Michael Wegmann

The NLRP3 inflammasome is a multi‐protein complex that plays a crucial role in inflammatory processes mediated by the innate immune system. Dysregulated NLRP3 activation has been implicated in age‐related inflammatory diseases, making it a promising therapeutic target. Here, we report that the synthetic membrane‐active antimicrobial peptide Pep19‐2.5 directly inhibits NLRP3 inflammasome activation. Through cellular, biophysical, and biochemical analyses, we find that Pep19‐2.5 suppresses NLRP3 inflammasome signaling downstream of NLRP3 activation. Pep19‐2.5 interacts with macrophage membranes, supporting a membrane‐targeting mechanism for its anti‐inflammatory effects. Mechanistically, Pep19‐2.5 binds to phosphatidylinositol (PI)‐containing lipid membranes and dispersed trans‐Golgi network (dTGN) structures, which could potentially affect NLRP3 recruitment to the dTGN. We demonstrate a strong and NLRP3‐dependent induction of IL‐1β secretion from human macrophages by house dust mite (HDM) extract, which can be inhibited by Pep19‐2.5. In line with these findings, therapeutic application of Pep19‐2.5 via the nasal aerosol route reduces IL‐1β levels, eosinophil infiltration in bronchoalveolar lavage and significantly improved lung function in an in vivo HDM‐mouse model of allergic airway inflammation. Our findings highlight the therapeutic potential of targeting NLRP3 activation by the small membrane‐active peptide Pep19‐2.5 for the treatment of NLRP3‐driven inflammatory diseases.

Environmental factors are often detrimental; however, certain environments enhance immune resilience. Notably, children raised on traditional farms show reduced allergies and asthma prevalence. Here, we investigated how a beneficial environment, using farm dust (FD) extract, influenced lung immune function in ovalbumin-induced allergic inflammation. FD exposure reduced allergic lung inflammation and increased monocyte-derived macrophage (MDM) recruitment. Single-cell RNA sequencing revealed that FD-exposed MDMs had altered gene expression, including dampened Ccl8 and major histocompatibility complex class II expression, impairing eosinophil recruitment and antigen presentation. RNA sequencing and assay for transposase-accessible chromatin using sequencing confirmed FD-induced epigenetic reprogramming ex vivo, on bone marrow–derived macrophages. This modulation, seen in both human and murine cells, relied on histone deacetylase activity sustained by peroxisome proliferator–activated receptor γ signaling. These findings suggest that beneficial environmental exposures can reprogram immune cells and may offer a previously unidentified strategy for asthma prevention.

Question: 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? Materials and Methods: 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). Results: 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. Answer: 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.

Epigenetics regulates gene expression and thus cellular processes that underlie the pathogenesis of chronic lung diseases such as chronic obstructive pulmonary disease (COPD), asthma, and idiopathic pulmonary fibrosis (IPF). Environmental factors (e.g., air pollution, smoking, infections, poverty), but also conditions such as gastroesophageal reflux, induce epigenetic changes long before lung disease is diagnosed. Therefore, epigenetic signatures have the potential to serve as biomarkers that can be used to identify younger patients who are at risk for premature loss of lung function or diseases such as IPF. Epigenetic analyses also contribute to a better understanding of chronic lung disease. This can be used directly to improve therapies, as well as for the development of innovative drugs. Here, we highlight the role of epigenetics in the development and progression of chronic lung disease, with a focus on DNA methylation.

Question: Epidemiological studies suggest 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? Material & Methods: Exacerbations of ovalbumin-induced experimental allergic asthma were elicited in wildtype and IL-6 deficient mice by intra-nasal (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. Human bronchial epithelial cells were exposed to poly(I:C) or infected with human rhinovirus-16, with IL6 expression and DNA methylation quantified. Genome-wide DNA methylation was assessed in airway 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). Results: 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. Answer: 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.

Introduction: Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the IL-1 Family, is a key mediator ofautoinflammatory diseases associated with the development of macrophage activation syndrome (MAS).High levels of IL-18 correlate with MAS and COVID-19 severity and mortality, particularly in COVID-19patients with MAS. As an inflammation inducer, IL-18 binds its receptor IL-1 Receptor 5 (IL-1R5), leadingto the recruitment of the co-receptor, IL-1 Receptor 7 (IL-1R7). This heterotrimeric complex subsequentlyinitiates downstream signaling, resulting in local and systemic inflammation.Methods: We reported earlier the development of a novel humanized monoclonal anti-human IL-1R7 antibody whichspecifically blocks the activity of human IL-18 and its inflammatory signaling in human cell and wholeblood cultures. In the current study, we further explored the strategy of blocking IL-1R7 inhyperinflammation in vivo using animal models.Results: We first identified an anti-mouse IL-1R7 antibody that significantly suppressed mouse IL-18 andlipopolysaccharide (LPS)-induced IFNg production in mouse splenocyte and peritoneal cell cultures. Whenapplied in vivo, the antibody reduced Propionibacterium acnes and LPS-induced liver injury and protectedmice from tissue and systemic hyperinflammation. Importantly, anti-IL-1R7 significantly inhibited plasma,liver cell and spleen cell IFNg production. Also, anti-IL-1R7 downregulated plasma TNFa, IL-6, IL-1b,MIP-2 production and the production of the liver enzyme ALT. In parallel, anti-IL-1R7 suppressed LPSinducedinflammatory cell infiltration in lungs and inhibited the subsequent IFNg production andinflammation in mice when assessed using an acute lung injury model.Discussion: Altogether, our data suggest that blocking IL-1R7 represents a potential therapeutic strategy to specificallymodulate IL-18-mediated hyperinflammation, warranting further investigation of its clinical application intreating IL-18-mediated diseases, including MAS and COVID-19.

Chronic airway inflammation is the cornerstone on which bronchial asthma arises, and in turn, chronic inflammation arises from a complex interplay between environmental factors such as allergens and pathogens and immune cells as well as structural cells constituting the airway mucosa. Airway epithelial cells (AECs) are at the center of these processes. On the one hand, they represent the borderline separating the body from its environment in order to keep inner homeostasis. The airway epithelium forms a multi-tiered, self-cleaning barrier that involves an unstirred, discontinuous mucous layer, the dense and rigid mesh of the glycocalyx, and the cellular layer itself, consisting of multiple, densely interconnected cell types. On the other hand, the airway epithelium represents an immunologically highly active tissue once its barrier has been penetrated: AECs play a pivotal role in releasing protective immunoglobulin A. They express a broad spectrum of pattern recognition receptors, enabling them to react to environmental stressors that overcome the mucosal barrier. By releasing alarmins—proinflammatory and regulatory cytokines—AECs play an active role in the formation, strategic orientation, and control of the subsequent defense reaction. Consequently, the airway epithelium is of vital importance to chronic inflammatory diseases, such as asthma.

Background Asthma is the most common chronic pediatric disease. Exacerbations are potentially lethal in children and can be induced by viral infections. Interleukin-6 (IL-6) has been shown to be an important cytokine produced by epithelial cells (ECs) and is upregulated in viral-induced asthma exacerbations. Regulation of IL-6 expression by transcription factors (TFs) such as the majorly repressive PR domain zinc finger protein 1 (PRDM1) has been subject of research. However, transcriptional control of IL-6 during exacerbation in asthma remains not well understood. We aim to examine the transcriptional regulation of IL-6 after viral analog stimulation in ECs. Methods ENCODE data was used to identify TFs for the IL-6 promoter. Chromatin immunoprecipitation with sequencing (ChIP) of PRDM1 and of Interferon regulatory factor 1 (IRF1) was conducted in diseased bronchial ECs 3h after poly(I:C) stimulation. Analysis was done via unpaired double-sided t-Test. HeLa cells were stimulated with 10 µg/mL polyinosinic:polycytidylic (poly(I:C)) and harvested every 30 minutes for 4.0h. An approximated time point of stationary phase beginning was determined by intersection point calculation of linear graphs. Results TFs IFR1 (p<0.001, r2=0.85) and PRDM1 (p<0.001, r2=0.86) were associated with IL-6 expression in a regression model. ChIP revealed that PRDM1 binds in the promotor and 3’UTR region of the IL-6 gene, while IRF1 binds only in the 3’UTR region. Spearman correlation of expression kinetics disclosed a stronger association between PRDM1 and IL-6 (p<0.001) compared to IRF1 and IL-6 (p<0.05). While expression kinetics of IL-6 and PRDM1 appear to be of similar sigmoid shape, IRF1 resembles a linear graph. Intersection point calculation revealed PRDM1 expression (x=1.76h) to reach the stationary phase earlier than IL-6 (x=1.99h). Conclusion Both transcription factors bind significantly in the IL-6 gene. While there is a steep increase in IRF1 expression after poly(I:C) stimulation expression kinetics of PRDM1 and IL-6 are of similar shape and timeline which may suggest a synchronized transcriptional regulation instead of a repressive function of PRDM1 on IL-6 expression. Further studies on the gene regulation of IL-6 and PRDM1 in the context of virally induced exacerbations are needed and may provide deeper insight into therapeutical approaches. Article published online: 09 March 2023 © 2023. Thieme. All rights reserved. Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany