Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes

Abstract

Context: Osteoarthritis (OA) is an invalidating articular disease characterized by cartilage degradation and inflammatory events. In horses, OA is associated with up to 60% of lameness and leads to reduced animal welfare along with extensive economic losses; currently, there are no curative therapies to treat OA. The mesenchymal stromal cell (MSC) secretome exhibits anti-inflammatory properties, making it an attractive candidate for improving the management of OA. In this study, we determined the best storage conditions for conditioned media (CMs) and tested whether priming MSCs with cytokines can enhance the properties of the MSC secretome.Methods: First, properties of CMs collected from bone-marrow MSC cultures and stored at −80°C, −20°C, 4°C, 20°C or 37°C were assessed on 3D cultures of equine articular chondrocytes (eACs). Second, we primed MSCs with IL-1β, TNF-α or IFN-γ, and evaluated the MSC transcript levels of immunomodulatory effectors and growth factors. The primed CMs were also harvested for subsequent treatment of eACs, either cultured in monolayers or as 3D cell cultures. Finally, we evaluated the effect of CMs on the proliferation and the phenotype of eACs and the quality of the extracellular matrix of the neosynthesized cartilage.Results: CM storage at −80°C, −20°C, and 4°C improved collagen protein accumulation, cell proliferation and the downregulation of inflammation. The three cytokines chosen for the MSC priming influenced MSC immunomodulator gene expression, although each cytokine led to a different pattern of MSC immunomodulation. The cytokine-primed CM had no major effect on eAC proliferation, with IL-1β and TNF-α slightly increasing collagen (types IIB and I) accumulation in eAC 3D cultures (particularly with the CM derived from MSCs primed with IL-1β), and IFN-γ leading to a marked decrease. IL-1β-primed CMs resulted in increased eAC transcript levels of MMP1, MMP13 and HTRA1, whereas IFNγ-primed CMs decreased the levels of HTRA1 and MMP13.Conclusion: Although the three cytokines differentially affected the expression of immunomodulatory molecules, primed CMs induced a distinct effect on eACs according to the cytokine used for MSC priming. Different mechanisms seemed to be triggered by each priming cytokine, highlighting the need for further investigation. Nevertheless, this study demonstrates the potential of MSC-CMs for improving equine OA management.

Pro-Inflammatory Cytokine Priming and Purification Method Modulate the Impact of Exosomes Derived from Equine Bone Marrow Mesenchymal Stromal Cells on Equine Articular Chondrocytes

“…IL1β- and TNFα-exos tended to induce a stronger increase in type I and IIB collagens accumulation than naive- and IFNγ-exos, while also increasing Htra1 levels. This observation suggests the diversity of the molecular pathways associated with the pro-inflammatory stimulation of MSCs, confirming our previous results on CMs derived from primed BM-MSCs [ 24 ]. Because a single cytokine does not recreate the whole pro-inflammatory environment that occurs in OA joints, associating several immunomodulatory markers could potentialize their priming potential and improve MSC-exos therapeutic capacities.…”

“…However, the therapeutic potential of exosomes derived from naive BM-MSCs can be enhanced through various stimulation techniques, including the use of pro-inflammatory cytokines. In a previous study, we demonstrated that the priming of BM-MSCs with equine IL-1β, TNF-α, or IFN-γ can modulate the effects mediated by their secretome on cartilaginous organoids [ 24 ]. Here, we aimed to investigate whether exosomes isolated from primed BM-MSCs can also mediate similar effects.…”

“…As part of this effort, two studies led by our research team recently demonstrated that the equine BM-MSC secretome can increase eAC migration and collagen synthesis [ 21 ]. Moreover, we were able to modulate this therapeutic potential by stimulating BM-MSCs with equine pro-inflammatory cytokines (IL-1β, TNF-α, and IFN-γ) [ 24 ]. Despite these encouraging results, it is now recognized that the use of exosomes instead of the whole secretome ensures better safety, prevents some standardization issues, and offers valuable targeting capacities [ 25 , 26 , 27 ].…”

“…Among these methods, pro-inflammatory cytokines hold great promise in stimulating MSC therapeutic properties because MSCs are sensitive to immune stimulation, leading to the modulation of their secretome [ 23 ]. Recently, our research group highlighted the potential of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) in promoting the ability of MSC-CM to significantly regenerate cartilage tissue [ 24 ]. Overall, cytokine priming triggers the stimulation of the secretion of immunomodulatory mediators in MSCs, enhances the chondroprotective effects of the MSC secretome and downregulates cartilage catabolism-associated markers such as matrix metalloproteases (MMPs) [ 22 ].…”

“…Following up on our previous work [ 21 , 22 , 24 ], in this study we aimed to investigate the presence of exosomes in MSC-CM and their implication in the effects attributed to the MSC secretome on equine articular chondrocyte (eAC) phenotype. For this purpose, we isolated exosomes using different isolation methods—i.e., PEG precipitation, MAC and SEC—whose capacities to generate consistent EV samples were compared using TEM, nanotracking analysis (NTA), and Western blotting.…”