Molecular clocks play an important role in coordinating circadian rhythms in the body and intracellular circadian networks in peripheral cells. It is thought that the coordinated network establishes coupling and uncoupling of pathways to support cellular function in parallel and anti-parallel fashion. To our knowledge, molecular clock proteins have not been investigated in primary synovial material of patients with OA and RA but similarly not in other rheumatic diseases.
There are three reports that linked molecular clocks to rheumatology. In mouse chondrocytes, CLOCK, Period 1 and Period 2 were found to exhibit biological rhythms after serum shock [29
]. The authors found CLOCK upregulated after mechanical stress which identifies CLOCK as a possible mechanosensitive gene [29
]. Another report demonstrated that melatonin inhibited Cryptochrome 1 in a mouse model of experimental arthritis [30
]. However, no functional tests were performed to understand the link between melatonin, cryptochrome 1 and proinflammatory mechanisms [30
In a third study, the authors investigated mice with collagen type II-induced arthritis using synovial and spleen cells [23
]. They found marked upregulation of Period 2 independent of the light dark cycle in arthritic animals. This is somewhat similar to the upregulation observed in RA patients compared to OA (Figure ). In addition, they found that daily rhythm of Period 1/2 mRNA shifted six hours back and that of Bmal-1 mRNA remained constantly low indicating dampened rhythm [23
]. They further demonstrated that mice deficient in Cryptochrome 1 and Cryptochrome 2 demonstrated arthritis aggravation, which might be due to upregulation of TNF [23
]. They concluded that the lack of Cryptochrome gene function abrogates normal biological clock function and aggravates pathological changes in arthritis.
Our report adds more information on human subjects with OA and RA. All clock proteins are ubiquitously present in synovial cells of these patients and in trauma controls (all cells are positive). It seems that density of molecular clock-positive cells was higher in RA compared to OA, which was significant for BMAL-1. Since we have not corrected for overall cellular density, this difference might depend on increased cellular infiltration in RA compared to OA though density is relatively similar in the chronic phase of both diseases. In addition, a possible undulation of density of molecular clock-positive cells did not exist in synovial tissue. This might depend on several mechanisms, such as heterogeneous chronotypes of investigated patients (two chronotypes are known: lark and owl), different medication in the various patients that might influence molecular clock levels, short observation period from 8:00 am to 4:00 pm, and low numbers of investigated patients (type II error). Since several studies demonstrated that inflammation disturbs molecular clock oscillation in different cell types in vivo
and in vitro
], it might well be that typical oscillations are abrogated in inflamed tissue in OA and RA.
A closer investigation of molecular clock mRNA demonstrated that synovial fibroblasts of OA and RA patients are capable of starting the typical oscillation. However, a long-standing rhythm was not established because the signal rapidly died down. Time series analysis did not demonstrate regularity in OA and RA but in healthy fibroblasts. This can depend on the contact to the proinflammatory environment to which these cells have been adapted prior to serum shock synchronization in OA and RA [31
When comparing the hardly visible oscillation in these synovial fibroblasts of OA and RA patients, fibroblasts of normal rats demonstrate excellent rhythmic oscillation [15
]. From this first study in rat fibroblasts, this cell type was one of the typical peripheral cell types to study daily rhythms of the intracellular molecular clock network [37
]. This is also true for normal human fibroblasts of the skin [40
], and we also found a rhythm in fibroblasts of healthy individuals. Thus, one would have expected similar rhythms in OA and RA fibroblasts. The question appears whether or not proinflammatory factors can influence molecular clock mRNA.
Our study demonstrated that proinflammatory cytokines decreased or tended to decrease mRNA of molecular clocks in OA synovial fibroblasts. This was opposite in RA synovial fibroblast which might also explain the higher density of molecular clock - positive cells in RA synovial tissue. In these experiments, we used a dose of 1 ng/ml of IL-1β or TNF. Although this is a typical cytokine concentration to stimulate cells in vitro, it does not give us a dose-response effect and it is difficult to simply translate it to a situation in vivo. Nevertheless, these concentrations appear in the proximity of cytokine-producing cells. In order to study the subject more carefully, we applied different doses in the MH7A RA synovial fibroblast cell line. Here a clear dose-response was observable.
In addition, IL-1β and TNF both changed the time point of the first BMAL-1 mRNA peak by induction of a delay of approximately three to six hours. Similar shifts of molecular clock peaks have been demonstrated in a model of experimental arthritis [23
], which indicates that inflammation can directly interfere with rhythmicity of cells. This is supported by a TNF-induced inhibition of Period 1 to 3 in NIH 3T3 fibroblasts and in mice which depends on a direct influence of BMAL-1 and CLOCK binding to the E-box [36
]. In addition, expression of molecular clock genes was markedly inhibited in peripheral blood leukocytes in surgical ICU patients [41
In further time series analyses, according to the first peak of mRNA rhythm, four different groups of molecular clocks were identified. Period 1, Period 2, Period 3, cryptochrome 1 and cryptochrome 2 (blue in Additional files 4
, Figures S4 and S5) demonstrated the first peak immediately after the serum shock. BMAL-1 and CLOCK demonstrated an intermediate pattern with a peak at 8 to 17 hours (pink and red in Additional files 4
, Figures S4 and S5). Furthermore, the peak of Rev-Erbα mRNA appeared late between 18 and 19 hours (green in Additional files 4
, Figures S4 and S5). This sequential increase of mRNA peaks is indicative of a first successful attempt to start oscillation in these OA and RA synovial fibroblasts. However, this phenomenon is impermanent, leading to rapid destruction of the rhythm.