RNA-seq analysis of IL-1B and IL-36 responses in epidermal keratinocytes identifies a shared MyD88-dependent gene signature

IL-36 cytokines have recently emerged as mediators of inflammation in autoimmune conditions including psoriasis vulgaris (PsV) and generalized pustular psoriasis (GPP). This study used RNA-seq to profile the transcriptome of primary epidermal keratinocytes (KCs) treated with IL-1B, IL-36A, IL-36B or IL-36G. We identified some early IL-1B-specific responses (8 hours post-treatment), but nearly all late IL-1B responses were replicated by IL-36 cytokines (24 hours post-treatment). Type I and II interferon genes exhibited time-dependent response patterns, with early induction (8 hours) followed by no response or repression (24 hours). Altogether, we identified 225 differentially expressed genes (DEGs) with shared responses to all 4 cytokines at both time points (8 + 24 hours). These involved up-regulation of ligands (IL1A, IL1B, IL36G) and activating proteases (CTSS), but also up-regulation of inhibitors such as IL1RN and IL36RN. Shared IL-1B/IL-36 DEGs overlapped significantly with genes altered in PsV and GPP skin lesions, as well as genes near GWAS loci linked to autoimmune and autoinflammatory diseases (e.g., PsV, psoriatic arthritis, IBD, primary biliary cholangitis). Inactivation of MyD88 adapter protein using CRISPR/Cas9 completely abolished expression responses of such DEGs to IL-1B and IL-36G stimulation. These results provide a global view of IL-1B and IL-36 expression responses in epidermal KCs with fine-scale characterization of time-dependent and cytokine-specific response patterns. Our findings support an important role for IL-1B and IL-36 in autoimmune or autoinflammatory conditions and show that MyD88 adaptor protein mediates shared IL-1B/IL-36 responses. Cultures were treated with truncated recombinant human IL-1B, IL-36A, IL-36B or IL-36G (10 ng/ml for IL-1B; 2000 ng/ml for IL-36 cytokines). Three cell lines were used (lines A, B and C) with samples processed in 4 batches. Samples within the same batch are comparable. Experiments were performed with 8 and 24 hours of cytokine treatment (n = 2-3 per time point).

IL-36细胞因子（IL-36 cytokines）近年来被证实为寻常型银屑病（psoriasis vulgaris, PsV）、泛发性脓疱型银屑病（generalized pustular psoriasis, GPP）等自身免疫性疾病的炎症介导因子。本研究通过RNA测序（RNA-seq）对经IL-1B、IL-36A、IL-36B或IL-36G处理的原代表皮角质形成细胞（primary epidermal keratinocytes, KCs）的转录组进行了谱分析。我们鉴定出IL-1B特异性的早期应答（处理后8小时），但几乎所有IL-1B的晚期应答（处理后24小时）均可被IL-36细胞因子复现。I型与II型干扰素基因呈现时间依赖性应答模式：于处理后8小时早期诱导表达，至24小时则无应答或被抑制。综上，我们在两个时间点（8小时与24小时）均鉴定出225个对全部4种细胞因子存在共同应答的差异表达基因（differentially expressed genes, DEGs）。此类基因涵盖配体（IL1A、IL1B、IL36G）与激活蛋白酶CTSS的上调表达，同时也包含IL1RN、IL36RN等抑制剂的上调。IL-1B与IL-36的共同差异表达基因，与PsV、GPP皮肤病变中发生表达改变的基因，以及与自身免疫性、自身炎症性疾病（如PsV、银屑病关节炎、炎症性肠病、原发性胆汁性胆管炎）相关的全基因组关联研究（Genome-Wide Association Study, GWAS）位点附近的基因存在显著重叠。通过CRISPR/Cas9技术敲除MyD88衔接蛋白后，此类差异表达基因对IL-1B与IL-36G刺激的表达应答完全消失。本研究结果全面解析了表皮角质形成细胞中IL-1B与IL-36的表达应答特征，并精细刻画了时间依赖性与细胞因子特异性的应答模式。本研究的发现证实了IL-1B与IL-36在自身免疫性或自身炎症性疾病中的关键作用，并表明MyD88衔接蛋白介导了IL-1B与IL-36的共同应答通路。实验中采用截短型重组人IL-1B、IL-36A、IL-36B或IL-36G进行细胞处理，其中IL-1B的作用浓度为10 ng/ml，IL-36细胞因子的作用浓度为2000 ng/ml。本研究使用3株细胞系（A、B、C株），样本分为4批进行处理，同批次内的样本具有可比性。细胞因子处理时长分别设置为8小时与24小时，每个时间点设置2-3个生物学重复（n=2-3 per time point）。