HIF-1α Stabilization in Flagellin-Stimulated Human Bronchial Cells Impairs Barrier Function.

Ivan Ramirez-Moral,Riekelt H Houtkooper,Natasja A Otto,Bianca L Ferreira,Menno D De Jong,Alex F De Vos,Joe M Butler,Tom Van Der Poll,Michel Van Weeghel,Xiao Yu

doi:10.3390/cells11030391

Abstract

The respiratory epithelium provides a first line of defense against pathogens. Hypoxia-inducible factor (HIF)1α is a transcription factor which is stabilized in hypoxic conditions through the inhibition of prolyl-hydroxylase (PHD)2, the enzyme that marks HIF1α for degradation. Here, we studied the impact of HIF1α stabilization on the response of primary human bronchial epithelial (HBE) cells to the bacterial component, flagellin. The treatment of flagellin-stimulated HBE cells with the PHD2 inhibitor IOX2 resulted in strongly increased HIF1α expression. IOX2 enhanced the flagellin-induced expression of the genes encoding the enzymes involved in glycolysis, which was associated with the intracellular accumulation of pyruvate. An untargeted pathway analysis of RNA sequencing data demonstrated the strong inhibitory effects of IOX2 toward key innate immune pathways related to cytokine and mitogen-activated kinase signaling cascades in flagellin-stimulated HBE cells. Likewise, the cell–cell junction organization pathway was amongst the top pathways downregulated by IOX2 in flagellin-stimulated HBE cells, which included the genes encoding claudins and cadherins. This IOX2 effect was corroborated by an impaired barrier function, as measured by dextran permeability. These results provide a first insight into the effects associated with HIF1α stabilization in the respiratory epithelium, suggesting that HIF1α impacts properties that are key to maintaining homeostasis upon stimulation with a relevant bacterial agonist.

Highlights

Under normal conditions, the human airways are protected from potentially harmful bacteria from the environment by a complex interplay between the respiratory epithelium and tissue-resident immune cells [1]
The addition of IOX2 to human bronchial epithelial (HBE) cells prior to stimulation with flagellin resulted in the accumulation of HIF1α (Figure 1D)
Sequencing showed the increased expression of genes implicated in hypoxia and anaerobic metabolism in HBE cells exposed to flagellin in the presence of IOX2 when compared with cells stimulated with flagellin alone (Figure 1E)

Summary

Introduction

The human airways are protected from potentially harmful bacteria from the environment by a complex interplay between the respiratory epithelium and tissue-resident immune cells [1]. One common feature of chronic respiratory diseases is areas of hypoxia in the airways [2,3]. Composed of mucus-secreting and ciliated cells on the most apical side, and secretory cells able to produce antimicrobial mediators intercalated, the human respiratory epithelium acts as a physical barrier preventing bacterial translocation and dissemination in the host [5]. The respiratory epithelium is equipped with a repertoire of pattern-recognition receptors (PRRs), which sense conserved microbial components; among these, Toll-like receptor 5 (TLR5) is of great importance in the epithelium [6]. Flagellin is the unique natural TLR5 agonist, and it is expressed by several mucosal pathogens, including

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