NOX1 Regulates Collective and Planktonic Cell Migration: Insights From Patients With Pediatric-Onset IBD and NOX1 Deficiency.
NOX1 调节集体和浮游细胞迁移: 来自儿科发病的 IBD 和 NOX1 缺乏患者的见解。
- 作者列表："Khoshnevisan R","Anderson M","Babcock S","Anderson S","Illig D","Marquardt B","Sherkat R","Schröder K","Moll F","Hollizeck S","Rohlfs M","Walz C","Adibi P","Rezaei A","Andalib A","Koletzko S","Muise AM","Snapper SB","Klein C","Thiagarajah JR","Kotlarz D
BACKGROUND:Genetic defects of pediatric-onset inflammatory bowel disease (IBD) provide critical insights into molecular factors controlling intestinal homeostasis. NOX1 has been recently recognized as a major source of reactive oxygen species (ROS) in human colonic epithelial cells. Here we assessed the functional consequences of human NOX1 deficiency with respect to wound healing and epithelial migration by studying pediatric IBD patients presenting with a stop-gain mutation in NOX1. METHODS:Functional characterization of the NOX1 variant included ROS generation, wound healing, 2-dimensional collective chemotactic migration, single-cell planktonic migration in heterologous cell lines, and RNA scope and immunohistochemistry of paraffin-embedded patient tissue samples. RESULTS:Using exome sequencing, we identified a stop-gain mutation in NOX1 (c.160C>T, p.54R>*) in patients with pediatric-onset IBD. Our studies confirmed that loss-of-function of NOX1 causes abrogated ROS activity, but they also provided novel mechanistic insights into human NOX1 deficiency. Cells that were NOX1-mutant showed impaired wound healing and attenuated 2-dimensional collective chemotactic migration. High-resolution microscopy of the migrating cell edge revealed a reduced density of filopodial protrusions with altered focal adhesions in NOX1-deficient cells, accompanied by reduced phosphorylation of p190A. Assessment of single-cell planktonic migration toward an epidermal growth factor gradient showed that NOX1 deficiency is associated with altered migration dynamics with loss of directionality and altered cell-cell interactions. CONCLUSIONS:Our studies on pediatric-onset IBD patients with a rare sequence variant in NOX1 highlight that human NOX1 is involved in regulating wound healing by altering epithelial cytoskeletal dynamics at the leading edge and directing cell migration.
背景: 儿童发病的炎症性肠病 (IBD) 的遗传缺陷为控制肠道稳态的分子因素提供了重要的见解。NOX1 最近被认为是人结肠上皮细胞活性氧 (ROS) 的主要来源。在这里，我们通过研究在 NOX1 中呈现停止增益突变的儿童 IBD 患者，评估了人类 NOX1 缺陷对伤口愈合和上皮迁移的功能影响。 方法: NOX1 变体的功能表征包括 ROS 生成、伤口愈合、二维集体趋化迁移、异源细胞系中的单细胞浮游迁移、和石蜡包埋患者组织样本的 RNA 范围和免疫组织化学。 结果: 使用外显子组测序，我们在儿科发病的 IBD 患者中发现了 NOX1 (c.160C> T，p.54R> *) 的停止增益突变。我们的研究证实 NOX1 的功能丧失导致 ROS 活性消失，但也为人类 NOX1 缺乏提供了新的机制见解。NOX1-mutant 的细胞显示伤口愈合受损，并减弱二维集体趋化迁移。迁移细胞边缘的高分辨率显微镜显示 NOX1-deficient 细胞中丝状突起密度降低，粘着斑改变，伴随 p190A 磷酸化减少。单细胞浮游向表皮生长因子梯度迁移的评估表明，NOX1 缺乏与迁移动力学改变、方向性丧失和细胞-细胞相互作用改变有关。 结论: 我们对具有 NOX1 罕见序列变异的儿科发病 IBD 患者的研究强调，人类 NOX1 通过改变前沿的上皮细胞骨架动力学和引导细胞迁移参与调节伤口愈合。
METHODS::Chronic diseases, including inflammatory bowel disease (IBD) urgently need new biomarkers as a significant proportion of patients, do not respond to current medications. Inflammation is a common factor in these diseases and microbial sensing in the intestinal tract is critical to initiate the inflammation. We have identified ELMO1 (Engulfment and Cell Motility Protein-1) as a microbial sensor in epithelial and phagocytic cells that turns on inflammatory signals. Using a stem-cell-based "gut-in-a-dish" coculture model, we studied the interactions between microbes, epithelium and monocytes in the context of IBD. To mimic the in-vivo cell physiology, enteroid-derived monolayers (EDMs) were generated from the organoids isolated from WT and ELMO1-/- mice and colonic biopsies of IBD patients. The EDMs were infected with the IBD-associated microbes to monitor the inflammatory responses. ELMO1-depleted EDMs displayed a significant reduction in bacterial internalization, a decrease in pro-inflammatory cytokine productions and monocyte recruitment. The expression of ELMO1 is elevated in the colonic epithelium and in the inflammatory infiltrates within the lamina propria of IBD patients where the higher expression is positively correlated with the elevated expression of pro-inflammatory cytokines, MCP-1 and TNF-α. MCP-1 is released from the epithelium and recruits monocytes to the site of inflammation. Once recruited, monocytes require ELMO1 to engulf the bacteria and propagate a robust TNF-α storm. These findings highlight that the dysregulated epithelial ELMO1→MCP-1 axis can serve as an early biomarker in the diagnostics of IBD and other inflammatory disorders.
METHODS:BACKGROUND:Peripheral blood eosinophilia (PBE) is a biomarker of an aggressive multiyear natural history in adults with inflammatory bowel diseases (IBDs). Additionally, PBE at diagnosis is associated with higher disease activity in pediatric-onset IBD. We sought to determine if PBE can function as a biomarker of long-term disease severity in pediatric-onset IBD patients who are followed into adulthood. METHODS:We analyzed a consented, prospective, natural history IBD registry at an adult tertiary center from 2009 to 2018. Prevalence of PBE was evaluated in both pediatric- and adult-onset IBD patients. Demographics, clinical characteristics, and health care utilization data were compared in patients with and without PBE. RESULTS:Among 2800 adult IBD patients, 23.4% had pediatric-onset disease. PBE was found in 34% of the pediatric-onset patients compared with 26.8% of the adult-onset IBD patients (P < 0.001). In the pediatric-onset IBD cohort, PBE was associated with higher rates of allergies (P < 0.0001), but not of asthma, allergic rhinitis, or primary sclerosing cholangitis. In the adult IBD patients with pediatric-onset disease, PBE was associated with higher rates of C-reactive protein elevation (P < 0.0001), erythrocyte sedimentation rate elevation (P < 0.0001), higher health care utilization, and higher average health care charges per year (P < 0.00001). CONCLUSIONS:Peripheral blood eosinophilia was more prevalent in adult IBD patients with pediatric-onset compared with adult-onset disease. Among all IBD patients with long-term follow-up, PBE defined a subgroup with more severe illness. These data suggest that PBE may be a biomarker for a high-risk subgroup with high cost trajectory and long-term severity in pediatric-onset IBD that persists into adulthood.
METHODS::Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex pathogenesis, affecting people of all ages. They are characterized by alternating phases of clinical relapse and remission, depending on the fine balance between immune cells and the gut microbiota. The cross talk between cells of the immune system and the gut microbiota can result in either tolerance or inflammation, according to multifactorial triggers, ranging from environmental factors to genetic susceptibility. Glucocorticoid (GC) administration remains the first-line treatment for IBDs, although long-term use is limited by development of serious adverse effects. Recently, new alternative pharmacological therapies have been developed, although these are not always effective in IBD patients. There is a constant demand for effective new drug targets to guarantee total remission and improve the quality of life for IBD patients. The glucocorticoid-induced leucine zipper (GILZ) has been implicated as a promising candidate for this purpose, in view of its powerful anti-inflammatory effects that mimic those of GCs while avoiding their unwanted adverse reactions. Here we present and discuss the latest findings about the involvement of GILZ in IBDs.