Nadatani Yuji, Huo Xiaofang, Zhang Xi, Yu Chunhua, Cheng Edaire, Zhang Qiuyang, Dunbar Kerry B, Theiss Arianne, Pham Thai H, Wang David H, Watanabe Toshio, Fujiwara Yasuhiro, Arakawa Tetsuo, Spechler Stuart J, Souza Rhonda F
Esophageal Diseases Center, VA North Texas Health Care System and University of Texas Southwestern Medical Center, Dallas, Texas; Department of Medicine, VA North Texas Health Care System and University of Texas Southwestern Medical Center, Dallas, Texas; Department of Gastroenterology, Osaka City University Graduate School of Medicine, Osaka, Japan.
Esophageal Diseases Center, VA North Texas Health Care System and University of Texas Southwestern Medical Center, Dallas, Texas; Department of Medicine, VA North Texas Health Care System and University of Texas Southwestern Medical Center, Dallas, Texas.
Cell Mol Gastroenterol Hepatol. 2016 Mar 19;2(4):439-453. doi: 10.1016/j.jcmgh.2016.03.006. eCollection 2016 Jul.
BACKGROUND & AIMS: Microbial molecular products incite intestinal inflammation by activating Toll-like receptors (TLRs) and inflammasomes of the innate immune system. This system's contribution to esophageal inflammation is not known. Gram-negative bacteria, which dominate the esophageal microbiome in reflux esophagitis, produce lipopolysaccharide (LPS), a TLR4 ligand. TLR4 signaling produces pro-interleukin (IL)1β, pro-IL18, and NOD-like receptor protein 3 (NLRP3), which prime the NLRP3 inflammasome. Subsequent NLRP3 inflammasome activation cleaves caspase-1, inducing secretion of proinflammatory cytokines and pyroptosis (inflammatory cell death). We explored LPS effects on NLRP3 inflammasome priming and activation in esophageal cells.
We exposed esophageal squamous and Barrett's epithelial cells to LPS and measured the following: (1) TLR4, pro-IL1β, pro-IL18, and NLRP3 expression; (2) caspase-1 activity; (3) tumor necrosis factor-α, IL8, IL1β, and IL18 secretion; (4) lactate dehydrogenase (LDH) release (a pyroptosis marker); and (5) mitochondrial reactive oxygen species (ROS). As inhibitors, we used acetyl-Tyr-Val-Ala-Asp-CHO for caspase-1, small interfering RNA for NLRP3, and (2-(2,2,6,6,-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride for mitochondrial ROS.
Squamous and Barrett's cells expressed similar levels of TLR4, but LPS induced TLR4 signaling that increased tumor necrosis factor-α and IL8 secretion only in Barrett's cells. Barrett's cells treated with LPS showed increased expression of pro-IL18, pro-IL1β, and NLRP3, and increased mitochondrial ROS levels, caspase-1 activity, IL1β and IL18 secretion, and LDH release. Acetyl-Tyr-Val-Ala-Asp-CHO, NLRP3 small interfering RNA, and Mito-TEMPO all blocked LPS-induced IL1β and IL18 secretion and LDH release.
In Barrett's cells, LPS both primes and activates the NLRP3 inflammasome, causing secretion of proinflammatory cytokines and pyroptosis. By triggering molecular events promoting inflammation, the esophageal microbiome might contribute to inflammation-mediated carcinogenesis in Barrett's esophagus.
微生物分子产物通过激活天然免疫系统的Toll样受体（TLR）和炎性小体引发肠道炎症。该系统对食管炎症的作用尚不清楚。在反流性食管炎中占主导地位的革兰氏阴性菌可产生脂多糖（LPS），一种TLR4配体。TLR4信号传导产生前白细胞介素（IL）-1β、前IL-18和NOD样受体蛋白3（NLRP3），后者启动NLRP3炎性小体。随后NLRP3炎性小体激活可切割半胱天冬酶-1，诱导促炎细胞因子分泌和细胞焦亡（炎性细胞死亡）。我们探讨了LPS对食管细胞中NLRP3炎性小体启动和激活的影响。
我们将食管鳞状上皮细胞和巴雷特上皮细胞暴露于LPS中，并检测以下指标：（1）TLR4、前IL-1β、前IL-18和NLRP3的表达；（2）半胱天冬酶-1活性；（3）肿瘤坏死因子-α、IL-8、IL-1β和IL-18的分泌；（4）乳酸脱氢酶（LDH）释放（细胞焦亡标志物）；（5）线粒体活性氧（ROS）。作为抑制剂，我们使用乙酰-Tyr-Val-Ala-Asp-CHO抑制半胱天冬酶-1，使用小干扰RNA抑制NLRP3，并使用（2-(2,2,6,6-四甲基哌啶-1-氧化-4-基氨基)-2-氧代乙基）三苯基氯化鏻抑制线粒体ROS。
鳞状上皮细胞和巴雷特细胞表达相似水平的TLR4，但LPS诱导的TLR4信号传导仅在巴雷特细胞中增加肿瘤坏死因子-α和IL-8的分泌。用LPS处理的巴雷特细胞显示前IL-18、前IL-1β和NLRP3的表达增加，线粒体ROS水平、半胱天冬酶-1活性、IL-1β和IL-18的分泌以及LDH释放增加。乙酰-Tyr-Val-Ala-Asp-CHO、NLRP3小干扰RNA和Mito-TEMPO均阻断了LPS诱导的IL-1β和IL-18分泌以及LDH释放。
在巴雷特细胞中，LPS既能启动又能激活NLRP3炎性小体，导致促炎细胞因子分泌和细胞焦亡。通过触发促进炎症的分子事件，食管微生物群可能在巴雷特食管的炎症介导的致癌过程中发挥作用。
