Role of Neuroendocrine Stress Response in Inflammatory and Behavioral Illness.

神经内分泌免疫学和行为学部(SNIB)的研究集中在炎症、感染和情绪障碍中糖皮质激素抵抗的机制，使用细菌毒素抑制糖皮质激素受体(GR)和其他核激素受体(NHR)作为环境诱导的糖皮质激素抵抗研究的模型。我们最初发现炭疽杆菌致死毒素(LeTx)抑制GR和其他NHR，目前正在研究其他细菌毒素对GR和NHR的影响以及这种作用在炎症性休克中的作用(项目1)。在项目2中，我们正在研究孕酮对先天炎症细胞反应的影响，特别是树突状细胞，以确定另一种核激素受体-孕酮受体(PR)的状态是否有助于女性对这种情况的易感性。在一项临床研究(项目3)中，我们验证了一种测量正常对照组汗液中压力和免疫生物标记物的方法，目前正在将这种验证扩展到其他人群的临床研究，包括患有严重抑郁障碍(MDD)的女性。 在项目1中，我们正在扩大我们的研究，基于我们的初步发现，炭疽杆菌LeTx是一种有效的和选择性的核激素受体(NHR)抑制物，包括糖皮质激素和孕激素受体(GR和PR)。这些发现对炭疽、LeTx和其他细菌毒素的毒性和致死性的治疗和预防具有重要意义，也将有助于阐明环境诱导的糖皮质激素耐药的分子机制。我们目前的研究集中在(A)阐明这种效应的分子机制；(B)确定这种效应延伸到其他细菌毒素的程度；(C)确定这种体外效应的体内相关性；以及(D)临床和治疗意义。分子研究表明，细菌毒素对NHR的抑制既依赖于受体又依赖于启动子，与简单的GRE启动子相比，LeTx对复杂启动子(如MMTV启动子)的抑制作用更强。在我们最近的研究中，我们已经确定NHR的抑制作用延伸到其他细菌毒素，包括索氏梭菌致死毒素(TcsL)和艰难梭菌毒素A和B(TcdA和TcdB)。与LeTx一样，这种影响是高度敏感的(ng/ml浓度)，而且是非竞争性的。由于不影响p38MAPK途径的细菌毒素不表现出这种作用，我们推测p38在细菌毒素抑制NHR中发挥作用。此外，体外研究表明，TcsL以剂量依赖的方式阻止地塞米松抑制脾细胞产生TNF-α，提示TcsL的GR抑制作用可能与其预防糖皮质激素抗炎作用有关。目前的研究旨在进一步明确糖皮质激素抵抗系统中涉及的分子途径，以及确定体外效应是否与这些毒素的体内效应相关，并在体外和体内系统中测试潜在的治疗药物。在一项平行的人体组织研究中，我们也在评估糖皮质激素受体的多态性，我们和其他人已经证明，在各种临床条件下，糖皮质激素抵抗与糖皮质激素抵抗有关。 为了确定PR的细菌毒素抑制是否与炎症有关，项目2正在评估黄体酮对树突状细胞(DC)功能和成熟的影响。我们发现，孕酮在妊娠相关浓度下，通过PR介导的机制，抑制成熟DC产生促炎细胞因子、共刺激分子表达和这些细胞的T细胞增殖，但对未成熟DC抗原摄取几乎没有影响。这些效应可与糖皮质激素对DC功能的这些参数的影响相媲美，提示孕酮可能在女性激素周期的先天免疫和获得性免疫调节中发挥重要作用。目前的研究集中于确定这些体外研究结果是否在体内条件下被修改，例如在啮齿类动物的整个发情周期中，以及与已知孕酮敏感的器官(子宫)的DC相比，淋巴器官(骨髓、脾、淋巴结)中的DC对孕酮的影响是否存在差异。 在项目3中，我们验证了一种测量汗液中免疫和应激神经肽生物标记物的方法，该方法使用皮肤汗液贴片收集，使用循环免疫亲和层析进行检测，并与Terry Phillips，NIBIB，NIH合作，通过质谱仪和MALDI-TOF进行确认。目前的研究正在将这种方法应用于各种精神障碍的更多受试者，包括门诊环境中的MDD和强迫症。我们最近的发现(2007年在内分泌学会上发表的综述和摘要中的手稿)表明，患有MDD的女性在一系列促炎症细胞因子以及汗液和血浆中几个应激神经肽生物标志物的同步变化中显示出显著的升高。这些发现对包括骨质疏松症和心血管疾病在内的炎症相关疾病MDD发病率的增加具有重要意义。

The research of the Section on Neuroendocrine Immunology and Behavior (SNIB) focuses on mechanisms of glucocorticoid resistance in inflammatory, infectious and mood disorders, using repression of the glucocorticoid receptor (GR) and other nuclear hormone receptors (NHRs) by bacterial toxins as a model for study of environmentally induced glucocorticoid resistance. We initially showed that Bacillus anthracis (anthrax) lethal toxin (LeTx) repressed the GR and other NHRs and are currently studying the effects of other bacterial toxins on the GR and NHRs and the role of this effect in inflammatory shock (Project 1). In Project 2 we are studying the effects of progesterone on innate inflammatory cell responses, specifically dendritic cells, to determine whether the status of another nuclear hormone receptor, the progesterone receptor (PR) contributes to females susceptibility to such conditions. In a clinical study (Project 3) we have validated a method to measure stress and immune biomarkers in sweat in normal controls and are currently extending this validation to clinical studies in other populations, including women with Major Depressive Disorder (MDD). In Project 1, we are extending our research based on our initial finding that B. anthracis LeTx is a potent and selective repressor of nuclear hormone receptors (NHRs), including the glucocorticoid and progesterone receptors (GR and PR). These findings have important implications for treatment and prevention of toxicity and lethality from the anthrax LeTx and other bacterial toxins but also will help to elucidate molecular mechanisms of environmentally induced glucocorticoid resistance. Our current studies focus on (a) elucidation of the molecular mechanisms of this effect; (b) determination of the degree to which this effect extends to other bacterial toxins; (c) determination of the in vivo correlates of this in vitro effect; and (d) clinical and therapeutic implications. Molecular studies indicate that bacterial toxin repression of NHRs is both receptor and promoter dependent, with LeTx showing a greater repression of complex promoters (such as the MMTV promoter) compared to a simple GRE promoter. In our most recent studies we have determined that the NHR repressive effect extends to other bacterial toxins, including Clostridium sordellii lethal toxin (TcsL) and Clostridium difficile Toxins A and B (TcdA and TcdB). As with LeTx, this effect is highly sensitive (ng/ml concentrations) and non-competitive. Since bacterial toxins that do not affect the p38 MAP kinase pathway do not exhibit this effect, we postulate that p38 plays a role in bacterial toxin repression of NHRs. Furthermore, in vitro studies show that TcsL prevents dexamethasone suppression of TNF-alpha production by splenocytes in a dose-related manner, indicating that the GR repressive effect of TcsL may have functional relevance in preventing glucocorticoids anti-inflammatory effects. Current studies are aimed at further defining the molecular pathways involved in glucocorticoid resistance in this system, as well as at determining whether the in vitro effects are relevant to in vivo effects of these toxins, and testing potential therapeutic agents in both in vitro and in vivo systems. In a parallel human tissue study we are also evaluating glucocorticoid receptor polymorphisms, which we and others have shown are associated with glucocorticoid resistance in a variety of clinical conditions. In order to determine whether the bacterial toxin repression of PR might have functional relevance to inflammation, Project 2 is evaluating the effects of progesterone on dendritic cell (DC) function and maturation. We have found that progesterone, in pregnancy-associated concentrations and through a PR-mediated mechanism, suppresses mature DC production of pro-inflammatory cytokines, co-stimulatory molecule expression, and T cell proliferation by these cells but has little effect on immature DC antigen uptake. These effects, which are comparable to the effects of glucocorticoids on these parameters of DC function, suggest that progesterone may play an important role in regulation of innate and adaptive immunity during the hormonal cycle in females. Current studies are focusing on determining whether these in vitro findings are modified in such in vivo conditions, e.g. throughout the estrus cycle in rodents, and if there are differences in progesterone effects on DCs from lymphoid organs (bone marrow, spleen, lymph node) compared to DCs from organs with known progesterone sensitivity (uterus). In Project 3 we have validated a method for measuring immune and stress neuropeptide biomarkers in sweat, collected using skin sweat patches and assayed using recycling immunoaffinity chromatography and confirmed with mass spectrometry and MALDI-TOF, in collaboration with Terry Phillips, NIBIB, NIH. Current studies are applying this method to larger numbers of subjects in a variety of psychiatric disorders, including MDD and obsessive compulsive disorder in ambulatory settings. Our recent findings, (manuscript in review and abstract presented at the Endocrine Society, 2007) indicate that women with MDD show significant elevations in a battery of pro-inflammatory cytokines as well as concurrent changes in several stress neuropeptide biomarkers in sweat and plasma. These findings have important implications for the increased morbidity in MDD of inflammation-related illnesses, including osteoporosis and cardiovascular disease.