KLF4介导的MDSCs募集与分化调控皮肤屏障形成的机制研究

Skin barrier defect or damage is the primary etiology or clinical manifestation of various skin diseases. KLF4 is known to be involved in building skin barrier; however, its mechanisms have not been clearly appreciated. We previous found that MDSCs participated in the formation of the epidermal barrier, and KLF4 knockout in MDSCs reduced the recruitment and differentiation of MDSCs in the wound site, which inhibited the wound healing. In this study, we will further address the primary signalling pathways regulated by KLF4 in such a process. We have bred the first KLF4-/-(FSP-1) mice, specifically knocking out KLF4 in MDSCs. We will use HE, IHC, WB and flow cytometry techniques to investigate the integrity of the skin barrier in KLF4-/-(FSP-1) mice. We will sort out MDSCs from bone marrow, perform proteomics analysis to probe differentially expressed proteins and employ the chromatin coprecipitation sequencing assay to find the genes whose promoter regions are specifically bound by KLF4; together with the two methods, we will find the key genes regulated by KLF4 in MDSCs. Furthermore, we try to overexpress or inhibit these particular genes in MDSCs, which are involved in the downstream signaling pathways of KLF4. We will biologically validate the proteins that play central roles. This research has the potential to reveal the molecular mechanism of KLF4-mediated MDSCs recruitment and differentiation, and its roles in building skin barrier, which theoretically contributes to the clinical therapy of skin barrier defects.

皮肤屏障缺陷或损伤是多种皮肤疾病的病因或临床表现，已知KLF4可参与调控皮肤屏障形成，但其作用机制尚不清楚。我们前期研究发现MDSCs参与表皮屏障的构成，敲除KLF4可显著减少MDSCs向伤口的募集和分化，阻碍伤口愈合。本研究拟进一步明确KLF4在该过程中的核心信号转导机制。我们已首次培育出在MDSCs中特异性敲除KLF4的KLF4-/-(FSP-1)小鼠。我们将采用HE、IHC、WB和流式细胞技术观察KLF4-/-(FSP-1)小鼠皮肤屏障的完整性；从骨髓中分选出MDSCs，用蛋白质组学手段筛选差异蛋白，并采用染色质免疫共沉淀测序技术筛选KLF4特异性结合的基因，两者结合筛选出调控MDSCs的关键基因X；在MDSCs中过表达和抑制基因X，加以生物学验证，完善下游信号通路。本课题有望首次揭示KLF4介导MDSCs募集与分化调控皮肤屏障形成的分子机制，为治疗皮肤屏障缺陷相关疾病提供理论依据。

皮肤不仅是物理屏障，还是免疫屏障，维持皮肤的免疫功能稳态至关重要，皮肤屏障缺陷或损伤是多种皮肤疾病的病因或临床表现。已知KLF4是建立皮肤屏障不可或缺的基因，但KLF4调控皮肤屏障形成的分子机制尚不清楚。课题组在前期的创伤愈合研究中发现MDSCs募集在创口处，而KLF4的敲除可显著减少MDSCs向伤口的募集和分化，阻碍伤口愈合，影响皮肤屏障的恢复。课题组拟进一步研究KLF4在皮肤屏障构建中的核心信号转导机制，提出KLF4调控MDSCs募集和分化参与皮肤屏障的形成。课题组培育出在MDSCs中特异性敲除KLF4的KLF4-/-(FSP-1)小鼠，观察到小鼠的皮肤屏障不完整，MDSCs中敲除KLF4后影响MDSCs向皮肤的趋化和募集；MDSCs不仅具有增殖分化的能力，影响皮肤基质和细胞的构成，还发挥免疫调节的功能，影响皮肤中炎症因子的释放和免疫细胞的募集。KLF4-/-(FSP-1)小鼠的皮肤中MDSCs功能受损，无法发挥免疫抑制调节功能，导致皮肤中促炎免疫细胞Th17和γδ17细胞明显增多，释放的促炎细胞因子IL-1、IL-6、TNF-α和IL-17显著增加，而在使用抗IL-17的抗体后，KLF4-/-(FSP-1)小鼠的皮肤损伤状态明显好转，皮肤中浸润的Th17和γδ17细胞显著减少，MDSCs增殖能力和免疫抑制功能增强。本项目初步揭示了KLF4介导MDSCs募集与分化调控皮肤屏障形成的分子机制，为治疗皮肤屏障缺陷相关疾病提供理论依据，团队研发的抗IL-17的抗体也有望进入临床试验。