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Updated: Aug 17, 2025
Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity
Published on: March 30, 2018
Conditioned media of mouse macrophages modulates neuronal dynamics in mouse hippocampal cells
Ayla Batu Öztürk1, Nail Can Öztürk2, Furkan Ayaz3
1Department of Histology and Embryology, Faculty of Medicine, Mersin University, Mersin, Turkey.
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.
Insights
Peripheral macrophages can be a less invasive therapeutic target than microglia for neuroinflammation. This study explored how macrophage-conditioned media affects hippocampal neuron viability and gene expression, revealing significant cellular communication pathways.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Neurodegenerative diseases often involve neuroinflammation and reduced hippocampal neuron production.
- Targeting microglia for neuroinflammation is challenging due to invasive procedures.
- Peripheral monocytes/macrophages offer a less invasive immunotherapeutic strategy, but their interaction with neurons is poorly understood.
Purpose of the Study:
- To investigate the response of hippocampal neuronal cells (HT-22) to soluble factors from macrophages (RAW264.7).
- To establish an experimental platform for studying neuron-macrophage communication.
- To assess the impact of macrophage-conditioned media on neuronal viability and gene expression.
Main Methods:
- HT-22 cells were cultured with conditioned media (CM) from unstimulated and lipopolysaccharide-stimulated RAW264.7 macrophages.
- Cell viability was monitored using a real-time cell analyzer.
- Immunocytochemistry and quantitative reverse transcription polymerase chain reaction (q-RT-PCR) were used to assess protein and gene expression (CR, CB, Ascl1, Bdnf, CB, Grn, Nrf2, Rac1).
Main Results:
- Real-time monitoring provided physiological evidence of how treatments affected HT-22 cell viability.
- Unstimulated macrophage CM significantly increased CR expression and mRNA levels of Ascl1, Bdnf, CB, and Grn.
- Stimulated macrophage CM decreased the expression of these markers.
Conclusions:
- HT-22 cells are responsive to the secretome of RAW264.7 macrophages.
- The study establishes a valuable platform for deciphering molecular mechanisms of immune cell-neuron communication.
- Findings suggest potential for utilizing peripheral macrophage interactions in understanding and treating neurodegenerative conditions.
