The role of complement in epithelial cell interactions with the gut microbiome

肠上皮是一个高度动态的器官，具有持续的细胞分裂，成熟为不同的细胞类型，以及替换，所有这些都在被微生物严重污染的环境中。我们对上皮细胞如何感知这些微生物并将抗微生物防御释放到管腔中的理解还远未完成。事实上，在这种关系中，我们最大的抗微生物系统补体的贡献几乎完全被忽视了。补体是一种蛋白质网络，激活后以级联方式裂解，片段组装成具有新特性的分子。新的特性包括标记生物体，让我们的白色血细胞摧毁，直接摧毁微生物和垂死的细胞，以及炎症。炎症由C3 a和C5 a引发。在NSERC的资助下，我已经成为补体和肠道科学的领导者，包括发现上皮细胞具有C3 a和C5 a的受体并对其做出反应。尽管取得了这一进展，但尚不清楚肠道是否完全装备了补体的所有成分。 这种更新将通过两种方法来解决我们知识中的这一差距; 1）测量由细胞生长为“类器官”所产生的补体分子，这是一种生长没有所有其他细胞类型的肠上皮细胞的新方法，以及2）确定补体如何在使用细菌感染结肠的小鼠中被激活。类器官是在皮氏培养皿中生长的来自肠干细胞的完全成熟的小肠或大肠上皮的三维管状网络。我将从小鼠中培养类器官，并测量补体分子和受体的水平，以确定哪些存在。当我知道哪些补体分子存在时，我将从缺乏这些分子的小鼠中培养类器官，并测量对细胞发育的影响。当我知道存在哪些分裂补体分子的受体时，我将培养类器官并添加结合受体的分子，并测量细胞的类型和数量及其抗微生物分泌物。重要的是，我将使用类器官研究潘氏细胞，这是一种上皮细胞，可以保护我们免受细菌的侵害，但在其他方面很难研究。然后，我将使用缺乏某些补体分子的小鼠，并用结肠细菌病原体感染它们。我已经知道感染触发补体激活，测量为增加C3 a和C5 a，但我不知道如何或什么补体分子在级联导致这些增加。我将使用选定的补体缺陷小鼠来鉴定哪些分子被感染激活。我未来的计划是利用这些信息进行实验，以发现补体如何影响肠道中的细菌（我们的微生物组）。

The intestinal epithelium is a highly dynamic organ with ongoing cell division, maturation into different cell types, and replacement, all in an environment heavily polluted with microbes. Our understanding of how epithelial cells sense these microorganisms and discharge anti-microbial defenses into the lumen is far from complete. In fact, the contribution of our single-greatest anti-microbial system, complement, has been almost totally neglected in this relationship. Complement is a network of proteins that upon activation become cleaved in a cascade fashion with the fragments assembling into molecules with new properties. The new properties include tagging the organisms for our white blood cells to destroy, the direct destruction of the microbes and dying cells, and inflammation. Inflammation is triggered by C3a and C5a. With NSERC funding I have emerged as a leader in the science of complement and the gut, including by discovering that epithelial cells possess receptors for and respond to C3a and C5a. Despite this progress it is unclear whether the intestine is fully armed with all the components of complement.  This renewal will address this gap in our knowledge through 2 approaches; 1) measure the complement molecules made by cells growing as “organoids”, a new way to grow intestinal epithelial cells devoid of all other cell types, and 2) determine how complement becomes activated in mice using a bacterial infection of their colons. Organoids are 3-dimensional tubular networks of fully mature small or large intestinal epithelium derived from intestinal stem cells, growing in petri dishes. I will grow organoids from mice and measure levels of complement molecules and receptors to determine which are present. When I know which complement molecules are present I will grow organoids from mice lacking these molecules and measure the impact on cell development. When I know which receptors for split complement molecules are present I will grow organoids and add the molecule that binds the receptor and measure the types and numbers of cells and their anti-microbial secretions. Importantly, using organoids I will study Paneth cells, a cell in the epithelium that protects us from bacteria but that is otherwise very difficult to study. Then, I will use mice deficient in certain complement molecules and infect them with a bacterial pathogen of the colon. I already know that the infection triggers complement activation, measured as increased C3a and C5a, but I do not know how or what complement molecules are in the cascade leading to these increases. I will use selected complement deficient mice to identify which molecules are activated by the infection. My future plan is to use this information to conduct experiments to discover how complement affects the bacteria living in the intestine (our microbiome).