Original Research
NOD-Like Receptor Protein 3 Inflammasome Priming and Activation in Barrett’s Epithelial Cells

https://doi.org/10.1016/j.jcmgh.2016.03.006Get rights and content
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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.

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.

Keywords

IL1β
Pyroptosis
Esophageal Squamous Cell
GERD
Cytokine

Abbreviations used in this paper

Ac-YVAD-CHO
acetyl-Tyr-Val-Ala-Asp-CHO
AIM
absent in melanoma
ASC
apoptosis-associated speck-like protein containing a caspase recruitment domain
ATP
adenosine triphosphate
DAMP
damage-associated molecular pattern
GAPDH
glyceraldehyde-3-phosphate dehydrogenase
GERD
gastroesophageal reflux disease
IL
interleukin
LPS
lipopolysaccharide
LDH
lactate dehydrogenase
mRNA
messenger RNA
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
pro
protein
PRRs
pattern-recognition receptors
qPCR
quantitative reverse-transcription polymerase chain reaction
ROS
reactive oxygen species
RT-PCR
reverse-transcription polymerase chain reaction
siRNA
small interfering RNA
TLR
Toll-like receptor
TNF
tumor necrosis factor

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Conflicts of interest The authors disclose no conflicts.

Funding Supported by Merit Review Award BX002666 from the US Department of Veterans Affairs Biomedical Laboratory Research Program (S.J.S.), the National Institutes of Health (R01-DK63621 to R.F.S. and S.J.S., K12 HD-068369-01 and K08-DK099383 to E.C., and R01-DK097340 to D.H.W.). This material is the result of work supported with resources and the use of facilities at the Dallas VA Medical Center. The contents do not represent the views of the US Department of Veterans Affairs or the United States Government.

Authors share co-first authorship.