Enhancing the therapeutic effect of MSC in osteoarthritis: role of PPARb/d – PPAROA

To achieve its goals, the PPAROA project is structured into four work packages:

WP0: Project management, communication, and dissemination.

WP1: Studying the effect of PPARβ/δ on the immune-regulating properties of BM-MSCs and AT-MSCs, focusing on macrophages and T lymphocytes.

WP2: Analyzing the role of PPARβ/δ in the cartilage-protective properties of human MSCs and predicting their therapeutic potential.

WP3: Evaluating MSCs with PPARβ/δ modulation in the onset and progression of OA in mouse models.

WP1: Role of PPARβ/δ in the Immunomodulatory Effect of MSCs from Bone Marrow and Adipose Tissue

In this phase, PPARβ/δ levels and its target genes (ANGPTL4, PDK4) were analyzed in MSCs from bone marrow and adipose tissue, collected from healthy donors and mice. The effects of activating or inhibiting PPARβ/δ (using specific molecules: GSK3787 to inhibit and G501516 to activate) on gene expression and key immunoregulatory factors of MSCs were studied. Finally, the ability of modified MSCs to reduce T lymphocyte activity and influence macrophage polarization was assessed.

WP2: Role of PPARβ/δ in Cartilage Protection by MSCs

This phase aimed to determine whether PPARβ/δ levels in human and mouse MSCs can predict their ability to protect chondrocytes, the cells of cartilage. To test this, MSCs (pre-treated with an activator or inhibitor of PPARβ/δ) were evaluated for their effects on (i) Chondrocyte proliferation (ii) Resistance to cell death (iii) Cell state (mature, hypertrophic, or fibrotic).

WP3: Role of PPARβ/δ in the Therapeutic Effects of MSCs in Osteoarthritis Models

In this final phase, the impact of PPARβ/δ modulation on the anti-osteoarthritis properties of MSCs was tested. Different mouse models were used, including a collagenase-induced osteoarthritis model. Additionally, an innovative humanized mouse model of osteoarthritis was developed to evaluate MSC effectiveness.

The project highlighted the role of PPARβ/δ in the immunomodulatory and therapeutic properties of mesenchymal stem/stromal cells (MSCs) from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs), particularly in the context of osteoarthritis (OA).

Key Findings:

- PPARβ/δ expression increases in human MSCs (BM-MSCs and AT-MSCs) in response to pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β). This regulation is linked to a decrease in target gene expression (ANGPTL4 and PDK4).

- Inhibiting PPARβ/δ in human MSCs enhances the effects of pro-inflammatory cytokines and increases the expression of immunoregulatory mediators (**COX2, TSG6, IDO, PGE2**) but does not improve their ability to suppress T lymphocyte proliferation.

- PPARβ/δ modulation affects interactions with macrophages, promoting an anti-inflammatory M2 phenotype.

- Transcriptomic analysis identified ANGPTL4 as the main differentially expressed gene after PPARβ/δ modulation.

Effects on Chondrocytes and Cartilage Protection:

- Modulating PPARβ/δ in MSCs does not significantly alter the expression of IL6 and COX2 in chondrocytes co-cultured with MSCs.

- Activating PPARβ/δ protects MSCs from oxidative stress and enhances their anti-apoptotic effect on chondrocytes exposed to H₂O₂-induced oxidative stress. This protective role is mediated by ANGPTL4.

Zebrafish Model Findings:

- In zebrafish, PGE2, a factor produced by PPARβ/δ-deficient MSCs, plays a role in caudal fin regeneration and macrophage recruitment.

- MSC injection promoted regeneration, but this effect was blocked by indomethacin, a PGE2 inhibitor.

Therapeutic Effects in Osteoarthritis Mouse Models:

- PPARβ/δ-activated MSCs significantly improved cartilage parameters compared to unmodified MSCs.

- PPARβ/δ activation enhances the therapeutic potential of MSCs, protecting cartilage from breakdown and slowing osteoarthritis progression.

The project has generated promising prospects in scientific, medical, and translational fields, particularly in regenerative medicine, inflammatory disease management, and osteoarthritis treatment. The project made significant progress in understanding the molecular mechanisms regulating mesenchymal stem/stromal cells (MSCs), especially the role of PPARβ/δ signaling.

It was demonstrated that PPARβ/δ modulation affects MSC immunoregulatory properties, macrophage polarization, and MSC survival under stress conditions. The identification of key mediators such as Prostaglandin E2 (PGE2) and Angiopoietin-like Protein 4 (ANGPTL4) has opened new possibilities to enhance MSC functionality.

The study also highlighted differences between mouse and human MSC biology, emphasizing the need for tailored approaches to improve therapeutic effectiveness. These findings could be applied to other degenerative or inflammatory diseases beyond osteoarthritis, expanding the potential of MSC-based therapies.

The integration of various models (zebrafish, mouse, and human) demonstrates the value of multidisciplinary approaches in regenerative medicine research. This methodological framework provides a strong foundation for future studies and innovations in the field.

Impact and Future Perspectives: The project has made dual contributions: advancing fundamental scientific knowledge while laying the groundwork for innovative therapeutic applications. By doing so, it offers hope for improved patient outcomes and contributes to the development of regenerative medicine.

Bodic B, Boyer C, Metayer B, Masson M, Vignes C, Lesoeur J, Veziers J, Daguin V, Haspot F, Maugars Y, Djouad F, Guicheux J, and Vinatier C. « Development and characterization of a humanized mouse model of steoarthritis”. Osteoarthritis and cartilage, Submitted.
V. Delplace, M.A. Boutet, C. Le Visage, Y. Maugars, J. Guicheux, C. Vinatier. Osteoarthritis: from upcoming treatments to treatments yet to come. Joint Bone Spine 2021
Contreras-Lopez R, Elizondo-Vega R, Paredes MJ, Luque-Campos N, Torres MJ, Tejedor G, Vega-Letter AM, Figueroa-Valdés A, Pradenas C, Oyarce K, Jorgensen C, Khoury M, Garcia-Robles MLA, Altamirano C, Djouad F, Luz-Crawford PHIF1a-dependent metabolic reprogramming governs mesenchymal stem/stromal cell immunoregulatory functions. FASEB J. 2020
Contreras-Lopez RA, Elizondo-Vega R, Torres MJ, Vega-Letter AM, Luque-Campos N, Paredes-Martinez MJ, Pradenas C, Tejedor G, Oyarce K, Salgado M, Jorgensen C, Khoury M, Kronke G, Garcia-Robles MA, Altamirano C, Luz-Crawford P, Djouad F. PPARß/d-dependent MSC metabolism determines their immunoregulatory properties. Sci Rep. 2020
Contreras-Lopez R, Elizondo-Vega R, Luque-Campos N, Torres MJ, Pradenas C, Tejedor G, Paredes-Martínez MJ, Vega-Letter AM, Campos-Mora M, Rigual-Gonzalez Y, Oyarce K, Salgado M, Jorgensen C, Khoury M, Garcia-Robles MLÁ, Altamirano C, Djouad F, Luz-Crawford P. The ATP synthase inhibition induces an AMPK-dependent glycolytic switch of mesenchymal stem cells that enhances their immunotherapeutic potential. Theranostics. 2021

Osteoarthritis (OA) is the most common inflammatory, degenerative and painful joint disorder. Despite the increase in the incidence of OA, there is still no effective treatment capable of restoring the structure of articular cartilage and function of damaged joints. Consequently, mesenchymal stromal/stem cell (MSC)-based therapies have become a thriving area of research. Pre-clinical and phase I or II clinical studies have shown that intra-articular MSC injection is well-tolerated. In this context, partner 1 (INSERM U1229-RMES/Nantes) coordinates, since 2013, a clinical trial “Arthrostem” (NCT: 01879046) that aims at assessing the immunomodulatory and anti-osteoarthritic properties of bone marrow-MSC (BM-MSC) and adipose tissue-MSC (AT-MSC) harvested from the same osteoarthritic patient. Currently, thanks to this clinical trial, a biocollection of MSC from 30 OA patients has been constituted. For its part, Partner 2 (INSERM U1183-IRMB/Montpellier), within an ongoing H2020 program, coordinates ADIPOA2 a randomized controlled study to validate the efficacy of the intra-articular injection of AT-MSC in mild to moderate knee OA. Moreover, partner 2 has demonstrated the capacity of human MSC, to reduce the clinical severity of OA by decreasing inflammation and protecting chondrocytes from apoptosis and loss of phenotype. However and despite their clinical efficacy in human OA, MSC display poor in vivo survival and engraftment rate. This in vivo ephemeral presence of MSC after injection has raised the possibility that their therapeutic effects in OA were mediated through the release of chondro-protective molecules and/or the education of immune cells towards a regulatory phenotype. In line with this hypothesis, partner 2 has shown that besides their capacity to protect chondrocytes, MSC control the fate of macrophages by switching their phenotype from pro-inflammatory M1 to anti-inflammatory M2. In OA patients and as described above, although MSC are considered safe when intra-articularly administrated, their therapeutic effects remain to be further improved to warrant long-term benefits and turn them into a routine OA treatment in clinical practice. Interestingly, data have suggested a role for PPARß/d in the onset and severity of murine OA and partner 2 has recently demonstrated that PPARß/d expression level predicts MSC immunomodulatory potential and more importantly that its inhibition stimulates their therapeutic effects in murine arthritis. Considering the safety and promising therapeutic effect of MSC in OA as well as the potential role of PPARß/d in the immunomodulatory properties of MSC, the overall objective of the PPAROA project is to investigate whether a modulation of PPARß/d can improve the MSC therapeutic properties in OA. Particular attention will be paid to (i) deciphering the role of PPARß/d in the therapeutic effects of BM-MSC and AT-MSC collected from Arthrostem OA patients and (ii) investigating whether PPARß/d modulation in MSC may enhance and prolong their anti-OA therapeutic effect. To address these issues, PPAROA will be organized in 3 scientific work packages (WP): •WP1: Role of PPARß/d in the immunomodulatory properties of BM-MSC and AT-MSC on cells of innate and adaptive immune system •WP2:Role of PPARß/d in the chondro-protective properties of BM-MSC and AT-MSC •WP3: Role of PPARß/d on the therapeutic effects of BM-MSC and AT-MSC in OA preclinical models. In case of success, this 4-year ambitious project will undoubtedly lead us to consider clinical trials and thus pave the way of new therapeutic avenue in the management of OA patients.