Developmental changes in hypothalamic toll-like-receptor 4 mRNA expression and the effects of lipopolysaccharide on such changes in female rats

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Short communication

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Developmental changes in hypothalamic toll-like-receptor 4 mRNA expression and the effects of lipopolysaccharide on such changes in female rats

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Takeshi Iwasa a,∗ , Toshiya Matsuzaki a , Altankhuu Tungalagsuvd a , Munkhsaikhan Munkhzaya a , Takako Kawami a , Mikio Yamasaki a , Masahiro Murakami b , Takeshi Kato a , Akira Kuwahara a , Toshiyuki Yasui c , Minoru Irahara a a Department of Obstetrics and Gynecology, The University of Tokushima Graduate School, Institute of Health Biosciences, 3-18-15 Kuramoto-Cho, Tokushima 770-8503, Japan b Department of Obstetrics and Gynecology, Shikoku Medical Center for Children and Adults, Senyu-cho 2-1-1, Zentsuji City, Kagawa 765-8507, Japan c Department of Reproductive Technology, Institute of Health Biosciences, The University of Tokushima Graduate School, Japan

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Article history: Received 30 September 2014 Received in revised form 19 October 2014 Accepted 19 October 2014 Available online xxx

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Keywords: TLR4 IL-1␤ TNF-␣ IL-6 Hypothalamus

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1. Introduction

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Hypothalamic pro-inflammatory cytokine expression exhibits a weaker response to lipopolysaccharides (LPS) during the early neonatal period than during the later developmental period. Although toll-like receptor 4 (TLR4), which recognizes bacterial molecules, activates pro-inflammatory cytokine responses, the developmental changes in hypothalamic TLR4 expression have not been evaluated. In this study, the hypothalamic TLR4 mRNA levels of saline-injected and LPS-injected rats were measured during the neonatal, pre-pubertal, and post-pubertal periods. The rats’ hypothalamic TLR4 mRNA levels gradually increased from the neonatal to pubertal period and were altered by the injection of LPS at all examined ages (postnatal day (PND) 5, 15, 25, and 42). LPS injection resulted in decreased hypothalamic TLR4 mRNA expression at PND5, whereas it increased hypothalamic TLR4 mRNA expression at PND15, 25, and 42. After the injection of LPS, the hypothalamic mRNA levels of the pro-inflammatory cytokines interleukin (IL)-1␤, tumor necrosis factor ␣, and IL-6 were attenuated during the early developmental period and increased acutely on PND42. The expression profiles of these pro-inflammatory cytokines exhibited similar, but not entirely consistent, changes to those displayed by TLR4 during the developmental period. Hypothalamic TLR4 mRNA expression gradually increased throughout the developmental period, whereas the mRNA expression levels of the pro-inflammatory cytokines increased acutely at PND42. Thus, it is assumed that hypothalamic TLR4 hypoactivity contributes to the low sensitivity of pro-inflammatory cytokines to LPS during the early developmental period. © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

The pathophysiological responses to various stressors are not fully established during the first 2–3 weeks of postnatal life, which is known as the stress hyporesponsive period (Walker et al., 1986; Witek-Janusek, 1988; Widmaier, 1989, 1990). Such hyporesponsiveness is mainly caused by the low sensitivity of hypothalamic stress-related factors, such as corticotropin-releasing factor and pro-inflammatory cytokines, to stressors during the early neonatal period (Witek-Janusek, 1988; Widmaier, 1989, 1990; Iwasa et al., 2011). In addition, such hyporesponsiveness might be associated

∗ Corresponding author. Tel.: +81 88 633 7177. E-mail address: [email protected] (T. Iwasa).

with the high lethality rate and/or weak anorectic response seen under immune stress conditions during the same period (WitekJanusek, 1988; Iwasa et al., 2011). In previous studies, we have reported that the hypothalamic expression levels of interleukin-1␤ (IL-1␤) and tumor necrosis factor-␣ (TNF-␣) (representative pro-inflammatory cytokines) exhibited weaker responses to lipopolysaccharides (LPS) during the early neonatal period than during the later developmental period (Iwasa et al., 2011). In the brain, IL-1␤ and TNF-␣ suppress appetite under immune stress conditions (Wisse et al., 2007); therefore, the weaker anorectic response to LPS observed during the early neonatal period might be partially caused by the hyporesponsiveness of these cytokines. Toll-like-receptors (TLR) on macrophages and other types of cells play important roles in the regulation of the innate immune response (Takeda and Akira, 2001). To date, 11

http://dx.doi.org/10.1016/j.ijdevneu.2014.10.002 0736-5748/© 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Iwasa, T., et al., Developmental changes in hypothalamic toll-like-receptor 4 mRNA expression and the effects of lipopolysaccharide on such changes in female rats. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.10.002

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members of the TLR family have been identified, and TLR4 recognizes bacterial molecules, e.g., LPS (Blatteis et al., 2004). After the activation of TLR4, a variety of inflammatory molecules, including 55 56Q3 pro-inflammatory cytokines (Luheshi, 1998; Dinarello, 1999) and cyclooxygenase-2 (Khan et al., 2012), are activated, and these alter57 ations can have adverse health effects. Although the developmental 58 changes in TLR4 expression that occur in peripheral tissues/cells 59 have been described in previous studies (Harju et al., 2001; Rouzic 60 et al., 2012), the equivalent changes in hypothalamic TLR4 expres61 sion have not been reported. In addition, the responses of TLR4 62 mRNA expression in peripheral organs, tissues, and macrophages to 63 LPS have been examined in several studies (Matsuguchi et al., 2000; 64 Yang et al., 2002; Rouzic et al., 2012), whereas the effects of LPS on 65 hypothalamic TLR4 mRNA expression have been fully evaluated. 66 Only one previous report have shown that TLR4 is exist in medial 67 preoptic area, one of the hypothalamic nuclei, and that TLR4 mRNA 68 level is decreased by LPS injection. We speculate that the hypothal69 amic TLR4 mRNA expression level and its sensitivity to LPS varies 70 from the neonatal to pubertal period and that these alterations 71 are involved in the developmental changes in the responses of 72 inflammatory factors to stressors. To evaluate this hypothesis, the 73 hypothalamic TLR4 mRNA levels of rats that had been injected with 74 saline or LPS were examined during the neonatal, pre-pubertal, and 75 post-pubertal periods. The mRNA levels of IL-1␤, TNF-␣, and IL-6 76 (potent inflammatory factors that are regulated by TLR4) were also 77 measured. 78 53

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2. Materials and methods

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Pregnant Sprague–Dawley rats were purchased (Charles River Japan Inc., Tokyo, Japan) and housed under controlled lighting (14 h light, 10 h dark) and temperature (24 ◦ C) conditions. The day on which the pups were born was defined as postnatal day (PND) 1. After birth, 10–12 female pups were randomly assigned to each dam. To ensure that the litter size per dam remained constant, pups were culled or moved to other dams and fostered until weaning (PND21). At PND5, 15, 25, and 42, 13–16 rats were randomly selected from each dam and divided into two groups. The rats in one group were intraperitoneally injected (ip) with 1 mg/kg LPS (0111:B4) (Sigma, St. Louis, MO, USA) and those in the other group were injected with sterile saline between 0900 h and 1100 h. Injection volume did not exceed 0.1 ml. The rats were killed by decapitation at 6 h after the injections, and their whole brains were harvested, snap frozen, and stored at −80 ◦ C. As previous study revealed that hypothalamic pro-inflammatory cytokine reached peak levels around 6 h after LPS injection (Iwasa et al., 2011), this time point was selected in present study. Hypothalamic explants were dissected from the frozen brains as described previously (Iwasa et al., 2011, 2014a–d). Total RNA was isolated from the hypothalamus using a TRIzol® reagent kit (Invitrogen Corp., Carlsbad, CA, USA) and an RNeasy® mini kit (Qiagen GmbH, Hilden, Germany). cDNA was synthesized with oligo(deoxythymidine) primers at 50 ◦ C using the SuperScript IIITM first-strand synthesis system for RT-PCR (Invitrogen Co.). PCR analysis was performed using the StepOnePlusTM RT-PCR system (PE Applied Biosystems, Foster City, CA, USA) and FAST SYBR® green. Standard curves, which were generated by serially diluting an abundant sample at least 4 times, were used for the relative quantification of each mRNA expression level. The mRNA expression levels of the target molecules were normalized to the mRNA expression level of GAPDH. The following forward and reverse primers were used: TLR4: F: 5 -CAT GAA GGC CTC CCT GGT GTT-3 , R: 5 TGC CAG AGC GGC TAC TCA GAA-3 ; IL-1␤: F: 5 -GCT GTG GCA GCT ACC TAT GTC TTG-3 , R: 5 -AGG TCG TCA TCA TCC CAC GAG-3 ; TNF-␣: F: 5 -AGC CCT GGT ATG AGC CCA TGT A-3 , R: 5 -CCG GAC TCC GTG ATG TCT AAG T-3 ; IL-6: F: 5 -TCC TAC CCC AAC TTC CAA TGC TC-3 , R: 5 -TTG GAT GGT CTT GGT CCT TAG CC-3 ; GAPDH: F: 5 -ATGGCA CAG TCA AGG CTG AGA-3 , R: 5 -CGC TCC TGG AAG ATG GTG AT-3 . The PCR conditions were as follows: initial denaturation and enzyme activation at 95 ◦ C for 20 s, followed by 45 cycles of denaturation at 95 ◦ C for 3 s and annealing and extension at 68 ◦ C for 30 s (TLR4), 61 ◦ C for 30 s (IL-1␤), 65.5 ◦ C for 30 s (TNF-␣), 66 ◦ C for 30 s (IL-6), or 64 ◦ C for 30 s (GAPDH). Data were analyzed using one-way analysis of variance (ANOVA) followed by the Tukey–Kramer post hoc test or the Student’s t test. All results are presented as mean ± SEM values.

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Under the basal (saline injection) conditions, hypothalamic TLR4 mRNA expression differed significantly among the age groups

(one-way ANOVA; F(3, 29) = 34.1, P < 0.01) (Fig. 1A). Specifically, the hypothalamic TLR4 mRNA level was lowest at PND5 and increased thereafter. At PND5, the LPS-injected rats exhibited significantly lower TLR4 mRNA levels than the saline-injected rats (Fig. 1A). On the other hand, the TLR4 mRNA levels of the LPS-injected rats were significantly higher than those of the saline-injected rats at PND15, 25, and 42. Among the LPS-injected rats, the hypothalamic mRNA levels of IL-1␤ (one-way ANOVA; F(3, 29) = 19.2, P < 0.01), TNF-␣ (one-way ANOVA; F(3, 29) = 14.3, P < 0.01), and IL-6 (one-way ANOVA; F(3, 29) = 3.2, P < 0.05) differed significantly among the age groups (Fig. 1B–D). Furthermore, the LPS-injected rats’ mRNA levels of IL-1␤, TNF-␣, and IL-6 were significantly higher at PND42 than at PND5, 15, and 25. 4. Discussion It has been established that TLR4, which recognizes bacterial molecules, plays a pivotal role in the regulation of innate immune responses (Takeda and Akira, 2001). Whilst some previous studies have investigated the developmental changes in peripheral TLR4 expression, the developmental changes in hypothalamic TLR4 expression have not been evaluated. In addition, the response of the central TLR4 system to inflammatory stress has not been examined. In the present study, we found that hypothalamic TLR4 mRNA expression gradually increased from the neonatal to the pubertal period and that it was affected by the injection of LPS at all examined ages. Interestingly, the changes in hypothalamic TLR4 mRNA expression induced by LPS injection differed between the early neonatal period (PND5) and the later time points (PND15, 25, and 42); i.e., LPS injection caused a reduction in hypothalamic TLR4 mRNA at PND5 whereas it induced increased hypothalamic TLR4 mRNA expression at the other (later) time points. This observation is contrast with previously observed decrease in TLR4 mRNA during LPS injected condition (Laflamme and Rivest, 2001). We speculate that the difference of LPS dose, 1 mg/kg in present study and 40–100 ␮g/kg, might induce such discrepancies. Previous studies have shown that TLR4 mRNA expression is increased in the lungs, but not the liver, during the prenatal and postnatal developmental periods (Harju et al., 2001; Rouzic et al., 2012). In addition, TLR4 mRNA expression was found to increase in vascular endothelial cells, but not macrophages, after the injection of LPS (Matsuguchi et al., 2000; Yang et al., 2002). These results show that the developmental changes in the response of TLR4 expression to inflammatory stress differ among organs, tissues, and cell types. In lung tissue, inflammatory responses to microbial toxins are attenuated during the immature period, and it is assumed that the weak TLR4 activity seen during the early developmental period contributes to this attenuation. As hypothalamic TLR4 mRNA expression was found to be downregulated during the early developmental period in the present study, we speculated that hypothalamic inflammatory responses might also be attenuated in this period. Therefore, we measured the hypothalamic mRNA levels of pro-inflammatory cytokines whose expression is upregulated by TLR4 activation (IL-1␤, TNF-␣, and IL-6) at PND5, 15, 25, and 42. As a result, we found that the hypothalamic mRNA levels of IL-1␤, TNF-␣, and IL-6 increased in an age-dependent manner under the LPS-injected conditions. The changes in the expression profiles of these pro-inflammatory cytokines were similar, but not entirely consistent, with the changes in the expression profile of TLR4. The hypothalamic TLR4 mRNA level increased gradually, whereas those of the pro-inflammatory cytokines increased acutely on PND42. These data do not support the fact that the reduced inflammatory response to LPS at neonatal period could be ascribed to a reduced level of TLR4. One possibility is that the functions of other factors that regulate the activity of pro-inflammatory cytokines are established around PND42. Another is that weak peripheral TLR4 activity

Please cite this article in press as: Iwasa, T., et al., Developmental changes in hypothalamic toll-like-receptor 4 mRNA expression and the effects of lipopolysaccharide on such changes in female rats. Int. J. Dev. Neurosci. (2014), http://dx.doi.org/10.1016/j.ijdevneu.2014.10.002

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Fig. 1. Developmental changes in hypothalamic TLR4 (A), IL-1␤ (B), TNF-␣ (C), and IL-6 (D) mRNA expression in saline- () or LPS (1 mg/kg) ()-injected rats (n = 6–8 per group). The mRNA expression levels of the target molecules were normalized to the mRNA expression level of GAPDH. Values are expressed as the mean + SEM. Different letters (a–c) indicate a significant difference (P < 0.05) within the same treatment (saline or LPS) group. *P < 0.01 vs. each other.

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results in attenuated pro-inflammatory cytokine responses in the hypothalamus. As pro-inflammatory cytokines from peripheral tissues can reach the brain and activate inflammatory responses in the hypothalamus, weak peripheral pro-inflammatory cytokine responses due to the attenuation of TLR4 expression might result in the incomplete induction of hypothalamic pro-inflammatory cytokine production. Further studies focusing on both central and peripheral tissues are needed to examine these hypotheses. In this study, protein levels of TLR4 and pro-inflammatory cytokines were not measured. Thus, it is possible that mRNA levels and protein levels are mismatched. In future studies, protein levels should be measured under same experimental condition. In addition, accessory proteins of TLR4, such as myeloid differentiation factor 2, were not evaluated in this study. Because adaptations could easily have occurred in these systems, developmental changes of these factors should be focused in future examinations. References Harju, K., Glumoff, V., Hallman, M., 2001. Ontogeny of toll-like receptors Tlr2 and Tlr4 in mice. Pediatr. Res. 49, 81–83. Iwasa, T., Matsuzaki, T., Murakami, M., Kinouchi, R., Gereltsetseg, G., Nakazawa, H., Yamamoto, S., Kuwahara, A., Yasui, T., Irahara, M., 2011. Changes in responsiveness of appetite, leptin and hypothalamic IL-1␤ and TNF-␣ to lipopolysaccharide in developing rats. J. Neuroimmunol. 236, 10–16. Iwasa, T., Matsuzaki, T., Munkhzaya, M., Tungalagsuvd, A., Kawami, T., Murakami, M., Yamasaki, M., Kato, T., Kuwahara, A., Yasui, T., Irahara, M., 2014a. Changes in the responsiveness of hypothalamic prokineticin 2 mRNA expression to food deprivation in developing female rats. Int. J. Dev. Neurosci. 34, 76–78. Iwasa, T., Matsuzaki, T., Munkhzaya, M., Tungalagsuvd, A., Kawami, T., Murakami, M., Yamasaki, M., Kato, T., Kuwahara, A., Yasui, T., Irahara, M., 2014b. Pre-pubertal serum leptin levels and sensitivity to central leptin injection of prenatally undernourished female rats. Int. J. Dev. Neurosci. 35, 52–54.

Iwasa, T., Matsuzaki, T., Munkhzaya, M., Tungalagsuvd, A., Kawami, T., Murakami, M., Yamasaki, M., Kato, T., Kuwahara, A., Yasui, T., Irahara, M., 2014c. Prenatal exposure to glucocorticoids affects body weight, serum leptin levels, and hypothalamic neuropeptide-Y expression in pre-pubertal female rat offspring. Int. J. Dev. Neurosci. 36, 1–4. Iwasa, T., Matsuzaki, T., Munkhzaya, M., Tungalagsuvd, A., Kawami, T., Yamasaki, M., Murakami, M., Kato, T., Kuwahara, A., Yasui, T., Irahara, M., 2014d. Changes in the responsiveness of hypothalamic PK2 and PKR1 gene expression to fasting in developing male rats. Int. J. Dev. Neurosci. 38C, S0736–S5748. Khan, M.M., Gandhi, C., Chauhan, N., Stevens, J.W., Motto, D.G., Lentz, S.R., Chauhan, A.K., 2012. Alternatively-spliced extra domain A of fibronectin promotes acute inflammation and brain injury after cerebral ischemia in mice. Stroke 43, 1376–1382. Laflamme, N., Rivest, S., 2001. Toll-like receptor 4: the missing link of the cerebral innate immune response triggered by circulating gram-negative bacterial cell wall components. FASEB J. 15, 155–163. Matsuguchi, T., Musikacharoen, T., Ogawa, T., Yoshikai, Y., 2000. Gene expressions of Toll-like receptor 2, but not Toll-like receptor 4, is induced by LPS and inflammatory cytokines in mouse macrophages. J. Immunol. 165, 5767–5772. Takeda, K., Akira, S., 2001. Roles of Toll-like receptors in innate immune responses. Genes Cells 6, 733–742. Rouzic, V.L., Wiedinger, K., Zhou, H., 2012. Attenuated mRNA expression of inflammatory mediators in neonatal rat lung following lipopolysaccharide treatment. J. Inflamm. Res. 5, 99–109. Walker, C.D., Perrin, M., Vale, W., Rivier, C., 1986. Ontogeny of the stress response in the rat: role of the pituitary and the hypothalamus. Endocrinology 118, 1445–1451. Widmaier, E.P., 1989. Development in rats of the brain–pituitary–adrenal response to hypoglycemia in vivo and in vitro. Am. J. Physiol. 257, E757–E763. Widmaier, E.P., 1990. Change in responsiveness of the hypothalamicpituitaryadrenocortical axis to 2-deoxy-d-glucose in developing rats. Endocrinology 126, 3116–3123. Wisse, B.E., Ogimoto, K., Tang, J., Harris Jr., M.K., Raines, E.W., Schwartz, M.W., 2007. Evidence that lipopolysaccharide-induced anorexia depends upon central, rather than peripheral, inflammatory signals. Endocrinology 148, 5230–5237. Witek-Janusek, L., 1988. Pituitary–adrenal response to bacterial endotoxin in developing rats. Am. J. Physiol. 255, E525–E530. Yang, Q., Zhu, P., Wang, Z., Jiang, J., 2002. Lipopolysaccharide upregulates the expression of Toll-like receptor 4 in human vascular endothelial cells. Chin. Med. J. 115, 286–289.

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