Abstract
Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections.
Highlights
The gastrointestinal epithelium serves as an initial interface between the host and luminal microbiota [1] and initiates innate immune responses to infection
We studied a molecule known as APE-1 in gastric and intestinal cells, which is activated upon encounter of reactive oxygen species (ROS)
Our results show that APE1 limits the production of ROS in cells that form the lining of the gastrointestinal tract
Summary
The gastrointestinal epithelium serves as an initial interface between the host and luminal microbiota [1] and initiates innate immune responses to infection. Gastric and intestinal epithelial cells infected by microbial pathogens or commensal microbiota typically activate Rho GTPases leading, amongst other effects, to the production of reactive oxygen species (ROS) [2,3] that arise from the activation of the NADPH oxidase complex (Nox1) [4]. Nox family proteins are the catalytic, electron transporting subunits of Nox in non-phagocytic cells that produce superoxide [5,6]. While production of microbicidal levels of ROS in professional phagocytes via Nox is well-studied, information on ROS generation by gastric and intestinal epithelial cells in response to microbial signals via epithelial Nox is limited. The levels of ROS produced by epithelial cells are much lower than in phagocytes, and are more important in redox-sensitive signaling than direct antimicrobial killing. While NADPH oxidase can be activated in epithelial cells throughout the gut, little is known about its responses to enteric infection
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