Poster BB19 - Gut Microbial Metabolite Mediated Immunomodulation to Promote Cartilage Tissue Regeneration

Introduction: Osteoarthritis (OA) is a leading cause of disability for aging populations, with an ever-growing burden affecting over 7% of the global population [1]. OA is a chronic, progressively degenerative joint disease, mediated by inflammatory agents that deteriorate cartilage [2]. Localized accumulation of major pro-inflammatory cytokines activate catabolic pathways that progress OA pathogenesis. Despite progress, the majority of the cartilage tissue engineering studies are conducted in the absence of OA and the associated inflammatory conditions. Controlling local inflammation while promoting tissue regeneration is an unmet clinical need for cartilage tissue engineering. In this study, we are proposing Urolithin-A (UroA), a gut microbial metabolite derived from ellagic acid/ellagitannin-rich diets (e.g., pomegranates and berries), as a potent anti-inflammatory agent [3]. However, UroA’s role as an immunomodulator for OA cartilage is unexplored. Through this study, we hypothesize that gut metabolite, UroA will balance OA-induced local inflammation while promoting new cartilage matrix formation. The objective here is to establish the dual role of UroA, so it could be effectively used in cartilage tissue/ osteochondral tissue engineering in OA condition.

Materials and

Methods: Our inflammation model treats Human articular chondrocytes (C28/I2) cultured in DMEM media (2.5% FBS and 1% P/S) with 10 µg/ml LPS (Escherichia coli O111:B4) for 48 hrs to simulate inflammation relevant to OA pathogenesis. A dose-dependent study with UroA was conducted to select an effective dose. MTS assay was used to assess cell viability with the chosen UroA concentration. Pro-inflammatory cytokines (IL-1β, TNF-α, IL-6 and IL-8) were measured using ELISA. Chondrogenic differentiation was assessed qualitatively using Alcian Blue staining and quantitatively via Dimethyl-methylene Blue (DMMB) assay on day 1, 2, 4, and 7 with media changes every 2 days containing LPS with or without UroA.

Results, Conclusions, and Discussions: Results &

Discussion: The dose-dependent cell viability testing established 5µM UroA as an effective concentration. Treatment with LPS (10 µg/ml) led to decrease in cell viability by ~50%. However, UroA (5 µM) treatment protected against LPS-induced cell death. This indicates UroA can maintain healthy cell activity when introduced to inflammatory environments. Further, UroA treatment significantly reduced LPS-induced proinflammatory cytokines (Figure 1A-D). In this study, we strategically treated chondrocytes with LPS then UroA to investigate whether this metabolite can negate pro-inflammatory cytokine accumulation. Our data suggests that UroA can locally modulate chondrocyte’s immune response to prevent from developing a microenvironment dominated by catabolic factors. UroA was also assessed for glycosaminoglycans (sGAG) concentration and ECM deposition, as shown from DMMB (Figure 2A), and Alcian blue staining (Figure 2B). LPS treatment caused sGAG inhibition and depleted ECM deposition in chondrocytes, while UroA treatment restored the sGAG production similar to control group. On Day 4, sGAG concentration for the LPS and UroA group was higher than the control group. Similarly, UroA maintained new matrix formation similar to the control group. It can be assumed that the predominance of pro-inflammatory factors in the microenvironment of LPS-treated cells prevented new matrix synthesis. This indicates that UroA exhibits chondroprotective behavior, which is also reflected in UroAs ability to maintain healthy metabolic activity. Collectively, these findings demonstrate UroA’s immunomodulatory nature, which was instrumental in creating a healthy environment for chondrocyte growth, differentiation, and new matrix development.

Conclusion: We have screened and developed gut metabolites that are naturally available in the body as potential immunomodulators. Our results demonstrate that the gut metabolite UroA exhibits synergistic anti-inflammatory and chondroprotective nature. These findings suggest gut metabolite mediated immunomodulation to promote cartilage tissue repair and regeneration in OA condition.

Acknowledgements (Optional):

References:
[1] GBD 2021 Osteoarthritis Collaborators. Global, regional, and national burden of osteoarthritis, 1990-2020 and projections to 2050: a systematic analysis for the Global Burden of Disease Study 2021. Lancet Rheumatol. 2023 Aug 21;5(9):e508-e522. doi: 10.1016/S2665-9913(23)00163-7. PMID: 37675071; PMCID: PMC10477960.
[2] A. De Roover, A. Escribano-Núñez, S. Monteagudo, and R. Lories, “Fundamentals of osteoarthritis: Inflammatory mediators in osteoarthritis,” Osteoarthritis and Cartilage, vol. 31, no. 10, pp. 1303–1311, Oct. 2023. doi:10.1016/j.joca.2023.06.005
[3] Singh, R., Chandrashekharappa, S., Bodduluri, S.R. et al. Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway. Nat Commun 10, 89 (2019). https://doi.org/10.1038/s41467-018-07859-7

Acknowledgement: National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health (#R01EB030060 & R01EB020640)