gms | German Medical Science

Introduction: Rheumatoid arthritis (RA) is characterized by recurrent inflammation of synovial joints. This is associated with progressive bone destruction. The resulting increase in calcium and phosphate concentrations impairs local mineral homeostasis. These conditions facilitate the formation of amorphous structures, called calciprotein particles (CPPs) [1], [2]. In vitro studies on monocytes revealed the pro-inflammatory effect of the particles. Cellular internalization of CPPs leads to activation of the NLRP3 inflammasome. The subsequent release of interleukin 1β (IL-1β) could contribute to inflammatory progression of RA [3]. This study visualizes and characterizes the process of CPP uptake at single cell level. The aim is to analyze the relationship between particle internalization and the following inflammatory response.

Methods: CPP uptake was studied on primary monocytes from RA patients compared with age-matched healthy controls. Monocytes were obtained via ficoll density gradient centrifugation and subsequently stimulated with fluorescently labeled CPPs. To visualize cellular particle uptake, imaging flow cytometry (Amnis^® ImageStream^®X Mk II) and fluorescence microscopy (ZEISS AxioObserver) were performed. Analysis with different image processing programs and strategies (IDEAS 6.2, ZEN, Fiji/ImageJ) allowed a qualitative evaluation and quantification of CPP uptake into the cells. To assess the pro-inflammatory response to CPP internalization, the level of IL-1β secretion was determined.

Results: Fundamental for the analysis was to evaluate diverse staining methodologies for CPPs in the context of cellular imaging. The use of fluorescently labeled bisphosphonates to detect CPPs in combination with advanced imaging analysis allowed reliable visualization of the CPP uptake into the endolysosomal system. Interestingly, imaging analysis revealed that monocytes from RA patients showed enhanced internalization of CPPs compared to healthy controls. The increased particle uptake was associated with a significantly increased pro-inflammatory response.

Conclusion: This study presents a novel qualitative, as well as quantitative, investigation of the CPP uptake in monocytes and demonstrates its association with NLRP3 inflammasome activation. The increased pro-inflammatory response of monocytes from RA patients underlines the pathogenetic relevance of uptake and subsequent lysosomal digestion of CPPs.