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

描述（由申请人提供）：有研究表明，一系列感染引起的母体免疫激活（MIA）可影响胎儿大脑发育，从而影响年轻和成年后代的行为。最近，Smith和他的同事（2007）报告说，母体血清中IL-6的增加在改变胎儿大脑发育和损害后代的社会互动行为方面起着关键作用，一直到成年。有趣的是，这些作用可以通过阻断IL-6，使用抗IL-6抗体和/或IL-6缺陷小鼠来减弱。我们的初步研究表明，柑橘类生物黄酮木犀草素可以抑制小鼠神经元样细胞（N2a）和原代培养的小鼠重组IL-6蛋白刺激的神经元细胞中JAK2/STAT3的磷酸化。为了验证这些发现，我们接下来研究了STAT3抑制剂（S31-201）和二硫米（一种结构类似木犀草素的类黄酮）在体内对JAK2/STAT3信号传导的影响。当这两种药物同时注射IL-6给孕鼠时，新生小鼠脑匀浆中JAK2/STAT3磷酸化和促炎细胞因子均显著降低。我们进一步发现，在饲料中口服地奥司明[10 mg/kg/天（NIH31对照饲料中0.005%地奥司明）]可显著改变IL-6/MIA成年后代的社会交往行为缺陷，并降低脑组织中的促炎细胞因子。我们还发现，地臭草明降低了中枢神经系统中与JAK2/STAT3信号通路抑制一致的促炎细胞因子水平。众所周知，改变胎儿大脑发育的风险与产前母体感染有关，特别是与中枢神经系统中细胞因子相关的炎症事件有关。推测地奥米明分子抑制JAK2/STAT3通路可能参与改善IL-6/MIA成年后代的异常社会互动。在本研究中，我们将研究JAK2/STAT3信号通路激活是否与il -6诱导的MIA的脑组织异常有关（可能导致成年后代出现模仿自闭症特征的异常行为）。此外，我们打算通过减弱JAK2/STAT3信号通路，充分表征和鉴定地臭虫明在改善IL-6/MIA后代成年期异常、自闭症样社会行为方面的潜在作用。这些研究可以为将来的孤独症临床试验奠定基础。

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.