Avian biological clock – Immune system relationship

Accepted Manuscript Avian biological clock - immune system relationship Magdalena Markowska, Paweł M. Majewski, Krystyna Skwarło-Sońta PII:

S0145-305X(16)30169-0

DOI:

10.1016/j.dci.2016.05.017

Reference:

DCI 2643

To appear in:

Developmental and Comparative Immunology

Received Date: 4 December 2015 Revised Date:

23 May 2016

Accepted Date: 23 May 2016

Please cite this article as: Markowska, M., Majewski, P.M., Skwarło-Sońta, K., Avian biological clock - immune system relationship, Developmental and Comparative Immunology (2016), doi: 10.1016/ j.dci.2016.05.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Avian biological clock - immune system relationship Magdalena Markowska*, Paweł M. Majewski, Krystyna Skwarło-Sońta University of Warsaw, Faculty of Biology, Institute of Zoology, Department of Animal Physiology, Miecznikowa 1 str., 02-096, Warsaw, Poland

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*Corresponding author: Magdalena Markowska [email protected] Abstract

Biological rhythms in birds are driven by the master clock, which includes the

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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

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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

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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

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regulate Aanat.

Key words: pineal gland, chicken, immunity, peritonitis, seasonality, photoperiod

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1. Introduction

The pineal gland, a neuroendocrine organ existing solely in vertebrate species, is involved in the control and synchronization of several behaviors and physiological processes through the circadian synthesis of its main hormone, melatonin (Falcon, 2007). Melatonin synthesized during the night is immediately secreted into the cerebrospinal fluid and into the blood where it circulates and acts as a signal to the body that it is dark, regardless of the diurnal or nocturnal pattern of the animal’s locomotor activity (Challet, 2007). The role of the pineal gland in the regulation of functions varies according to species. In particular, there is a huge difference between mammals and birds in the importance of the pineal gland in 1

ACCEPTED MANUSCRIPT the control of reproduction, which is one of the best known seasonally variable physiological processes in the species living in the temperate zone (Ikegami and Yoshimura, 2012; Sharp, 2005). Apart from reproduction, several other functions exhibit well expressed seasonality, including metabolism and immunity. In avian species, migration and singing are also highly seasonal (Wang et al., 2014). All of these processes remain under the control of an

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endogenous biological clock, which is organized differently in mammals and birds (Bell-

Pedersen et al., 2005). The biological clock is involved not only in the temporal organization of an organism’s functions but also in their synchronization with the diurnal and seasonal

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changes in the external conditions, as perceived by a specialized receptor system. 2. Pineal gland as a component of the avian master clock

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In mammals the master clock is located within the hypothalamic suprachiasmatic nuclei (SCN), which controls downstream physiological processes via the autonomic nervous system and neurohormonal pathways. In contrast, the avian master circadian clock is much more complex. It is composed of the avian equivalent of the SCN, the pineal gland and the retina, and the impact of each of these components varies according to the bird species (Underwood et al., 2001). The circadian clock operating within the avian pineal gland

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belongs, at the molecular level, to a group of highly conserved mechanisms composed of a set of genes collectively called “clock genes”, containing E-box elements in the promoter region (Cassone, 2014). Rhythmic transcriptional/translational interplay of the “positive” and

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“negative” elements makes up a universal feedback loop leading to the diurnal (or circadian) variations in the clock function, as well as in the downstream regulation of the majority of

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rhythmic metabolic processes (Karaganis et al., 2009). “Positive element” proteins include BMAL1-2 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1) and CLOCK (circadian locomotor output cycles kaput) , while CRY1-2 (cryptochrom) and PER1-3 (period) are “negative”. To date, however, PER1 has not been demonstrated in birds. Clock genes regulate the expression of the so-called clock controlled genes (CCGs), which in turn adjust the metabolic functions of an organism to the particular phase of the diurnal cycle. CCGs differ according to the tissue and its metabolic function. In the avian pineal gland CCGs are represented by the genes encoding the enzymes of the melatonin biosynthesis pathway (Cassone, 2014; Piesiewicz et al., 2015).

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ACCEPTED MANUSCRIPT The main environmental cue synchronizing endogenous clock is light perceived by the retinal photoreceptor system. In this system, the melanopsin-containing intrinsically photoreceptive retinal ganglion cells are directly connected, via the retino-hypothalamic tract, to the SCN master clock (Berson et al., 2002). These melanopsin receptors are maximally sensitive to blue light at a wavelength of approximately 480 nm, which does not overlap with the vision-

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related sensitivity of the rod and cone photoreceptors (for more details see (Stevens and Zhu, 2015). This blue light corresponds to the color of the sky on a clear morning and is the best signal for an organism of the transition from day to night, allowing the melanopsincontaining photoreceptors to keep the organisms’ physiology in strict synchrony with the

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sun (Stevens and Zhu, 2015). Photic information from the SCN is sent to the pineal gland where it controls the magnitude and duration of nocturnal melatonin synthesis, which is

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strictly correlated with night length. For these reasons, melatonin is considered as “a clock and calendar”, translating the actual environmental lighting conditions into the chemical messages understood by the cells and organs within the body (Reiter, 1993). Differences between avian and mammalian pineal glands include the direct photosensitivity of the former, which has been lost through evolution in the latter. Therefore, environmental

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information about light reaches the avian pineal gland directly through the photosensitive pinealocytes of the gland, which are located between the brain hemispheres and the cerebellum and just beneath the largely translucent skull (Falcon, 2007). As in mammals, avian pineal gland also receive multi-synaptic sympathetic innervation, going throughout the

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superior cervical ganglia (Claustrat et al., 2005). The pathway for sympathetic transmission of light information is the same in mammals and birds. However, adrenergic regulation of

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pineal gland function differs between mammals and birds. Specifically, in mammals, noradrenaline released from the postsynaptic adrenergic terminals at the transition to dark stimulates β- and α1 adrenergic receptors present on the pinealocytes. This results in an increase in the intracellular cAMP level, eventually leading to elevated melatonin biosynthesis (see the discussion later). In contrast, avian pinealocytes are equipped with α-2 adrenergic receptors, which are stimulated by the postsynaptic noradrenaline released at the transition to light, evoking a sequence of reactions that leads to the inhibition of melatonin biosynthesis during the day (Zatz and Mullen, 1988). Therefore, the circadian rhythm of melatonin synthesis and release in birds and mammals is the same, although with

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ACCEPTED MANUSCRIPT quite different receptor-mediated mechanisms of adrenergic regulation (Skwarlo-Sonta et al., 2003). The biosynthesis of melatonin is a well-characterized sequence of enzymatic reactions. It starts with the hydroxylation of the essential amino acid tryptophan (TRP) to 5hydroxytryptophan (5-HTP), catalyzed by tryptophan hydroxylase (TPH; E.C. 1.14.16.4),

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which in chickens is encoded by the tryptophan hydroxylase 1 gene (Tph1). The next

enzyme, aromatic amino acid decarboxylase (AADC; E.C.4.1.1.28), encoded by the dopa decarboxylase gene (Ddc), converts 5-HTP to 5-hydroxytryptamine (serotonin, 5-HT).

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Serotonin is then transformed to N-acetylserotonin (NAS) by arylalkylamine-N-

acetyltransferase (AANAT; E.C. 2.3.1.87), encoded by the arylalkylamine-N-acetyltransferase gene (Aanat). Finally, the hydroxyindole-O-methyltransferase (HIOMT; E.C.2.1.1.4), encoded

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by the acetylserotonin O-methyltransferase gene (Asmt), converts NAS to melatonin (Klein et al., 2010). The pineal AANAT is the key enzyme controlling the rhythm of melatonin biosynthesis and its properties are extensively explored. In more recent times HIOMT appears to control the nocturnal amplitude of melatonin in mammals (Simonneaux and Ribelayga, 2003). Generally it has been little interest in the activities of the enzymes TPH and

(Huang et al., 2008).

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AADC, although the former has been proposed as a key enzyme in serotonin biosynthesis

The most frequently examined genes in the melatonin biosynthesis pathway are Aanat and

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Asmt, the transcription of which is regulated via numerous endogenous factors. Among them are the clock genes working through E-box and D-box promoter binding sites and the

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cAMP-responsive elements and inducible cAMP early repressor that interact with a binding protein induced by cAMP (CREB), mediating positive and negative transcriptional regulation, respectively (Shimizu and Fukada, 2007). 3. Peculiarities of the avian immune system Despite an evolutionary proximity between birds and mammals, occupation of similar habitats and biological niches, and challenges by a similar spectrum of pathogens, their immune systems differ. While avian immune system seem to be simpler, birds and mammals achieve the same overall responses, although sometimes via different ways. For example, avian B lymphocytes, responsible for the humoral immune response, develop not in the 4

ACCEPTED MANUSCRIPT bone marrow as in mammals but in the bursa of Fabricius (BF), a unique avian organ (Glick, 1994; Olah and Nagy, 2013). The repertoire of B lymphocyte receptors and antibodies is more limited than in mammals because the birds have only a single functional copy of the genes for the V and J segments for both the light and heavy chain, and these segments are produced by a mechanism called gene conversion rather than via gene rearrangement

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(Kaiser, 2012). The genome region encoding the major histocompatibility complex, highly polymorphic in mammals, is minimal in birds and contains only two class I and two class II genes. In addition to the BF, the avian immune system has several unique features: a) it lacks lymph nodes, and the antigens are presented at the site of infection, probably as the

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lymphoid aggregates, b) functional eosinophils are lacking and many components of the mammalian Th2 response are absent, c) only IgA, IgM and IgY immunoglobulins (the

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functional equivalent of mammalian IgG) are present, d) neutrophils are replaced by heterophils, and e) there are no residential leukocytes in the body cavities. After the chicken genome sequence became available (International Chicken Genome Sequencing, 2004), data about differences in the repertoire of synthesized chemokines, cytokines, and their receptors as well as the pattern recognition receptors started to be published. It seems to be

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a trend that immune messengers have fewer representatives in the chicken genome than in the same immune gene families in mammals, with some exceptions. Because of this observation, it was proposed that birds (chickens, specifically) have a “minimal essential cytokine, chemokine repertoire” and strengthens the proposed idea of the redundancy of

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mammalian cytokines (Kaiser, 2012). As found for many immune gene families, the toll-like receptor (TLR) and chemokine/chemokine receptor repertoires are different in chickens than

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in mammals. An interesting observation is that entire subfamilies of the tumor necrosis factor (TNF) superfamily are also absent from the chicken genome. In mammals, TNF-α is the main pro-inflammatory cytokine and its lack in chickens may suggest an impairment of the inflammatory response, but this is not known for certain. The features of the avian immune system described above are thoroughly reviewed in several papers (Kaiser, 2010, 2012; Kaufman, 2015; Wu and Kaiser, 2011). 4. Thioglycollate-induced peritonitis as a model of inflammatory reaction To investigate neuroendocrine control of immunity in vivo, it is essential to develop a good model of an active immune system, such as the inflammatory reaction. In mammals 5

ACCEPTED MANUSCRIPT lipopolysaccharide (LPS) is the agent commonly used to evoke inflammation. LPS is an endotoxin found in the outer membrane of gram-negative bacteria, and it can be injected intravenously (i.v.) or intraperitoneally (i.p.). The effects following LPS injections are well known and usually result in the synthesis of pro-inflammatory cytokines, including TNF-α, interleukin 1 β (IL 1 β) and IL 6 (Sweet and Hume, 1996). On the other hand, thioglycollate

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(TG), is an agent that recruits a large number of cells to the site of inflammation and is used to study phagocytic cell properties (Baron and Proctor, 1982; Leijh et al., 1984). In the murine model of TG induced peritonitis, neutrophil numbers starts to increase in the peritoneal cavity and reach a peak between 4 and 24 hours after treatment, while

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macrophage numbers start to increase at 24 hours and reach a peak level at 3 to 4 days. The influx of leukocytes is accompanied by an increase in several chemoattractants and

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inflammatory mediators in the peritoneal exudate and blood. However, increases in the concentrations of the majority of mediators are higher in exudates than in blood (Lam et al., 2013). Moreover, it has been suggested that TG-elicited peritonitis is driven by leukotrienes and complement component C5a (Segal et al., 2002).

Fifteen years ago, initiating comparative studies on neuroendocrine regulation of

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inflammatory reaction in vertebrates, we established a model of the inflammatory response in chickens based on TG-induced peritonitis (Majewski et al., 2005b; Majewski et al., 2002). In birds, the influx of heterophils is observed as early as 3-4 hours hour after an i.p. TG injection, and it ends 24 hours later, which is much earlier than observed in mammals

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(Majewski et al., 2002). This short development time allowed us to study the circadian control of the inflammation. Leukocyte influx into the peritoneum is accompanied by an

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increase in peritoneal vascular permeability (4 h after initiation of peritonitis) (Majewski et al., 2002). It has to be mentioned that the injection of saline into control animals also induced an increase in the number of peritoneal leukocytes (PTLs). However, this increase was smaller and shorter lasting than that observed after TG injection. The basal concentration of reactive oxygen species (ROS) and their induction by phorbol 12-myristate 13-acetate is higher in PTLs isolated from TG-injected animals than from the controls (Majewski et al., 2005a; Skwarlo-Sonta et al., 2002). The effects of TG injection are also observed at the periphery, as the number of white blood cells increases until 12 h after the initiation of peritonitis (Majewski et al., 2002) and both spontaneous and mitogen6

ACCEPTED MANUSCRIPT stimulated splenocyte proliferation is higher in chickens that have developed inflammation (Majewski et al., 2005a; Majewski et al., 2005b). Immune mediators of chicken TG-elicited peritonitis are poorly described. Recently, Turkowska and co-workers found that peritonitis increased serum lysozyme concentrations and mRNA expression of pro-inflammatory Il 6

2015). 5. Circadian control of the chicken immunity

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and Il 18 in splenocytes, in a season- and time-of-day dependent manner (Turkowska et al.,

Biological rhythms are an essential part of animal physiology. It is not surprising, therefore,

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that the rhythms are also observed within the immune system. They include cyclic, usually circadian, changes in the number of circulating immune cells, cytokines synthesis and the

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development of the immune responses. Immune rhythms are generated by clock genes expressed in immune cells and/or by the central biological clock. They are regulated by neurotransmitters, such as noradrenaline, and hormones, such as glucocorticoids, testosterone, adrenaline and melatonin. These phenomena are present in mammals and, while they are not the main subject of this paper, they are described in a number of excellent review articles (Arjona et al., 2012; Berger, 2008; Cermakian et al., 2013;

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Cermakian et al., 2014; Geiger et al., 2015). However, a few studies that revealed the existence of the circadian control of immunity in chickens have to be mentioned in the following.

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Experiments on surgical pinealectomy (Px) in chicken embryos showed that the structural and functional development of immune and neuroendocrine systems is associated with the

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proper development of the pineal gland during embryogenesis (Jankovic et al., 1994). In addition, in our early experiments, surgical Px of newly hatched chickens disturbed the diurnal rhythm of immune parameters (e.g., granulocyte number and serum lysozyme concentration in the blood) and these changes were reversed by treatment with exogenous melatonin (Rosolowska-Huszcz et al., 1991; Skwarlo-Sonta et al., 1991; Skwarlo-Sonta et al., 1992). Thereafter, we proposed that the mitogenic response to phytohemagglutinin of splenocytes isolated in the middle of the day was stronger than that observed in splenocytes isolated during the night (Majewski et al., 2005b). We also observed diurnal differences in mRNA expression and in melatonin receptor affinity in the splenocytes (Wronka et al., 2008). The development of peritonitis evoked by TG also depends on the time of the i.p. 7

ACCEPTED MANUSCRIPT irritant injection. In chickens injected at the beginning of the light phase, the inflammatory reaction developed faster than in those injected just before the beginning of the dark phase (Skwarlo-Sonta et al., 2007). We have also shown that in chickens treated with melatonin before TG injection, the development of peritonitis is delayed but PTL numbers increased in the following phase (Majewski et al., 2005b). The second effect appeared to be caused by

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the stimulatory activity of melatonin-induced endogenous opioids (Skwarlo-Sonta et al., 2002). The intensity of an oxygen burst measured by ROS production in PTLs varied during the day and night (Skwarlo-Sonta et al., 2007). Recently, Turkowska and coworkers observed diurnal changes in the levels of IL 6 and IL 18 mRNA in chicken peripheral blood leukocytes

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(PBLs). These levels were partially abolished by constant light conditions and were restored by melatonin supplementations in the drinking water (Turkowska et al., 2013). The

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connection between, photoperiod, melatonin and immunity was also observed in Japanese quail (Siopes and Underwood, 2008), and the tropical bird Perdicula asiatica (Kharwar and Haldar, 2011; Yadav and Haldar, 2014). Taken together, significant evidence lead to the conclusion that endogenous melatonin is among the factors regulating rhythms in avian (chicken) immunity.

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6. Chicken pineal – immune axis

Our long-term observation, not published until now, was that TG injection induces sleepiness in the chickens, suggesting that activation of the immune system is perceived by the central

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nervous system and structures of the master biological clock including the pineal gland and its hormone melatonin , a phenomenon well known in mammals (Coogan and Wyse, 2008;

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Leone et al., 2006; Paladino et al., 2014). Therefore, we postulated the existence of bidirectional communication between the pineal gland and the immune system in birds (Skwarlo-Sonta et al., 2003) and began to experimentally test this hypothesis. We found that immunization with sheep red blood cells modified the activity of AANAT in a season- and sex-specific manner (Markowska et al., 2000). Subsequently, we have shown that TGinduced peritonitis decreased pineal AANAT activity and lead to the disappearance of its nocturnal peak (Majewski et al., 2005a). Among the molecular factors modified in the pineal gland after the induction of inflammation, we also observed a decrease in Aanat mRNA, the AANAT protein, and melatonin content. On the other hand, the expression of both Tph1 and Asmt genes increased, as did the activity of HIOMT, an enzyme encoded by Asmt and 8

ACCEPTED MANUSCRIPT involved in the regulation of the nocturnal surge of melatonin. However, these later changes did not lead to an increase in the level of pineal melatonin, the direct product of HIOMT activity. This was most likely due to the simultaneous inhibition of NAS biosynthesis. Finally, we concluded that the molecular target of the yet unknown inflammatory mediators in the chicken pineal gland is the gene encoding AANAT (Piesiewicz et al., 2012). These

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observations, collectively called the pineal-immune axis, confirmed our initial hypothesis and agreed with similar observation made in mammals by Regina Markus’s research group (Markus et al., 2007).

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7. Mechanisms of the down-regulation of pineal melatonin synthesis during peritonitis The mechanism of the pineal gland’s response to inflammation in chickens, including the

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involvement of receptors and downstream mechanisms, are still unknown and remain under investigation by our team. These mechanisms are much better recognized and understood in mammals. Some published data suggest direct regulation via immune-derived mediators acting on pinealocytes, as well as on the astrocytes, microglial cells and lymphocytes present in the pineal gland (Carvalho-Sousa et al., 2011; da Silveira Cruz-Machado et al., 2010; Fernandes et al., 2006; Kaur et al., 1997; Olah and Glick, 1984; Uede et al., 1981). It has been

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shown that lymphocytes in the pineal gland are distributed among pineal-associated lymphoid tissue (PALT), and in chickens their number changes over a 24-hour period (Mosenson and McNulty, 2006). The pineal gland is one of the circumventricular organs

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where the brain-blood-barrier (BBB) is not strictly impermeable to cells and inflammatory mediators. It has been demonstrated in both in vitro and in vivo studies that LPS may

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penetrate the pineal gland and probably directly activates NF-kB (nuclear factor kappa-lightchain-enhancer of activated B cells) signaling in pinealocytes and microglia via TLR4 receptors. In microglia, LPS induces TNF production, which in turn may activate the expression of its own receptor (TNFR1) in pinealocytes (da Silveira Cruz-Machado et al., 2010).

To investigate the mechanisms of Aanat down regulation in the chicken pineal gland which we observed during inflammatory reactions, we developed our own in vitro model of pinealimmune communication. The avian pineal gland is a very convenient experimental model for examining the function of the pineal clock. When cultured in vitro, in constant darkness and without any sympathetic stimuli, the diurnal rhythm of melatonin synthesis and release is 9

ACCEPTED MANUSCRIPT still maintained. The duration of this rhythmic function varies among species and its disappearance is related to the internal desynchronization of cultured pinealocytes (Underwood et al., 2001). Nevertheless, it is the best available illustration of endogenous pineal clock stability and synchronization (Karaganis et al., 2008). Therefore, this in vitro model allows for the study of the direct effects of any mediators/factors on the endogenous

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melatonin rhythm.

There are at least two possible scenarios for modulation of pineal melatonin biosynthesis by peritonitis: 1) the inflammatory mediators produced during the developing peritonitis reach

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the pineal gland and act directly on pinealocytes or on the PALT, or 2) the release of

neurotransmitters from neurons innervating the pineal gland is altered by inflammatory cytokines. We started by trying to verify the first scenario in an organotypic pineal gland

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culture. In our model, pineal glands were isolated from 16-days-old chickens kept in a lightdark cycle (LD=12:12, light on (L) at ZT 0 (zeitgeber time 0), light off (D) at ZT 12). Glands were isolated at the end of the light phase (ZT 11) and at the beginning of the dark phase (ZT 12) the whole organ in vitro pineal culture was started. The glands were cultured for 15 – 24 hours in 24-well plates (n=3/well), on special inserts, in constant darkness (DD), at 41°C and

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with a 5% CO2 atmosphere (Fig. 1). As the DD condition were kept for all the culture duration the respective time point of samples collection are defined as CT (circadian time). Pineal glands were collected every 3 hours from the start of the culture and analyzed for mRNA

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expression of the Aanat gene, AANAT enzymatic activity, and melatonin content in the medium. Under these culture conditions, Aanat gene expression was elevated in the middle of the subjective night. This was followed by an increase in AANAT activity and a release of

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melatonin into the medium (Fig. 1). Subsequently, we investigated whether the endogenous rhythm of melatonin synthesis observed in vitro is altered by peritonitis evoked in vivo. Pineal glands were isolated at the end of the light phase from both the normal chickens and birds with peritonitis induced by TG injection 6 h earlier (middle of the day). These glands were cultured in vitro for 18 hours. Aanat expression, AANAT activity and melatonin release were measured every 3 h. As shown in Fig. 2, expression of the Aanat gene was higher in pineal glands isolated from TG chickens compared to expression in normal chickens. However, the enzymatic activity of AANAT was decreased. Moreover, the melatonin content in the medium did not differ between the two groups. The results obtained from the in vitro 10

ACCEPTED MANUSCRIPT experiments differed from those observed in vivo (Piesiewicz et al., 2012). We expected a decrease of all measured parameters in the pineal glands from chickens with peritonitis when compared to the controls. Taking into the consideration the regulation of AANAT synthesis at the transcriptional level, and this enzyme’s activity at the posttranslational step (Schomerus and Korf, 2005; Zatz et al., 2000) we concluded that in the pineal glands of

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chickens from the TG group: 1) the increase in the Aanat expression was due a recovery effect from the influence of inhibitory inflammatory mediators circulating in vivo, 2) the decrease of AANAT activity was due to reductions in the level of its phosphorylated form as a result of the actions of inhibitory mediators in vivo, and 3) melatonin synthesis is

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compensated for by increased HIOMT activity crucially involved in the generation of the nocturnal amplitude of the hormone (Simonneaux and Ribelayga, 2003). The last two

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assumptions are consistent with the results obtained in the in vivo experiments. The level of AANAT protein was lower in chicken with peritonitis when detected using an antibody against its phosphorylated form, and Asmt expression and HIOMT activity were slightly increased (Piesiewicz et al., 2012).

To explain our first suggested conclusion, we formulated the following hypothesis:

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inflammatory mediators inhibit Aanat gene expression and/or the expression of clock gene(s) regulating the Aanat gene thereby lowering the synthesis of melatonin. When we started our in vitro experiments, avian cytokines were not commercially available and use of

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mammalian cytokines was useless as the gene sequence homology is only between 20 and 50 percent. Therefore, we decided to use chicken leukocytes as the source of endogenous cytokines. Our first choice was peritoneal leukocytes entering the peritoneal cavity after TG

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injection. We co-cultured pineal glands with different number of PTLs in the culture medium and, after 24 hours, measured pineal AANAT activity. As shown in Fig. 3, in the presence of PTLs, the activity of AANAT increased proportionally to the number of PTLs in the culture. In subsequent experiments, we also observed an increase in Aanat gene expression in the presence of PTLs (Markowska M et al., 2015) (manuscript in preparation). However, if we cocultured pineal glands with PBLs isolated from animals with peritonitis we observed a decrease in Aanat mRNA expression, AANAT activity and melatonin content in the medium (Markowska M et al., 2015) (manuscript in preparation). This suggests that these populations of leukocytes express different cytokines and immune mediators during the 11

ACCEPTED MANUSCRIPT development of peritonitis. Based on experiments performed in vivo, we knew that during the developing inflammation IL 6 and IL 18 may by candidates as mediators that modulate pineal melatonin synthesis. We monitored the expression of IL 6, IL 18 and IL 1 β in the cocultures and observed elevated mRNA levels for IL 18 in the PTLs and for IL 6 in PBLs. The expression of IL 1 β did not differ between the leukocyte populations studied (Markowska M

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et al., 2015) (manuscript in preparation). Based on these results, we formulated the

following hypothesis: 1) TG-elicited peritonitis induces synthesis of IL 6 in the PBLs, 2) this cytokine crosses the BBB, reaches the pineal gland and inhibits melatonin synthesis, 3) in

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parallel, IL 18 synthesized by PTLs acts locally at the site of inflammation.

We cannot rule out the possibility that, during inflammation, melatonin biosynthesis in the pineal gland is also connected with mechanisms other than immune mediators. Looking for

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other in vivo mediators of peritonitis-induced melatonin inhibition in the pineal gland, we turned our attention to stress. It is known that stress hormones and their synthetic analogues are able to alter pineal melatonin biosynthesis in mammals (Couto-Moraes et al., 2009) and in birds (Barriga et al., 2002). In rodents, stress hormones have been shown to stimulate pineal Asmt transcription, thus increasing the level and activity of HIOMT, which

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results in increased melatonin biosynthesis (Fernandes et al., 2009; Lopes et al., 2001). In the ring dove (Streptopelia risoria), stress was found to increase the serum melatonin level, even during the light phase. However, the influence of this stimulus on the transcription of genes

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encoding enzymes that participate in its biosynthesis remains unknown (Barriga et al., 2002). To test this hypothesis, we tested the activity of the hypothalamus-pituitary-adrenal axis in chickens with inflammation by measuring corticosterone in the serum and noradrenaline in

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the pineal glands (Piesiewicz et al., 2012). Peritonitis caused a decrease in serum corticosterone, whereas the pineal noradrenaline content was not affected. Therefore, it may be concluded that in our model of experimental peritonitis in the chicken, pineal melatonin biosynthesis is not regulated by the stress. 8. Seasonality of the reciprocal interrelationships between inflammation and the chicken pineal clock Regular observations of seasonality in the development of inflammation in chickens (Majewski et al., 2005a; Majewski et al., 2012; Turkowska et al., 2013) led us to test the

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ACCEPTED MANUSCRIPT following hypothesis: if this variability is related to pineal melatonin biosynthesis, then Px chickens may develop peritonitis independent of the season in which they hatch, and mimicking season with the appropriate melatonin supplementation should antagonize the effect of Px. We maintained functional Px chickens by keeping them in continuous illumination (LL) from the first day after hatch, as light is a factor that inhibits AANAT activity

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and eventually melatonin biosynthesis (John et al., 1994). In our protocol, experimental peritonitis was elicited at the beginning or towards the end of the subjective day, while

levels of exogenous melatonin in the drinking water strictly corresponded to the natural night duration (i.e., 8 or 16 hours during the summer and winter, respectively). Control

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chickens were kept in lighting conditions that reflected summer or winter i.e. LD = 16:8 and LD = 8:16 respectively . This experimental protocol allowed us to distinguish among those

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inflammatory parameters that were more and less closely related with melatonin (Turkowska et al., 2015; Turkowska et al., 2013). The observations presented here not only supported, once again, the notion that the immunomodulatory effects of melatonin are a complicated phenomenon (Carrillo-Vico et al., 2013) but also indicated that melatonin is not the only factor responsible for controlling the diurnal and seasonal variability of chicken

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inflammation.

Therefore, the next step of our research was to address the role of the core pineal clock genes in relation to inflammation in chickens. While there is good evidence that melatonin entrains the central oscillator in the avian SCN (Karaganis et al., 2009), less is known about

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the mechanisms by which it acts on the clock or the clock controlled genes in the peripheral tissues, including the immune system. The ability of melatonin to modulate immune function

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in relation to the clock and to cytokine gene expression has only recently been examined in the spleen of chicken with LPS-induced inflammation (Naidu et al., 2010). Moreover, transcriptomic analysis of the chicken pineal gland revealed (Bailey et al., 2003) the rhythmic oscillations of several factors related to the immune response and stress. Our experimental protocol differed from those above mentioned in using LD and LL (with melatonin supplementation, (Turkowska et al., 2014), instead of DD conditions (Bailey et al., 2003). Under continuous illumination, the diurnal profile of the transcription of particular core clock genes in the pineal gland appeared to be disturbed (in comparison with those observed in LD), but not eliminated, and it was generally synchronized by exogenous melatonin 13

ACCEPTED MANUSCRIPT supplied in drinking water. This melatonin effect was the most pronounced in the Per3 mRNA profile (see Table 1) and was also observed in the entrainment of the diurnal pattern of the chicken’s rest and activity schedule (Turkowska et al., 2014). This is the best illustration of the role of circulating melatonin in the downstream mechanisms controlled by the pineal clock, including both the peripheral control of behavior (sleep) and in the

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synchronization of clock gene expression in the pineal gland itself (Karaganis et al., 2009). Peritonitis elicited in this experimental method allowed us to detect cross-talk between the activated immune system and seasonal core clock gene expression in the pineal gland,

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indicating a role of melatonin in this information circuit . Namely, TG-induced transcription of the genes encoding IL 6 and IL 18 cytokines in the spleen and transcription of Bmal1 in the pineal gland during summer were increased in LL conditions. Melatonin supplementation

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significantly antagonized this effect (Turkowska et al., 2015). This means that LL conditions are stimulatory for the inflammatory mediators, which is in line with the comparable effects observed in chickens during stress (Shini et al., 2010) and in mice submitted to environmental circadian disruption (Adams et al., 2013). More interesting relationships between inflammation and the pineal clock were noted at night in the winter. During this

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period, IL 18 gene transcription in the spleen, stimulated by LL, was accompanied by an inhibition of Bmal1 and a stimulation of Per3 in the pineal gland. Once again, melatonin supplementation reversed the effects of LL (Table 1). Although these preliminary results deserve further research, they still support the anti-inflammatory role of melatonin that has,

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to date, been described using several experimental approaches.

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It is worth noting that the transcription of the positive (Bmal1) and negative (Per3) elements of the pineal clock were inversely related to that of the pro-inflammatory cytokine IL 18 (Table 1). Moreover, these relationships varied as a function of the season, indicating one possible mechanism responsible for the previously described seasonality of peritonitis in chickens. These findings also shed new light on a possible way by which the pineal clock and melatonin are reciprocally related to peripheral inflammation, where inflammation generates soluble mediators that are perceived by the coordination centers, including the master clock operating within the avian pineal gland. 9. Conclusions

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ACCEPTED MANUSCRIPT Bidirectional relationships between the immune system and the pineal biological clock in chickens are part of the mechanism that maintains organismal homeostasis. Melatonin is a well known anti-inflammatory agent (Carrillo-Vico et al., 2013; Majewski et al., 2005b) and the development of cytokines in response to inflammatory reactions inhibits its synthesis in the pineal gland. Based on our experiments, we propose that IL 6 decreases pineal

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expression of Aanat and modulates the Per3 and Bmal1 clock genes, which results in a

decrease in nocturnal melatonin levels (Fig. 4). This hypothesis seems to be supported if the activation of the immune system occurs at the end of light phase. The seasonality of immune processes is correlated with the period of pineal melatonin synthesis and expression of the

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well-studied core clock genes, as well as with IL 18 expressed within the immune cells (Table 1).

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10. Acknowledgments

We would like to thank Iwona Adamska, Olga Jarco, Hanna Markiewicz and Agnieszka Markowska-Zagrajek for their assistance in the laboratory work. This work was performed within the framework of Polish Ministry of Science and Higher Education Grants NN303 3177

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33, NN303 5034 38, NN303 5957 39 and NN303 5036 38.

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ACCEPTED MANUSCRIPT Zatz, M., Gastel, J.A., Heath, J.R., 3rd, Klein, D.C., 2000. Chick pineal melatonin synthesis: light and cyclic AMP control abundance of serotonin N-acetyltransferase protein. J. Neurochem. 74, 2315-2321. Zatz, M., Mullen, D.A., 1988. Norepinephrine, acting via adenylate cyclase, inhibits melatonin output but does not phase-shift the pacemaker in cultured chick pineal cells. Brain Res. 450,

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137-143.

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ACCEPTED MANUSCRIPT Figure captions Figure 1 Aanat expression (A), AANAT activity (B) and melatonin concentration (C) in pineal gland cultures in vitro. Pineal gland cultures were started at CT 12 (beginning of the subjective

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night) and were conducted for 19 h in DD conditions. Experimental variables were analyzed every 3 h in the subjective night (dark grey bar) and every 6 h in the subjective day (light grey bar). Aanat mRNA level was measured using real-time PCR and AANAT enzymatic activity was evaluated using a biphasic diffusion method in pineal glands (n=3, each parameter) as

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described by (Piesiewicz et al., 2012). Melatonin concentration was measured in the culture medium using ELISA (IBL) according to the manufacturer’s instructions. The results are

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presented as the mean ± SE, statistical analysis was performed by one-way ANOVA followed by Newman-Keuls multiple comparison test. * p < 0,05 vs. other time point. CT = circadian time (subjective hours during constant darkness).

Figure 2

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Aanat expression (A), AANAT activity (B) and melatonin concentration (C) in pineal gland isolated from intact chickens (INT) and birds with thioglycollate induced peritonitis (TG) and cultured in vitro. Some animals were injected i.p. with 3% TG at ZT 8 and sacrificed 4 hours

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later. Pineal glands were isolated and cultured in vitro starting at CT 12 (beginning of the subjective night). Pineal glands from intact chickens were used as controls. Cultures were

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conducted for 24 h in DD conditions and experimental variables were analyzed every 3 h in the subjective night (dark grey bar) and in the subjective day (light grey bar). Aanat mRNA level was measured using real-time PCR and the AANAT enzymatic activity was evaluated by a biphasic diffusion method in pineal glands (n=3, each parameter) as described by (Piesiewicz et al., 2012). Melatonin concentration was measured in the culture medium using RIA. The results are presented as the mean ± SE, statistical analysis was performed by two-way ANOVA. * p < 0,05 vs. control animals at the same time-point. CT = circadian time (subjective hours during constant darkness).

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ACCEPTED MANUSCRIPT Figure 3 AANAT activity in pineal glands co-cultured in vitro with peritoneal leukocytes (PTLs) isolated from chickens with thioglycollate-induced peritonitis. Some animals were injected i.p. with 3% TG at ZT 8, and sacrificed 4 hours later. The peritoneal cavity washed with PBS and PTLs collected. Pineal glands from control animals were co-cultured in DD conditions for 24 h with

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different numbers of PTLs in the medium, starting at CT 12 (beginning of the subjective night). AANAT enzymatic activity was evaluated by a biphasic diffusion method in pineal glands (n=3) as described by (Piesiewicz et al., 2012). The results are presented as the mean

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± SE, statistical analysis was performed by one-way ANOVA followed by Newman-Keuls

multiple comparison test. *** p < 0,001 vs. other time point. CT = circadian time (subjective

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hours during constant darkness).

Figure 4

Proposed mechanism of the inhibition of pineal melatonin synthesis during development of TG-induced peritonitis in the chickens. BMAL and CLOCK proteins (positive elements of the

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biological clock loop) stimulate the transcription of Aanat gene which results in the rhythmic melatonin synthesis at night (A). During development of inflammatory reaction, interleukins synthesized at the periphery reach the pineal gland, increase PER3 expression which in turn inhibits binding of BMAL/CLOCK heterodimer to the E-box sequences in the Aanat gene

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promotor and the rhythm of melatonin synthesis is abolished. In parallel interleukin 6 can directly decrease the Aanat expression and/or activity which results in the decrease of

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melatonin synthesis (B).

Table 1. Interrelationships between transcription of the pro-inflammatory cytokine gene IL18 in the spleen and the core clock genes Bmal1 and Per3 in the pineal gland of chickens with experimental peritonitis in relation to the time of day and season of hatch. Explanations: LL- chickens were kept in continuous illumination from the day of hatch. LL MEL - chicken from the LL group supplied with melatonin in their drinking water for the time corresponding to the summer or winter day length. ↑ - increase in transcription level in 25

ACCEPTED MANUSCRIPT relation to the respective LD group; ↓ - decrease in transcript level in relation to the respective LD group; = no effect. For details, see Turkowska et al., 2015.

Abbreviations

AADC – aromatic amino acid decarboxylase AANAT – arylalkylamine-N-acetyltransferase Aanat – arylalkylamine-N-acetyltransferase gene

BBB – brain-blood-barrier BF – bursa of Fabricius

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Asmt – acetylserotonin O-methyltransferase gene

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5-HTP – 5-hydroxytryptophan

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5-HT – 5-hydroxytryptamine, serotonin

BMAL - brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 CCGs – clock controlled genes

CLOCK – circadian locomotor output cycles kaput protein

CRY – cryptochrom CT – circadian time

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DD – constant darkness

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CREB – binding protein induced by cAMP

Ddc – dopa decarboxylase gene

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HIOMT – hydroxyindole-O-methyltransferase i.p. – intraperitoneally i.v. – intravenously IL – interleukin

LD – light/dark photoperiod LL – constant light LPS – lipopolysaccharide NAS – N-acetylserotonin NF-kB – nuclear factor kappa-light-chain-enhancer of activated B cells 26

ACCEPTED MANUSCRIPT PALT – pineal-associated lymphoid tissue PBLs – peripheral blood leukocytes PER – period

PTLs – peritoneal leukocytes

ROS – reactive oxygen species SCN – hypothalamic suprachiasmatic nuclei TG – thioglycollate

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TLR – toll-like receptor TNF – tumor necrosis factor

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TRP – tryptophan

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TPH – tryptophan hydroxylase Tph1 – tryptophan hydroxylase 1 gene

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Px – pinealectomy

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a. DAY

LL ↑ ↑ =

SUMMER LL MEL ↓ ↓ =

LL ↓ = ↑

WINTER LL MEL ↑ = ↓

b. NIGHT

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WINTER LL LL MEL ↑ ↓ ↓ ↑ ↑ ↓

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Gene/group IL 18 Bmal1 Per3

SUMMER LL LL MEL ↑ ↓ ↓ = = =

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Gene/group IL 18 Bmal1 Per3

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rhythmical expression

BMAL CLOCK

NORMAL MELATONIN BIOSYNTHESIS

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E-box B PER3

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BMAL CLOCK

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DECREASED MELATONIN BIOSYNTHESIS

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E-box

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decreased expression

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PER3

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DEVELOPING INFLAMMATION

IL6

ACCEPTED MANUSCRIPT Highlights 1. In the chickens peritonitis inhibits expression of Aanat gene and melatonin synthesis. 2. The inhibition of melatonin synthesis is mediated by interleukin 6 and/or interleukin 18.

AC C

EP

TE D

M AN U

SC

RI PT

3. Expression of Per3 and Bmal1 clock genes in pineal gland is modulated by peritonitis.