In this prospective observational pilot study, we examined the rhythmicity of the 15 most common clock and clock-associated genes in 17 patients with septic shock and compared them with young, healthy men. We were able to show a decreased mean gene expression of almost every clock gene in patients with septic shock, except for CRY2, which was upregulated. The positive regulators CLOCK and ARNTL (BMAL1) had the lowest rhythmicity score and were most arrhythmic, while the negative regulators NR1D1, NR1D2 and CRY2 were the most rhythmic. No association with secondary clinical parameters was found. To the best of our knowledge, no other study investigated a comparable amount of clock genes in septic shock patients.
Other studies also investigated the circadian rhythm in critically ill patients. Coiffard et al. included 38 severe trauma patients, of whom at least 13 developed a septic episode . All trauma patients had disrupted rhythms of cortisol, leukocytes and cytokines. Of the three measured clock genes BMAL1, PER2 and PER3 only the last two were down-regulation. Diaz et al. investigated the circadian rhythm in 11 patients in neurological intensive care units . On the day of admission, all patients had a rhythmic expression of CLOCK, BMAL1, CRY1 and PER2, whereas after one week the clock gene CLOCK showed a disrupted circadian rhythm . A recent study of Acuna-Fernandez et al. compared BMAL1, CLOCK, PER2 and CRY1 expression in healthy volunteers, ICU and sepsis patients . In line with our results, the authors found disrupted circadian rhythms in sepsis patients. In addition, they showed that conditions in ICUs, such as permanently high light levels, do not disrupt circadian rhythms in sepsis patients, because non-sepsis ICU-patients expressed clock genes rhythmically. In comparison to these studies, we investigated a larger panel of clock genes and were able to show a downregulation of BMAL1 and PER2 as well. Additionally, we found that NR1D1, NR1D2, CRY1, CRY2, PER1, PER2 and DBP had altered rhythms in at least some patients with septic shock.
Secondary outcome parameters including SOFA, SAPS II and TISS-28, lactate, time of mechanical ventilation, ICU and hospital length of stay and mortality did not show a linear correlation with rhythmicity scores. The core body temperature measurements coincident with clock gene measurements were highly variable with strong trends and therefore, could not be associated with clock gene expression amplitudes. As already known, body temperature varies rhythmically with its peak in the evening and trough in the morning hours . However, the core body temperature troughed at different times during the day in different patients, which is in agreement with our finding of disrupted circadian rhythmicity. Generally, due to the small number of patients, associations of rhythmicity scores with outcome parameters might have gone undetected. Larger studies should examine whether disrupted rhythmicity is associated with mortality.
As another clinical secondary outcome parameter, we investigated the correlation between circadian rhythmicity and the expression of mHLA-DR on monocytes, which is one of the most studied markers of immunosuppression. A decreased mHLA-DR expression is associated with higher mortality in critically ill as well as sepsis patients . Due to the robust intrinsic circadian rhythm of monocytes , we hypothesized that in patients with septic shock the grade of immunosuppression as measured by mHLA-DR expression is associated with a loss of circadian rhythm. In our study population, 11 out of 14 patients had levels of HLA-DR of 15.000 molecules per monocyte or less, which is associated with immunosuppression and immunopraralysis . However, HLA-DR expression on monocytes did not show an association or significant correlation with the rhythmicity score. Nearly all patients showed immunosuppression and no rhythmicity in the expression of some clock genes. This could be attributed to the small study population not capturing the multitude of factors influencing clock gene expression.
It is recognized that melatonin helps restoring and maintaining circadian homeostasis . Additionally, melatonin was reported to have immunomodulatory and antioxidative effects . Lorente et al. found that melatonin levels in non-surviving sepsis patients were higher than in survivors as high melatonin levels correlate with worse clinical conditions of the patients . The increased melatonin levels are compatible with the reported disruption of circadian rhythmicity in critically ill patients . Melatonin is used as a supporting drug in septic infants and could be a promising therapeutic option in sepsis patients to promote stable circadian rhythms, improve immunity and outcomes . However, further research is needed to analyze the impact of the circadian rhythm on sepsis mortality and its translational potential to find promising treatment options for improving sepsis care . Understanding circadian rhythmicity in sepsis and the implications from and on the immune response are fundamental to develop new diagnostic and therapeutic tools and to individualize sepsis treatment.
This study has several limitations. First, only septic shock patients were included in our study. Thus, no conclusion can be drawn as to whether the disruption of the circadian rhythm favors sepsis or whether sepsis is the cause of the arrhythmia. Second, the inability of the analysis to classify clock genes as rhythmic or arrhythmic was due to a limited number of samples in the time series, i.e. we measured only 7 time points for each subject. When the rhythms are partially disturbed as is the case here, more data points are needed to detect arrhythmicity than to show rhythmicity. Third, the differences in mean expression levels of clock genes can both cause the changes in rhythmicity observed in septic shock patients and affect our statistical ability to quantify rhythms. Finally, samples from septic shock patients and BOTI patients were measured using slightly different Nanostring™ technologies, which – although unlikely - may result in differences in quantified expression levels.