Supplementary Material for: AhR Regulates Peptidoglycan-Induced Inflammatory Gene Expression in Human Keratinocytes

Bacterial peptidoglycan (PGN) stimulates toll-like receptor 2 (TLR2) on the surface of keratinocytes (KCs), triggering signaling pathways that promote an innate immune response. However, excessive TLR2 activation can lead to inappropriate inflammation, which contributes to skin conditions such as rosacea. To better treat these conditions, there is a need to understand the molecular mechanisms that regulate the cellular response to TLR2 activation in the skin. Aryl hydrocarbon receptor (AhR) is a transcription factor that modulates the immune response in KCs and is a promising therapeutic target for inflammatory skin diseases. Here, we investigated the role of the AhR in regulating the transcriptional response of human KCs to PGN. We performed whole-transcriptome sequencing in wild-type and AhR-depleted KCs after PGN stimulation. AhR depletion altered the expression of 72 genes in response to PGN, leading to increased expression of 48 genes and repression of 24 genes, including interleukin (IL)-1β. Chromatin immunoprecipitation showed that PGN stimulation resulted in AhR binding the promoters of IL-1β and IL-6 to activate them. More broadly, AhR promoted inflammatory gene expression by increasing JNK/mitogen-activated protein kinase signaling and FosB expression. Finally, we observed that AhR depletion increased TLR2 expression itself, raising the hypothesis that AhR may serve to restrain TLR2-mediated inflammation in KCs through negative feedback. Viewed together, our findings demonstrate a significant and complex role for AhR in modulating the expression of inflammatory genes in KCs in response to PGN.

细菌肽聚糖（peptidoglycan, PGN）可激活角质形成细胞（keratinocytes, KCs）表面的Toll样受体2（toll-like receptor 2, TLR2），进而触发可促进固有免疫应答的信号通路；然而过度的TLR2激活会引发异常炎症，进而诱发玫瑰痤疮等皮肤病症。为优化此类病症的治疗方案，亟需明确皮肤中调控角质形成细胞对TLR2激活产生细胞应答的分子机制。芳香烃受体（aryl hydrocarbon receptor, AhR）是一类可调控角质形成细胞免疫应答的转录因子，亦是炎症性皮肤病颇具前景的治疗靶点。本研究探讨了AhR在调控人角质形成细胞对PGN产生转录应答过程中的作用。我们对PGN刺激后的野生型与AhR敲减的角质形成细胞开展了全转录组测序，结果显示AhR敲减会改变72个基因对PGN刺激的表达水平，其中48个基因表达上调、24个基因表达下调，包括白细胞介素（interleukin, IL）-1β。染色质免疫沉淀（chromatin immunoprecipitation）实验表明，PGN刺激可促使AhR结合至IL-1β与IL-6的启动子区域并激活其转录；进一步研究发现，AhR可通过增强JNK/丝裂原活化蛋白激酶信号通路活性与上调FosB的表达，促进炎症相关基因的表达。此外我们观察到，AhR敲减会提升TLR2自身的表达水平，由此提出假说：AhR可通过负反馈机制抑制角质形成细胞中TLR2介导的炎症反应。综上，本研究结果证实，AhR在调控角质形成细胞对PGN刺激产生的炎症基因表达过程中发挥着关键且复杂的作用。