Microglia inflammatory responses are controlled by an intrinsic circadian clock

Abstract

The circadian system regulates many physiological functions including inflammatory responses. For example, mortality caused by lipopolysaccharide (LPS) injection varies depending on the time of immunostimulation in mammals. The effects of more subtle challenges on the immune system and cellular mechanisms underlying circadian differences in neuroinflammatory responses are not well understood. Here we show that adult male Sprague-Dawley rats injected with a sub-septic dose of LPS during the light phase displayed elevated sickness behaviors and hippocampal cytokine production compared to rats injected during the dark phase. Microglia are the primary central nervous system (CNS) immune cell type and may mediate diurnal differences in sickness response, thus we explored whether microglia demonstrate temporal variations in inflammatory factors. Hippocampal microglia isolated from adult rats rhythmically expressed inflammatory factors and circadian clock genes. Microglia displayed robust rhythms of TNFα, IL1β and IL6 mRNA, with peak cytokine gene expression occurring during the middle of the light phase. Microglia isolated during the light phase were also more reactive to immune stimulation; such that, ex vivo LPS treatment induced an exaggerated cytokine response in light phase-isolated microglia. Treating microglia with corticosterone ex vivo induced expression of the circadian clock gene Per1. However, microglia isolated from adrenalectomized rats maintained temporal differences in clock and inflammatory gene expression. This suggests circadian clock gene expression in microglia is entrained by, but oscillates in the absence of, glucocorticoids. Taken together, these findings demonstrate that microglia possess a circadian clock that influences inflammatory responses. These results indicate time-of-day is an important factor to consider when planning inflammatory interventions such as surgeries or immunotherapies.

Keywords: Circadian; Clock gene; Cytokines; Glucocorticoids; Inflammation; Microglia.

Figure 1. Circadian timing of LPS injection affects sickness behavior

Adult male Sprague-Dawley rats were injected with LPS (100 ug/kg) at either ZT6 or ZT16 and (A) social investigation, (B) sucrose anhedonia, and (C) changes in body mass were evaluated (n=4). Data are expressed as mean ± SEM. *p<0.05 between ZT16 and ZT6 LPS groups, †p<0.05 between LPS and saline

Figure 2. Temporal differences in hippocampal cytokine expression following an LPS injection

Adult male Sprague-Dawley rats were injected with LPS (100 ug/kg) at either ZT6 or ZT16 and hippocampal tissue was collected after 3 or 24 h to evaluate (A) IL1β, (E) TNFα, (C) IL6, (D) IL10 mRNA expression and (E) IL1β, (F) TNFα, (G) IL6, (H) IL10 protein concentrations (n=4). Data are expressed as mean ± SEM. *p<0.05 between ZT16 and ZT6, †p<0.05 between LPS and saline.

Figure 3. Circadian clock gene expression in microglia

Hippocampal microglia were isolated from adult male Sprague-Dawley rats every 6h (n=5). Microglia were plated for 3 h prior to RNA extraction. mRNA concentrations are expressed relative to β-actin and presented as mean ± SEM.

Figure 4. Circadian gene expression of inflammatory factors in microglia

Hippocampal microglia were obtained from adult male Sprague Dawley rats every 6 h (n=5). Microglia were then plated with media for 3 h and expression of (A) IL1β, (B) TNFα, (C) IL6, (D) IL1R1, (E) IKB, (F) MHCII, (G) NLRP3, and (H) TLR4 mRNA were evaluated. To assess microglia priming (I) IL1β and (J) TNFα mRNA expression were also evaluated in microglia stimulated for 3 h ex vivo with 0, 10, or 100 ng of LPS. mRNA concentrations are expressed relative to β-actin and presented as mean ± SEM.

Figure 5. Microglia rhythms in circadian clock genes are entrained by but not dependent on glucocorticoids

Hippocampal microglia were isolated at ZT6 or ZT16 from rats that underwent either an ADX or sham surgery (n=4). Microglia were plated with media for 3 h and mRNA expression of (A) Per1, (B) BMAL1, and (C) CLOCK were evaluated. In a separate cohort of rats hippocampal microglia were isolated at ZT6 or ZT16 and stimulated ex vivo with 0, 10, 100, or 1000 nM CORT for 2 h prior to extracting mRNA to measure whether CORT induces (D) Per1, (E) BMAL1, and (F) CLOCK expression (n=3). mRNA concentrations are expressed relative to β-actin and presented as mean ± SEM. *p<0.05 between ZT6 and ZT16, †p<0.05 between CORT concentrations.

Figure 6. Time-of-day differences in microglia inflammatory potential are not mediated by glucocorticoids

Hippocampal microglia were isolated at ZT6 or ZT16 from rats that underwent either an ADX or sham surgery (n=4). Microglia were plated with 0, 10, or 100 ng of LPS for 3 h and mRNA expression of (A) IL1 and (B) TNF were evaluated. mRNA concentrations are expressed relative to β-actin and presented as mean ± SEM. *p<0.05 between ZT6 and ZT16.