基于单细胞测序分析人结直肠癌白细胞抗原D受体抗原高表达CD209和CD163低表达型巨噬细胞诱导免疫抑制的机制

Mechanism of CD14hi HLA-DRlo CD209lo CD163lo macrophages in colorectal cancer inducing negative immune regulation

目的基于生物信息学分析结直肠癌(CRC)中肿瘤相关巨噬细胞(TAM)促进趋化因子C-C基序趋化因子配体3(CCL3)的分泌引导免疫负调节的机制。方法获取人CRC和正常组织(CON)中的scRNA-seq(单细胞转录组测序)数据进行降维、聚类、细胞注释,分析不同表型TAM、调节T细胞(Tregs)UMAP分布;分析CRC-TAM基因差异表达和富集通路情况。String分析免疫负调控基因的蛋白-蛋白互作(PPI)。分析免疫负调控相关基因之间pearson相关性,基于celltalker和cellphoneDB工具评估TAM分泌CCL3与Tregs之间的相互作用。卡方检验和Wilcoxon检验用于组间差异显著性分析。结果CRC-TAM中CCL3表达量显著高于CON组(2.566±1.877比0.000±0.000 log_(2)TPM,P<0.01),且是免疫负调节途径(P<0.01)的核心基因;与免疫负调控基因包括IRF1(2.550±1.882比0.605±0.802 log_(2)TPM,t=13.221,r=0.82,P<0.05)、TNFAIP3(2.550±1.882比1.037±1.039 log_(2)TPM,t=22.419,r=0.90,P<0.01)、IFI16(0.451±0.671比0.605±0.802 log_(2)TPM,t=5.914,r=0.80,P<0.05)等的表达呈中强正相关。CCL3主要在Mf1(100%,CD14hiHLA-DRloCD209^(lo)CD163^(lo))和Mf2(93%,CD14^(hi/lo)HLA-DR^(hi)CD209^(lo)CD163^(lo))表达。CCL3阳性Mf2(CCL3+Mf2)与Treg表面配体-受体对IL1B-IL1R2/ICAM1-IL2RA/FN1-CD79A/CD14-ITGB1/CD14-ITGA4(R>3,P<0.01)有强相互作用。结论CRC中CD14^(hi/lo)HLA-DR^(hi)CD209^(lo)CD163^(lo)型TAM可能分泌CCL3募集Tregs,诱导免疫负调控。

Objective To analyze the mechanism of tumor-associated macrophage(TAM)in colorectal cancer(CRC)promoting the secretion of chemokine C-C motif chemokine ligand 3(CCL3)to induce negative immune regulation based on bioinformatics.Methods Single cell ribonucleic acid sequencing(scRNA-seq)data in human CRC and normal tissues were subjected to UMAP dimensionality reduction,cell clustering and annotation of regulatory T cells(Tregs)and different phenotypes of TAM.The differential gene expression and signal enrichment of TAM were analyzed,and the key genes involved in negative immune regulation were screened by protein protein interaction(PPI)analysis.Pearson correlation between genes related to negative immune regulation was analyzed.Interaction between CCL3 positive TAM(CCL3+TAM)and Tregs was evaluated based on celltalker and cellphoneDB tools.Chi-square test and Wilcoxon rank sum test were used to analyze the significance of pearson correlation and ligand receptor interaction.Results CCL3 was significantly up-regulated in TAM of CRC patients(2.566±1.877 vs.0.000±0.000 log_(2)TPM,P<0.01)compared with controls.CCL3 was the core gene of negative regulation of immune system process(P<0.01).CCL3 was significantly and positively correlated with IRF1(2.550±1.882 vs.0.605±0.802log_(2)TPM,t=13.221,r=0.82,P<0.05),TNFAIP3(2.550±1.882 vs.1.037±1.039log_(2)TPM,t=22.419,r=0.90,P<0.01),IFI16(0.451±0.671 vs.0.605±0.802log_(2)TPM,t=5.914,r=0.80,P<0.05)and so on.CCL3 was mainly expressed in Mf1(100%,CD14hiHLA-DR^(ho)CD209^(lo)CD163^(lo))and Mf2(93%,CD14^(hi/lo)HLA-DR^(ho)CD209^(lo)CD163^(lo)).CCL3+TAM ligand had strong interaction with Treg surface receptors such as IL1B-IL1R2/ICAM1-IL2RA/FN1-CD79A/CD14-ITGB1/CD14-ITGA4(R>3,P<0.01).Conclusion CD14^(hi/lo)HLA-DR^(ho)CD209^(lo)CD163^(lo) cells in CRC could secrete CCL3 to induce negative regulation of immunity and promote Tregs recruitment.