Microglia are brain resident macrophages with essential roles in neural circuit function in physiology and disease (Priller and Prinz, 2019; Hammond et al., 2018; Vainchtein and Molofsky, 2020). Microglia respond in sexually dimorphic ways in a variety of contexts, including autism, stroke, neurodegenerative diseases, and interestingly in the microglial contribution to pain processing (Mogil, 2020; Villa et al., 2018; Weinhard et al., 2018; Sorge et al., 2011; Inyang et al., 2019; Rosen et al., 2019; Kodama and Gan, 2019; Guneykaya et al., 2018). For example, although male and female microglia are competent to induce pain (Yi et al., 2021), pharmacologic ablation or chemogenetic inhibition of microglia reverses peripheral nerve injury-induced mechanical hypersensitivity only in male mice (Sorge et al., 2015; Saika et al., 2020). In contrast, inhibition of microglia is sufficient to reverse injury-induced hypersensitivity in B- and T-cell deficient female mice (Sorge et al., 2015). Taken together, these data imply that there are sex-specific differences in how the innate and adaptive immune compartments interact to regulate neuropathic pain.

We previously identified microglial activation via release of the myeloid survival factor, colony-stimulating factor 1 (CSF1), from injured sensory neurons as a mechanism contributing to nerve injury-induced pain (Guan et al., 2016). Here, we show that intrathecal administration of CSF1 is sufficient to induce pain (mechanical hypersensitivity) in male, but not female mice. Transcriptomic profiling of dorsal horn microglia and morphologic analyses demonstrated that this sex-specific effect correlates with robust microglial activation in male but not female mice. Furthermore, intrathecal CSF1 markedly expanded lymphocytes and myeloid cells in the spinal cord meninges, and resulted in a preferential expansion of regulatory T-cells (Tregs), in female mice. Finally, we demonstrate that Treg depletion (FoxP3^DTR) in female mice promotes CSF1-induced microglial activation and is sufficient to induce CSF1-induced pain hypersensitivity equivalent to males. Our findings reveal novel cross-regulatory interactions between Tregs and spinal cord microglia that modulate a sex-specific pain phenotype.

CSF1 is de novo expressed in injured sensory neurons (Guan et al., 2016), and in the spinal cord, parenchymal microglia are the only cells expressing CSF1 receptor (CSF1R). We first analyzed injury-induced mechanical hypersensitivity in female Avil^Cre:Csf1^fl/fl mice (Adv-CSF1) in which CSF1 is specifically deleted from sensory neurons. We found that female Adv-CSF1 mice developed normal mechanical hypersensitivity after peripheral nerve injury (Figure 1—figure supplement Figure 1—figure supplement 1A, B), in contrast to male rats and mice, in which hypersensitivity was CSF1-dependent (Guan et al., 2016; Okubo et al., 2016). Thus, CSF1 is not required to induce mechanical hypersensitivity in females.

We next assessed whether selective administration of CSF1, via an intrathecal route, is sufficient to induce mechanical hypersensitivity. Three consecutive injections of CSF1 provoked profound mechanical hypersensitivity in male, but not in female mice (Figure 1A–C), even at very high doses (30 ng; Figure 1—figure supplement 1C). Furthermore, after intrathecal CSF1, male microglia acquired a robust amoeboid morphology, characterized by loss of ramification, but in females, microglia acquired a highly ramified morphology, consistent with a persistent homeostatic phenotype (Figure 1D–E). Fluorescence-activated cell sorting (FACS) analysis also revealed larger numbers of microglia in males and higher expression of cell surface activation markers, CD11b/CD45 (Figure 1F–H, Figure 1—figure supplement 1D). Taken together, these data demonstrate a male-specific impact and sufficiency of CSF1 for microglia activation and pain hypersensitivity.

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To determine whether there was a differential impact of CSF1 on male versus female microglia, we transcriptionally profiled flow-sorted microglia from the lumbar dorsal horn. Sex differences were modest at baseline (86 genes, p_Adj<0.01), and CSF1 induced robust gene expression changes in both male and female microglia (Figure 2A, PC1, 56% of variance). However, CSF1 induced an 8.3-fold increase in differentially expressed genes (both upregulated and downregulated) in male microglia (Figure 2B, Supplementary file 1; adjusted p-value<0.01: males 1350 genes, females 165 genes). As CSF1 is an essential survival factor for microglia and myeloid cells, these sex-specific microglia responses to CSF1 were surprising. Neither the protein nor transcriptomic CSF1R levels differed between males and females (Figure 2—figure supplement 1A, B).

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We next examined these gene expression changes in the context of published microglial transcriptomic data sets in homeostasis and disease (Friedman et al., 2018; Figure 2C). Both male and female microglia responded to CSF1 with a decrease in homeostatic gene expression and an increase in proliferative genes, which were more prominent in males than females. Most prominent in male microglia was a striking upregulation of pathology-associated microglial activation genes (Figure 2C; Neurodegeneration module) (Friedman et al., 2018; Keren-Shaul et al., 2017). Gene ontology (GO) enrichment analysis (Figure 2D) revealed that male microglia induced genes and GO terms that are linked to classical immune activation and recruitment pathways, including many (Itgax, Lpl, Ccl3, Cybb, Clec7a, and Ctsb) associated with the ‘disease associated microglia’ DAM phenotype identified in single-cell sequencing experiments (Butovsky and Weiner, 2018). Some of these genes, for example, Ctsb, have been linked to chronic pain (Sun et al., 2012). In addition, male microglia downregulated genes facilitating responsiveness to extracellular signals as well as some supportive functions, for example, extracellular matrix regulation (Figure 2D). Taken together, intrathecal CSF1 not only triggers pain hypersensitivity in male mice, but also induces robust transcriptomic changes associated with inflammatory activation in male but not female microglia.

Our findings suggest that other immune cells contribute to amplify or suppress the microglial response to CSF1. The CNS meninges have a rich population of immune cells that mirrors the composition of tissue resident immune cells in other organs (Alves de Lima et al., 2020; Figure 3B). Meningeal lymphocyte-derived cytokines also impact CNS function in both normal and pathologic settings (Liu et al., 2020; Pasciuto et al., 2020; Ribeiro et al., 2019). We examined the immune cell composition of spinal cord meninges using 11-parameter flow cytometry of dissociated meninges (Figure 3—figure supplement 1A-C, Figure 3A–C). As expected, intrathecal CSF1 expanded meningeal macrophages (Figure 3—figure supplement 1B), but we also observed a marked increase in lymphocytes, 6.5-fold in males and 9-fold in females (Figure 3—figure supplement 1C). Further examination of lymphocyte subsets demonstrated a similar increase of CD4⁺ FoxP3 T cells, CD8⁺ T cells, B cells, and ILC2 cells in male and female meninges, but also revealed a significantly greater expansion of regulatory T cells and natural killer (NK) cells in female mice (Figure 3B–C).

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As NK cells are traditionally considered pro-inflammatory including in the context of pain (Greisen et al., 1999; Das et al., 2018) and microglial activation (Garofalo et al., 2020), whereas Tregs are potent suppressors of inflammation, we next asked whether Tregs in females counter the CSF1-induced microglial activation and pain. To acutely deplete Tregs, we administered diphtheria toxin to FoxP3^DTR mice (Sakaguchi et al., 2008; Ali et al., 2017; Da Costa et al., 2019; Kim et al., 2007; Figure 3—figure supplement 1D,E). From these mice, we transcriptionally profiled female microglia after CSF1 intrathecal injection in the control or Treg depleted setting (Figure 3D–F/Supplementary file 3). We found that female microglia expressed many of the male-specific CSF1 induced genes, including genes involved in immune activation and recruitment (Clec7a, Il1rn, Ccl3, Ccl4, and Ctsb; Figure 3E–F). We also observed alterations of genes that are unique to the Treg-depleted context (Figure 3—figure supplement 1F). We conclude that Treg depletion partly restores the pro-inflammatory microglial response to CSF1 in female mice.

Finally, we tested whether Tregs suppress CSF1-induced mechanical hypersensitivity in female mice. We depleted Tregs in FoxP3^DTR mice by administering diphtheria toxin prior to CSF1 injection (Figure 3G). Compared to wild-type (WT) females, Treg depletion in females led to a 33% increase in mechanical hypersensitivity (Figure 3H–I; summarizes D3 timepoint from Figure 1A). This effect was phenocopied in Rag1^−/−, which lack T- and B-cells from birth but retain innate lymphocytes, such as NK cells (Figure 3J) and the findings are reminiscent of those reported in Rag1^−/− female after peripheral nerve injury (Sorge et al., 2015). Of note, depleting Tregs in males did not alter their mechanical hypersensitivity (Figure 3—figure supplement 1G). Acute antibody blockade of CD4⁺ T-cells, which include both suppressive (Tregs) and inflammatory subsets (Th1/Th2), also phenocopied this increase in mechanical hypersensitivity (Figure 3K; Figure 3—figure supplement 1H-I). Taken together, we demonstrate that this difference reflects a suppressive effect of Tregs on the CSF1-mediated immune activation in female mice, rather than a direct pain-mediating effect of T-cells on dorsal horn pain circuitry.

Our identification of a sex-specific interaction between spinal cord microglia and Tregs that mediates male/female differences in a model of neuropathic pain has several important implications. First, we defined the immune activation profile of CSF1 on microglia in vivo and demonstrated robust expansion of lymphocytes within the spinal cord meninges in response to CSF1. These results are consistent with a model in which one function of CSF1-stimulated myeloid cells is to recruit other immune cells that in turn release cytokines and chemokines to impact microglial function. However, the nature of this immune response is strikingly sex-specific. In males, the balance tips toward pro-inflammatory signaling. In females, Tregs suppress inflammatory activation and limit mechanical hypersensitivity development, despite expansion of the myeloid and lymphoid compartments. As intrathecal CSF1 induces mechanical hypersensitivity in Treg-depleted female mice, we concur that female microglia are indeed competent to contribute to pain hypersensitivity (Yi et al., 2021; Sorge et al., 2015). However, our results demonstrate that CSF1-mediated cross-talk between spinal cord microglia and lymphocytes can either amplify or suppress pain phenotypes.

Our findings also introduce spinal cord meninges as a potentially relevant source of immune cells that coordinate microglial responses in the setting of neuropathic pain. Importantly, in contrast with a previous report (Costigan et al., 2009), we rarely detected lymphocytes, including T-cells, in the spinal cord, even after nerve injury (Figure 3—figure supplement 2). However, we found that immune cells markedly expand within the spinal cord meninges, even when absent from the parenchyma. As lymphocytes act primarily via secreted cytokines, we suggest that release of meningeal-derived cytokines impacts microglial function as well as directly impacts nociceptors (Liu et al., 2014). Although our report focuses on the contribution of Tregs, we also detected a female-specific increase in meningeal NK cells in response to CSF1. NK cells are classically associated with pro-inflammatory responses, however, recent studies highlight their more diverse functions. These include instruction of anti-inflammatory astrocytes from meningeal NK cells (Sanmarco et al., 2021), beneficial effects after peripheral nerve injury (Davies et al., 2019), and a negative correlation between NK cells in the cerebrospinal fluid and mechanical pain sensitivity in chronic neuropathic pain patients (Lassen et al., 2021). The function of meningeal NK cells in CSF1-induced pain in mice remains to be determined.

In the setting of injury, inflammatory signaling at multiple access points (e.g., injury site, nerve, and DRG) activates nociceptive circuits (Yu et al., 2020). However, our finding that intrathecal activation of myeloid cells is sufficient to activate meningeal immunity raises the possibility that modulating the meninges is a potential therapeutic avenue of neuropathic pain management, by suppressing meningeal Treg expansion-mediated microglial activation or by the release of intrathecal immune modulators that override peripheral inflammatory cues. Given that human genetic analyses and other studies indicate a contribution of Tregs and their dominant cytokines in neuropathic and inflammatory pain models (Davoli-Ferreira et al., 2020; Fischer et al., 2019; Milligan et al., 2006; Eijkelkamp et al., 2016; Echeverry et al., 2009; Kringel et al., 2018), further investigations of Treg localization and impact on microglia will be relevant to understanding the generation and conceivably the treatment of nerve-injury-induced chronic pain.

RNA sequencing data are available through GEO accession #GSE 184801 All data generated or analysed during this study and required for conclusions to be drawn are included in the manuscript and supporting files. The upload can be identified at the following link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE184801.

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Acceptance summary:

This manuscript is of broad relevance to the research areas of pain and neuroimmunology, and is of particular interest for its investigation of sex differences in pain sensation. The data support the major conclusions, and highlight a previously unappreciated role for specific types of immune cells residing in the spinal cord envelope and how they can influence the nervous system by acting on microglia within the spinal cord.

Decision letter after peer review:

Thank you for submitting your article "Regulatory T-cells inhibit microglia-induced pain hypersensitivity in female mice" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kate Wassum as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Claire E Le Pichon (Reviewer #1); Long-Jun Wu (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. We are pleased to say that all agree this is an interesting and exciting study that would be suitable for eLife after a few revisions. The consensus is that some additional information should be added to help clarify a few important details. Specifically, the reviewers ask for evidence relating to the role of Treg in microglia-mediated pain control in males and that you discuss how meningeal Treg remotely communicate with microglia in the parenchyma of spinal cord. Ideally, these answers would be given through the inclusion of new data, or where not possible, at least discussed. Please refer to the specific reviewers' comments for details. We look forward to reading your revised manuscript when it is ready.

Reviewer #1:

The authors start with the discovery of a sex difference in intrathecally injected (i.t.) CSF1-induced pain behavior. Females do not develop the hypersensitivity observed in males after this treatment. The authors then go after the mechanism that would explain this sex difference and focus on one facet of how this occurs (a sex-specific role of regulatory T cells).

In previous work (Guan et al. 2016), the same authors had investigated the mechanism(s) underlying pain sensitivity following nerve injury. They had shown that nerve injury causes upregulation of the cytokine Csf1 in sensory neurons and a microglial response in the spinal cord, and that deletion of this gene in these neurons prevents the microglial activation and the pain behavior. They hypothesized that CSF1 from sensory neurons is secreted at their spinal terminals where microglia expressing the CSF1 receptor (Csf1r) engage in signaling that ultimately results in pain hypersensitivity behavior. They also showed the sufficiency of CSF1 to produce at least transient pain behavior by i.t. injection of CSF1 protein in the absence of a nerve injury. However all this was performed only in male mice.

In the current study, the authors reproduce this data in males and discover an intriguing sex difference. In females, i.t. CSF1 (in the absence of nerve injury) does not cause microglial activation nor pain hypersensitivity as it does in males. An advance over their previous study is a more in-depth characterization of the nature of the microglial activation, using transcriptional profiling and morphological analysis. By both these readouts, the microglia are significantly less activated in females. The authors hypothesize this dampening of microglial activation in females results from regulation of microglia by other immune cells residing in the meninges. The authors investigate the lymphocyte composition in the spinal cord meninges after i.t. CSF1 and observe that regulatory T cells (Treg) increase to a greater extent in females than males, along with other changes in lymphocyte populations that are differentially altered by sex. They ask whether Tregs are responsible for dampening the microglial activation in females. Remarkably, Treg depletion by multiple methods results in females now resembling males, with an increased microglial activation and pain behavior following i.t. CSF1.

Overall, this data in this study convincingly supports the major conclusions that (1) the immune response to i.t. CSF1 differs between sexes, and (2) meningeal lymphocytes play important roles in regulating microglial function. The methods employed are well suited to test the authors' hypotheses, and the authors consistently use multiple methods to converge on a given result (e.g. microglial activation in Figures 1 and 2 or Treg depletion in Figure 3G-K) which makes the data very convincing overall. The microglial profiling by sex is of high interest and provides a resource for the community to mine and to dig deeper into how the microglial activation might alter von Frey reflexive behavior.

The follow-up investigation of how this microglial activation may be repressed in females is only partial. It is restricted just to the role of Tregs and only in female mice, which may provide an incomplete picture of the mechanisms involved. However, it is nevertheless convincing as an investigation of one aspect of meningeal lymphocyte-dependent regulation of microglial function that then impacts neural circuits and behavior.

In summary, this well-written and -presented study opens up many interesting questions and directions for future work in the pain field and more broadly for other neurological conditions investigating the influence of meningeal lymphocytes on parenchymal inflammation that may be differentially regulated by sex.

Comments for the authors:

One useful control would be to actually show independent data for the citation of reference 32 (Costigan et al) that "lymphocytes including T cells are only rarely detected in the spinal cord, even after nerve injury". Demonstrating this would help solidify the authors' novel finding that lymphocytes do not infiltrate the parenchyma, but rather signal from the meninges.

Another recommendation is an experiment that would clarify to what extent the i.t. CSF1 mirrors CSF1 upregulation in the context of SNI (and therefore validity for neuropathic pain). The authors could examine SNI-induced meningeal lymphocyte composition. One hypothesis for how i.t. CSF1 and SNI may differ mechanistically is the exact location of CSF1 release (parenchymal vs meningeal) and thus which CSF1R-expressing myeloid cells it is able to act on. In other words – does CSF1 originating from neurons actually cause meningeal lymphocyte proliferation? (In Figure S3 the authors show that myeloid cells increase in the meninges after i.t. CSF1.)

An acknowledgement of the complexity of the many differences shown in Figure 3B would be a welcome addition before focusing on the Tregs.

Along similar lines, is it possible the CSF-1 dependent increase in NK cells in females shown in Figure 3B might influence microglial function despite the pro-inflammatory role that is cited (line 189-190)?

A control experiment depleting Tregs in males is not included so it would be helpful to hear from the authors about why this may or may not have helped their study. From Figure 3B, Tregs do increase in males, although not to the same extent as in females. Similarly, would it be possible to induce Treg proliferation in males and test whether this now renders them female-like? A comment on the feasibility of these experiments would be appreciated, especially since the authors allude to conceptually similar strategies to potentially tune pain up or down (lines 257-259).

Reviewer #2:

The concept of crosstalk between spinal cord microglia and meningeal lymphocytes to control the sense of pain is intriguing and trendy in the field. The phenotypes they show in gender difference and Treg dependent pain control in females are stunning. This is a well-written and enjoyable manuscript for readers.

It will be reader's interest to know whether Tregs play a role in pain control only in females or also alleviate male's mechanical hypersensitivity at certain level. Although depletion of Treg in females recapitulates mechanical hypersensitivity induced by CSF1 in males, evidence suggests Treg may also play a role in males. For example, in figures 3 and 4, males show an increased Treg population after CSF1 injection compared to the saline control. In figure 3D, the PCA shows that microglial gene expression profiles in Treg depleted, CSF1 treated females are closer to "basal levels (saline treated)" in males, but are distant from "CSF1 treated" male microglia. The authors should include the male CSF1 treated, Treg depleted dataset to support the idea that Treg's effect is gender biased.

Reviewer #3:

In this study, Kuhn et al. investigated the sexual dimorphism in the contribution of microglia to chronic pain in mice. The study nicely extended their previous work on microglial CSF1 signaling in pain to sex dependence. Here, the authors started with an interesting phenomenon that CSF1 induced pain in female but not male mice. They further showed that CSF1 induced more immune activation in male than female mice. More interestingly, the study described CSF1-mediated cross talk between spinal cord microglia and lymphocytes from spinal cord meninges, and Treg can suppress the pain phenotype in female mice. Overall, the study is well designed and performed using multidisciplinary approaches, including behavioral test, FACs, RNAseq, and DTR mediated Treg ablation. Although it is relatively brief and lacks detailed mechanistic exploration, the results are exciting to understand an intriguing cellular mechanism of sex differences in neuropathic pain.

Reviewer #1:

[…] Comments for the authors:

One useful control would be to actually show independent data for the citation of reference 32 (Costigan et al) that "lymphocytes including T cells are only rarely detected in the spinal cord, even after nerve injury". Demonstrating this would help solidify the authors' novel finding that lymphocytes do not infiltrate the parenchyma, but rather signal from the meninges.

To validate the absence of substantial T cell infiltration upon sciatic nerve injury, we performed SNI in male and female mice. Seven days post injury, we only occasionally observed a CD3+ T cell in the lumbar region of the dorsal spinal cord. We added these data as Supplementary Figure 4 to the manuscript.

Another recommendation is an experiment that would clarify to what extent the i.t. CSF1 mirrors CSF1 upregulation in the context of SNI (and therefore validity for neuropathic pain). The authors could examine SNI-induced meningeal lymphocyte composition. One hypothesis for how i.t. CSF1 and SNI may differ mechanistically is the exact location of CSF1 release (parenchymal vs meningeal) and thus which CSF1R-expressing myeloid cells it is able to act on. In other words – does CSF1 originating from neurons actually cause meningeal lymphocyte proliferation? (In Figure S3 the authors show that myeloid cells increase in the meninges after i.t. CSF1.)

For a variety of reasons, we cannot provide information as to changes in meningeal Tregs after sciatic nerve injury. Even after intrathecal CSF1 we are detecting very few Tregs in the spinal cord meninges (Figures 3B,C). In contrast to intrathecal injections, sciatic nerve injury would induce a much more localized effect limited to the ipsilateral lumbar spinal cord making it very unlikely that we could detect a change in Tregs with a reasonable number of animals after SNI. For this reason, we respectfully request that this experiment not be required for the revision.

An acknowledgement of the complexity of the many differences shown in Figure 3B would be a welcome addition before focusing on the Tregs.

We modified the text to address this question.

Along similar lines, is it possible the CSF-1 dependent increase in NK cells in females shown in Figure 3B might influence microglial function despite the pro-inflammatory role that is cited (line 189-190)?

We added a short paragraph about NK cells to the discussion.

A control experiment depleting Tregs in males is not included so it would be helpful to hear from the authors about why this may or may not have helped their study.

To address this question, we repeated the experiment originally performed in females and now examined male mice. In contrast to female mice, ablation of Tregs using diphtheria toxin in Foxp3-DTR male mice did not alter mechanical withdrawal thresholds in response to intrathecal CSF1 (Suppl. Figure 3G). We conclude that Tregs do not modulate intrathecal CSF1-induced pain behavior in male mice.

From Figure 3B, Tregs do increase in males, although not to the same extent as in females. Similarly, would it be possible to induce Treg proliferation in males and test whether this now renders them female-like? A comment on the feasibility of these experiments would be appreciated, especially since the authors allude to conceptually similar strategies to potentially tune pain up or down (lines 257-259).

To induce Treg proliferation in spinal cord meninges of male mice, we injected i.p. and i.t. a combination of IL2/IL2RA antibodies, a well-established system to drive Treg proliferation. Unfortunately, injection of IL2/IL2RA alone resulted in a significant drop in mechanical thresholds compared to saline-treated mice. Intrathecal CSF1 after IL2/IL2RA treatment did not induce an additional drop in pain thresholds. Although this experiment might suggest that Tregs can inhibit CSF1-induced pain in male mice, we cannot draw this conclusion due to the lower pain thresholds in the IL2/IL2RA treated mice.

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Reviewer #2:

[…] It will be reader's interest to know whether Tregs play a role in pain control only in females or also alleviate male's mechanical hypersensitivity at certain level. Although depletion of Treg in females recapitulates mechanical hypersensitivity induced by CSF1 in males, evidence suggests Treg may also play a role in males. For example, in figures 3 and 4, males show an increased Treg population after CSF1 injection compared to the saline control. In figure 3D, the PCA shows that microglial gene expression profiles in Treg depleted, CSF1 treated females are closer to "basal levels (saline treated)" in males, but are distant from "CSF1 treated" male microglia. The authors should include the male CSF1 treated, Treg depleted dataset to support the idea that Treg's effect is gender biased.

To better understand the role of Tregs in regulating CSF1-induced pain in male mice, we depleted Tregs in Foxp3-DTR male mice with diphtheria toxin, prior to intrathecal CSF1 injections. In contrast to female mice, we did not observe a difference in mechanical withdrawal thresholds between control and Treg depleted male mice (Suppl. Figure 3G), indicating that Treg depletion in males is not sufficient to alter pain thresholds in this model, even though we detected an increase in meningeal Tregs in response to CSF1. Based on this experiment, we believe that an additional microglia RNASeq gene expression analysis from Treg depleted male mice is unlikely to reveal key insights into sex specific pain signaling. For this reason, we hope that this experiment is not a requirement for the revision.

Author details

1. Julia A Kuhn

   Department of Anatomy, University of California San Francisco, San Francisco, United States

   Present address

   Genentech, South San Francisco, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft

   Contributed equally with

   Ilia D Vainchtein

   Competing interests

   Patent approved on use of CSF1 blockade to treat neuropathic pain (Publication Number WO/2016/057800).

2. Ilia D Vainchtein

   Department of Psychiatry and Behavioral Sciences/Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Julia A Kuhn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3190-8524

3. Joao Braz

   Department of Anatomy, University of California San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

4. Katherine Hamel

   Department of Anatomy, University of California San Francisco, San Francisco, United States

   Contribution

   Data curation, Investigation, Methodology, Validation, Visualization

   Competing interests

   No competing interests declared

5. Mollie Bernstein

   Department of Anatomy, University of California San Francisco, San Francisco, United States

   Contribution

   Data curation, Investigation, Methodology, Validation, Visualization

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2327-5771

6. Veronica Craik

   Department of Anatomy, University of California San Francisco, San Francisco, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

7. Madelene W Dahlgren

   Department of Laboratory Medicine, University of California, San Francisco, San Francisco, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization

   Competing interests

   No competing interests declared

8. Jorge Ortiz-Carpena

   Department of Laboratory Medicine, University of California, San Francisco, San Francisco, United States

   Contribution

   Data curation, Investigation, Methodology, Validation, Visualization

   Competing interests

   No competing interests declared

9. Ari B Molofsky

   Department of Laboratory Medicine, University of California, San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

10. Anna V Molofsky

    Department of Psychiatry and Behavioral Sciences/Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, United States

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing

    For correspondence

    Anna.Molofsky@ucsf.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-4709-2411

11. Allan I Basbaum

    Department of Anatomy, University of California San Francisco, San Francisco, United States

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing

    For correspondence

    allan.basbaum@ucsf.edu

    Competing interests

    Reviewing editor, eLife

    "This ORCID iD identifies the author of this article:" 0000-0002-1710-6333

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