Conditioned media of mouse macrophages promote neuronal differentiation of mouse hippocampal cells

Abstract

Understanding how the immune system associates with the pathophysiological hippocampal neurobiology in the neurodegenerative diseases is an emerging topic. Since the cellular population of hippocampal neurogenic niche is extraordinarily diverse, there is still a large gap in our understanding of the interactions between the immune cells and hippocampal neurons under even normal conditions, setting aside the disease conditions. To examine how the hippocampal neurons receive and respond to the cellular factors from the immune cells, we introduced the conditioned cell culture media (CM) of unstimulated, and lipopolysaccharide stimulated RAW 264.7 macrophages to HT22 hippocampal cells during and after they were exposed to neuronal differentiation environment to mimic the states during and after the initiation of the neurogenesis. Then, HT22 cell responses were further examined by assessing immunocytochemical expression of CR protein and mRNA levels of Ascl1, Bdnf, Nrf2 and Rac1 genes via semi quantitative image analysis and q-RT-PCR, respectively. Our results suggest that culturing the HT22 cells with the unstimulated CM of macrophages substantially increased calretinin protein expression, while the latter media containing quite high levels of inflammatory cytokines, measured by ELISA, resulted in decreased protein levels for Calretinin. Additionally, Ascl1 gene expression levels in HT22 cells had similar trends as calretinin protein levels in the presence of CM of stimulated versus unstimulated media. Whereas there was no substantial difference in the gene expression levels for the Bdnf, Nrf2 and Rac1 genes. Overall, our results imply that utilization of HT22 cells and macrophage interplay can be instructive to decipher the molecular mechanisms of cellular communication between the immune system cells and neurons. Our overarching aim is developing immunotherapy strategies by using non-CNS derived immune system cells to modulate the neurogenesis in disease conditions such as Multiple Sclerosis, Alzheimer’s and ALS to enable the recovery in the patients.