Establishing an in vitro cell differentiation assay using the disease synovial fluid to characterize the immunological milieu in osteoarthritis

Purpose: Current osteoarthritis (OA) therapies mostly target pain and impaired joint-function alleviation with minimal effectiveness. In order to develop efficient disease modifying therapies, comprehensive knowledge of OA patho-physiology as well as to develop competent in vitro models to test the newly invented drug is imperative. Increasing body of evidence reflects close involvement of immune system in the pathology; synovitis, especially in its chronic low-grade form is thought to activate innate and adaptive immune responses and plays a vital role in OA progression and cartilage loss. Indeed, increased infiltration of monocytes and macrophages typically found in OA synovium have been attributed to pathological synovitis. A good way to understand the effect of inflammatory micro-environment on cellular phenotypic changes is to perform in vitrocell differentiation assay using human blood derived monocytes, haematopoetic stem cells and cells of different lineages, in to macrophages under the influence of OA synovial fluid (SF). Methods: For the cell differentiation assay, the three types of cells were treated with 10% of OA SFs of different grades for 9 days. Complete media change was performed after every 3 days. After 9 days, the cells were harvested without enzymatic digestion and were detected using IFN-ɣ, IL-10 and CD44 antibodies to analyse the differentiation. For each marker, the median florescence intensity was measures by Attune Nxt Acoustic Flow cytometer and graphs were plotted. Human THP1 monocytes supplemented with 10% FBS and phorbol 12-myristate 13-acetate (PMA) was used as control. Results: After the SF treatment, the monocytes revealed a grade-wise increase in the number of differentiated cells as exhibited by CD44+ and CD16+ phenotype. Hematopoetic stem cells but not mesenchymal cells showed a clear differentiation in to the cells with PMCF+ and FCεRI+ phenotype. Conclusions: The results showed that SF from advanced grade OA can potentially differentiate the circulating cells into effector cell types and ultimately escalates the inflammatory ecosystem of the affected joint. Further, the presence of OA SF, which holds a plethora of cytokines and chemokines released by activated immune cells, has succeeded inducing a biased differentiation of monocytes and hematopoietic stem cells in to macrophages and mast-like cells, respectively. It further revealed the role of the inflammatory microenvironment, imposed by the activated immune cells on cellular and structural damage in OA. The results are prominent for both the differentiation parameters and functional aspects of both in terms of phagocytosis and degranulation. This experimental set up is useful to understand the pathobiological action of cytokine cocktail present in OA SF and further can serve as an excellent platform as a model system to test the drug efficacy, those are claimed to modulate the immune environment in OA. The data will be further useful to understand differentiation and polarity bias created by inflammatory milieu at cellular level. This information can be utilized in other autoimmune and inflammatory diseases as well.