Conditioned media of mouse macrophages modulates neuronal dynamics in mouse hippocampal cells.

Abstract

Many neurodegenerative diseases display both neuroinflammation and impaired neuron production in hippocampus. Although immunotherapeutic strategies indicate a promising avenue for combating neuroinflammation-induced diseases, directly targeting microglia, principle immune cells of CNS for such therapeutic purposes might be problematic due to invasive procedures. Instructing monocytes/macrophages from the periphery can be a less invasive and advantageous strategy compared to reaching microglia. But interplay between CNS neurons and macrophages even under normal conditions is poorly understood. To explore the experimental platform of how CNS derived neuronal cells respond to overall soluble factors of a non-CNS derived immune cell type, we introduced the conditioned media (CM) of unstimulated, and lipopolysaccharide stimulated RAW264.7 mouse macrophages to immortalized HT-22 mouse hippocampal cells during and after they were exposed to neuronal differentiation media. First, we recorded the cell viability of HT-22 cell study groups by using a real time cell analyzer. Then, we assessed the immunocytochemical expression of CR and CB proteins and mRNA levels of Ascl1, Bdnf, CB, Grn, Nrf2 and Rac1 genes via semi quantitative image analysis and q-RT-PCR among the different groups of HT-22 cells. Real time cell monitoring provided a solid physiological evidence regarding how various cell culture treatments affected the cell viability of HT-22 cells over time. Our further findings suggested that culturing HT-22 cells with unstimulated CM of macrophages markedly increased the immunocytochemical expression of CR and mRNA expression of Ascl1, Bdnf, CB and Grn genes, while the latter media resulted in decreases of those expressions. Overall, our results imply that HT-22 cells are meaningfully responsive to the secretome of RAW264.7 macrophages and using the interaction of macrophage with CNS derived neuronal cells is an instructive platform for deciphering the molecular mechanisms of cellular communication between immune system cells and neurons.