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. 2013 Aug 21;33(34):13820-33.
doi: 10.1523/JNEUROSCI.1671-13.2013.

Stress-induced recruitment of bone marrow-derived monocytes to the brain promotes anxiety-like behavior

Eric S Wohleb  1 , Nicole D Powell, Jonathan P Godbout, John F Sheridan
Affiliations

Stress-induced recruitment of bone marrow-derived monocytes to the brain promotes anxiety-like behavior

Eric S Wohleb et al. J Neurosci. .

Abstract

Social stress is associated with altered immunity and higher incidence of anxiety-related disorders. Repeated social defeat (RSD) is a murine stressor that primes peripheral myeloid cells, activates microglia, and induces anxiety-like behavior. Here we show that RSD-induced anxiety-like behavior corresponded with an exposure-dependent increase in circulating monocytes (CD11b(+)/SSC(lo)/Ly6C(hi)) and brain macrophages (CD11b(+)/SSC(lo)/CD45(hi)). Moreover, RSD-induced anxiety-like behavior corresponded with brain region-dependent cytokine and chemokine responses involved with myeloid cell recruitment. Next, LysM-GFP(+) and GFP(+) bone marrow (BM)-chimeric mice were used to determine the neuroanatomical distribution of peripheral myeloid cells recruited to the brain during RSD. LysM-GFP(+) mice showed that RSD increased recruitment of GFP(+) macrophages to the brain and increased their presence within the perivascular space (PVS). In addition, RSD promoted recruitment of GFP(+) macrophages into the PVS and parenchyma of the prefrontal cortex, amygdala, and hippocampus of GFP(+) BM-chimeric mice. Furthermore, mice deficient in chemokine receptors associated with monocyte trafficking [chemokine receptor-2 knockout (CCR2(KO)) or fractalkine receptor knockout (CX3CR1(KO))] failed to recruit macrophages to the brain and did not develop anxiety-like behavior following RSD. Last, RSD-induced macrophage trafficking was prevented in BM-chimeric mice generated with CCR2(KO) or CX3CR1(KO) donor cells. These findings indicate that monocyte recruitment to the brain in response to social stress represents a novel cellular mechanism that contributes to the development of anxiety.

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Figures

Figure 1.
Figure 1.
RSD-induced anxiety-like behavior was paralleled by an increased percentage of circulating monocytes (CD11b+/SSClo/Ly6Chi) and macrophages (CD11b+/CD45hi) in the brain. Male C57BL/6 mice were subjected to one, three, or six cycles of social defeat (RSD) or left undisturbed as controls (CON). Anxiety-like behavior was determined 14 h after the final cycle of RSD (n = 6–9, 3 independent experiments). A, B, Time to enter the center of the open field (A) and total time spent in the open field (B). C, Time to enter the dark zone. D, Total time spent in the light zone. Following behavioral testing, blood and enriched brain CD11b+ cells were collected (n = 6–9, 3 independent experiments). E, Representative flow bivariate dot plots of SSC/CD11b and SSC/Ly6C labeling in the blood. Myeloid subsets were gated into Ly6C− monocyte (m: CD11b+/SSClo/Ly6C−), granulocyte (G: CD11b+/SSChi/Ly6Cint), and Ly6Chi monocyte (M: CD11b+/SSClo/Ly6Chi) subsets. F–H, The percentage of blood myeloid cells (F, CD11b+), granulocytes (G, G), and monocytes (H, m: Ly6C− or M: Ly6Chi). I, Representative flow bivariate dot plots of CD11b/CD45 labeling in Percoll-isolated microglia/macrophages and side scatter/forward scatter (SSC/FSC) properties are shown in RSD mice. J, The percentage of brain macrophages (CD11b+/CD45hi) are shown. Bars represent the mean ± SEM. Means with asterisk are significantly different from CON (p < 0.05) and means with number sign (#) tended to be different from CON (p < 0.06–0.10).
Figure 2.
Figure 2.
RSD-induced anxiety was associated with increased recruitment of LysM-GFP+ monocytes from circulation into the brain. LysM-GFP+ mice were subjected to six cycles of social defeat (RSD) or left undisturbed as controls (CON). Anxiety-like behavior was tested 14 h after RSD. Following behavioral testing, blood and enriched brain CD11b+ cells were collected (n = 6–8, 2 independent experiments). A, Time to enter the center of the open field. B, Time spent in the open field. C, Average spleen weight. D, Representative flow bivariate dot plots of SSC/Ly6C on CD11b+ cells. E, The percentage of CD11b+/SSClo/Ly6Chi monocytes in the blood. F, Representative bivariate dot plots of GFP/CD11b labeling of Percoll-isolated microglia/macrophages. G, The percentage of GFP+/CD11b+ cells in the brain. H, The number of GFP+/CD11b+ cells that were CD45hi or CD45lo. I, Representative histograms of GFP expression in blood monocytes (CD11b+/Ly6Chi) and brain macrophages (CD11b+/CD45hi). J, Relative MFI of GFP+/CD11b+ cells in the blood or brain. Bars represent the mean ± SEM. Means with asterisk are significantly different from CON (p < 0.05).
Figure 3.
Figure 3.
LysM-GFP+ macrophages were present in the PVS of the brain following RSD. LysM-GFP+ mice were subjected to six cycles of social defeat (RSD) or left undisturbed as controls (CON). Brains were collected 14 h after RSD for histology (n = 4–6, 2 independent experiments). A, B, Representative images of LysMGFP (green), merged LysMGFP/Iba-1 (red), and merged LysMGFP/Iba-1/Ly6C (blue) in the PFC of CON and RSD mice. C, D, Representative images of LysMGFP (green), merged LysMGFP/Iba-1 (red), and merged LysMGFP/Iba-1/Ly6C (blue) immunofluorescence in the HPC of CON and RSD mice. White arrowheads point to perivascular LysM-GFP+ macrophages. Scale bars, 100 μm.
Figure 4.
Figure 4.
Peripheral GFP+/CD11b+ macrophages were recruited into the brain of GFP+ BM-chimeric mice after RSD. GFP+ BM-chimeric mice were subjected to six cycles of social defeat (RSD) or left undisturbed as controls (CON). Brains were collected 14 h after RSD and enriched CD11b+ cells were assessed with flow cytometry (n = 4–5). A, Representative flow bivariate dot plots of GFP/CD11b labeling of Percoll-isolated microglia/macrophages. B, The percentage of GFP+/CD11b+ cells in the brain. C, The number of GFP+/CD11b+ cells that were CD45lo or CD45hi. Bars represent the mean ± SEM. Means with asterisk (*) are significantly different from CON (p < 0.05). Another group of mice were perfused and brains were collected for histology (n = 4–6, 2 independent experiments). D, E, Representative images of GFP (green), merged GFP/Iba-1 (red), and merged GFP/Iba-1/Ly6C (blue) immunofluorescence in the HPC of CON and RSD mice. F–H, In separate studies, mice that received intraocular injury [(+) Control], RSD, or busulfan treatment were retro-orbitally injected with Evans blue dye (1% solution in 100 μl). Mice were perfused and brains were collected for histology 2 h after Evans blue dye injection. Representative images of the whole brain (inset) and in the HPC of (+) Control (F), RSD (G), and busulfan-treated (H) mice. Scale bars, 100 μm.
Figure 5.
Figure 5.
RSD promoted infiltration of peripheral macrophages (ramified GFP+/Iba-1+) into the parenchyma of brain regions associated with fear and anxiety. GFP+ BM-chimeric mice were subjected to six cycles of social defeat (RSD) or left undisturbed as controls (CON). The neuroanatomical distribution of GFP+ cells was determined 14 h after the final cycle of RSD (n = 3–4). A, B, Representative images of GFP (green), merged GFP/Iba-1 (red), and merged GFP/Iba-1/Ly6C (blue) immunofluorescence in the PFC of CON and RSD mice. C, D, Representative images of GFP (green), merged GFP/Iba-1 (red), and merged GFP/Iba-1/Ly6C (blue) immunofluorescence in the AMYG of CON and RSD mice. E, Representative images of GFP (green), merged GFP/Iba-1 (red), and merged GFP/Iba-1/Ly6C (blue) immunofluorescence in the M1 CTX of RSD mice. Scale bars, 100 μm.
Figure 6.
Figure 6.
Recruitment of ramified GFP+ macrophages in the brain required repeated cycles of RSD. GFP+ BM-chimeric mice were subjected to one, three, or six cycles of social defeat (RSD) or left undisturbed as controls (CON). The neuroanatomical distribution of GFP+ cells was determined 14 h after the final cycle of RSD (n = 3–7, 3 independent experiments). A–D, Quantification of the total number of perivascular and parenchymal GFP+ cells in the PFC (A), PVN (B), AMYG (C), and HPC (D). E, Region-specific quantification of the HPC (CA1, CA2, CA3, and DG) is shown. Bars represent the mean ± SEM. Means with asterisk (*) are significantly different from CON (p < 0.05) and means with number sign (#) tend to be different from CON (p = 0.06–0.10). F, Representative illustration of GFP+ cell distribution throughout the brain following RSD. Areas in dark gray indicate significant distribution of ramified GFP+ macrophages. OB, Olfactory bulb; NAc, nucleus accumbens; V, dorsal lateral ventricle; Pir, piriform cortex.
Figure 7.
Figure 7.
RSD increased the proportion of circulating CD11b+/SSClo/Ly6Chi monocytes independent of CCR2 and CX3CR1 expression. In two separate experiments, WT and CCR2KO or CX3CR1HET and CX3CR1KO C57BL/6 mice were subjected to six cycles of social defeat (RSD) or left undisturbed as controls (CON). Spleens and blood were collected 14 h after the final cycle of RSD (n = 3–8, 3 independent experiments). A, Average spleen weight. B, The percentage of myeloid cells (CD11b+) in the blood. C, Representative flow bivariate dot plots of Ly6C/CD11b labeling. D, The percentage of blood monocytes (CD11b+/Ly6Chi). Bars represent the mean ± SEM. Means with asterisk (*) are significantly different from genotype CON (p < 0.05).
Figure 8.
Figure 8.
RSD-induced macrophage recruitment to the brain and anxiety-like behavior required expression of CCR2 and CX3CR1. WT and CCR2KO or CX3CR1HET and CX3CR1KO C57BL/6 mice were subjected to six cycles of social defeat (RSD) or left undisturbed as controls (CON). Anxiety-like behavior was determined 14 h after RSD. Following behavioral testing, brains were collected for Percoll isolation of brain CD11b+ cells (n = 7–12, 4 independent experiments). A, Time to enter the open field. B, Total time spent in the open field. C, Representative flow bivariate dot plots of CD11b/CD45 labeling of Percoll-isolated microglia/macrophages. D, The percentage of brain macrophages. Bars represent the mean ± SEM. Means with asterisk (*) are significantly different from genotype CON (p < 0.05).
Figure 9.
Figure 9.
Expression of CCR2 and CX3CR1 on peripheral myeloid cells was required for RSD-induced macrophage infiltration into the brain parenchyma. WT recipient mice received either CCR2HET(+/RFP), CCR2KO(RFP/RFP), CX3CR1HET(+/GFP), or CX3CR1KO(GFP/GFP) BM-derived donor cells. BM-chimeric mice were subjected to six cycles of social defeat (RSD) or left undisturbed as controls (CON). The neuroanatomical distribution of GFP+ cells was determined 14 h after the final cycle of RSD (n = 3–6, 2 independent experiments). A, Representative merged images of Iba-1 (green)/CCR2 (red)/Ly6C (blue) immunofluorescence in the PFC of CON or RSD mice that received CCR2HET and CCR2KO donor BM. B–D, Quantification of the total number of perivascular and parenchymal cells in the PFC (B), AMYG (C), and HPC (D). E, Representative merged images of CX3CR1 (green)/Iba-1 (red)/Ly6C (blue) immunofluorescence in the AMYG of CON or RSD mice that received CX3CR1HET or CX3CR1KO donor BM. F–H, Quantification of the total number of perivascular and parenchymal GFP+ cells in the PFC (F), AMYG (G), and HPC (H). Bars represent the mean ± SEM. Means with asterisk (*) are significantly different from CON (p < 0.05) and means with number sign (#) tend to be different from CON (p = 0.06).
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