【摘要】：無論是在天然免疫還是適應(yīng)性免疫中,對于負(fù)向調(diào)控分子和負(fù)向調(diào)控機(jī)制的研究是近年來免疫學(xué)研究領(lǐng)域的前沿?zé)狳c(diǎn),通過開展這方面的研究既能夠更好地認(rèn)識免疫系統(tǒng)的作用方式,也能夠?yàn)橄嚓P(guān)疾病的治療提供新的思路。我們前期的研究表明,LRRFIP2與CDllb對于免疫應(yīng)答具有負(fù)向調(diào)控作用,本課題就這兩個分子發(fā)揮各自免疫應(yīng)答反應(yīng)的調(diào)控功能與相應(yīng)的作用機(jī)制開展了研究。 第一部分LRRFIP2負(fù)向調(diào)控NLRP3炎性復(fù)合體的活化及其分子機(jī)制研究 炎性細(xì)胞因子IL-1β在天然免疫中發(fā)揮著重要的作用,它的產(chǎn)生過程中既需要編碼蛋白的基因轉(zhuǎn)錄表達(dá)產(chǎn)生前體,也需要一類叫做炎性復(fù)合體(inflammasome)的分子復(fù)合物將它剪切成為成熟體。炎性復(fù)合體種類有很多,目前研究最為集中的是一類叫做NLRP3的炎性復(fù)合體(NLRP3inflammasome),它是由模式識別受體NLRP3與接頭分子ASC以及效應(yīng)分子Caspase-1所構(gòu)成的,在一些病原體相關(guān)分子或危險信號相關(guān)分子的刺激下三者形成復(fù)合體并活化Caspase-1,使其行使剪切IL-1β的功能。為了進(jìn)一步研究NLRP3炎性復(fù)合體的活化與條件過程,我們利用免疫共沉淀技術(shù)與液相色譜聯(lián)用質(zhì)譜技術(shù),尋找和NLRP3炎性復(fù)合體相互作用的蛋白,并找到了LRRFIP2分子在LPS+ATP刺激后能夠與NLRP3炎性復(fù)合體的接頭蛋白ASC結(jié)合。在本研究中,我們發(fā)現(xiàn)在小鼠腹腔巨噬細(xì)胞中用siRNA干擾LRRFIP2表達(dá)后,NLRP3炎性復(fù)合體相關(guān)刺激引起的細(xì)胞分泌IL-1β會增加,而且這種增加是由于NLRP3炎性復(fù)合體活化水平升高導(dǎo)致IL-1p剪切加快所引起的,而對IL-1p前體合成過程沒有影響。通過免疫共沉淀實(shí)驗(yàn)我們發(fā)現(xiàn)LRRFIP2能夠在活化NLRP3炎性復(fù)合體的刺激后通過其N端氨基酸序列和NLRP3結(jié)合,進(jìn)而和整個NLRP3炎性復(fù)合體相互作用。在THP-1中過表達(dá)LRRFIP2的實(shí)驗(yàn)表明,其N端序列和中部的Coil motif對其負(fù)向調(diào)控NLRP3炎性復(fù)合體活化是必須的。接著我們用免疫共沉淀實(shí)驗(yàn)證實(shí)LRRFIP2能夠通過Coil motif與Flightless Ⅰ分子結(jié)合。進(jìn)一步研究Flightless Ⅰ在炎性復(fù)合體中的功能,我們發(fā)現(xiàn)用siRNA干擾小鼠腹腔巨噬細(xì)胞中的Flightless Ⅰ后,活化炎性復(fù)合體的刺激引起的IL-1β的分泌水平與Capase-1的活化水平都升高。通過干擾與免疫共沉淀實(shí)驗(yàn),我們發(fā)現(xiàn)LRRFIP2能夠在NLRP3炎性復(fù)合體活化之后促進(jìn)Flightless Ⅰ與NLRP3炎性復(fù)合體的結(jié)合并促進(jìn)其發(fā)揮抑制炎性復(fù)合體活性的功能。最后,當(dāng)我們在THP-1細(xì)胞中干擾掉FlightlessI的表達(dá)之后,再過表達(dá)LRRFIP2就不能抑制NLRP3炎性復(fù)合體的活性,證明了LRRFIP2發(fā)揮抑制功能需要依賴于Flightless Ⅰ。綜上所述,本研究結(jié)果提示,LRRFIP2是NLRP3炎性復(fù)合體的一個負(fù)向調(diào)控分子,能夠通過N端與NLRP3相互作用并促進(jìn)Flightless Ⅰ與NLRP3炎性復(fù)合體的結(jié)合來抑制NLRP3炎性復(fù)合體的活化以及IL-1β的剪切成熟。 第二部分整合素cD11b通過促進(jìn)巨噬細(xì)胞產(chǎn)生IL-10減輕炎癥反應(yīng)及其機(jī)制的研究 整合素在炎癥反應(yīng)與淋巴細(xì)胞遷移方面有重要作用,作為高表達(dá)于單核巨噬細(xì)胞上的整合素的α亞基CD11b在調(diào)控免疫反應(yīng)方面的功能研究還不是很深入。在本實(shí)驗(yàn)室之前的工作中我們發(fā)現(xiàn)整合素CD11b能夠通過活化Syk引起TRIF和MyD88的降解來負(fù)向調(diào)控Toll樣受體(TLR)信號通路活化引起的炎癥反應(yīng)。白細(xì)胞介素10(IL-10)是天然免疫與適應(yīng)性免疫中發(fā)揮重要作用的調(diào)節(jié)性細(xì)胞因子,它能夠在TLR通路活化后上調(diào)表達(dá)從而參與炎癥反應(yīng)的調(diào)控過程,關(guān)于CD11b與TLR活化誘導(dǎo)的IL-10上調(diào)表達(dá)之間的關(guān)系還沒有被報道過。在本研究中我們首先發(fā)現(xiàn)CD11b缺陷小鼠的腹腔巨噬細(xì)胞在Toll樣受體3,4,9(TLR3,4,9)的配體Poly(I:C), LPS和CpG-ODN刺激下IL-10的表達(dá)上升明顯受到抑制。進(jìn)一步利用抑制劑與過表達(dá)實(shí)驗(yàn)發(fā)現(xiàn)CDllb主要通過磷酸化巨噬細(xì)胞中的酪氨酸激酶Src從而活化PI3K/Akt通路來來增強(qiáng)TLR活化引起的IL-10上調(diào)表達(dá)。Src活化后,能夠與P13K的p85亞基結(jié)合并使其磷酸化并促進(jìn)E3泛素連接酶c-Cb1介導(dǎo)的p85的降解,從而引起PI3K/Akt信號通路的活化。PI3K/Akt的活化能夠引起下游分子GSK3a/β的磷酸化與活性的抑制,從而解除其對轉(zhuǎn)錄因子CREB轉(zhuǎn)錄活性的抑制功能,進(jìn)而促進(jìn)IL-10的轉(zhuǎn)錄表達(dá)。同時我們發(fā)現(xiàn)在DSS誘導(dǎo)的腸炎模型中,CD11b缺陷小鼠有更嚴(yán)重的炎癥反應(yīng),同時血清中IL-10的含量以及腸系膜淋巴結(jié)與脾臟中CD4+Foxp3+的調(diào)節(jié)性T細(xì)胞比例低于同窩對照�？偠灾�,在通過本研究,我們發(fā)現(xiàn)了巨噬細(xì)胞上的CDllb對TLR活化引起的IL-10表達(dá)上調(diào)有促進(jìn)作用,而這種促進(jìn)作用是通過CDllb活化其下游激酶Src經(jīng)由PI3K/Akt通路以及其下游的GSKα/β的一系列信號傳遞來實(shí)現(xiàn)的,另外我們還發(fā)現(xiàn)在小鼠DSS誘導(dǎo)的炎性腸病中,CDllb缺陷小鼠炎性腸病更加嚴(yán)重,同時血清中IL-10的表達(dá)更低,腸系膜淋巴結(jié)與脾臟中調(diào)節(jié)性T細(xì)胞數(shù)量也更少。而這一現(xiàn)象很可能是由于巨噬細(xì)胞IL-10產(chǎn)生減少導(dǎo)致調(diào)節(jié)性T細(xì)胞數(shù)量無法維持,而缺少了調(diào)節(jié)型T細(xì)胞對免疫反應(yīng)的調(diào)控,使得腸炎無法得到有效控制。本研究為整合素參與細(xì)胞因子的表達(dá)調(diào)控提出了新的機(jī)制解釋,也為控制炎性腸病提供了新的思路。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R392

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4 李s，

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