Table 1.
Primers used for real time PCR analysis and respective gene targets.
Fig 1.
LPS induces a phase shift in synchronized peritoneal macrophages and impairs the expression of core circadian genes in peritoneal macrophages.
(A) Peritoneal macrophages from mPer2luciferase transgenic mice were seeded in 96-well plate (white) for 24 hours. After 2h serum shock, cells were kept in luminescence buffer in presence or absence of LPS (20ng/ml) and bioluminescence was recorded every 2 hours which starts from Time 0. (B) Oscillation curves were analyzed by cosinor (time 0 and 24h represented by 0° and 360°, respectfully) and acrophase determined (mean ± SEM, n = 8, t-test, * p<0.05, versus Control). (C) Peritoneal macrophages were synchronized and mRNA levels of Bmal1 and 18S were assessed every 8h with qRT-PCR in presence or absence of LPS (20ng/ml). Transcript abundance (ΔΔCt) was reported relative to Time 0 in the control group (mean ± SEM, n = 5, one-way ANOVA with Bonferroni post hoc correction, *p<0.05, versus control).
Fig 2.
LPS promotes a circadian phase-shift at low concentrations.
Varying doses of LPS (0, 5, 20 and 100ng/ml) were added into the luminescence buffer at Time 0 after serum shock. (A) Left panel: Bioluminescence were recorded every 2h continuing for 68 hours. Right panel: Relative cell numbers were measured via a cell viability assay at the end of the luminescence measurements (mean ± SEM, n = 4, one-way ANOVA, ns versus Control). (B) Oscillation curves were analyzed by cosinor and acrophase compared by one-way Anova with Bonferroni post hoc correction (mean ± SEM, n = 8, *p<0.05, versus control, # p<0.05, versus LPS 5ng/ml).
Fig 3.
Inhibition of TLR4 reverses the LPS-induced phase-shift.
Peritoneal macrophages were isolated from mPer2luciferase transgenic mice and subjected to synchronization via dexamethasone shock. 20ng/ml LPS with or without LPS-RS (5μg/ml) was added to the luminescence buffer at Time 0. (A) Bioluminescence were recorded every 2h continuing for 68 hours. (B) Oscillation curves were analyzed by cosinor and acrophase compared by one-way Anova with Bonferroni post hoc correction (mean ± SEM, n = 8, *p<0.05, versus Control, ns p>0.05, versus LPS-RS).
Fig 4.
LPS stimulates ROS release from peritoneal macrophage and elevated ROS impairs the function of circadian transcription factors.
(A) Peritoneal macrophages were isolated from mPer2luciferase transgenic mice and subjected to different treatments over 24h (LPS 20ng/ml, LPS-RS 5μg/ml, gp91 ds-tat 1μM). Unstimulated or basal superoxide release was monitored using L-012 chemiluminescence (mean ± SEM, n = 5, one-way ANOVA with Bonferroni post hoc correction, *p<0.05, versus Control). (B) Peritoneal macrophages from mPer2luciferase mice were subjected to different treatments (LPS 20ng/ml, PEG-SOD, 100U/ml, Tempol 0.4mM) and bioluminescence recorded every 2h for 68 hours (mean ± SEM, n = 5, acrophase were compared via one-way ANOVA with Bonferroni post hoc correction, *p<0.05, versus control. # p<0.05, versus LPS). (C) U2OS Bma1luciferase cells were transduced with active or inactive Nox5 adenovirus (15 MOI) and the oscillation of expressed luciferase activity recorded every 2h after serum shock. Oscillation curves were analyzed by cosinor and acrophase compared by one-way Anova with Bonferroni post hoc correction (mean ± SEM, n = 6, *p<0.05, versus Control, # p<0.05, versus Nox5 active). (D) Per1 promoter transactivation was assessed by a dual luciferase assay in transfected COS cells expressing BMAL1, BMAL1+CLOCK in the presence or absence of the ROS generator NOX5 or an inactive NOX5 enzyme (H268Q), (mean ± SEM, n = 5, one-way ANOVA with Bonferroni post hoc correction, *p<0.05, versus Bmal1 alone. # p<0.05, versus Nox5 active). (E) SOD-sensitive superoxide production was monitored by L-012 chemiluminescence. Results are presented as mean ± SEM, n = 6, one-way ANOVA with Bonferroni post hoc correction, *p<0.05, versus Bmal1 alone.
Fig 5.
LPS increases NO release from peritoneal macrophage and iNOS-derived NO has no effect on circadian transcription factor activity.
(A) NO release was measured by chemiluminescence detection of NO2− (mean ± SEM, n = 5, one-way ANOVA with Bonferroni post hoc correction, *p<0.05, versus control. # p<0.05, versus LPS). (B) Dual luciferase assay in COS cells expressing the Per1 promoter luciferase and BMAL1 and CLOCK or BMAL1, BMAL1+NPAS2+iNOS in the presence or absence of L-NAME (2mM), mean ± SEM, n = 6, one-way ANOVA with Bonferroni post hoc correction, *p<0.05, versus Bmal1 alone). (C) Peritoneal macrophages were synchronized as described and exposed to LPS (20ng/ml) with or without L-NAME (2mM). Bioluminescence was recorded every 2h for 48 hours. Oscillation curves were analyzed by cosinor and acrophase compared by one-way Anova with Bonferroni post hoc correction (mean ± SEM, n = 5, *p<0.05, versus Control, ns versus LPS).
Fig 6.
Circadian genes knockout alters the balance ROS and NO release from peritoneal macrophages.
Peritoneal macrophages were isolated from WT mice and circadian gene knockout mice (Bmal1-KO mice and Per-TKO mice), and cells were subjected to different treatments over 24h. (A) Unstimulated or basal superoxide release was monitored using L-012. NO release was measured by NO-specific chemiluminescence of NO2−. The data was normalized by residual NO2− detected in the presence of L-NAME (mean ± SEM, n = 6, two-way ANOVA with Bonferroni post hoc correction, *p<0.05, versus control. # p<0.05, versus WT). (B) Peritoneal macrophages were isolated from WT or circadian clock knockout mice (Bmal1 KO and Per-TKO), exposed to vehicle or LPS (20ng/ml, 24h) and lysed in Laemmli sample buffer. Cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies to gp91phox (NOX2), iNOS and GAPDH. Results are representative of 3 experiments. (C) Peritoneal macrophages were isolated from WT or circadian clock knockout mice (Bmal1 KO and Per-TKO), exposed to vehicle or LPS (20ng/ml, 24h) and lysed in TRIZOL for mRNA extraction. Relative mRNA expression levels of Tnfα and Il-6 were measured by qRT-PCR (ΔΔCt) normalized to GAPDH (mean ± SEM, n = 6, two-way ANOVA with Bonferroni post hoc correction, *p<0.05 versus control, # p<0.05 versus WT).
Fig 7.
Circadian clock disruption increases LDL uptake by macrophages.
Peritoneal macrophages were isolated from WT, Bmal1 KO or Per-TKO mice and cells exposed to oxLDL (50μg/ml) for 72h. (A) Oil red O staining for lipid uptake in macrophages. The extent of staining was quantitated by measurement of fluorescent intensity. (mean ± SEM n = 4, t-test, *p<0.05 versus WT). (B) Measurement of cholesterol ester and free cholesterol content from macrophages. (mean ± SEM n = 4, t-test, *p<0.05 versus WT
Fig 8.
Circadian clock disruption alters the migration and adhesion of macrophages.
(A) The effect of circadian disruption on macrophage migration was evaluated using the Oris Cell Migration Assay. Peritoneal macrophages were isolated from WT and Per-TKO mice and subjected to the indicated treatment (LPS 100ng/ml) for 24h, cells stained with calcein AM for 30 min and the fluorescence signal in the migration zone quantified. (mean ± SEM n = 4–5, two-way ANOVA with Bonferroni post hoc correction, *p<0.05 versus control, # p<0.05 versus WT). (B) Fluorescently labeled peritoneal macrophages were incubated with activated adherent human aortic endothelial cells for 15 minutes at 37°C and the degree of cell adhesion assay was quantified (mean ± SEM n = 6, two-way ANOVA with Bonferroni post hoc correction, *p<0.05 versus control, # p<0.05 versus WT).