Pain is a main symptom of inflammatory diseases and often persists beyond clinical remission. Although we have a good understanding of the mechanisms of sensitization at the periphery during inflammation, little is known about the mediators that drive central sensitization. Recent reports have identified hematopoietic colony-stimulating factors as important regulators of tumor- and nerve injury-associated pain. Using a mouse model of colitis, we identify the proinflammatory cytokine granulocyte-colony-stimulating factor (G-CSF or Csf-3) as a key mediator of visceral sensitization. We report that G-CSF is specifically up-regulated in the thoracolumbar spinal cord of colitis-affected mice. Our results show that resident spinal microglia express the G-CSF receptor and that G-CSF signaling mediates microglial activation following colitis. Furthermore, healthy mice subjected to intrathecal injection of G-CSF exhibit pronounced visceral hypersensitivity, an effect that is abolished by microglial depletion. Mechanistically, we demonstrate that G-CSF injection increases Cathepsin S activity in spinal cord tissues. When cocultured with microglia BV-2 cells exposed to G-CSF, dorsal root ganglion (DRG) nociceptors become hyperexcitable. Blocking CX3CR1 or nitric oxide production during G-CSF treatment reduces excitability and G-CSF-induced visceral pain in vivo. Finally, administration of G-CSF-neutralizing antibody can prevent the establishment of persistent visceral pain postcolitis. Overall, our work uncovers a DRG neuron-microglia interaction that responds to G-CSF by engaging Cathepsin S-CX3CR1-inducible NOS signaling. This interaction represents a central step in visceral sensitization following colonic inflammation, thereby identifying spinal G-CSF as a target for treating chronic abdominal pain.

Highlights d Hypothalamic microglia survey the embryonic environment by touching radial glia cells d Embryonic microglia upregulate MERTK and AXL in response to exogenous insults d Embryonic microglia show a remarkable responsiveness to insults during gestation

Background: Although historically microglia were thought to be immature in the fetal brain, evidence of purposeful interactions between these immune cells and nearby neural progenitors is becoming established. Here, we examined the influence of embryonic microglia on gliogenesis within the developing tuberal hypothalamus, a region later important for energy balance, reproduction, and thermoregulation. Methods: We used immunohistochemistry to quantify the location and numbers of glial cells in the embryonic brain (E13.5-E17.5), as well as a pharmacological approach (i.e., PLX5622) to knock down fetal microglia. We also conducted cytokine and chemokine analyses on embryonic brains in the presence or absence of microglia, and a neurosphere assay to test the effects of the altered cytokines on hypothalamic progenitor behaviors. Results: We identified a subpopulation of activated microglia that congregated adjacent to the third ventricle alongside embryonic Olig2+ neural progenitor cells (NPCs) that are destined to give rise to oligodendrocyte and astrocyte populations. In the absence of microglia, we observed an increase in Olig2+ glial progenitor cells that remained at the ventricle by E17.5 and a concomitant decrease of these Olig2+ cells in the mantle zone, indicative of a delay in migration of these precursor cells. A further examination of maturing oligodendrocytes in the hypothalamic grey and white matter area in the absence of microglia revealed migrating oligodendrocyte progenitor cells (OPCs) within the grey matter at E17.5, a time point when OPCs begin to slow their migration. Finally, quantification of cytokine and chemokine signaling in ex vivo E15.5 hypothalamic cultures +/− microglia revealed decreases in the protein levels of several cytokines in the absence of microglia. We assayed the influence of two downregulated cytokines (CCL2 and CXCL10) on neurosphere-forming capacity and lineage commitment of hypothalamic NPCs in culture and showed an increase in NPC proliferation as well as neuronal and oligodendrocyte differentiation. Conclusion: These data demonstrate that microglia influence gliogenesis in the developing tuberal hypothalamus.