Defining the mechanism of skin barrier restoration by bacterial lysates

背景：AKT1控制皮肤屏障形成的多个方面：Ryan O‘Shaughnessy已经证明，AKT1基因敲除会导致物理屏障和免疫屏障的缺陷。它防止丝状颗粒的加工，并抑制核降解，这是形成坚韧的保护性角化包膜的关键部分。AKT1基因敲除的角质形成细胞降低了组织蛋白酶H(CTSH)的表达。CTSH是细丝蛋白加工和Th2细胞因子控制所必需的，Ctsh-/-小鼠表现出Th2细胞因子TSLP的表达增加。细菌裂解物对表皮屏障的影响：Catherine O‘Neill是乳杆菌和双歧杆菌裂解物有益效果方面的专家。它们加强紧密连接并促进微丝蛋白的表达，这两个都是健康皮肤屏障的关键成分。SB已经开发出一种包含这些细菌裂解物之一的局部配方，用于人体研究，在人体研究中，它们显示出减少了人类志愿者(SB个人交流)的经皮水分损失(屏障的体内标记)。该公司希望从机械上了解其裂解物是如何在屏障上产生影响的。为此，SB开发了专有技术来生产乳酸菌裂解物组分，并在其定制的分析中对其进行了测试。我们假设这些裂解物具有与上述过程相关的多种额外的有益皮肤屏障修复功能。总体方法：我们将在屏障缺陷和有能力的角质形成细胞和器官型培养中测试SB开发的微生物裂解物及其部分，以了解它们如何恢复或增强皮肤屏障。学生将进行蛋白质组学，以确定微生物裂解物功效的生物标记物。这项工作将对微生物制剂中活性物质的下游鉴定以及对皮肤屏障生物学研究的关键，不仅提高对皮肤屏障功能的了解，而且对细菌-宿主相互作用的了解也是当前皮肤生物学研究的热点。目的：1.测试细菌裂解物拯救物理和免疫皮肤屏障功能的能力。测定经细菌裂解物处理的角质形成细胞的蛋白质组图谱，以确定与屏障增强相关的途径和生物标记物。裂解物的分级和分析生物标志物以确定细菌裂解物中的关键活性物质。跨学科方面和附加值：该项目结合了细胞生物学、蛋白质组学/生物信息学和SB在微生物学和分离技术方面的独特专业知识，以生产具有生物活性的裂解物，并对皮肤屏障功能和细菌与宿主的相互作用获得新的见解。

Background: AKT1 controls multiple aspects of skin barrier formation: Ryan O'Shaughnessy has shown that AKT1 knockdown leads defects in both the physical barrier and immune barrier. It prevents filaggrin processing, and inhibits nuclear degradation, a key part of the formation of a tough, protective cornified envelope. AKT1 knockdown keratinocytes have reduced expression of a protease Cathepsin H (CTSH). CTSH is required for filaggrin processing, and control of Th2 cytokines, with Ctsh -/- mice showing increased expression of the Th2 cytokine TSLP.Effects of bacterial lysates on epidermal barrier: Catherine O'Neill is an expert on the beneficial effects of lactobacillus and bifidobacterial lysates. They enhance tight junctions and promote filaggrin expression, both key components of healthy skin barrier. SB has developed a topical formulation containing one of these bacterial lysates for use in a human study, where they showed reduced transepidermal water loss (an in vivo marker of the barrier) in human volunteers (SB personal communication). The company wishes to understand mechanistically how its lysate is inducing its effects on the barrier. To this end SB developed propriatory techniques to produce lactobacillus lysate fractions and tested them in their bespoke assays.We hypothesise that these lysates have multiple additional beneficial skin barrier restorative functions related to the processes described aboveOverall approach: We will test microbial lysates and fractions thereof developed by SB in barrier deficient and competent keratinocytes and organotypic cultures to understand how they restore or enhance skin barrier. The student will perform proteomics to identify biomarkers of efficacy of the microbial lysates. This work will be critical to SB in their aim of the downstream identification of actives within the microbial preparations, and to skin barrier biology research by improving understanding not only of skin barrier function, but also bacteria-host interaction, currently a "hot-topic" in skin biology research.Objectives:1. Testing the ability of bacterial lysates to rescue physical and immune skin barrier function2. Determining the proteomic profile of keratinocytes treated with bacterial lysates to identify pathways and biomarkers associated with barrier enhancement3. Fractionation of lysates and assaying biomarkers to identify key actives within the bacterial lysates.Interdisciplinary aspect and added value: This project combines cell biology, proteomics/bioinformatics , and SB's unique expertise in microbiology and the use of separation techniques to produce lysates with biological activity and to gain new insights into barrier function and bacteria-host interactions in skin.