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, 9 (1), 20217

Experimental Colitis Reduces Microglial Cell Activation in the Mouse Brain Without Affecting Microglial Cell Numbers

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Experimental Colitis Reduces Microglial Cell Activation in the Mouse Brain Without Affecting Microglial Cell Numbers

Hoda M Sroor et al. Sci Rep.

Abstract

Inflammatory bowel disease (IBD) patients frequently suffer from anxiety disorders and depression, indicating that altered gut-brain axis signalling during gastrointestinal inflammation is a risk factor for psychiatric disease. Microglia, immune cells of the brain, is thought to be involved in a number of mental disorders, but their role in IBD is largely unknown. In the current work, we investigated whether colitis induced by dextran sulphate sodium (DSS), a murine model of IBD, alters microglial phenotypes in the brain. We found that colitis caused a reduction of Iba-1 and CD68 immunoreactivity, microglial activation markers, in specific brain regions of the limbic system such as the medial prefrontal cortex (mPFC), while other areas remained unaffected. Flow cytometry showed an increase of monocyte-derived macrophages during colitis and gene expression analysis in the mPFC showed pronounced changes of microglial markers including cluster of differentiation 86 (CD86), tumour necrosis factor-α, nitric oxide synthase 2, CD206 and chitinase-like protein 3 consistent with both M1 and M2 activation. Taken together, these findings suggest that experimental colitis-induced inflammation is propagated to the brain altering microglial function.

Conflict of interest statement

This work was supported by the Austrian Science fund (FWF); grant number: P 25912-B23.

Figures

Figure 1
Figure 1
Colitis decreases Iba-1 immunoreactivity in the infralimbic cortex (ILC), independently of stress exposure. Panels a-d show representative images of Iba-1 immunohistochemical micrographs for the 4 treatment groups under study: VEH/CO (a), VEH/WAS (b), DSS/CO (c) and DSS/WAS (d); The selected counting area within the region of interest is indicated by a red rectangle (300 × 300 µm). Abbreviations: DP-dorsal peduncular cortex, fmi-forceps minor corpus callosi, ILC-infralimbic cortex.
Figure 2
Figure 2
Brain region-specific effects of colitis and water avoidance stress (WAS) on Iba-1 immunoreactivity in the limbic system. DSS-induced colitis decreases Iba-1 immunoreactivity in the infralimbic cortex (ILC; a), cingulate cortex (CC; b) and medial amygdala (MeA; c), while WAS had no effect. In the hippocampus, both colitis and WAS failed to alter Iba-1-immunoreactivity in Cornu ammonis area 1 (CA1; d) and Cornu ammonis area 3 (CA3; e), but colitis reduced Iba-1 signals in the dentate gyrus (DG; f) independently of stress exposure. In the hypothalamus colitis decreased Iba-1 levels in the paraventricular nucleus of the hypothalamus (PVH; g) independently of stress exposure, but both colitis and WAS did not alter Iba-1 immunoreactivity in the lateral hypothalamus (LH; h). Two-way analysis of variance (ANOVA), **p < 0.01, *p < 0.05 DSS main factor effect.
Figure 3
Figure 3
Brain region-specific effects of colitis and water avoidance stress (WAS) on CD68 immunoreactivity in the limbic system. Both DSS-induced colitis and WAS did not alter CD68 immunoreactivity in the infralimbic cortex (ILC; a) and cingulate cortex (CC; b), but colitis reduced CD68 levels in the medial amygdala (MeA; c) and Cornu ammonis area 1 (CA1; d) independently of stress exposure. WAS and colitis failed to alter CD68 immunoreactivity in other hippocampal and hypothalamic brain regions including Cornu ammonis area 3 (CA3; e), dentate gyrus (DG; f), paraventricular nucleus of the hypothalamus (PVH; g) and lateral hypothalamus (LH; h). Two-way analysis of variance (ANOVA), *p < 0.05 DSS main factor effect.
Figure 4
Figure 4
Colitis decreases CD68 immunoreactivity in the Cornu ammonis area 1 (CA1), independently of stress exposure. Panels a–d show representative images of CD68 immunohistochemical micrographs for the 4 treatment groups under study: VEH/CO (a), VEH/WAS (b), DSS/CO (c) and DSS/WAS (d); The selected counting area within the region of interest is indicated by a red rectangle (300 × 300 µm). Abbreviations: CA1- Cornu ammonis area 1, CCal-corpus callosum, DG-dendate gyrus.
Figure 5
Figure 5
Colitis induces pronounced changes in microglial marker expression in the medial prefrontal cortex. Panels a-k show relative mRNA expression levels of ionized calcium-binding adapter molecule 1 (Iba-1; a), cluster of differentiation 68 (CD68; b), cluster of differentiation 11b (CD11b; c), chitinase-like protein 3 (Chil3; d), cluster of differentiation 206 (CD206; e), arginase 1 (Arg1; f), cluster of differentiation 86 (CD86; g), tumour necrosis factor-α (TNF-α; h), interleukin 1β (IL-1ß; i), nitric oxide synthase 2 (Nos2; j) and indoleamine 2,3-dioxygenase 1 (IDO-1; k). The data presented are means + SEM, n = 7–8/group; t-tests, *p < 0.05, **p < 0.01, ***p < 0.001 vs. VEH.
Figure 6
Figure 6
Colitis increases the number of monocyte-derived macrophages in the brain. (a) Representative polychromatic dot plots demonstrating the gating strategy employed to identify microglia and monocyte-derived macrophages in the brains of mice. Starting at the top left, a size gate was applied followed by gating on live (PI) and CD45+ cells. Finally, microglia cells were defined as CD11b+/CD45med and monocyte-derived macrophages were identified as CD11b+/CD45high (b,c). Colitis increased the number of monocyte-derived macrophages, but did not alter the number of microglial cells. Cell counts normalized to tissue weight. Data are presented as means + SEM, n = 10/group; t-tests, *p < 0.05, vs. VEH.

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