Molecular determinants of macrophages with a proregenerative phenotype supporting axonal growth

Abstract

Although intrinsic capacity of CNS axon regeneration is highly limited, injuries to the peripheral nerves frequently result in successful axon regeneration. We have previously shown that macrophage activation in the dorsal root ganglia (DRGs) following peripheral nerve injury contributes to the enhanced axon regeneration capacity. We further demonstrated that neuron-macrophage interaction occurring in the DRGs is essential linking injury-triggered neuronal chemokine expression with the proregenerative macrophage activation. However, it is not known how activated macrophages, which we defined as regeneration-associated macrophages (RAMs), in turn contribute to the enhanced capacity of axon regeneration in DRG neurons. The present study sought to characterize molecular signatures of RAMs and to identify key signaling pathways that drive the activation of the proregenerative phenotype in RAMs. To generate RAMs, we employed an in vitro neuron-macrophage interaction model where co-cultures of DRG neurons and peritoneal macrophages are treated with cAMP. In this model, conditioned medium of primed macrophages promotes robust neurite outgrowth. We performed transcriptomic profiling using RAMs obtained in this co-culture paradigm. Ingenuity Pathway Analysis indicated that genes related to cancer and wound healing processes, which are known to increase in tumor-associated macrophages, were highly upregulated in RAMs. Furthermore, analysis of upstream transcriptional networks suggested that beta-catenin and Hif1-alpha could play a role in shaping the transcriptomic signatures. To verify the upregulated genes in an in vivo neuron-macrophage interaction setting, CX3CR1-GFP mice were utilized to isolate activated macrophages in DRGs following peripheral nerve injury using Fluorescence-Activating Cell Sorting (FACS). Ongoing experiments involving analysis of the FACS-isolated macrophages with immunohistochemistry and real time PCR will characterize the molecular determinants of RAMs promoting capacity of axon regeneration in vivo.