Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability.

Kejie Chen,Nanda Kumar N Shanmugam,Bobby J Cherayil,Bryan P Hurley,Michael A Pazos,Irving Coy Allen

doi:10.1371/journal.pone.0160937

Abstract

Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis.

Highlights

The inflammasome is a cytosolic, multi-subunit protein complex that is involved in the processing and secretion of the pro-inflammatory cytokine interleukin-1ß (IL-1ß)
Infection of mouse bone marrow-derived macrophages (BMDMs) with B. infantis or B. fragilis for 1 hour followed by overnight incubation resulted in the appearance of appreciable levels of IL-1ß in the cell supernatants as determined by ELISA (Fig 1A)
The amounts of secreted IL-1ß induced by B. infantis and B. fragilis were significantly less than the amount induced by infection with Citrobacter rodentium, a Gram-negative bacterial enteropathogen that is known to activate the nucleotide-binding/leucine-rich repeat (NLR)-pyrin domain-containing 3 (NLRP3) inflammasome (Fig 1A) [23]

Summary

Introduction

The inflammasome is a cytosolic, multi-subunit protein complex that is involved in the processing and secretion of the pro-inflammatory cytokine interleukin-1ß (IL-1ß). It is assembled in response to a number of exogenous and endogenous stimuli associated with infection or tissue damage [1,2] Such stimuli are usually sensed by a specific member of the nucleotide-binding/leucine-rich repeat (NLR) family of proteins and trigger the formation of the inflammasome oligomer, which consists of the NLR, the inactive cysteine-aspartate protease pro-caspase 1 and, often, the adaptor ASC (apoptosis-associated speck-like protein with a caspase recruitment domain). Of this complex leads to the proximity-induced autoproteolytic activation of pro-caspase 1. Of the NLRs, NLR-pyrin domain-containing 3 (NLRP3) is one of the best studied, and has been shown to induce inflammasome formation in response to a number of stimuli, including potassium efflux, lysosomal destabilization and generation of reactive oxygen species [1,5]

Methods

Results

Conclusion