Infection–induced Bystander-Apoptosis of Monocytes Is TNF-alpha-mediated

Phagocytosis induced cell death (PICD) is crucial for controlling phagocyte effector cells, such as monocytes, at sites of infection, and essentially contributes to termination of inflammation. Here we tested the hypothesis, that during PICD bystander apoptosis of non-phagocyting monocytes occurs, that apoptosis induction is mediated via tumor necrosis factor-alpha (TNF-α and that TNF-α secretion and -signalling is causal. Monocytes were infected with Escherichia coli (E. coli), expressing green fluorescent protein (GFP), or a pH-sensitive Eos-fluorescent protein (EOS-FP). Monocyte phenotype, phagocytic activity, apoptosis, TNF-receptor (TNFR)-1, -2-expression and TNF-α production were analyzed. Apoptosis occured in phagocyting and non-phagocyting, bystander monocytes. Bacterial transport to the phagolysosome was no prerequisite for apoptosis induction, and desensitized monocytes from PICD, as confirmed by EOS-FP expressing E. coli. Co-cultivation with non-infected carboxyfluorescein-succinimidyl-ester- (CFSE-) labelled monocytes resulted in significant apoptotic cell death of non-infected bystander monocytes. This process required protein de-novo synthesis and still occurred in a diminished way in the absence of cell-cell contact. E. coli induced a robust TNF-α production, leading to TNF-mediated apoptosis in monocytes. Neutralization with an anti-TNF-α antibody reduced monocyte bystander apoptosis significantly. In contrast to TNFR2, the pro-apoptotic TNFR1 was down-regulated on the monocyte surface, internalized 30 min. p.i. and led to apoptosis predominantly in monocytes without phagocyting bacteria by themselves. Our results suggest, that apoptosis of bystander monocytes occurs after infection with E. coli via internalization of TNFR1, and indicate a relevant role for TNF-α. Modifying monocyte apoptosis in sepsis may be a future therapeutic option.

吞噬诱导细胞死亡（Phagocytosis induced cell death, PICD）是控制感染部位吞噬细胞效应细胞（如单核细胞）的关键机制，同时对炎症终止具有重要意义。本研究旨在验证下述假说：在PICD过程中，未发生吞噬作用的单核细胞会出现旁观者凋亡，且该凋亡诱导过程由肿瘤坏死因子-α（tumor necrosis factor-alpha, TNF-α）介导，同时TNF-α的分泌与信号传导是凋亡发生的核心诱因。研究人员将表达绿色荧光蛋白（green fluorescent protein, GFP）或pH敏感型Eos荧光蛋白（Eos-fluorescent protein, EOS-FP）的大肠杆菌（Escherichia coli, E. coli）感染单核细胞，随后对单核细胞表型、吞噬活性、凋亡水平、肿瘤坏死因子受体（tumor necrosis factor receptor, TNFR）-1与-2的表达情况以及TNF-α的产生量进行了检测分析。实验结果显示，发生吞噬作用与未发生吞噬作用的单核细胞均出现了凋亡，其中未吞噬的单核细胞即为旁观者细胞。细菌转运至吞噬溶酶体并非凋亡诱导的必要条件；经脱敏处理的单核细胞可免受PICD影响，这一结论通过表达EOS-FP的大肠杆菌得到了验证。将未感染且经羧基荧光素琥珀酰亚胺酯（carboxyfluorescein-succinimidyl-ester, CFSE）标记的单核细胞与感染组共培养后，未感染的旁观者单核细胞出现了显著的凋亡死亡。该凋亡过程依赖于新生蛋白的合成，且即使在缺乏细胞间直接接触的情况下，仍会以减弱的程度发生。大肠杆菌可诱导单核细胞产生大量TNF-α，进而介导单核细胞的凋亡。采用抗TNF-α抗体进行中和处理，可显著降低单核细胞的旁观者凋亡水平。与TNFR2不同，促凋亡的TNFR1会在单核细胞表面表达下调，并于感染后30分钟内发生内化，且主要在未自身吞噬细菌的单核细胞中介导凋亡。本研究结果表明，大肠杆菌感染后，单核细胞通过TNFR1内化引发旁观者单核细胞凋亡，同时证实了TNF-α在该过程中的关键作用。靶向调节脓毒症中的单核细胞凋亡，或可成为未来的潜在治疗策略。