IL-6-mediated Jak2/Stat3 signaling and Brain Development

描述（由申请人提供）：已经表明，由一系列感染引起的母体免疫激活（MIA）可以影响胎儿大脑发育，从而影响年幼和成年后代的行为。最近，Smith及其同事（2007）报道，母体血清中IL-6的增加在改变胎儿大脑发育和损害后代社会交往行为方面起着关键作用，一直到成年。有趣的是，这些作用可以通过使用抗IL-6抗体和/或IL-6缺陷的小鼠阻断IL-6来减弱。我们的初步研究表明，柑橘黄酮，木犀草素，抑制神经元JAK 2/STAT 3磷酸化在小鼠神经元样（N2 a）细胞和原代培养的神经元细胞与小鼠重组IL-6蛋白的挑战。作为这些发现的验证，我们接下来检查了STAT 3抑制剂（S31-201）和地奥司明（一种结构上类似于毛地黄黄酮的类黄酮）对体内JAK 2/STAT 3信号传导的影响。当将任一种药物应用于注射IL-6的妊娠小鼠时，新生小鼠脑匀浆中JAK 2/STAT 3磷酸化和促炎细胞因子均显著降低。我们进一步表明，在食物中口服给予地奥司明[10 mg/kg/天（NIH 31对照食物中0.005%地奥司明）]显著改变了IL-6/MIA成年后代在社会交往中的行为缺陷，并减少了脑组织中的促炎细胞因子。我们还发现地奥司明降低了促炎细胞因子的CNS水平，这与JAK 2/STAT 3信号通路抑制一致。众所周知，改变胎儿脑发育的风险与产前母体感染有关，特别是与CNS中细胞因子相关的炎症事件有关。地奥司明对JAK 2/STAT 3通路的分子抑制可能参与改善IL-6/MIA成年后代的异常社会互动。 在这个提议中，我们将研究JAK 2/STAT 3信号通路激活是否可以特异性地参与IL-6诱导的MIA的脑组织学异常（可能导致成年后代出现模仿自闭症特征的异常行为）。此外，我们打算通过JAK 2/STAT 3信号通路的衰减，充分表征和鉴定地奥司明对改善IL-6/MIA后代成年期异常、自闭症样社会行为的潜在作用。这些研究可以为不久的将来使用地奥司明饮食补充剂进行自闭症临床试验奠定基础。 公共卫生相关性：有人认为，感染激活孕妇的免疫系统会影响发育中的胎儿的大脑，从而影响年轻和成年后代的行为。最近，据报道，母亲血液中增加的IL-6（免疫系统的化学信使）在改变胎儿大脑发育和损害后代社会互动行为方面起着关键作用。有趣的是，这些影响可以通过用抗体化学阻断IL-6或通过遗传改变小鼠使其缺乏IL-6来避免。我们的初步研究表明，天然柑橘类分子，毛地黄黄酮，抑制神经元中的信号传导机制（JAK 2/STAT 3），该机制将IL-6的信号传递到细胞核。这发生在神经元样（N2 a）细胞和从小鼠脑中取出并培养的神经元中，然后用小鼠IL-6蛋白激发。作为这些发现的验证，我们接下来检查了人工STAT 3抑制剂和地奥司明（一种结构上类似于木犀草素的天然柑橘分子）对小鼠JAK 2/STAT 3信号传导的影响。当将任一种药物应用于妊娠小鼠并注射IL-6以激活母亲的免疫系统时，新生小鼠脑中的JAK 2/STAT 3激活和炎性细胞因子均显著减少。我们进一步表明，地奥司明口服混合在小鼠食物中显著改善了社会交往中的行为问题，并减少了注射IL-6的母亲的后代脑组织中的炎性细胞因子。此外，地奥司明特异性地减少JAK 2/STAT 3信号传导触发的脑炎症细胞因子。据推测，地奥司明抑制JAK 2/STAT 3通路可能参与改善IL-6注射母亲后代的异常社会互动。在这项提案中，我们将研究JAK 2/STAT 3信号通路激活是否可能特异性地参与IL-6诱导的母体免疫系统激活中的脑结构异常（可能导致成年后代出现模仿自闭症特征的异常行为）。此外，我们打算通过用地奥司明阻断JAK 2/STAT 3信号通路来全面测试地奥司明对改善IL-6免疫激活母亲的后代的异常、自闭症样行为的潜在作用。这些研究可以为地奥司明作为高危母亲的产前补充剂奠定基础，以避免孩子患自闭症，就像叶酸目前用于避免神经管缺陷一样。

DESCRIPTION (provided by applicant): It has been suggested that maternal immune activation (MIA) by a range of infections can affect fetal brain development and thus behavior of young and adult offspring. Most recently, Smith and colleagues (2007) reported that increased IL-6 in the maternal serum plays a key role in altering fetal brain development and impairing behaviors in social interactions in the offspring, all the way into adulthood. Interestingly, these effects could be attenuated by blocking IL-6, using anti-IL-6 antibody and/or mice deficient in IL-6. Our preliminary studies showed that the citrus bioflavonoid, luteolin, inhibits neuronal JAK2/STAT3 phosphorylation in both murine neuron-like (N2a) cells and primary cultured neuronal cells challenged with mouse recombinant IL-6 protein. As a validation of these findings, we next examined the effects of a STAT3 inhibitor (S31-201) and diosmin, a flavanoid structurally similar to luteolin, on JAK2/STAT3 signaling in vivo. When either agent was applied to pregnant mice with an injection of IL-6, JAK2/STAT3 phosphorylation and pro-inflammatory cytokines were both significantly reduced in brain homogenates from newborn mice. We further showed that diosmin administered orally in chow [10 mg/kg/day (0.005% diosmin in NIH31 control chow)] significantly changes behavioral deficits in social interaction and reduces pro-inflammatory cytokines in brain tissues of IL-6/MIA adult offspring. We also found that diosmin reduces the CNS levels in pro- inflammatory cytokines consistent with JAK2/STAT3 signal pathway inhibition. It's well known that the risk of altering fetal brain development is associated with prenatal maternal infection, specifically, with cytokine- related inflammatory events in the CNS. Presumably, diosmin molecular inhibition of JAK2/STAT3 pathway could be involved in improving abnormal social interaction in IL-6/MIA adulthood offspring. In this proposal, we will examine whether or not JAK2/STAT3 signal pathway activation could be specifically involved in brain histological abnormalities in IL-6-induced MIA (presumably resulting in development of abnormal behavior in adult offspring that mimics features of autism). In addition, we intend to fully characterize and qualify diosmin's potential effect on improving abnormal, autistic like social behaviors in IL-6/MIA offspring in adulthood via its attenuation of the JAK2/STAT3 signal pathway. These studies could lay the foundation for autism clinical trials with diosmin diet supplementation in the near future. PUBLIC HEALTH RELEVANCE: It has been suggested that activation of pregnant mother's immune system by infections can affect the brain of the developing fetus and thus behavior of young and adult offspring. Most recently, it was reported that increased IL-6 (a chemical messenger of the immune system) in the mother's blood plays a key role in altering fetal brain development and impairing behaviors in social interactions in the offspring. Interestingly, these effects could be avoided by blocking IL-6 chemically with an antibody or by genetically altering mice so they are deficient in IL-6. Our preliminary studies showed that the natural citrus molecule, luteolin, inhibits the signaling mechanism (JAK2/STAT3) in neurons which carries IL-6's signal to the nucleus. This occurred in neuron-like (N2a) cells and neurons removed and cultured from mouse brains and then challenged with mouse IL-6 protein. As a validation of these findings, we next examined the effects of an artificial STAT3 inhibitor and diosmin, a structurally similar natural citrus molecule to luteolin, on JAK2/STAT3 signaling in mice. When either agent was applied to pregnant mice with an injection of IL-6 to activate the mother's immune system JAK2/STAT3 activation and inflammatory cytokines were both significantly reduced in the brains of the newborn mice. We further showed that diosmin given orally mixed in mouse food significantly improves behavioral problems in social interaction and reduces inflammatory cytokines in brain tissues of the offspring of the IL-6 injected mothers. Further, diosmin specifically reduces the brain inflammatory cytokines triggered by JAK2/STAT3 signaling. Presumably, diosmin inhibition of JAK2/STAT3 pathway could be involved in improving abnormal social interaction in offspring of IL-6 injected mothers. In this proposal, we will examine whether or not JAK2/STAT3 signal pathway activation could be specifically involved in brain structural abnormalities in IL-6-induced activation of the maternal immune system (presumably resulting in development of abnormal behavior in adult offspring that mimics features of autism). In addition, we intend to fully test diosmin's potential effect on improving abnormal, autistic like behaviors of offspring of IL-6 immune activated mothers by the JAK2/STAT3 signal pathway blocking with diosmin. These studies could lay the foundation for diosmin as a prenatal supplement in at-risk mothers to avoid autism development in their children, much like folate is currently in use to avoid neural tube defects.