Photoperiod-dependent effect of inflammation on nocturnal gene expression of proinflammatory cytokines and their receptors in pars tuberalis of ewe

Pituitary gland plays an important role in the maintaining homeostasis since it is involved in the regulation of numerous physiological processes including reproduction (Perez-Castro et al., 2012). The pituitary gland is functionally and anatomically connected with the hypothalamus by the median eminence (ME), therefore the pituitary gland is an intermediary organ for physiological signal exchanges between the hypothalamus and peripheral organs (Perez-Castro et al., 2012). The adenohypophysis is composed of hormone-secreting epithelial cells and divided into three discrete parts such as:


Introduction
Pituitary gland plays an important role in the maintaining homeostasis since it is involved in the regulation of numerous physiological processes including reproduction (Perez-Castro et al., 2012).The pituitary gland is functionally and anatomically connected with the hypothalamus by the median eminence (ME), therefore the pituitary gland is an intermediary organ for physiological signal ex-changes between the hypothalamus and peripheral organs (Perez-Castro et al., 2012).The adenohypophysis is composed of hormone-secreting epithelial cells and divided into three discrete parts such as: . The PT develops from the lateral lobes of the Rathke's pouch (Lafarque et al., 2004;Yasuo and Korf, 2011).The PT plays an important role in the hypothalamic-pituitary interaction because this pituitary region surrounds the infundibular stalk and is the exclusive pituitary structure in close anatomical contact with the medial-basal hypothalamus, ME and the third ventricle (Guerra et al., 2010).The PT is bathed in the cerebrospinal fluid (Guerra et al., 2010;Yasuo and Korf, 2011) and possessed by intercellular channels needed for direct communication with the subarachnoid space (Morgan and Williams, 1996;Lafarque et al., 2004;Dupré, 2011).The PT is composed of the follicular cells -small cells without secretory features, PT-specific secretory cells and PD-like cells (Dupré, 2011).The PT-specific secretory cells contain a small number of secretory granules and aggregate or scatter glycogen particles (Morgan and Williams, 1996;Yasuo and Korf, 2011).It was established that gonadotrophs from the PT may contain and secrete some amount of the luteinizing hormone (LH) and follicle stimulating hormone (FSH) (Mignot and Skinner, 2005;Perez-Castro et al., 2012).Colocalization of LH and other hormones like prolactine, growth hormone (GH) and thyroid-stimulating hormone (TSH) was immunocytochemically demonstrated by Mignot and Skinner (2005).Additionally, some studies showed that the PT can release factors which can modulate the activity of the PD lactotrophs (Lafarque et al., 2004).For that reasons, it is suggested that the PT supports the PD functions (Lafarque et al., 2004).However, some studies on melatonin receptors in the pituitary gland showed that melatonin-binding sites of high density are present in the PT, whereas they are absent in the PD (Morgan and Williams, 1996).Therefore, it is suggested that the PT may mediate the melatonin effect on the neuroendocrine function and may be involved in the photoperiodic regulation of pituitary hormones secretion (Morgan and Williams, 1996;Lafarque et al., 2004).Photoperiod influences the reproductive function of seasonal breeders including sheep, however the exact mechanism of which photoperiod affects reproductive system is not completely understood (Goodman et al., 2010).Moreover, both seasonal (Vázquez et al., 2007) and diurnal rhythms (Mattern et al., 1993) occur in the gonadotropins secretion even in non-seasonal breeders.
Many studies showed that both acute and prolonged inflammation affect endocrine system functioning in numerous animal species, including sheep (Herman and Tomaszewska-Zaremba, 2010;Fergani et al., 2012;Danek and Żurek, 2014;Herman et al., 2014a).Circulating inflammatory mediators acting both at the hypothalamic (Herman et al., 2014b) and pituitary level (Tsagarakis et al., 1998) play an important role in the induction of endocrine disorders during inflammation.Due to rich vascularization of the PT, the cells located in this pituitary region are an easy target for blood born inflammatory mediators such as interleukin-1 (IL-1β, IL1B for gene), interleukin-6 (IL-6, IL6 for gene) and tumor necrosis factor (TNF, TNF for gene) α which may affect the activity of cells located in the PT acting their corresponding receptors.This local action of the inflammatory mediators in the PT could have a profound effect on the secretory activity of the whole pituitary gland.It is worth mentioning that photoperiod influences immune system affecting the synthesis of proinflammatory cytokines (Haldar and Ahmad, 2010).It was described that there is diurnal fluctuation in both proand anti-inflammatory cytokines synthesis (Lange et al., 2010).
The aim of the study was to determine the effect of acute inflammation induced by intravenous lipopolysaccharide (LPS) injection on the nocturnal mRNA expression of proinflammatory cytokines and their corresponding receptors in the PT of ewe under different photoperiodic condition.

Animals and experimental design
The experiments were carried out on Blackhead ewe (n = 24) during long night (16:8, October; n = 12) and short night (8:16, June; n = 12) periods.The animals were maintained indoors in individual pens and were exposed to natural daylight present at 52°N latitude and 21°E longitude.The ewe were maintained in good conditions, i.e. their body condition was estimated at 3 in a five-point scale (Russel, 1991) and they were adapted to the experimental conditions for one month.The ewe had constant visual contact with each other in order to avoid isolation stress.The animals were fed constant diet of commercial concentrates with hay and water available , according to the recommendations proposed by the National Research Institute of Animal Production for adult ewe (IZ PIB-INRA, 2009).
The study included two analogical experiments.The animals (n = 12) in each photoperiod were divided into two subgroups: control (n = 6) and LPS-treated (n = 6).Two hours after the sunset an appropriate volume of LPS from 055:B5 (400 ng • kg -1 ) (Sigma-Aldrich, St Louis, MO, USA) dissolved in saline (0.9% w/v NaCl; Baxter, Deerfield, IL, USA) was injected intravenously (i.v.) into the jugular vein.The maximum volume of injected LPS solution (10 mg • l -1 ) has never exceeded 2.5 ml.The control group received the same volume of NaCl (based on their body weight).The efficiency of the LPS treatment to induce an inflammatory response in the animal was estimated basing on the measurement of the body temperature.All procedures were conducted in the darkness with the use of red light.Three hours after the LPS or saline injection blood samples were collected by intrajugular catheter and then all animals were euthanized by decapitation.The brain was immediately removed from the skulls, and the was dissected, immediately frozen in liquid nitrogen and stored at -80 °C until further assay.
All procedures were performed in agreement with the Local Ethics Committee of Warsaw University of Life Sciences -SGGW.

Assays
Radioimmunoassay for melatonin.Melatonin concentration in plasma was analysed by radioimmunoassay double-antibody method according to the method of Fraser et al. (1983), and modified in our laboratory, using anti-ovine melatonin serum (AB/S/01, Stockgrand Ltd., Surrey, UK).Synthetic melatonin as a standard (Sigma-Aldrich, St. Louis, MO, USA), and [O-methyl-3H]-melatonin (Amersham PLC, Amersham, UK) as a tracer, were used.The sensitivity of the assay was 16.8 ± 8.0 pg • ml -1 and the intra-and interassay coefficients of variation were 10.5 and 13.2%, respectively.
Determination of the relative gene expression.The NucleoSpin ® RNA kit (MACHEREY-NAGEL GmbH and Co, Düren, Germany) was used to isolate the total RNA from the PT fragments.All isolation steps were conducted in accordance with the manufacturer instruction.The purity and concentration of the isolated RNA was quantified spectrophotometrically with the use of NanoDrop 1000 instrument (Thermo Fisher Scientific Inc.; Waltham, MA, USA).The integrity of isolated RNA was confirmed by electrophoresis with the use of 1% agarose gel stained with ethidium bromide.The Maxima ™ First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific Inc.; Waltham, MA, USA) was used to perform cDNA synthesis.As a starting material for cDNA reversed transcription reaction (RT) synthesis 2 µg of total RNA was used.Real-Time RT-PCR was carried out with the use of the HOT FIREPol EvaGreen ® qPCR Mix Plus (Solis BioDyne; Tartu, Estonia) and HPLC-grade oligonucleotide primers (Genomed; Warszawa, Poland).The primer sequences were designed using Primer 3 software (Herman et al., 2014b) (Table 1).One reaction mixture of total volume amounting 20 µl contained: 4 µl of PCR Master Mix (5×), 14 µl of RNase-free water, 1 µl of primers (0.5 µl each primer, 0.25 µM working concentration) and 1 μl of the cDNA template.The reactions were conducted on Rotor-Gene 6000 instrument (Qiagen; Dusseldorf, Germany) with the following protocol: 95 °C for 15 min and 30 cycles of 95 °C for 10 s for denaturation, 60 °C for 20 s for annealing and 72 °C for 10 s for extension.The specificity of the amplification was confirmed by a final melting curve analysis.
The relative gene expression was calculated using the comparative quantification option (Rasmussen, 2001) of the Rotor Gene 6000 software 1.7.(Qiagen; Dusseldorf, Germany).Three housekeeping genes were examined: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB) and cyclophilin C (PPIC).GAPDH were chosen for further analysis based on the result analysis performed on BestKeeper software (Pfaffl et al., 2004).The average relative quantity of gene expression in the control group of the PT collected during SN was set to 1.0.

Statistical analysis
The statistical analysis was performed using the STATISTICA 10 software, 2010 (Stat Soft.Inc.; Tulsa, OK, USA).The results of gene expression were analysed using two-way analyses of variances (ANOVA) to identify significant influence of two parameters (photoperiod and LPS-treatment) and followed by a post-hoc Tukey's test.The results are presented as mean ± SEM.Statistical significance was set at P < 0.05.

Results
Nocturnal melatonin concentration in control ovine serum was higher (P < 0.05) during long night (LN) than short night (SN) period.Endotoxin injection decreased (P < 0.05) the melatonin concentration only in ewe kept under SN condition (Figure 1).
Intravenous injections of endotoxin increased (P < 0.05) the relative gene expression of IL1B in the collected during SN (9-fold) and LN (16-fold) when compared with this gene expression in the corresponding control groups (Figure 2A).lipopolysaccharide (LPS) did not affect the interleukin-1 receptor (IL-1R, IL1R for gene) of mRNA expression in the PT (Figure 2B).Moreover, no photoperiod effect on the expression of both these genes was found.
Endotoxin injection elevated (P < 0.05) gene expression of IL6 during SN (170-fold,) and LN (496-fold) compared with the proper control group (Figure 3A).It was found that LPS more strongly (P < 0.05) stimulated IL6 gene expression during LN than SN period.Administration of endotoxin did not influence the gene expression of interleukin-6 receptor (IL-6R, IL6R for gene) regardless the photoperiod (Figure 3B) but elevated (P < 0.05) the level of glycoprotein (gp) 130 (IL6ST for gene) mRNA in the PT during LN (2-fold) compared with the control group (Figure 3C).It is worth noting that the influence of LPS on the level of IL6 and IL6ST mRNA in the PT was stronger (P < 0.05) during LN than SN.Endotoxin injection did not affect TNF gene expression but the level of TNF transcript determined in control group in LN period was higher (P < 0.05) when compared to the control group from SN photoperiod (Figure 4A).No LPS-induced changes in the gene expression of both tumour necrosis factor receptor (TNFR) type 1 (TNFSF1A for gene) and type 2 (TNFSF1B for gene) were observed during SN (Figures 4B and C).However, endotoxin stimulated (P < 0.05) both TNFRSF1A (2-fold) and TNFRSF1B (4-fold) mRNA expression during LN period in comparison with the corresponding control group.

Discussion
Our study showed that cells located in the (PT) expressed mRNA encoding receptors for proinflammatory cytokines such as IL-1β, IL-6 and TNFα.As the existence of cytokine receptors on the membrane is essential for the transduction of the cytokine signal into the cell, obtained data suggests that proinflammatory cytokines may influence the PT cells activity.It is worth mentioning that this is the first scientific report describing the occurrence of transcript encoding these interleukins and their corresponding in the PT.However, based on PCR assays it is impossible to judge in which type of the PT cells those mRNAs were expressed.The expression of IL-1R in the mouse pituitary gland had been previously shown by Takao et al. (1993) who found that LPS treatment enhanced IL-1β synthesis but down-regulated IL-1R expression.Although the IL-6 signalling pathway includes IL-6R and signal transducer gp130, only IL6R transcript was detected in the rat anterior pituitary (Tsagarakis et al., 1998).In our study, LPS enhanced IL6ST gene expression during LN but did not affect IL6R mRNA level.Our results are partially different from the studies performed on rats.It was demonstrated that LPS injection increased both IL6ST and IL6R transcription in the rat pituitary (Vallières and Rivest, 1997).On the other hand, the study on the human hepatocytes performed by Bauer et al. (1989) showed that LPS down-regulates IL6R mRNA synthesis in these cells.Probably, the way in which LPS regulates IL6R transcription may be dependent upon the kind of cells expressing this receptor as well as used animal model species.Our study showed that inflammation induced by bacterial endotoxin stimulated the TNF receptors gene expression in the PT only during LN period.It is the first scientific report showing the stimulatory influence of LPS on the TNF receptors mRNA expression in the pituitary tissue.However, increased expression of TNFRSF1A and TNFRSF1B may not result from direct effect of LPS on the PT cells but may be induced by the ligand of these receptors, which circulating concentration is significantly increased during LPS-induced inflammation (Danek and Żurek, 2014).It was showed that TNFα stimulated TNFRSF1B mRNA and its protein expression in human pancreatic cancer cells (Kalthoff et al., 1993).We also found that the basal gene expression proinflammatory cytokines receptors in the PT was similar during LN and SN.The fact that LPS promotes both TNF receptors gene expression and IL6ST gene expression in LN but not in SN period may result from the different level of melatonin during these two photoperiods, because the PT is exclusive pituitary structure expressing melatonin binding sites (Cogé et al., 2009).
It is well known that immune function, like other common physiological and behavioural process, undergoes daily variation.Enhanced immune functions are generally observed in short days (Haldar and Ahmad, 2010).All laboratory studies of photoperiodic changes in immune parameters of mammals demonstrated enhanced immune function in short winter-like photoperiod, for example in mice significantly increased lymphocyte, neutrophil and white blood cell counts occur in short photoperiods (Blom et al., 1994).Moreover, the increased activity of lymphoproliferative immune cells and changes in spleen morphology occur in hamsters under short day (Nelson, 2004).Photoperiodic information from the environment is conveyed to the organism by a circadian rhythm of melatonin production by the pineal gland.The circulating level of melatonin depends on both daytime and season.Our studies showed that circulating level of melatonin is higher during LN than SN.Such result is fully consistent with previousely published data, which also showed higher secretion of melatonin in sheep during winter months when night period is longer (Todini et al., 2011).
Because time of melatonin secretion is longer during LN, its proinflammatory action can be stronger than during SN period (Mauriz et al., 2013).It is well established that melatonin takes part in immune response stimulation or regulation of immunodeficiencies secondary to acute stress and viral diseases (Esquifino et al., 2004).Melatonin was also identified as stimulator of the inflammation by activating monocytes and enhancing production of IL-1, IL-6 and TNFα (Haldar and Ahmad, 2010).It is generally accepted that immune function is enhanced in LN period (Haldar and Ahmad, 2010;Weil et al., 2014).However, the role of melatonin in the regulation of the course of the inflammatory response may be more ambiguous.On the one hand melatonin might promote early phases of inflammation, but on the other hand contribute to its attenuation in order to avoid complications of chronic inflammation (Herman et al., 2015).It was also found that melatonin administration normalize IL1, IL6 and TNF mRNA levels during cardiac inflammation which supports the thesis about dual role of melatonin in modulation of the inflammation (Mauriz et al., 2013).
Although, we did not state the influence of LPS on the expression of mRNA encoding IL1R1 and IL6R in the PT, it does not preclude that these receptors may be involved in the immune-endocrine interaction.Probably relatively high basal expression of mRNA for these receptors in the PT caused that endotoxin did not affect these genes expression, but the expression of these receptor is enough to enable the of inflammatory signals carried out by their ligands during an LPS-induced immune/ inflammatory challenge.Our study not only suggests that proinflammatory cytokines such as IL-1β, IL-6 and TNFα may act on the PT cells due to the existence of their corresponding receptors but also that these molecules are locally synthesized in this pituitary structure because significant gene expression of IL1B, IL6 and TNF was found in the PT.It is worth mentioning that the expression of mRNAs encoding these cytokines in the PT has not been described yet.Although obtained data concern only transcription of the genes encoding proinflammatory cytokines in the PT, taking into account that cytokines are not accumulated in the cell but they are continuously released during stimulation (Zagury et al., 2001), it is assumed that the changes in the cytokines expression are generally parallel to their gene transcription.The synthesis of proinflammatory cytokines mRNA have been previously found in other parts of the pituitary gland.In rat IL-1 mRNA was detected in pituicytes and thyrotrops (Koenig et al., 1990), while in the human expression of this mRNA was noted only in pituitary tumor cells (Tsagarakis et al., 1998).Additionaly studies carried out on rats and mice anterior pituitary showed that IL-1, IL-6 and TNFα are secreted by folliculo-stellate cells (Jovanović et al., 2014), and the presence of stellate cells is necessary for IL-6 secretion (Vankelecom et al., 1989).Expression of proinflammatory cytokines can be modulated by numerous factors including LPS or other cytokines (Gabay and Kushner, 1999).It is well established that IL-6 synthesis is induced by IL-1, as well as LPS, both and (Muramami et al., 1993).Moreover, it was proved that IL-6 secretion is inhibited by glucocorticoids (Tsagarakis et al., 1998).TNFα is a main stimulator of IL-1, which in turn can regulate its own receptor (Gabay and Kushner, 1999).Our study showed that endotoxin-induced expression of IL6 and TNF gene in the ovine PT was higher during LN than SN period.This difference may also result from the stimulatory effect of melatonin on the proinflammatory cytokines synthesis.
The results of the present study suggest that the immune-endocrine interaction occurs at the level of the PT.The local synthesis of proinflammatory cytokines and expression of their corresponding receptors in the PT may have a profound effect on the pituitary hormones secretion because the changes occurring in the PT may influence the secretory activity of the PD cells (Lafarque et al., 2004).However, there is no data concerning the influence of proinflammatory mediators on the hormone secretion from the PT, these cytokines may affect the pituitary hormones release from the anterior pituitary cells (Arzt et al., 1998).It was also found that IL-1β increased the adrenocorticotropic hormone, LH, GH and TSH secretion in primary cultures of rat pituitary cells, while IL-6 stimulated prolactin, GH, FSH and LH release from rat pituicytes culture (Tsagarakis et al., 1998).On the other hand, the study performed on the sheep pituitary explants showed that IL-1β suppressed LH secretion (Herman et al., 2013).

Conclusions
The study showed that mRNAs for proinflammatory cytokines and their receptors are expressed in the Photoperiod particularly influenced the response of proinflammatory cytokines on lipopolysaccharide treatment, however minor influence was observed on basal gene expression of proinflammatory cytokines in PT.Obtained results indicate that the PT may be one of the gateways for immune-endocrine interactions.The fact that the photoperiod influences to some extend the nocturnal synthesis of proinflammatory cytokines and their corresponding receptors in the PT during inflammatory condition suggests that these interactions could be influenced by the circulating melatonin.

Table 1 .
All genes analysed by real-time PCR are listed with their full names and abbreviations