Microtomographical, histological and molecular analysis of the role of the CCR5 receptor, and possible cooperative role of CCR2 receptor, in macrophage migration and its impact on the alveolar repair process post extraction in mice

Abstract

Macrophages play important roles in bone repair, including the control of immune response and inflammation, and regulating the transition between granulation tissue to osteogenesis and formation of new bone after an injury. In this context, chemokine receptors, such as CCR5 and CCR2, seems to be key players in the chemotaxis of monocytes/macrophages to sites of tissue injury. Consequently, the blockage or absence of such receptors could interfere the recruitment of these cells to the site alveolar bone repair, elucidating its regulatory role in this process. In fact, previous results of our research group (process FAPESP 2012 / 03636-0, manuscript for publication in final drafting stage) demonstrated the involvement of the CCR2 receptor in alveolar bone repair in mice through comparative studies between WT-C57BL/6 and CCR2KO strains. However, CCR2KO animals still displayed monocyte/macrophage F4/80+ and CCR5+ cells in the repair site, demonstrating the possible involvement of CCR5 in the remaining migration of monocytes/macrophages in CCR2KO mice, and therefore a cooperative role of CCR2 and CCR5 in the process of alveolar bone repair. The objective of this project is investigate simultaneously the role of CCR5 and CCR2 receptors in the cell migration and its subsequent impact on the alveolar repair process in mice, through a microtomographic, histological and molecular characterization. Mice frin C57BL/6 WT and CCR5KO strains will be submitted to the upper incisor extraction process, and maintained under control conditions (no further treatment) or treated with the CCR2 antagonist (RS504393 - Santa Cruz Biotechnology) to enable analysis of the effect of blocking (genetic or pharmacological) individually or simultaneously of the CCR5 and CCR2 receptors. The animals of the different experimental groups will be submitted to the extraction process of the upper incisor, and subsequently euthanized (periods of 0, 7, 14 and 21 days), the specimens will be removed and submitted to microtomographic analysis, histology (histomorphometry, immunohistochemistry and analysis birefringence) as well as molecular analysis using PCRArray for quantification of different markers involved in the repair process -bone markers, matrix of markers, growth factors, cytokines, endogenous controls, markers of MSCs, chemokines and their receptors (as previously described by our group Vieira et al., 2015 PlosOne, in press).The data will be statistically analyzed and compared among the four groups in the different experimental periods, with adequate testing to number of groups/samples and distribution of normality. The joint analysis of such data will contribute to a better understanding of the roleofCCR5+ and CCR2+cells in the alveolar bone repair process, as well as to deepen our knowledge of the regulatory mechanisms that intervene in bone homeostasis and metabolism.