Knockdown of interleukin-1 receptor type-1 on endothelial cells attenuated stress-induced neuroinflammation and prevented anxiety-like behavior

Abstract

Interleukin-1β (IL-1β) is an inflammatory cytokine that plays a prominent role in stress-induced behavioral changes. In a model of repeated social defeat (RSD), elevated IL-1β expression in the brain was associated with recruitment of primed macrophages that were necessary for development of anxiety-like behavior. Moreover, microglia activation and anxiety-like behavior associated with RSD did not occur in IL-1 receptor type-1 knock-out (IL-1R1(KO)) mice. Therefore, the objective of this study was to examine the role of IL-1 signaling in RSD-induced macrophage trafficking to the brain and anxiety-like behavior. Initial studies revealed that RSD did not increase circulating myeloid cells in IL-1R1(KO) mice, resulting in limited macrophage trafficking to the brain. In addition, IL-1R1(KO) bone marrow-chimera mice showed that IL-1R1 expression was essential for macrophage trafficking into the brain. To differentiate cellular mediators of stress-induced IL-1 signaling, endothelial-specific IL-1R1 knock-down (eIL-1R1kd) mice were used. Both wild-type (WT) and eIL-1R1kd mice had increased circulating monocytes, recruitment of macrophages to the brain, and altered microglia activation after RSD. Nonetheless, RSD-induced expression of IL-1β, TNF-α, and IL-6 mRNA in brain CD11b(+) cells was attenuated in eIL-1R1kd mice compared with WT. Moreover, anxiety-like behavior did not develop in eIL-1R1kd mice. Collectively, these findings demonstrated that there was limited RSD-induced priming of myeloid cells in IL-1R1(KO) mice and disrupted propagation of neuroinflammatory signals in the brain of eIL-1R1kd mice. Furthermore, these data showed that transduction of IL-1 signaling by endothelial cells potentiates stress-induced neuroinflammation and promotes anxiety-like behavior.

Keywords: anxiety; blood-brain barrier; interleukin-1; microglia; neuroinflammation; stress.

Figures

Figure 1.

IL-1R1KO mice do not have increased myeloid cells in the blood and brain after RSD. WT and IL-1R1KO C57BL/6 mice were subjected to RSD or left undisturbed as CON mice. Spleen, blood, and brain CD11b+ cells were collected 14 h after the final cycle of RSD. A, Average spleen weight. B, Average percentage of blood CD11b+ cells. C, Average percentage of blood monocytes (CD11b+/Ly6Chi). D, Representative bivariate dot plots of CD11b/CD45 labeling in each experimental group. E, Average proportion of brain macrophages (CD11b+/CD45hi). Bars represent average ± SEM. *p < 0.05, different from CON; #p < 0.10, different from CON.

Figure 2.

Expression of IL-1R1 is required for RSD-induced recruitment of myeloid cells into the brain of GFP+ BM-chimeric mice. GFP+ BM-chimeric mice were generated in WT or IL-1R1KO C57BL/6 mice and subjected to six cycles of RSD or left undisturbed as CON mice. Brains were collected 14 h after RSD for histology (n = 4–6). Representative images of GFP labeling (top) and GFP/Iba-1 co-labeling (bottom) in the AMYG WT–CON (A), WT–RSD (B), and IL-1R1KO–RSD (C) mice are shown. Quantification of perivascular and parenchymal GFP+ macrophages in the PFC (D), AMYG (E), and HPC (F) are shown. Average proportional area of Iba-1+ labeling in the PFC (G), AMYG (H), and HPC (I) are shown. Bars represent average ± SEM. *p < 0.05, significantly different from CON.

Figure 3.

RSD increased spleen weight, circulating myeloid cells, and enhanced the inflammatory potential of CD11b+ splenocytes in eIL-1R1kd mice. WT and eIL-1R1kd FVB mice were subjected to RSD or left undisturbed as CON mice. A, Average spleen weight. B, Plasma corticosterone levels. C, Percentage of CD11b+ cells in the blood. D, Representative flow bivariate dot plots of CD11b/Ly6C labeling. E, Average percentage of CD11b+/Ly6Chi monocytes are shown. Bars represent average ± SEM. *p < 0.05, significantly different from CON. #p < 0.10, different from CON.

Figure 4.

RSD promoted recruitment of peripheral macrophages and increased activation markers on brain CD11b+ cells in WT and eIL-1R1kd mice. WT and eIL-1R1kd FVB mice were subjected to RSD or left undisturbed as CON. Brains were collected 14 h after the final cycle and brain CD11b+ cells (microglia/macrophages) were isolated. A, Representative flow bivariate dot plots of CD11b/CD45 labeling. B, Average percentage of CD11b+/CD45hi brain macrophages. C, Average proportion of CD14+ macrophages. D, Average proportion of CD14+ microglia in the brain. Bars represent average ± SEM. *p < 0.05, significantly different from CON.

Figure 5.

RSD enhanced Iba-1 proportional area in the PFC, PVN, AMYG, and HPC of eIL-1R1kd mice. WT and eIL-1R1kd FVB mice were subjected to RSD or left undisturbed as CON. Brains were collected 14 h after RSD for histology. A, Representative images of Iba-1 labeling in the AMYG of WT–CON, WT–RSD, and eIL-1R1kd–RSD mice are shown. Average Iba-1 proportional area in the AMYG (B), PFC (C), PVN (D), and HPC-DG (E) are shown. Enlarged image of Iba-1+ microglia indicated by dashed outline. Bars represent average ± SEM. *p < 0.05, significantly different from CON.

Figure 6.

Endothelial knockdown of IL-1R1 prevented RSD-induced anxiety-like behavior. WT and eIL-1R1kd FVB mice were subjected to RSD or left undisturbed as CON. Anxiety-like behavior was tested 14 h after the final cycle of RSD. A, Representative motion plots of each experimental group in the open field. B, Average time to enter the open-field center. C, Average time spent in the open-field center. D, Average time to enter the dark zone. E, Average time spent in the dark zone of the light/dark preference paradigm are shown. Bars represent average ± SEM. *p < 0.05, significantly different from CON.

Figure 7.

IL-1R1 expression mediates central and peripheral immune responses that are critical in development of RSD-induced anxiety-like behavior. A, WT mice subjected to RSD have increased primed myeloid cells in circulation, leading to robust macrophage trafficking in the brain that corresponded with microglia activation. These brain CD11b+ cells have increased proinflammatory cytokine production (IL-1β, TNF-α, IL-6) that promoted development of anxiety-like behavior after RSD. B, IL-1R1KO mice subjected to RSD do not develop primed myeloid cells in circulation, leading to reduced macrophage trafficking in the brain and limited microglia activation. These mice fail to development anxiety-like behavior after RSD. C, eIL-1R1kd mice subjected to RSD have increased primed myeloid cells in circulation, leading to robust macrophage trafficking in the brain that corresponded with microglia activation. Limited IL-1 signaling at the vascular interface attenuated proinflammatory cytokine production (IL-1β, TNF-α, IL-6), preventing development of anxiety-like behavior after RSD.