Data Sheet 1_An off-target effect of class A CpG-oligonucleotides on suppressing the cyclic GMP-AMP synthase signaling in fibroblastic reticular cells.pdf

BackgroundClass A CpG-oligonucleotides (ODNs), a Toll-like receptor 9 (TLR9) agonist, have been applied for treating inflammatory diseases and cancer in preclinical studies and clinical trials. A recent study has reported that class A ODNs can activate the Cyclic GMP-AMP synthase (cGAS) signaling to regulate the inflammatory response in human monocytes. However, it remains unknown whether class A ODNs can activate the cGAS pathways in other cell types, such as fibroblastic reticular cells (FRC), which play critical roles in modulating the immune environments during inflammatory diseases and cancer.
MethodsTo understand the role of class A ODN in regulating the cGAS signaling in FRC, we treated mouse FRC and human fibroblast with class A ODN, a cGAS agonist (HT-DNA), and combined class A and HT-DNA.
ResultsUnexpectedly, we found that class A ODNs suppress the cGAS level and downstream signaling in human and murine FRC. The class A ODN-induced suppression effect on cGAS is limited in FRC, but not other immune cell types, and is independent of TLR9. Performing pulldown assay and Mass spectrum, we found that class A ODNs regulate the cGAS level post translationally by interacting with cGAS and ZNF598, an E3 ubiquitin ligase.
ConclusionOur data reveal an unrecognized off-target effect of class A ODN on suppressing the cGAS signaling in FRCs, which should be considered when designing class A ODN regimens for inflammatory diseases and cancer.

【背景】A型CpG寡核苷酸（CpG-oligonucleotides, ODNs）作为Toll样受体9（Toll-like receptor 9, TLR9）激动剂，已在临床前研究与临床试验中被用于治疗炎症性疾病与癌症。近期有研究报道，A型ODN可激活环GMP-AMP合酶（Cyclic GMP-AMP synthase, cGAS）信号通路，以调控人单核细胞中的炎症应答。然而，目前尚不清楚A型ODN能否在其他细胞类型中激活cGAS通路，例如成纤维网状细胞（fibroblastic reticular cells, FRC）——这类细胞在炎症性疾病与癌症进程中对免疫微环境的调控发挥关键作用。
【方法】为阐明A型ODN对成纤维网状细胞中cGAS信号通路的调控作用，我们分别用A型ODN、cGAS激动剂（HT-DNA）以及二者联合处理小鼠成纤维网状细胞与人成纤维细胞。
【结果】出乎意料的是，我们发现A型ODN可在人源与鼠源成纤维网状细胞中抑制cGAS的表达水平及其下游信号通路。A型ODN对cGAS的抑制效应仅局限于成纤维网状细胞，而非其他免疫细胞类型，且该效应不依赖于TLR9。通过下拉实验（pulldown assay）与质谱（Mass spectrum）分析，我们发现A型ODN通过与cGAS及ZNF598（一种E3泛素连接酶，E3 ubiquitin ligase）相互作用，在翻译后层面调控cGAS的表达水平。
【结论】本研究揭示了A型ODN此前未被认知的脱靶效应：其可抑制成纤维网状细胞中的cGAS信号通路，该效应在设计针对炎症性疾病与癌症的A型ODN治疗方案时需予以考量。