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Summer Research Fellowship Programme of India's Science Academies

In-vitro generation of osteoclast from polarized macro-phage population

Roshan Kumar Roy

Amity Institute of Virology and Immunology

Anbazhagan Kolandaswamy

St. Johns Research Institute, Bengaluru

Abstract

Bone metabolism is amongst the most important dynamic process that maintains architectural integrity of living body. This dynamic process is broadly contributed by mesenchymal stem cell derived osteoblast and haematopoietic stem cell derived osteoclast, via bone formation and resorption respectively. Osteoclasts are multinuclear giant macrophages residing near bone which are capable of degrading bone matrix with the help of lysozymes and proteases which include cathepsin k, matrix metalloproteinases (MMP). In-vitro osteoclast genesis is achieved by culturing monocytes with M-CSF (monocyte colony stimulating factor) and RANKL (receptor activator of nuclear factor kappa beta ligand). Sequence of events expected to be included are macrophage differentiation from monocytes by MCSF and multinucleation of differentiated macrophages to form osteoclast with the help of RANKL, but in in-vivo setting we only observe osteoclast and monocyte stages. With no clear evidence of macrophage intermediate stage observed in in-vivo settings, this lineage commitment here arises the most important question that which subset among M0, M1, M2, moDC are more committed to form osteoclasts. If all subsets are capable to generate osteoclast, then what is difference among the osteoclasts formed from respective subsets. By this work we tried to address these questions and differentiated the macrophages from monocytes (PBMC). Followed by polarization of differentiated macrophages to M0, M1, M2, moDC and GM-CSF population. Once polarized population were obtained, they were supplemented with RANKL and expression of CX3CR1 was analysed on both subset specific osteoclasts and polarized population. CX3CR1 was chosen because of its higher expression on pre-osteoclast and direct relation to RANK expression, it has normal expression on monocytes, macrophages. As expected with the huge variability that macrophages had, there was variable expression of CX3CR1 among osteoclasts produced by polarized population of monocytes (macrophages). Results indicated that MO macrophages were more committed for osteoclast genesis with higher expression of CX3CR1 among all kinds of population, osteoclasts generated from M2 macrophages were higher in CX3CR1 expression than M1. We also showed that CX3CR1 expression is directly co-related with multinucleation, as seen in M1 population with low CX3CR1 expression and low multinucleation.

Keywords: monocyte, polarisation, macrophage, osteoclast.

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