Microglial recruitment of IL-1β-producing monocytes to brain endothelium causes stress-induced anxiety

Abstract

Psychosocial stress contributes to the development of anxiety and depression. Recent clinical studies have reported increased inflammatory leukocytes in circulation of individuals with stress-related psychiatric disorders. Parallel to this, our work in mice shows that social stress causes release of inflammatory monocytes into circulation. In addition, social stress caused the development of prolonged anxiety that was dependent on inflammatory monocytes in the brain. Therefore, we hypothesize that chronic stress drives the production of inflammatory monocytes that are actively recruited to the brain by microglia, and these monocytes augment neuroinflammatory signaling and prolong anxiety. Here we show that repeated social defeat stress in mice activated threat appraisal centers in the brain that spatially coincided with microglial activation and endothelial facilitation of monocyte recruitment. Moreover, microglial depletion with a CSF1R antagonist prior to stress prevented the recruitment of monocytes to the brain and abrogated the development of anxiety. Cell-specific transcriptional profiling revealed that microglia selectively enhanced CCL2 expression, while monocytes expressed the pro-inflammatory cytokine interleukin-1β (IL-1β). Consistent with these profiles, the recruited inflammatory monocytes with stress adhered to IL-1R1⁺ neurovascular endothelial cells and this interaction was blocked by microglial depletion. Furthermore, disruption of IL-1β signaling by caspase-1^KO specifically within bone marrow-derived cells revealed that monocytes promoted anxiogenesis through stimulation of neurovascular IL-1R1 by IL-1β. Collectively, the development of anxiety during stress was caused by microglial recruitment of IL-1β-producing monocytes, which stimulated brain endothelial IL-1R1. Thus, monocyte IL-1β production represents a novel mechanism that underlies behavioral complications associated with stress-related psychiatric disorders.

Figure 1. Monocyte recruitment to threat appraisal centers is dependent on neuronal and microglial activation

Male C57BL/6 mice received either minocycline (Mino) in their drinking water, clonazepam (CZP) injections i.p., or vehicle treatment daily two days before and during repeated social defeat (Stress). Mice were perfused and the brain was PFA fixed 14 h after the last cycle of stress. Neuronal activation (ΔFosB), microglial activation (Iba-1), vascular endothelial activation (ICAM-1), and the presence of monocytes (CD45⁺) in the vasculature (ICAM-1/CD45) were assessed. Representative images within the prelimbic cortex of A) ΔFosB, C) Iba1, and E) ICAM-1/CD45 labeling. Arrows indicate the cells used in insets. B) The number of ΔFosB+ cells (Stress × Intervention interaction, F(2,33)=12.02, p=0.0002), D) Iba-1 proportional area (Stress × Drug interaction, F(2,36)=11.21, p=0.0002), and F) ICAM-1 proportional area (Stress × Intervention interaction; F(2,30)=5.23, p=0.012). G) The number of CD45⁺ cells in threat appraisal centers (Stress × Intervention interaction; F(2,35)=3.31, p=0.05). Number of CD45⁺ cells associated with H) ICAM-1 positive blood vessels (Stress × Intervention interaction; F(2,35)=17.57, p<0.0001) and I) VCAM-1 positive blood vessels (Stress × Intervention interaction; F(2,35)=2.95, p=0.067). In a separate experiment, male C57BL/6 mice received minocycline or vehicle in their drinking water two days before and during social defeat (Stress). Anxiety-like behavior was determined 14 h after the last cycle of stress and then samples (bone marrow, blood, and brain) were collected for cell or mRNA analyses. J) CD45^hi macrophages in the brain (main effect of Stress; F(1,49)=7.37, p=0.0093 & main effect of Minocycline; F(1,49)=7.91, p=0.0072). K) IL-1β mRNA levels in enriched CD11b⁺ cells from the brain (Stress × Minocycline interaction; F(1,25)=7.76, p=0.011). L) Latency to enter the center of the open field (Stress × Minocycline interaction; F(1,76)=4.043, p=0.048) and M) time spent in the center of the open field (main effect of minocycline; F(1,75)=4.094, p=0.047). Total number of samples per group (n) and number of replicates (rep): A-I (n=4–6, rep=2), J (n=12–16, rep=2), K (n=6, rep=2), L-M (n=18–20, rep=4). Bars represent the mean ± SEM. Means with asterisk (*) are significantly different from the corresponding control mice (p<0.05), and means with (#) tended to be different from control mice (p<0.1), according to F-protected post hoc analysis.

Figure 2. Microglial depletion with CSF1R antagonist does not prevent stress-induced threat appraisal or endothelial activation

Male C57BL/6 mice were provided ad libitum diets of PLX5622 (1200 ppm chow) or vehicle chow for 14 days. Brain samples were collected to determine microglial ablation. A) Representative flow bivariate dot plots of CD11b and CD45 labeling on enriched microglia and macrophages. B) Number of CD11b⁺CD45^lo microglia (main effect of PLX, F(1,7)=123.30, p<0.0001). C) CX₃CR1 mRNA levels in a coronal brain section (main effect of PLX, F(1,18)=896.65, p<0.0001). A separate set of mice provided with PLX5622 or vehicle chow for 14 days were then exposed to repeated social defeat (Stress). Mice were perfused and brains were PFA fixed 14 h after the last cycle of stress. Neuronal activation (ΔFosB), microglial activation (Iba-1) and vascular endothelial activation (ICAM-1) were assessed. Representative images within the prelimbic cortex (PrL) of D) ΔFosB, F) Iba-1, H) ICAM-1/CD45 labeling. Arrows indicate the cells used in insets. E) Number of ΔFosB+ cells (main effect of Stress; F(1,15)=26.00, p=0.0003), G) Iba-1 proportional area (Stress × PLX interaction; F(1,15)=21.37, p=0.0006), and I) ICAM-1 proportional area (main effect of Stress; F(1,16)=27.19, p=0.0002). In a separate experiment, mice were provided with a diet of PLX5622 (1200 ppm chow) or vehicle chow for 14 days and then exposed to social defeat (Stress). Anxiety-like behavior was determined 14 h after the last cycle of stress and then samples (blood and brain) were collected for cell or mRNA analyses. J) Percentage of circulating CD11b⁺/Ly6C^hi monocytes (main effect of Stress; F(1,30)=44.18, p<0.0001). K) Representative flow bivariate dot plots of CD11b and CD45 labeling on enriched brain microglia and macrophages. L) Number of CD11b⁺CD45^lo microglia (main effect of PLX; F(1,44)=164.89, p<.0001) and M) CD11b⁺CD45^hi macrophages (Stress × PLX interaction; F(1,38)=3.33, p=0.0767) in the brain. N) IL-1β mRNA levels in the brain (Stress × PLX interaction; F(1,28)=40.95, p<0.0001). O) Latency to enter the center of the open field (main effect of Stress; F(1,46)=4.78, p=0.0343) and P) time spent in the center of the open field (main effect of Stress; F(1,46)=9.69, p=0.0033 & main effect of PLX; F(1,46)=3.99, p=0.0522). Total number of samples per group (n) and number of replicates (rep): B (n=4, rep=1), C (n=4, rep=1), D-I (n=4–5, rep=1), J-M (n=8–12, rep=2), and O&P (n=8–12, rep=2). Bars represent the mean ± SEM. Means with asterisk (*) are significantly different (p<0.05), and means with (#) tended to be different (p=0.1) from the corresponding control mice, according to F-protected post hoc analysis.

Figure 3. Transcriptional profiling of FAC-Sorted microglia and monocytes

Male C57BL/6 mice were subjected to 6 cycles of repeated social defeat (stress) or left undisturbed as controls. A) Microglia (CD11b⁺/CD45^lo) and brain macrophages (CD11b⁺/CD45^hi) were Percoll-enriched and FAC-Sorted 14 h after the last cycle of stress. Then, mRNA copy number was determined by nanoString analysis. Cell-specific mRNA copy number for microglia or monocytes is shown for B) signature transcripts, C) cytokines/chemokines, D) adrenergic receptors, E) cytokine receptors, F) glucocorticoid signaling, and G) immune signaling. Total number of samples per group (n) and number of replicates (rep): n=6, rep=2). Bolded and grey shaded groups of transcripts indicate significant main effect of cell type (p<0.05 from MANOVA). All groups, n= 6. Means with asterisk (*) are significantly different (p<0.05), and means with (#) tended to be different (p<0.1) from the corresponding control mice.

Figure 4. Microglial activation increases brain endothelial IL-1R1 expression during stress

A) Diagram of exon XI of the wild type IL-1R1^+/+ allele (top) and the IL-1R1^GR/GR targeted mutation allele. IL-1R1^GR/GR allele includes 3 hemagglutinin (HA) tagging sequences within exon XI and includes an internal ribosomal entry sequence (IRES) followed by tdTomato sequence. IL-1R1 transcription results in tdTomato fluorescence and translated IL-1R1 proteins detectable by antibody labeling of HA. Representative images showing tdTomato fluorescence (red) co-labeled with B) anti-HA antibody (green) and C) anti-IL1R1 antibody (green). Male IL-1R1^GR/GR mice were subjected to social defeat (Stress) or left undisturbed as controls. Mice were perfused and the brain was PFA fixed 14 h after the last cycle of stress. D) Representative images of tdTomato fluorescence in the prelimbic cortex. E) tdTomato proportional area in the PrL after stress (F(1,4)=17.33, p=0.0252). In related experiments, male C57B/L6 wild type mice were subjected to social defeat (Stress) or left undisturbed as controls. Samples were collected 14 h later and either brain IL-1R1 mRNA or IL-1R1⁺ cells and CD45⁺ cells were determined in the PrL. F) IL-1R1 mRNA levels in a coronal section after stress (F(1,7)=33.75, p=0.0011). G) Representative images of CD45 and IL-1R1 antibody labeling within the prelimbic cortex. H) IL-1R1 proportional area after stress (F(1,7)=51.06, p=0.0004), and I) Percentage of CD45 positive cells associated with IL-1R1 positive blood vessels (F(1,7)=8.05, p=0.0297). Next, male C57B/L6 mice were provided ad libitum diets of PLX5622 (1200 ppm chow) or vehicle chow for 14 days and then exposed to repeated social defeat (Stress). J) Representative images of IL-1R1 antibody labeling within the PrL. K) IL-1R1 proportional area in the PrL (Stress × PLX interaction; F(1,15)=15.88, p=0.0018), CeA (Stress × PLX interaction; F(1,16)=11.61, p=0.0047), CA3 (Stress × PLX interaction; F(1,16)=8.23, p=0.0132) and Motor cortex. L) IL-1R1 mRNA levels in a coronal brain section (Stress × PLX interaction; F(1,38)=4.86, p=0.0339). Total number of samples per group (n) and number of replicates (rep): E (n=3, rep=1), F (n=4, rep=1), H-I (n=4, rep=1), K (n=4, rep=1), and L (n=8–10, rep=3). Bars represent the mean ± SEM. Means with asterisk (*) are significantly different (p<0.05), and means with (#) tended to be different (p<0.1) from the corresponding control mice, according to F-protected post hoc analysis.

Figure 5. Bone marrow-derived monocyte production of active IL-1β protein in the brain mediates anxiety

Bone marrow cells were collected from male C57BL/6 WT and Caspase-1^KO (Casp1^−/−) mice and treated with ex vivo LPS (1μg/mL) for 18 hours. A) IL-1β protein levels (Genotype × Treatment interaction; F(1,22)=488.48, p<0.0001) and B) IL-1β mRNA levels were determined (main effect of Genotype; F(1,23)=5.12, p=0.0350 and main effect of LPS; F(1,23)=1197.51, p<0.0001). C) In another experiment, Casp1^−/− bone marrow-chimeric mice were generated. C57BL/6 CD45.1 mice were reconstituted with bone marrow from wildtype CD45.2 mice or Casp1^−/− CD45.2 mice to produce WT BM chimera (BM^{Casp1 WT→WT}) and Casp1^−/− BM chimera (BM^{Casp1 KO→WT}), respectively. The chimeric mice were then exposed to repeated social defeat (Stress) or left undisturbed as controls. D) Percentage of CD11b⁺CD45.2⁺ blood cells in WT chimera and Casp1^−/− chimera mice after exposure to repeated social defeat (Stress). E) Percentage of bone marrow monocytes (main effect of Stress; F(1,44)=34.44, p<.0001), granulocytes (main effect of Stress; F(1,43)=30.71, p<.0001), lymphocytes (main effect of Stress; F(1,45)=26.39, p<.0001) and erythrocytes (main effect of Stress; F(1,44)=29.82, p<.0001). F) Percentage of circulating Ly6C^hi monocytes in the blood (main effect of Stress; F(1,42)=7.25, p=0.0104). G) Representative bivariate dot plots of CD11b and CD45 labeling on enriched brain macrophages and microglia. H) Percentage of CD45^hi macrophages in the brain (main effect of Stress; F(1,35)=21.22, p<.0001). I) Heat-map representing activity in the open field box. J) Latency to enter the center of the open field (Stress × Genotype interaction; F(1,66)=3.98, p< 0.05) and K) time spent in the center (main effect of Stress; F(1,66)=6.56, p < 0.01). L) IL-1β mRNA (main effect of Stress; F(1,26)=9.67, p < 0.005) and M) IL-6 mRNA levels in a coronal brain section (Stress × Genotype interaction; F(1,22)=11.06, p=0.0036 & main effect of Genotype; F(1,22)=32.31, p<0.0001). Total number of samples per group (n) and number of replicates (rep): A-B (n=5–6, rep=1), D-H (n=6, rep=1), J&K (n=19–23, rep=3), L&M (n=6, rep=1). Bars represent the mean ± SEM. Means with asterisk (*) are significantly different from the corresponding control mice (p<0.05), and means with (#) tended to be different from control mice (p<0.1), according to F-protected post hoc analysis.

Figure 6. Microglial recruitment of IL-1β-producing monocytes to brain endothelium causes stress-induced anxiety

This figure illustrates the key findings of this study. First, exposure to repeated social defeat (Stress) in mice caused significant neuronal activation (ΔFosB) within threat appraisal regions of the brain (e.g., frontal cortex, amygdala, and hippocampus). This threat appraisal was associated with increased endothelial cell adhesion molecule (CAM) expression, activation of microglia, and the release of monocytes into circulation. Second, inhibition of threat appraisal with clonazepam (CZP) prevented endothelial CAM induction, microglial activation, and monocyte recruitment to the brain. Third, stress-induced microglial activation was associated morphological restructuring, increased chemokine expression (CCL2), and induction of IL-1R1 on endothelial cells. For example, inhibition (minocycline; Mino) and depletion (Plexxikon 5622; PLX) of microglia prevented stress-induced monocyte recruitment, endothelial IL-1R1 induction, and anxiety-like behavior. Notably, induction of social avoidance with repeated social defeat was blocked by CZP, but was unaffected by the microglial interventions, PLX or Mino. Fourth, monocytes recruited to the brain with stress expressed a high level of IL-1β. Inflammatory monocytes recruited to the brain with stress stimulated IL-1R1+ endothelial cells (eIL-1R1) to promote anxiety. For example, the inability of monocytes to make functional IL-1β in caspase-1 KO bone marrow (Casp1^−/− BM) chimeras prevented the augmentation of neuroinflammatory signaling and the development of anxiety in response to stress. Overall, monocyte IL-1β production represents a novel cellular mechanism by which the immune system communicates with the brain to influence behavior.