Madelene W. Dahlgren

<h2>Summary</h2> Type 2 lymphocytes promote both physiologic tissue remodeling and allergic pathology, yet their physical tissue niches are poorly described. Here, we used quantitative imaging to define the tissue niches of group 2 innate lymphoid cells (ILC2s), which are critical instigators of type 2 immunity. We identified a dominant adventitial niche around lung bronchi and larger vessels in multiple tissues, where ILC2s localized with subsets of dendritic and regulatory T cells. However, ILC2s were most intimately associated with adventitial stromal cells (ASCs), a mesenchymal fibroblast-like subset that expresses interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP). <i>In vitro</i>, ASCs produced TSLP that supported ILC2 accumulation and activation. ILC2s and IL-13 drove reciprocal ASC expansion and IL-33 expression. During helminth infection, ASC depletion impaired lung ILC2 and Th2 cell accumulation and function, which are in part dependent on ASC-derived IL-33. These data indicate that adventitial niches are conserved sites where ASCs regulate type 2 lymphocyte expansion and function.

Peripheral nerve injury-induced neuropathic pain is a chronic and debilitating condition characterized by mechanical hypersensitivity. We previously identified microglial activation via release of colony-stimulating factor 1 (CSF1) from injured sensory neurons as a mechanism contributing to nerve injury-induced pain. Here, we show that intrathecal administration of CSF1, even in the absence of injury, is sufficient to induce pain behavior, but only in male mice. Transcriptional profiling and morphologic analyses after intrathecal CSF1 showed robust immune activation in male but not female microglia. CSF1 also induced marked expansion of lymphocytes within the spinal cord meninges, with preferential expansion of regulatory T-cells (Tregs) in female mice. Consistent with the hypothesis that Tregs actively suppress microglial activation in females, Treg deficient (Foxp3DTR) female mice showed increased CSF1-induced microglial activation and pain hypersensitivity equivalent to males. We conclude that sexual dimorphism in the contribution of microglia to pain results from Treg-mediated suppression of microglial activation and pain hypersensitivity in female mice.

Allergic immunity is orchestrated by group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells prominently arrayed at epithelial- and microbial-rich barriers. However, ILC2s and Th2 cells are also present in fibroblast-rich niches within the adventitial layer of larger vessels and similar boundary structures in sterile deep tissues, and it remains unclear whether they undergo dynamic repositioning during immune perturbations. Here, we used thick-section quantitative imaging to show that allergic inflammation drives invasion of lung and liver non-adventitial parenchyma by ILC2s and Th2 cells. However, during concurrent type 1 and type 2 mixed inflammation, IFNγ from broadly distributed type 1 lymphocytes directly blocked both ILC2 parenchymal trafficking and subsequent cell survival. ILC2 and Th2 cell confinement to adventitia limited mortality by the type 1 pathogen Listeria monocytogenes. Our results suggest that the topography of tissue lymphocyte subsets is tightly regulated to promote appropriately timed and balanced immunity.

<h2>Summary</h2> Aberrant tissue-immune interactions are the hallmark of diverse chronic lung diseases. Here, we sought to define these interactions in emphysema, a progressive disease characterized by infectious exacerbations and loss of alveolar epithelium. Single-cell analysis of human emphysema lungs revealed the expansion of tissue-resident lymphocytes (TRLs). Murine studies identified a stromal niche for TRLs that expresses <i>Hhip</i>, a disease-variant gene downregulated in emphysema. Stromal-specific deletion of <i>Hhip</i> induced the topographic expansion of TRLs in the lung that was mediated by a hyperactive hedgehog-IL-7 axis. 3D immune-stem cell organoids and animal models of viral exacerbations demonstrated that expanded TRLs suppressed alveolar stem cell growth through interferon gamma (IFNγ). Finally, we uncovered an IFNγ-sensitive subset of human alveolar stem cells that was preferentially lost in emphysema. Thus, we delineate a stromal-lymphocyte-epithelial stem cell axis in the lung that is modified by a disease-variant gene and confers host susceptibility to emphysema.

The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection.

Perivascular, mammalian adventitial cuffs are enriched in specialized stromal cells and tissue-resident immune cells, acting as outposts for regional tissue immunity. Adventitial cuffs are dynamic environments that channel interstitial fluid from tissue parenchyma to lymphatics. Stromal cells are heterogenous and shape discrete functional niches in tissues. Stromal cell subset(s) can support the differentiation and maintenance of innate lymphocytes in peripheral tissues. Inflammation must be effective, while limiting excessive tissue damage. To walk this line, immune functions are grossly compartmentalized by innate cells that act locally and adaptive cells that function systemically. But what about the myriad tissue-resident immune cells that are critical to this balancing act and lie on a spectrum of innate and adaptive immunity? We propose that mammalian perivascular adventitial ‘cuffs’ are conserved sites in multiple organs, enriched for these tissue-resident lymphocytes and dendritic cells, as well as lymphatics, nerves, and subsets of specialized stromal cells. Here, we argue that these boundary sites integrate diverse tissue signals to regulate the movement of immune cells and interstitial fluid, facilitate immune crosstalk, and ultimately act to coordinate regional tissue immunity. Inflammation must be effective, while limiting excessive tissue damage. To walk this line, immune functions are grossly compartmentalized by innate cells that act locally and adaptive cells that function systemically. But what about the myriad tissue-resident immune cells that are critical to this balancing act and lie on a spectrum of innate and adaptive immunity? We propose that mammalian perivascular adventitial ‘cuffs’ are conserved sites in multiple organs, enriched for these tissue-resident lymphocytes and dendritic cells, as well as lymphatics, nerves, and subsets of specialized stromal cells. Here, we argue that these boundary sites integrate diverse tissue signals to regulate the movement of immune cells and interstitial fluid, facilitate immune crosstalk, and ultimately act to coordinate regional tissue immunity. the outermost layer of connective tissue supporting larger vessels or other tubular structures. small lymphoid aggregates that develop postnatally in the small intestinal lamina propria. professional antigen-presenting cells with the capacity to process and present antigen and prime naïve T cells. Subpopulations of DCs traffic from peripheral tissues to draining lymph nodes to initiate adaptive immune responses. nonclassical lymphoid aggregates (TLOs) found in adipose tissues in both human and mouse. heterogenous nonhematopoietic stromal cells residing in secondary lymphoid organs (SLOs), defined by the expression of PDGFRα and gp38 (podoplanin) and the absence of the endothelial marker CD31 (PECAM-1). Phenotypically and functionally similar populations of FRC-like stromal cells are recognized in diverse non-SLO tissues. ectopic lymphoid tissue (TLOs) induced by infection or inflammation in both human and mouse lungs. lymphocytes that are predominantly tissue-resident, developmentally deployed, lack the expression of antigen-specific receptors, and rapidly respond to tissue-derived signals. Divided into three major subgroups (ILC1–3) that functionally resembles the Th1, Th2, and Th17 subsets of corresponding CD4+ T helper cells. reabsorb excess interstitial fluid from peripheral tissue, transporting immune cells and antigen to draining lymph nodes; and can expand in response to inflammation. Lymphatic endothelial cells produce signals that can influence immune cell activation, survival, and trafficking. a group of group 3 innate lymphoid cells that are essential for the formation of lymph nodes and intestinal Peyer’s patches via crosstalk with specialized stromal cells. heterogenous adult, nonhematopoietic stromal cells with the potential to differentiate into all mesodermal lineages (bone, muscle, cartilage, fat) in vitro. Adult mesenchymal cells do not all have multipotency and the in vivo multipotency of many tissue MSCs is not well established. MSCs are widely distributed in tissues and are studied for their immunomodulatory and reparative properties. perivascular stromal cells that wrap around endothelial cells of small arterioles, venules, and capillaries to maintain vascular integrity and to regulate blood flow. organized lymphoid structures found in the submucosal layer of the small intestine. include spleen and lymph nodes that are embryonically formed and interconnected via the lymphatic system and blood circulation, allowing adaptive lymphocytes to recirculate systemically. SLOs are organized by specialized stromal cells to facilitate antigen encounter and coordinate efficient immune responses. loosely defined group of nonhematopoietic cells, often derived from the mesenchyme or mesodermal layer during development. Stromal cells lack the obvious cell polarity of an epithelial cell and produce extracellular fibers and ECM components. ‘Mesenchymal cells’ (Box 2) is a term often used interchangeably with ‘stromal cells’. inflammation-induced lymphocyte aggregates that form postnatally in tissues, often in perivascular areas, and sometimes bear tissue-specific names (iBALT in lung, FALCs in fat, SALT in skin). They range in size from small (loose lymphoid aggregates), to fully organized structures (with B cell follicles that resemble SLOs). Also referred to as tertiary lymphoid tissue. subpopulations of CD4+ and CD8+ effector memory T cells that remain in tissues following antigen clearance, and provide increased protection against subsequent challenges. Commonly identified by CD69 expression, absence of CCR7, and variable expression of CD103. They adapt to their immediate environment and display striking similarities to tissue-resident innate lymphocytes. enhanced responsiveness of previously activated cells that lacks antigen-specific receptors, including macrophages, innate lymphocytes, and stromal cells, upon rechallenge. subset of CD4+ T cells that express FoxP3 and CD25 and suppress effector T cells, maintain tolerance to self-antigens, and prevent autoimmune responses.

Systemic immunization with soluble flagellin (sFliC) from Salmonella Typhimurium induces mucosal responses, offering potential as an adjuvant platform for vaccines. Moreover, this engagement of mucosal immunity is necessary for optimal systemic immunity, demonstrating an interaction between these two semi-autonomous immune systems. Although TLR5 and CD103+CD11b+ cDC2 contribute to this process, the relationship between these is unclear in the early activation of T cells and the development of antigen-specific B cell responses. In this work, we use TLR5-deficient mice and CD11c-cre.Irf4fl/fl mice (which have reduced numbers of cDC2, particularly intestinal CD103+CD11b+ cDCs), to address these points by studying the responses concurrently in the spleen and the mesenteric lymph nodes (MLN). We show that CD103+CD11b+ cDC2 respond rapidly and accumulate in the MLN after immunization with sFliC in a TLR5-dependent manner. Furthermore, we identify that whilst CD103+CD11b+ cDC2 are essential for the induction of primary T and B cell responses in the mucosa, they do not play such a central role for the induction of these responses in the spleen. Additionally, we show the involvement of CD103+CD11b+ cDC2 in the induction of Th2-associated responses. Since in CD11c-cre.Irf4fl/fl mice showed a reduced primary FliC-specific IgG1 responses, but enhanced Th1-associated IgG2c responses. These data expand our current understanding of the mucosal immune responses promoted by sFliC and highlights the potential of this adjuvant for vaccine usage by taking advantage of the functionality of mucosal CD103+CD11b+ cDC2.

Antibody responses induced at mucosal and nonmucosal sites demonstrate a significant level of autonomy. Here, we demonstrate a key role for mucosal interferon regulatory factor-4 (IRF4)-dependent CD103+CD11b+ (DP), classical dendritic cells (cDCs) in the induction of T-dependent immunoglobulin G (IgG) and immunoglobulin A (IgA) responses in the mesenteric lymph node (MLN) following systemic immunization with soluble flagellin (sFliC). In contrast, IRF8-dependent CD103+CD11b- (SP) are not required for these responses. The lack of this response correlated with a complete absence of sFliC-specific plasma cells in the MLN, small intestinal lamina propria, and surprisingly also the bone marrow (BM). Many sFliC-specific plasma cells accumulating in the BM of immunized wild-type mice expressed α4β7+, suggesting a mucosal origin. Collectively, these results suggest that mucosal DP cDC contribute to the generation of the sFliC-specific plasma cell pool in the BM and thus serve as a bridge linking the mucosal and systemic immune system.

Type I interferons (IFNs) are essential for antiviral immunity, appear to represent a key component of mRNA vaccine-adjuvanticity, and correlate with severity of systemic autoimmune disease. Relevant to all, type I IFNs can enhance germinal center (GC) B cell responses but underlying signaling pathways are incompletely understood. Here, we demonstrate that a succinct type I IFN response promotes GC formation and associated IgG subclass distribution primarily through signaling in cDCs and B cells. Type I IFN signaling in cDCs, distinct from cDC1, stimulates development of separable Tfh and Th1 cell subsets. However, Th cell-derived IFN-γ induces T-bet expression and IgG2c isotype switching in B cells prior to this bifurcation and has no evident effects once GCs and bona fide Tfh cells developed. This pathway acts in synergy with early B cell-intrinsic type I IFN signaling, which reinforces T-bet expression in B cells and leads to a selective amplification of the IgG2c+ GC B cell response. Despite the strong Th1 polarizing effect of type I IFNs, the Tfh cell subset develops into IL-4 producing cells that control the overall magnitude of the GCs and promote generation of IgG1+ GC B cells. Thus, type I IFNs act on B cells and cDCs to drive GC formation and to coordinate IgG subclass distribution through divergent Th1 and Tfh cell-dependent pathways.

Group 2 innate lymphoid cells (ILC2s) cooperate with adaptive Th2 cells as key organizers of tissue type 2 immune responses, while a spectrum of innate and adaptive lymphocytes coordinate early type 3/17 immunity. Both type 2 and type 3/17 lymphocyte associated cytokines are linked to tissue fibrosis, but how their dynamic and spatial topographies may direct beneficial or pathologic organ remodelling is unclear. Here we used volumetric imaging in models of liver fibrosis, finding accumulation of periportal and fibrotic tract IL-5 + lymphocytes, predominantly ILC2s, in close proximity to expanded type 3/17 lymphocytes and IL-33 high niche fibroblasts. Ablation of IL-5 + lymphocytes worsened carbon tetrachloride-and bile duct ligation-induced liver fibrosis with increased niche IL-17A + type 3/17 lymphocytes, predominantly γδ T cells. In contrast, concurrent ablation of IL-5 + and IL-17A + lymphocytes reduced this progressive liver fibrosis, suggesting a cross-regulation of type 2 and type 3 lymphocytes at specialized fibroblast niches that tunes hepatic fibrosis.

Cholera toxin (CT) binds to GM1-ganglioside receptors present on all nucleated cells. Despite this, it is a very potent mucosal adjuvant that has a dramatic impact on immune cells, as well as nerve and epithelial cells, causing diarrhea. This fact has hampered our understanding of whether the adjuvanticity of CT is direct or indirect, as cells that bind CT may or may not be involved in its adjuvant function. The mucosal barrier is maintained by tight junctions between epithelial cells but dendritic cells (DCs) can protrude luminal dendrites. Here we investigated which cells are involved in the immune augmenting effect of CT. We explored oral immunizations with ovalbumin (OVA) and CT in bone marrow chimeric mice deficient in GM1-ganglioside in defined cellular subsets. We found that chimeric mice lacking GM1 in nonhematopoietic cells, including epithelial cells, mounted an unaltered intestinal IgA response. In contrast, chimeric mice lacking GM1-expressing hematopoietic cells in general, or specifically GM1-expressing conventional DCs (cDCs), largely failed to elicit anti-OVA adaptive immune responses. Therefore, the adjuvanticity of CT does not require epithelial activation, but is directly dependent on the binding of CT to gut cDCs via GM1-ganglioside. These results could have important implications for the generation of novel oral adjuvants.

Development of long-lived humoral immunity is dependent on CXCR5-expressing T follicular helper (Tfh) cells, which develop concomitantly to effector Th cells that support cellular immunity. Conventional dendritic cells (cDCs) are critical APCs for initial priming of naive CD4(+) T cells but, importantly, also provide accessory signals that govern effector Th cell commitment. To define the accessory role of cDCs during the concurrent development of Tfh and effector Th1 cells, we performed high-dose Ag immunization in conjunction with the Th1-biased adjuvant polyinosinic:polycytidylic acid (pI:C). In the absence of cDCs, pI:C failed to induce Th1 cell commitment and IgG2c production. However, cDC depletion did not impair Tfh cell differentiation or germinal center formation, and long-lived IgG1 responses of unaltered affinity developed in mice lacking cDCs at the time point for immunization. Thus, cDCs are required for the pI:C-driven Th1 cell fate commitment but have no crucial accessory function in relation to Tfh cell differentiation.

Abstract The innate immune system plays essential roles in brain synaptic development, and immune dysregulation is implicated in neurodevelopmental diseases. Here we show that a subset of innate lymphocytes (group 2 innate lymphoid cells, ILC2s) is required for cortical inhibitory synapse maturation and adult social behavior. ILC2s expanded in the developing meninges and produced a surge of their canonical cytokine Interleukin-13 (IL-13) between postnatal days 5-15. Loss of ILC2s decreased cortical inhibitory synapse numbers in the postnatal period where as ILC2 transplant was sufficient to increase inhibitory synapse numbers. Deletion of the IL-4/IL-13 receptor ( Il4ra ) from inhibitory neurons phenocopied the reduction inhibitory synapses. Both ILC2 deficient and neuronal Il4ra deficient animals had similar and selective impairments in adult social behavior. These data define a type 2 immune circuit in early life that shapes adult brain function. One sentence summary Type 2 innate lymphoid cells and Interleukin-13 promote inhibitory synapse development.

Fibroblasts coordinate the response to tissue injury, directing organ regeneration versus scarring. In the central nervous system (CNS), fibroblasts are uncommon cells enriched at tissue borders, and their molecular, cellular, and functional interactions after brain injury are poorly understood. Here we define the fibroblast response to sterile brain damage across time and space. Early pro-fibrotic myofibroblasts infiltrated CNS lesions and were functionally and spatially organized by fibroblast TGF β signaling, pro-fibrotic macrophages and microglia, and perilesional brain glia that activated TGF β via integrin α v β 8 . Early myofibroblasts subsequently transitioned into a variety of late states, including meningeal and lymphocyte-interactive fibroblasts that persisted long term. Interruption of this dynamic fibroblast-macrophage-glial coordination impaired brain wound healing and the resolution of neuroinflammation, disrupted generation of late de novo CNS lymphocyte niches, and increased mortality in a stroke model. This work highlights an unexpected role of fibroblasts as coordinate regulators of CNS healing and neuroinflammation after brain injury.

Group 2 innate lymphoid cells (ILC2) are a subset of innate lymphocytes that responds to local, tissue-derived signals and initiates allergic immune responses. ILC2 activation promotes the recruitment of eosinophils, polarization of alternatively activated macrophages, and tissue-remodeling, processes associated with the ‘weep and sweep’ response to helminthic worm colonization and infection. ILC2s also coordinate both physiologic and pathologic type 2 allergic immune responses, including promoting normal tissue development and remodeling and driving allergic pathology such as atopic dermatitis and allergic asthma. In this review we summarize recent advances in ILC2 biology, particularly focusing on how local cells and signals coordinately regulate ILC2s, how this may influence physiologic processes, and how ILC2 cooperate with adaptive T helper type 2 cells to drive pathologic allergic inflammation.

The protective effect of most currently available vaccines is dependent on high-affinity antibodies and long lived B cell memory, which develop within organized structures of secondary lymphoid organs called germinal centers (GCs). GC formation is supported by T follicular helper (Tfh) cells, a subset of CD4+ T cells that develop in parallel to other effector T cells and specifically localizes to B cell follicles. The aim for the work presented in this thesis was to define how the GC response and Tfh cell differentiation are regulated by innate effectors, such as conventional dendritic cells (cDCs) and type I interferons (IFNs), in response to protein immunization adjuvanted by the dsRNA-analogue poly(I:C). In paper I, we show that Tfh cells and GC B cell responses of the IgG1 isotype can develop normally in the absence of cDCs when a sufficient amount of antigen, allowing initial T cell priming, is provided. In contrast, the concurrent Th1 cell differentiation is impaired together with a selective loss of IgG2c production. We also find that B cells, monocytes and possibly plasmacytoid DCs (pDCs) redundantly can prime CD4+ T cells in the absence of cDCs, and thereby support early expression of the Tfh cell-associated chemokine receptor CXCR5. In paper II we find that type I IFNs predominantly promote IgG2c+ GC B cell differentiation and in this context function through both B cell intrinsic and extrinsic signaling. While we provide evidence for that direct IFN-γ signaling in B cells controls switching to IgG2c, our results indicate that type I IFNs regulate IgG2c-associated GC responses beyond isotype switching, as the magnitude of the GC B cell response is reduced when type I IFNs, but not when IFN-γ, are absent. In contrast to IgG2c, switching to IgG1 is unaffected by type I IFN deficiency, although the overall magnitude and quality of the IgG1 response also is affected when type I IFN signaling is abrogated. Additionally, the formation of B cell memory appears to be impaired in the absence of type I IFNs. In paper III, we demonstrate how clonal competition selectively affects Tfh cell differentiation, GC responses and generation of IgG1+ GC B cells, while the generation of IgG2c+ GC B cells appears to be less affected. Taken together, the work included in this thesis increase our understanding of how GC B cell responses and Tfh cell development are regulated, and furthermore, it suggests that switching to IgG1 and IgG2c associated GC B cell fate commitment may be differentially dependent on Tfh cells. (Less)

Abstract Mobilization of type 2 immune responses is critical to both physiologic tissue remodeling and allergic pathology such as allergic asthma. However, whether there are physical tissue niches with unique cellular constituents that mediate type 2 immunity is unknown. We used quantitative 3D-imaging to define tissue niches of group 2 innate lymphoid cells (ILC2), critical instigators of type 2 immunity. We identified a dominant adventitial ‘cuff’ niche around vessels, airways, and ducts, present in the lung and multiple other tissues. ILC2s, as well as subsets of other tissue-resident hematopoietic cells, localized to these sites. However, ILC2s were most intimately associated with fibroblast-like adventitial stromal cells (ASCs) that were functionally distinct and sufficient to support ILC2 survival and activation. Here we discuss data supporting a model where ILC2s engage in a conversation with micro-anatomically and functionally distinct adventitial mesenchymal cells and other niche components, creating dynamic type 2-biased tissue foci that regulate tissue allergic immunity.

Abstract Type I interferons (IFNs) play an essential role in antiviral immunity, correlate with severity of systemic autoimmune disease, and are likely to represent a key component of mRNA vaccine-adjuvanticity. Relevant to all, type I IFNs can enhance germinal center (GC) B cell responses but underlying signaling pathways are incompletely understood due to pleiotropic effects in multiple cell types. Here, we demonstrate that a succinct type I IFN response promotes GC formation and associated IgG subclass distribution primarily through signaling in cDCs and B cells. Type I IFN signaling in cDCs, distinct from cDC1, stimulates development of separable Tfh and Th1 cell subsets. However, Th cell-derived IFN-γ induces T-bet expression and IgG2c isotype switching prior to this bifurcation and has no evident effects once GCs and bona fide Tfh cells developed. This pathway acts in synergy with early B cell-intrinsic type I IFN signaling, which reinforces T-bet expression in B cells and leads to a selective amplification of the IgG2c + GC B cell response. Despite the strong Th1 polarizing effect of type I IFNs, the Tfh cell subset develops into IL-4 producing cells that control the overall magnitude of the GCs and promote generation of IgG1 + GC B cells. Thus, type I IFNs act on B cells and cDCs to drive GC formation and to coordinate IgG subclass distribution through parallel Th1 and Tfh cell-dependent pathways.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Peripheral nerve injury-induced neuropathic pain is a chronic and debilitating condition characterized by mechanical hypersensitivity. We previously identified microglial activation via release of colony-stimulating factor 1 (CSF1) from injured sensory neurons as a mechanism contributing to nerve injury-induced pain. Here, we show that intrathecal administration of CSF1, even in the absence of injury, is sufficient to induce pain behavior, but only in male mice. Transcriptional profiling and morphologic analyses after intrathecal CSF1 showed robust immune activation in male but not female microglia. CSF1 also induced marked expansion of lymphocytes within the spinal cord meninges, with preferential expansion of regulatory T-cells (Tregs) in female mice. Consistent with the hypothesis that Tregs actively suppress microglial activation in females, Treg deficient (Foxp3DTR) female mice showed increased CSF1-induced microglial activation and pain hypersensitivity equivalent to males. We conclude that sexual dimorphism in the contribution of microglia to pain results from Treg-mediated suppression of microglial activation and pain hypersensitivity in female mice. Introduction 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 (FoxP3DTR) 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. Results 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 AvilCre:Csf1fl/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. Figure 1 with 1 supplement see all Download asset Open asset CSF1 induces pain hypersensitivity and microglial activation in male but not female mice. (A) Schematic depicting 3 days of CSF1 intrathecal injection (i.t.) paradigm with von Frey assay. (B, C) Change in mechanical pain threshold in males and females after saline or CSF1 injection. N=5–7 mice per condition, repeated measures ANOVA. (D) Representative immunohistochemistry of lumbar spinal cord sections after 3 days of CSF1 i.t. injection. Insets indicate single microglia and binary images used for subsequent quantifications. Scale bar=50 µm. (E) Ramification calculated by Scholl analysis in males (blue, top) and females (red, bottom). N=3 mice/condition, 25 cells/group; dots represent individual microglia, Student’s t-test. (F) Representative flow cytometry plot demonstrating right-shift of the CD11b+/CD45+ population in lumbar spinal cord. Insets indicate microglia population gated on CD11b+CD45+Ly-6C−. (G) Microglial activation index calculated from flow-cytometry data as a sum of mean fluorescence intensity of CD11b and CD45 fluorescence intensity. Dots represent individual mice. One-way ANOVA with Tukey’s multiple comparisons. (H) Microglial numbers calculated by flow cytometry data. Dots represent individual mice. One-way ANOVA with Tukey’s multiple comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CSF1, colony-stimulating factor 1. 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, pAdj<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). Figure 2 with 1 supplement see all Download asset Open asset CSF1 promotes immune activation in male but not female microglia. (A) Principal component analysis of genes expressed by microglia isolated by flow cytometry from male and female mice after 3 days of saline or CSF1 i.t. Dots represent individual mice. (B) Number of differentially expressed genes (DEGs) per comparison (adjusted p-value<0.01). (C) Heatmap of DEGs in male and female microglia after CSF1 overlaid with microglia activation modules curated by Friedman et al., 2018. (D) Four-way plot depicting DEGs (adjusted p-values<0.01) that are male-specific (blue), female-specific (red), or male-female shared (green). Inset highlights gene ontology terms identified in the respective categories. CSF1, colony-stimulating factor 1; i.t., intrathecal injection. 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). Figure 3 with 2 supplements see all Download asset Open asset Regulatory T-cells restrict microglial activation and pain behavior in female mice. (A) Schematic of spinal cord meninges. (B) UMAP plot of lymphoid, non-myeloid cells (CD45+CD11b−) isolated from spinal cord meninges. Image is a pool of all samples colored by cell type specific markers as indicated. Bar graph shows fold-change in indicated populations in males and females after CSF1. Dots in bar graph: individual samples. N=5 mice per group. (C) Quantification of regulatory T-cells (Tregs; CD4+FoxP3+) from (B). (D) Principal component analysis (PCA) of microglial gene expression profiles in select conditions. Red=female, blue=male, green=Treg deficient female (FoxP3DTR). Dots: individual mice. PCA consists of two experiments. The first experiment is depicted in Figure 2A and complemented with a second experiment consisting of WT females with CSF1 and Treg deficient females treated with CSF1. (E) Volcano plot depicting DEGs (adjusted p-values<0.05; green) between female Treg KO mice after CSF1 versus female mice after CSF1. N=4 mice per group. (F) Gene ontology terms for upregulated and downregulated genes from volcano plot in (E). (G) Schematic depicting the approach of using Rag1 KO mice (no T/B cells), antibody against CD4 (aCD4) to deplete T-cells and FoxP3DTR mice, in which Tregs are depleted using diphtheria toxin. (H, I) Change in mechanical hypersensitivity at day 3 after i.t. CSF1 in WT female mice (data from day 3, Figure 1B) or in females lacking regulatory T-cells (FoxP3DTR). Dots: individual mice. (J) Change in mechanical hypersensitivity at day three after CSF1 i.t. in Rag1−/−. Dots: individual mice. (K) Change in mechanical hypersensitivity at day 3 after CSF1 in female mice injected with a CD4 blocking antibody 1 day prior to CSF1 injections. Dots: individual mice. In (I–K) unpaired two-tailed t-test and (C) one-way ANOVA with Tukey’s multiple testing correction. *p<0.05, **p<0.01, ****p<0.0001. DEG, differentially expressed gene; WT, wild-type. 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 FoxP3DTR 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 FoxP3DTR 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. Discussion 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. Materials and methods Key resources table Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional informationGene (Mus musculus)Csf1MGIMGI:1339753NCBI Gene: 12,977Gene (M. musculus)Foxp3MGIMGI:1891436NCBI Gene: 20,371Gene (M. musculus)AvilMGIMGI:1333798NCBI Gene: 11,567Strain, strain background (M. musculus, male and female)C57BL/6 JThe Jackson LaboratoryRRID:IMSR_JAX:000664Strain, strain background (M. musculus, male and female)B6.129S7-Rag1tm1Mom/JThe Jackson LaboratoryRRID:IMSR_JAX:002216Strain, strain background (M. musculus, male and female)B6.129(Cg)-Foxp3tm3(DTR/GFP)Ayr/JThe Jackson LaboratoryRRID:IMSR_JAX:016958Strain, strain background (M. musculus, male and female)AvilCreZurborg et al., 2011Strain, strain background (M. musculus, male and female)Csf1fl/flHarris et al., 2012Peptide, recombinant proteinCSF1(M. musculus)Thermo Fisher ScientificCat: #PMC204415 ng or 30 ng in 5 µl (i.t.)Peptide, recombinant proteinDiphtheria Toxin(Corynebacterium diphtheriae)Sigma-AldrichCat: #D056430 ng/g in 200 µl (i.p.)AntibodyMonoclonal rat anti-mouse CD4Clone: GK1.5Bio X CellCat: #BE0003-1250 µg in 200 µl (i.p.)AntibodyPolyclonal Rabbit anti-mouse Iba1WAKOCat: #019-19741IF: (1:2000)AntibodyMonoclonal Alexa 647-coupled rat anti-mouse CD45(clone 30-F11)BioLegendCat: #103123IF: (1:200)AntibodyMonoclonal hamster anti-mouse CD3 (clone 145-2C11)BD BioscienceCat: #553058IF: (1:200)AntibodyMonoclonal PE anti-mouse CD11b (clone M01/70)eBioscienceCat: #12-0112-81FACS (1:200)AntibodyMonoclonal PE/Cy7 anti-mouse CD11b (clone M01/70)eBioscienceCat: #25-0112-81FACS (1:200)AntibodyMonoclonal Brilliant Violet 605-conjugated anti-CD11b (M1/70)Thermo Fisher ScientificCat: #BDB563015FACS (1:400)AntibodyMonoclonal FITC anti-mouse CD45 (clone 30-F11)eBioscienceCat: #11-0451-81FACS (1:200)AntibodyMonoclonal BUV395 anti-mouse CD45 (clone 30-F11)BD BiosciencesCat: #564279FACS (1:400)AntibodyMonoclonal PE/Cy7 anti-mouse CD45 (clone 30-F11)eBioscienceCat: #25-0451-82FACS (1:200)AntibodyMonoclonal APC anti-mouse Ly-6C (clone HK1.4)BioLegendCat: #128016FACS (1:150)AntibodyMonoclonal APC/Cy7 anti-mouse Ly-6C (clone HK1.4)BioLegendCat: #128025FACS (1:150)AntibodyMonoclonal PE anti-mouse CSF1R (clone AFS98)BioLegendCat: #135505FACS (1:100)AntibodyMonoclonal Brilliant Violet 421-conjugated anti-Thy1.2 (clone 53-2.1)BioLegendCat: #140327FACS (1:400)AntibodyMonoclonal PEDazzle594-conjugated anti-CD19 (6D5)BioLegendCat: #115553FACS (1:400)AntibodyMonoclonal Brilliant Violet 711-conjugated anti-CD4 (RM4-5)BioLegendCat: #100549FACS (1:200)AntibodyMonoclonal Brilliant Violet 785-conjugated anti-CD8a (53-6.7)BioLegendCat: #100749FACS (1:200)AntibodyMonoclonal Brilliant Violet 650-conjugated anti-NK1.1 (PK136)BioLegendCat: #108735FACS (1:400)AntibodyMonoclonal Alexa Fluor 700-conjugated anti-CD3 (17A2)BioLegendCat: #100215FACS (1:200)AntibodyMonoclonal AF488-conjugated anti-FoxP3 (FJK-16s)eBioscienceCat: #53-5773-82FACS (1:200)AntibodyMonoclonal PE-conjugated anti-Gata3 (TWAJ)eBioscienceCat: #12-9966-42FACS (1:100)AntibodyMonoclonal anti-mouse CD16/32 antibodyeBioscienceCat: #14-0161-82FACS (1:200)Commercial assay or kitFoxp3/Transcription Factor Staining Buffer SeteBioscience (Thermo Fisher Scientific)Cat. no.: 00-5523-00Commercial assay or kitRNeasy Plus Micro KitQiagenCat. no./ID: 74034Commercial assay or kitAgilent RNA 6000 Pico KitAgilentPart no.: 5067-1513Commercial assay or kitOvation RNA-Seq System V2 KitNuGenPart no.: 7102Commercial assay or kitTrio RNA-Seq KitNuGenPart no.: 0506Commercial assay or kitQubit dsDNA HS Assay KitThermo Fisher ScientificCat no.: Q32851Software, algorithmFiji (ImageJ)Schindelin et al., 2012RRID:SCR_002285Software, algorithmFastQCBabraham InstituteRRID:SCR_011106Software, algorithmSTAR(version 2.5.4b)Dobin et al., 2013Software, algorithmHTSeq(version 0.9.0)Anders et al., 2015RRID:SCR_005514Software, algorithmDESeq2(version 1.24.0)Love et al., 2014RRID:SCR_015687Software, algorithmLimmaRitchie et al., 2015RRID:SCR_010943Software, algorithmMetascapeZhou et al., 2019RRID:SCR_016620OtherZombie NIR(fixable viability dye)BioLegendCat: #423105FACS1:1000OtherDAPISigma-AldrichCat: #95421:1000OtherRLT+QiagenCat: # 1053393 Mice All mouse experiments were approved by UCSF Institutional Animal Care and Use Committee and conducted in accordance with the guidelines established by the Institutional Animal Care and Use Committee and Laboratory Animal Resource Center. All mice were between 8 and 14 weeks old when experiments were performed. Littermate controls were used for all experiments when feasible and all experiments were performed in male and female mice. WT (C57BL/6J) and Rag1 knockout (B6.129S7-Rag1tm1Mom/J; Stock no.: 002216) mice were purchased from The Jackson Laboratory. The following previously described strains were used and bred in house: Csf1fl/fl (Harris et al., 2012), AvilCre (Zurborg et al., 2011), and FoxP3DTR (B6.129(Cg)-Foxp3tm3(DTR/GFP)Ayr/J) (Kim et al., 2007). Injury, injections, and behavioral analysis Request a detailed protocol Spared Nerve Injury (SNI) was performed by ligation and transection of the sural and superficial peroneal branches of the sciatic nerve, leaving the tibial nerve intact (Shields and Eckert, 2003). CSF1 (Life Technologies; PMC2044) was injected intrathecally at low dose (15 ng) or high dose (30 ng) in a total volume of 5 µl for three times over 3 days (24 hr between injections). Behavioral analysis was done 2 hr after injections; mice were euthanized for analysis about 4 hr after the last injection. All Von Frey behavioral experiments were performed during the light cycle as previously reported (Guan et al., 2016) in a blinded manner. Intraperitoneal injection of anti-CD4 (250 µg) (InVivoPlus; Bio X Cell) and Diphtheria toxin (30 ng/g) (Sigma-Aldrich) were all in a volume of 200 µl per injection. Anti-CD4 was given 1 day prior to the start of CSF1 injections, and on day 2 of the CSF1 injections. Diphtheria toxin was given 2 days (two subsequent injections) before the start of the CSF1 injections, and on day 2 of the CSF1 injections. Immunohistochemistry and analysis Request a detailed protocol Avertin-anesthetized mice were transcardially perfused with 1× phosphate-buffered saline (PBS) (~10 ml) followed by 4% (weight/volume) paraformaldehyde (PFA) diluted in PBS (~10 ml). Spinal cord tissue was dissected out and post-fixed in 4 % PFA for 4 hr and then transferred to a 30% sucrose solution overnight. Subsequently, spinal cords were sectioned coronally at 25 µm using a cryostat (Thermo Fisher Scientific). Spinal cord sections were incubated in a blocking solution consisting of 10% normal goat (Thermo Fisher Scientific) and 0.4% Triton (Sigma-Aldrich) diluted in 1× PBS. Primary antibodies included: rabbit anti-mouse Iba1 (WAKO, 1:2000); Alexa 647-coupled mouse anti-CD45 (BioLegend, 1:200); and hamster anti-CD3 (BD BioScience, 1:200). Antibodies were diluted in 10% normal goat with 0.4% Triton in PBS and incubated on a shaker overnight at 4oC. Secondary antibodies (Thermo Fisher Scientific, 1:1000) were diluted in 0.4% Triton in PBS and spinal cord sections were incubated on a shaker for 2 hr at room temperature. Spinal cord sections were mounted on coverslips with DAPI containing Fluoromount-G (Thermo Fisher Scientific). Slides were imaged on an LSM700 (Zeiss) confocal microscope using 63× objectives and z-stacks with a step size of 1 µm were collected. In Fiji (Schindelin et al., 2012) (ImageJ), maximum intensity images were generated and binary, thresholded images for morphology analysis were created. Subsequently, Scholl analysis (Ferreira et al., 2014) was done in Fiji (ImageJ) on microglia from the binary images with a step size of 2.5 µm. Fluorescence-activated cell sorting of microglia Request a detailed protocol To isolate microglia, we followed a previously described method (Galatro et al., 2017). Briefly, lumbar dorsal horn spinal cords were mechanically dissociated in isolation medium (HBSS, 15 mM HEPES, 0.6% glucose, 1 mM EDTA pH 8.0) using a glass tissue homogenizer (VWR). Next, the suspension was filtered through a 70 µm filter and then pelleted at 300×g for 10 min at 4oC. The pellet was resuspended in 22% Percoll (GE Healthcare) and centrifuged at 900×g for 20 min (acceleration set to 4 and deceleration set to 1). The myelin free pelleted cells were then incubated in blocking solution consisting of anti-mouse CD16/32 antibody (eBioscience) for 5 min on ice, followed for 30 min in a mix of PE or PE/Cy7-conjugated anti-mouse CD11b (eBioscience), FITC or PE/Cy7-conjugated anti-mouse CD45 (eBioscience), and APC or APC/Cy7-conjugated anti-mouse Ly-6C (BioLegend) in isolation medium that did not contain phenol red. For flowcytometric analysis of CSF1R expressed by microglia, PE-conjugated anti-mouse CSF1R (BioLegend) was added. The cell suspension was centrifuged at 300×g for 10 min at 4oC and the pellet was incubated with DAPI (Sigma-Aldrich) before sorting. Microglia were sorted on a BD FACS Aria III and gated on forward/side scatter, live cells by DAPI, and CD11bhigh, CD45low, and Ly-6Cneg. After sorting, cells were spun down at 500×g, 4oC for 10 min and the pellet was lysed with RLT+ (Qiagen). Isolation of spinal cord meningeal cells Request a detailed protocol Single-cell suspensions were prepared by digesting dissected spinal cord meninges with Liberase TM (0.208 WU/ml) and DNase I (40 ug/ml) in 1.0 ml cRPMI (RPMI supplemented with 110% (vol/vol) fetal bovine serum (FBS), 1% (vol/vol) Hepes, 1% (vol/vol) Sodium Pyruvate, 1% (vol/vol) penicillin-streptomycin) for 30–40 min at 37°C, 220 RPM. Digested samples were then passed over a 70 µm cell strainer and any remaining tissue pieces macerated with a plunger. Cell strainers were additionally flushed with FACS wash buffer (FWB, PBS w/o Mg2+ and Ca2+ supplemented with 3% FBS and 0.05% NaN3). Single-cell suspensions were washed and resuspended in FWB. Flow cytometry of spinal cord meningeal cells Request a detailed protocol To exclude dead cells from the analysis, single-cell suspensions were stained with a fixable viability dye (Zombie NIR, BioLegend), followed by staining for surface antigens with a combination of the following fluorescence-conjugated mAbs: Brilliant Violet 421-conjugated anti-Thy1.2 (53-2.1) (BioLegend), PEDazzle594-conjugated anti-CD19 (6D5) (BioLegend), Brilliant Violet 605-conjugated anti-CD11b (M1/70) (Thermo Fisher Scientific), Brilliant Violet 711-conjugated anti-CD4 (RM4-5) (BioLegend), Brilliant Violet 785-conjugated anti-CD8a (53-6.7) (BioLegend), Brilliant Violet 650-conjugated anti-NK1.1 (PK136) (BioLegend), Alexa Fluor 700-conjugated anti-CD3 (17A2) (BioLegend), and BUV395-conjugated anti-CD45