Chronic alcohol-induced neuroinflammation involves CCR2/5-dependent peripheral macrophage infiltration and microglia alterations.

Alcohol induces infiltration of peripheral macrophages into the CNS. a Alcohol-fed mice received 5% (v/v) alcohol in Lieber-DeCarli liquid diet ad libitum (EtOH) for 6 weeks while pair-fed animals received a calorie-matched liquid diet (PF). b mRNA expression of Ccl2, Ccr2, and Ccr5 was measured from the hippocampus of PF and EtOH mice. c Brain immune cells from wild-type mice fed chronic alcohol (EtOH) or a pair-fed (PF) control diet were isolated and quantified by flow cytometry gating for live, single cells. Microglia (CD11b+ CD45lo) were differentiated from peripheral macrophages (CD11b+ CD45hi) based on surface marker staining and infiltrating macrophages in pair- vs alcohol-fed mice. d Quantification of microglia (CD11b+ CD45lo) and peripheral macrophages (CD11b+ CD45hi) in PF and EtOH mice. Data are mean ± SEM, n = 4–7 mice/group. *p < 0.05 by Student’s t test

Alcohol-induced peripheral macrophages differentially infiltrate the CNS. a CX3CR1eGFP/+ CCR2RFP/+ mice were generated by crossing CX3CR1eGFP/eGFP CCR2+/+ and CX3CR1+/+ CCR2RFP/RFP mice to allow visualization of resident microglia (GFP+; green) and brain infiltrating macrophages (RFP+; red) in the cortex, cerebellum, and hippocampus of mice fed chronic alcohol or a calorie-matched diet. b Acquired × 10 images of ethanol-fed CX3CR1eGFP/+ CCR2RFP/+ mice were stitched together to provide the larger representative images shown. c, d Quantification of resident CX3CR1-eGFP+ microglia and CCR2-RFP+ brain infiltrating macrophages in the cortex, cerebellum, and hippocampus. Data are mean ± SEM, n = 4–7 mice/group. *p < 0.05; n.s., not significant by Student’s t test

Inhibition of CCR2/5 signaling reduces CNS macrophage infiltration without altering activation marker expression of infiltrating macrophages. a Mice received a pair-fed diet (PF) or chronic alcohol (EtOH), and some alcohol-fed mice received 6 weeks of daily preventive subcutaneous CVC injection (EtOH + 6wk CVC) or 3 weeks of daily CVC treatment (EtOH + 3wk CVC). PF and EtOH mice received daily vehicle control injections. b Representative flow cytometry plots of peripheral macrophages (CD11b+ CD45hi) in EtOH, EtOH + 6wk CVC and EtOH + 3wk CVC-treated mice. c Quantification of infiltrating brain macrophages (CD11b+ CD45hi) in pair-fed, alcohol-fed, and treatment groups. d Expression of various activation markers were measured by flow cytometry in CD11b+ CD45hi infiltrating macrophages including CD86, CD68, MHC-II, CD163, and CD206. Data are mean ± SEM, n = 6–7 mice/group. *p < 0.05 by one-way ANOVA

Chronic alcohol induces inflammatory gene expression changes in multiple brain regions. a Proinflammatory cytokine proteins TNFα, IL-1β, and IL-6 were measured from the hippocampus of pair- (PF) and alcohol-fed (EtOH) as well as alcohol-fed mice treated with CVC for 3 weeks (EtOH +3wk CVC) by ELISA. b Inflammatory gene expression of pair- and alcohol-fed mice was analyzed using the Nanostring nCounter Immunology Panel and revealed 17 genes significantly altered in the cerebellum (green, expression increased; red, expression decreased). c Gene expression changes of genes found by Nanostring to be altered as well as some common inflammatory markers, and CCR2/5 axis genes were measured in the hippocampus, cortex, and cerebellum by qPCR in a larger cohort of pair- and alcohol-fed mice as well as mice treated with 3 or 6 weeks CVC (purple, > 3-fold increase in expression; cyan > 3-fold decrease in the expression compared to PF; complete gene expression profile data found in Tables 3, 4, and 5). Data are mean ± SEM, n = 4–6 mice/group. *p < 0.05 by one-way ANOVA

Alcohol-induced morphologic changes in microglia are partially restored by CCR2/5 inhibition. a Representative images of hippocampal microglia (CX3CR1-GFP+; green) displaying altered morphologies in pair-fed (PF) vs alcohol-fed (EtOH) mice, acquired at × 63 magnification. b Quantification of average hippocampal microglia soma area (pixel2) and c perimeter distance (pixel length) in mice fed control or alcohol diet or alcohol-fed mice treated with CCR2/5 inhibitor. d Expression of various activation markers were measured by flow cytometry in CD11b+ CD45lo microglia including CD86, CD68, MHC-II, CD163, and CD206. Data are mean ± SEM, n = 3 mice/group and 3–10 sections/mouse for a–c and n = 6–7 mice/group for d. *p < 0.05 by one-way ANOVA.

Alcohol downregulates the microglial expression of CD68. a Representative images of the hippocampal sections stained for Iba1 (green), CD68 (red), and DAPI (blue) from pair-fed (PF), alcohol-fed (EtOH), and CVC-treated alcohol-fed mice, acquired at × 63 magnification. b Quantification of Iba1 and CD68 colocalization, normalized to total Iba1+ area per image in the hippocampal CA1, CA3, and DG regions and throughout the HPF. c Flow cytometry data showing the percent of microglia (defined as CD11b+ CD45lo) that are positive for CD68 expression. Data are mean ± SEM, n = 3–4 mice/group with 4–5 images per region per mouse. *p < 0.05; n.s., not significant by one-way ANOVA