Diel Variation in CC Chemokine Gene Expression in the Japanese Pufferfish Takifugu rubripes.

CC chemokines are key molecules in the regulation of leukocyte trafficking to the site of injury, infection, or inflammation. In recent years, some mammalian chemokines have been shown to exhibit rhythmic expression, regulated by clock genes. However, the rhythmic expression of chemokines in teleost fish remains unknown. In the present study, the diel variation of teleost CC chemokine genes was investigated using the model fish, Fugu (Takifugu rubripes). Diel variation analysis revealed that clock (bmal1, clock1, per2, rorα, and rev-erbß) and CC chemokine (ccl18l, ccl19, and ccl25l) genes show diel expression under 12:12 light-dark cycle (LD12:12) conditions. CC chemokine genes, which exhibit diel expression, contain RORE (ccl18l, ccl19, ccl25l) and/or E-box (ccl25l) motifs in their transcription regulatory region. Moreover, in vitro head kidney stimulation with lipopolysaccharide (LPS) at different zeitgeber times (ZT) under LD12:12 conditions affected the degree of ccl18l, ccl19, and ccl25l expression; high and low responsiveness to LPS stimulation at ZT12 and ZT0 (ccl25l), and ZT16 and ZT4 (ccl18l and ccl19), respectively, were observed. These results suggest that the expression of some fish CC chemokines is affected by the diel variation regulated by clock proteins, and that responsiveness against bacterial infection depends on the time zone.

Circadian clock components Bmal1 and Clock1 regulate tlr9 gene expression in the Japanese medaka (Oryzias latipes).

Currently, circadian regulation of immune molecules in lower vertebrates, particularly, diurnal oscillation in the immune status of a fish, is not well understood. In this study, the diurnal oscillation of toll-like receptor (Tlr) 9, which plays a role in pathogen recognition, was investigated in the Japanese medaka fish (Oryzias latipes). We confirmed the expression of tlr9 and clock genes (bmal1 and clock1) in the central and peripheral tissues of medaka. These genes were expressed in a diurnal manner in medaka acclimated to a 12-h:12-h light-dark (12:12 LD) cycle. In addition, increased tlr9 expression was detected in medaka embryo cells (OLHdrR-e3) overexpressing both bmal1 and clock1 genes; however, this result was not obtained when only one or neither of the genes was overexpressed. This suggests that the increase in expression was mediated by the Bmal1 and Clock1 proteins together. In vitro stimulation of the head kidney with CpG-oligodeoxynucleotides (CpG-ODNs) at different zeitgeber times (ZTs; ZT0 = light on, ZT12 = light off) affected the degree of tlr9 gene expression, showing high and low responsiveness to CpG-ODN stimulation at ZT6/10 and ZT18/22, respectively. Similarly, bacterial infection at different ZT points induced a difference in the expression of Tlr9 signaling pathway-related genes (tlr9 and myd88). These results suggested that fish tlr9 exhibits diurnal oscillation, which is regulated by clock proteins, and its responsiveness to immune-stimulation/pathogen infection depends on the time of the day.

Circadian oscillation of TNF-α gene expression regulated by clock gene, BMAL1 and CLOCK1, in the Japanese medaka (Oryzias latipes).

To date, little information is available on the effects of circadian oscillation on immune regulation in lower vertebrates, such as teleost fish. In the present study, regulation of circadian oscillation of inflammatory cytokine TNF-α gene expression by clock gene was investigated using model fish medaka (Oryzias latipes). Firstly, structural analysis of clock genes was performed, which revealed that medaka BMAL1 and CLOCK1 conserve functionally important domains, such as basic helix-loop-helix (bHLH) and period-aryl hydrocarbon receptor nuclear translocator-single-minded (PAS), seen in their counterparts in other vertebrates. Expression of medaka Bmal1, Clock1, and Per1 genes was confirmed in central and peripheral tissues. Moreover, the expression of these clock genes and TNF-α genes in medaka acclimated to a 12:12 light (L) - dark (D) cycle showed circadian oscillation. In addition, higher expression of TNF-α gene was detected in medaka embryo cells (OLHdrR-e3) overexpressing Bmal1 and Clock1 genes. It was suggested that this increase was mediated by transcriptional regulation by clock proteins, which target E-box sequence in the cis-element of TNF-α gene as was detected by luciferase reporter gene assay. Moreover, in vitro head kidney stimulation with LPS at different zeitgeber time (ZT) under LD12:12 condition affected the degree of TNF-α gene expression, which shows high and low responsiveness to LPS stimulation at ZT18 and ZT10, respectively. These results suggested that fish TNF-α exhibited circadian oscillation regulated by clock proteins and its responsiveness against immune-stimulation depends on time zone.