Mucosal Inflammation Program, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;
Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045.
Proc Natl Acad Sci U S A. 2021 May 18;118(20). doi: 10.1073/pnas.2023871118.
Metabolic changes associated with tissue inflammation result in significant extracellular acidosis (EA). Within mucosal tissues, intestinal epithelial cells (IEC) have evolved adaptive strategies to cope with EA through the up-regulation of SLC26A3 to promote pH homeostasis. We hypothesized that EA significantly alters IEC gene expression as an adaptive mechanism to counteract inflammation. Using an unbiased RNA sequencing approach, we defined the impact of EA on IEC gene expression to define molecular mechanisms by which IEC respond to EA. This approach identified a unique gene signature enriched in cyclic AMP response element-binding protein (CREB)-regulated gene targets. Utilizing loss- and gain-of-function approaches in cultured epithelia and murine colonoids, we demonstrate that EA elicits prominent CREB phosphorylation through cyclic AMP-independent mechanisms that requires elements of the mitogen-activated protein kinase signaling pathway. Further analysis revealed that EA signals through the G protein-coupled receptor GPR31 to promote induction of FosB, NR4A1, and DUSP1. These studies were extended to an in vivo murine model in conjunction with colonization of a pH reporter strain that demonstrated significant mucosal acidification in the TNFΔARE model of murine ileitis. Herein, we observed a strong correlation between the expression of acidosis-associated genes with bacterial reporter sfGFP intensity in the distal ileum. Finally, the expression of this unique EA-associated gene signature was increased during active inflammation in patients with Crohn's disease but not in the patient control samples. These findings establish a mechanism for EA-induced signals during inflammation-associated acidosis in both murine and human ileitis.
与组织炎症相关的代谢变化导致显著的细胞外酸中毒（EA）。在黏膜组织中，肠上皮细胞（IEC）通过上调 SLC26A3 来促进 pH 平衡，从而进化出适应性策略来应对 EA。我们假设 EA 会显著改变 IEC 的基因表达，作为一种适应机制来对抗炎症。我们采用无偏 RNA 测序方法，定义了 EA 对 IEC 基因表达的影响，以确定 IEC 对 EA 反应的分子机制。这种方法确定了一个独特的基因特征，富含环 AMP 反应元件结合蛋白（CREB）调节的基因靶标。在培养的上皮细胞和鼠结肠类器官中，我们利用失活和功能获得方法，证明 EA 通过环 AMP 非依赖性机制引起明显的 CREB 磷酸化，该机制需要丝裂原活化蛋白激酶信号通路的元件。进一步的分析表明，EA 通过 G 蛋白偶联受体 GPR31 信号传递，促进 FosB、NR4A1 和 DUSP1 的诱导。这些研究在 TNFΔARE 模型的鼠回肠炎中与 pH 报告菌株的定植相结合，在体内鼠模型中得到了扩展。在此，我们观察到在 TNFΔARE 模型的鼠回肠炎中，酸相关性基因的表达与细菌报告 sfGFP 强度之间存在很强的相关性。最后，在克罗恩病患者的活动性炎症期间，这种独特的与 EA 相关的基因特征的表达增加，但在患者对照样本中没有增加。这些发现为在鼠和人类回肠炎中炎症相关酸中毒期间 EA 诱导的信号建立了一种机制。
