Avian biological clock - Immune system relationship.

Biological rhythms in birds are driven by the master clock, which includes the suprachiasmatic nucleus, the pineal gland and the retina. Light/dark cycles are the cues that synchronize the rhythmic changes in physiological processes, including immunity. This review summarizes our investigations on the bidirectional relationships between the chicken pineal gland and the immune system. We demonstrated that, in the chicken, the main pineal hormone, melatonin, regulates innate immunity, maintains the rhythmicity of immune reactions and is involved in the seasonal changes in immunity. Using thioglycollate-induced peritonitis as a model, we showed that the activated immune system regulates the pineal gland by inhibition of melatonin production at the level of the key enzyme in its biosynthetic pathway, arylalkylamine-N-acetyltransferase (AANAT). Interleukin 6 and interleukin 18 seem to be the immune mediators influencing the pineal gland, directly inhibiting Aanat gene transcription and modulating expression of the clock genes Bmal1 and Per3, which in turn regulate Aanat.

Daily profiles of melatonin synthesis-related indoles in the pineal glands of young chickens (Gallus gallus domesticus L.).

The aim of the present study was to characterize the daily profiles of melatonin synthesis-related indoles in the pineal glands of male Hy-Line chickens hatched in the winter and reared under controlled light (L:D 12:12) conditions. The pineal glands were isolated from 16-day-old birds immediately after decapitation every 2h over a 24-h period. The indole contents were measured using HPLC with fluorescence detection. According to the obtained data, in chicken pineal glands tryptophan occurred at the highest level among the investigated compounds, showing rhythmical, daily changes with decreased levels during the scotophase. Rhythmical fluctuations were also observed for all studied tryptophan derivatives, except serotonin, 5-hydroxyindole acetic acid and 5-hydroxytryptophol. The highest levels of 5-hydroxytryptophan, N-acetylserotonin and melatonin were observed during the night, whereas the highest levels of 5-methoxytryptophol and 5-methoxyindoleacetic acid were observed during the day. The most interesting results concerned serotonin and the associated oxidative deamination products. The serotonin content did not exceed 5% of all investigated indoles, suggesting that there is no reserve pool of this indoleamine in the chicken pineal gland and that the activity of aromatic L-amino acid decarboxylase might be an important factor of melatonin synthesis regulation. In contrast to serotonin, 5-hydroxyindole acetic acid occurred at high levels in the investigated pineal gland, suggesting intensive oxidative deamination and explaining the observed low content of this indoleamine.

Cell Wall Anchoring of the Campylobacter Antigens to Lactococcus lactis.

Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein - CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type C. jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analyzed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ Lactic Acid Bacteria (LAB) strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered as an alternative vector to deliver heterologous antigens to the bird immune system. Additionally, the analysis of the structure and immunogenicity of the generated rCjaAD hybrid protein showed that the CjaA antigen can be considered as a starting point to construct multiepitope anti-Campylobacter vaccines.

Seasonal postembryonic maturation of the diurnal rhythm of serotonin in the chicken pineal gland.

Previously, we have demonstrated the postembryonic development of chicken (Gallus gallus domesticus L.) pineal gland functions expressed as changes in melatonin (MEL) biosynthesis. Pineal concentrations of MEL and its precursor serotonin (5-HT) were shown to increase between the 2nd and 16th day of life. We also found that levels of the mRNAs encoding the enzymes participating in the final two steps of MEL biosynthesis from 5-HT: arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), as well as their enzymatic activities, were raised during postembryonic development. Moreover, the manner of these changes was season-of-hatch dependent, even in animals kept under constant laboratory conditions (L:D 12:12). The most pronounced changes were seen in the concentrations of 5-HT and MEL, as well as in Aanat mRNA level and its enzymatic activity. The high daily variability in 5-HT content suggested that season- and age-dependent changes in the activity of the chicken pineal gland might rely on the availability of 5-HT, i.e. it may be limited by changes in pineal tryptophan (TRP) and/or 5-hydroxytryptophan (5-HTP) levels as well as by the activity of tryptophan hydroxylase (TPH) and aromatic l-amino acid decarboxylase (AADC): two enzymes participating in the conversion of TRP to 5-HT. The present study was undertaken with the following objectives: (1) to examine whether the pineal concentration of the 5-HT precursors TRP and 5-HTP exhibit age- and season-related changes; (2) to look for season-related differences in the transcription of the Tph1 and Ddc genes encoding enzymes TPH and AADC; (3) to identify the step(s) in postembryonic development in which these season-related variations in pineal gland function are most pronounced. Male Hy-line chickens hatched in the summer or winter, from eggs laid by hens held in L:D 16:8 conditions were kept from the day of hatch in L:D 12:12 conditions. At the age of 2 or 9 days, animals were sacrificed every 2 or 4 h over a 24-h period and their pineal glands were isolated under dim red light and processed for the measurement of (i) the pineal content of TRP, 5-HTP and 5-HT, and (ii) the level of Tph1 and Ddc mRNAs. Circadian rhythmicity of all the measured parameters was evaluated by the cosinor method. The pineal levels of TRP and 5-HT as well as the Tph1 and Ddc transcripts changed during postembryonic development in a season-related way. Whereas, the 5-HTP concentration did not vary between animals from both age groups, regardless of the season. Circadian rhythmicity of all the measured parameters was dependent on both the age and the season of hatch, and was greatest in older animals in the summer. These findings indicated that the efficiency of season-related MEL biosynthesis, reported previously, is limited by 5-HT availability and this limitation depends on the transcription of both the Tph1 and Ddc genes. Moreover, Ddc mRNA level in 9-d-old birds changed rhythmically, even though this gene is generally considered to be arrhythmic.

Pineal oscillator functioning in the chicken--effect of photoperiod and melatonin.

The avian pineal gland, apart from the hypothalamic master clock (suprachiasmatic nuclei, SCN) and retina, functions as an independent circadian oscillator, receiving external photic cues that it translates into the rhythmical synthesis of melatonin, a biochemical signal of darkness. Functional similarity to the mammalian SCN makes the avian pineal gland a convenient model for studies on biological clock mechanisms in general. Pineal melatonin is produced not only in a light-dependent manner but also remains under the control of the endogenous oscillator, while the possible involvement of melatonin in maintaining cyclic expression of the avian clock genes remains to be elucidated. The aim of the present study was to characterize the diurnal profiles of main clock genes transcription in the pineal glands of chickens exposed to continuous light (LL) and supplemented with exogenous melatonin. We hypothesized that rearing chickens from the day of hatch under LL conditions would evoke a functional pinealectomy, influencing, in turn, pineal clock function. To verify this hypothesis, we examined the diurnal transcriptional profiles of selected clock genes as well as the essential parameters of pineal gland function: transcription of the genes encoding arylalkylamine N-acetyltransferase (Aanat), a key enzyme in melatonin biosynthesis, and the melatonin receptor (Mel1c), along with the blood melatonin level. Chickens hatched in summer or winter were maintained under LD 16:8 and 8:16, corresponding to the respective photoperiods, as the seasonal control groups. Another set of chickens was kept in parallel under LL conditions and some were supplemented with melatonin to check the ability of exogenous hormone to antagonize the effects evoked by continuous light. Twelve-day-old chickens were sacrificed every 3 h over a 24-h period and the mRNAs of selected clock genes, Bmal1, Cry1, Per3, E4bp4, together with those of Aanat and Mel1c, were quantified in the isolated pineal glands. Our results indicate that the profiles of clock gene transcription are not dependent on the duration of the light phase, while LL conditions decrease the amplitude of diurnal changes, but do not abolish them entirely. Melatonin supplied in drinking water to the birds kept in LL seems to desynchronize transcription of the majority of clock genes in the summer, while in the winter, it restores the pattern, but not the diurnal rhythmicity. Rhythmic expression of Bmal1 appears to provide a direct link between the circadian clock and the melatonin output pathway, while the availability of cyclic melatonin is clearly involved in the canonical transcription pattern of Per3 in the chicken pineal gland. Regardless of the experimental conditions, a negative correlation was identified between the transcription of genes involved in melatonin biosynthesis (Aanat) and melatonin signal perception (Mel1c receptor).

Cooperative role of NF-{kappa}B and poly(ADP-ribose) polymerase 1 (PARP-1) in the TNF-induced inhibition of PHEX expression in osteoblasts.

Reduced bone mass is a common complication in chronic inflammatory diseases, although the mechanisms are not completely understood. The PHEX gene encodes a zinc endopeptidase expressed in osteoblasts and contributes to bone mineralization. The aim of this study was to determine the molecular mechanism involved in TNF-mediated down-regulation of Phex gene transcription. We demonstrate down-regulation of the Phex gene in two models of colitis: naive T-cell transfer and in gnotobiotic IL-10(-/-) mice. In vitro, TNF decreased expression of Phex in UMR106 cells and did not require de novo synthesis of a transrepressor. Transfecting UMR-106 cells with a series of deletion constructs of the proximal Phex promoter identified a region located within -74 nucleotides containing NF-κB and AP-1 binding sites. After TNF treatment, the RelA/p50 NF-κB complex interacted with two cis-elements at positions -70/-66 and -29/-25 nucleotides in the proximal Phex promoter. Inhibition of NF-κB signaling increased the basal level of Phex transcription and abrogated the effects of TNF, whereas overexpression of RelA mimicked the effect of TNF. We identified poly(ADP-ribose) polymerase 1 (PARP-1) binding immediately upstream of the NF-κB sites and showed that TNF induced poly(ADP-ribosyl)ation of RelA when bound to the Phex promoter. TNF-mediated Phex down-regulation was completely abrogated in vitro by PARP-1 inhibitor and overexpression of poly(ADP-ribose) glucohydrolase (PARG) and in vivo in PARP-1(-/-) mice. Our results suggest that NF-κB signaling and PARP-1 enzymatic activity cooperatively contribute to the constitutive and inducible suppression of Phex. The described phenomenon likely contributes to the loss of bone mass density in chronic inflammatory diseases, such as inflammatory bowel disease.

Tumor necrosis factor and interferon-gamma down-regulate Klotho in mice with colitis.

BACKGROUND & AIMS: Klotho (KL) is an anti-inflammatory protein that protects the endothelium from nitric oxide (NO)-induced dysfunction, reduces the expression of endothelial adhesion molecules, and potentially regulates T-cell functions. KL deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis, which can lead to inflammatory bowel disease (IBD)-associated osteopenia/osteoporosis. We investigated the changes in renal expression of Kl as a consequence of colitis. METHODS: We studied 3 mouse models of IBD: colitis induced by trinitrobenzene sulfonic acid, colitis induced by microflora (in gnotobiotic interleukin-10(-/-)), and colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells. Effects of the tumor necrosis factor (TNF) and interferon (IFN)-gamma on Kl expression and the activity of its promoter were examined in renal epithelial cells (mpkDCT4 and mIMCD3). RESULTS: Renal expression of Kl messenger RNA (mRNA) and protein was reduced in all 3 models of IBD. Reduced level of KL correlated with the severity of colitis; the effect was reversed by neutralizing antibodies against TNF. In vitro, TNF inhibited Kl expression, an effect potentiated by IFN-gamma. The combination of TNF and IFN-gamma increased expression of inducible nitric oxide synthase (iNOS) and increased NO production. The effect of IFN-gamma was reproduced by exposure to an NO donor and reversed by the iNOS inhibitor. In cells incubated with TNF and/or IFN-gamma, Kl mRNA stability was unaffected, whereas Kl promoter activity was reduced, indicating that these cytokines regulate Kl at the transcriptional level. CONCLUSIONS: The down-regulation of KL that occurs during inflammation might account for the extraintestinal complications such as abnormalities in bone homeostasis that occur in patients with IBD.