In vivo endotoxin synchronizes and suppresses clock gene expression in human peripheral blood leukocytes*

Abstract

Objectives The intravenous administration of a bolus dose of endotoxin to healthy human subjects triggers acute systemic inflammatory responses that include cytokine production and dynamic changes in gene expression in peripheral blood leukocytes (PBL). This study sought to determine the state of clock gene expression in human PBL, and leukocytes subpopulations, challenged with in vivo endotoxin at two circadian/diurnal phases of the clock. Design Clinical and laboratory investigation. Setting University-based research laboratory and clinical research center Subjects Human volunteers. Interventions Human subjects were administered a standard dose of endotoxin (2ng/kg) or saline at either 09:00 or 21:00 h. Blood samples were collected at selected time points pre- and post-infusion. Measurements and Mains results Clock gene expression was determined in human PBL, neutrophils, and monocytes, by quantitative real-time polymerase chain reaction. The fold change for each gene was determined using the 2(-ΔΔCt) method. We show that endotoxin causes profound suppression of circadian clock gene expression, clearly manifested in human PBL, neutrophils, and monocytes. Clock, Cry1-2, Per3, CSNK1ε, Rora and Rev-erb gene expression were all reduced by 80-90% with the nadir between 3 to 6 hours post-infusion. Per1 and Per2 reached an expression nadir between 13 and 17 hours post-infusion. The levels of plasma interleukin-6 and tumor necrosis factor peaked and then returned to baseline within 6 hours. In contrast, clock gene expression remained suppressed for up to 17 hours, irrespective of the phase of the clock at the time of the endotoxin challenge. Endotoxin did not perturb the melatonin secretory rhythm. Conclusions Circadian clock gene expression in PBL is dramatically altered, and possibly uncoupled from the activity of the central clock, during periods of acute systemic inflammation. The realignment of the central and peripheral clocks may constitute a previously unappreciated key factor affecting recovery from disease in humans.

Evaluation of suppressive and pro-resolving effects of EPA and DHA in human primary monocytes and T-helper cells

“…For Th-cell assays, PBMC (1 × 10 6 /ml) were incubated without or with increasing concentrations of EPA or DHA (25,50, 100 µM) for 19 h. Subsequently, cells were alloreactively stimulated with PMA (2.5 ng/ml; induces cytokine production by activation of protein kinase C) and ionomycin (0.5 µg/ml; potentiates activation as calcium ionophore) in the presence of brefeldin A (5 µg/ml; increases sensitivity of intracellular cytokine detection by interfering with the function of the Golgi apparatus) for another 5 h. Supernatants were frozen at Ϫ 80°C until lipid mediator analysis. For some experiments, cells were preincubated for 30 min with different concentrations of the selective peroxisome proliferatoractivated receptor (PPAR) ␥ inhibitor T0070907 (0.4 or 2 µM) before 100 µM EPA or DHA and the stimulation cocktail were added as described above.…”

Timing Matters: Circadian Rhythm in Sepsis, Obstructive Lung Disease, Obstructive Sleep Apnea, and Cancer

“…Circadian rhythm may be suppressed during acute inflammatory illness, as evidenced by suppression of circadian clock genes in peripheral leukocytes for at least 17 hours after inoculation of endotoxin in mice (34). This finding may explain why diurnal variation of melatonin was blunted in ICU patients with sepsis compared with ICU patients without sepsis (35).…”

Diurnal and seasonal changes in <i>IL-6</i> and <i>IL-18</i> gene expression in blood leukocytes of male chickens with experimental peritonitis: the impact of lighting conditions and melatonin

“…These circadian clock mechanisms, which can operate autonomously even ex vivo in the spleen and lymph nodes, are based on the expression of major clock genes, like Bmal, Per, Clock and Rev-erb α (Keller et al, 2009). Moreover, clock gene expression in human immune cells seems to be very sensitive to a damaging challenge, since a single injection with endotoxin was found to suppress the expression of these genes in the PBLs of healthy humans volunteers (Haimovich et al, 2010). Constant light applied to the chickens in the present study desynchronized or shifted the circadian rhythm of the transcription of selected clock genes in the pineal gland, while melatonin supplementation restored the diurnal pattern in a season-dependent manner (data not shown).…”