Single cell RNA sequencing reveals endothelial cell killing and resolution pathways in experimental malaria-associated acute respiratory distress syndrome.

Plasmodium parasites cause malaria, a global health disease that is responsible for more than 200 million clinical cases and 600 000 deaths each year. Most deaths are caused by various complications, including malaria-associated acute respiratory distress syndrome (MA-ARDS). Despite the very rapid and efficient killing of parasites with antimalarial drugs, 15% of patients with complicated malaria succumb. This stresses the importance of investigating resolution mechanisms that are involved in the recovery from these complications once the parasite is killed. To study the resolution of MA-ARDS, P. berghei NK65-infected C57BL/6 mice were treated with antimalarial drugs after onset of symptoms, resulting in 80% survival. Micro-computed tomography revealed alterations of the lungs upon infection, with an increase in total and non-aerated lung volume due to edema. Whole body plethysmography confirmed a drastically altered lung ventilation, which was restored during resolution. Single-cell RNA sequencing indicated an increased inflammatory state in the lungs upon infection, which was accompanied by a drastic decrease in endothelial cells, consistent with CD8+ T cell-mediated killing. During resolution, anti-inflammatory pathways were upregulated and proliferation of endothelial cells was observed. MultiNicheNet interactome analysis identified important changes in the ligand-receptor interactions during disease resolution that warrant further exploration in order to develop new therapeutic strategies. In conclusion, our study provides insights in pro-resolving pathways that limit inflammation and promote endothelial cell proliferation in experimental MA-ARDS. This information may be useful for the design of adjunctive treatments to enhance resolution after Plasmodium parasite killing by antimalarial drugs.

Impact of a Polymer-Based Nanoparticle with Formoterol Drug as Nanocarrier System In Vitro and in an Experimental Asthmatic Model.

The implementation of nanotechnology in pulmonary delivery systems might result in better and more specific therapy. Therefore, a nano-sized drug carrier should be toxicologically inert and not induce adverse effects. We aimed to investigate the responses of a polymer nano drug carrier, a lysine poly-hydroxyethyl methacrylate nanoparticle (NP) [Lys-p(HEMA)], loaded with formoterol, both in vitro and in vivo in an ovalbumin (OVA) asthma model. The successfully synthesized nanodrug formulation showed an expectedly steady in vitro release profile. There was no sign of in vitro toxicity, and the 16HBE and THP-1 cell lines remained vital after exposure to the nanocarrier, both loaded and unloaded. In an experimental asthma model (Balb/c mice) of ovalbumin sensitization and challenge, the nanocarrier loaded and unloaded with formoterol was tested in a preventive strategy and compared to treatment with the drug in a normal formulation. The airway hyperresponsiveness (AHR) and pulmonary inflammation in the bronchoalveolar lavage (BAL), both cellular and biochemical, were assessed. The application of formoterol as a regular drug and the unloaded and formoterol-loaded NP in OVA-sensitized mice followed by a saline challenge was not different from the control group. Yet, both the NP formulation and the normal drug application led to a more deteriorated lung function and increased lung inflammation in the OVA-sensitized and -challenged mice, showing that the use of the p(HEMA) nanocarrier loaded with formoterol needs more extensive testing before it can be applied in clinical settings.

Sampling strategies and analytical techniques for assessment of airborne micro and nano plastics.

The atmosphere is pervasively polluted by microplastics and nano plastics (M/NPs) released into indoor and outdoor areas. However, various methodologies and their limitations along with non-standardization make the comparison of information concerning their prevalence difficult. Such diversity in techniques greatly limits the interpretation of results. Herein, We extracted data from publications on PubMed and Embase database up to the year 2022 regarding sampling strategies, identification methods, and reporting data for M/NPs quantification. In this review, 5 major areas for measuring airborne M/NPs have been identified including pre-sampling/ sampling/ post-sampling/ analysis/ and contamination avoidance. There are many challenges specific to each of those sections that need to be resolved through further method development and harmonization. This review mainly focuses on the different methods for collecting atmospheric M/NPs and also the analytical tools which have been used for their identification. While passive sampling is the most user-friendly method, the most precise and reproducible approach for collecting plastic particles is an active method which is directly followed by visual counting as the most common physical analysis technique. Polymers collected using visual sorting are most frequently identified by spectroscopy (FTIR; Raman). However, destructive analytical techniques (thermal degradation) also provide precise chemical information. In all cases, the methods were screened for advantages, limitations, and fieldwork abilities. This review outlines and critiques knowledge gaps, and recommendations to support standardized and comparable future research.

Peribronchial Inflammation Resulting from Regulatory T Cell Deficiency Damages the Respiratory Epithelium and Disturbs Barrier Function.

Regulatory T cells (Tregs) that express the transcription factor Foxp3 have a critical role in limiting inflammatory processes and tissue damage. Whether Tregs are functional in maintaining epithelial barriers and in control of tight junction expression has not yet been explored. In this study, we investigated the effect of Treg deficiency on the airway epithelial barrier in an experimental murine model in which diphtheria toxin was repeatedly injected in Foxp3-diphtheria toxin receptor (DTR) mice to deplete Tregs. This resulted in spontaneous peribronchial inflammation and led to a systemic and local increase of IL-4, IL-5, CCL3, IFN-γ, and IL-10 and a local (lung) increase of IL-6 and IL-33 and decreased amphiregulin levels. Moreover, Treg depletion increased airway permeability and decreased epithelial tight junction (protein and mRNA) expression. CTLA4-Ig treatment of Treg-depleted mice almost completely prevented barrier dysfunction together with suppression of lung inflammation and cytokine secretion. Treatment with anti-IL-4 partly reversed the effects of Treg depletion on tight junction expression, whereas neutralization of IL-6 of IFN-γ had either no effect or only a limited effect. We conclude that Tregs are essential to protect the epithelial barrier at the level of tight junctions by restricting spontaneous T cell activation and uncontrolled secretion of cytokines, in particular IL-4, in the bronchi.

Chlorine exposure and intensive exercise induces airway hyperreactivity in a 3-week murine exercise model.

RATIONALE: Exercise-induced bronchoconstriction (EIB) is defined as acute narrowing of the airways during or immediately after exercise. EIB has a high prevalence in elite swimmers probably due to the high ventilation rate and exposure to the chlorine by-products. It is still puzzling which pathophysiological mechanisms drive EIB. OBJECTIVE: In this study, we evaluated airway hyperreactivity, permeability, integrity and inflammation in a murine swimmers EIB model with and without chlorine exposure. METHODS: Mice performed a 3-week swimming protocol in a swimming pool with counter current. Three hours after the last swimming session, airway hyperreactivity to methacholine was assessed. Cytokine levels and cellular differential analysis was performed in BAL fluid. Airway permeability and tight junction expression was measured in serum and lung tissue. T-, B-, dendritic and innate lymphoid cells were determined in lung tissue via flow cytometry. RESULTS: A significant higher airway resistance (Rn; P < 0.0001) was observed in mice swimming in chlorinated water (mean Rn = 1.26 cmH2O.s/ml) compared to mice swimming in tap water (mean Rn = 0.76 cmH2O.s/ml) and both inhalation groups in the absence of cellular inflammation. No significant differences were found in lung immune cell populations or in lung tight junction mRNA expression. Experiments in SCID, Rag2-/-γc-/- or Cpa3cre/+ mice showed a limited involvement of the innate, adaptive immune system or the mast cells. CONCLUSION: Our 3-week swimming murine model mimics intensive swimming in chlorinated water with the presence of airway hyperreactivity in mice swimming in chlorinated water in the absence of airway inflammation and airway epithelial damage.

Occupational Asthma Caused by Low-Molecular-Weight Chemicals Associated With Contact Dermatitis: A Retrospective Study.

BACKGROUND: Occupational asthma (OA) may have different etiologies, but it is not clear whether the etiologic agents influence the clinical presentation, especially the co-occurrence of skin lesions. OBJECTIVE: To determine the impact of different asthmagens on the characteristics of OA, with a focus on the occurrence of prior or concomitant skin disorders. METHODS: In a retrospective analysis of patients who visited the Occupational and Environmental Disease Clinic of a tertiary referral hospital from 2009 to 2019, we classified patients into definite, probable, or possible OA according to prespecified diagnostic guidelines. In multivariate logistic regression with sensitivity analysis, we examined the relation of high- and low-molecular-weight (HMW and LMW) agents with the clinical presentation. RESULTS: Of 209 cases of OA, 66 were caused by HMW agents and 143 by LMW agents. Patients with OA exposed to LMW agents had higher odds of having (had) allergic contact dermatitis (odds ratio, 5.45 [1.80-23.70]; P < .01), compared with patients exposed to HMW agents. Conversely, HMW agents were associated with higher odds of rhinitis symptoms (odds ratio of LMW/HMW, 0.33 [0.17-0.63]; P < .001) and high total IgE (odds ratio of LMW/HMW, 0.35 [0.17-0.70]; P < .01). Risk factors for having coexisting contact dermatitis included construction work, hairdressing, and exposure to metals or epoxy resins. CONCLUSIONS: Among patients with OA, exposure to specific LMW agents was associated with a high frequency of contact dermatitis. Different types of asthmagens within HMW or LMW agents appear to determine the phenotype and comorbidity of OA.

Effect of Graphene and Graphene Oxide on Airway Barrier and Differential Phosphorylation of Proteins in Tight and Adherens Junction Pathways.

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.

Involvement of Innate Lymphoid Cells and Dendritic Cells in a Mouse Model of Chemical-induced Asthma.

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.

An alternative method to assess permeation through disposable gloves.

Previously, we have demonstrated the capability of activated charcoal cloth (ACC) to assess dermal exposure to VOCs. Here we investigated whether ACC patches can be used as an under-glove indicator to evaluate the ingress of toluene through disposable gloves in a controlled environment, and compared these results to the amount of toluene ingress determined from the standardized test methods for determining chemical permeation through PPE. In a test chamber, with plugs for air sampling, five to six ACC patches were placed on a mannequin hand underneath disposable gloves (latex, nitrile, neoprene, polymer laminate). Three work-exposure scenarios were simulated to assess toluene ingress through the different gloves: vapor exposure; spray exposure, and immersion. The standard permeation test, using a diffusion cell, was carried with glove material of the palm, with continuous contact conditions. In all of ACC test, the order of toluene ingress was latex > neoprene > nitrile > Barrier, but for the standardized testing, the order of the neoprene and nitrile was reversed, and nitrile had higher levels of toluene ingress. These results show the need to think beyond standard testing techniques for occupational exposure to hazardous substances, and the added value of "application style" testing.

Cobalt exposure via skin alters lung immune cells and enhances pulmonary responses to cobalt in mice.

Cobalt has been associated with allergic contact dermatitis and occupational asthma. However, the link between skin exposure and lung responses to cobalt is currently unknown. We investigated the effect of prior dermal sensitization to cobalt on pulmonary physiological and immunological responses after subsequent challenge with cobalt via the airways. BALB/c mice received epicutaneous applications (25 µL/ear) with 5% CoCl2*6H2O (Co) or the vehicle (Veh) dimethyl sulfoxide (DMSO) twice; they then received oropharyngeal challenges with 0.05% CoCl2*6H2O or saline five times, thereby obtaining four groups: Veh/Veh, Co/Veh, Veh/Co, and Co/Co. To detect early respiratory responses noninvasively, we performed sequential in vivo microcomputed tomography (µCT). One day after the last challenge, we assessed airway hyperreactivity (AHR) to methacholine, inflammation in bronchoalveolar lavage (BAL), innate lymphoid cells (ILCs) and dendritic cells (DCs) in the lungs, and serum IgE. Compared with the Veh/Veh group, the Co/Co group showed increased µCT-derived lung response, increased AHR to methacholine, mixed neutrophilic and eosinophilic inflammation, elevated monocyte chemoattractant protein-1 (MCP-1), and elevated keratinocyte chemoattractant (KC) in BAL. Flow cytometry in the Co/Co group demonstrated increased DC, type 1 and type 2 conventional DC (cDC1/cDC2), monocyte-derived DC, increased ILC group 2, and natural cytotoxicity receptor-ILC group 3. The Veh/Co group showed only increased AHR to methacholine and elevated MCP-1 in BAL, whereas the Co/Veh group showed increased cDC1 and ILC2 in lung. We conclude that dermal sensitization to cobalt may increase the susceptibility of the lungs to inhaling cobalt. Mechanistically, this enhanced susceptibility involves changes in pulmonary DCs and ILCs.

Innate lymphoid cells in isocyanate-induced asthma: role of microRNA-155.

BACKGROUND: Occupational asthma, induced by workplace exposures to low molecular weight agents such as toluene 2,4-diisocyanate (TDI), causes a significant burden to patients and society. Little is known about innate lymphoid cells (ILCs) in TDI-induced asthma. A critical regulator of ILC function is microRNA-155, a microRNA associated with asthma. OBJECTIVE: To determine whether TDI exposure modifies the number of ILCs in the lung and whether microRNA-155 contributes to TDI-induced airway inflammation and hyperresponsiveness. METHODS: C57BL/6 wild-type and microRNA-155 knockout mice were sensitised and challenged with TDI or vehicle. Intracellular cytokine expression in ILCs and T-cells was evaluated in bronchoalveolar lavage (BAL) fluid using flow cytometry. Peribronchial eosinophilia and goblet cells were evaluated on lung tissue, and airway hyperresponsiveness was measured using the forced oscillation technique. Putative type 2 ILCs (ILC2) were identified in bronchial biopsies of subjects with TDI-induced occupational asthma using immunohistochemistry. Human bronchial epithelial cells were exposed to TDI or vehicle. RESULTS: TDI-exposed mice had higher numbers of airway goblet cells, BAL eosinophils, CD4+ T-cells and ILCs, with a predominant type 2 response, and tended to have airway hyperresponsiveness. In TDI-exposed microRNA-155 knockout mice, inflammation and airway hyperresponsiveness were attenuated. TDI exposure induced IL-33 expression in human bronchial epithelial cells and in murine lungs, which was microRNA-155 dependent in mice. GATA3+CD3- cells, presumably ILC2, were present in bronchial biopsies. CONCLUSION: TDI exposure is associated with increased numbers of ILCs. The proinflammatory microRNA-155 is crucial in a murine model of TDI asthma, suggesting its involvement in the pathogenesis of occupational asthma due to low molecular weight agents.

Skin Exposure Contributes to Chemical-Induced Asthma: What is the Evidence? A Systematic Review of Animal Models.

It is generally assumed that allergic asthma originates primarily through sensitization via the respiratory mucosa, but emerging clinical observations and experimental studies indicate that skin exposure to low molecular weight (LMW) agents, i.e. "chemicals," may lead to systemic sensitization and subsequently develop asthma when the chemical is inhaled. This review aims to evaluate the accumulating experimental evidence that adverse respiratory responses can be elicited upon inhalation of an LMW chemical sensitizer after previous sensitization by dermal exposure. We systematically searched the PubMed and Embase databases up to April 15, 2017, and conducted forward and backward reference tracking. Animal studies involving both skin and airway exposure to LMW agents were included. We extracted 6 indicators of "selective airway hyper-responsiveness" (SAHR)-i.e. respiratory responses that only occurred in previously sensitized animals-and synthesized the evidence level for each indicator into strong, moderate or limited strength. The summarized evidence weight for each chemical agent was graded into high, middle, low or "not possible to assess." We identified 144 relevant animal studies. These studies involved 29 LMW agents, with 107 (74%) studies investigating the occurrence of SAHR. Indicators of SAHR included physiological, cytological/histological and immunological responses in bronchoalveolar lavage, lung tissue and airway-draining lymph nodes. Evidence for skin exposure-induced SAHR was present for 22 agents; for 7 agents the evidence for SAHR was inconclusive, but could not be excluded. The ability of a chemical to cause sensitization via skin exposure should be regarded as constituting a risk of adverse respiratory reactions.

Assessment of the absorbed dose after exposure to surgical smoke in an operating room.

Surgical smoke produced by electrosurgery contains various chemical substances such as volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs). The aim of this study is to investigate airborne concentrations of VOCs and PAHs during electrosurgery in an operating room, in relation to metabolites in urine in order to assess the absorbed dose. A 5-day exposure study was set up in a general surgery operation room including surgeons, scrub assistants and circulation nurses (n = 15). Stationary and personal air sampling for VOCs and PAHs were carried out. Pre-, mid- and end-shift analysis of urinary S-phenylmercapturic acid (SPMA), o-cresol, mandelic acid and 1-hydroxypyrene was performed to assess the internal exposure to respectively benzene, toluene, styrene and PAHs. Several VOCs (styrene, ethyl benzene, benzene and toluene), ranging from 0.7 to 3.27 µg/m3 were detected in the air samples, along with one PAH (naphthalene, ranging from 0.012 to 0.39 µg/m3). There was no significant correlation between air monitoring and urinary biomonitoring. O-cresol levels were increased, especially among assistants and nurses at mid- and end-shift, exceeding current biological exposure indices several times. External and internal exposure for assistants and nurses was substantially more, compared to surgeons. This study confirms the presence of VOCs and PAHs in surgical smoke and shows the presence of their metabolites in urine, but the association is unclear. Urinary biomonitoring shows especially high concentrations of o-cresol.

Dermal exposure determines the outcome of repeated airway exposure in a long-term chemical-induced asthma-like mouse model.

BACKGROUND: Exposure to diisocyanates is an important cause of occupational asthma (OA) in the industrialized world. Since OA occurs after long-term exposure to diisocyanates, we developed a chronic mouse model of chemical-induced asthma where toluene diisocyanate (TDI) was administered at two different exposure sites. OBJECTIVES: Evaluating the effect of long-term respiratory isocyanate exposure - with or without prior dermal exposure- on sensitization, inflammatory responses and airway hyperreactivity (AHR). METHODS: On days 1 and 8, BALB/c mice were dermally treated (20 µl/ear) with 0.5% 2,4-toluene diisocyanate TDI or the vehicle acetone olive oil (AOO) (3:2). Starting from day 15, mice received intranasal instillations with 0.1% TDI of vehicle five times in a week, for five successive weeks. One day after the last instillation airway hyperreactivity (AHR) to methacholine was assessed, followed by an evaluation of pulmonary inflammation and structural lung changes. Immune-related parameters were assessed in the lungs (BAL and tissue), blood, cervical- and auricular lymph nodes. RESULTS: Mice repeatedly intranasally exposed to TDI showed systemic sensitization and a mixed Th1/Th2 type immune response, without the presence of AHR. However, when mice are first dermally sensitized with TDI, followed by repeated intranasal TDI challenges, this results in a pronounced Th2 response and AHR. CONCLUSION: Dermal exposure to TDI determines airway hyperreactivity after repeated airway exposure to TDI.

Exposure to Polycyclic Aromatic Hydrocarbons Leads to Non-monotonic Modulation of DNA and RNA (hydroxy)methylation in a Rat Model.

Besides genetic modifications, rapidly growing evidence has linked environmental pollutants with epigenetic variations. To date, only a few studies have been performed on DNA methylation changes of polycyclic aromatic hydrocarbons (PAH), which showed contradictory results. These discrepancies might be partially explained by differences in used agents. Generally in in vitro studies, a single compound is used, while in humans environmental studies, multi-residue exposure is investigated. The present study aimed to study epigenetic alterations induced by multi-residue exposure to PAH. Female Long Evans rats were exposed to a mixture of 16 US-EPA priority PAH, 3 times per week over a 90-day period. The livers were used to assess the (hydroxy)methylation status of genomic DNA/RNA, together with reduced and oxidized forms of glutathione. The results of this study demonstrate that a multi-residue exposure to PAH affects glutathione status, DNA (hydroxy)methylation, and RNA (hydroxy)methylation, together with DNA PAH-adducts formation. In addition, a non-monotonic response relationship was demonstrated between PAH concentration, the levels of glutathione and DNA (hydroxy)methylation levels at environmental relevant doses. This hormetic response gives a novel insight concerning the toxicity of environmental pollutants such as PAH and the biological response that may be different depending on the level of exposure.

Single-walled and multi-walled carbon nanotubes induce sequence-specific epigenetic alterations in 16 HBE cells.

Recent studies have identified carbon nanotube (CNT)-induced epigenetic changes as one of the key players in patho-physiological response. In the present study, we investigated whether CNT exposure is associated with epigenetic changes in human bronchial epithelial cells (16 HBE), in vitro. We focused on global DNA methylation, methylation of LINE-1 elements and promoter sequence of twelve functionally important genes (SKI, DNMT1, HDAC4, NPAT, ATM, BCL2L11, MAP3K10, PIK3R2, MYO1C, TCF3, FGFR 1 and AGRN). Additionally, we studied the influence of CNT exposure on miRNA expression. Using a LC-MS/MS method and pyrosequencing for LINE-1, we observed no significant changes in global DNA methylation (%) between the concentrations of multi-walled and single-walled CNT (MWCNT and SWCNT, respectively). Significant changes in sequence-specific methylation was observed in at least one CpG site for DNMT1 (SWCNT), HDAC4 (MWCNT), NPAT/ATM (MWCNT and SWCNT), MAP3K10 (MWCNT), PIK3R2 (MWCNT and SWCNT) and MYO1C (SWCNT). While changes in DNA methylation of the genes were relatively small, these changes were associated with changes in RNA expression, especially for MWCNT. However, the study did not reveal any significant alteration in the miRNA expression, associated with MWCNT and SWCNT exposure. Based on our results, mainly MWCNT influence DNA methylation and expression of the studied genes and could have significant impact on several critical cellular processes.

Global and gene-specific DNA methylation effects of different asbestos fibres on human bronchial epithelial cells.

Inhalation exposure to asbestos is associated with lung and pleural diseases in humans and remains a major public health issue worldwide. Human bronchial epithelial cells (16HBE) were exposed to UICC amosite, crocidolite and chrysotile. Cytotoxicity, genotoxicity, global DNA methylation on cytosine residues (using LC-MS/MS) were investigated at different doses (2.5-100 µg/ml). Gene-specific DNA methylation alterations at the whole genome were investigated using a microarray that interrogates >450 thousand CpG sites. Subsequently, gene functional analyses (KEGG pathway, Gene Ontology and functional classification) were performed on genes with differentially methylated gene promoters. At non-cytotoxic doses, global DNA methylation was altered after 24 h exposure to amosite and crocidolite (>2.5 µg/ml). Exposure to amosite and crocidolite (amphibole type asbestos) induced both hypomethylation and hypermethylation at single CpG site and gene promoter levels whereas exposure to chrysotile (serpentine type asbestos) induced hypomethylation at the gene promoter level. Gene functional classification analyses revealed that all types of asbestos fibres induce alterations on GO-clusters i.e. on regulation of Rho-protein signal transduction, nucleus, (e.g. homeobox genes), ATP-binding function and extracellular region (e.g. WNT-group of genes). These differentially methylated genes might contribute to asbestos-related diseases in bronchial cells.

TRPV4 activation triggers protective responses to bacterial lipopolysaccharides in airway epithelial cells.

Lipopolysaccharides (LPS), the major components of the wall of gram-negative bacteria, trigger powerful defensive responses in the airways via mechanisms thought to rely solely on the Toll-like receptor 4 (TLR4) immune pathway. Here we show that airway epithelial cells display an increase in intracellular Ca2+ concentration within seconds of LPS application. This response occurs in a TLR4-independent manner, via activation of the transient receptor potential vanilloid 4 cation channel (TRPV4). We found that TRPV4 mediates immediate LPS-induced increases in ciliary beat frequency and the production of bactericidal nitric oxide. Upon LPS challenge TRPV4-deficient mice display exacerbated ventilatory changes and recruitment of polymorphonuclear leukocytes into the airways. We conclude that LPS-induced activation of TRPV4 triggers signaling mechanisms that operate faster and independently from the canonical TLR4 immune pathway, leading to immediate protective responses such as direct antimicrobial action, increase in airway clearance, and the regulation of the inflammatory innate immune reaction.