In spite of many compounds identified as antifibrotic in preclinical studies, pulmonary fibrosis remains a life-threatening condition for which highly effective treatment is still lacking. Towards improving the success-rate of bench-to-bedside translation, we investigated in vivo µCT-derived biomarkers to repeatedly quantify experimental silica-induced pulmonary fibrosis and assessed clinically relevant readouts up to several months after silicosis induction. Mice were oropharyngeally instilled with crystalline silica or saline and longitudinally monitored with respiratory-gated-high-resolution µCT to evaluate disease onset and progress using scan-derived biomarkers. At weeks 1, 5, 9 and 15, we assessed lung function, inflammation and fibrosis in subsets of mice in a cross-sectional manner. Silica-instillation increased the non-aerated lung volume, corresponding to onset and progression of inflammatory and fibrotic processes not resolving with time. Moreover, total lung volume progressively increased with silicosis. The volume of healthy, aerated lung first dropped then increased, corresponding to an acute inflammatory response followed by recovery into lower elevated aerated lung volume. Imaging results were confirmed by a significantly decreased Tiffeneau index, increased neutrophilic inflammation, increased IL-13, MCP-1, MIP-2 and TNF-α concentration in bronchoalveolar lavage fluid, increased collagen content and fibrotic nodules. µCT-derived biomarkers enable longitudinal evaluation of early onset inflammation and non-resolving pulmonary fibrosis as well as lung volumes in a sensitive and non-invasive manner. This approach and model of non-resolving lung fibrosis provides quantitative assessment of disease progression and stabilization over weeks and months, essential towards evaluation of fibrotic disease burden and antifibrotic therapy evaluation in preclinical studies.

After a recovery period of 14 days an intranasal (i.n.) challenge with 0.003% active chlorine (in ClO) or vehicle (distilled water, HO) was given, followed by assessment of the breathing frequency. One day later, pulmonary function, along with pulmonary inflammation was determined. Lung permeability was assessed by means of total broncho-alveolar lavage (BAL) protein content and plasma surfactant protein (SP)-D levels. In vivo micro-CT imaging revealed enlargement of the lungs and airways early after NA treatment, with a return to normal at day 14. When challenged i.n. with ClO, NA-pretreated mice immediately responded with a sensory irritant response. Twenty-four hours later, NA/ClO mice showed airway hyperreactivity (AHR), accompanied by a neutrophilic and eosinophilic inflammation. NA administration followed by ClO induced airway barrier impairment, as shown by increased BAL protein and plasma SP-D concentrations; histology revealed epithelial denudation. These data prove that NA-induced lung impairment renders the lungs of mice more sensitive to an airway challenge with ClO, confirming the hypothesis that incomplete barrier repair, followed by irritant exposure results in airway hypersensitivity.

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The airway epithelium is continuously exposed to environmental irritants, which can cause adverse effects such as irritant-induced asthma (IIA). Mast cells are located near airway epithelia and are able to respond to a variety of stimuli. We aimed to investigate whether mast cells influence the response of the epithelium upon irritant exposure. Two cell lines and three different seeding conditions, that is, bronchial epithelial cells (16HBE) only, 16HBE with mast cells (HMC-1’s) basolaterally, and 16HBE with HMC-1’s apically, were established. Upon exposure to the environmental irritants, graphene (G), graphene oxide (GO), diesel exhaust particles (DEPs) or hypochlorite (ClO−), transepithelial electrical resistance (TEER) and paracellular flux of fluorescent-labeled dextrans were determined, along with the release of mediators. Identical experiments were conducted with the Ca2+ ionophore ionomycin. Exposure to G and GO induced a significant and permanent decrease of approximately 70% in TEER after 3 h of exposure, whereas DEP and ClO− exposure resulted in a transient decrease of approximately 20% in TEER. This response pattern was similar in all the different seeding conditions. After 24 h of exposure, fluorescein isothiocyanate–dextran transport was 10-fold greater for G and 5-fold greater for GO in each of the tested seeding conditions, while DEP and ClO− induced no change compared to the control. Upon exposure to the irritants, 16HBE did not release thymic stromal lymphopoietin, interleukin 33 (IL-33), or IL-1α, and HMC-1 cells did not release histamine, IL-6, or IL-8. Epithelial barrier integrity upon treatment with ionomycin was not affected by the presence of HMC-1 cells. A limited amount of IL-6 and IL-8 was released by ionomycin-exposed HMC-1 cells. To conclude, we found that the studied environmental irritants do not directly or indirectly activate HMC-1 cells. These mast cells did not influence the epithelial barrier function upon environmental exposure, and thus currently do not provide additional information for the underlying mechanism of IIA.

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Via inhalation we are continuously exposed to environmental and occupational irritants which can induce adverse health effects, such as irritant-induced asthma (IIA). The airway epithelium forms the first barrier encountered by these agents. We investigated the effect of environmental and occupational irritants on the airway epithelial barrier in vitro. The airway epithelial barrier was mimicked using a coculture model, consisting of bronchial epithelial cells (16HBE) and monocytes (THP-1) seeded on the apical side of a permeable support, and human lung microvascular endothelial cells (HLMVEC) grown on the basal side. Upon exposure to graphene (G) and graphene oxide (GO) in a suspension with fetal calf serum (FCS), ammonium persulfate (AP), sodium persulfate (SP) and hypochlorite (ClO−), the transepithelial electrical resistance (TEER) and flux of fluorescent labelled dextran (FD4-flux), was determined. Exposure to graphene nanoparticles (GNPs) induced an immediate negative effect on the epithelial barrier, whereas ClO− only had a negative impact after 24 h of exposure. AP and SP did not affect the barrier properties. The tight junctions (TJ) network showed less connected zonula occludens 1 (ZO-1) and occludin staining in GNP-exposed cocultures. Functional analysis of the phosphoproteomic data indicated that proteins in the adherens junction (AJ) and TJ pathways showed an altered phosphorylation due to GNP exposure. To conclude, the negative effect of GNPs on the epithelial barrier can be explained by the slightly altered the TJ organization which could be caused by alterations in the phosphorylation level of proteins in the AJ and TJ pathway.

Purpose Exposure to low concentrations of toluene diisocyanate (TDI) leads to immune-mediated chemical-induced asthma. The role of the adaptive immune system has already been thoroughly investigated; nevertheless, the involvement of innate immune cells in the pathophysiology of chemical-induced asthma is still unresolved. The aim of the study is to investigate the role of innate lymphoid cells (ILCs) and dendritic cells (DCs) in a mouse model for chemical-induced asthma. Methods On days 1 and 8, BALB/c mice were dermally treated (20 µL/ear) with 0.5% TDI or the vehicle acetone olive oil (AOO; 2:3). On days 15, 17, 19, 22 and 24, the mice received an oropharyngeal challenge with 0.01% TDI or AOO (1:4). One day after the last challenge, airway hyperreactivity (AHR) to methacholine was assessed, followed by an evaluation of pulmonary inflammation and immune-related parameters, including the cytokine pattern in bronchoalveolar lavage fluid, lymphocyte subpopulations of the lymph nodes and their ex vivo cytokine production profile, blood immunoglobulins and DC and ILC subpopulations in the lungs. Results Both DC and ILC2 were recruited to the lungs after multiple airway exposures to TDI, regardless of the prior dermal sensitization. However, prior dermal sensitization with TDI alone results in AHR and predominant eosinophilic airway inflammation, accompanied by a typical type 2 helper T (Th2) cytokine profile. Conclusions TDI-induced asthma is mediated by a predominant type 2 immune response, with the involvement of adaptive Th2 cells. However, from our study we suggest that the innate ILC2 cells are important additional players in the development of TDI-induced asthma.