SIRT2通过组蛋白H4K8去乳酸化修饰调控巨噬细胞趋化功能
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SIRT2 regulates macrophage chemotaxis by de-modifying histone H4K8 lactylation
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Corresponding authors: CAO Qing, E-mail:caoqing@scmc.com.cn.
Received: 2023-04-18 Accepted: 2023-05-06 Online: 2023-08-28
目的·探讨沉默信息调节因子2(silent information regulator 2,SIRT2)通过组蛋白H4第8位赖氨酸(H4K8)去乳酸化修饰对早期感染后巨噬细胞免疫表型的调节作用及相应机制。方法·使用佛波醇-12-肉豆蔻酸酯-13-乙酸酯(phorbol-12-myristate-13-acetate,PMA)诱导人单核细胞白血病THP-1细胞,使其分化为具有巨噬细胞特性的人血单核细胞株(PMA-primed THP-1,pTHP-1),再以脂多糖(lipopolysaccharide,LPS)刺激建立巨噬细胞感染模型。将未经LPS处理的巨噬细胞(pTHP-1)设为对照(CTRL)组,经过LPS处理的巨噬细胞设为感染(LPS)组。通过蛋白质印迹法(Western blotting)检测巨噬细胞中组蛋白乳酸化各位点修饰水平、组蛋白乙酰化各位点修饰水平及SIRT2蛋白水平;通过实时荧光定量PCR(RT-qPCR)方法检测2组间糖酵解限速酶乳酸脱氢酶A(lactate dehydrogenase A,LDHA)、肝脏磷酸果糖激酶(phosphofructokinase liver type,PKFL),糖酵解调剂因子低氧诱导因子1α(hypoxia inducible factor 1α,HIF-1α),以及Sirtuin家族基因和HDAC家族基因表达水平;通过Transwell方法检测巨噬细胞趋化能力;使用慢病毒包装及细胞感染方法建立SIRT2过表达细胞系;使用RNA测序技术(RNA sequencing,RNA-seq)与染色质免疫共沉淀测序技术(chromatin immunoprecipitation sequencing,ChIP-seq)交互分析方法对组蛋白H4第8位赖氨酸乳酸化(lactylation of histone H4 lysine 8,H4K8la)特异性结合的基因进行差异性分析及通路富集分析。结果·相较于CTRL组,LPS组巨噬细胞糖酵解上调,组蛋白H4K8位点乳酸化水平显著增加(P<0.05),而组蛋白其余位点乙酰化水平未见显著变化。所有已知的具有去乳酸化修饰功能的酶中,仅SIRT2在LPS处理后出现显著降低(P<0.05),且SIRT2过表达可显著抑制巨噬细胞中组蛋白H4K8位点的乳酸化水平(P<0.05),但不影响组蛋白H4K8位点的乙酰化水平(P>0.05)。ChIP-seq与RNA-seq交互分析发现,组蛋白H4K8位点乳酸化修饰可调控巨噬细胞趋化相关基因,并且巨噬细胞的趋化能力在SIRT2过表达、H4K8la修饰水平下调后显著下降(P<0.05)。结论·SIRT2可通过去修饰组蛋白H4K8位点乳酸化改变趋化相关靶基因表达,从而降低巨噬细胞趋化能力。靶向SIRT2及H4K8la修饰将有助于控制巨噬细胞介导的炎症反应。
关键词:
Objective ·To explore the regulatory role of silent information regulator 2 (SIRT2) in modulating the immune phenotype of macrophages after infection by removing the lactylation at H4K8 site of histone and the corresponding mechanism. Methods ·Human THP-1 leukemia cells were induced by phorbol 12-myristate 13-acetate (PMA) and stimulated by lipopolysaccharide (LPS) to establish a macrophage infection model. Macrophages without LPS treatment (pTHP-1) were set as the control (CTRL) group, and macrophages with LPS treatment were set as the infected (LPS) group. Western blotting was used to detect the level of histone modification and SIRT2 protein in macrophages. RT-qPCR was used to detect the expression level of glycolytic key enzymes [phosphofructokinase liver type (PFKL), lactate dehydrogenase A (LDHA)] and modulators genes hypoxia inducible factor 1α (HIF-1α), and the expression level of Sirtuin genes and HDAC genes between the two groups. Transwell was used to detect the ability of macrophage chemotaxis. Lentivirus packaging and cell infection were used to construct SIRT2 overexpression cell line. The interaction analysis method of RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) was used to analyze the difference and pathway enrichment of the genes specifically bound to H4K8 lactylation (H4K8la). Results ·Compared to the CTRL group, macrophage glycolysis was upregulated and the level of H4K8la was significantly increased in the LPS group (P<0.05), while the level of lactylation in other sites remained unchanged. Among all known enzymes with deacetylation modification function, only SIRT2 showed a significant decrease after LPS treatment (P<0.05), and overexpression of SIRT2 could significantly inhibit the level of H4K8la modification, while the level of H4K8ac remained unchanged (P>0.05). The interactive analysis of ChIP-seq and RNA-seq revealed that chemotaxis-related genes were regulated by H4K8la, and macrophage chemotaxis ability significantly decreased after the overexpression of SIRT2 and downregulation of H4K8la (P<0.05). Conclusion ·SIRT2 can change the expression of target genes related to chemotaxis by removing H4K8la modification, thereby reducing the chemotaxis ability of macrophages. Targeting SIRT2 and H4K8la modification may help control inflammation mediated by macrophages.
Keywords:
本文引用格式
宋文汀, 陶悦, 潘艺, 莫茜, 曹清.
SONG Wenting, TAO Yue, PAN Yi, MO Xi, CAO Qing.
组蛋白翻译后修饰是表观遗传调控最重要的方式之一,其可通过影响染色质构象调控下游基因转录,进而在生理病理过程中发挥作用[7]。近年来,已有多种组蛋白修饰被证明与固有免疫相关,可调节巨噬细胞功能并参与感染进程[8]。2019年,有研究[9]首次发现乳酸可作为反应底物在巨噬细胞中修饰组蛋白H3第18位赖氨酸(H3K18)位点,并在感染晚期调节巨噬细胞向M2型极化。但组蛋白乳酸化修饰是否可以调控感染早期的巨噬细胞功能,其在巨噬细胞内又是如何被调控的尚不清楚。因此本研究通过建立巨噬细胞感染早期模型,明确感染早期巨噬细胞不同位点组蛋白乳酸化修饰的变化及其机制,揭示组蛋白乳酸化对感染早期巨噬细胞的功能调控作用,旨在为感染性疾病宿主控制炎症和改善预后提供新的靶点。
1 材料与方法
1.1 细胞和质粒
人单核细胞白血病THP-1细胞及人胚胎肾上皮细胞系HEK 293T均购自中国科学院上海细胞库。SIRT2过表达(SIRT2 overexpression,SIRT2 OE)质粒由上海儿童医学中心感染研究室李畅技术员提供。
1.2 主要试剂和仪器
佛波醇-12-肉豆蔻酸酯-13-乙酸酯(phorbol-12-myristate-13-acetate,PMA;上海碧云天生物技术有限公司),重组人单核细胞趋化蛋白-1(recombinant human monocyte chemotactic protein-1,rhMCP-1;Peprotech,美国),脂多糖(lipopolysaccharide,LPS;上海碧云天生物技术有限公司),TRIzol试剂(Life Technologies,美国),Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR和SYBR Green Realtime PCR Master Mix(上海翌圣生物科技有限公司),RPMI-1640培养基(Hyclon,美国),10%胎牛血清(Gemini,美国),0.25%胰酶(Gibco,美国),LIPOFECTAMINE 3000和puromycin(Life Technologies,美国),乙酰化及乳酸化抗体(杭州景杰生物科技股份有限公司),Anti-Rabbit/Mouse IgG抗体(CST,美国),抗SIRT2抗体(CST,美国),辣根过氧化物酶(horseradish peroxidase,HRP)-conjugated GAPDH抗体和HRP-conjugated β-actin抗体(上海翌圣生物科技有限公司),Anti-Rabbit-488抗体(Invirtrogen,美国),染色质免疫共沉淀(chromatin immunoprecipitation,ChIP)试剂盒、结晶紫和4%多聚甲醛(上海碧云天生物技术有限公司),16%甲醛(CST,美国),ECL发光试剂盒(Millipore,美国)。
生物安全柜[力新仪器(上海)有限公司],二氧化碳培养箱(Thermo Scientific,美国),电热恒温水槽(上海一恒科技有限公司),荧光倒置显微镜(Olympus,日本),水平脱色摇床(海门市其林贝尔仪器制造有限公司),涡旋振荡器(Scientific Industries,美国),冷冻离心机(Eppendorf,德国),生物分子成像仪(GE,美国),自动聚焦超声波破碎仪(Covaris,美国)。
1.3 实验方法
1.3.1 巨噬细胞感染早期细胞模型建立
THP-1细胞采用添加10%胎牛血清的RPMI-1640培养基,置于5%CO2、37 ℃恒温湿润培养箱培养。将THP-1细胞加入含100 ng/mL PMA的RPMI-1640培养基,孵育48 h后更换普通RPMI-1640培养基继续培养24 h,使其刺激分化为具有巨噬细胞特性的人血单核细胞株(PMA-primed THP-1,pTHP-1)。使用含1 μg/mL LPS处理pTHP-1细胞12 h,获得巨噬细胞早期感染的细胞模型。将未经LPS处理的巨噬细胞(pTHP-1)设为对照组(CTRL组),经LPS处理的巨噬细胞设为感染组(LPS组)。
1.3.2 蛋白质印迹法
取CTRL组及LPS组巨噬细胞,使用1.25×Loading Buffer进行裂解,99 ℃煮10 min,Nanodrop检测蛋白浓度。采用15%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)分离总蛋白,并转移至聚偏氟乙烯(polyvinylidene fluoride,PVDF)膜;5%脱脂奶粉室温封闭1 h;添加一抗包括乙酰化、乳酸化抗体(1∶1 000)、抗SIRT2抗体(1∶1 000)、抗甘油醛-3-磷酸脱氢酶(glyceraldehyde 3-phosphate dehydrogenase,GAPDH)抗体(1∶40 000)、抗β-actin抗体(1∶50 000),4 ℃孵育过夜;聚对苯二甲酸-共-丁二酸丁二醇酯[poly(butylene succinate-co-terephthalate),PBST]洗涤后加入HRP偶联的二抗(1∶3 000)孵育1 h;PBST洗涤后使用ECL发光检测试剂盒显色后成像分析。
1.3.3 RNA提取及实时荧光定量PCR
取LPS组巨噬细胞,按TRIzol试剂盒说明书提取总RNA;取总RNA 1 μg,使用反转录试剂盒合成cDNA;取cDNA 1 μL进行实时荧光定量PCR(RT-qPCR)反应,引物序列见表1。反应条件:95 ℃,30 s;95 ℃ 5 s,60 ℃ 30 s,共39个循环;再95 ℃ 15 s。
表1 RT-qPCR引物序列
Tab 1
Gene | Forward primer (5´→3´) | Reverse primer (5´→3´) |
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HIF-1α | GAACGTCGAAAAGAAAAGTCTCG | CCTTATCAAGATGCGAACTCACA |
PFKL | GTACCTGGCGCTGGTATCTG | CCTCTCACACATGAAGTTCTCC |
LDHA | ATGGCAACTCTAAAFFATCAGC | CCAACCCCAACAACTGTAATCT |
SIRT1 | AGGCCACGGATAGGTCCATA | GTGGAGGTATTGTTTCCGGC |
SIRT2 | TGCGGAACTTATTCTCCCAGA | GAGAGCGAAAGTCGGGGAT |
SIRT3 | TGCTCATCAACCGGGACTTG | TTGTCTGGTCCATCAAGCCTA |
SIRT5 | CTCAAGATGCCAGCATCCCA | AGGAAGTGCCCACCACTAGA |
HDAC1 | CATCGCTGTGAATTGGGCTG | ACCCTCTGGTGATACTTTAGCAG |
HDAC2 | TCTGCTACTACTACGACGGTGA | TCATTTCTTCGGCAGTGGCT |
HDAC3 | CATGACGGTGTCCTTCCACA | CAGAGTCAGCTCCACACTGG |
GAPDH | TCTCCTCTGACTTCAACAGCGACA | CCCTGTTGCTGTAGCCAAATTCGT |
1.3.4 Transwell趋化实验
Transwell小室提前1 h置于含RPMI-1640培养基的24孔板中孵育。取CTRL组及LPS组巨噬细胞,洗涤后取同等密度细胞悬液200 μL加入Transwell小室上室,下室加入趋化因子rhMCP-1,于37 ℃培养箱培养16 h;取出小室,4%多聚甲醛固定30 min,洗涤后结晶紫染色10 min,显微镜下观察计数。
1.3.5 慢病毒包装及细胞感染
在HEK 293T细胞中使用Lipo3000试剂盒进行SIRT2 OE质粒转染,48 h后收集病毒液并过滤;将THP-1细胞与病毒液1∶1混合,培养6 h后使用含10%胎牛血清的RPMI-1640培养基换液,继续培养48 h,使用杀稻瘟菌素(blasticidin)筛选细胞并检测细胞过表达效率。
1.3.6 RNA测序
RNA测序(RNA sequencing,RNA-seq)文库构建和Illumina测序由明码生物科技(上海)有限公司完成;使用基因本体(gene ontology,GO)数据库、京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)数据库进行基因功能注释;使用DESeq2软件分析各细胞之间的差异基因表达。
1.3.7 ChIP测序
ChIP测序技术(chromatin immunoprecipitation sequencing,ChIP-seq)是ChIP后结合高通量测序的方法。取LPS组巨噬细胞,使用1%甲醛交联10 min后加入1×甘氨酸终止交联;SDS裂解液重悬细胞后超声法破碎DNA,取10%DNA产物作为Input,其余DNA产物用于后续免疫沉淀(immunoprecipitation,IP)实验,分别加入抗组蛋白H4第8位赖氨酸乳酸化(lactylation of histone H4 lysine 8,H4K8la)抗体及抗IgG抗体,4 ℃低速混合孵育过夜;每个样本中加入30 μL protein A/G beads,4 ℃低速混合孵育2 h;按说明书洗涤DNA产物,并使用Elute buffer洗脱;加入无水乙醇沉淀产物后,使用乙二胺四乙酸(ethylenediaminetetra-acetic acid,EDTA)、三羟甲基氨基甲烷(trihydroxymethyl aminomethane,Tris)及Proteinase K解交联;使用DNA纯化试剂盒纯化DNA。后续文库构建和测序由安诺优达基因科技有限公司完成。
1.4 统计学分析
使用GraphPad Prism 9进行统计分析。每组实验进行3次独立生物学重复,组间比较采用配对t检验。P<0.05表示差异具有统计学意义。
2 结果
2.1 巨噬细胞经LPS处理后糖酵解增强
与CTRL组相比,LPS组巨噬细胞可观察到糖酵解限速酶肝脏磷酸果糖激酶(phosphofructokinase liver type,PFKL)和乳酸脱氢酶A(lactate dehydrogenase A,LDHA)显著升高,糖酵解调剂因子低氧诱导因子1α(hypoxia inducible factor 1α,HIF-1α)也显著升高(均P<0.05,图1),提示感染早期的巨噬细胞存在糖酵解通路激活。
图1
图1
RT-qPCR检测巨噬细胞糖酵解限速酶及糖酵解调节因子表达
Note: ①P=0.027, ②P=0.015, ③P=0.049, compared with the CTRL group.
Fig 1
Expression of glycolytic rate-limiting enzyme and transcription factors in macrophages detected by RT-qPCR
2.2 LPS处理显著上调巨噬细胞组蛋白H4K8乳酸化水平
与CTRL组相比,LPS组巨噬细胞组蛋白H4K8位点乳酸化修饰水平显著升高(P<0.05),而其余位点乳酸化水平均无显著升高;与此同时,12 h LPS处理也不能改变组蛋白相应位点的乙酰化水平(图2)。结果提示LPS处理可以特异性上调巨噬细胞H4K8la修饰水平。
图2
图2
蛋白质印迹法检测巨噬细胞组蛋白修饰水平
Note: A. Detection of histone lactylation (left) and acetylation (right) levels by Western blotting. B. Expression of histone lactylation (above) and acetylation (below). ①P=0.000,②P=0.002, compared with the CTRL group.
Fig 2
Histone modification levels in macrophages detected by Western blotting
2.3 SIRT2是巨噬细胞中H4K8la修饰的去修饰酶
图3
图3
LPS处理后巨噬细胞Sirtuin家族和 HDAC 家族的RNA表达水平
Note: A. Expression of Sirtuin family mRNA. B. Expression of HDAC family mRNA. C. Detection of SIRT2 protein by Western blotting. ①P=0.010, compared with the CTRL group.
Fig 3
RNA expression levels of Sirtuin family and HDAC family of macrophages after LPS treatment
图4
图4
Western blotting检测巨噬细胞SIRT2和H4K8la水平
Note: A. Detection of SIRT2 protein by Western blotting. B. Detection of H4K8la by Western blotting. ①P=0.001.
Fig 4
The levels of SIRT2 and H4K8la in macrophages detected by Western blotting
2.4 SIRT2通过去修饰H4K8la调控巨噬细胞趋化功能
图5
图5
测序结果概述
Note: A. Genomic distribution of H4k8la signal peaks. B. Volcano plot of differential genes after LPS stimulation. MERTK—MER proto-oncogene, tyrosine kinase; APBB1IP—amyloid beta precursor protein binding family B member 1 interacting protein; SLC7A8—solute carrier family 7 member 8; LRP1—LDL receptor related protein 1; ADAMTS8—ADAM metallopeptidase with thrombospondin type 1 motif 8; RAB7B—RAB7B, member RAS oncogene family; MSR1—macrophage scavenger receptor 1; SPNS2—SPNS lysolipid transporter 2, sphingosine-1-phosphate; CHST13—carbohydrate sulfotransferase 13; PRAG1—PEAK1 related, kinase-activating pseudokinase 1; SHANK3—SH3 and multiple ankyrin repeat domains 3; DHRS3—dehydrogenase/reductase 3; SPP1—secreted phosphoprotein 1; LGI2—leucine rich repeat LGI family member 2; LOXL4—lysyl oxidase like 4; TAC4—tachykinin precursor 4; P2RY12—purinergic receptor P2Y12; TRAF1—TNF receptor associated factor 1; CACNA1E—calcium voltage-gated channel subunit alpha1 E; PARM1—prostate androgen-regulated mucin-like protein 1; EBI3—Epstein-Barr virus induced 3; ABTB2—ankyrin repeat and BTB domain containing 2; CCL5—C-C motif chemokine ligand 5; PERP—p53 apoptosis effector related to PMP22; IL1B—interleukin 1 beta; CKB—creatine kinase B; RFTN1—raftlin, lipid raft linker 1; TNFAIP2—TNF alpha induced protein 2; MT2A—metallothionein 2A; ECE1—endothelin converting enzyme 1; CHST2—carbohydrate sulfotransferase 2; GIMAP8—GTPase, IMAP family member 8; TNF—tumor necrosis factor; SLAMF7—SLAM family member 7; NRP2—neuropilin 2; SERPINE2—serpin family E member 2; KCNQ4—potassium voltage-gated channel subfamily Q member 4; ADA—adenosine deaminase; SOCS3—suppressor of cytokine signaling 3; DLL4—delta like canonical Notch ligand 4; HIVEP2—HIVEP zinc finger 2; TFPI2—tissue factor pathway inhibitor 2; PIM2—Pim-2 proto-oncogene, serine/threonine kinase; KCNN2—potassium calcium-activated channel subfamily N member 2; DTX4—deltex E3 ubiquitin ligase 4; TFPI2—tissue factor pathway inhibitor 2; PTGS2—prostaglandin-endoperoxide synthase 2; MCOLN2—mucolipin TRP cation channel 2; EVA1A—eva-1 homolog A, regulator of programmed cell death; RIN3—Ras and Rab interactor 3; SLC7A2—solute carrier family 7 member 2; TNFAIP6—TNF alpha induced protein 6; IGFBP5—insulin like growth factor binding protein 5; SERPINB7—serpin family B member 7; IDO1—indoleamine 2,3-dioxygenase 1; SERPINA9—serpin family A member 9; CCL1—C-C motif chemokine ligand 1; CCR7—C-C motif chemokine receptor 7; CSF2—colony stimulating factor 2; LAMP3—lysosomal associated membrane protein 3.
Fig 5
Overview of sequencing results
图6
图6
RNA-seq及ChIP-seq交互分析
Note: A. Gene set enrichment analysis of macrophages after LPS stimulation. NES—normalized enrichment score. B. Heatmap for macrophage chemotaxis- related genes bound to H4K8la. CTRL1, CTRL2, and CTRL3 were three replicate samples from the control group, while LPS1, LPS2, and LPS3 were three replicate samples from the LPS-treated infection group. PIK3CB—phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta; DPYSL3—dihydropyrimidinase like 3; JAG1—jagged canonical Notch ligand 1; FMNL3—formin like 3; CXCL1—C-X-C motif chemokine ligand 1; PRKCD—protein kinase C delta; SMAD3—SMAD family member 3; PTAFR—platelet activating factor receptor; CDH2—cadherin 2.
Fig 6
Interaction analysis of RNA-seq and ChIP-seq
图7
图7
SIRT2过表达抑制LPS诱导的巨噬细胞趋化作用(×10)
Note: Detection of macrophage chemotaxis ability by Transwell.①P=0.008, ②P=0.766.
Fig 7
SIRT2 overexpression inhibits LPS-induced macrophage chemotaxis ability (×10)
3 讨论
作为糖酵解的代谢产物,乳酸多年来被认为是细胞代谢的废物。循环中乳酸水平升高被称为“乳酸性酸中毒”,是脓毒症患者的常见特征,已被用作指示脓毒症患者严重程度和结局的指标。同时,乳酸被证明是通过HIF-1α依赖性重编程抑制巨噬细胞极化的主要介质,可以通过剂量依赖性方式增加M2型巨噬细胞标志物的表达,并通过抑制核因子κB(nuclear factor-κB,NF-κB)活化来抑制M1型巨噬细胞[3,13-14]。某些情况下,乳酸也具有促炎作用。例如,在脓毒症情况下,乳酸也被证明会诱导中性粒细胞迁移及中性粒细胞胞外诱捕网(neutrophil extracellular trap,NET)形成[15-16],也可增加LPS刺激后巨噬细胞中白细胞介素-6(interleukin-6,IL-6)、基质金属蛋白酶1(matrix metalloproteinase 1,MMP1)及IL-1的产生[5-6],但直到最近乳酸才被发现可以直接修饰组蛋白并通过表观遗传调控的方式调节巨噬细胞功能[9]。
表观遗传修饰包括组蛋白修饰、DNA甲基化、RNA修饰、染色质重塑等方式。其中,组蛋白修饰是一种受环境因素(如代谢物、组蛋白修饰转移酶和去修饰酶等)调控的可遗传的表观修饰。由于其对免疫细胞的长久影响,感染过程中的组蛋白修饰相关调控机制受到越来越多的关注[17]。研究[9,18]发现乳酸作为一种表观遗传调控分子,可以通过组蛋白乳酸化来调控M2极化相关基因的表达。随后,陆续有研究[19-21]提示,巨噬细胞中的乳酸化修饰可通过增加下游基因表达参与损伤修复及组织纤维化过程,但现有研究[22-23]多集中在H3K18位点修饰水平及总体乳酸化修饰水平,或集中在巨噬细胞向M2方向极化,而组蛋白乳酸化是否可以调控感染早期巨噬细胞的炎症反应尚不清楚。本研究发现,当使用LPS处理巨噬细胞12 h模拟其感染早期状态时,可观察到巨噬细胞中糖酵解显著激活,且H4K8位点乳酸化修饰水平特异性显著升高,而相应位点的乙酰化水平则无明显改变,提示H4K8la可能在巨噬细胞感染早期发挥作用。
LPS诱导的H4K8la修饰水平升高,一方面可能源于糖酵解增强引起的乳酸水平升高,但另一方面也可能是H4K8la修饰的调节蛋白水平变化,修饰酶水平的升高和/或去修饰酶水平的降低导致的。现有研究[9,24]表明,组蛋白乳酸化的修饰酶是p300,去修饰酶包含HDAC1~3和SIRT1/2/3/5等多个蛋白。HDAC家族可以通过改变赖氨酸残基的乙酰化状态影响组蛋白和非组蛋白的翻译后修饰,同样被提示在体外具有去乳酸化活性[24]。Sirtuin是一类高度保守的烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD)依赖的蛋白质家族,在进化过程中高度保守,以其抗炎和抗氧化特性而闻名[25]。本研究发现,在所有的去修饰酶中,只有SIRT2的表达量在LPS处理后显著降低。SIRT2是Sirtuin家族的成员,其主要特点是可以实现乙酰赖氨酸底物的去乙酰化,并可通过该方式参与感染和炎症过程[26]。脓毒症是一种由宿主对感染的反应失调引起的危及生命的器官功能障碍,SIRT2被发现在早期脓毒症患者体内水平低于健康志愿者,且在休克患者体内水平更低[27]。一些报告[28]表明,SIRT2可通过增加NF-κB去乙酰化、调节巨噬细胞极化和增加M2相关的抗炎途径来防止炎症过程的发展。2022年,有研究[29]报道SIRT2可以作为组蛋白乳酸化的去修饰酶参与调控抑制神经母细胞瘤细胞的增殖及迁移。本研究也证实,在巨噬细胞中过表达SIRT2可显著降低组蛋白H4K8la水平,表明SIRT2确实可以在巨噬细胞中去修饰H4K8la。LPS处理巨噬细胞引起的H4K8la修饰水平增加可能是底物(乳酸)水平升高和去修饰酶(SIRT2)水平降低共同作用的结果。
为进一步探索SIRT2调控H4K8la修饰在巨噬细胞中的功能,本研究针对H4K8la抗体的ChIP-seq和LPS处理的RNA-seq进行了交互分析,并发现该修饰在调控巨噬细胞趋化功能中发挥重要作用。当SIRT2过表达时,巨噬细胞内H4K8位点乳酸化水平显著降低,同时伴有巨噬细胞趋化能力的显著降低。多项研究已证实,巨噬细胞的趋化能力在宿主的抗感染免疫中占据重要地位。例如,巨噬细胞趋化能力的增强可提高宿主的病原清除能力,并进一步激活局部的炎症反应[30]。但固有免疫系统的过度激活会导致巨噬细胞等免疫细胞破坏性侵入组织,伴随细胞因子、趋化因子及脂质介质等的过度释放,也会引起严重的组织损伤,甚至导致死亡。
综上所述,本研究首次发现SIRT2可通过去修饰组蛋白H4K8la减弱巨噬细胞趋化能力。感染早期巨噬细胞中H4K8位点的乳酸化修饰水平特异性升高,SIRT2可作为去乳酸化修饰酶降低组蛋白H4K8la修饰水平,进而抑制巨噬细胞的趋化能力,这将为控制感染性疾病中的过度炎症反应提供新的治疗靶点。
作者贡献声明
宋文汀和莫茜参与实验设计;宋文汀和潘艺完成实验操作;宋文汀、莫茜、陶悦和曹清参与论文的写作和修改。所有作者均阅读并同意了最终稿件的提交。
AUTHOR's CONTRIBUTIONS
The study was designed by SONG Wenting and MO Xi. The experimental operation was completed by SONG Wenting and PAN Yi. The manuscript was drafted and revised by SONG Wenting, MO Xi, TAO Yue and CAO Qing. All the authors have read the last version of paper and consented for submission.
利益冲突声明
本研究不存在利益冲突。
COMPETING INTERESTS
All authors disclose no relevant conflict of interests.
参考文献
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