Abstract
The circadian clock plays a critical role in regulating the immune system. Our previous publication revealed that a mutation in the circadian gene period1b (per1b) in zebrafish significantly decreased proinflammatory gene expression, particularly under constant darkness (DD) conditions; however, the underlying mechanisms remain unclear. In this study, using per1b-null mutant zebrafish and a larval tail fin injury model, we observed that the loss of per1b resulted in the downregulation expression of proinflammatory cytokines, such as IL-6 and TNF-α, at protein level.Furthermore, the loss of per1b downregulated ERK phosphorylation and inhibited p65 phosphorylation, leading to reduced NF-κB activation, which could downregulate the expression of proinflammatory cytokines, such as IL-6 and TNF-α, in zebrafish.These results provided insight into the communication between the circadian clock and immune functions.
Key words: per1b; cytokines; ERK; NF-κB; zebrafish
1. Introduction
The circadian clock is an evolutionarily conserved mechanism [1, 2] that organisms use to coordinate their internal systems to adapt to surrounding environments [3, 4].Most physiological processes, such as the sleep cycle and hormone production,exhibited oscillations with a period length of approximately 24 h [5]. A group of interlocked genes, which are controlled by auto-regulatory oscillations through a transcriptional feedback system, entrain a stable circadian clock [6, 7]. Period (per) 46 genes, including per1, per2 and per3, are regarded as core clock genes in mammals and zebrafish [8, 9].
Previous studies have demonstrated that the circadian clock played regulatory roles in many physiological activities, such as metabolism [10], reproduction [11],regeneration [12], and immunity [13]. In addition, published studies have indicated that various components, activities, processes and functions of the immune system exhibited robust circadian changes [9]. The available evidence demonstrated that the sensitivity and vulnerability of mice to acute inflammatory insult by pathogens displayed clear rhythmicity, peaking at night [14-17]. Many immune components in mice and rats, including macrophages, dendritic cells, natural killer (NK) cells, and even spleens and lymph nodes, exhibited robust clock genes and cytokine oscillations [18-21]. The serum lysozyme activity, peroxidase activity and total serum globulin level in Oreochromis mossambicus, all exhibited daily oscillations [22]. The innate immune defenses in shelter medicine Nile tilapia (Oreochromis niloticus) showed circadian rhythmicity and differential temporal sensitivity to bacterial endotoxin [23]. In zebrafish, studied indicated that the innate immune cell responses was light-regulated [24-26]. Intriguingly, proinflammatory cytokines, such as TNF-α BB94 and IL-6, were significantly upregulated in macrophages and fibroblasts of double Cry 1−/−; Cry2−/−mice [19, 27], suggesting a regulatory role for the circadian clock in the inflammatory response.
Although zebrafish, a diurnal species, have increasingly been used in circadian studies over a period of several decades [28], studies on the circadian immune functions in zebrafish are rare. Our previous study demonstrated that a mutation in the clock gene per1b downregulated the circadian expression of proinflammatory cytokines in zebrafish, particularly under constant darkness (DD) conditions [29]. In this study, we found downregulation of the NF-κB signalling pathway and decreased ERK phosphorylation in per1b mutant zebrafish. Furthermore, the blocking of ERK activity with the inhibitor PD0325901 suppressed proinflammatory cytokine production and NF-κB activation, and these effects were partly rescued by an ERK agonist. Together, our study findings demonstrated the mechanism underlying the regulation of proinflammatory cytokines by a per1b mutant in zebrafish.
2. Materials and methods
2.1 Zebrafish maintenance
Wild-type (AB) and per1b-/- zebrafish embryos were harvested from naturally matched at 28.5°C. Embryos were maintained in 14/10 light/dark (LD) conditions at 28.5°C in E3 embryo medium for 5 days and then tested at ZT13 (ZT0=light on) conditions. Larval manipulations were performed after anaesthesia with tricaine methane-sulfonate (MS-222, Sigma, USA) Fluimucil Antibiotic IT solution, and all efforts were made to minimize suffering. All zebrafish manipulations were conducted in strict accordance with the guidelines and regulations set by the University of Science and Technology of China (USTC) Animal Resources Center and the University Animal Care and Use Committee. The protocol was approved by the Committee on the Ethics of Animal Experiments of the USTC (Permit Number: USTCACUC1103013).
2.2 Pharmacological treatment
According to previous studies [30], the effect of ERK activity on cytokine secretion was evaluated. The 5-dpf larvae were pre-treated (1 h before injury) and treated (after injury) with PD0325901 (20 μM) (Sigma, PZ0162, USA) to inhibit ERK activation according to previous study [30]. The 5-dpf fish were pre-treated (1 h before injury) and treated (after injury) with EGF (1 μg/mL) (Sigma, E9644, USA) to upregulate ERK activity. Then, the treated samples were collected for ELISA and western-blot examination.
2.3 Analysis of zebrafish cytokine levels by ELISA
Tail fins were transected at the end of the spinal cord to induce acute inflammation.For TNF-α, IL-1β, IL-6 and IL-8 detection, 50 embryos were collected from the cloacal orifice to the injury end and lysed 3 h after injury, and the lysed embryos were then subjected to ELISA kits (Boster, China) assay according to a previous study [25].The experimental procedures were performed according to the manufacturer’s instructions (Boster, China): primary antibody (sourced from mouse) incubation for 1.5 h, secondary antibody incubation for 1 h, solution development for 0.5 h, addition of stopping solution and detection at 460 nm with a microplate reader.
2.4 Separation of the nucleus and cytoplasm
To evaluate the p65 content in the cell nucleus, we conducted an experiment in which the nucleus and cytoplasm were separated as previously described [31, 32] with a minor modification using commercial kit (Beyotime, China). Larvae were washed with PBS, homogenized on ice for 15 min with 400 μl of solution A (200 μl of 10 mM KCl, 2 ml of 10 mM HEPES (pH 7.9), 20 μl of 0.1 mM EDTA, 1.2 ml of 1.5 mM MgCl2, 200 μl of 1 mM DTT, 100 μl pf 0.5 mM PMSF, and 16.28 ml of ddH2O),vortexed for 10 s and then centrifuged for 1 min at 12000 g. The supernatant was then transferred into a new tube as the cytoplasmic fraction, and the precipitate was resuspended with 30 μl of solution B (5 ml of 25% glycerinum, 4 ml of 20 mM HEPES (pH 7.9), 4 ml of 400 mM NaCl, 200 μl of 1 mM EDTA, 200 μl of 1 mM DTT, 200 μl of 1 mM PMSF, and 6.4 ml of ddH2O) on ice for 20 min. The resuspended precipitate was centrifuged at 12000 g for 10 min, and the supernatant was collected as the nuclear fraction. The collected protein was used for Western blot analysis.
2.5 Western blotting
The cytoplasmic and nuclear proteins obtained as described above were used for the detection of NF-κB and IκB expression. The proteins were incubated overnight at 4°C with NF-κB (ANASPEC, 55482S, USA) and IκB (ANASPEC, 55481S, USA) primary antibodies sourced from rabbits at 1:1000 dilution. One hour after injury,larvae were homogenized and centrifuged, and proteins were collected for the detection of p-ERK (CST, 4379S, USA) and ERK (CST, 4695S, USA) expression after treatment with PD0325901 (20 μM) and EGF (1 μg/ml). The proteins were incubated overnight at 4°C with p-ERK and ERK primary antibodies sourced from rabbits at 1:1000 dilution. β-actin (CST, 4967S, USA) and TNF-α monoclonal antibodies sourced from rabbits were also used at 1:1000 dilution. The HRP-conjugated goat anti-rabbit secondary antibody (Sangon, D111042-0100, China) was used at 1:10000 dilution, and the incubation was performed for 2 h at room temperature.
2.6 Statistical analysis
Three samples were used for the PCR, ELISA and Western blot experiments. All experiments were independently repeated three times. The PCR and Western blot experimental data were standardized against β-actin. The integrated density values of the Western blot data were analysed using ImageJ software (NIH, USA). The results were analysed through unpaired t-tests or one-way ANOVA using GraphPad Prism version 5.0 (Prism, USA) and are shown as the means ± SEM. The level of significance was set to P<0.05. , , and indicate P<0.05, P<0.01, and P<0.001,respectively.zebrafish tail fin tissue.
3. Results
3.1 Expression of TNF-α, IL-1β, IL-6 and IL-8 is upregulated in wounded
The zebrafish larval tail fin injury model was used to induce an acute inflammatory response according to previous studies [33, 34]. It has been shown that Cxcl8 (IL-8) was upregulated under acute conditions in carp and zebrafish [33, 35]. In this study,we analysed the expression of four typical proinflammatory cytokines 3 h after injury using zebrafish at 5 days post-fertilization (dpf). The results indicated that the TNF-α and IL-1β levels were significantly upregulated in wounded tail fin tissue (Fig. 1A,1B), and the expression of IL-6 and IL-8 was also i (Fig. 1C, 1D).
3.2 Mutatnt per1b inhibits TNF-α, IL-1β and IL-8 expression in acute inflammation.
Through a Q-PCR analysis, our previous study indicated that a per1b gene mutation significantly decreased cytokine expression at ZT13 (ZT0 corresponding to lights on) under LD conditions. Here, we assessed whether the per1b mutant gene also affected cytokine expression at the protein level at ZT13. The cytokine expression was assayed using ELISA and Western blot methods. The results indicated that the per1b gene mutation significantly decreased the TNF-α protein level (Fig. 2A, 2B). The ELISA results also showed decreased IL-1β and IL-8 production in the per1b mutant fish (Fig.2C, 2D).
3.3 NF–κB signalling is downregulated in per1b gene mutant zebrafish.
We further attempted to study the upstream signalling involved in this process. Many studies have demonstrated that the NF–κB pathway regulated cytokine production [27,36]. The per1b mutation resulted in an apparent deficiency of IκB degradation (Fig.3A, 3B), which is necessary for NF–κB activation. Hence, we separated the nuclear and cytoplasmic proteins and evaluated the expression of p65, a subunit of NF–κB.We found that the per1b mutant zebrafish showed decreased p65 expression in the nucleus but not in the cytoplasm (Fig. 3C, 3D, 3E). Together, these results demonstrated that NF–κB signalling was suppressed in per1b mutant zebrafish.
3.4 Decreased ERK activity in per1b-/- zebrafish contributes to aberrant IκB degradation.
Given the role of ERK protein in NF–κB activation and cytokine production [37-40],we hypothesized that the per1b mutation may influence ERK activity in this model.Our experiment indicated that the absence of per1b resulted in a significant decrease in ERK phosphorylation post-injury (Fig. 4A, 4B), and we thus wondered whether ERK activity affects IκB degradation. Here, the PD0325901 inhibitor was used to suppress ERK phosphorylation according to previous studies in mice and zebrafish [30, 34]. A Western blot analysis showed that ERK activity was almost completely inhibited at the protein level after PD0325901 treatment (Fig. 4C, 4D). Moreover, we found that the inhibition of ERK activation could suppress IκB degradation during acute inflammation (Fig. 4E, 4F). These data collectively demonstrated that the decreased ERK activity in per1b mutant zebrafish modulated aberrant IκB degradation.
3.5 ERK regulates TNF-α and IL-1β production during acute inflammation.
Finally, we aimed to investigate whether ERK activity affects the injury-induced production of cytokines. ELISA results demonstrated that the TNF-α and IL-1β levels were significantly decreased following treatment with the ERK inhibitor during acute inflammation (Fig. 5A, 5B). EGF was then used to rescue the p-ERK deficiency in per1b-/- zebrafish (Fig. 5C, 5D) [41]. To some extent, EGF was able to increase the production of TNF-α in per1b mutant zebrafish (Fig. 5E).
Discussion
Numerous studies have suggested molecular links between circadian rhythms and the immune system of rodents [27, 42-45]; however, few studies have examined this process in diurnal animals. Like mammals, adult zebrafish owns an immune system including almost all lymphoid organs and immune cell types [46, 47]. Only innate immunity, mainly including macrophages and neutrophils, is relied upon at the early developmental stages of embryos, [46]. Our previous study showed that the per1b gene regulated the circadian expression of cytokines in larval zebrafish. Here, we continued our study to evaluate how the circadian gene per1b regulates the larval cytokine expression.
Our previous work examined circadian cytokine changes at the mRNA level in per1b mutant fish. In this study, we found that the per1b gene mutation also decreased the protein expression of TNF-α, IL-1β, and IL-8 (Fig. 2) in the inflammatory model.Hence, we could summary that circadian gene per1b suppressed proinflammatory cytokines expression at both mRNA and protein level. Because NF–κB signalling was vital for cytokine expression [27], we attempted to investigate whether the per1b mutation could regulate NF–κB activity in zebrafish. The results showed that the per1b mutation suppressed the activation of p65 and IκB (Fig. 3). Previous studies have demonstrated that ERK participated in many inflammatory processes [34, 48,49], and accumulating evidence indicated that ERK phosphorylation exhibited rhythmic variation and that circadian genes could regulate ERK activity [50-52]. Our results showed that the mutant per1b gene significantly suppressed ERK activation,even though a direct interaction between them was not demonstrated in our experiment (Fig. 4A). A previous study verified that the per1b mutation notably affected the expression of other clock genes [53]; therefore, we hypothesized that decreased ERK activation may be both a direct regulatory effect of per1b and a result of changes in other clock genes induced by the per1b mutation. Finally, we demonstrated that ERK could regulate cytokine expression (Fig. 5) and IκB activity (Fig. 4) during acute inflammation. In summary, the results showed that ERK-dependent NF–κB activation contributed to the decreased secretion of proinflammatory cytokines, such as TNF-α, IL-1β and IL-8, in per1b mutant zebrafish.Here, we did not determine the circadian-immune relations in adult fish. We supposed the results are likely to be different in adult fish, as the immune system of adult zebrafish is more complex than larval fish by developing the adaptive immunity [47].In other fish species such as Oreochromis mossambicus and Oreochromis niloticus,the innate immune activities also showed circadian rhythmicity [23, 24]. In rodents,circadian gene mutations also resulted in increased cytokine expression [27, 54].Combined with our study, this finding suggests that the phenomenon and mechanism diurnal and nocturnal animals exhibit complexity. In conclusion, deletion of a key circadian gene causes immune dysfunction, which may provide clues to the mechanisms of circadian-related diseases.