AMELIORATION OF SEPSIS BY MACROPHAGE ACTIVATION

在上一个资助期间，我们的研究集中在了解早期细胞内信号传导事件，这些事件参与了脓毒症综合征的发生和传播。我们发现在脓毒症早期抑制促炎细胞内信号传导与改善预后密切相关。具体而言，我们研究了葡聚糖配体在多微生物脓毒症中的保护功效，并确定葡聚糖的受体识别调节细胞内信号传导途径，使得对脓毒症的炎症反应“钝化”。我们确定葡聚糖预处理或后处理将减弱脓毒症诱导的组织NF κ B和NF-IL 6活化以及降低促炎细胞因子基因转录。抑制转录因子活性和细胞因子基因表达的早期增加与发病率和死亡率的降低密切相关。我们确定葡聚糖处理通过降低MEKK 1、NIK和IKK α/β激酶活性以及降低Ikappa-B α磷酸化和降解来减弱LPS诱导的NF κ B活性。我们证实了巨噬细胞上存在多个葡聚糖结合位点，确定了葡聚糖的分子结构，并证实了七糖（7个葡萄糖亚基）聚合物是最小的结合单位。初步数据表明，Toll受体（TLR）2，可能还有CR 3，赋予葡聚糖反应性。该延续提案的假设是，卢坎配体通过与Toll样受体（TLR）和/或CR 3（CD 11 b/CD 18）结合位点相互作用调节炎症反应，从而改善多微生物脓毒症的脓毒症后遗症。有四个具体目标。1.我们将使用表面等离子体共振方法表征葡聚糖配体与TLR 1、2和4、3型补体受体（CR 3）和CD 14的受体介导的相互作用。2.我们将通过研究TLR 2和TLR 4基因敲除小鼠的CLP脓毒症来确定TLR 2和TLR 4在葡聚糖抗脓毒症作用中的作用。3.我们将通过研究CR 3基因敲除小鼠的CLP脓毒症来研究CR 3（CD 11b/CD 18）在葡聚糖抗脓毒症作用中的作用。4.在上一个资助期间，我们在理解（1-3）-β-D-葡聚糖的基本化学方面取得了很大进展。利用这些知识，我们将合成化学纯的、高度均匀的、水溶性的（1-3）-β-D-葡聚糖配体，这些配体具有特定的结构特征。我们将制备一个小分子量（1-3）-β-D-葡聚糖库，用于制备更大的聚合物。将使用CLP模型在受体结合研究、体外细胞内信号传导研究和体内保护研究中评价合成聚合物。这些目标的成功完成将促进我们对脓毒症综合征相关的细胞和分子事件的理解，并可能导致新疗法的开发。

During the previous grant period our research focused on understanding the early intracellular signaling events which are involved in the initiation and propagation of sepsis syndrome. We found that suppressing pro-inflammatory intracellular signaling events during the early phase of sepsis strongly correlated with improved outcome. Specifically, we investigated the protective efficacy of glucan ligands in polymicrobial sepsis and determined that receptor recognition of glucan modulates intracellular signaling pathways such that the inflammatory response to sepsis is "blunted". We established that glucan pre- or post-treatment would blunt sepsis induced tissue NFkappaB and NF-IL6 activation as well as decrease pro-inflammatory cytokine gene transcription. Blunting early increases in transcription factor activity and cytokine gene expression strongly correlated with decreased morbidity and mortality. We determined that glucan treatment blunted LPS induced NFkappaB activity through decreased MEKK1, NIK and IKKalpha/beta kinase activity as well as decreased Ikappa-Balpha phosphorylation and degradation. We confirmed the existence of multiple glucan binding sites on macrophages, defined the molecular structure of a glucan and confirmed that a heptaose (7 glucose subunit) polymer was the minimum binding unit. Preliminary data suggest that Toll receptor (TLR) 2, and perhaps CR3, confers responsiveness to glucan. The hypothesis for this continuation proposal is that lucan ligands ameliorate septic sequelae in polymicrobial sepsis by modulating inflammatory responses via interactions with Toll-like receptors (TLR) and/or CR3 (CD11b/CD18) binding sites. There are four specific aims. 1. We will characterize the receptor mediated interaction of glucan ligands with TLR 1, 2 and 4, the type 3 complement receptor (CR3) and CD 14 using a surface plasmon resonance approach. 2. We will establish the role of TLR2 and TLR4 in the anti-sepsis effect of glucan by studying CLP sepsis in TLR2 and TLR4 knockout mice. 3. We will investigate the role of CR3 (CD11b/CD18) in the anti-sepsis effect of glucan by studying CLP sepsis in CR3 knockout mice. 4. During the last grant period we made great strides in understanding the basic chemistry of (1-3)-beta-D-glucans. Using this knowledge, we will synthesize chemically pure, highly uniform, water soluble (1-3)-beta-D-glucan ligands which have specific structural characteristics. We will prepare a library of small molecular weight (1-3)-beta-D-glucans which will be used to prepare larger polymers. The synthetic polymers will be evaluated in receptor binding studies, in vitro intracellular signaling studies and in vivo protection studies using the CLP model. The successful completion of these aims will advance our understanding of the cellular and molecular events associated with sepsis syndrome and may lead to the development of new therapeutics.