A circadian clock in macrophages controls inflammatory immune responses

Abstract

Time of day-dependent variations of immune system parameters are ubiquitous phenomena in immunology. The circadian clock has been attributed with coordinating these variations on multiple levels; however, their molecular basis is little understood. Here, we systematically investigated the link between the circadian clock and rhythmic immune functions. We show that spleen, lymph nodes, and peritoneal macrophages of mice contain intrinsic circadian clockworks that operate autonomously even ex vivo. These clocks regulate circadian rhythms in inflammatory innate immune functions: Isolated spleen cells stimulated with bacterial endotoxin at different circadian times display circadian rhythms in TNF-alpha and IL-6 secretion. Interestingly, we found that these rhythms are not driven by systemic glucocorticoid variations nor are they due to the detected circadian fluctuation in the cellular constitution of the spleen. Rather, a local circadian clock operative in splenic macrophages likely governs these oscillations as indicated by endotoxin stimulation experiments in rhythmic primary cell cultures. On the molecular level, we show that >8% of the macrophage transcriptome oscillates in a circadian fashion, including many important regulators for pathogen recognition and cytokine secretion. As such, understanding the cross-talk between the circadian clock and the immune system provides insights into the timing mechanism of physiological and pathophysiological immune functions.

Fig. 1.

Fully competent circadian clocks in tissues and cells of the immune system. (Left) Circadian clock genes Per2 (filled circles) and Rev-Erbα (open circles) are rhythmically expressed in murine spleen cells, lymph nodes, and peritoneal macrophages. Tissues and cells were harvested at regular intervals over the course of the first 2 days after transfer of the mice from a LD cycle to DD. Gray and black bars refer to the previous light and dark periods, respectively. CT 0 corresponds to the time in DD when the light would have turned on in the prior LD cycle. Transcript levels were analyzed by using quantitative RT-PCR. Displayed are the means ± SEM. (spleen: n = 3–6; lymph nodes: n = 3–4 except for n = 2 at CT 6/day 2; macrophages: n = 3–4) normalized to nonoscillating Gapdh expression levels. (Right) Autonomous clock gene oscillation in in vitro conditions. A small piece of spleen, superficial inguinal lymph nodes as well as peritoneal macrophages from PER2::LUC knockin mice (20) were cultured in medium containing luciferin. Circadian bioluminescence was continuously recorded for ≈1 week by using photomultiplier tubes. Representative time series for at least three independent experiments are shown.

Fig. 2.

Circadian cytokine secretion upon challenge with bacterial endotoxin. (A) Spleens from C57BL/6 mice transferred in DD were harvested at regular 4-h intervals. After stimulation with LPS, TNF-α (Left) and IL-6 (Right) secretion was determined by ELISA. Gray and black bars refer to the previous light and dark periods, respectively. Presented are the means ± SEM (n = 4–5). Circadian oscillations are statistically significant as analyzed with CircWave (TNF-α and IL-6: P < 0.05). (B) Cellular composition of the spleen is time-of-day dependent. The same samples as in A were analyzed with cell-counting chamber and flow cytometry. CD19, CD90.2, and CD11b in combination with CD14 were used as characteristic surface markers of B cells, T cells, and monocytes/macrophages, respectively (for FACS gate settings and surface marker expression levels, see Fig. S4). (C) Cytokine response as in A with respect to numbers of CD11b/CD14-positive spleen cells from B lower right.

Fig. 3.

A macrophage intrinsic clockwork regulates circadian TNF-α and IL-6 secretion upon LPS stimulation. (A) Circadian modulation of LPS-induced cytokine response is independent of systemic cortisol. Spleens from adrenalectomized C57BL/6 mice were harvested and analyzed as described in Fig. 2. TNF-α and IL-6 cytokine secretion per macrophage was determined via ELISA by taking the absolute number of monocytes/macrophages of the spleen into account (see also Figs. S2 and S4A and Materials and Methods). Circadian oscillations are statistically significant as analyzed with CircWave (P < 0.001 and P < 0.05 for TNF-α and IL-6, respectively). (B) TNF-α response upon LPS stimulation is regulated by a cell-intrinsic, local clock. Spleen cells from 20 C57BL/6 mice were harvested, pooled, and plated for tissue culture. Individual wells were stimulated for 4 h with LPS at indicated times, and supernatants were collected thereafter. TNF-α levels in supernatant were determined by ELISA and tested for statistical significance with CircWave (presented are means ± SEM, n = 15, P < 0.0001).

Fig. 4.

Eight percent of all transcripts in macrophages are expressed with a circadian rhythm. (A) Phase-sorted heat map of genes transcribed in a circadian manner in peritoneal macrophages. Cells harvested via peritoneal lavage from four C57BL/6 mice every 4 h were magnetically purified for CD11b surface expression (see Fig. S5). Three individual RNA samples of each time were pooled and subjected to global gene transcription measurement by using Affymetrix chips. The analysis on circadian rhythmicity was done with CircWaveBatch. Lfdrs were determined and cutoff value was arbitrarily set to 0.1 as a measure for rhythmic versus nonrhythmic transcripts (see Fig. S7). Genes expressed in a circadian manner were plotted phase-sorted in a heat-map style (colors indicate min–max normalized relative expression: green, minimum expression; red, maximum expression). (B) Canonical clock gene expression in peritoneal macrophages. Individual datasets from A were plotted (filled circles) together with data obtained by a quantitative RT-PCR assay of the same samples (open circles, means ± SEM, n = 4, except for times CT 24 and 28, n = 3). Statistical analysis for qPCR data and chip data were performed with CircWave and CircWaveBatch, respectively (microarray: P ≤ 0.0001: Bmal1, Cry1, Per2; P ≤ 0.01: Rev-Erbα, Dbp, Cry2; P ≤ 0.05: Per1 and Clock; qPCR: P ≤ 0.0001: Bmal1, Cry1, Per1/2, Clock, Rev-Erbα, Dbp; P ≤ 0.001: Cry2).

Fig. 5.

Circadian transcription of genes contributing to LPS response. (A) Gene regulatory network forming the LPS-triggered cytokine response. Dark gray boxes indicate circadian transcriptional regulation of the respective gene (P ≤ 0.05). Arrows indicate molecular interaction of genes involved in LPS response generation. For detailed information about circadian transcripts in this network, see Table S2. (B) Selected circadian transcriptional profiles of genes participating in LPS-triggered signaling cascade. Individual datasets from microarray analysis were plotted (filled circles) together with data obtained by a quantitative RT-PCR assay of the same samples (open circles, means ± SEM, n = 4, except of times CT 24 and 28, n = 3). Statistical analysis for qPCR data and chip data were performed with CircWave and CircWaveBatch, respectively (microarray: P ≤ 0.0001: Jun, Adam17, Cd180; P ≤ 0.01: IκBα, MD-1, Elavl1, Fos, Erk1; qPCR: P ≤ 0.0001: Adam17, Elavl1, Cd180, Erk1, MD-1; P ≤ 0.01: IκBα; P ≤ 0.05: Jun).