Date Published: September 28, 2018
Publisher: Public Library of Science
Author(s): Simona Moravcová, Dominika Pačesová, Barbora Melkes, Hana Kyclerová, Veronika Spišská, Jiří Novotný, Zdenka Bendová, Henrik Oster.
The circadian clock in the suprachiasmatic nucleus (SCN) regulates daily rhythms in physiology and behaviour and is an important part of the mammalian homeostatic system. Previously, we have shown that systemic inflammatory stimulation with lipopolysaccharide (LPS) induced the daytime-dependent phosphorylation of STAT3 in the SCN. Here, we demonstrate the LPS-induced Stat3 mRNA expression in the SCN and show also the circadian rhythm in Stat3 expression in the SCN, with high levels during the day. Moreover, we examined the effects of LPS (1mg/kg), applied either during the day or the night, on the rhythm in locomotor activity of male Wistar rats. We observed that recovery of normal locomotor activity patterns took longer when the animals were injected during the night. The clock genes Per1, Per2 and Nr1d1, and phosphorylation of kinases ERK1/2 and GSK3β are sensitive to external cues and function as the molecular entry for external signals into the circadian clockwork. We also studied the immediate changes in these clock genes expressions and the phosphorylation of ERK1/2 and GSK3β in the suprachiasmatic nucleus in response to daytime or night-time inflammatory stimulation. We revealed mild and transient changes with respect to the controls. Our data stress the role of STAT3 in the circadian clock response to the LPS and provide further evidence of the interaction between the circadian clock and immune system.
The temporal organisation of behavioural and physiological functions in mammals mostly depends on the timing signals generated by the circadian clock in the suprachiasmatic nucleus (SCN). The SCN integrates external timing cues with an endogenous molecular clockwork and synchronises circadian oscillations in other brain parts and peripheral tissues . The morphology of the SCN varies across species, but its basic structure is shared by all mammals. It consists of two main parts: the shell or the dorsomedial part (dmSCN), which contains intrinsically rhythmic cells, and the core or the ventrolateral part (vlSCN), which receives most of the regulatory inputs from other parts of the brain . The molecular basis of a circadian clock involves several interlocking transcriptional loops of clock genes, such as Clock, Bmal1, Period 1 (Per1), Per2, Cryptochrome 1 (Cry1) and Cry2, Casein kinase 1 epsilon (CK1ε), RevErbα (Nr1d1) and Rorα. The stability of these loops is supported by posttranslational modifications of clock protein by several kinases, including glycogen synthase kinase-3beta (GSK3β) and p42/44 mitogen-activated protein kinase (ERK1/2) [3–7]. This kinase also plays a role in photic entrainment of the circadian clock in the SCN [8, 9]; thus, it is an integral part of the circadian clock in the SCN.
Several reports have focused on the effect of systemic inflammation on various aspects of the circadian physiology [12, 16, 19–22, 33–35]. Most of these studies, however, only used one daytime period for inflammatory stimulation, although it is well understood that the susceptibility of organisms to inflammatory stimuli is affected by the circadian clock. Previously, we have shown that LPS applied either during the day or at night affects the phosphorylation of STAT3 in the SCN differently . In this study, we followed the changes in the Stat3 mRNA and several selected markers that are known for their sensitivity to extra-clock events and may serve as probes to the main oscillatory loops and posttranslational events. The daytime point of infection coincides with high levels of STAT3 protein and of clock genes Per1, Per2 and Nr1d1 mRNAs [1, 24, 36, 37], and was chosen to deduce a possible LPS-induced decrease of their levels. The night point coincides with low levels of selected gene transcripts, which allows for measuring a possible induction in response to LPS. We used 1 mg/kg of LPS, which has been shown to suppress the expression of Per1 gene in the SCN the day following the injection at ZT1  and to induce the phosphorylation of STAT3 in the SCN astrocytes .
The present study shows that the time of LPS administration affects the recovery rate of locomotor activity rhythm and induces the transient changes in clock gene expression and the levels of pERK1/2 and pGSK3β in the rat SCN that may be a part of the steady-state function of the clock in mild pathological conditions. We also provide the first report on the circadian rhythmicity of Stat3 gene expression in the SCN, and we demonstrate that the LPS administration induces not only phosphorylation of STAT3 that has been shown previously, but also its transcription and regulate thus significantly Stat3 mRNA level in the SCN.