TNF SIGNALING IN THE ABSENCE OF FUNCTIONAL TNF RECEPTORS.

描述（由申请人提供）：我们先前报道了耐药C57 BL/6小鼠（B6.TNF-/-）中TNF的遗传缺失使其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，我们发现基因缺失已知TNF受体TNFR 1或p55和TNFR 2或p75中的一种或两种的B6小鼠对致命的HSV感染具有抗性。这一令人惊讶的结果表明，TNF介导的对HSV感染的抗性是由不同于已知TNF p55（TNFR 1）和p75（TNFR 2）受体的受体介导的。 我们证实了TNF信号介导的小鼠保护作用，通过显示用仅结合TNF而不结合LPS-（LT-）的可溶性TNFR 1制剂或靶向TNF的单克隆抗体（mAb）治疗HSV感染的双敲除B6.p55-/-p75-/-（dKO）小鼠增加了小鼠的死亡率。 相同程度，包括在野生型（wt）对照B6小鼠中，这证实了TNF信号传导在dKO小鼠中的保护作用。TNF及其受体以膜结合和可溶性蛋白的形式存在，因此这些方法的警告是，我们不能排除由sTNFR和/或抗TNF mAb以某种方式损害抗性引发的通过膜TNF的反向信号传导的可能性。因此，我们设计了一种体内siRNA方法来特异性下调TNF而不影响LT-。体内siTNF处理在HSV感染的dKO和B6 wt小鼠中以相同程度增加死亡率，而乱序对照siTNF没有影响。因此，TNF保护dKO小鼠免受致死性HSV感染是通过新型TNF受体的信号传导介导的。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）缀合的TNF与dKO小鼠中的各种免疫细胞类型的特异性结合，其模式与野生型和p55-/- B6小鼠中观察到的模式不同。 我们建议使用双功能交联剂耦合到TNF交联新的TNFR（nTNFR）从dKO小鼠腹腔巨噬细胞。将纯化的TNF-nTNFR复合物酶促片段化，并通过对所得胰蛋白酶肽进行液相色谱-串联质谱（LC-MS/MS）分析，确定nTNFR的身份。nTNFR将根据其表达模式、配体结合（包括LT-α和颗粒蛋白前体，新鉴定的p75的配体（TNFR 2））、与p55和p75的结构关系、在人体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来表征。这些研究的结果将显著增强对先天免疫背景下TNF信号传导的理解，在TNF发挥核心作用的各种自身免疫性疾病以及炎症调节中。

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.