Abstract 1755 Elevated inflammatory responsiveness in aged murine tenocytes

Objective: This study aims to elucidate disparities in cellular reactions to inflammatory cues between tenocytes of differing ages, aiming to enhance our understanding of how inflammation influences age-related histone reorganization. Methods: Mouse tenocytes were isolated from young (<5 weeks) and mature (>45 weeks) tail tendons and cultured in TNFα-containing media for various durations. Fixed cells were immunostained for either super-resolution imaging (for Histone-H2B or TNFaR2) for density quantification, or immunofluorescence (for histone modification markers or NFκB) for abundance measurements. Results: Super-resolution H2B localizations revealed a global elevation of nanoscale H2B density within mature nuclei, which continued to increase with TNFα treatment. Young tenocytes exhibit a gradual increase in chromatin condensation, in contrast to the sharp increase observed in mature cells. Examining histone modification status, the marker associated with gene repression (e.g., H3K27me3) increased in intensity with increasing treatment duration in both groups, with more substantial changes seen in mature cells. Interestingly, the marker associated with gene activation (e.g., H3K9ac) exhibited an increase in young cells but a decrease in mature cells in response to inflammation. Upon TNFα treatment, NFκB, a downstream effector of TNFα signaling, swiftly translocated into the nucleus of mature cells compared to their younger counterparts. Additionally, mature cells exhibited an elevation in TNFαR2 receptor density, indicating heightened sensitivity to TNFα treatment in mature cells. Conclusions: This study reveals age-related differences in tenocyte responses to inflammation. TNFα induces global chromatin compaction in both groups, with mature cells showing a substantial increase after 6-hours of treatment. The interplay between changes in epigenetic marks (H3K27me3 and H3K9ac) between young and mature cells likely contributes to the observed global chromatin condensation changes. Given that chromatin conformation influences gene expression patterns, these findings suggest that the inflammatory microenvironment typical of aged tendons may drive abnormal tenocyte phenotypes, leading to degeneration. Elevated TNFαR2 levels in mature tenocytes may contribute to age-associated inflammatory responsiveness. This research informs the development of improved treatment options for patients with tendinopathy in the future. Ongoing studies are investigating the downstream effects of chromatin conformational changes in the context of tendon aging and disease. This work was supported by the NIH (P50 AR080581, K01 AR077087) and NSF (CMMI-1548571).