Combined phosphoproteome analysis of the response to mycobacterial cord factor reveals a dichotomy of Mincle-dependent and -independent signaling in macrophages

Sensing of Mycobacterium tuberculosis by the immune system relies on recognition by macrophages. Mycobacterial cord factor, trehalose-6,6’-dimycolate (TDM), is the most abundant cell wall glycolipid and binds to the C-type lectin receptor Mincle. To explore the kinase signaling linking the TDM-Mincle interaction to gene expression, we employed quantitative phosphoproteome analysis using dimethyl-labeling and high-resolution mass spectrometry. TDM caused upregulation of 6.7% and suppressed 3.8% of the 14k phospho-sites identified in 3727 proteins. Mincle-dependent phosphorylation was observed for several canonical players of CLR signaling (e.g. PLC, PKC), and was enriched for PKC and GSK3 kinase motifs. Mincle-dependent activation of the PI3K-AKT-GSK3 pathway contributed to TDM-induced inflammatory gene expression and required the PI3K regulatory subunit p85. Unexpectedly, a substantial fraction of TDM-induced phosphorylation was Mincle-independent, a finding which was paralleled by RNAseq-based transcriptome data. GO and pathway enrichment analysis of both datasets concurred in the requirement for Mincle in “innate immune response”. In contrast, Mincle-independent phosphorylation and transcriptome responses to TDM were linked to “cell cycle” and to the DNA damage response. Collectively, our global analyses show substantial reprogramming of macrophages by mycobacterial cord factor and reveal a dichotomy of Mincle-dependent and –independent signaling linked to distinct biological responses

免疫系统识别结核分枝杆菌（Mycobacterium tuberculosis）的过程依赖于巨噬细胞的介导作用。分枝杆菌索状因子——海藻糖-6,6'-双霉菌酸酯（trehalose-6,6’-dimycolate, TDM）——是丰度最高的细胞壁糖脂，可结合巨噬细胞诱导型C型凝集素受体（Mincle）。为探究TDM与Mincle的相互作用如何调控下游激酶信号通路以介导基因表达，本研究采用二甲基标记法结合高分辨质谱技术开展定量磷酸化蛋白质组学分析。在3727个蛋白质所对应的14000个磷酸化位点中，TDM处理可上调6.7%的位点、下调3.8%的位点。多种C型凝集素受体（C-type lectin receptor, CLR）信号通路的经典效应分子（如磷脂酶C（PLC）、蛋白激酶C（PKC））均出现了Mincle依赖型磷酸化，且这些磷酸化位点显著富集于PKC和糖原合成激酶3（GSK3）的激酶识别基序。PI3K-AKT-GSK3通路的Mincle依赖型激活参与了TDM诱导的炎症基因表达调控，且该过程依赖于磷脂酰肌醇3-激酶（PI3K）的调节亚基p85。出乎意料的是，大部分TDM诱导的磷酸化事件并不依赖于Mincle，这一发现与基于RNA测序（RNAseq）的转录组学数据结果一致。对两组数据集开展基因本体论（GO）和通路富集分析后发现，Mincle在"天然免疫应答"过程中发挥了不可或缺的作用。与之相反，TDM诱导的Mincle非依赖型磷酸化及转录组应答则与"细胞周期"和"DNA损伤应答"密切相关。综上，本研究的全景分析表明分枝杆菌索状因子可显著重编程巨噬细胞的生物学功能，并揭示了Mincle依赖型与非依赖型信号通路分别介导不同生物学应答的二元调控机制。