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Eosinophils were previously reported to play a role in intestinal inflammation and fibrosis. Whether this is as a bystander or as an active participant is still up for debate. Moreover, data describing a causal relationship between eosinophils and intestinal fibrosis are scarce. We here aimed to elucidate the role of eosinophils in the pathogenesis of intestinal inflammation and fibrosis. Therefore, we stimulated fibroblasts with (active) eosinophils or with Eosinophil Cationic Protein (ECP), and assessed fibroblast activation via flow cytometry and immunocytochemistry. We observed decreased fibroblast activation when fibroblasts were co-cultured with active eosinophils or after stimulation with ECP in comparison to monoculture conditions, but not in case of co-culturing with inactivated eosinophils. Furthermore, eosinophil depletion in a RAG −/− chronic DSS colitis model resulted in decreased inflammation, but increased development of fibrosis. In this model, we could show increased expression of the anti-inflammatory protein IL-10 and the pro-fibrotic factors IL-1β, FGF-21 and TGF-β3 in the eosinophil-depleted mice compared to the control mice. In conclusion, our in vitro data revealed an anti-fibrotic role for eosinophils. In line, in a chronic murine colitis model, we observed a pro-inflammatory, but an anti-fibrotic, role for eosinophils. Furthermore, we identified an increased presence of anti-inflammatory and pro-fibrotic cytokines in the eosinophil depleted group.

There is growing evidence that neurogenic inflammation contributes to the pathophysiology of upper airway diseases, with nasal hyperreactivity (NHR) being a key symptom. The rare neuroendocrine cells (NECs) in the epithelium have been linked to the pathophysiology of bronchial and intestinal hyperreactivity, however their presence in the nasal mucosa and their potential role in NHR remains unclear. Therefore, we studied the presence of NECs in the nasal epithelium of controls, allergic rhinitis patients and chronic rhinosinusitis with nasal polyps patients, and their link to NHR. The expression of typical NECs markers, CHGA, ASCL1 and CGRP, were evaluated on gene and protein level in human samples using real-time quantitative PCR (RT-qPCR), western blot, immunohistochemistry fluorescence staining, RNA scope assay, flow cytometry and single cell RNA-sequencing. Furthermore, the change in peak nasal inspiratory flow after cold dry air provocation and visual analogue scale scores were used to evaluate NHR or disease severity, respectively. Limited gene expression of the NECs markers CHGA and ASCL1 was measured in patients with upper airway diseases and controls. Gene expression of these markers did not correlate with NHR severity nor disease severity. In vitro , CHGA and ASCL1 expression was also evaluated in primary nasal epithelial cell cultures from patients with upper airway disease and controls using RT-qPCR and western blot. Both on gene and protein level only limited CHGA and ASCL1 expression was found. Additionally, NECs were studied in nasal biopsies of patients with upper airway diseases and controls using immunohistochemistry fluorescence staining, RNA scope and flow cytometry. Unlike in ileum samples, CHGA could not be detected in nasal biopsies of patients with upper airway diseases and control subjects. Lastly, single cell RNA-sequencing of upper airway tissue could not identify a NEC cluster. In summary, in contrast to the bronchi and gut, there is only limited evidence for the presence of NECs in the nasal mucosa, and without correlation with NHR, thereby questioning the relevance of NECs in upper airway pathology.

Background Exercise-induced bronchoconstriction (EIB) is a transient airway narrowing, occurring during or shortly after intensive exercise. It is highly prevalent in non-asthmatic outdoor endurance athletes suggesting an important contribution of air pollution in the development of EIB. Therefore, more research is necessary to investigate the combination of exercise and pollutants on the airways. Methods Balbc/ByJ mice were intranasally challenged 5 days a week for 3 weeks with saline or 0.2 mg/ml diesel exhaust particles (DEP), prior to a daily incremental running session or non-exercise session. Once a week, the early ventilatory response was measured and lung function was determined at day 24. Airway inflammation and cytokine levels were evaluated in bronchoalveolar lavage fluid. Furthermore, innate lymphoid cells, dendritic cells and tight junction mRNA expression were determined in lung tissue. Results Submaximal exercise resulted in acute alterations of the breathing pattern and significantly improved FEV 0.1 at day 24. DEP exposure induced neutrophilic airway inflammation, accompanied with increased percentages of CD11b + DC in lung tissue and pro-inflammatory cytokines, such as IL-13, MCP-1, GM-CSF and KC. Occludin and claudin-1(Cldn-1) expression were respectively increased and decreased by DEP exposure. Whereas, exercise increased Cldn-3 and Cldn-18 expression. Combining exercise and DEP exposure resulted in significantly increased SP-D levels in the airways. Conclusion DEP exposure induced typical airway neutrophilia, DC recruitment and pro-inflammatory cytokine production. Whereas, intensive exercise induced changes of the breathing pattern. The combination of both triggers resulted in a dysregulation of tight junction expression, suggesting that intensive exercise in polluted environments can induce important changes in the airway physiology and integrity.

Cold air and air pollution are known triggers to induce symptoms in exercise-induced bronchoconstriction (EIB). Mast cells are hypothesized to be a key player in the pathogenesis of EIB. This study aims to investigate the role of mast cells using mast cell deficient (Cpa3 ) mice and with mast cell stabilizers (Doxantrazole) in an exposure-associated mouse model of EIB. Male Cpa3 mice and wild type littermates or BALB/c mice were exposed to a submaximal running protocol in cold environment (4°C) or resting period (room temperature) 5 days for 3 weeks after oropharyngeal challenge with saline or 0.1 mg/ml diesel exhaust particles (DEP). BALB/c mice were intraperitoneally injected with 16.5 mg/kg Doxantrazole or placebo (0.5% NaHCO ) during the last week. Twenty-four hours after the last running or resting session, lung function, lung inflammation and immune mediated response was determined. Inhibition of mast cells by Doxantrazole or mice lacking functional mast cells (Cpa3 ), resulted in blunting of bronchial hyperreactivity, both in acute breathing pattern and in hyperreactivity to increasing doses of methacholine. Neutrophilic inflammation was still present after Doxantrazole treatment, but not in Cpa3 mice. These results were similar in neutrophil extracellular traps and neutrophil-linked cytokines and chemokines. Macrophage activaty was also reduced in the absence of functional mast cells. Mast cells are crucial in the development of bronchial hyperreactivity and macrophage activation. Additionally, they have a complex interplay with neutrophilic inflammation. These findings highlight the potential of mast cell modulation as therapeutic strategy in exposure-associated EIB.

Primary human mast cells (MC) obtained through culturing of blood-derived MC progenitors are the preferred model for the ex vivo study of MRGPRX2- vs. IgE-mediated MC activation. In order to assess the impact of culture conditions on functional MRGPRX2 expression, we cultured CD34 + -enriched PBMC from peripheral whole blood (PB) and buffy coat (BC) samples in MethoCult medium containing stem cell factor (SCF) and interleukin (IL)-3, modified through variations in seeding density and adding or withholding IL-6, IL-9 and fetal bovine serum (FBS). Functional expression of MRGPRX2 was assessed after 4 weeks via flow cytometry. We found similar proportions of CD34 + MC-committed progenitors in BC and PB. Higher seeding densities (≥ 1x10 5 cells/mL) and exposure to IL-9 and FBS suppressed functional MRGPRX2 expression at 4 weeks, while leaving MC yield largely unaffected. IL-6 had no impact on MRGPRX2 expression. MRGPRX2-expressing MC upregulated CD63 upon stimulation with polyclonal anti-IgE, substance P and compound 48/80 at 4 weeks. Ketotifen and dasatinib but not cromolyn sodium inhibited both IgE- and MRGPRX2-dependent pathways. Our results confirm the feasibility of functional MC activation studies on PB-derived MC after a short 4-week culture and highlight the impact of culture conditions on functional MRGPRX2 expression.

Non-allergic asthma is driven by multiple endotypes of which neutrophilic and pauci-granulocytic asthma have been best established. However, it is still puzzling what drives inflammation and airway hyperreactivity (AHR) in these patients and how it can be treated effectively. Recently, a potential role of the innate immune system and especially the innate lymphoid cells (ILC) has been proposed. In this study, we investigated the effects of LPS inhalation on airway inflammation and AHR as a potential model for elucidating the pathogenesis of non-allergic asthma. Wild-type (BALB/c), SCID, IL-17A , and Rag2 γC mice were endonasally exposed to lipopolysaccharide (LPS, 2 µg) on four consecutive days. Twenty-four hours after the last exposure, AHR to methacholine was assessed. Cytokine levels and ILC subpopulations were determined in lung tissue. Cellular differential analysis was performed in BAL fluid. In this study, we developed a murine model for non-allergic neutrophilic asthma. We found that repeated endonasal applications of low-dose LPS in BALB/c mice led to AHR, BAL neutrophilia, and a significant increase in lung ILC3 as well as a significant increase in lung chemokines KC and MIP-2 and cytokines IL-1β, IL-17A, IL-22, and TNF. The adoptive transfer of ILC in Rag2 γC mice showed that ILC played a causal role in the induction of AHR in this model. Antagonising IL-1β, but not IL-17A or neutrophils, resulted in a partial reduction in LPS-induced AHR. In conclusion, we report here a murine model for neutrophilic asthma where ILC are required to induce airway hyperreactivity.

[This corrects the article DOI: 10.3389/fimmu.2022.849155.].[This corrects the article DOI: 10.3389/fimmu.2022.849155.].

Background Leukocyte- and platelet-rich fibrin (L-PRF) is an autologous platelet concentrate, prepared by centrifugation of blood and consisting of a dense fibrin network with incorporated leukocytes and platelets. This study aims to perform an in-depth analysis of the cells, growth factors, and transcriptome of L-PRF. Methods and results Fresh, 1 week and 2 weeks cultured human L-PRF membranes and liquid L-PRF glue were characterized on cellular and transcriptional level using flow cytometry ( n  = 4), single-cell RNA sequencing ( n  = 5) and RT-qPCR. Growth factor kinetics were investigated using ELISA (EGF, VEGF, PDGF-AB, TGF-β1, bFGF). L-PRF contained a large number of viable cells (fresh 97.14 ± 1.09%, 1 week cultured 93.57 ± 1.68%), mainly granulocytes in fresh samples (53.9 ± 19.86%) and T cells in cultured samples (84.7 ± 6.1%), confirmed with scRNA-seq. Monocytes differentiate to macrophages during 1 week incubation. Specifically arterial L-PRF membranes were found to release significant amounts of VEGF, EGF, PDGF-AB and TGF-β1. Conclusion We characterized L-PRF using in vitro experiments, to obtain an insight in the composition of the material including a possible mechanistic role for tissue healing. This was the first study characterizing L-PRF at a combined cellular, proteomic, and transcriptional level.

RationaleParticipation in high-intensity exercise in early life might act as stressor to the airway barrier.ObjectivesTo investigate the effect of intense exercise and associated exposure to air pollution on the airway barrier in adolescent elite athletes compared with healthy controls and to study exercise-induced bronchoconstriction (EIB) in this population.MethodsEarly-career elite athletes attending ‘Flemish-Elite-Sports-Schools’ (12–18 years) of 4 different sport disciplines (n=90) and control subjects (n=25) were recruited. Presence of EIB was tested by the eucapnic voluntary hyperventilation (EVH) test. Markers at mRNA and protein level; RNA-sequencing; carbon load in airway macrophages were studied on induced sputum samples.Results444 genes were differentially expressed in sputum from athletes compared with controls, which were related to inflammation and epithelial cell damage and sputum samples of athletes contained significantly more carbon loaded airway macrophages compared with controls (24%, 95% CI 20% to 36%, p<0.0004). Athletes had significantly higher substance P (13.3 pg/mL, 95% CI 2.0 to 19.2) and calprotectin (1237 ng/mL, 95% CI 531 to 2490) levels as well as IL-6, IL-8 and TNF-α mRNA levels compared with controls (p<0.05). The incidence of EIB in athletes was 9%. The maximal fall in forced expiratory volume in 1 s (%) after EVH test in athletes was significantly associated with prior PM10 and PM2.5 exposure.ConclusionEarly-career elite athletes showed increased markers of air pollution exposure, epithelial damage and airway inflammation compared with controls. Acute exposure to increased air pollution PM10 levels was linked to increased airway hyper-reactivity.Trial registration numberNCT03587675.

Background Asthma is characterized by a heterogeneous inflammatory profile and can be subdivided into T(h)2-high and T(h)2-low airway inflammation. Profiling of a broader panel of airway cytokines in large unselected patient cohorts is lacking. Methods Patients ( n  = 205) were defined as being “cytokine-low/high” if sputum mRNA expression of a particular cytokine was outside the respective 10 th /90 th percentile range of the control group ( n  = 80). Unsupervised hierarchical clustering was used to determine clusters based on sputum cytokine profiles. Results Half of patients ( n  = 108; 52.6%) had a classical T(h)2-high (“IL-4-, IL-5- and/or IL-13-high”) sputum cytokine profile. Unsupervised cluster analysis revealed 5 clusters. Patients with an “IL-4- and/or IL-13-high” pattern surprisingly did not cluster but were equally distributed among the 5 clusters. Patients with an “IL-5-, IL-17A-/F- and IL-25- high” profile were restricted to cluster 1 ( n  = 24) with increased sputum eosinophil as well as neutrophil counts and poor lung function parameters at baseline and 2 years later. Four other clusters were identified: “IL-5-high or IL-10-high” ( n  = 16), “IL-6-high” ( n  = 8), “IL-22-high” ( n  = 25). Cluster 5 ( n  = 132) consists of patients without “cytokine-high” pattern or patients with only high IL-4 and/or IL-13. Conclusion We identified 5 unique asthma molecular phenotypes by biological clustering. Type 2 cytokines cluster with non-type 2 cytokines in 4 out of 5 clusters. Unsupervised analysis thus not supports a priori type 2 versus non-type 2 molecular phenotypes. www.clinicaltrials.gov NCT01224938. Registered 18 October 2010.