TNF SIGNALING IN THE ABSENCE OF FUNCTIONAL TNF RECEPTORS.

描述(申请人提供)：我们早些时候报道了耐药的C57BL/6小鼠(B6.TNF-/-)的肿瘤坏死因子基因缺失使它们对致死性单纯疱疹病毒1型(HSV)感染易感。在进一步的研究中，我们发现，已知的肿瘤坏死因子受体TNFR1或P55和TNFR2或P75中的一个或两个的基因缺失的B6小鼠对致命性HSV感染具有抵抗力。这一令人惊讶的结果表明，肿瘤坏死因子介导的对HSV感染的抵抗是由一种不同于已知的肿瘤坏死因子p55(TNFR1)和p75(TNFR2)受体的受体介导的。我们证实了肿瘤坏死因子信号在小鼠中的保护作用，方法是用只结合肿瘤坏死因子而不结合淋巴毒素(LT-)的可溶性TNFR1制剂或针对肿瘤坏死因子的单抗(MAb)治疗单纯疱疹病毒感染的双基因敲除B6.p55-/-p75-/-(DKO)小鼠，使死亡率增加到相同程度，包括在野生型(Wt)对照B6小鼠中，这证实了肿瘤坏死因子信号在dKO小鼠中的保护作用。肿瘤坏死因子及其受体既以膜结合蛋白的形式存在，又以可溶性蛋白的形式存在，因此这些方法需要注意的是，我们不能排除sTNFR和/或抗肿瘤坏死因子单抗在某种程度上损害了耐药性而通过膜上的肿瘤坏死因子启动反向信号传递的可能性。因此，我们设计了一种体内siRNA方法，在不影响LT-的情况下特异性下调肿瘤坏死因子。在体内，siTNF治疗对HSV感染的dKO和B6wt小鼠的死亡率有相同程度的增加，而混杂的对照siTNF没有影响。因此，肿瘤坏死因子对dKO小鼠致死性单纯疱疹病毒感染的保护作用是通过一种新的肿瘤坏死因子受体介导的。为了支持这一假设，我们提供了大量的初步数据，证明了与Alexa 488或生物素(Alexa-TNF/Bio)结合的肿瘤坏死因子在dKO小鼠中与各种免疫细胞类型的特异性结合，其模式与wt和p55-/-B6小鼠不同。我们建议使用双功能交联剂与肿瘤坏死因子偶联，将新的肿瘤坏死因子受体(NTNFR)与dKO小鼠的腹腔巨噬细胞进行交联。纯化的肿瘤坏死因子-nTNFR复合体将被酶切，并将通过对得到的胰蛋白酶多肽进行LC-MS/MS分析来确定nTNFR的身份。NTNFR将从其表达模式、配体结合包括LT-a和原颗粒蛋白、新发现的p75(TNFR2)的配体、与p55和p75的结构关系、在人类中的保守性以及在宿主防御、炎症反应和自身免疫中的作用等方面进行表征。这些研究的结果将显著提高对在天然免疫、在各种自身免疫性疾病中发挥核心作用的肿瘤坏死因子信号以及在炎症调节中的理解。

DESCRIPTION (provided by applicant): We reported earlier that genetic deletion of TNF in resistant C57BL/6 mice (B6.TNF-/-) rendered them susceptible to fatal herpes simplex virus type 1 (HSV) infection. In further studies, we showed that B6 mice genetically deleted for either or both of the known TNF receptors TNFR1 or p55 and TNFR2, or p75 were resistant to fatal HSV infection. This surprising result suggested that TNF mediated resistance to HSV infection was mediated by a receptor distinct from the known TNF p55 (TNFR1) and p75 (TNFR2) receptors. We confirmed that TNF signaling mediated protection in mice by showing that Treatment of HSV infected double knockout B6.p55-/-p75-/- (dKO) mice with either a soluble TNFR1 preparation that binds only TNF and not lymphotoxin- (LT-)or a monoclonal antibody (mAb) targeting TNF increased mortality to the same extent, including in wild type (wt) control B6 mice which confirms the protective role of TNF signaling in dKO mice. Both TNF and its receptors exist as both membrane bound and as soluble proteins, hence a caveat with these approaches is that we could not exclude the possibility of reverse signaling through membrane TNF initiated by sTNFR and/or the anti-TNF mAb somehow impaired resistance. We therefore devised an in vivo siRNA approach to specifically down regulate TNF without affecting LT-. In vivo siTNF treatment increased mortality to the same extent in HSV infected dKO and B6 wt mice while the scrambled control siTNF had no effect. Thus, TNF protection against lethal HSV infection in dKO mice is mediated by signaling via a novel TNF receptor. In support of this hypothesis we present extensive preliminary data demonstrating specific binding of TNF conjugated to Alexa 488 or biotin (Alexa-TNF/Bio) to various immune cell types in dKO mice in a pattern distinct from that observed for wt and p55-/- B6 mice. We propose using a bifunctional crosslinker coupled to TNF to crosslink the new TNFR (nTNFR) on peritoneal macrophages from dKO mice. The purified TNF-nTNFR complex will be enzymatically fragmented and the identity of nTNFR will be established by liquid chromatography - tandem mass spectrometry (LC- MS/MS) analysis of the resulting tryptic peptides. nTNFR will characterized in term of its pattern of expression, ligand binding including LT-a and Progranulin, the newly identified ligand for p75 (TNFR2), structural relationship to p55 and p75, conservation in humans and role in host defense, inflammatory responses and autoimmunity. Results from these studies will significantly enhance understanding TNF signaling in the context of innate immunity, in various autoimmune diseases in which TNF plays a central role as well as in regulation of inflammation.