The nuclear receptor REV-ERBα mediates circadian regulation of innate immunity through selective regulation of inflammatory cytokines

Abstract

Diurnal variation in inflammatory and immune function is evident in the physiology and pathology of humans and animals, but molecular mechanisms and mediating cell types that provide this gating remain unknown. By screening cytokine responses in mice to endotoxin challenge at different times of day, we reveal that the magnitude of response exhibited pronounced temporal dependence, yet only within a subset of proinflammatory cytokines. Disruption of the circadian clockwork in macrophages (primary effector cells of the innate immune system) by conditional targeting of a key clock gene (bmal1) removed all temporal gating of endotoxin-induced cytokine response in cultured cells and in vivo. Loss of circadian gating was coincident with suppressed rev-erbα expression, implicating this nuclear receptor as a potential link between the clock and inflammatory pathways. This finding was confirmed in vivo and in vitro through genetic and pharmacological modulation of REV-ERBα activity. Circadian gating of endotoxin response was lost in rev-erbα(-/-) mice and in cultured macrophages from these animals, despite maintenance of circadian rhythmicity within these cells. Using human macrophages, which show circadian clock gene oscillations and rhythmic endotoxin responses, we demonstrate that administration of a synthetic REV-ERB ligand, or genetic knockdown of rev-erbα expression, is effective at modulating the production and release of the proinflammatory cytokine IL-6. This work demonstrates that the macrophage clockwork provides temporal gating of systemic responses to endotoxin, and identifies REV-ERBα as the key link between the clock and immune function. REV-ERBα may therefore represent a unique therapeutic target in human inflammatory disease.

Fig. 1.

Circadian gating of murine cytokine responses to LPS. (A) Serum cytokines were quantified 4 h after i.p. LPS administration at either CT0 or CT12. IL-6, IL-12(p40), CCL5, CXCL1, and CCL2 (but not TNF-α) showed significantly higher levels after CT12 challenge vs. CT0 (n = 7–9, two-way ANOVA, post hoc Bonferroni). (B) mRNA was isolated from PECs harvested 30 min after LPS treatment at either CT0 or CT12. Levels of cytokine mRNA were quantified (relative to β-actin) and are presented in relation to expression levels in PECs harvested at CT0 from vehicle-treated mice (n = 6–7, t test).

Fig. 2.

Generation of LysM-bmal^−/− mice. (A) PECs cultured from LysM-bmal^−/− on a PER2::luc background are arrhythmic in contrast to WT mice. Arrow indicates application of dexamethasone (representative of four trials). (B) Western blotting confirms loss of BMAL protein in targeted macrophages. (C) Isolated PECs from LysM-bmal^−/− mice show altered expression of core clock genes in culture (n = 3); transcripts are reported relative to time 0 in WT mice. (D) In vivo LPS challenge at CT0 and CT12 elicits comparable levels of IL-6 release in LysM-bmal^−/− mice, whereas WT mice retain the gated responses (n = 8–9, one-way ANOVA, Bonferroni t test). (E) In vitro stimulation of PECs from LysM-bmal^−/− mice at opposing time points confirmed loss of gating in these cells (n = 3–4, t test).

Fig. 3.

Loss of gating in rev-erbα^−/− mice. (A) IL-6 release after in vivo LPS does not differ between CT0 and CT12 administration in rev-erbα^−/− mice, unlike WT littermates (n = 6–7, ANOVA and Bonferroni). (B) PECs isolated from rev-erbα^−/− mice show loss of a gated response to LPS (n = 3–4). (C) Isolated PECs from rev-erbα^−/− mice on a PER2::luc background show circadian oscillations (period 24.1 ± 0.4) comparable to WTs (period 23.7 ± 0.4; n = 4). (D) Circadian profiling shows that bmal, rev-erbβ, and dbp mRNA expression in PECs remains circadian despite the absence of rev-erbα; transcripts are reported relative to time 0 in WT mice (n = 3).

Fig. 4.

Circadian gating in human cells. (A) In human MDMs, il6 mRNA response to LPS peaks 16 h after serum synchronization (values are mean ± SD, n = 4). (B) In response to LPS, IL-6 release (but not IL-8) is inhibited by the REV-ERB ligand GSK4112 in both MDMs (n = 3) and primary alveolar macrophages (n = 18). Cells were incubated with LPS and GSK4112 for 16 h before harvest (one-way ANOVA, post hoc Bonferroni). (C) IL-6 protein (but not IL-8) expression by THP-1 cells in response to LPS is significantly attenuated by application of GSK4112 (one-way ANOVA, post hoc Bonferroni). (D) il6 mRNA expression after LPS application is reduced by both GSK4112 and hemin in THP-1 cells (n = 3, one-way ANOVA, post hoc Bonferroni); transcript abundance is reported relative to LPS alone. (E) shRNA knockdown of rev-erbα in THP-1 cells increases il6 (but not il8) mRNA after LPS challenge compared with controls (n = 3, t test). (F) Knockdown of rev-erbα abolishes the inhibition of il6 mRNA abundance seen with GSK4112 (n = 3, t test).

Fig. 5.

Confirmation of the microarray study in human primary MDMs. Transcription of a selection of cytokines, but not il8, after LPS challenge is attenuated by coapplication of the REV-ERB ligand GSK4112 (n = 6, one-way ANOVA, post hoc Bonferroni); transcript abundance is reported relative to LPS-treated cells.