Chronic social defeat stress induces meningeal neutrophilia via type I interferon signaling in male mice

Abstract

Inflammation is increasingly recognized as a risk factor for psychiatric disorders. Animal models of stress and stress-related disorders are associated with blood neutrophilia. The mechanistic relevance of this to symptoms or behavior is unclear. We characterized the immune response to chronic social defeat (CSD) stress at brain border regions in male mice. Here we show that chronic, but not acute, stress causes neutrophil accumulation in the meninges-i.e., "meningeal neutrophilia"- but not the brain. CSD promotes neutrophil trafficking to meninges via vascular channels originating from skull bone marrow (BM). Transcriptional analysis suggests CSD increases type I interferon (IFN-I) signaling in meningeal neutrophils. Blocking this pathway via the IFN-I receptor (IFNAR) protects against the negative behavioral effects of CSD stress. Our identification of IFN-I signaling as a putative mediator of meningeal neutrophil recruitment may facilitlate development of new therapies for stress-related disorders.

Fig. 1. Meningeal neutrophils are elevated following chronic social defeat (CSD) stress, which causes depressive-like behavioral changes.

a CSD mice were behaviorally tested on days 10-13, and tissue harvested at day 14. A fluorescently labeled CD45 antibody was retro-orbitally injected to discern blood-exposed meningeal cells. CSD causes expected sexual (b; ****p < 0.0001, U = 13, Hodges-Lehmann estimate = −24.5, n_HC = 16, n_CSD = 13. N = 5 experiments.) and social (c; *p = 0.011, U = 323, Hodges-Lehmann estimate = −4.0, n_HC = 34, n_CSD = 30. N = 10 experiments) anhedonia in USM and SI tests, respectively. In (b), pink arrowheads indicate female stimulus urine, blue arrowheads indicate representative urine scent marks from male test mice. Heatmaps (c, d) indicate animal tracking; red color indicates greater time spent in that location. CSD causes increased anxiety-like behavior in the OF (d; ****p < 0.0001, U = 65, Hodges-Lehmann estimate = −3.4, n_HC = 23, n_CSD = 21. N = 8 experiments) and LD box (e; ***p = 0.0007, U = 38, Hodges-Lehmann estimate = −11.0, n_HC = 15, n_CSD = 16. N = 7 experiments) tests. Flow cytometric analysis shows CSD causes an increase in: (f) iv^- meningeal neutrophils (CD45iv^-; CD11b⁺; Ly6G⁺; Ly6C^int), both as a percent of live CD45⁺ cells (****p < 0.0001, U = 573, Hodges-Lehmann estimate = −6.6, n_HC = 52, n_CSD = 44. N = 19 experiments), and in absolute cell counts (Unpaired t test, *p = 0.038, t = 2.2, df = 22, 95% CI [128.4, 4107], n_HC = 14, n_CSD = 10. N = 4 experiments); (g) iv⁺ meningeal neutrophils (CD45iv⁺)^, as a percentage (Unpaired t test, **p = 0.0024, t = 3.1, df = 94, 95% CI [0.24, 1.08], n_HC = 52, n_CSD = 44), but not absolute counts (n_HC=8, n_CSD = 4. N = 1 experiment); and (h) blood neutrophils (CD45iv⁺), in both percentage (****p < 0.0001, U = 146, Hodges-Lehmann estimate=34.2, n_HC = 47, n_CSD = 37. N = 17 experiments) and absolute counts (Unpaired t test, *p = 0.021, t = 2.6, df = 17, 95% CI [11.6, 122.8], n_HC = 12, n_CSD = 7. N = 3 experiments). Data points represent individual mice. Two-tailed tests were used in (b–h); all tests were Mann-Whitney tests unless otherwise indicated. No adjustments for multiple comparisons were made. Additional data shown in Figs. S1–4. Gating strategy shown in Fig. S2. CSD = chronic social defeat stress, HC = home cage, LD = light/dark, OF = open field, SI = social interaction (S = social, NS = non-social), USM = urine scent marking. Data shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data provided in Source Data file. Schematics from BioRender. Kigar, S. (2025) https://BioRender.com/v98waxl.

Fig. 2. Elevated neutrophil levels are associated with the negative behavioral sequelae of CSD stress.

Plots show standardized effect sizes for neutrophil levels on behavioral outcomes shown in Fig. 1. Left to right: USM marking (n_HC = 16, n_CSD = 17. N = 5 experiments), SI social approaches (n_HC = 22, n_CSD = 26. N = 10 experiments), OF novel arena exploration (n_HC = 21, n_CSD = 23. N = 9 experiments), and L/D box crosses to light (n_HC = 15, n_CSD = 17. N = 7 experiments). Mixed generalized linear regression modeling with maximum likelihood estimation (MLE) revealed negative relationships between USM marking behavior and blood (**FDR = 0.0087, β = -5.12, z = -2.98, 95% CI [-4.47, -1.12]), iv^- (*FDR = 0.016, β = -4.60, z = -2.55, 95% CI [-4.50, -0.85]), and iv⁺ (*FDR = 0.021, β = -4.74, z = -2.30, 95% CI [-4.74, −0.65]) meningeal neutrophils. Blood (**FDR = 0.0053, β = −2.59, t = −3.50, 95% CI [−0.82, −0.24]) and iv⁺ (*FDR = 0.020, β = −2.88, t = −2.61, 95% CI [−1.06, −0.13]) meningeal neutrophils are significantly associated with OF anxiety-like behavior. There were no significant relationships between neutrophils and SI or LD. See Tables S1–S4 for statistics. Unless otherwise indicated, ordinary least squares (OLS) regression was used for modeling. Significance levels reflect FDR-adjusted p values. LD light/dark, OF open field, SI social interaction, USM urine scent marking. *p < 0.05, **p < 0.01. Source data provided in Source Data file.

Fig. 3. Chronic, but not acute, social defeat stress elevates meningeal neutrophils; prolonged elevation of meningeal neutrophils relative to blood in recovery.

a Left: Schematic of acute vs chronic stress study; red arrows indicate time points in days at which mice were killed and tissue was harvested. Right: There was a main effect of the number of encounters for meningeal neutrophils (*P = 0.030, F_(5,48) = 2.7), but only the CSD day-14 group showed a significant increase by post hoc analysis. In contrast, there was a significant increase in blood neutrophils overall (****P < 0.0001, F_(5,48) = 10.5) and at each time point when compared to HC (subscript indicates days of defeat: n_HC = 8, n₁ = 7; n₂ = 7; n₄ = 9; n₈ = ₄; n₁₄ = 10. N = 2 experiments). Results for other cell types are shown in Fig. S8A. b Left: Schematic of post-CSD recovery study; red arrows indicate time points in hours (blue ticks) or days (black ticks) at which mice were killed and tissue was harvested. Gray shading indicates shared HC and CSD animals with (a), as experiments were done contemporaneously. Right: There was a main effect of time post-CSD for meningeal neutrophils (***P = 0.0001, F_(6,39) = 6.1); 16 and 24 h post-CSD, meningeal neutrophils were still significantly elevated relative to HC. In contrast, blood neutrophil levels showed an effect of time (****P < 0.0001, F_(6,40) = 11.4) and remained significantly elevated for up to 4 h post-CSD, then recovered to HC-like levels thereafter (subscript indicates time post-CSD: n_4h = 3, n_8h = 5; n_16h = 4; n_1d = 4; n_7d = 4. N = 1 experiment). See Tables S5–S8 for post-hoc statistics. Data points represent individual mice. All statistical tests were run as 1-way ANOVAs. Significance levels shown are from post-hoc testing, with adjustment for multiple comparisons. Results for other cell types are shown in Fig. S8B. All defeated groups are shown in red; open bars indicate fewer defeat encounters than the standard CSD paradigm, shaded bars indicate animals that underwent the complete CSD paradigm and were given time to recover. CSD = chronic social defeat stress, HC = home cage. Data shown as mean ± SEM. *p < 0.05, ****p < 0.0001. Source data are provided as a Source Data file. Schematics created in BioRender. Kigar, S. (2025) https://BioRender.com/djwvueb.

Fig. 4. CSD stress leads to increased numbers of LysM⁺ myeloid cells in vascular channels connecting skull BM to meninges.

a LysM^gfp/+ mice were subjected to CSD stress and behaviorally phenotyped (Fig. S8). Before TomL intravascular (iv) injection, blood was drawn for flow cytometry (Fig. S9). Dorsal meninges and brain (Fig. S10) were prepared for imaging. A separate cohort of mice was used for tissue clearing. b Flow cytometry data from blood shows high GFP expression in neutrophils compared to other cell types. c Left: Representative image for dorsal whole-mount meningeal tissue. Scale bar = 1 mm. Right: LysM-GFP⁺ cells adjacent to a blood vessel; merge and individual channels. Scale bar = 10μm. d Representative images from HC and CSD mice showing hand-counted cells, normalized to indicated area. Inset box represents size of area shown in (c, Right). e Quantification of total meningeal LysM-GFP⁺ cells shows an increase with CSD stress (Unpaired t test, *p = 0.046, t = 2.2, df = 23, 95% CI [0.019, 0.86], n_HC = 13, n_CSD = 12. N = 2 experiments). ^‡natural log-transformed. f LysM-GFP⁺ cells from (e) were separated into three categories based on their location in the tissue: >10μm away from a blood vessel (“non-vascular”), ≤10μm away from a blood vessel (“abluminal”), and intravascular (“iv”). CSD led to an overall increase in LysM-GFP⁺ cells (RM^-ANOVA: group, *p = 0.043, F_(1,23) = 4.6, n_HC = 13, n_CSD = 12); asterisk indicates main effect of CSD, but there were no significant post-hoc comparisons. ^†log₁₀ -transformed. g Representative image from cleared skulls showing vascular channels between skull BM and the meninges. Scale bar = 100μm. Left: Individual channels. Middle: Merged image, HC mouse. Right: Merged image, CSD mouse. Quantification of LysM-GFP⁺ cells per channel (h) indicates increased egress from skull BM following CSD stress (Unpaired t test, *p = 0.049, t = 2.4, df = 7, 95% CI [0.0028, 0.70]), and (i) no differences in the number of channels counted between groups (n_HC = 3, n_CSD = 6. N = 2 experiments). Data points represent individual mice. T tests were two-tailed. BM = bone marrow, BV = blood vessel, CSD = chronic social defeat stress, HC = home cage, iv = intravascular, SSC = side scatter complexity, TomL = tomato lectin. Data shown as mean ± SEM. *p < 0.05. Source data are provided as a Source Data file. Schematic created in BioRender. Kigar, S. (2025) https://BioRender.com/p6bsp7f.

Fig. 5. CSD leads to widespread tissue increase in neutrophils, relationships present between skull and meningeal neutrophils support microscopy data.

a Flow cytometry shows increased neutrophil levels throughout the body following CSD stress in C57BL/6 J mice (REML model, main effect of group, ****p < 0.0001, F_(1,15) = 112.8, n_HC = 8, n_CSD = 9; for blood, n_HC = 7). See Table S9 for post-hoc statistics. b Unsupervised hierarchical clustering of data from (a) shows significant relationships between BM (Skull: ***FDR = 0.0005, r = 0.83, t(15) = 5.81. Tibia: **FDR = 0.009, r = 0.75, t(15) = 4.36) and iv^- meningeal neutrophils, but not with blood. Blood and iv⁺ meningeal neutrophils were significantly correlated (*FDR = 0.018, r = 0.72, t(15) = 3.97). Asterisks in heatmap indicate significant Pearson correlation after Bonferroni correction for multiple comparisons. See Table S10 for correlation coefficients and FDR values. Data points represent individual mice; data represent 4 independent experiments. Multiple tissues were collected from individuals; repeat measures were accounted for in (a). BM = bone marrow, CSD = chronic social defeat stress, HC = home cage, iv = intravascular. Data shown as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001. Source data are provided as a Source Data file.

Fig. 6. Single-cell RNA sequencing (scRNAseq) of meningeal tissue validates CSD neutrophilia; biological assays support transcriptional findings.

a Timeline for sample collection and processing for scRNAseq (N = 1 experiment). b C57BL/6 J mice (n_HC = 8, n_CSD = 8) were behaviorally phenotyped prior to scRNAseq; a subset with representative phenotypes for anhedonic- (SI: *p = 0.022, t = 2.6, df = 14, 95% CI [−1.78, −0.16]. USM: Fisher’s exact test, ***p = 0.0002), and anxiety-like (OF: **p = 0.0022, t = 3.6, df = 17, 95% CI [−15.10, −3.93]) behavior were selected (shown in order from left to right). Data points represent individual mice. c Left: Visualization of recovered cells as a proportion of total cells recovered per group (n_HC=8, n_CSD = 4). Right: Meningeal scRNAseq reveals 20 distinct immune cell clusters; data points represent individual cells. Plot shown previously. d Volcano plot showing DEG between CSD and HC in the neutrophil cluster (excludes preneutrophil cluster). Indicated points represent DGE with LFC > 0.5 and FDR p < 0.001. e Flow cytometric validation of reduced iv^- meningeal neutrophil MHCII expression in (d). Left: Contour plots from representative samples; points represent cells from one mouse. Right: data points represent individual mice (Mann Whitney, ***p = 0.0006, U = 64.50, Hodges-Lehmann estimate = −0.40, n_HC = 22, n_CSD = 16. N = 5 experiments). f Gene set enrichment analysis (GSEA) revealed several enriched pathways related to cell size and the cytoskeleton (for complete list, see Fig. S15). g Imaging flow cytometry was used to visualize neutrophils. Representative images from individual meningeal samples. Two differently sized populations emerged, as depicted (white bar = ‘small’, black bar = ‘large’). There were nearly 3x more enlarged neutrophils in CSD meninges compared to HC (*p = 0.025, t = 3.5, df = 4, 95% CI [0.44, 3.81], n_HC = 3, n_CSD = 3; N = 1 experiment), consistent with actin- and cytoskeleton-related pathway enrichment in (f). Data points represent individual mice; for more details, see Fig. S16. Two-tailed tests were used in (b, d, g); all tests were unpaired t tests unless otherwise indicated. BAM = border associated macrophage, BF = brightfield, cDC = conventional dendritic cell, CSD = chronic social defeat stress, HC = home cage, NES = normalized enrichment score, pDC = plasmacytoid dendritic cell, PVM = perivascular macrophage, SSC = side scatter complexity. Data shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Source data provided in Source Data file. Schematics from BioRender. Kigar, S. (2025) https://BioRender.com/v98waxl.

Fig. 7. Migration of IFNAR⁺ neutrophils from skull BM to the meninges may underlie the type I interferon neutrophil signature seen in CSD stressed mice.

a UMAP shows expression of Ifitm2 and Ifitm3, the leading-edge genes for enrichment of the GO: “Response to type I interferon” pathway in neutrophils, in CSD compared to HC animals. Data points represent individual cells from the indicated group. b Dot plot showing gene expression in each neutrophil subcluster, which represents neutrophil maturation (see Fig. S13) for all genes comprising this pathway. Expression is scaled to mean ± standard deviation. c Confocal microscopy showing skull bone marrow neutrophils; IFNAR⁺ staining in more ‘mature’ neutrophil, based on nuclear morphology. Scale = 5μm. NB: these data are not quantified. d Flow cytometry data for IFNAR⁺ neutrophils, normalized to total neutrophils, for blood, skull, and tibia. The population of IFNAR⁺ skull BM neutrophils was decreased in CSD stressed mice (Unpaired t test, two-tailed, *p = 0.017, t = 2.7, df = 17, 95% CI [−3.12, −0.36]) and may represent a migration event to the meninges. Data points represent individual mice (n_HC = 10, n_CSD = 10; for skull, n_HC = 9. N = 2 experiments). See Fig. S21 for more detail. BM = bone marrow, CSD = chronic social defeat, HC = home cage, IFNAR = interferon-α/β receptor. Data shown as mean ± SEM. *p < 0.05. Source data are provided as a Source Data file.

Fig. 8. Type I interferon receptor (IFNAR) blockade may improve CSD-stress related behavioral anhedonia and prevent meningeal neutrophil accumulation.

a Schematic showing drug-delivery schedule for IFNAR blocking antibodies. Mice were injected with either matched IgG control antibody or anti-IFNAR antibody on the days indicated with yellow arrows. b USM results from LysM^gfp/+ mice; see Fig. S22 for comparison with HC+IgG mice and for more detail. CSD+anti-IFNAR treated mice showed behavioral improvement compared to CSD+IgG mice (Mann Whitney, *p = 0.014, U = 36, Hodges-Lehmann estimate=21.35, n_CSD+IgG = 12, n_CSD+IFNAR = 12. N = 5 experiments). Hereafter we considered these LysM^gfp/+ mice as two separate groups – those that marked in the USM test (+, indicated with closed circles) and those that didn’t (-, indicated with open circles). cLeft: CSD+anti-IFNAR(+) mice showed improvement in the OF task for anxiety-like behavior compared to CSD+IgG-treated mice (*p = 0.046, t = 2.2, df = 16, 95% CI [0.10, 9.40]). Right: No improvement in OF behavior overall for CSF+anti-IFNAR-treated mice. n_CSD+IgG = 12, n_CSD+IFNAR = 12 (within IFNAR-treated, n_USM+=6, n_USM-=6). dTop: Representative images for dorsal whole-mount meningeal tissue from CSD+IgG and CSD+anti-IFNAR treated mice. Scale bar = 1 mm. Bottom: Images from HC and CSD mice showing hand-counted LysM-GFP⁺ cells, normalized to indicated area. e Left: Quantification of total nonvascular (>10μm away from a blood vessel) meningeal LysM-GFP⁺ cells shows reduced myeloid cell accumulation in CSD+anti-IFNAR(+) treated mice (*p = 0.048, t = 2.2, df = 11, 95% CI [−0.69, −0.0043]). Right: No improvement in LysM-GFP⁺ cells accumulation overall for CSF+anti-IFNAR-treated mice. n_CSD+IgG = 9, n_CSD+IFNAR = 10 (within IFNAR-treated, n_USM+=4, n_USM-=6). ^†square root^-transformed values. f In C57BL/6 J wild type mice there was an IFNAR-mediated rescue in meningeal neutrophil accumulation following CSD stress, with expected differences in IgG control groups (2-way ANOVA, *P = 0.033, F_(1,27) = 5.0, n_CSD+IFNAR = 7, otherwise n = 8. N = 2 experiments). See Table S11 for post-hoc statistics. Significance levels shown are from post-hoc testing adjusted for multiple comparisons. There was no effect of anti-IFNAR treatment on iv⁺ meningeal or blood neutrophils or other cell types; see Fig. S23. Data points represent individual mice. Two-tailed tests were used in (b, c, e); all tests were unpaired t tests unless otherwise indicated. CSD = chronic social defeat, HC = home cage. Data shown as mean ± SEM. *p < 0.05, **p < 0.01. Source data provided in Source Data file. Schematics from BioRender. Kigar, S. (2025) https://BioRender.com/rvrmii4.

Fig. 9. Proposed model for how chronic, but not acute, stress leads to dysregulation of the meningeal environment.

a At baseline, both skull and tibia BM contain a small population of IFNAR⁺ neutrophils (orange), further divisible into IFNAR^lo and IFNAR^hi populations. Relatively mature IFNAR^hi cells express more Ly6C and may thus be more proinflammatory. In blood, B cell numbers greatly outnumber neutrophils; in the meninges these two populations are roughly equal. b Acute stress exposure leads to repetitive release of neutrophils into the blood but is not sufficient for accumulation of neutrophils in the meninges (Figs. 3a, S6A, S7A). Prolonged exposure to psychosocial stress leads to a decline in meningeal B cells (Fig. S2C) that precedes an increase in meningeal neutrophils (Figs. S6A, S7A) and is prolonged for up to a week of recovery post-CSD (Fig. 3b, S6B, S7B). Whereas CSD-associated meningeal neutrophilia appears to be mediated by IFN-I signaling, meningeal B cell depletion does not (Figs. 8f, S23A). CSD stress causes random neurovascular damage—potentially via stalled and rigid ROS-producing neutrophils in brain capillaries (Figs. S10, S16E, S18)—which amplifies neutrophil recruitment from adjacent skull bone marrow (Fig. 4g, h). Increased expression of the neutrophil chemoattractant CXCL1 in the liver, increased CXCL1 protein in plasma, and reduced BM expression of CXCL12—which promotes retention of neutrophils—may contribute to the elevated circulating pool of neutrophils in blood (Fig. S17), though there were no detectable changes related to this pathway in the meninges. BBB = blood brain barrier, CSD = chronic social defeat, CXCL = C-X-C motif chemokine ligand, HC = home cage, IFN-I = type I interferon, IFNAR = interferon-α/β receptor, ROS = reactive oxygen species, SSC = side scatter complexity. Fig. created in BioRender. Kigar, S. (2025) https://BioRender.com/yf2zm8x.