# Immune Training of the Interleukin 6 Gene in Airway Epithelial Cells is Central to Asthma Exacerbations

## TL;DR

The study shows that repeated immune stimulation trains airway cells to release more IL-6, worsening asthma, and that this is linked to epigenetic changes in asthma patients.

## Contribution

The study identifies immune training of IL-6 in airway epithelial cells as a novel mechanism driving asthma exacerbations, linked to epigenetic hypomethylation.

## Key findings

- Exaggerated IL-6 release in airway epithelium is central to asthma exacerbations in mice.
- Repeated viral or poly(I:C) exposure trains human epithelial cells to release more IL-6.
- IL6 gene hypomethylation in asthma patients correlates with higher IL-6 expression and frequent exacerbations.

## Abstract

Epidemiological studies suggest that respiratory viral infections are major triggers of asthma exacerbations, and clinical studies have suggested the involvement of an increased interleukin‐6 (IL‐6) release. What is the pathophysiological role of IL‐6 in asthma exacerbation, and which mechanisms lead to enhanced IL‐6 release?

Exacerbations of ovalbumin‐induced experimental allergic asthma were elicited in wild‐type and IL‐6‐deficient mice by intranasal (i.n.) application of poly(I:C). Airway inflammation, cytokine expression and release, mucus production and airway hyperresponsiveness were measured. IL‐6 was neutralised by i.n. anti‐IL‐6 antibody application. The human bronchial epithelial cell line, BEAS‐2B, was stimulated with poly(I:C) and infected with human rhinovirus‐16 in vitro, followed by quantification of IL6 gene expression and DNA methylation. Genome‐wide DNA methylation was assessed in bronchial epithelial cells from adults with asthma (cohort I, n = 54) and in nasal epithelial cells from children and adults in the All‐Age‐Asthma cohort (ALLIANCE, n = 53 and n = 108 respectively).

Poly(I:C)‐induced experimental exacerbations in mice were preceded and paralleled by exaggerated IL‐6 release in the airway epithelium, with IL‐6 neutralisation completely preventing experimental exacerbations. Repetitive infection/stimulation with RV16 or poly(I:C) resulted in training of the IL‐6 release in human respiratory epithelial cells. In patients, hypomethylation at the
IL6
gene methylation was associated with high
IL6
expression and future exacerbations.

An exaggerated IL‐6 release is required for exacerbation of experimental asthma, potentially the result of viral PAMP‐induced immune training of airway epithelial cells. Additionally, patients with asthma carrying the epigenetic signature of a trained IL‐6 response exacerbate more frequently. These findings open new avenues to identify and treat exacerbation‐prone patients.

This study aims to investigate how immune activation influences the epigenetic regulation and expression of the Interleukin‐6 (IL‐6) gene during asthma exacerbations. By examining molecular mechanisms of immune training, we seek to elucidate how IL‐6‐driven inflammatory pathways contribute to airway dysfunction and worsening asthma symptoms. We deployed a multilevel, translational pipeline to identify the role of IL‐6, its gene methylation and downstream signalling in a murine model of experimental asthma exacerbation, in vitro tissue culture models and human cohorts. Murine models triangulated the central role of IL‐6 expression by using IL6 knockouts and mimicked a therapeutic approach using a neutralising antibody. The tissue culture models were conducted using a bronchial epithelial cell line (BEAS‐2B), and primary nasal and bronchial epithelial cells. In BEAS‐2B cells, repeated viral infections or stimulations with poly(I:C) resulted in increased IL‐6 release and epigenetic training was observed. Primary epithelial cells were used to confirm the effects of DNA methylation (DNAm) on IL‐6 transcription and secretion. Human cohorts were used to establish the molecular intercorrelation of DNAm, IL‐6 expression and IL‐6 trans‐signalling (IL6TS) in asthmatics. Nasal and bronchial specimens served to prove the transferability of the intercorrelation of DNAm and IL6TS between upper and lower airways. Nasal specimen data were used to identify clinical outcomes (exacerbations) to confirm the mouse model findings. In the paediatric cohort, a 36‐month follow‐up design allows for first associations with disease trajectory outcomes. This study investigates how immune activation influences the epigenetic regulation and expression of the IL‐6 gene during asthma exacerbations. Poly(I:C)‐induced excessive IL‐6 release, driven by training of innate immunity in the respiratory epithelium, is essential for the exacerbation of experimental asthma. Repeated RV16 or poly(I:C) exposure trained IL‐6 release in human respiratory epithelial cells. Asthma patients with a trained IL‐6 response, characterised by IL6 gene hypomethylation, exhibit more frequent exacerbations.