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. This study sought to determine the state of clock gene expression in human peripheral blood leukocytes, and leukocyte 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 (2 ng/kg) or saline at either 0900 or 2100 hrs. Blood samples were collected at selected time points pre- and postinfusion.

Measurements and main results: Clock gene expression was determined in human peripheral blood leukocytes, neutrophils, and monocytes by quantitative real-time polymerase chain reaction. The fold change for each gene was determined by use of the 2 method. We show that endotoxin causes profound suppression of circadian clock gene expression, clearly manifested in human peripheral blood leukocytes, neutrophils, and monocytes. Clock, Cry1-2, Per3, CSNK1epsilon, Rora, and Rev-erb gene expression were all reduced by 80% to 90% with the nadir between 3 and 6 hrs postinfusion. Per1 and Per2 reached an expression nadir between 13 and 17 hrs postinfusion. The levels of plasma interleukin-6 and tumor necrosis factor peaked and then returned to baseline within 6 hrs. In contrast, clock gene expression remained suppressed for up to 17 hrs 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 peripheral blood leukocytes 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.

Figure 1. Clock gene expression in peripheral blood leukocytes obtained from human subjects challenged with endotoxin or saline at 09:00 h

(A) Blood was collected at the indicated time points post-endotoxin (closed symbols; n=4 subjects) or post-saline (open symbols; n=2 subjects) infusion. Gene expression levels were analyzed by qRT-PCR and are expressed as fold change relative to the 09:00 h infusion time, taken to be time 0. The relationship between the time from infusion and the time-of-day is illustrated in the lower right hand corner. Data are expressed as mean fold change ± SD. (B) Plasma cortisol levels (n=4) as a function of time post-endotoxin were monitored by direct radioimmunoassay. (C) TNFα (n=4) and (D) IL-6 (n=4) levels were determined by enzyme-linked immunoassay.

Figure 1. Clock gene expression in peripheral blood leukocytes obtained from human subjects challenged with endotoxin or saline at 09:00 h

(A) Blood was collected at the indicated time points post-endotoxin (closed symbols; n=4 subjects) or post-saline (open symbols; n=2 subjects) infusion. Gene expression levels were analyzed by qRT-PCR and are expressed as fold change relative to the 09:00 h infusion time, taken to be time 0. The relationship between the time from infusion and the time-of-day is illustrated in the lower right hand corner. Data are expressed as mean fold change ± SD. (B) Plasma cortisol levels (n=4) as a function of time post-endotoxin were monitored by direct radioimmunoassay. (C) TNFα (n=4) and (D) IL-6 (n=4) levels were determined by enzyme-linked immunoassay.

Figure 2. Clock gene expression in peripheral blood leukocytes obtained from human subjects challenged with endotoxin at 21:00 h

Blood was collected at the indicated times prior to (open symbols) and post-endotoxin infusion (closed symbols) (n=3 subjects). Gene expression levels were analyzed by qRT-PCR and are expressed as fold change relative to the 21:00 h infusion time, taken to be time 0. The relationship between the time from infusion and the time-of-day is illustrated in the lower right hand corner.

Figure 3. Clock gene expression in peripheral blood leukocytes, neutrophils and monocytes obtained from human subjects challenged with endotoxin at 09:00 h

Blood was collected at time 0, 6 and 24 hours post-endotoxin infusion. Total peripheral blood leukocytes, neutrophils and monocytes subpopulations (n=3 for each) were obtained prior to RNA isolation. Gene expression levels were analyzed by qRT-PCR and are expressed as fold change relative to the 09:00 h infusion time, taken to be time 0. The relationship between the time from infusion and the time-of-day is illustrated in the lower right hand corner. The data shown are averages (n=3) (all data sets are presented in Fig. 2S).

Figure 4. Endotoxin does not alter the melatonin secretory rhythm

(A) Plasma melatonin concentrations were determined in blood samples obtained from control subjects (n=8 subjects) at 22:00 h, 02:00 h, 06:00 h, 09:00 h and 10:00 h (open squares). Samples were also obtained at the indicated times of the day pre- and post-saline infusion (open circles; n=2 subjects), and pre- and post-endotoxin infusion (closed diamonds; n=4 subjects). Arrow indicates the 09:00 h infusion time. (B) Samples were obtained at the indicated times of the day pre- and post-endotoxin infusion (closed diamonds; n=4 subjects). Arrow indicates the 21:00 h infusion time.

Figure 5. Clock gene expression in human peripheral blood leukocytes

The relative gene expression values for each gene post-endotoxin infusion were combined for all day (n=4 subjects) and night subjects (n=3 subjects), and are presented as an average. Where shown, bars represent standard error of the mean. The line that is featured in each panel was drawn based on the mean expression values of Clock, Cry1, Cry2, and Per3, which exhibited correlated expression in both the day and night subjects.