Role Of Monocytes In Immunopathology

单核细胞在免疫病理学中的作用

• 批准号: 8148618
• 负责人: LARRY M WAHL
• 金额: $ 87.68万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148618
• 关键词: Role; immunopathology; monocyte

Research in the Immunopathology Section focuses on the biological mediators and signal transduction pathways involved in the modulation of human monocyte and lymphocyte functions that may contribute to the immunopathology associated with various inflammatory lesions. Monocytes/macrophages are prominent in many inflammatory diseases, such as periodontal disease, rheumatiod arthritis, atherosclerosis, and cancer. The pathology associated with these diseases involves alterations in the integrity of the connective tissue framework implicating a role for matrix metalloproteinases (MMPs). MMPs are comprised of a family of zinc dependent endopeptidases divided into major subgroups that include the interstitial collagenases, the gelatinases, stromelysins, membrane type MMPs and others. Collectively these enzymes are capable of degrading all the extracellular matrix components. Because MMPs and tissue inhibitors of MMPs (TIMPs) are believed to play a major role in the destruction and remodeling of connective tissue, a major emphasis has been placed on how these enzymes and inhibitors are regulated in the human monocyte/macrophage as well as in the reciprocal interaction between monocytes/macrophages and tumor cells. Interaction between monocytes/macrophages and human oral squamous carcinoma cells (HNSCC) in the production and activation of MMPs The stromal cells surrounding cancer sites are thought to play a major role in the degree of tumor progression. A significant portion of these stromal cells are comprised of monocytes and macrophages that have been polarized by the specific cytokines present at the site into subtypes known as M1 and M2 macrophages. The MMPs produced by these subtypes of macrophages may be important determinates in the extent of cancer metastasis through the digestion of specific extracellular matrix surrounding the cancer site. Our previous qPCR studies have identified the differential expression levels of the various members of the MMP family and TIMPs by monocytes, macrophages and their subtypes in the presence or absence of specific cytokines and endotoxin. In addition to the levels of MMPs produced by stromal cells, HNSCCs also produce MMPs and TIMPs the levels of which tend to vary depending on the specific HNSCCs. To examine the base line levels and variability of MMPs and TIMPs of HNSCCs we examined five HNSCC lines. We found considerable differences in the expression profile between these HNSCC lines. For example NH12 and OSCC3 cell lines express very low to undetectable levels of MMP-1, -2, -3, -10 and -11 compared to HN30, Cal27 and HN4 cell lines which express high levels of these MMPs. Our next step in this study is to determine the effect of co-cultures of human oral squamous carcinoma cells (HNSCC) on the expression MMPs and TIMPs by monocytes and macrophages. HNSCCs and monocytes/macrophages are being cultured in wells containing inserts to separate the populations. Expression levels of MMPs and TIMPs in the individual populations are being determined by qPCR. We are addressing the following questions in this study. Do HNSCCs instruct stromal cells to express MMPs and TIMPs? Are those HNSCC lines that produce low levels of specific MMPs compensating by inducing the production of those MMPs by monocytes/macrophages? Conversely, do monocytes/macrophages induce HNSCCs to produce MMPs that are normally expressed at low or non-detectable levels? Are monocyte/macrophages involved in the activation of pro-MMPs produced by HNSCCs? Regulation of monocyte/macrophage MMP production by inhibitors used to target signaling pathways in tumor cells. The PI3K/AKT/mTOR pathway is major signal transduction pathway involved in tumor progression and considerable effort has been aimed at designing pharmacological inhibitors to interrupt this pathway. In addition to modulating tumor cell signaling these agents also have a potential impact on signaling in stromal cells and their regulation of connective tissue turnover. Our recent studies have targeted the role of AKT in the regulation of MMP production by monocytes. Stimulation of monocytes with LPS results in the activation of AKT as demonstrated by the phosphorylation of Ser 473 and Thr 308. We have shown that monocytes in which AKT has been inhibited have enhanced expression and production of MMP-1. This is in contrast to previous findings in which we demonstrated that MMP-9 production was inhibited when AKT activation was blocked. Examination of the mechanism involved in enhancement of monocyte MMP-1 production as a result of inhibition of AKT revealed that it is unrelated to downstream activation of mTOR since rapamycin failed in increase MMP-1 in activated monocytes. However, inhibition of AKT resulted in an increase in the activation of p38 MAPK and a decrease in ERK1/2 MAPK phosphorylation. Based on previous findings from our laboratory this may explain, in part, the differential regulation of MMP-1 and -9. We are currently focusing on glycogen synthase kinase -3 (GSK-3β), a downstream target of AKT that is inactivated when phosphorylated (pS9GSK-3β) by AKT. pS9GSK-3β is known to exert both pro- and anti-inflammatory effects through its interaction with transcription factors. We are currently examining the role of GSK-3β in differential regulation of MMPs through the use of specific inhibitors. Involvement of TLR2 and TLR4 in cell responses to Rickettsia akari In an ongoing collaboration with investigators at the University of Maryland we have been examining the role of TLRs in endotoxin signaling which leads to endotoxin tolerance. TLRs are primary sensors of microbial pathogens that activate innate immune cells, as well as initiate and orchestrate adaptive immune responses. Our recent studies have focused on the role of TLR2 and TLR4 in the recognition of Rickettsia akari, a causative agent of rickettsialpox. The findings in this paper demonstrate that TLR2 and TLR4 are innate immune sensors closely involved in recognition of heat killed (HK) and live R. akari. This conclusion is supported by several lines of evidence. These include enabling cell responsiveness to HK R. akari upon complementation of TLR2/4-negative HEK293T cells with TLR2 or TLR4, compromised ability of mutants TLR2 and TLR4 to mediate NF-κB activation in response to HK R. akari, and inhibition of HK R. akari-elicited TNF-α secretion in human monocytes treated with blocking TLR2 or TLR4 Ab. In contrast to HK R. akari using only TLR2 and TLR4, infection with live R. akari activated IRAK1 and p38 MAPK in TLR2/4-negative HEK293T cells and showed lower TLR2/4 dependence in human monocytes and mouse macrophages, indicating the use of TLR2, TLR4, and other TLRs and/or NLRs for recognition. Experiments are ongoing to identify putative TLR2 and TLR4 agonists expressed by R. akari and to delineate innate immune sensors involved in the recognition of live bacterium.

免疫病理学部分的研究重点关注于调节人单核细胞和淋巴细胞功能的生物学介质和信号转导途径，这些途径可能有助于与各种炎症性病变相关的免疫病理学。单核细胞/巨噬细胞在许多炎症性疾病中很突出，例如牙周疾病，风湿性关节炎，动脉粥样硬化和癌症。 与这些疾病相关的病理涉及结缔组织框架完整性的改变，这暗示了基质金属蛋白酶（MMP）的作用。 MMP由锌依赖性内肽酶家族组成，分为主要亚组，其中包括间质胶原酶，明胶酶，曲折蛋白，膜型MMP等。这些酶能够降解所有细胞外基质成分。由于据信MMP（TIMP）的MMP和组织抑制剂在结缔组织的破坏和重塑中起主要作用，因此对这些酶和抑制剂在人类单核细胞/巨噬细胞中如何调节这些酶和抑制剂的主要重点是如何调节的。 单核细胞/巨噬细胞与人口腔鳞状癌细胞（HNSCC）之间的相互作用在MMP的生产和激活中 据认为，周围癌症部位的基质细胞在肿瘤进展程度中起主要作用。 这些基质细胞的很大一部分由单核细胞和巨噬细胞组成，这些单核细胞和巨噬细胞已被位点上的特定细胞因子偏振成亚型，称为M1和M2巨噬细胞。 这些巨噬细胞亚型产生的MMP在癌症转移的程度上通过消化围绕癌症部位的特定细胞外基质而言是重要的决定。 我们以前的QPCR研究已经通过单核细胞，巨噬细胞及其亚型在存在或不存在特定细胞因子和内毒素的情况下通过单核细胞，巨噬细胞及其亚型确定了MMP家族和TIMP的差异表达水平。 除了基质细胞产生的MMP水平外，HNSCC还产生MMP和TIMP，其水平倾向于根据特定的HNSCCS变化。 为了检查HNSCC的MMP和TIMP的基本线水平和可变性，我们检查了五个HNSCC线。 我们发现这些HNSCC线之间的表达曲线有很大差异。 例如，与HN30，Cal27和HN4细胞系相比，NH12和OSCC3细胞系的MMP -1，-2，-3，-10和-11表达的MMP -1，-2，-3，-10和-11水平很低，这些细胞表达了这些MMP的高水平。 这项研究的下一步是确定人口腔鳞状癌细胞（HNSCC）对单核细胞和巨噬细胞表达MMP和TIMP的共同培养的影响。 HNSCC和单核细胞/巨噬细胞正在包含插入物的井中培养以分离人群。 通过qPCR确定了个体种群中MMP和TIMP的表达水平。 我们正在解决本研究中的以下问题。 HNSCC是否指示基质细胞表达MMP和TIMP？那些通过单核细胞/巨噬细胞诱导这些MMP的产生而产生低水平的特定MMP的HNSCC系列？相反，单核细胞/巨噬细胞是否会诱导HNSCC产生通常在低或不可检测水平上表达的MMP？单核细胞/巨噬细胞是否参与HNSCCS产生的Pro-MMP的激活？ 通过用于靶向肿瘤细胞信号通路的抑制剂对单核细胞/巨噬细胞MMP的调节。 PI3K/AKT/MTOR途径是参与肿瘤进展的主要信号转导途径，旨在设计药理抑制剂以中断该途径。 除了调节肿瘤细胞信号外，这些药物还可能对基质细胞的信号传导及其调节结缔组织更新。 我们最近的研究针对AKT在单核细胞调节MMP生产中的作用。 用LPS刺激单核细胞会导致AKT的激活，如Ser 473和Thr 308的磷酸化所证明。我们已经表明，抑制AKT的单核细胞增强了MMP-1的表达和产生。 这与以前的发现相反，在这种发现中，我们证明当Akt激活被阻断时，MMP-9的产生受到抑制。 检查因AKT抑制而涉及增强单核细胞MMP-1产生的机制表明，它与MTOR的下游激活无关，因为雷帕霉素在活化的单核细胞中增加了MMP-1。 然而，对AKT的抑制作用导致p38 MAPK的激活增加，而ERK1/2 MAPK磷酸化的降低。 根据我们实验室的先前发现，这可能部分解释了MMP -1和-9的差异调节。 我们目前正在关注糖原合酶激酶-3（GSK-3β），这是Akt的下游靶标，当Akt磷酸化（PS9GSK-3β）时，AKT被灭活。 已知PS9GSK-3β通过与转录因子的相互作用而发挥促炎和抗炎作用。 我们目前正在研究GSK-3β通过使用特定抑制剂在MMP的差异调节中的作用。 TLR2和TLR4参与细胞对立克Akari的反应 在与马里兰大学的研究人员进行的持续合作中，我们一直在研究TLR在内毒素信号传导中的作用，从而导致内毒素耐受性。 TLR是激活先天免疫细胞的微生物病原体的主要传感器，并启动和编排适应性免疫反应。我们最近的研究集中在TLR2和TLR4在Ricketsia Akari识别的识别中的作用，Rickettsialpox的病因剂。 本文中的发现表明，TLR2和TLR4是与生俱来的免疫传感器，与识别杀死的热量（HK）和Live R. Akari紧密相关。该结论得到了几条证据的支持。这些包括在与TLR2或TLR4的TLR2/4阴性HEK293T互补后，使细胞对HK R. Akari的反应能力，突变体TLR2和TLR4的能力损害了NF-κB激活HKR。用阻止TLR2或TLR4 AB。与仅使用TLR2和TLR4的HK R. Akari相反，在TLR2/4阴性HEK293T细胞中，Live R. Akari激活了Irak1和P38 MAPK的感染，显示了较低的TLR2/4在人类和小鼠巨噬细胞中的依赖性依赖于TLR2，并指示了TLR2和TLR4和其他tlr4和其他TLR4，以及其他tlr4，以及其他。正在进行实验，以识别R. akari表达的推定的TLR2和TLR4激动剂，并描述参与识别活细菌的先天免疫传感器。