【作者】 陈琦；

【导师】 赖玉平；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2020， 硕士

【摘要】 银屑病的原位复发是银屑病领域的难题。虽然记忆样γδT细胞、组织常驻记忆T细胞被认为是引起银屑病复发的主要细胞,但这些记忆细胞在病灶部位聚集的同时,也会迁移到皮肤中的其他部位,因此,这些记忆细胞并非银屑病原位复发的始作俑者。而已知基底层角质形成细胞（keratinocytes,KCs）具有固有免疫记忆功能,但是KCs的记忆功能产生的机制以及能否导致银屑病的原位复发仍未知。为了解答上述关键科学问题,我们首先检测银屑病皮损中的炎性微环境是否能够诱导KCs的固有免疫记忆。因此,我们利用银屑病皮损中高表达的细胞因子刺激KCs,然后检测标志固有免疫记忆的组蛋白第三亚基四号赖氨酸三甲基化修饰（H3K4me3）。通过一系列的筛选,我们发现IL-1β、IL-36γ、IL-17A、IL-17D、IL-17E、IL-17F均可以促进H3K4me3。接着,我们用这些诱导H3K4me3的细胞因子刺激KCs,在细胞静息一段时间后进行poly（I:C）的二次刺激,通过检测poly（I:C）诱导的IL-36γ的表达来衡量KCs的固有免疫记忆功能。我们发现细胞因子IL-17D明显诱导KCs的固有免疫记忆,而组蛋白甲基转移酶的抑制剂5’-脱氧-5’-腺苷（MTA）能够抑制IL-17D促进的H3K4me3和固有免疫记忆。并且,我们通过染色质免疫共沉淀实验证明IL-17D促进H3K4me3富集在IL-36γ启动子附近,而Il17d^–/–银屑病小鼠皮损中H3K4me3水平明显低于WT银屑病小鼠。确定IL-17D通过促进H3K4me3诱导KCs的固有免疫记忆后,我们进一步探究IL-17D调节KCs固有免疫记忆的功能机制。由于糖代谢参与巨噬细胞中H3K4me3介导的固有免疫记忆,因此,我们假设IL-17D可能通过调节KCs的代谢来诱导KCs的记忆。为了证明这个假设,我们分离了WT和Il17d^–/–小鼠的KCs进行代谢组学分析,分析结果显示IL-17D调节糖代谢产物丙酮酸、乳酸和影响糖代谢的谷氨酰胺等氨基酸的生成,并且糖代谢的抑制剂2-脱氧-D-葡萄糖（2-DG）能抑制IL-17D促进的H3K4me3和固有免疫记忆。值得一提的是,在IL-17D调节的所有糖代谢产物中,只有乳酸在银屑病中是增加的,而且IL-17D能够诱导KCs中的乳酸脱氢酶A（Lactate dehydrogenase A,LDHA）的表达。因此,我们进一步确定乳酸是否是IL-17D诱导的能够促进KCs固有免疫记忆的主要代谢产物。我们用乳酸刺激KCs,发现乳酸能诱导KCs的H3K4me3和固有免疫记忆,而LDHA的抑制剂草氨酸盐能抑制IL-17D诱导的乳酸和固有免疫记忆。这些研究结果显示,IL-17D通过诱导LDHA的表达促进乳酸的增加,从而调节KCs的H3K4me3和固有免疫记忆。综上所述,本研究发现了细胞因子IL-17D通过诱导LDHA的表达促进乳酸的产生,增加的乳酸通过诱导H3K4me3促进KCs的固有免疫记忆的形成,为银屑病的复发提供了理论基础。更多还原

【Abstract】 Psoriasis relapse in the same locations is always a condundrum.So far,memory-likeγδT cells or tissue-resident memory T cells are considered as the main drivers of psoriasis relapsing.However,these memory cells not just reside in the site of the lesion,but also migrate to other parts of the skin.Therefore,they are probably not the only reason that causes psoriasis relapsing in the same location.Besides memory T cells,basal keratinocytes（KCs）have been show to have the ability to aquire innate immune memory.However,the underlying mechanisms by which KCs acquire innate immune memory and whether KCs with innate immune memory would be involved in the relapse of psoriasis remain unknown.To address these questions,we first examined whether the inflammatory cytokines highly expressed in psoriatic lesions could induce innate memory of KCs.We stimulated KCs with a panel of cytokines known to highly expressed in psoriatic lesions and detect the level of H3K4me3,a histone modification that marks innate immune memory.We found that IL-1β,IL-36γ,IL-17A,IL-17D,IL-17E and IL-17F increased the level of H3K4me3 in KCs.Next,we used poly（I:C）as a secondary stimulus to stimulated KCs which originally stimulated by these cytokines,and measured innate immune memory by detecting the expressing of poly（I:C）-induced-IL-36γ.It was found that IL-17D induced innate immune memory significantly.Moreover,histone methyltransferase inhibitor MTA inhibited IL-17D-induced H3K4me3 and the expression of IL-36γinduced by poly（I:C）as second stimulation.CHIP assay further confirmed that IL-17D increased the enrichment of H3K4me3 on IL-36γpromoter.Furthermore,the level of H3K4me3 was lower in Il17d^-/-psoriatic mice compared to WT controls.Next we explore the mechanisms by which IL-17D regulated innate immune memory of KCs.Given that glucose metabolism is involved in H3K4me3-mediated innate immune memory in macrophages,we hypothesized that IL-17D might induce innate immune memory of KCs through glucose metabolism.To address this,we isolated KCs from WT and Il17d^-/-mice and showed that the deficiency of Il17d in keratinocytes led to decreased glucose metabolism and amino acid metabolism,that is,Il17d^-/-keratinocytes had less production of pyruvate,lactate,and glutamine which also involves in glucose metabolism.Furthermore,the glucose metabolism inhibitor 2-DG inhibited IL-17D-indued H3K4me3 and innate immune memory in KCs.Notably,among these metabolites regulated by IL-17D,we found that only lactate,a production of glycolysis,was increased in lesional skin of psoriasis compared to normal skin.We thereby further determined whether IL-17D-indcued innate immune memory of KCs would be dependent on lactate.KCs stimulated by lactate showed increased H3K4me3and innate immune memory,whereas the inhibitor of lactate dehydrogenase A（LDHA）oxamate impaired lactate production and innate immune memory induced by IL-17D in KCs.These results suggest that IL-17D induces LDHA to promote lactate production,thereby modulating H3K4me3 and innate immune memory of KCs.In summary,our study demonstrates that IL-17D promotes the production of lactate by LDHA,and the increased lactate induces innate immune memory in KCs by increasing H3K4me3,which provides new insighits into the relapse of psoriasis.更多还原