Analysis of spatial mRNA expression differences in the skin of Casp-8F/-K5-Cre or Casp-8F/+K5-Cre mice

Expression of enzymatically inactive caspase-8, or deletion of caspase-8 from basal epidermal keratinocytes, triggers chronic skin inflammation in mice. Unlike similar inflammation resulting from arrest of NF-kB activation in the epidermal cells, the effect induced by caspase-8 deficiency did not depend on TNF, IL1, dermal macrophage function, or expression of the Toll-like receptor adapter proteins MyD88 or TRIF. Both interferon regulatory factor (IRF)3 and TANK-binding kinase were constitutively phosphorylated in the caspase-8-deficient epidermis, and knockdown of IRF3 in the epidermis-derived cells from these mice abolished the expression of upregulated genes. Temporal and spatial analyses of the alterations in gene expression that result from caspase-8 deficiency reveal that the changes are initiated before birth, around the time that cornification develops, and occur mainly in the suprabasal layer. Finally, we found that caspase-8-deficient keratinocytes display an enhanced response to gene activation by transfected DNA. Our findings suggest that an enhanced response to endogenous activators of IRF3 in the epidermis, presumably generated in association with keratinocyte differentiation, contributes to the skin inflammatory process triggered by caspase-8 deficiency. Total RNA was extracted from the dermis or epidermis derived from Casp-8F/-K5-Cre and Casp-8F/+K5-Cre mice at P3. RNA from two or three mice of each genotype was pooled prior to sample processing for microarray analysis. The appropriate pairs of RNA samples suited for a direct comparison of the two different genotypes under examination were differently labeled and subsequently co-hybridized onto one microarray (dual color design).在小鼠体内，酶失活型半胱天冬酶-8（caspase-8）的表达，或表皮基底角质形成细胞中caspase-8的基因缺失，均可诱发慢性皮肤炎症。与表皮细胞中核因子κB（NF-κB）活化受阻所引发的同类炎症不同，caspase-8缺乏诱导的炎症效应并不依赖于肿瘤坏死因子（TNF）、白细胞介素1（IL1）、真皮巨噬细胞功能，亦不依赖于Toll样受体衔接蛋白MyD88或TRIF的表达。在caspase-8缺陷的表皮组织中，干扰素调节因子3（IRF3）与TANK结合激酶均呈现组成型磷酸化状态；而在源自该类小鼠的表皮细胞中敲低IRF3的表达，可完全消除上调基因的表达水平。对caspase-8缺乏所致基因表达变化的时空分析显示，这些变化在出生前、角质化进程启动阶段即已发生，且主要局限于基上层。本研究还发现，caspase-8缺陷的角质形成细胞对转染DNA介导的基因活化展现出增强的响应能力。上述结果提示，表皮内IRF3的内源性激活剂响应增强（该激活剂可能伴随角质形成细胞分化过程产生），参与了caspase-8缺乏触发的皮肤炎症进程。研究人员于出生后第3天（P3），从Casp-8F/-K5-Cre与Casp-8F/+K5-Cre小鼠的真皮或表皮组织中提取总RNA。将每种基因型的2~3只小鼠的RNA混合后，进行样本处理以用于基因芯片（microarray）分析。将可直接比对两种待测基因型的合适RNA样本进行差异化标记，随后共同杂交至一张基因芯片上，即采用双色标记设计。