Abstract

The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteria-induced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERK-specific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.

Highlights

  • Grants R01DK071604, DK089763, and AI064462. □S The on-line version of this article contains supplemental Figs. 1– 4. 1 Supported by Grant K01DK081481 from the NIH. 2 To whom correspondence should be addressed: Whitehead Bldg., Rm. 105F, wise indigestible complex carbohydrates, and competitive exclusion of pathogens [3, 4]

  • We investigated the extent to which commensal bacteria or fMLF activated formyl peptide receptors (FPRs) situated on the apical surface of epithelial cells and mediated an analogous reactive oxygen species (ROS) response

  • Both fMLF- and Lactobacillus rhamnosus GG (LGG)-induced ROS generation was abrogated in cells pretreated with Boc2, a competitive inhibitor of fMLF binding to FPRs [21, 22], and by Pertussis toxin (PTx), an inhibitor the G␣i subunit of G protein-coupled receptors [23] (Fig. 1A)

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Summary

Introduction

Grants R01DK071604, DK089763, and AI064462. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1– 4. 1 Supported by Grant K01DK081481 from the NIH. 2 To whom correspondence should be addressed: Whitehead Bldg., Rm. 105F, wise indigestible complex carbohydrates, and competitive exclusion of pathogens [3, 4]. To evaluate whether inhibitors of G protein-coupled receptors or FPRs could block observed ROS generation, cells were pretreated with NAC (20 mM), diphenyliodinium (DPI) (40 mM), PTx (1 ␮g/ml), or Boc2 (100 ng/ml) 30 min prior to LGG or fMLF treatment.

Results
Conclusion

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