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Abstract

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.

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  • 03 February 2016

    In the supplementary information originally posted online, the symbols were switched in the key to Supplementary Figure 8. The error has been corrected in the HTML and PDF versions as of 3 February 2016.

References

  1. 1.

    , , & Cellular and molecular mechanisms of pain. Cell 139, 267–284 (2009).

  2. 2.

    , & Deconstructing the neuropathic pain phenotype to reveal neural mechanisms. Neuron 73, 638–652 (2012).

  3. 3.

    & Future directions in neuropathic pain therapy: closing the translational loop. Oncologist 15 (suppl. 2), 24–29 (2010).

  4. 4.

    & Beyond stem cells: self-renewal of differentiated macrophages. Science 342, 1242974 (2013).

  5. 5.

    & Sublime microglia: expanding roles for the guardians of the CNS. Cell 158, 15–24 (2014).

  6. 6.

    , & P2X4R+ microglia drive neuropathic pain. Nat. Neurosci. 15, 1068–1073 (2012).

  7. 7.

    , & Glia and pain: is chronic pain a gliopathy? Pain 154 (suppl. 1), S10–S28 (2013).

  8. 8.

    & Fractalkine/CX3CR1 signaling during neuropathic pain. Front. Cell. Neurosci. 8, 121 (2014).

  9. 9.

    , , & Pathological pain and the neuroimmune interface. Nat. Rev. Immunol. 14, 217–231 (2014).

  10. 10.

    , & The effect of site and type of nerve injury on spinal glial activation and neuropathic pain behavior. Exp. Neurol. 157, 289–304 (1999).

  11. 11.

    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).

  12. 12.

    & Chemokine mediated neuron-glia communication and aberrant signaling in neuropathic pain states. Curr. Opin. Pharmacol. 12, 67–73 (2012).

  13. 13.

    et al. Neuronal CCL21 up-regulates microglia P2X4 expression and initiates neuropathic pain development. EMBO J. 30, 1864–1873 (2011).

  14. 14.

    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).

  15. 15.

    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).

  16. 16.

    et al. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 424, 778–783 (2003).

  17. 17.

    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).

  18. 18.

    , & Characterization of cell proliferation in rat spinal cord following peripheral nerve injury and the relationship with neuropathic pain. Pain 135, 37–47 (2008).

  19. 19.

    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).

  20. 20.

    , , , & Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat. Neurosci. 10, 1538–1543 (2007).

  21. 21.

    , , , & Patterns of pain: meta-analysis of microarray studies of pain. Pain 152, 1888–1898 (2011).

  22. 22.

    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).

  23. 23.

    , , & Effects of colony stimulating factors on isolated microglia in vitro. J. Neuroimmunol. 30, 111–120 (1990).

  24. 24.

    et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010).

  25. 25.

    et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 336, 86–90 (2012).

  26. 26.

    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).

  27. 27.

    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).

  28. 28.

    , , , & NPY and pain as seen from the histochemical side. Peptides 28, 365–372 (2007).

  29. 29.

    et al. Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene. J. Leukoc. Biol. 75, 612–623 (2004).

  30. 30.

    et al. Meox2Cre-mediated disruption of CSF-1 leads to osteopetrosis and osteocyte defects. Bone 50, 42–53 (2012).

  31. 31.

    et al. Generation and characterization of an Advillin-Cre driver mouse line. Mol. Pain 7, 66 (2011).

  32. 32.

    , & Spared nerve injury model of neuropathic pain in the mouse: a behavioral and anatomic analysis. J. Pain 4, 465–470 (2003).

  33. 33.

    et al. BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature 438, 1017–1021 (2005).

  34. 34.

    et al. The microglial sensome revealed by direct RNA sequencing. Nat. Neurosci. 16, 1896–1905 (2013).

  35. 35.

    , , & A DAP12-dependent signal promotes pro-inflammatory polarization in microglia following nerve injury and exacerbates degeneration of injured neurons. Glia 63, 1073–1082 (2015).

  36. 36.

    et al. DAP12-deficient mice fail to develop autoimmunity due to impaired antigen priming. Immunity 13, 345–353 (2000).

  37. 37.

    & Heritability of symptoms in an experimental model of neuropathic pain. Pain 42, 51–67 (1990).

  38. 38.

    , , & Identification of genes preferentially expressed by microglia and upregulated during cuprizone-induced inflammation. Glia 55, 777–789 (2007).

  39. 39.

    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).

  40. 40.

    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).

  41. 41.

    , & Macrophage-colony stimulating factor as an inducer of microglial proliferation in axotomized rat facial nucleus. J. Neurochem. 115, 1057–1067 (2010).

  42. 42.

    , , , & 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).

  43. 43.

    et al. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345, 442–444 (1990).

  44. 44.

    , , & 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).

  45. 45.

    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).

  46. 46.

    & Neuronal CC chemokines: the distinct roles of CCL21 and CCL2 in neuropathic pain. Front. Cell. Neurosci. 8, 210 (2014).

  47. 47.

    & The myeloid cells of the central nervous system parenchyma. Nature 468, 253–262 (2010).

  48. 48.

    et al. IRF8 is a critical transcription factor for transforming microglia into a reactive phenotype. Cell Reports 1, 334–340 (2012).

  49. 49.

    et al. Transcription factor IRF5 drives P2X4R+-reactive microglia gating neuropathic pain. Nat. Commun. 5, 3771 (2014).

  50. 50.

    et al. Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain. Nature 424, 938–942 (2003).

  51. 51.

    et al. Altered hippocampal synaptic potentiation in P2X4 knock-out mice. J. Neurosci. 26, 9006–9009 (2006).

  52. 52.

    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).

  53. 53.

    et al. Forebrain GABAergic neuron precursors integrate into adult spinal cord and reduce injury-induced neuropathic pain. Neuron 74, 663–675 (2012).

  54. 54.

    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).

  55. 55.

    et al. Primary afferent and spinal cord expression of gastrin-releasing peptide: message, protein, and antibody concerns. J. Neurosci. 35, 648–657 (2015).

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Acknowledgements

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.

Author information

Author notes

    • Bradley Colquitt
    • , Zoe Evans-Reinsch
    •  & Stavros Lomvardas

    Present addresses: Department of Physiology, University of California San Francisco, San Francisco, California, USA (B.C.), Master Program in Translational Medicine, University of California Berkeley and University of California San Francisco, San Francisco, California, USA (Z.E.-R.), Department of Biochemistry and Molecular Biophysics, Mortimer B. Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, New York, USA (S.L.).

    • Zhonghui Guan
    •  & Julia A Kuhn

    These authors contributed equally to this work.

Affiliations

  1. Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, California, USA.

    • Zhonghui Guan
  2. Department of Anatomy, University of California San Francisco, San Francisco, California, USA.

    • Julia A Kuhn
    • , Xidao Wang
    • , Bradley Colquitt
    • , Carlos Solorzano
    • , Smitha Vaman
    • , Andrew K Guan
    • , Zoe Evans-Reinsch
    • , Joao Braz
    • , Stavros Lomvardas
    •  & Allan I Basbaum
  3. Department for Cell and Animal Biology, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.

    • Marshall Devor
  4. Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.

    • Sherry L Abboud-Werner
  5. Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California, USA.

    • Lewis L Lanier

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Contributions

Z.G. and A.I.B. designed the experiments and with J.A.K., wrote the manuscript. Z.G. performed and organized experiments to which J.A.K., X.W., C.S., S.V., A.K.G., Z.E.-R. and J.B. contributed. J.A.K. completed many of the neuroanatomical studies. B.C. performed RNA-Seq analysis. M.D. provided spinal cord tissue from HA and LA rats. S.L.A.-W. provided Csf1fl/fl mice. L.L.L. provided Tyrobp−/− mice. L.L.L. and S.L. contributed to experimental design and interpretation of results.

Competing interests

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.

Corresponding authors

Correspondence to Zhonghui Guan or Allan I Basbaum.

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DOI

https://doi.org/10.1038/nn.4189