Although microglia have been implicated in nerve injury–induced neuropathic pain, the manner by which injured sensory neurons engage microglia remains unclear. We found that peripheral nerve injury induced de novo expression of colony-stimulating factor 1 (CSF1) in injured sensory neurons. CSF1 was transported to the spinal cord, where it targeted the microglial CSF1 receptor (CSF1R). Cre-mediated sensory neuron deletion of Csf1 completely prevented nerve injury–induced mechanical hypersensitivity and reduced microglial activation and proliferation. In contrast, intrathecal injection of CSF1 induced mechanical hypersensitivity and microglial proliferation. Nerve injury also upregulated CSF1 in motoneurons, where it was required for ventral horn microglial activation and proliferation. Downstream of CSF1R, we found that the microglial membrane adaptor protein DAP12 was required for both nerve injury– and intrathecal CSF1–induced upregulation of pain-related microglial genes and the ensuing pain, but not for microglial proliferation. Thus, both CSF1 and DAP12 are potential targets for the pharmacotherapy of neuropathic pain.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Basbaum, A.I., Bautista, D.M., Scherrer, G. & Julius, D. Cellular and molecular mechanisms of pain. Cell 139, 267–284 (2009).
von Hehn, C.A., Baron, R. & Woolf, C.J. Deconstructing the neuropathic pain phenotype to reveal neural mechanisms. Neuron 73, 638–652 (2012).
Backonja, M. & Woolf, C.J. Future directions in neuropathic pain therapy: closing the translational loop. Oncologist 15 (suppl. 2), 24–29 (2010).
Sieweke, M.H. & Allen, J.E. Beyond stem cells: self-renewal of differentiated macrophages. Science 342, 1242974 (2013).
Salter, M.W. & Beggs, S. Sublime microglia: expanding roles for the guardians of the CNS. Cell 158, 15–24 (2014).
Beggs, S., Trang, T. & Salter, M.W. P2X4R+ microglia drive neuropathic pain. Nat. Neurosci. 15, 1068–1073 (2012).
Ji, R.R., Berta, T. & Nedergaard, M. Glia and pain: is chronic pain a gliopathy? Pain 154 (suppl. 1), S10–S28 (2013).
Clark, A.K. & Malcangio, M. Fractalkine/CX3CR1 signaling during neuropathic pain. Front. Cell. Neurosci. 8, 121 (2014).
Grace, P.M., Hutchinson, M.R., Maier, S.F. & Watkins, L.R. Pathological pain and the neuroimmune interface. Nat. Rev. Immunol. 14, 217–231 (2014).
Colburn, R.W., Rickman, A.J. & DeLeo, J.A. The effect of site and type of nerve injury on spinal glial activation and neuropathic pain behavior. Exp. Neurol. 157, 289–304 (1999).
Zhuang, Z.Y. et al. Role of the CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injury–induced cleavage of fractalkine. Brain Behav. Immun. 21, 642–651 (2007).
Old, E.A. & Malcangio, M. Chemokine mediated neuron-glia communication and aberrant signaling in neuropathic pain states. Curr. Opin. Pharmacol. 12, 67–73 (2012).
Biber, K. et al. Neuronal CCL21 up-regulates microglia P2X4 expression and initiates neuropathic pain development. EMBO J. 30, 1864–1873 (2011).
Jung, H. et al. Visualization of chemokine receptor activation in transgenic mice reveals peripheral activation of CCR2 receptors in states of neuropathic pain. J. Neurosci. 29, 8051–8062 (2009).
Calvo, M. et al. Neuregulin-ErbB signaling promotes microglial proliferation and chemotaxis contributing to microgliosis and pain after peripheral nerve injury. J. Neurosci. 30, 5437–5450 (2010).
Tsuda, M. et al. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 424, 778–783 (2003).
Ulmann, L. et al. Up-regulation of P2X4 receptors in spinal microglia after peripheral nerve injury mediates BDNF release and neuropathic pain. J. Neurosci. 28, 11263–11268 (2008).
Echeverry, S., Shi, X.Q. & Zhang, J. Characterization of cell proliferation in rat spinal cord following peripheral nerve injury and the relationship with neuropathic pain. Pain 135, 37–47 (2008).
Priller, J. et al. Targeting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment. Nat. Med. 7, 1356–1361 (2001).
Ajami, B., Bennett, J.L., Krieger, C., Tetzlaff, W. & Rossi, F.M. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat. Neurosci. 10, 1538–1543 (2007).
LaCroix-Fralish, M.L., Austin, J.S., Zheng, F.Y., Levitin, D.J. & Mogil, J.S. Patterns of pain: meta-analysis of microarray studies of pain. Pain 152, 1888–1898 (2011).
Perkins, J.R. et al. A comparison of RNA-seq and exon arrays for whole genome transcription profiling of the L5 spinal nerve transection model of neuropathic pain in the rat. Mol. Pain 10, 7 (2014).
Suzumura, A., Sawada, M., Yamamoto, H. & Marunouchi, T. Effects of colony stimulating factors on isolated microglia in vitro. J. Neuroimmunol. 30, 111–120 (1990).
Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010).
Schulz, C. et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 336, 86–90 (2012).
Wang, Y. et al. IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia. Nat. Immunol. 13, 753–760 (2012).
Tsujino, H. et al. Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: a novel neuronal marker of nerve injury. Mol. Cell. Neurosci. 15, 170–182 (2000).
Hökfelt, T., Brumovsky, P., Shi, T., Pedrazzini, T. & Villar, M. NPY and pain as seen from the histochemical side. Peptides 28, 365–372 (2007).
Burnett, S.H. et al. Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene. J. Leukoc. Biol. 75, 612–623 (2004).
Harris, S.E. et al. Meox2Cre-mediated disruption of CSF-1 leads to osteopetrosis and osteocyte defects. Bone 50, 42–53 (2012).
Zurborg, S. et al. Generation and characterization of an Advillin-Cre driver mouse line. Mol. Pain 7, 66 (2011).
Shields, S.D., Eckert, W.A. III & Basbaum, A.I. Spared nerve injury model of neuropathic pain in the mouse: a behavioral and anatomic analysis. J. Pain 4, 465–470 (2003).
Coull, J.A. et al. BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature 438, 1017–1021 (2005).
Hickman, S.E. et al. The microglial sensome revealed by direct RNA sequencing. Nat. Neurosci. 16, 1896–1905 (2013).
Kobayashi, M., Konishi, H., Takai, T. & Kiyama, H. A DAP12-dependent signal promotes pro-inflammatory polarization in microglia following nerve injury and exacerbates degeneration of injured neurons. Glia 63, 1073–1082 (2015).
Bakker, A.B. et al. DAP12-deficient mice fail to develop autoimmunity due to impaired antigen priming. Immunity 13, 345–353 (2000).
Devor, M. & Raber, P. Heritability of symptoms in an experimental model of neuropathic pain. Pain 42, 51–67 (1990).
Bédard, A., Tremblay, P., Chernomoretz, A. & Vallières, L. Identification of genes preferentially expressed by microglia and upregulated during cuprizone-induced inflammation. Glia 55, 777–789 (2007).
Otero, K. et al. Macrophage colony-stimulating factor induces the proliferation and survival of macrophages via a pathway involving DAP12 and beta-catenin. Nat. Immunol. 10, 734–743 (2009).
Raivich, G. et al. Regulation of MCSF receptors on microglia in the normal and injured mouse central nervous system: a quantitative immunofluorescence study using confocal laser microscopy. J. Comp. Neurol. 395, 342–358 (1998).
Yamamoto, S., Nakajima, K. & Kohsaka, S. Macrophage-colony stimulating factor as an inducer of microglial proliferation in axotomized rat facial nucleus. J. Neurochem. 115, 1057–1067 (2010).
Dubois, N.C., Hofmann, D., Kaloulis, K., Bishop, J.M. & Trumpp, A. Nestin-Cre transgenic mouse line Nes-Cre1 mediates highly efficient Cre/loxP mediated recombination in the nervous system, kidney, and somite-derived tissues. Genesis 44, 355–360 (2006).
Yoshida, H. et al. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345, 442–444 (1990).
Raivich, G., Moreno-Flores, M.T., Möller, J.C. & Kreutzberg, G.W. Inhibition of posttraumatic microglial proliferation in a genetic model of macrophage colony-stimulating factor deficiency in the mouse. Eur. J. Neurosci. 6, 1615–1618 (1994).
Elmore, M.R. et al. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron 82, 380–397 (2014).
Biber, K. & Boddeke, E. Neuronal CC chemokines: the distinct roles of CCL21 and CCL2 in neuropathic pain. Front. Cell. Neurosci. 8, 210 (2014).
Ransohoff, R.M. & Cardona, A.E. The myeloid cells of the central nervous system parenchyma. Nature 468, 253–262 (2010).
Masuda, T. et al. IRF8 is a critical transcription factor for transforming microglia into a reactive phenotype. Cell Reports 1, 334–340 (2012).
Masuda, T. et al. Transcription factor IRF5 drives P2X4R+-reactive microglia gating neuropathic pain. Nat. Commun. 5, 3771 (2014).
Coull, J.A. et al. Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain. Nature 424, 938–942 (2003).
Sim, J.A. et al. Altered hippocampal synaptic potentiation in P2X4 knock-out mice. J. Neurosci. 26, 9006–9009 (2006).
Cavanaugh, D.J. et al. Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli. Proc. Natl. Acad. Sci. USA 106, 9075–9080 (2009).
Bráz, J.M. et al. Forebrain GABAergic neuron precursors integrate into adult spinal cord and reduce injury-induced neuropathic pain. Neuron 74, 663–675 (2012).
Wang, X. et al. Excitatory superficial dorsal horn interneurons are functionally heterogeneous and required for the full behavioral expression of pain and itch. Neuron 78, 312–324 (2013).
Solorzano, C. et al. Primary afferent and spinal cord expression of gastrin-releasing peptide: message, protein, and antibody concerns. J. Neurosci. 35, 648–657 (2015).
We thank N. Shah (University of California, San Francisco) for providing Nestin-Cre mice, and D.L. Davies (University of Southern California) and F. Rassendren (Inst. Genomique Functionnelle) for providing the P2X4−/− mice. We also thank E.K. Unger for illustrating Supplementary Figure 13 and J. Leff for technical support. This work was supported by grants from the Foundation for Anesthesia Education and Research (FAER) MRTG-BS-02/15/2010-G and the US National Institutes of Health (K08NS078050) to Z.G., the DFG (KU 3039/1-1) to J.A.K., NIH-DE022001 to A.I.B. and S.L., NIH-NS14627 and the Wellcome Trust to A.I.B., NIH-AG045040 to S.L.A.-W. and NIH-AI068129 to L.L.L.
The authors have submitted a patent, “Targeted disruption of a CSF1-DAP12 pathway member gene for the treatment of neuropathic pain” (PCT/US2015/054704), based on the work described in this article.
Integrated supplementary information
(a) Double immunolabeling for ATF3 and CSF1 in sensory neurons at different time points after nerve injury; (b) Percentage of ATF3-expressing neurons that co-express CSF1. CSF1 is detected in a subset of ATF3- expressing neurons within 18 h of nerve injury and persists for at least 3 weeks. At least 150 ATF3-expressing neurons were counted in each mouse. N = 3 mice per time point. Scale bar represents 100 µm. Data are presented as mean ± s.e.m.
Supplementary Figure 2 CSF1 is induced in injured sensory neurons and co-transported with NPY to the spinal cord, where CSF1R is expressed in microglia.
(a) CSF1 induction in DRG neurons ipsilateral to the nerve injury co-localizes with NPY (Inset), a neuropeptide that is only expressed in sensory neurons after injury (1 d). Scale bars represent 200 μm and 10 μm (Inset); (b) Accumulation of CSF1 and NPY at the dorsal root ligature (4d post injury). Co-localization of CSF1 with NPY establishes that the CSF1 transport is intra-axonal. Dashed line denotes ligature. Scale bar represents 200 μm; (c) CSF1R (immunostaining) co-localizes with the microglial marker CD11b and both markers are induced in the dorsal horn after nerve injury (3 d post injury). Scale bar represents 100 μm; (d) There is complete CSF1R-GFP co-localization with the microglial marker Iba1, and none with the neuronal marker, NeuN. Both CSF1R-GFP and Iba1 are induced in the dorsal horn after nerve injury (3 d post injury). White square shows enlarged region. Scale bar represents 100 μm; (e) Quantification of CSF1R immunostaining in CD11b positive cells in the superficial dorsal horn 3 days after nerve injury; (f) Quantification of GFP intensity in Iba1 positive cells in the superficial dorsal horn 3 days after nerve injury. N = 3–4 mice/group. Data are presented as mean ± s.e.m. *p≤0.05, ***p≤0.001.
Supplementary Figure 3 CSF1 is both necessary and sufficient for microglia activation in the dorsal horn.
(a) Quantification of dorsal horn microglial Iba1 immunoreactivity in control and Adv-CSF1 KO mice 3 days after nerve injury; (b) Iba1 quantification in dorsal horn microglia 3 days after intrathecal injection of CSF1 or PBS. N = 3 mice/group. Data are presented as mean ± s.e.m. *p≤0.05, **p≤0.01.
Supplementary Figure 4 Csf1 deletion from sensory neurons prevents nerve injury-induced mechanical hypersensitivity for at least 4 weeks.
(a) Mice with Advillin-Cre-mediated deletion of Csf1 from sensory neurons do not develop mechanical hypersensitivity for at least 4 weeks after nerve injury (n = 4–5 mice/group). These mutant mice have normal body weight (n = 7 mice/group) (b), normal Rotarod motor function (n = 7 mice/group) (c), normal behavioral responses to noxious heat (n = 7 and 6 mice/group) (d,e), normal nocifensive behavior in an inflammatory pain (hindpaw formalin) model (n = 6 mice/group) (f), and normal numbers of CGRP- and NF200-expressing DRG neurons (n = 3 mice/group) (g,h). In the box plots, the box limits show the first and third quartile, the center line is the median and the whiskers represent the minimum and maximum values. Other data are presented as mean ± s.e.m. *** p≤0.001, **** p≤0.0001.
(a) At 2 hours after CSF1 intrathecal injection, we observed an amoeboid morphology of activated dorsal horn microglia (inset reveals shorter, thickened processes, with larger cell body), and (b) a slight, but significant increase of Iba1 expression (n = 6 for PBS, n = 4 for CSF1). (c) The microglia inhibitor, minocycline, prevents the mechanical hypersensitivity produced by intrathecal CSF1 (n = 6). Scale bar represents 100 µm. In the box plots, the box limits show the first and third quartile, the center line is the median and the whiskers represent the minimum and maximum values. Other data are presented as mean ± s.e.m. * p≤0.05, ** p≤0.01.
(a) Upregulation of Tyrobp mRNA in the dorsal horn persists 7 d post-nerve injury (n = 3 mice/group). (b) CSF1 induction is preserved in injured (ATF3+) sensory neurons of Tyrobp–/– mice (1 d post injury). Scale bar represents 50 μm. Tyrobp–/– mice have normal Rotarod motor activity (c), normal responses to noxious heat in the Hargreaves (d) and hot plate tests (e). N = 6 mice/group (c-e). Data are presented as mean ± s.e.m. ***p≤0.001.
Supplementary Figure 7 Increased expression of DAP12 mRNA correlates with a higher incidence of autotomy in the rat.
Tyrobp mRNA levels in the spinal cord are elevated in a rat strain with high autotomy scores (self-mutilation of a denervated limb) compared to a rat strain with low autotomy scores. These differences are present both before and after denervation (n = 4 rats/group). The box limits show the first and third quartile, the center line is the median and the whiskers represent the minimum and maximum values. * p≤0.05.
Supplementary Figure 8 Microglia–enriched genes are induced in the dorsal cord after nerve injury; monocyte–specific genes are not.
qRT-PCR illustrates that microglia-enriched genes are induced in the ipsilateral dorsal cord 3 d after nerve injury; the levels of monocyte specific genes remain undetectable. (n = 3 mice/group). Data are presented as mean ± s.e.m. **** p≤0.0001.
Supplementary Figure 9 Nerve injury and CSF1-induced microglia proliferation in the dorsal horn is DAP12-independent.
(a) Advillin-Cre-mediated deletion of Csf1 from sensory neurons decreases nerve injury-induced dorsal horn microglia proliferation (3 d post injury; 3 mice per group); (b) Microglial proliferation 3 d post injury persists in Tyrobp–/– mice (3 d post injury, n = 4 mice); (c) Dorsal horn microglia proliferation after intrathecal CSF1 (3 d); (d) Intrathecal CSF1-induced microglia proliferation persists in Tyrobp–/– mice. Scale bar represents 100 μm.
Nerve injury induces CSF1 expression in the injured (ATF3-expressing) motoneurons within 18 h of the injury (left) and the induction persists for at least 3 weeks (right). Scale bar represents 100 μm.
Supplementary Figure 11 Nerve injury-induced CSF1 in injured sensory neurons is preserved in Nestin-Cre; Csf1fl/fl mice.
CSF1 induction in DRG neurons ipsilateral to the nerve injury is preserved in Nestin-Cre;Csf1fl/fl mice (8 d post injury), indicating that Nestin-Cre is not expressed in DRG neurons. Scale bar represents 50 μm.
Supplementary Figure 12 DAP12 is required for CSF1-induced upregulation of neuropathic pain-related microglial genes.
(a) Intrathecal CSF1 upregulates Irf8, Irf5, and P2X4 in the spinal cord and (b) this induction is completely prevented in Tyrobp–/– mice (n = 3–4 mice/group). Data are presented as mean ± s.e.m. * p≤0.05, ** p≤0.01.
Supplementary Figure 13 De novo CSF1 expression in injured sensory neurons triggers a DAP12-independent self-renewal of microglia and a DAP12-dependent upregulation of microglial genes that contribute to the neuropathic pain phenotype.
CSF1 is induced in injured (ATF3-positive) sensory neurons within 1 d of injury and is transported to the spinal cord, where it interacts with microglial CSF1R. Stimulated microglia, in turn, undergo a DAP12-independent proliferation/self-renewal and a DAP12-dependent neuropathic pain–associated gene induction, including BDNF and cathepsin S (CatS). The microglial–derived BDNF contributes to reduced GABAergic inhibitory control and a consequent hyperexcitability of dorsal horn pain transmission neurons. By cleaving CX3CL1 (fractalkine) from neuronal cell membranes, cathepsin S amplifies the activation of microglia. Whether the neuropathic pain phenotype is exacerbated by the concurrent CSF1-induced microglia self-renewal/proliferation and whether DAP12 contributes to that process remains to be determined.
About this article
Cite this article
Guan, Z., Kuhn, J., Wang, X. et al. Injured sensory neuron–derived CSF1 induces microglial proliferation and DAP12-dependent pain. Nat Neurosci 19, 94–101 (2016). https://doi.org/10.1038/nn.4189
Inhibition of autotaxin activity ameliorates neuropathic pain derived from lumbar spinal canal stenosis
Scientific Reports (2021)
Cytokine receptor clustering in sensory neurons with an engineered cytokine fusion protein triggers unique pain resolution pathways
Proceedings of the National Academy of Sciences (2021)
Neuroscience & Biobehavioral Reviews (2021)
Brainstem local microglia induce whisker map plasticity in the thalamus after peripheral nerve injury
Cell Reports (2021)
Biochemical Pharmacology (2021)