An EZH2-dependent transcriptional complex promotes aberrant epithelial remodelling after injury.

Unveiling the molecular mechanisms of tissue remodelling following injury is imperative to elucidate its regenerative capacity and aberrant repair in disease. Using different omics approaches, we identified enhancer of zester homolog 2 (EZH2) as a key regulator of fibrosis in injured lung epithelium. Epithelial injury drives an enrichment of nuclear transforming growth factor-ß-activated kinase 1 (TAK1) that mediates EZH2 phosphorylation to facilitate its liberation from polycomb repressive complex 2 (PRC2). This process results in the establishment of a transcriptional complex of EZH2, RNA-polymerase II (POL2) and nuclear actin, which orchestrates aberrant epithelial repair programmes. The liberation of EZH2 from PRC2 is accompanied by an EZH2-EZH1 switch to preserve H3K27me3 deposition at non-target genes. Loss of epithelial TAK1, EZH2 or blocking nuclear actin influx attenuates the fibrotic cascade and restores respiratory homeostasis. Accordingly, EZH2 inhibition significantly improves outcomes in a pulmonary fibrosis mouse model. Our results reveal an important non-canonical function of EZH2, paving the way for new therapeutic interventions in fibrotic lung diseases.

Neutralization of both IL-1α/IL-1ß plays a major role in suppressing combined cigarette smoke/virus-induced pulmonary inflammation in mice.

Smoking is an important risk factor for the development of chronic obstructive pulmonary disease (COPD) and viral infections are believed to be major triggers of exacerbations, which periodically lead to a worsening of symptoms. The pro-inflammatory IL-1 family members IL-1α and IL-1ß are increased in COPD patients and might contribute to disease pathology. We investigated whether individual or combined inhibition of these cytokines reduced lung inflammation in cigarette smoke (CS)-exposed and H1N1-infected BALB/c mice. Animals were treated with individual or combined antibodies (Abs) directed against IL-1α, IL-1ß or IL-1R1. Cells in BAL fluid and cytokines/chemokines in lung homogenate were determined. The viral load was investigated. Blocking IL-1α had significant suppressive effects on total cells, neutrophils, and macrophages. Furthermore, it reduced KC levels significantly. Blocking of IL-1ß did not provide significant activity. In primary human bronchial epithelial air-liquid-interface cell cultures infected with H1N1, IL-1α Abs but not IL-1ß Abs reduced levels of TNF-α and IL-6. Concomitant usage of Abs against IL-1α/IL-1ß revealed strong effects in vivo and reduced total cells, neutrophils and macrophages. Additionally, levels of KC, IL-6, TNF-α, MCP-1, MIP-1α and MIP-1ß were significantly reduced and ICAM-1 and MUC5 A/C mRNA expression was attenuated. The viral load decreased significantly upon combined IL-1α/IL-1ß Ab treatment. Blocking the IL-1R1 provided significant effects on total cells, neutrophils and macrophages but was inferior compared to inhibiting both its soluble ligands IL-1α/IL-1ß. Our results suggest that combined inhibition of IL-1α/IL-1ß might be beneficial to reduce CS/H1N1-induced airway inflammation. Moreover, combined targeting of both IL-1α/IL-1ß might be more efficient compared to individual neutralization IL-1α or IL-1ß or inhibition of the IL-1R1.

Cigarette smoke exposure reduces hemorrhagic shock induced circulatory dysfunction in mice with attenuated glucocorticoid receptor function.

Introduction: We previously showed that attenuated glucocorticoid receptor (GR) function in mice (GRdim/dim) aggravates systemic hypotension and impairs organ function during endotoxic shock. Hemorrhagic shock (HS) causes impaired organ perfusion, which leads to tissue hypoxia and inflammation with risk of organ failure. Lung co-morbidities like chronic obstructive pulmonary disease (COPD) can aggravate tissue hypoxia via alveolar hypoxia. The most common cause for COPD is cigarette smoke (CS) exposure. Therefore, we hypothesized that affecting GR function in mice (GRdim/dim) and pre-traumatic CS exposure would further impair hemodynamic stability and organ function after HS. Methods: After 3 weeks of CS exposure, anesthetized and mechanically ventilated GRdim/dim and GR+/+ mice underwent pressure-controlled HS for 1h via blood withdrawal (mean arterial pressure (MAP) 35mmHg), followed by 4h of resuscitation with re-transfusion of shed blood, colloid fluid infusion and, if necessary, continuous intravenous norepinephrine. Acid-base status and organ function were assessed together with metabolic pathways. Blood and organs were collected at the end of the experiment for analysis of cytokines, corticosterone level, and mitochondrial respiratory capacity. Data is presented as median and interquartile range. Results: Nor CS exposure neither attenuated GR function affected survival. Non-CS GRdim/dim mice had a higher need of norepinephrine to keep target hemodynamics compared to GR+/+ mice. In contrast, after CS exposure norepinephrine need did not differ significantly between GRdim/dim and GR+/+ mice. Non-CS GRdim/dim mice presented with a lower pH and increased blood lactate levels compared to GR+/+ mice, but not CS exposed mice. Also, higher plasma concentrations of some pro-inflammatory cytokines were observed in non-CS GRdim/dim compared to GR+/+ mice, but not in the CS group. With regards to metabolic measurements, CS exposure led to an increased lipolysis in GRdim/dim compared to GR+/+ mice, but not in non-CS exposed animals. Conclusion: Whether less metabolic acidosis or increased lipolysis is the reason or the consequence for the trend towards lower catecholamine need in CS exposed GRdim/dim mice warrants further investigation.

IL36 is a critical upstream amplifier of neutrophilic lung inflammation in mice.

IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we combined in vivo and in vitro approaches using primary mouse and human cells, as well as, acute and chronic mouse models of lung inflammation to provide mechanistic insight into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. IL-36 receptor deficient mice exposed to cigarette smoke or cigarette smoke and H1N1 influenza virus had attenuated lung inflammation compared with wild-type controls. We identified neutrophils as a source of IL-36 and show that IL-36 is a key upstream amplifier of lung inflammation by promoting activation of neutrophils, macrophages and fibroblasts through cooperation with GM-CSF and the viral mimic poly(I:C). Our data implicate IL-36, independent of other IL-1 family members, as a key upstream amplifier of neutrophilic lung inflammation, providing a rationale for targeting IL-36 to improve treatment of a variety of neutrophilic lung diseases.