Cytokine effects on Dbp, Per1, Per2, and Per3 in NIH 3T3 fibroblasts. Confluent cells were synchronized with 50% horse serum for 2 h (ZT 0–2). After serum shock, cells were kept in serum-free medium with TNF-α (10 ng/ml), IL-1β (10 ng/ml), IL-6 (10 ng/ml), IFN-α (10 ng/ml), IFN-γ (20 ng/ml) or without cytokines and analyzed at ZT 24 with quantitative real-time RT-PCR. Results are shown as percent of expression to noncytokine-treated fibroblast cultures; one representative experiment of three; mean values of triplicates ± SD; independent sample t test; *, P ≤ 0,05; **, P ≤ 0,005; ***, P ≤ 0.0005.

Impaired expression of clock genes in livers of TNF-α-infused mice. Animals were entrained to a LD cycle during 2 weeks, and their locomotor activity was constantly monitored via a passive infrared sensor and a running wheel. Mice were then implanted with osmotic minipumps delivering TNF-α (light gray bars) or saline as control (dark gray bars). On the third day after the operation, mice were killed at ZT = 11.5 or = 15.5 (ZT = 0, light on; ZT = 12, light off). Livers were extracted and gene expression assessed by real-time PCR. (A) Per1/2/3, RevErbα, and PAR bZip family genes, Dbp, Hlf, and Tef, are down-regulated at both time points, the suppression of PAR bZip family genes is highly significant. In contrast to fibroblast data, RevErbα is strongly down-regulated. Results are shown as percentages relative to the mean of the control group. One representative experiment of three is shown (n = 6 per group; Mann–Whitney test. *, P ≤ 0,05; **, P ≤ 0,005.)

TNF-α impairs expression of clock genes in synchronized NIH 3T3 fibroblasts. (A) TNF-α attenuates circadian Per1/2/3 and PAR bZIP family (Dbp, Tef, and Hlf) gene expression. After serum shock (ZT 0–2), cells were kept in serum-free medium with TNF-α (10 ng/ml; open circles) or without the cytokine (filled circles) and analyzed every 4 h with quantitative real-time RT-PCR. Results are shown as x-fold variations to nonsynchronized fibroblast cultures at ZT 0; three independent experiments; mean values ± SD. (B) Per and PAR bZIP family genes are significantly down-regulated at the 24-h peak (TNF-α: gray bars; controls: black bars), whereas Clock and RevErbα are not affected. Expression of Rev-Erbα was assessed at its peak at ZT 20. Data show one representative experiment done in triplicate (mean ± SD) of four experiments. (C) The amplitude of Dbp expression in serum-shocked NIH 3T3 fibroblasts was attenuated during 60 h in the presence of TNF-α (open circles) compared with controls (filled circles). (D) Withdrawal of TNF-α after the first peak at ZT 24 shows that the suppression of Dbp, Per1, and Per2 at the second peak at ZT 48 is reversible. After serum shock, cells were treated with TNF-α for 24 h (gray bars), or for 48 h with or without a withdrawal of TNF-α after 24 h (white and black bars, respectively). Gene expression was compared with mock-treated cells at the respective ZT (100% expression). Data show one representative experiment done in triplicates (mean ± SD) of three experiments. (E) The suppression of the expression of Dbp, Per1, and Per2 in synchronized fibroblasts at ZT 24 is dose-dependent being significant (P < 0.005) at doses higher than 1 ng/ml TNF-α. Data show the mean ± SD of three independent experiments performed in triplicates. For BDE, we used the independent-sample t test; *, P ≤ 0,05; **, P ≤ 0,005; ***, P ≤ 0.0005.

TNF-α suppresses E-box-mediated transcription of clock genes. (A) Schematic representation of the luciferase reporter genes used. mPer1-luc (3-kb promoter fragment) and mPer3-luc (1.7-kb promoter fragment) contain promoter sequences upstream of their genes in the pGL3basic vector. pBmal1-luc is regulated by a 1-kb promoter fragment and a 1-kb sequence of the 3′UTR from mBmal1 gene. E54-TK consists of the three E-boxes of the mouse Per1 gene and their immediate flanking sequences in front of a TK promoter. DBP-Ebox857-luc contains one of four E-boxes of the Dbp promoter regulating the activity of a SV40 promoter. (B) Native clock gene promoters bearing an E-box are affected by TNF-α. Percent luciferase expression of mPer1-luc, mPer3-luc, and pBmal1-luc transfected NIH 3T3 cells after treatment with 10 ng/ml TNF-α overnight compared with untreated controls (100% luciferase expression) is shown. (C) E-boxes of mPer1 are affected by TNF-α treatment. TNF-α significantly reduces E54-TK-dependent luciferase expression but not pGL3-TK lacking E-boxes and flanking sequences as shown by raw data (RLU, relative light units; Left) and percent inhibition (Right). (D) The E-box of Dbp gene at position +857 is repressed by TNF-α even when CLOCK and BMAL1 are overexpressed. Cotransfection with CLOCK and BMAL1 leads to four to five times higher relative luciferase activity of 857wt compared with the mutated E-box vector (857mut) indicating that the interaction of CLOCK:BMAL1 with the E-box is functional. Luciferase activity is efficiently suppressed by overnight treatment with TNF-α as shown by raw data (Left) and percent inhibition (Right). TNF-α leads to a higher repression in 857wt than in 857mut. For all assays, mean ± SD of triplicates from one representative experiment of three; independent sample t test.

TNF-α reduces locomotor activity of mice but does not alter their circadian rest-activity cycles in free running conditions. (A) Reduced locomotor activities in mice with constant TNF-α infusions are detected from days 1–4. Boxplot showing the changes of locomotor activity for each day after minipump implantation (TNF-α, 1.5 μg/day, for 6 days) or saline as control, compared with the baseline (mean of 3 days immediately before the operation). The TNF-α-treated group is shown as light gray bars and the control group as dark gray bars; circles are outliers, and triangles are extreme values. Six mice per group were analyzed. Data show one experiment (ANOVA for repeated measures, followed by independent-sample t tests; *, P ≤ 0.05). The effect of TNF-α to impair locomotor activity was confirmed at day 3 in two independent experiments. (B) TNF-α reduces locomotor activity in the second half of the dark phase. Actograms (as measured by infrared and running wheel) show data from six individual mice before and after the minipump insertion. Each line represents one individual mouse per treatment, 1 day before the implantation of minipumps (baseline, BL) and 3 days after the operation (OP).