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不同来源腹腔巨噬细胞在子宫内膜异位症发病中的作用机制研究
中文摘要

研究背景: 子宫内膜异位症(Endometriosis,EM)在生育期妇女的发病率约为10~15%,且有逐年增高的趋势。主要症状包括不孕和各种盆腔内脏疼痛,严重影响患者的日常生活和工作,EM治疗后复发率高,给患者带来巨大的身心痛苦。然而EM的发病机理目前尚不明确,现有发病机制学说包括经血逆流种植学说、子宫内膜干细胞种植学说、体腔上皮化生学说和苗勒管残留学说等,目前学界广泛认可的为子宫内膜经血逆流种植学说,然而该学说无法解释以下两个主要现象:1)多达90%的女性存在经血逆流,但只有少部分女性患有EM;2)女性月经初潮后就开始出现子宫内膜碎片的逆流,但EM患者发病年龄主要集中在25~45岁,该学说无法解释在人体中子宫内膜逆流开始到发病之间延迟的10年。最新研究表明经血逆流可能只是诱因,而免疫功能异常在EM的种植、粘附及生长过程中发挥着重要作用。EM的发病机制研究仍停留在初级阶段,发病机制不明确导致缺乏针对病因的特异性治疗措施,在既有发病学说的基础上,进一步阐明EM的发病机制是首要任务。 巨噬细胞是固有免疫系统的重要组成部分,有抗原递呈、细胞吞噬以及合成释放细胞因子等多重功能,腹腔中的巨噬细胞在EM的发生和进展中发挥决定性作用。子宫内膜组织逆流进入腹腔后,腹腔巨噬细胞被激活并向异位组织聚集、浸润,活化的巨噬细胞分泌大量细胞因子参与形成异位病灶的新生血管和神经纤维。临床研究表明EM患者腹腔巨噬细胞数量、功能及其相关细胞因子(IL-1β, IL-6,IL-8,IL-10等)表达均存在异常。EM动物模型实验表明:1)药物剔除腹腔巨噬细胞可以抑制异位病灶的生长;2)使用LPS诱导腹腔巨噬细胞转化为M1型巨噬细胞可以抑制异位病灶的生长;3)利用IL-4诱导巨噬细胞向M2极化后则促进异位病灶的生长。因此,腹腔巨噬细胞的功能状态很可能决定EM的发生和发展过程。然而目前关于腹腔巨噬细胞在EM病程中的极化和功能状态的研究存在诸多矛盾:1)Bacci等在EM患者异位病灶和EM小鼠模型研究中发现巨噬细胞CD163和CD206表达量升高,提示巨噬细胞向M2转化。与前者相反, Takebayashi等通过免疫组织化学技术发现EM患者的CD206的表达量比对照组的子宫内膜组织低。2)在体外细胞实验中,用子宫内膜细胞的条件培养基刺激巨噬细胞(THP-1诱导来源)后,在巨噬细胞培养上清中检测到M1和M2代表性指标均有表达增加。类似前者,Beste等分离EM患者腹腔巨噬细胞进行分离纯化和体外培养,通过高通量多因子检测技术探宄其细胞因子的分泌模式,发现EM患者来源的腹腔巨噬细胞M1特异性细胞因子(IL-1β,IL-12)和M2特异性因子(IL-10)都表达升高。综上所述,腹腔巨噬细胞在EM发病中功能状态尚不明确、具体作用机制尚不清楚。 最新研究表明,巨噬细胞根据其来源不同可以划分为组织特异性原住巨噬细胞(如:大脑中的小胶质细胞、肝脏中的Kupffer细胞)和外周血单核细胞来源的巨噬细胞。两者不仅表型不同,而且功能存在显著差异,表现为不同来源的巨噬细胞亚群在接受相同的刺激因子作用后表现出不同的功能极化状态。腹腔中的巨噬细胞有两种来源:胚胎早期卵黄囊来源的腹腔大巨噬细胞(1arge peritoneal macrophage,LPM)和单核细胞来源的腹腔小巨噬细胞(small peritoneal macrophage,SPM),两者表型和功能属性不同。既往关于巨噬细胞在EM中的研究完全忽略并混淆了两种巨噬细胞的异质性,导致研究结论不一致。对巨噬细胞功能评价体系的不完善是导致研究结论不一致的另一个重要原因。巨噬细胞对其外界刺激因子反应敏感,被激活后能够发生大规模的转录调控变化,在既往研究中多将巨噬细胞的功能状态简单划分为M1型或者M2型巨噬细胞,但是巨噬细胞功能变化非常复杂,M1-M2极化是一个连续的功能变化过程,因此既往研究结果不能真实的反映EM发病过程中腹腔巨噬细胞的功能特点,需要采用高通量的实验方法筛选更加全面的指标来衡量巨噬细胞的即时功能状态。 实验目的: 在本课题中,针对既往研究中的难点和疑点,以腹腔巨噬细胞为研究核心,从以下几个方面展开探索:1)采用高通量的技术手段全面探究腹腔巨噬细胞亚群在EM进程中的功能状态变化,完善腹腔巨噬细胞在EM中的作用机制研究; 2)进一步对腹腔巨噬细胞相关T细胞和肠道微生物进行检测,从个多层面评估EM对机体的影响;3)通过干预调节巨噬细胞功能,初步探索基于巨噬细胞的EM治疗新策略。 试验方法: 本实验基于小鼠EM模型。1)利用流式细胞术检测腹腔巨噬细胞亚群在EM小鼠模型中的变化规律。通过流式分选技术获取微量LPM和SPM,采用微量mRNA测序分析全面检测两者的功能状态。2)利用流式细胞术检测巨噬细胞相关T细胞亚群的变化规律,通过16S rRNA测序分析EM疾病进程中肠道微生物丰度和构成变化规律。3)通过两种巨噬细胞调控手段(CD47抗体阻断和视黄酸诱导腹腔巨噬细胞亚群转化)探索EM治疗新靶点。 实验结果和结论: 1)实验结果表明LPM占总腹腔巨噬细胞比例在EM早期明显下降,后期逐渐增加,而SPM占总腹腔巨噬细胞比例在EM早期明显升高,后期呈下降趋势。 EM造模两周后,腹腔总巨噬细胞出现双向极化现象,即NOS2和CD206的比例均增加。进一步分析发现,LPM细胞是M1型巨噬细胞的主要来源,SPM细胞是M2型巨噬细胞的来源,即LPM和SPM在EM中的功能分化方向不同导致腹腔总巨噬细胞呈现出双向功能分化。流式分选微量LPM和SPM细胞行mRNA测序全面分析比较两者的功能差异,结果表明EM小鼠中LPM和SPM的基因表达模式分别发生大规模改变,且EM小鼠中LPM和SPM之间功能特征不同。 KEGG pathway分析提示LPM和SPM的代谢相关通路发生明显变化,巨噬细胞的代谢状态改变与其功能分化/极化密切相关,细胞代谢状态的重塑是为巨噬细胞功能转化提供所需要的能量支持,通过直接调控细胞代谢关键信号通路可以影响巨噬细胞功能分化,进一步提示两种巨噬细胞功能分化不同,进而在EM的发生发展中发挥不同作用。对关键细胞代谢通路进行干预调节,可以挖掘新的EM治疗靶点。 2)小鼠腹腔巨噬细胞由来源、表型和功能不同的LPM和SPM组成。其中LPM通过细胞吞噬途径在清除组织环境中凋亡细胞方面发挥主要作用,SPM主要参与腹腔感染引起的炎症。且LPM在EM进程中有向M1型巨噬细胞趋化的趋势,M1型巨噬细胞能够促进炎症。而SPM则向M2型巨噬细胞分化,M2型巨噬细胞具有抑制炎症、促进修复组织损伤的作用。因此,LPM在EM进程中可能具有更强的抑制病灶子宫内膜细胞生长的作用。在本实验中,通过流式细胞术检测发现LPM的细胞吞噬能力是SPM的两倍。视黄酸在LPM发育成熟中起重要作用,能够诱导SPM向LPM转化,给予EM小鼠视黄酸诱导后,SPM的标记物向LPM转化,而且视黄酸诱导的SPM具有更强的细胞吞噬能力。 CD47-SIRPα通路是固有免疫中重要的自我保护机制,巨噬细胞表达SIRPα,与目标细胞表面的CD47结合后会抑制巨噬细胞吞噬目标细胞。阻断CD47-SIRPα信号通路在巨噬细胞免疫治疗癌症中已经取得进展。本研究中探索了阻断CD47对异位子宫内膜细胞生存能力的影响,首先通过组织免疫荧光染色技术检测异位子宫内膜组织中子宫内膜细胞的CD47分子表达量。进一步利用流式细胞术和活细胞标记技术检测,结果提示中和抗体法阻断子宫内膜细胞表面的CD47分子后,巨噬细胞吞噬子宫内膜细胞能力增强,子宫内膜细胞的凋亡增加。 3)腹腔免疫环境非常复杂,包含多种免疫细胞,如巨噬细胞、T细胞、B细胞等,腹腔巨噬细胞是EM发病中的关键因素,巨噬细胞被激活后,通过直接或者间接途径与其他免疫细胞相互作用,可以影响辅助性T细胞(Th细胞)和调节性T细胞(Treg细胞)的分化。LPM还能够通过作用于腹腔中B1细胞进一步调控肠粘膜相关淋巴组织合成IgA,进而影响肠道微生物。在本研究中,我们利用流式细胞术多时间点检测EM发病过程中Th细胞、Treg细胞的比例变化情况,结果提示EM小鼠造模后6周腹腔中Th1和Treg细胞比例升高,Th17细胞在EM造模后第4周升高并持续至第6周,Th2细胞比例无明显改变。其中Th1和Th17是促进炎性免疫细胞,而Treg细胞是抑制炎症的免疫细胞,两类细胞比例都升高,这与腹腔总巨噬细胞的双向极化相吻合,与LPM和SPM功能分化方向不同相关。本研究中首次利用高通量16S rRNA测序技术手段对EM相关肠道微生物变化进行多时间点的检测评估。实验结果提示在EM造模后6周内, EM并未引起小鼠体重改变和肠道炎症。α多样性分析显示EM造模小鼠6周内与对照组小鼠无明显差异。但β多样性分析显示EM造模后第6周,小鼠肠道菌群构成发生了明显变化,各组间基于门分类水平的UPGMA分析和PCOA分析显示造模后6周EM小鼠的肠道微生物组成与其他各组存在明显差异。通过LEFse分析明确了造成EM小鼠菌群变化的主要因素为Firmicutes和Bacteroidetes的变化,EM小鼠的Firmicutes/Bacteroidetes比值是对照组的两倍,该比值是衡量肠道菌群失调的重要参考指标。提示EM小鼠存在菌群失调。 本课题完善了不同来源腹腔巨噬细胞在EM发病中的数量变化规律和功能状态研究,初步通过干预巨噬细胞探索了EM治疗新策略。并从肠道微生物等全新角度明确EM对机体的影响。 关键词:子宫内膜异位症;腹腔巨噬细胞;免疫代谢;肠道微生物;视黄酸

英文摘要

Background: The incidence of endometriosis (EM) in women at the reproductive stage is approximately 10% to 15% and tends to increase yearly. The main symptoms of EM include infertility and various pelvic visceral pains, which seriously affect the daily life and work of the patients. In addition, the high recurrence rate after EM treatment causes great pain to the patient’s body and mind. However, the pathogenesis of EM remains unclear. The existing hypothesis of EM pathogenesis includes retrograde menstruation theory, endometrial stem cell implantation theory, coelomic metaplasia theory, and Mullerian residue theory. At present, retrograde menstruation theory is widely recognized by scholars but cannot explain these two main phenomena: 1) as many as 90% of women have menstrual reflux, but only a minority of them suffers from EM; and 2) the reflux of endometrial debris began to appear in women after menarche, but the age at onset of patients with EM is 25-45 years. This theory cannot explain the 10 years delay between the onset of endometrial reflux and the disease. The latest research shows that menstrual reflux may be the only possible incentive, and immune dysfunction plays an important role in EM planting, adhesion, and growth. Understanding of EM pathogenesis is still at its infancy, and the unclear pathogenesis leads to the lack of specific etiological therapy. Based on existing EM etiology, the pathogenesis of this disease must be further elucidated. Macrophage is an important component of the innate immune system and exhibits multiple functions, including antigen presentation, phagocytosis, and cytokine synthesis and release; peritoneal macrophages play a decisive role in the occurrence and progression of endometriosis (EM). After the endometrial tissue flows counter-currently into the peritoneal cavity, peritoneal macrophages are activated, enriched, and infiltrated into the ectopic tissue. The activated macrophages secrete a large number of cytokines to participate in the formation of neovascularization and nerve fibers in ectopic lesions. Clinical studies reported abnormalities in the function and quantity of peritoneal macrophages and in the expression of related cytokines [interleukin (IL)-lβ, IL-6, 1L-8, and IL-10] in patients with EM. Experiments using animal models of EM revealed the following: 1) drug elimination of peritoneal macrophages can suppress the growth of ectopic lesions; 2) lipopolysaccharide-induced conversion of peritoneal macrophages into Ml inhibits the growth of ectopic foci; and 3) M2 polarization of macrophages induced by IL-4 can promote the growth of ectopic foci. Therefore, the functional status of peritoneal macrophages could likely determine the occurrence and development of EM. However, contradicting results were reported by studies on the polarization and functional status of peritoneal macrophages in the course of EM. 1) Bacci et al. found that the expression levels of CD 163 and CD206 in macrophages increased in the ectopic lesions of patients with EM and in mouse models of EM. This finding indicates that CD 163 and CD206 were converted into M2. By contrast, Takebayashi et al. conducted immunohistochemistry analysis and found that the CD206 expression in patients with EM was lower than that in the control group. 2) In ex-vivo experiments, the expression of representative M1 and M2 markers increased in macrophage culture supernatants after stimulation of THP-1 induced macrophages with conditioned medium derived from endometrial cells. Beste et al. isolated, purified, and cultured peritoneal macrophages from patients with EM in vitro. A high-throughput multifactor detection technology was used to determine cytokine secretion pattern; results indicated that Ml-specific cytokines (IL-lβ, 1L-12) and M2-specific cytokines (IL-10) were upregulated in peritoneal macrophages derived from patients with EM. In summary, the functional status of peritoneal macrophages in the pathogenesis of EM and the specific mechanism remain unclear. Macrophages can be divided depending on their origins into tissue-specific macrophages (e.g., microglia in the brain and Kupffer cells in the liver) and macrophages derived from peripheral monocytes. These macrophages possess different phenotypes and functions, that is, different subpopulations of macrophages exhibit different functional polarization states after receiving the same peripheral stimuli. The two sources of macrophages in the peritoneal cavity are large peritoneal macrophage (LPM) derived from the yolk sac and small peritoneal macrophage (SPM) derived from hematopoietic stem cell monocytes. LPM and SPM also have different phenotypes and functional properties. Previous studies on macrophages in EM neglected and were confused with regard to the heterogeneity of the two macrophages, leading to inconsistent results and conclusions. The imperfection of macrophage function evaluation system also leads to inconsistent research results. Macrophages are sensitive to their external stimulatory factors and undergo large-scale transcriptional regulation changes after activation. The functional status of macrophages is simply classified as Ml or M2-type macrophages. However, their metergasis is extremely complicated. Ml-M2 polarization is a continuous process of functional change. Therefore, most research results cannot accurately reflect the functional characteristics of peritoneal macrophages during the pathogenesis of EM. In this regard, high-throughput experimental methods must be developed to screen comprehensive indicators of the immediate functional status of macrophages. Objective: In this study, we focus on the difficulties encountered and the contradicting findings of previous works. We will use peritoneal macrophages as the core of our research and explore the following aspects. 1) High-throughput techniques were used to comprehensively assess changes in the functional status of peritoneal macrophage subpopulations in the course of EM and improve our understanding on the mechanism of peritoneal macrophages in EM. 2) We will assess the effect of EM on the body from multiple layers by further examination of peritoneal macrophage-associated T cells and gut microbiota. 3) Finally, we will explore new approaches of EM therapy by regulating the function of macrophages. Methods: A mouse model of EM was used in this study. 1) Flow cytometry was used to detect changes in peritoneal macrophage subpopulations. Trace amounts of LPM and SPM were obtained by flow sorting technology, and their functional status was detected by mRNA sequencing analysis. 2) Flow cytometry was also used to detect changes in peritoneal macrophage-associated T cells. 16S rRNA sequencing was conducted to analyze intestinal microbiological abundance and compositional changes during the course of EM progression. 3) Two intervention strategies based on peritoneal macrophage (CD47 antibody blockade and retinoic acid-induced intraperitoneal macrophage subpopulation transformation) were explored for treatment of EM. Results and conclusions: 1)Experimental results showed that the proportion of LPM in total peritoneal macrophages considerably decreased in the early stage of EM and gradually increased in the later period. By contrast, the proportion of SPM in the total peritoneal macrophages increased significantly in the early EM period and showed a decreasing trend in the later period. After 2 weeks of EM modeling, bipolar polarization occurred in total peritoneal macrophages, and the proportions of NOS2- and CD206-positive cells increased. Further analysis indicated that LPM cells were the main source of Ml macrophages, and SPM cells were the source of M2 macrophages. These findings indicate that LPM and SPM have different functional differentiation directions in EM, leading to biphasic functional differentiation of total peritoneal macrophages. Flow sorting technology and mRNA sequencing of trace amounts of LPM and SPM were performed to comprehensively analyze and compare their functional differences. The results showed large-scale changes in the gene expression of LPM and SPM in both EM and control mice. LPM and SPM exhibited different functions in EM mice. Analysis of the KEGG pathway suggested that the metabolic pathways of LPM and SPM considerably changed. Alteration in the metabolic state of macrophages could be related to their functional differentiation/polarization. Remodeling of the metabolic state provided the required energy to transform the function of macrophages. Directly modulating the key signal pathways of cell metabolism can affect the functional differentiation of macrophages. This finding further indicates that the two macrophages exhibit different levels of functional differentiation and thus play different roles in the occurrence and development of EM. Interventional regulation of key cellular metabolic pathways can excavate new therapeutic targets in EM. 2)The peritoneal macrophages of mice consist of LPM and SPM with different sources, phenotypes, and functions. LPM plays a major role in eliminating apoptotic cells in the tissue environment, and SPM is mainly involved in inflammation caused by abdominal infection. LPM tends to differentiate into Ml macrophages during EM progression, thereby promoting inflammation. SPM differentiates into M2 macrophages, which inhibit inflammation and promote the repair of damaged tissues. Therefore, LPM may play a major role in inhibiting the growth of endometrial cells in lesions by strong direct phagocytosis and indirect Ml macrophage differentiation during EM progression. Based on the flow cytometry data, the phagocytic capacity of LPM was found to be twice that of SPM. Retinoic acid plays an important role in LPM development and can induce the transformation of SPM into LPM. After administration of retinoic acid to EM mice, the markers of SPM were transformed into LPM, and retinoic acid-induced SPM exhibited strong phagocytic capacity. The CD47-SIRPα pathway is an important self-protection mechanism in innate immunity. Macrophages express SIRPα, which after binding to CD47 on the target cell surface, will inhibit macrophages from phagocytosing the target cell. The blockade of the CD47-SIRPα signaling pathway has contributed to development of macrophage immunotherapy for cancers. The effect of blocking CD47 on the survival of ectopic endometrial cells was also investigated in this study. First, tissue immunofluorescence staining was conducted. The CD47 expression in glandular epithelial cells in the ectopic endometrium was higher than that in the eutopic endometrium. Flow cytometry and live cell labeling techniques were then conducted. After blocking the CD47 molecule on the surface of endometrial cells, the ability of macrophages to engulf endometrial cells was enhanced, and the apoptosis of endometrial cells was increased. 3)The peritoneal immune environment is very complex and includes a variety of immune cells, such as macrophages. T cells, and B cells. Peritoneal macrophages are key factors in the pathogenesis of EM. Once activated, macrophages can affect the differentiation of T-helper cells (Th) and T-regulatory cells (Treg) through direct or indirect interactions with other immune cells. LPM can regulate the synthesis of IgA in the intestinal mucosa-associated lymphoid tissue by regulating B1 cells in the peritoneal cavity, thereby affecting the microorganisms in the intestinal tract. Flow cytometry was performed at multiple time points to detect changes in the proportion of helper Th and Treg cells during EM pathogenesis. The results suggest that after EM modeling, the proportion of Thl and Treg cells increased in peritoneal cavity in the 6th week, the proportion of Thl 7 cells increased in the 4th week and continued until the 6th week, but Th2 cells ratio did not change significantly. Wherein Thl and Th17 cells are proinflammatory immune cells, but Treg cells are anti-inflammatory immune cells. The ratio of both types of cells is increased, which is consistent with the bipolar polarization of total peritoneal macrophages and may be related to the different directions of LPM and SPM functional differentiation. This study is the first to apply high-throughput 16S rRNA sequencing technology for multiple time point assessments of EM-related intestinal microbial changes. The experimental results suggested that within 6 weeks after EM modeling, EM did not cause body weight changes and intestinal inflammation in mice. Analysis of a diversity showed no remarkable difference between the EM models and control mice within 6 weeks. However, β diversity analysis showed that the composition of the intestinal microflora in mice changed significantly in the 6th week after EM modeling. Furthermore, UPGMA and PCOA analyses at the phylum classification level revealed that the microbial composition in the intestine of the EM mice significantly differed from that in the other groups in the 6th week. LEFse analysis showed that the change of Firmicutes and Bacleroidetes was the main factor that altered the microbial flora in EM mice. The Firmicutes/Bacteroidetes ratio, which is an important reference for measuring the dysbacteriosis of intestinal flora, in EM mice was found to be twice that of the control group. Hence, EM mice developed dysbacteriosis. This work improved our understanding on the quantitative changes and functional status of peritoneal macrophages derived from different sources in the pathogenesis of EM. We also initially explored new strategies for EM treatment by intervening macrophages. Overall, the results clarified the effect of EM on the body from the perspective of intestinal microbiota. Keywords: Endometriosis, Peritoneal macrophage, Immune metabolism, Gut microbiota, Retinoic acid

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