title: NOD-Like Receptor Protein 3 Inflammasome Priming and Activation in Barrett's Epithelial Cells.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: 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.
authors: Yuji
authors: Y
authors: Nadatani
authors: 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.
authors: Xiaofang
authors: X
authors: Huo
authors: 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.
authors: Xi
authors: X
authors: Zhang
authors: 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.
authors: Chunhua
authors: C
authors: Yu
authors: Esophageal Diseases Center, VA North Texas Health Care System and University of Texas Southwestern Medical Center, Dallas, Texas; Department of Pediatrics, Children's Medical Center, University of Texas Southwestern Medical Center, Dallas, Texas.
authors: Edaire
authors: E
authors: Cheng
authors: 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.
authors: Qiuyang
authors: Q
authors: Zhang
authors: 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.
authors: Kerry B
authors: KB
authors: Dunbar
authors: Baylor Research Institute, Baylor University Medical Center, Dallas, Texas.
authors: Arianne
authors: A
authors: Theiss
authors: Esophageal Diseases Center, VA North Texas Health Care System and University of Texas Southwestern Medical Center, Dallas, Texas; Department of Surgery, VA North Texas Health Care System and University of Texas Southwestern Medical Center, Dallas, Texas.
authors: Thai H
authors: TH
authors: Pham
authors: 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; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas.
authors: David H
authors: DH
authors: Wang
authors: Department of Gastroenterology, Osaka City University Graduate School of Medicine, Osaka, Japan.
authors: Toshio
authors: T
authors: Watanabe
authors: Department of Gastroenterology, Osaka City University Graduate School of Medicine, Osaka, Japan.
authors: Yasuhiro
authors: Y
authors: Fujiwara
authors: Department of Gastroenterology, Osaka City University Graduate School of Medicine, Osaka, Japan.
authors: Tetsuo
authors: T
authors: Arakawa
authors: 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; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas.
authors: Stuart J
authors: SJ
authors: Spechler
authors: 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; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas.
authors: Rhonda F
authors: RF
authors: Souza
keywords: AIM, absent in melanoma; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; ATP, adenosine triphosphate; Ac-YVAD-CHO, acetyl-Tyr-Val-Ala-Asp-CHO; Cytokine; DAMP, damage-associated molecular pattern; Esophageal Squamous Cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GERD; GERD, gastroesophageal reflux disease; IL, interleukin; IL1β; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; NLRP3, NOD-like receptor protein 3; NOD, nucleotide-binding domain, leucine-rich repeat containing proteins; PAMP, pathogen-associated molecular pattern; PBS, phosphate-buffered saline; PRRs, pattern-recognition receptors; Pyroptosis; ROS, reactive oxygen species; RT-PCR, reverse-transcription polymerase chain reaction; TLR, Toll-like receptor; TNF, tumor necrosis factor; mRNA, messenger RNA; pro, protein; qPCR, quantitative reverse-transcription polymerase chain reaction; siRNA, small interfering RNA