跨膜型TNF-α抗体抵抗内毒素休克的作用及其机制的研究

Tumor necrosis factor-α exists transmembrane form (tmTNF-α) and secretory form (sTNF-α). It has been well known that sTNF-α, as a proinflammatory factor, plays an important role in endotoxin shock. Our previous work demonstrated that tmTNF-α acts as an anti-inflammatory factor and inhibition of ectdomain shedding of tmTNF-α by antisense oligonucleotides of TACE protects rats from endotoxin shock. Since TACE has a lot of substrates, inhibition of TACE may induce side effects. We made a monoclonal antibody specific to tmTNF-α which increases cell surface expression of tmTNF-α and decreases secretion of sTNF-α. Treatment with tmTNF-α antibody enhances survival rate of mice with endotoxin shock. Because LPS induces the production of a large number of inflammatory mediators through TLR4 signaling to participate in this pathological process, we hypothesized that tmTNF-α antibody may affect LPS/ TLR4 signaling and thus resist endotoxin shock by facilitating tmTNF-α expression. In this project, we shall investigate (1) the mechanism(s) by which tmTNF-α antibody works; (2) the effect of tmTNF-α antibody on LPS/ TLR4 signaling and its underlying mechanisms; (3) on which tmTNF-α signal pathways (forward or reverse signaling) tmTNF-α antibody acts to affect LPS/ TLR4 signaling. The results of this project will not only help to clarify the effects of tmTNF-α on endotoxin shock and underlying mechanisms, but also provide experimental evidences and a new clue to develop this antibody for the clinical treatment of this pathological process.

TNF-α有跨膜型（tmTNF-α）和分泌型(sTNF-α)。sTNF-α作为促炎细胞因子在内毒素休克中发挥重要作用，我们的前期工作证实tmTNF-α具有抑炎作用，抑制酶解tmTNF-α的TACE酶可保护大鼠抵抗内毒素休克。然而TACE底物众多，抑制之可能引起副作用。本室自制tmTNF-α单克隆抗体可促进tmTNF-α表达，抑制sTNF-α分泌，提高内毒素休克小鼠的存活率。由于LPS主要通过TLR4信号介导大量炎性介质产生而参与内毒素休克，我们推测此抗体可能通过影响LPS/TLR4信号通路发挥抵抗效应。本项目拟通过体内外实验明确此抗体作用的靶环节及其特征；探索其对LPS/TLR4信号的影响及分子机制；并阐明此抗体通过哪条tmTNF-α信号通路影响LPS/TLR4效应。本研究结果不但有利于阐明tmTNF-α在内毒素性休克中的作用及其机制，而且为开发该抗体的临床应用提供理论依据和实验证据。

本项目证实我们自主研发的tmTNF-α抗体通过与TACE竞争性结合tmTNF-α，抑制其酶解，促进tmTNF-α表达，抑制sTNF-α的分泌。tmTNF-α抗体通过这个作用在体外明显抑制单核／巨噬细胞对LPS的应答，抑制促炎因子IL-1β、IL-6、IFN-β和NO的产生；在体内对LPS或CLP诱导的内毒素休克小鼠具有保护作用，明显提高动物生存率，抑制上述促炎因子的产生，并发挥抗菌防御作用。机制研究揭示tmTNF-α抗体在LPS作用早期促进TLR4内化，在晚期促进Triad3A依赖的TLR4降解；此外，该抗体上调TLR4信号通路的负调分子A20和MCPIP1，进而促进TRAF6和TRAF3的脱泛素化与降解，使TLR4介导的MyD88与TRIF依赖的信号通路受到抑制。用外源性tmTNF-α证实其正向信号通过TNFR2抑制巨噬细胞对LPS的应答；此外，促进或抑制tmTNF-α反向信号可抑制或促进巨噬细胞对LPS的应答。本研究不但阐明了tmTNF-α在内毒素休克中的作用及其分子机制，而且证实tmTNF-α抗体具有抗内毒素休克作用，为进一步开发该抗体的临床应用提供了实验依据。