Table 1_CREB regulates Foxp3+ST-2+ TREGS with enhanced IL-10 production.docx

IntroductionRegulatory T-cells (Tregs) are characterized by the expression of Foxp3, a master regulator involved in the development and function of Tregs. Foxp3 expression is dependent on activity of the Treg specific demethylated site (TSDR), which contains a CREB binding site. We aimed to find out how Foxp3 specific CREB deletion affects Treg expression and function.
MethodsTregs from Foxp3creCREBfl/fl mice and wild type (CREBfl/fl) mice were analyzed by flow cytometry. Cytokine analysis was performed by flow cytometry, ELISA and RT-qPCR. Gene expression analysis was performed using Affymetrix HTA2 assays, ATAC-sequencing, and Methylation-assays. For functional relevance, a CD4 T cell mediated transfer colitis was performed.
Results and discussionFoxp3creCREBfl/fl mice showed increased frequencies of Tregs (CD25+/Foxp3+) in thymus, spleen and peripheral lymph nodes and in nonlymphoid organs including lung and colon, but decreased Foxp3 expression at the single cell level. Despite decreased Foxp3 expression, enhanced expression of the IL- 33 receptor (ST-2), IL-10, IL-13, and CREM was observed. CREB deficient Tregs were highly suppressive in vitro and prevented disease activity in a CD4 T cell mediated transfer colitis in an IL-10 dependent way. Mechanistically CREB fulfils dual roles in Tregs: (1) it promotes Foxp3 expression under Steady state conditions and (2) in cooperation with CREM, CREB restricts chromatin accessibility at the ST2 locus, thereby modulating IL-33 driven immune responses. This dual regulation balances FoxP3-dependent Treg stability with IL-10 mediated suppression of inflammation.

引言：调节性T细胞（Regulatory T-cells, Tregs）以Foxp3的表达为特征，Foxp3是参与Tregs发育与功能的核心调控因子。Foxp3的表达依赖于Treg特异性去甲基化区域（Treg specific demethylated site, TSDR）的活性，该区域包含CREB结合位点。本研究旨在探究Foxp3特异性CREB敲除对Treg表达与功能的影响。
方法：采用流式细胞术分析来自Foxp3creCREBfl/fl小鼠与野生型（CREBfl/fl）小鼠的Tregs。细胞因子检测采用流式细胞术、ELISA与逆转录实时定量PCR（RT-qPCR）。基因表达分析使用Affymetrix HTA2芯片、ATAC测序（ATAC-sequencing）以及甲基化检测实验。为验证功能相关性，构建了CD4 T细胞介导的转移性结肠炎模型。
结果与讨论：Foxp3creCREBfl/fl小鼠的胸腺、脾脏、外周淋巴结以及肺与结肠等非淋巴器官中，Tregs（CD25+/Foxp3+）的频率均有所升高，但单个细胞水平的Foxp3表达量却出现下降。尽管Foxp3表达降低，IL-33受体（ST-2）、IL-10、IL-13与CREM的表达却显著上调。CREB缺陷型Tregs在体外具有极强的免疫抑制活性，且可通过IL-10依赖的途径在CD4 T细胞介导的转移性结肠炎模型中抑制疾病活动。机制上，CREB在Tregs中发挥双重调控作用：（1）在稳态条件下促进Foxp3的表达；（2）与CREM协同作用，限制ST2基因座的染色质开放程度，从而调控IL-33介导的免疫应答。这种双重调控机制平衡了FoxP3依赖的Treg稳定性与IL-10介导的炎症抑制效应。