LRRFIP2和CD11b负向调控免疫应答及其作用机制研究

发布时间：2018-09-16 19:49

【摘要】：无论是在天然免疫还是适应性免疫中,对于负向调控分子和负向调控机制的研究是近年来免疫学研究领域的前沿热点,通过开展这方面的研究既能够更好地认识免疫系统的作用方式,也能够为相关疾病的治疗提供新的思路。我们前期的研究表明,LRRFIP2与CDllb对于免疫应答具有负向调控作用,本课题就这两个分子发挥各自免疫应答反应的调控功能与相应的作用机制开展了研究。第一部分LRRFIP2负向调控NLRP3炎性复合体的活化及其分子机制研究炎性细胞因子IL-1β在天然免疫中发挥着重要的作用,它的产生过程中既需要编码蛋白的基因转录表达产生前体,也需要一类叫做炎性复合体(inflammasome)的分子复合物将它剪切成为成熟体。炎性复合体种类有很多,目前研究最为集中的是一类叫做NLRP3的炎性复合体(NLRP3inflammasome),它是由模式识别受体NLRP3与接头分子ASC以及效应分子Caspase-1所构成的,在一些病原体相关分子或危险信号相关分子的刺激下三者形成复合体并活化Caspase-1,使其行使剪切IL-1β的功能。为了进一步研究NLRP3炎性复合体的活化与条件过程,我们利用免疫共沉淀技术与液相色谱联用质谱技术,寻找和NLRP3炎性复合体相互作用的蛋白,并找到了LRRFIP2分子在LPS+ATP刺激后能够与NLRP3炎性复合体的接头蛋白ASC结合。在本研究中,我们发现在小鼠腹腔巨噬细胞中用siRNA干扰LRRFIP2表达后,NLRP3炎性复合体相关刺激引起的细胞分泌IL-1β会增加,而且这种增加是由于NLRP3炎性复合体活化水平升高导致IL-1p剪切加快所引起的,而对IL-1p前体合成过程没有影响。通过免疫共沉淀实验我们发现LRRFIP2能够在活化NLRP3炎性复合体的刺激后通过其N端氨基酸序列和NLRP3结合,进而和整个NLRP3炎性复合体相互作用。在THP-1中过表达LRRFIP2的实验表明,其N端序列和中部的Coil motif对其负向调控NLRP3炎性复合体活化是必须的。接着我们用免疫共沉淀实验证实LRRFIP2能够通过Coil motif与Flightless Ⅰ分子结合。进一步研究Flightless Ⅰ在炎性复合体中的功能,我们发现用siRNA干扰小鼠腹腔巨噬细胞中的Flightless Ⅰ后,活化炎性复合体的刺激引起的IL-1β的分泌水平与Capase-1的活化水平都升高。通过干扰与免疫共沉淀实验,我们发现LRRFIP2能够在NLRP3炎性复合体活化之后促进Flightless Ⅰ与NLRP3炎性复合体的结合并促进其发挥抑制炎性复合体活性的功能。最后,当我们在THP-1细胞中干扰掉FlightlessI的表达之后,再过表达LRRFIP2就不能抑制NLRP3炎性复合体的活性,证明了LRRFIP2发挥抑制功能需要依赖于Flightless Ⅰ。综上所述,本研究结果提示,LRRFIP2是NLRP3炎性复合体的一个负向调控分子,能够通过N端与NLRP3相互作用并促进Flightless Ⅰ与NLRP3炎性复合体的结合来抑制NLRP3炎性复合体的活化以及IL-1β的剪切成熟。第二部分整合素cD11b通过促进巨噬细胞产生IL-10减轻炎症反应及其机制的研究整合素在炎症反应与淋巴细胞迁移方面有重要作用,作为高表达于单核巨噬细胞上的整合素的α亚基CD11b在调控免疫反应方面的功能研究还不是很深入。在本实验室之前的工作中我们发现整合素CD11b能够通过活化Syk引起TRIF和MyD88的降解来负向调控Toll样受体(TLR)信号通路活化引起的炎症反应。白细胞介素10(IL-10)是天然免疫与适应性免疫中发挥重要作用的调节性细胞因子,它能够在TLR通路活化后上调表达从而参与炎症反应的调控过程,关于CD11b与TLR活化诱导的IL-10上调表达之间的关系还没有被报道过。在本研究中我们首先发现CD11b缺陷小鼠的腹腔巨噬细胞在Toll样受体3,4,9(TLR3,4,9)的配体Poly(I:C), LPS和CpG-ODN刺激下IL-10的表达上升明显受到抑制。进一步利用抑制剂与过表达实验发现CDllb主要通过磷酸化巨噬细胞中的酪氨酸激酶Src从而活化PI3K/Akt通路来来增强TLR活化引起的IL-10上调表达。Src活化后,能够与P13K的p85亚基结合并使其磷酸化并促进E3泛素连接酶c-Cb1介导的p85的降解,从而引起PI3K/Akt信号通路的活化。PI3K/Akt的活化能够引起下游分子GSK3a/β的磷酸化与活性的抑制,从而解除其对转录因子CREB转录活性的抑制功能,进而促进IL-10的转录表达。同时我们发现在DSS诱导的肠炎模型中,CD11b缺陷小鼠有更严重的炎症反应,同时血清中IL-10的含量以及肠系膜淋巴结与脾脏中CD4+Foxp3+的调节性T细胞比例低于同窝对照。总而言之,在通过本研究,我们发现了巨噬细胞上的CDllb对TLR活化引起的IL-10表达上调有促进作用,而这种促进作用是通过CDllb活化其下游激酶Src经由PI3K/Akt通路以及其下游的GSKα/β的一系列信号传递来实现的,另外我们还发现在小鼠DSS诱导的炎性肠病中,CDllb缺陷小鼠炎性肠病更加严重,同时血清中IL-10的表达更低,肠系膜淋巴结与脾脏中调节性T细胞数量也更少。而这一现象很可能是由于巨噬细胞IL-10产生减少导致调节性T细胞数量无法维持,而缺少了调节型T细胞对免疫反应的调控,使得肠炎无法得到有效控制。本研究为整合素参与细胞因子的表达调控提出了新的机制解释,也为控制炎性肠病提供了新的思路。
[Abstract]:In recent years, the study of negative regulatory molecules and negative regulatory mechanisms has become a hot spot in the field of immunology, whether in natural or adaptive immunity. Through this study, we can not only better understand the mode of action of the immune system, but also provide new ideas for the treatment of related diseases. The results showed that LRRFIP2 and CDllb have negative effects on the immune response. In this paper, we studied the regulation function and the corresponding mechanism of these two molecules in the immune response.
Part one activation and molecular mechanism of LRRFIP2 negative regulation of NLRP3 inflammatory complex
Inflammatory cytokine IL-1beta plays an important role in innate immunity. It requires not only the transcription and expression of genes encoding proteins to produce precursors, but also a class of molecular complexes called inflammasomes to cut it into mature bodies. Inflammatory complex NLRP3 (NLRP3 inflammasome) consists of a pattern recognition receptor NLRP3 (NLRP3) with the junction molecule ASC and the effector molecule Caspase-1, which form complex and activate Caspase-1 under the stimulation of some pathogen-related molecules or risk signal-related molecules to perform the function of shearing IL-1 beta. To further study the activation and conditioning process of NLRP3 inflammatory complex, we used immunoprecipitation and liquid chromatography-mass spectrometry to find the proteins interacting with NLRP3 inflammatory complex, and found that LRRFIP2 could bind to ASC of NLRP3 inflammatory complex after LPS + ATP stimulation. It was found that the expression of LRRFIP2 was interfered by siRNA in mouse peritoneal macrophages, and the secretion of IL-1beta was increased by NLRP3 inflammatory complex-related stimuli. The increase was due to the increased activation level of NLRP3 inflammatory complex, which resulted in the acceleration of IL-1p shearing, but had no effect on the synthesis of IL-1p precursor. We found that LRRFIP2 could bind to NLRP3 through its N-terminal amino acid sequence after stimulation of NLRP3 inflammatory complex, and then interact with the whole NLRP3 inflammatory complex. Overexpression of LRRFIP2 in THP-1 showed that N-terminal sequence and central Coil motif were necessary to negatively regulate the activation of NLRP3 inflammatory complex. Then we used immunoprecipitation assay to confirm that LRRFIP2 could bind to Flightless I via Coil motif. To further study the function of Flightless I in inflammatory complex, we found that siRNA interfered with the secretion of IL-1 beta induced by stimulation of inflammatory complex in mouse peritoneal macrophages. Through interference and immunoprecipitation assay, we found that LRRFIP2 could promote the binding of Flightless I to the NLRP3 inflammatory complex after activation of the NLRP3 inflammatory complex and promote its function of inhibiting the activity of the inflammatory complex. Overexpression of LRRFIP2 does not inhibit the activity of NLRP3 inflammatory complex, which proves that LRRFIP2 is dependent on FlightlessI. In conclusion, our results suggest that LRRFIP2 is a negative regulator of NLRP3 inflammatory complex and can interact with NLRP3 through N-terminal and promote FlightlessI and NLRP3. The binding of the inflammatory complex inhibits the activation of NLRP3 inflammatory complex and the shear ripening of IL-1 beta.
The second part of integrin cD11b reduces inflammation by promoting macrophages to produce IL-10 and its mechanism.
Integrin plays an important role in inflammatory response and lymphocyte migration. The function of CD11b, an alpha subunit of integrin highly expressed on monocytes and macrophages, in regulating immune response is not well studied. Previous work in this laboratory has shown that integrin CD11b can induce the decrease of TRIF and MyD88 by activating Syk. Interleukin-10 (IL-10) is a regulatory cytokine that plays an important role in innate and adaptive immunity. It can up-regulate the expression of TLR after activation of TLR pathway and participate in the regulation of inflammation. About IL-10 induced by CD11b and TLR activation In this study, we first found that the expression of IL-10 was significantly inhibited by Toll-like receptor 3,4,9 (TLR3,4,9) ligands Poly (I:C), LPS and CPG-ODN in CD11b-deficient mice. Phosphorylation of tyrosine kinase Src in macrophages activates PI3K/Akt pathway to enhance IL-10 up-regulation induced by TLR activation. Activation of Src can bind and phosphorylate P13K p85 subunit and promote the degradation of p85 mediated by E3 ubiquitin ligase c-Cb1. Activation of PI3K/Akt signaling pathway can be induced. Inhibiting the phosphorylation and activity of downstream GSK3a/beta eliminates its inhibitory effect on transcription of transcription factor CREB and promotes the transcriptional expression of IL-10. We also found that CD11b-deficient mice had more severe inflammation, serum IL-10 levels and mesenteric lymph nodes in a DSS-induced enteritis model. In conclusion, we found that CDllb on macrophages can promote the up-regulation of IL-10 expression induced by TLR activation, and this effect is mediated by CDllb activating its downstream kinase Src via PI3K/Akt pathway and its downstream GSKa/beta. In addition, we also found that CDllb deficient mice had more severe inflammatory bowel disease, lower levels of IL-10 in serum, and fewer regulatory T cells in mesenteric lymph nodes and spleens in DSS-induced inflammatory bowel disease. This study provides a new mechanism for integrin to participate in the regulation of cytokine expression and provides a new idea for the control of inflammatory bowel disease.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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