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

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。

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 Singlecell 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-PCR and 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.