0.05 in samples versus PBMC alone (A) or control (D); #p < 0.05 between primed and unprimed samples.
Mentions: With the rationale in mind that priming eASCs will improve their immunosuppressive properties, we evaluated their capacities to inhibit the proliferation of ePBMCs. eASCs were pretreated or not with IFNγ for 24 h and then cultured with PHA-activated ePBMCs at different ratios for 3 days. When compared with ePBMCs alone, both eASCs- and IFNγ-primed eASCs were able to inhibit T cell proliferation in a dose-dependent manner (Figure 2A). The antiproliferative effect of IFNγ-primed eASCs was, however, significantly higher, whatever the eASC/ePBMC ratio. In order to determine whether this higher suppressive effect of IFNγ-primed eASCs was related to higher paracrine functions, expression of several known immunosuppressive mediators was quantified by RT-qPCR. We noticed that TGFβ1 expression levels were decreased in IFNγ-primed eASCs, while IL6 expression was increased (Figure 2B). No change in expression of COX-2 transcripts or PGE2 secretion was observed (Figures 2B,C).

View Article: PubMed Central - PubMed

ABSTRACT

Objective: Mesenchymal stem cells isolated from adipose tissue (ASC) have been shown to influence the course of osteoarthritis (OA) in different animal models and are promising in veterinary medicine for horses involved in competitive sport. The aim of this study was to characterize equine ASCs (eASCs) and investigate the role of interferon-gamma (IFN&gamma;)-priming on their therapeutic effect in a murine model of OA, which could be relevant to equine OA.

Methods: ASC were isolated from subcutaneous fat. Expression of specific markers was tested by cytometry and RT-qPCR. Differentiation potential was evaluated by histology and RT-qPCR. For functional assays, na&iuml;ve or IFN&gamma;-primed eASCs were cocultured with peripheral blood mononuclear cells or articular cartilage explants. Finally, the therapeutic effect of eASCs was tested in the model of collagenase-induced OA (CIOA) in mice.

Results: The immunosuppressive function of eASCs on equine T cell proliferation and their chondroprotective effect on equine cartilage explants were demonstrated in vitro. Both cartilage degradation and T cell activation were reduced by na&iuml;ve and IFN&gamma;-primed eASCs, but IFN&gamma;-priming enhanced these functions. In CIOA, intra-articular injection of eASCs prevented articular cartilage from degradation and IFN&gamma;-primed eASCs were more potent than na&iuml;ve cells. This effect was related to the modulation of eASC secretome by IFN&gamma;-priming.

Conclusion: IFN&gamma;-priming of eASCs potentiated their antiproliferative and chondroprotective functions. We demonstrated that the immunocompetent mouse model of CIOA was relevant to test the therapeutic efficacy of xenogeneic eASCs for OA and confirmed that IFN&gamma;-primed eASCs may have a therapeutic value for musculoskeletal diseases in veterinary medicine.

No MeSH data available.


Related in: MedlinePlus