Role of reactive oxygen species and the microbiome in intestinal barrier homeostasis

在正常和病理条件下，NOx酶是细胞内ROS的主要来源。到目前为止，已描述了7个哺乳动物NOX家族成员(NOX1-NOX5，DUOX1和DUOX2)。虽然NOX2主要存在于吞噬细胞中，但NOX1存在于结肠上皮细胞，DUOX2在小肠和大肠中都有表达。由于NOX2亚单位缺陷导致的吞噬细胞来源的ROS缺陷(gp91Phox、p47Phox、p67Phox、p22Phox、p40Phox)导致慢性CGD，这是一种以反复感染为特征的原发免疫缺陷，IBD的发生率几乎为50%。NOX2亚基中的单核苷酸多态(SNPs)和罕见的亚型变异，虽然会导致NOX2来源的ROS的一些减弱，但会导致成人和极早发病的IBD，而不会导致CGD。此外，NOX1和DUOX2错义变异体失活也与极早发病的IBD有关。与此同时，在小鼠身上的研究表明，NOX1衍生的ROS通过N-甲酰肽受体信号或共生乳酸菌的刺激，可以推动结肠上皮修复。总之，这些发现支持我们的目标，即确定NOX复合体衍生的ROS在IBD和肠道屏障动态平衡中的作用。 为此，我们重点研究CGD相关性IBD(CGD IBD)，以期首先确定吞噬细胞(NOX2)来源的ROS在IBD发病机制中的作用。我们开发了一种翻译研究模式，包括有针对性的体外研究和小鼠研究，以及对上下文中的人类样本的研究，同时为CGD IBD患者开发临床治疗方案。 我们在CGD小鼠身上的研究表明，虽然这些小鼠不会自发发展为结肠炎，但有菌株特有的结肠炎易感性模式，这在很大程度上是由出生时建立的肠道微生物群介导的。这些发现支持了对CGD患者的肠道微生物群的进一步研究，包括有和没有结肠炎的患者，在造血干细胞移植前后。我们正在进行的针对CGD患者的肠道微生物组研究已经帮助确定了肠道炎症的替代标志物，以及在存在或不存在IBD的情况下影响CGD微生物组特征的重要变量。 除了肠道微生物组的研究外，我们对CGD IBD的多方面研究还包括研究肠道造血细胞和非造血细胞在细胞类型特异性NOX复合体功能和ROS产生方面的免疫学作用。尽管目前在体外和小鼠模型中建立了我们的实验方法，但我们的实验方法将通过调查审查委员会(IRB)批准的临床方案进行优化，用于人类样本的研究。

NOX enzymes are the primary source of cellular ROS under normal and pathological conditions. To date, 7 mammalian NOX family members have been described (NOX1-NOX5, DUOX1 and DUOX2). While NOX2 is mostly present in phagocytes, NOX1 is present in the colonic epithelium and DUOX2 is expressed in both the small and large intestine. Deficiencies in phagocyte-derived ROS due to defective NOX2 subunits (gp91phox, p47phox, p67phox, p22phox, p40phox) lead to chronic CGD, a primary immunodeficiency presenting with recurrent infections and an almost 50% rate of IBD. Single-nucleotide polymorphisms (SNPs) and rare hypomorphic variants in NOX2 subunits, although causing some attenuation in NOX2-derived ROS, lead to adult and very early onset IBD without causing CGD. Moreover, inactivating missense variants in NOX1 and DUOX2 have also been associated with very early onset IBD. Meanwhile, studies in mice have shown that NOX1-derived ROS, through N-formyl peptide receptor signaling or stimulation by symbiotic lactobacilli, can drive colonic epithelial repair. Together, these findings support our goal of defining the role of NOX complex-derived ROS in IBD and intestinal barrier homeostasis. To do this, we have focused on the study of CGD-associated IBD (CGD IBD) in order to first determine the role of phagocyte (NOX2)-derived ROS in IBD pathogenesis. We have developed a translational research model including targeted in vitro and murine studies, and the study of contextualized human specimens while developing, in parallel, a clinical treatment protocol for patients with CGD IBD. Our studies in CGD mice have shown that although these mice do not spontaneously develop colitis, there are strain-specific patterns of colitis susceptibility, which are largely mediated by the intestinal microbiome established at birth. These findings have supported further investigation of the intestinal microbiome in patients with CGD, including patients with and without colitis, before and after hematopoietic stem cell transplantation. Our ongoing intestinal microbiome studies in patients with CGD have already helped identify surrogate markers of intestinal inflammation, as well as significant variables affecting the CGD microbiome signature in the presence or absence of IBD. In addition to the study of the intestinal microbiome, our multifaceted approach to the study of CGD IBD includes the investigation of the immunological role of intestinal hematopoietic and non-hematopoeitic cells in relation to cell-type specific NOX complex function and ROS production. Although currently being established in in vitro and murine models, our experimental methods will be optimized for the study of human specimens through an investigational review board (IRB)-approved clinical protocol.