【作者】 王璐；

【导师】 陆前进；

【作者基本信息】 中南大学 ， 临床医学（八年制）（专业学位）， 2025， 博士

【摘要】 背景:系统性红斑狼疮（Systemic lupus erythematosus,SLE）是一种经典的系统性自身免疫病,主要特征为T、B淋巴细胞功能紊乱和大量自身抗体的产生。其临床表现多样,常见于14至45岁的女性,可累及皮肤、肾脏等多个组织器官。尽管目前普遍认为SLE的发病主要源于免疫系统功能紊乱,但这一理论难以全面解释其复杂的临床表现,尤其是皮肤受累的高发性和早期性。角质形成细胞是表皮的主要组成细胞,不仅承担屏障防御功能,还具备一定的免疫调节能力。我们团队的前期研究首次证实,角质形成细胞的异常状态可直接驱动SLE发病。具体而言,SLE患者皮损基底层角质形成细胞过氧化物酶体增殖物激活受体γ（Peroxisome Proliferator-Activated Receptor Gamma,PPARγ）表达显著下调。模拟这种改变,免疫健全的小鼠会迅速出现SLE样自身免疫病表型。鉴于PPARγ是调控糖脂代谢的关键转录因子,我们推测在PPARγ低表达的角质形成细胞中可能存在显著的代谢紊乱,导致局部代谢产物异常积聚,进而激活下游免疫细胞,推动SLE的发病过程。目的:探讨PPARγ低表达角质形成细胞的代谢改变及其驱动SLE发病的分子机制,尤其是异常脂质代谢产物对SLE发生发展的影响。方法:（1）分析GEO数据库中已发表的SLE及其他自身免疫病皮损转录组数据,评估PPAR信号通路在自身免疫病皮损中的活化状态。结合免疫组化染色,验证PPARγ在健康对照皮肤及SLE和其他自身免疫病患者皮损中的蛋白表达水平和细胞定位特征。进一步通过单细胞转录组测序,解析SLE患者及角质形成细胞条件性Pparg敲除SLE小鼠,即Pparg^fl/fl;Krt5^{cre ERT2/+}小鼠的皮损中浸润免疫细胞的特征性群体,并结合流式细胞术与免疫荧光染色对关键细胞亚群进行验证和定位,明确潜在的致病免疫细胞亚群,即皮损内高度活化的树突状细胞（dendritic cell,DC）。筛选SLE患者与Pparg条件性敲除SLE小鼠皮损中共同高表达的趋化因子,解析异常角质形成细胞募集皮内DC的分子机制;（2）采集SLE患者及模型小鼠的皮损组织和血浆样本,开展脂质代谢组学分析,筛查共同升高的异常代谢产物。将异常代谢产物神经酰胺（Ceramide,Cer）,己糖神经酰胺（Hexosylceramide,Hex Cer）和鞘磷脂（Sphingomyelin,SM）腹腔注射至Pparg条件性敲除SLE小鼠中,观察狼疮样皮损、血清自身抗体、尿蛋白等SLE样表型的变化。接着,将SM从真皮注射至Pparg条件性敲除小鼠中,观察狼疮样皮损、血清自身抗体、尿蛋白等SLE样表型的变化,找出能影响SLE病情的皮内积累的异常代谢产物。随后,构建PPARγ敲除、敲降、过表达及敲除后回补的角质形成细胞模型（Ha Ca T）,检测不同处理条件下关键代谢物的变化。将异常代谢产物添加到DC的培养液中,并通过流式细胞术检测CD80和CD86的表达水平,以评估DC的激活状态,明确角质形成细胞来源的异常代谢产物是否可诱导DC的活化;（3）构建能靶向耗竭树突状细胞的SLE模型小鼠,即Pparg^fl/+;Krt5^{cre ERT2/+};Itgax^DTR/+小鼠。在诱导角质形成细胞Pparg条件性敲除的同时通过在真皮注射白喉毒素（diphtheria toxin,DT）选择性耗竭皮内DC,观察狼疮样皮损、血清自身抗体、尿蛋白等SLE样表型的变化;采用Pparg^fl/+;Krt5^{cre ERT2/+};Itgax^DTR/+小鼠,在诱导SLE的基础上,在真皮内注射DT和异常代谢产物。观察狼疮样皮损、血清自身抗体、尿蛋白等SLE样表型的变化,明确异常代谢产物是否通过活化局部DC推动疾病进展;（4）检测Pparg条件性敲除小鼠皮损局部DC的活化情况;检测皮内补充异常代谢产物后局部DC的活化情况;由小鼠骨髓诱导分化出未成熟的树突状细胞,利用原子力显微镜检测SM刺激后DC的细胞刚度变化,探索SM促进DC活化的具体机制;结果:（1）分析GEO数据库中系统性红斑狼疮、皮肌炎、银屑病关节炎和系统性硬化症患者皮损的转录组数据发现,PPAR信号通路在系统性红斑狼疮、皮肌炎、银屑病关节炎和系统性硬化症中均显著下调。免疫组化显示,系统性红斑狼疮、系统性硬化症、皮肌炎、成人斯蒂尔病和结节性多动脉炎患者皮损中角质形成细胞PPARγ蛋白表达明显低于健康对照,且在基底层的角质形成细胞中下降尤为明显。单细胞转录组测序和流式检测表明,致残性全硬化性硬斑病、系统性硬化症、SLE患者以及角质形成细胞条件性Pparg敲除的SLE模型小鼠皮损中均存在高度活化的DC群体。免疫荧光染色进一步验证这些DC主要定位于表皮和真皮浅层,靠近异常角质形成细胞区域。异常角质形成细胞促进DC活化可能在自身免疫病的发生发展中发挥重要作用;（2）分析SLE患者和小鼠的皮损单细胞转录组数据,发现角质形成细胞PPARγ缺失后显著上调包括CCL20、CXCL10在内的多个趋化因子,提示异常角质形成细胞可通过特定趋化因子-受体轴选择性募集DC。在健康小鼠真皮内注射CCL20蛋白可有效募集大量DC。而在角质形成细胞条件性Pparg敲除的SLE早期小鼠真皮内注射CCL20中和抗体来耗竭CCL20,则能有效抑制DC的皮肤浸润,并减轻SLE疾病表型,小鼠的皮损、尿蛋白、自身抗体、脾脏重量均显著缓解,表明皮内DC是角质形成细胞异常促进SLE样表型发生发展的关键介导细胞;（3）脂质代谢组学分析显示,SLE患者与角质形成细胞条件性Pparg敲除SLE小鼠皮损及血浆中共同升高了多种特征性代谢产物,尤其是鞘脂类代谢产物,包括Cer,Hex Cer和SM。将Cer、Hex Cer和SM分别按升高后的相应浓度腹腔注射至角质形成细胞条件性Pparg敲除SLE小鼠后,仅SM能明显推动疾病进展,包括小鼠生存率大大降低、皮损加重、血清抗ds DNA抗体升高及尿蛋白增加。通过构建PPARγ敲除、敲降、过表达及敲除后回补的角质形成细胞,发现低PPARγ的角质形成细胞培养液中SM含量升高。进一步通过真皮内注射方式,将相应浓度的上述异常代谢产物施加于模型小鼠的局部皮肤,发现亦仅SM能有效加重SLE疾病严重程度。这些结果表明异常角质形成细胞来源的SM富集足以推动SLE发生发展;（4）皮内积累的鞘磷脂可以通过活化DC加重SLE。疾病早期,基于角质形成细胞Pparg下调的SLE小鼠皮内DC活化即增加。将SM添加至DC培养体系中,流式细胞术结果显示,DC表面的CD80与CD86含量在SM处理后明显升高,并且这些SM处理的DC能进一步促进效应T细胞的分化。体内实验中,具有高SM含量的SLE小鼠皮损中DC的激活比例同样也升高,引流淋巴结中免疫促进性的效应T细胞分化增强。在Pparg^fl/+;Krt5^{cre ERT2/+};Itgax^DTR/+小鼠中敲除角质形成细胞Pparg,并通过DT选择性耗竭皮内DC后,SLE样皮损、血清自身抗体、尿蛋白及肾脏免疫沉积均明显减轻,表明皮内DC是角质形成细胞异常推动SLE样表型形成的关键介导者;在耗竭了皮内DC的Pparg^fl/+;Krt5^{cre ERT2/+};Itgax^DTR/+SLE小鼠的真皮内注射SM,小鼠皮损无明显加剧,血清自身抗体与尿蛋白水平亦无显著升高。与未耗竭DC的小鼠相比,疾病表型明显缓解,不同于单纯注射SM后疾病表型加重。表明SM需依赖皮内DC的存在以发挥致病作用;（5）利用SM探针EquinatoxinⅡ检测发现,成熟的活化DC的SM总含量更高,SM的积累主要发生在细胞的质膜水平。与体外实验一致,相较正常皮内普遍未活化的DC区室,SLE小鼠皮损内普遍活化的DC细胞膜SM含量明显升高。DC的刚度随着DC的成熟而增加,SM处理增加了DC的细胞刚度,降低流动性,促进DC成熟。结论:角质形成细胞内PPARγ降低会介导趋化因子CCL20的过量分泌和局部SM的积累。这些角质形成细胞来源的CCL20募集DC至近表皮处,并由SM活化,从而推动SLE的疾病发生发展。本研究还发现在皮肌炎和系统性硬化症等多种自身免疫病中同样存在角质形成细胞内PPARγ降低和皮内DC异常分布和活化。异常角质形成细胞通过激活DC驱动自身免疫病发生发展可能具有普适性。图59幅,表23个,参考文献95篇更多还原

【Abstract】 Background:Systemic lupus erythematosus（SLE）is a classic systemic autoimmune disease characterized by disturbances in T and B lymphocytes and the production of large amounts of autoantibodies.Its clinical manifestations are diverse,commonly seen in women of childbearing age between 14-45 years old,and can involve multiple organs and tissues such as the skin and kidneys.Although it is widely believed that the onset of SLE is mainly due to immune system dysfunction,this theory is difficult to fully explain its complex clinical manifestations,especially the high incidence and early stage of skin involvement.Keratinocytes are the main constituent cells of the epidermis,not only responsible for barrier defense functions,but also possessing certain immune regulatory abilities.Our team’s preliminary research has found for the first time that abnormal states of keratinocytes can directly drive the onset of SLE.Specifically,in the basal layer keratinocytes of SLE patients,the expression of peroxisome proliferator activated receptor gamma（PPARγ）is significantly downregulated.Simulating this change,immunocompetent mice will rapidly develop SLE like autoimmune disease phenotypes.Given that PPARγis a key transcription factor regulating glucose and lipid metabolism,we speculate that there may be significant metabolic disorders in keratinocytes with low PPARγexpression,leading to abnormal accumulation of local metabolites,which in turn activate downstream immune cells and promote the pathogenesis of SLE.Furthermore,considering that various autoimmune diseases can be accompanied by skin damage,it is worth further exploring whether this mechanism has broader applicability.Objective:Explore the metabolic changes of PPARγlow expression keratinocytes and their molecular mechanisms driving the onset of SLE,especially the influence of abnormal lipid metabolites on the occurrence and development of SLE.Methods:（1）Analyze the RNA sequencing data of SLE and other autoimmune disease skin lesions published in the GEO database to evaluate the activation status of the PPAR pathway in autoimmune disease skin lesions.Combined with immunohistochemical staining,verify the protein expression levels and cellular localization characteristics of PPARγin healthy control skin and skin lesions of patients with SLE and other autoimmune diseases.Further single-cell RNA Sequencing was used to analyze the characteristic population of infiltrating immune cells in the skin lesions of SLE patients and mice with conditional Pparg knockout of keratinocytes.Flow cytometry and immunofluorescence staining were used to validate and locate key cell subpopulations,identifying the potential pathogenic immune cell subpopulations,namely highly activated dendritic cells（DCs）in the skin lesions.Screening for chemokines overexpressed in the skin lesions of SLE patients and conditional Pparg knockout SLE mice,and elucidating the molecular mechanism of abnormal keratinocyte recruitment of intradermal DCs;（2）Collect skin lesion tissue and plasma samples from SLE patients and model mice,conduct lipidomics analysis,and screen for co-elevated abnormal metabolites.Inject ceramide（Cer）,hexosylceramide（Hex Cer）and sphingomyelin（SM）intraperitoneally into conditional Pparg knockout mice and observe changes in SLE like phenotypes such as lupus like skin lesions,serum autoantibodies,and urinary protein.Next,the abnormal metabolites were injected intradermally into conditional Pparg knockout mice,and changes in SLE like phenotypes such as lupus like skin lesions,serum autoantibodies,and urinary proteins were observed to identify the abnormal metabolites that could affect the intradermal accumulation of SLE.Subsequently,the keratinocyte models（Ha Ca T cells）were constructed to detect changes in key metabolites under different treatment conditions,including PPARγknockout,knockdown,overexpression,and replenishment after knockout（rescue）.Add abnormal metabolites to the culture medium of DCs and detect the expression levels of CD80 and CD86 by flow cytometry to evaluate the activation status of DCs and clarify whether abnormal metabolites derived from keratinocytes can induce their activation;（3）Construct Pparg^fl/+;Krt5^{cre ERT2/+};Itgax^DTR/+mice to induce conditional Pparg knockout in keratinocytes,and selectively deplete intradermal DCs by intradermal injection of diphtheria toxin（DT）.Observe changes in SLE like skin lesions,serum autoantibodies,urinary proteins,and other SLE like phenotypes;Pparg^fl/+;Krt5^{cre ERT2/+};Itgax^DTR/+mice were used to induce SLE,followed by intradermal injection of DT and abnormal metabolites.Observe changes in SLE like skin lesions,serum autoantibodies,urinary protein,and other SLE like phenotypes to determine whether abnormal metabolites promote disease progression by activating DCs;（4）To detect the activation of local DCs in the skin lesions of mice with conditional Pparg knockout.Detect the activation of local DCs after intradermal supplementation of abnormal metabolites.Inducing the differentiation of immature dendritic cells derived from mouse bone marrow,detecting the changes in cell stiffness of DCs after SM stimulation using atomic force microscopy,and exploring the specific mechanism of SM promoting DC activation;Results:（1）Analysis of RNA sequencing data of skin lesions from patients with SLE,dermatomyositis,psoriatic arthritis and systemic sclerosis in the GEO database revealed that the PPAR signaling pathway was significantly downregulated in systemic lupus erythematosus,dermatomyositis,psoriatic arthritis and systemic sclerosis.Immunohistochemistry showed that the expression of PPARγprotein in keratinocytes in the skin lesions of patients with SLE,systemic sclerosis,dermatomyositis,adult Still’s disease and polyarteritis nodosa was significantly lower than that in healthy controls,and the decrease was particularly obvious in keratinocytes in the basal layer.Single-cell RNA Sequencing and flow cytometry analysis showed that highly activated DCs were present in the skin lesions of patients with disabling pansclerotic morphea,systemic sclerosis,SLE,and keratinocyte conditional Pparg knockout mice.Immunofluorescence staining further confirmed that these DCs are mainly located in the superficial layers of the epidermis and dermis,near the area of abnormal keratinocytes.The promotion of DCs activation by abnormal keratinocytes may be a universal pathogenesis of autoimmune diseases;（2）Analyzing the single-cell RNA Sequencing data of skin lesions in patients and mice,it was found that the loss of PPARγin keratinocytes significantly upregulated multiple chemokines,including CCL20 and CXCL10,suggesting that abnormal keratinocytes can selectively recruit DCs through specific chemokine receptor axes.Intradermal injection of CCL20 protein in healthy mice can effectively recruit a large number of DCs.However,in preclinical mice with conditional Pparg knockout of keratinocytes,intradermal injection of CCL20 neutralizing antibodies to deplete CCL20 can effectively inhibit the skin infiltration of DCs and alleviate the SLE phenotype.The skin lesions,urinary protein,autoantibodies,and spleen weight of mice were significantly relieved,indicating that intradermal DCs are the key mediating cells for abnormal promotion of SLE like phenotype formation by keratinocytes;（3）Lipid metabolomics analysis showed that SLE patients and keratinocyte conditional Pparg knockout mice exhibited elevated levels of various characteristic metabolites in their skin lesions and plasma,particularly sphingolipid metabolites,including Cer,Hex Cer,and SM.After intraperitoneal injection of Cer,Hex Cer,and SM at corresponding elevated concentrations into keratinocyte conditional Pparg knockout mice,only SM significantly promoted disease progression,including significantly reduced survival rate,aggravated skin lesions,increased serum anti-ds DNA antibodies,and increased urinary protein.By constructing keratinocytes with PPARγknockout,knockdown,overexpression,and replenishment after knockout（rescue）,it was found that the SM content in the culture medium of keratinocytes with low PPARγwas increased.Furthermore,by intradermal injection,the corresponding concentrations of the abnormal metabolites were applied to the local skin of the model mice,and it was found that only SM could effectively exacerbate the severity of SLE.These results indicate that the enrichment of SM derived from abnormal keratinocytes is sufficient to promote the occurrence and development of SLE;（4）The accumulation of SM in the skin can exacerbate SLE by activating DCs.In preclinical stage,the activation of intradermal DCs in SLE mice based on downregulation of keratinocyte Pparg is increased.Adding SM to the DC culture system,flow cytometry results showed that the levels of CD80 and CD86 on the surface of DCs were significantly increased after SM treatment,and these SM treated DCs could further promote the differentiation of effector T cells.In vivo experiments,the activation ratio of DCs in the skin lesions of SLE mice with high SM content also increased,and the differentiation of immune promoting effector T cells in draining lymph nodes was enhanced.Conditional Pparg knockout of keratinocytes in Pparg^fl/+;Krt5^{cre ERT2/+};Itgax^DTR/+mice and selectively depleting intradermal DCs through DT significantly reduced SLE like skin lesions,serum autoantibodies,urinary protein,and renal immune deposition,indicating that intradermal DCs are key mediators of keratinocyte abnormalities driving SLE like phenotype formation;Intradermal injection of SM into Pparg^fl/+;Krt5^{cre ERT2/+};Itgax^DTR/+SLE mice depleted of intradermal DCs did not significantly worsen skin lesions,and serum autoantibodies and urinary protein levels did not significantly increase.Compared with mice without exhausted DCs,the disease phenotype was significantly alleviated,unlike the aggravation of the disease phenotype after injection of SM alone.Indicating that SM relies on the presence of intradermal DCs to exert its pathogenic effects;（5）Using the SM probe Equinatoxin II,it was found that mature activated DCs had a higher total SM content,and SM accumulation mainly occurred at the plasma membrane level of cells.Consistent with in vitro experiments,the SM content of activated DC cell membranes in SLE mouse skin lesions was significantly increased compared to the normally unactivated DC compartments.The stiffness of DCs increases with their maturation,and SM treatment increases the cellular stiffness of DCs,reduces fluidity,and promotes their maturation.Conclusion:The decrease of PPARγin keratinocytes mediates the excessive secretion of chemokine CCL20 and the accumulation of local SM.These CCL20 derived keratinocytes recruit DCs to the near epidermis and are activated by SM,thereby promoting the occurrence and development of SLE.This study also found that in various autoimmune diseases such as dermatomyositis and systemic sclerosis,there is also a decrease in PPARγwithin keratinocytes and abnormal distribution and activation of DCs within the dermis.Abnormal keratinocytes driving the occurrence and development of autoimmune diseases by activating DCs may be universal.更多还原