牙龈卟啉单胞菌来源的脂多糖诱导非酒精性脂肪肝的局部巨噬细胞免疫耐受表型

doi:10.7518/hxkq.2023.2023111

华西口腔医学杂志 ›› 2023, Vol. 41 ›› Issue (4): 385-394.doi: 10.7518/hxkq.2023.2023111

牙龈卟啉单胞菌来源的脂多糖诱导非酒精性脂肪肝的局部巨噬细胞免疫耐受表型

郭力嘉¹(), 刘奕彤², 陈颖怡², 徐骏疾², 刘怡²()

^1.首都医科大学附属北京口腔医院正畸科，北京 100050
^2.首都医科大学附属北京口腔医院牙周科，北京 100050

收稿日期:2023-04-11 修回日期:2023-05-01 出版日期:2023-08-01 发布日期:2023-07-21
通讯作者: 刘怡 E-mail:guolijia@mail.ccmu.edu.cn;lililiuyi@163.com
作者简介:郭力嘉，副主任医师，博士，E-mail：guolijia@mail.ccmu.edu.cn
基金资助:
国家重点研发计划(2022YFC2504200);国家自然科学基金(81600891);首都医科大学附属北京口腔医院创新团队建设项目(CXTD202202);首都医科大学附属北京口腔医院创新基金(21-09-18);首都医科大学附属北京口腔医院青年科技创新人才培育计划(YSP202105);中华口腔医学会青年临床科研基金(CSA-SIS2022-11);北京市医管局青年人才培养计划“青苗”计划(QML20181501);北京市医院管理局临床医学发展专项—“扬帆计划”(ZY-LX202121);北京市医院管理局“登峰”人才培养计划(DFL20-181501);北京市属医院科研培育计划(PX2023054)

Liver macrophages show an immunotolerance phenotype in nonalcoholic fatty liver combined with Porphyromonas gingivalis-lipopolysaccharide infection

Guo Lijia¹(), Liu Yitong², Chen Yingyi², Xu Junji², Liu Yi²()

^1.Dept. of Orthodontics, Beijing Stomatological Hospital, Capital Medical University, Beijing 100050, China
^2.Dept. of Periodontics, Beijing Stomatological Hospital, Capital Medical University, Beijing 100050, China

Received:2023-04-11 Revised:2023-05-01 Online:2023-08-01 Published:2023-07-21
Contact: Liu Yi E-mail:guolijia@mail.ccmu.edu.cn;lililiuyi@163.com
Supported by:
National Key R&D Program of China(2022YFC2504200);National Nature Science Foundation of China(81600891);Innovation Research Team Project of Beijing Stomatological Hospital, Capital Medical University(CXTD202202);Innovation Foundation of Beijing Stomatological Hospital, Capital Medical University(21-09-18);Young Scientist Program of Beijing Stomatological Hospital, Capital Medical University(YSP202105);Young Clinical Research Fund of Chinese Stomatological Association(CSA-SIS2022-11);Beijing Municipal Administration of Hospitals’ Youth Programme(QML20181501);Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support(ZYLX202121);Beijing Municipal Administration of Hospitals’ Ascent Plan(DFL20181501);Beijing Municipal Administration of Hospitals Incubating Program(PX2023054);Correspondence: Liu Yi, E-mail: lililiuyi@163.com

摘要/Abstract

摘要：

目的探索牙龈卟啉单胞菌感染的非酒精性脂肪肝局部巨噬细胞功能变化及潜在调控靶点。方法利用单细胞测序技术分析合并牙龈卟啉单胞菌感染的非酒精性脂肪性肝炎（NASH）小鼠肝脏中的各类细胞表型及其功能变化。进一步利用实时荧光定量聚合酶链反应、酶联免疫吸附和免疫荧光染色观察肝脏组织中的炎症程度及巨噬细胞抗原呈递功能标志物表达水平，利用油红染色观察NASH肝脏局部脂肪组织堆积情况。体外利用牙龈卟啉单胞菌来源的脂多糖干预小鼠腹腔巨噬细胞，通过实时荧光定量聚合酶链反应和转录组测序验证体内试验结果。结果与健康肝脏中的巨噬细胞相比，牙龈卟啉单胞菌感染的NASH小鼠肝脏巨噬细胞表现出了显著的异质性，其高表达包括C1qb、C1qc、Mafb、Apoe和Cd14在内的多个炎症基因，但与抗原提呈功能相关的基因Cd209a、H2-Aa、H2-Ab1和H2-DMb1等的表达相对较低。进一步的体内外研究表明，这些巨噬细胞的活化和浸润可能是由于局部牙龈卟啉单胞菌来源的脂多糖的积累和诱导造成的。结论牙龈卟啉单胞菌来源的脂多糖诱导非酒精性脂肪肝的局部巨噬细胞免疫耐受表型，可能是牙周炎致病菌感染促进NASH炎症和发病的关键机制。本研究结果进一步阐明巨噬细胞在NASH相关疾病发病过程中的功能障碍和调节机制，并为其临床治疗提供了几个潜在的调控靶点。

关键词: 牙龈卟啉单胞菌, 非酒精性脂肪性肝炎, 巨噬细胞, 抗原呈递, 单细胞测序

Abstract:

Objective This study aimed to explore the functions and potential regulatory targets of local macrophages in nonalcoholic fatty liver combined with Porphyromonas gingivalis (P. gingivalis)infection. Methods Single-cell RNA sequencing was used to analyze the phenotypes and functional changes in various cells in the liver tissue of nonalcoholic steatohepatitis (NASH) mice fed with P. gingivalis. Real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay, and immunofluorescence staining were applied to observe the inflammation and expression levels of macrophage antigen presenting functional markers in the NASH liver. Oil red staining was performed to observe the accumulation of local adipose tissue in the NASH liver. Results were verified through RT-PCRand RNA sequencing using P. gingivalis-lipopolysaccharide treated mouse peritoneal macrophages. Results In comparison with healthy livers with Kupffer cells, the NASH liver combined with P. gingivalis infection-related macrophages showed significant heterogeneity. C1qb, C1qc, Mafb, Apoe, and Cd14 were highly expressed, but Cd209a, H2-Aa, H2-Ab1, and H2-DMb1, which are related to the antigen presentation function, were weakly expressed. Further in vivo and in vitro investigations indicated that the activation and infiltration of these macrophages may be due to local P. gingivalis-lipopolysaccharide accumulation. Conclusion P. gingivalis-lipopolysaccharide induces a local macrophage immunotolerance phenotype in nonalcoholic fatty liver, which may be the key mechanism of periodontitis pathogen infection that promotes NASH inflammation and pathogenesis. This study further clarifies the dysfunction and regulatory mechanisms of macrophages in the pathogenesis of P. gingivalis-infected NASH, thereby providing potential therapeutic targets for its clinical treatment.

Key words: Porphyromonas gingivalis, nonalcoholic steatohepatitis, macrophage, antigen presentation, single-cell RNA sequencing

中图分类号:

R 780.2

郭力嘉, 刘奕彤, 陈颖怡, 徐骏疾, 刘怡. 牙龈卟啉单胞菌来源的脂多糖诱导非酒精性脂肪肝的局部巨噬细胞免疫耐受表型[J]. 华西口腔医学杂志, 2023, 41(4): 385-394.

Guo Lijia, Liu Yitong, Chen Yingyi, Xu Junji, Liu Yi. Liver macrophages show an immunotolerance phenotype in nonalcoholic fatty liver combined with Porphyromonas gingivalis-lipopolysaccharide infection[J]. West China Journal of Stomatology, 2023, 41(4): 385-394.

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参考文献 32

1	Xu X, Poulsen KL, Wu L, et al. Targeted therapeutics and novel signaling pathways in non-alcohol-associated fatty liver/steatohepatitis (NAFL/NASH)[J]. Signal Transduct Target Ther, 2022, 7(1): 287.
2	Zhang C, Yang M. Current options and future directions for NAFLD and NASH treatment[J]. Int J Mol Sci, 2021, 22(14): 7571.
3	Jayakumar S, Loomba R. Review article: emerging role of the gut microbiome in the progression of nonalcoho-lic fatty liver disease and potential therapeutic implications[J]. Aliment Pharmacol Ther, 2019, 50(2): 144-158.
4	Nagasaki A, Sakamoto S, Arai T, et al. Elimination of Porphyromonas gingivalis inhibits liver fibrosis and inflammation in NASH[J]. J Clin Periodontol, 2021, 48(10): 1367-1378.
5	Sumida Y, Yoneda M. Current and future pharmacolo-gical therapies for NAFLD/NASH[J]. J Gastroenterol, 2018, 53(3): 362-376.
6	Diskin C, Pålsson-McDermott EM. Metabolic modulation in macrophage effector function[J]. Front Immunol, 2018, 9: 270.
7	Barreby E, Chen P, Aouadi M. Macrophage functional diversity in NAFLD-more than inflammation[J]. Nat Rev Endocrinol, 2022, 18(8): 461-472.
8	Kazankov K, Jørgensen SMD, Thomsen KL, et al. The role of macrophages in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis[J]. Nat Rev Gastroenterol Hepatol, 2019, 16(3): 145-159.
9	Mehal WZ, Schuppan D. Antifibrotic therapies in the li-ver[J]. Semin Liver Dis, 2015, 35(2): 184-198.
10	Pellicoro A, Ramachandran P, Iredale JP, et al. Liver fibrosis and repair: immune regulation of wound healing in a solid organ[J]. Nat Rev Immunol, 2014, 14(3): 181-194.
11	Ramachandran P, Pellicoro A, Vernon MA, et al. Diffe-rential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis[J]. PNAS, 2012, 109(46): E3186-E3195.
12	Tacke F, Zimmermann HW. Macrophage heterogeneity in liver injury and fibrosis[J]. J Hepatol, 2014, 60(5): 1090-1096.
13	Karlmark KR, Weiskirchen R, Zimmermann HW, et al. Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis[J]. He-patology, 2009, 50(1): 261-274.
14	Duffield JS, Forbes SJ, Constandinou CM, et al. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair[J]. J Clin Invest, 2005, 115(1): 56-65.
15	Schuppan D, Surabattula R, Wang XY. Determinants of fibrosis progression and regression in NASH[J]. J Hepatol, 2018, 68(2): 238-250.
16	Eming SA, Wynn TA, Martin P. Inflammation and metabolism in tissue repair and regeneration[J]. Science, 2017, 356(6342): 1026-1030.
17	Murray PJ, Allen JE, Biswas SK, et al. Macrophage activation and polarization: nomenclature and experimental guidelines[J]. Immunity, 2014, 41(1): 14-20.
18	Hou R, Huang R, Zhou Y, et al. Single-cell profiling of the microenvironment in decidual tissue from women with missed abortions[J]. Fertil Steril, 2023, 119(3): 492-503.
19	Xia H, Liu Y, Xia G, et al. Novel isoquinoline alkaloid litcubanine A—A potential anti-inflammatory candidate[J]. Front Immunol, 2021, 12: 685556.
20	Caligiuri A, Gentilini A, Marra F. Molecular pathogenesis of NASH[J]. Int J Mol Sci, 2016, 17(9): E1575.
21	Poynard T, Munteanu M, Charlotte F, et al. Impact of steatosis and inflammation definitions on the performance of NASH tests[J]. Eur J Gastroenterol Hepatol, 2018, 30(4): 384-391.
22	Arrese M, Cabrera D, Kalergis AM, et al. Innate immunity and inflammation in NAFLD/NASH[J]. Dig Dis Sci, 2016, 61(5): 1294-1303.
23	Schuster S, Cabrera D, Arrese M, et al. Triggering and resolution of inflammation in NASH[J]. Nat Rev Gastroenterol Hepatol, 2018, 15(6): 349-364.
24	Guilliams M, Scott CL. Liver macrophages in health and disease[J]. Immunity, 2022, 55(9): 1515-1529.
25	刘崇武, 吴春芳, 骆凯, 等. 牙龈卟啉单胞菌脂多糖和白细胞介素-10对高脂血症兔肺巨噬细胞SR-A表达的影响[J]. 福建医科大学学报, 2014, 48(4): 213-216.
	Liu CW, Wu CF, Luo K, et al. The impact of Porphyromonas gingivalis lipopolysaccharide and IL-10 on the expression of SR-A in hyperlipidemic rabbit alveolar macrophages[J]. J Fujian Med Univ, 2014, 48(4): 213-216.
26	申道南, 吴亚菲, 赵蕾. 牙周致病菌在动脉粥样硬化发生发展中的作用研究[J]. 中华口腔医学杂志, 2021, 56(6): 584-590.
	Shen DN, Wu YF, Zhao L. Roles of periodontal pathogens in the pathogenesis of atherosclerosis[J]. Chin J Sto-matol, 2021, 56(6): 584-590.
27	Willems E, Alkema W, Keizer-Garritsen J, et al. Biosynthetic homeostasis and resilience of the complement system in health and infectious disease[J]. EBioMedicine, 2019, 45: 303-313.
28	Civeira-Marín M, Cenarro A, Marco-Benedí V, et al. A-POE genotypes modulate inflammation independently of their effect on lipid metabolism[J]. Int J Mol Sci, 2022, 23(21): 12947.
29	Tran MTN, Hamada M, Jeon H, et al. MafB is a critical regulator of complement component C1q[J]. Nat Commun, 2017, 8(1): 1700.
30	Wu Z, Zhang Z, Lei Z, et al. CD14: biology and role in the pathogenesis of disease[J]. Cytokine Growth Factor Rev, 2019, 48: 24-31.
31	Maurer M, von Stebut E. Macrophage inflammatory protein-1[J]. Int J Biochem Cell Biol, 2004, 36(10): 1882-1886.
32	Orci LA, Kreutzfeldt M, Goossens N, et al. Tolerogenic properties of liver macrophages in non-alcoholic steatohepatitis[J]. Liver Int, 2020, 40(3): 609-621.

基因名称	序列（5’-3’）
Gapdh	F：GAGAGGCCCTATCCCAACTC
Gapdh	R：TCAAGAGAGTAGGGAGGGCT
Tnfa	F：CCACCACGCTCTTCTGTCTA
Tnfa	R：TGGTTTGTGAGTGTGAGGGT
Nos2	F：CGGAGATCAATGTGGCTGTG
Nos2	R：GAAGGACTCTGAGGCTGTGT
Cd209a	F：CTGGGAGAGGAAGACTGTGC
Cd209a	R：CTTGCTAGGGCAGGAAGTTG
H2-Aa	F：TTCTTCAAGTGACCCCCAAC
H2-Aa	R：AATTCCAAGGGTGTGTGAGC
H2-Eb1	F：CTGGAACACCACAACCTCCT
H2-Eb1	R：TTCCGGAACCATCTGACTTC
H2-Ab1	F：TCTGCTCCGAATTCCTGACT
H2-Ab1	R：TCACAAGAGCTGAGGTGGTG
H2-DMb1	F：AACAAGGAGAAGACGGCTCA
H2-DMb1	R：GTGTAGACGTCCCCGTAGGA
Cd14	F：TCTCAGATCCGAAGCCAGAT
Cd14	R：CGTGTCCACACGCTTTAGAA

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牙龈卟啉单胞菌来源的脂多糖诱导非酒精性脂肪肝的局部巨噬细胞免疫耐受表型

Liver macrophages show an immunotolerance phenotype in nonalcoholic fatty liver combined with Porphyromonas gingivalis-lipopolysaccharide infection

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