背景:巨噬细胞(Mφs)是功能动态的免疫细胞,可桥接先天和适应性免疫反应;然而,控制Mφ可塑性和先天免疫功能的潜在表观遗传机制尚未得到很好的阐明。
目的:鉴定富含巨噬细胞的lncRNAs在调节极化和先天免疫应答中的新功能。
方法:使用RNAseq对从分化的单核细胞衍生的M1和M2Mφs中分离的总RNA进行lncRNAs表达谱。LRRC75A-AS1,GAPLINC和AL139099.5敲低对巨噬细胞分化的影响,偏振标记,吞噬作用,通过流式细胞术和荧光显微镜进行抗原处理。通过多重珠阵列检查细胞因子谱,并且通过基于PCR的阵列定量细胞骨架信号通路基因。从牙周健康和患病受试者收集牙龈活检以检查lncRNAs,M1/M2标志表达。
结果:M1和M2Mφs的转录组分析鉴定了数千个差异表达的已知和新的lncRNAs。我们在极化和先天免疫中表征了三种富含Mφ的lncRNAsLRRC75A-AS1,GAPLINC和AL139099.5。敲除LRRC75A-AS1和GAPLINC下调了Mφ分化标记,并通过减少M1标记而使Mφ极化偏斜,而对M2标记没有显着影响。LRRC75A-AS1和GAPLINC敲除也减弱了细菌的吞噬作用,Mφs中的抗原加工和炎性细胞因子分泌,支持它们在增强先天免疫功能中的功能作用。机械上,LRRC75A-AS1和GAPLINC敲低通过下调多个细胞骨架信号通路的表达而损害了Mφ的迁移,表明它们在调节Mφ迁移中的关键作用。最后,我们发现LRRC75A-AS1和GAPLINC在牙周炎中上调,它们的表达与较高的M1标记相关,提示它们在体内巨噬细胞极化中的作用.
结论:我们的结果表明,极化的Mφ获得了独特的lncRNA库,并鉴定了许多以前未知的lncRNA序列。LRRC75A-AS1和GAPLINC,在牙周炎中诱发,调节Mφ极化和先天免疫功能,支持它们在炎症中的关键作用。
BACKGROUND: Macrophages (Mφs) are functionally dynamic immune cells that bridge innate and adaptive immune responses; however, the underlying epigenetic mechanisms that control Mφ plasticity and innate immune functions are not well elucidated.
OBJECTIVE: To identify novel functions of macrophage-enriched lncRNAs in regulating polarization and innate immune responses.
METHODS: Total RNA isolated from differentiating monocyte-derived M1 and M2 Mφs was profiled for lncRNAs expression using RNAseq. Impact of LRRC75A-AS1, GAPLINC and AL139099.5 knockdown was examined on macrophage differentiation, polarization markers, phagocytosis, and antigen processing by flow cytometry and florescence microscopy. Cytokine profiles were examined by multiplex bead array and cytoskeletal signaling pathway genes were quantified by PCR-based array. Gingival biopsies were collected from periodontally healthy and diseased subjects to examine lncRNAs, M1/M2 marker expression.
RESULTS: Transcriptome profiling of M1 and M2 Mφs identified thousands of differentially expressed known and novel lncRNAs. We characterized three Mφ-enriched lncRNAs LRRC75A-AS1, GAPLINC and AL139099.5 in polarization and innate immunity. Knockdown of LRRC75A-AS1 and GAPLINC downregulated the Mφ differentiation markers and skewed Mφ polarization by decreasing M1 markers without a significant impact on M2 markers. LRRC75A-AS1 and GAPLINC knockdown also attenuated bacterial phagocytosis, antigen processing and inflammatory cytokine secretion in Mφs, supporting their functional role in potentiating innate immune functions. Mechanistically, LRRC75A-AS1 and GAPLINC knockdown impaired Mφ migration by downregulating the expression of multiple cytoskeletal signaling pathways suggesting their critical role in regulating Mφ migration. Finally, we showed that LRRC75A-AS1 and GAPLINC were upregulated in periodontitis and their expression correlates with higher M1 markers suggesting their role in macrophage polarization in vivo.
CONCLUSIONS: Our results show that polarized Mφs acquire a unique lncRNA repertoire and identified many previously unknown lncRNA sequences. LRRC75A-AS1 and GAPLINC, which are induced in periodontitis, regulate Mφ polarization and innate immune functions supporting their critical role in inflammation.