Selective Replacement of Cholesterol with Cationic Amphiphilic Drugs Enables the Design of Lipid Nanoparticles with Improved RNA Delivery.

The delivery of RNA across biological barriers can be achieved by encapsulation in lipid nanoparticles (LNPs). Cationic amphiphilic drugs (CADs) are pharmacologically diverse compounds with ionizable lipid-like features. In this work, we applied CADs as a fifth component of state-of-the-art LNPs via microfluidic mixing. Improved cytosolic delivery of both siRNA and mRNA was achieved by partly replacing the cholesterol fraction of LNPs with CADs. The LNPs could cross the mucus layer in a mucus-producing air-liquid interface model of human primary bronchial epithelial cells following nebulization. Moreover, CAD-LNPs demonstrated improved epithelial and endothelial targeting following intranasal administration in mice, without a marked pro-inflammatory signature. Importantly, quantification of the CAD-LNP molar composition, as demonstrated for nortriptyline, revealed a gradual leakage of the CAD from the formulation during LNP dialysis. Altogether, these data suggest that the addition of a CAD prior to the rapid mixing process might have an impact on the composition, structure, and performance of LNPs.

Evaluating ß2-agonists as siRNA delivery adjuvants for pulmonary surfactant-coated nanogel inhalation therapy.

The lung is an attractive target organ for inhalation of RNA therapeutics, such as small interfering RNA (siRNA). However, clinical translation of siRNA drugs for application in the lung is hampered by many extra- and intracellular barriers. We previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel (nanogel) core coated with Curosurf®, a clinically used pulmonary surfactant. The surfactant shell was shown to markedly improve particle stability and promote intracellular siRNA delivery, both in vitro and in vivo. However, the full potential of siRNA nanocarriers is typically not reached as they are rapidly trafficked towards lysosomes for degradation and only a fraction of the internalized siRNA cargo is able to escape into the cytosol. We recently reported on the repurposing of widely applied cationic amphiphilic drugs (CADs) as siRNA delivery enhancers. Due to their physicochemical properties, CADs passively accumulate in the (endo)lysosomal compartment causing a transient permeabilization of the lysosomal membrane, which facilitates cytosolic drug delivery. In this work, we assessed a selection of cationic amphiphilic ß2-agonists (i.e., salbutamol, formoterol, salmeterol and indacaterol) for their ability to enhance siRNA delivery in a lung epithelial and macrophage cell line. These drugs are widely used in the clinic for their bronchodilating effect in obstructive lung disease. As opposed to the least hydrophobic drugs salbutamol and formoterol, the more hydrophobic long-acting ß2-agonist (LABA) salmeterol promoted siRNA delivery in both cell types for both uncoated and surfactant-coated nanogels, whereas indacaterol showed this effect solely in lung epithelial cells. Our results demonstrate the potential of both salmeterol and indacaterol to be repurposed as adjuvants for nanocarrier-mediated siRNA delivery to the lung, which could provide opportunities for drug combination therapy.

Commensal bacteria of the lung microbiota synergistically inhibit inflammation in a three-dimensional epithelial cell model.

Patients with chronic lung disease suffer from persistent inflammation and are typically colonized by pro-inflammatory pathogenic bacteria. Besides these pathogens, a wide variety of commensal species is present in the lower airways but their role in inflammation is unclear. Here, we show that the lung microbiota contains several species able to inhibit activation of the pro-inflammatory NF-κB pathway and production of interleukin 8 (IL-8), triggered by lipopolysaccharide (LPS) or H2O2, in a physiologically relevant three-dimensional (3D) lung epithelial cell model. We demonstrate that the minimal dose needed for anti-inflammatory activity differs between species (with the lowest dose needed for Rothia mucilaginosa), and depends on the type of pro-inflammatory stimulus and read out. Furthermore, we evaluated synergistic activity between pairs of anti-inflammatory bacteria on the inhibition of the NF-κB pathway and IL-8 secretion. Synergistic anti-inflammatory activity was observed for 4/10 tested consortia. These findings indicate that various microbiota members can influence lung inflammation either alone or as a consortium. This information can contribute to a better understanding of the lung microbiota in chronic lung disease development and process, and could open up new avenues for treatment.

Trajectory of neutrophilic responses in a mouse model of pollutant-aggravated allergic asthma.

Experimental studies suggest that neutrophils could contribute to allergic asthma pathogenesis, that is mainly driven by type 2 immunity. Inhalation of diesel exhaust particles (DEP) is implicated in both exacerbation and development of asthma. Since exposure to DEP is associated with a neutrophilic component, we aimed to investigate how exposure to the combination of allergens and DEP modulates neutrophilic responses. Human bronchial epithelial cells (HBEC) were exposed to house dust mite (HDM), DEP or HDM + DEP in vitro to determine the expression of neutrophil-recruiting chemokines. Female (C57BL/6 J) mice were intranasally instilled with saline, DEP, HDM or combined HDM + DEP for 3 weeks (subacute) or 6 weeks (chronic). The neutrophilic responses were determined in lung tissue and bronchoalveolar lavage fluid (BALF). Simultaneous exposure to HDM + DEP resulted in increased CXCL1 and CXCL8 mRNA expression by HBEC in vitro. In mice, subacute exposure to HDM + DEP induced a strong mixed eosinophilic/neutrophilic inflammation in BALF and lung and was associated with higher expression of neutrophil-attracting chemokines and NET formation compared to the sole exposures. After chronic HDM + DEP exposure, a similar neutrophilic response was observed, however the NET formation was less pronounced. Interestingly, the increase of BALF eosinophils was also significantly attenuated after chronic HDM + DEP exposure compared to the subacute exposure. Subacute and chronic HDM + DEP exposure induced goblet cell hyperplasia and airway hyperresponsiveness. Our data suggest a role for neutrophils and NETs in pollutant-aggravated eosinophilic allergic asthma. Moreover, subacute exposure to HDM + DEP induces a mixed eosinophilic/neutrophilic response whereas upon chronic HDM + DEP exposure there is a shift in inflammatory response with a more prominent neutrophilic component.

Antiviral Responses of Tissue-resident CD49a+ Lung Natural Killer Cells Are Dysregulated in Chronic Obstructive Pulmonary Disease.

Rationale: Tissue-resident natural killer (trNK) cells have been identified in numerous organs, but little is known about their functional contribution to respiratory immunity, in particular during chronic lung diseases such as chronic obstructive pulmonary disease (COPD). Objectives: To investigate the phenotype and antiviral responses of trNK cells in murine cigarette smoke-induced experimental COPD and in human lung parenchyma from COPD donors. Methods: Mice were exposed to cigarette smoke for 12 weeks to induce COPD-like lung disease. Lung trNK cell phenotypes and function were analyzed by flow cytometry in both murine and human disease with and without challenge with influenza A virus. Measurements and Main Results: In the mouse lung, CD49a+CD49b+EOMES+ and CD49a+CD49b-EOMESlo NK cell populations had a distinct phenotype compared with CD49a- circulating NK cells. CD49a+ NK cells were more extensively altered earlier in disease onset than circulating NK cells, and increased proportions of CD49a+ NK cells correlated with worsening disease in both murine and human COPD. Furthermore, the presence of lung disease delayed both circulating and trNK cell functional responses to influenza infection. CD49a+ NK cells markedly increased their NKG2D, CD103, and CD69 expression in experimental COPD after influenza infection, and human CD49a+ NK cells were hyperactive to ex vivo influenza infection in COPD donors. Conclusions: Collectively, these results demonstrate that trNK cell function is altered in cigarette smoke-induced disease and suggests that smoke exposure may aberrantly prime trNK cell responsiveness to viral infection. This may contribute to excess inflammation during viral exacerbations of COPD.

RIPK1 kinase-dependent inflammation and cell death contribute to the pathogenesis of COPD.

BACKGROUND: Receptor-interacting protein kinase 1 (RIPK1) is a key mediator of regulated cell death (including apoptosis and necroptosis) and inflammation, both drivers of COPD pathogenesis. We aimed to define the contribution of RIPK1 kinase-dependent cell death and inflammation in the pathogenesis of COPD. METHODS: We assessed RIPK1 expression in single-cell RNA sequencing (RNA-seq) data from human and mouse lungs, and validated RIPK1 levels in lung tissue of COPD patients via immunohistochemistry. Next, we assessed the consequences of genetic and pharmacological inhibition of RIPK1 kinase activity in experimental COPD, using Ripk1 S25D/S25D kinase-deficient mice and the RIPK1 kinase inhibitor GSK'547. RESULTS: RIPK1 expression increased in alveolar type 1 (AT1), AT2, ciliated and neuroendocrine cells in human COPD. RIPK1 protein levels were significantly increased in airway epithelium of COPD patients compared with never-smokers and smokers without airflow limitation. In mice, exposure to cigarette smoke (CS) increased Ripk1 expression similarly in AT2 cells, and further in alveolar macrophages and T-cells. Genetic and/or pharmacological inhibition of RIPK1 kinase activity significantly attenuated airway inflammation upon acute and subacute CS exposure, as well as airway remodelling, emphysema, and apoptotic and necroptotic cell death upon chronic CS exposure. Similarly, pharmacological RIPK1 kinase inhibition significantly attenuated elastase-induced emphysema and lung function decline. Finally, RNA-seq on lung tissue of CS-exposed mice revealed downregulation of cell death and inflammatory pathways upon pharmacological RIPK1 kinase inhibition. CONCLUSIONS: RIPK1 kinase inhibition is protective in experimental models of COPD and may represent a novel promising therapeutic approach.

miR449 Protects Airway Regeneration by Controlling AURKA/HDAC6-Mediated Ciliary Disassembly.

Airway mucociliary regeneration and function are key players for airway defense and are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes and microRNA-449 (miR449). In vitro, miR449 was strongly increased during airway epithelial mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or infective insults. miR0449-/- mice (both alleles are deleted) showed impaired ciliated epithelial regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously in aged miR449-/- mice. We identified Aurora kinase A and its effector target HDAC6 as key mediators in miR449-regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is downregulated upon miR449 overexpression in vitro and upregulated in miR449-/- mouse lungs. Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and coverage in miR449-/- cells, consistent with its tubulin-deacetylating function. Altogether, our study establishes a link between miR449, ciliary dysfunction, and COPD pathogenesis.

Unlocking the secrets of long non-coding RNAs in asthma.

Asthma is a very heterozygous disease, divided in subtypes, such as eosinophilic and neutrophilic asthma. Phenotyping and endotyping of patients, especially patients with severe asthma who are refractory to standard treatment, are crucial in asthma management and are based on a combination of clinical and biological features. Nevertheless, the quest remains to find better biomarkers that distinguish asthma subtypes in a more clear and objective manner and to find new therapeutic targets to treat people with therapy-resistant asthma. In the past, research to identify asthma subtypes mainly focused on expression profiles of protein-coding genes. However, advances in RNA-sequencing technologies and the discovery of non-coding RNAs as important post-transcriptional regulators have provided an entire new field of research opportunities in asthma. This review focusses on long non-coding RNAs (lncRNAs) in asthma; these are non-coding RNAs with a length of more than 200 nucleotides. Many lncRNAs are differentially expressed in asthma, and several have been associated with asthma severity or inflammatory phenotype. Moreover, in vivo and in vitro functional studies have identified the mechanisms of action of specific lncRNAs. Although lncRNAs remain not widely studied in asthma, the current studies show the potential of lncRNAs as biomarkers and therapeutic targets as well as the need for further research.

Identification of asthma-associated microRNAs in bronchial biopsies.

BACKGROUND: Changes in microRNA (miRNA) expression can contribute to the pathogenesis of many diseases, including asthma. We aimed to identify miRNAs that are differentially expressed between asthma patients and healthy controls, and explore their association with clinical and inflammatory parameters of asthma. METHODS: Differentially expressed miRNAs were determined by small RNA sequencing on bronchial biopsies of 79 asthma patients and 82 healthy controls using linear regression models. Differentially expressed miRNAs were associated with clinical and inflammatory asthma features. Potential miRNA-mRNA interactions were analysed using mRNA data available from the same bronchial biopsies, and enrichment of pathways was identified with Enrichr and g:Profiler. RESULTS: In total, 78 differentially expressed miRNAs were identified in bronchial biopsies of asthma patients compared with controls, of which 60 remained differentially expressed after controlling for smoking and inhaled corticosteroid treatment. We identified several asthma-associated miRNAs, including miR-125b-5p and miR-223-3p, based on a significant association with multiple clinical and inflammatory asthma features and their negative correlation with genes associated with the presence of asthma. The most enriched biological pathway(s) affected by miR-125b-5p and miR-223-3p were inflammatory response and cilium assembly/organisation. Of interest, we identified that lower expression of miR-26a-5p was linked to more severe eosinophilic inflammation as measured in blood, sputum as well as bronchial biopsies. CONCLUSION: Collectively, we identified miR-125b-5p, miR-223-3p and miR-26a-5p as potential regulators that could contribute to the pathogenesis of asthma.

MiR-223 is increased in lungs of patients with COPD and modulates cigarette smoke-induced pulmonary inflammation.

Since microRNA (miR)-223-3p modulates inflammatory responses and chronic obstructive pulmonary disease (COPD) is associated with amplified pulmonary inflammation, we hypothesized that miR-223-3p plays a role in COPD pathogenesis. Expression of miR-223-3p was measured in lung tissue of two independent cohorts with patients with GOLD stage II-IV COPD, never smokers, and smokers without COPD. The functional role of miR-223-3p was studied in deficient mice and on overexpression in airway epithelial cells from COPD and controls. We observed higher miR-223-3p levels in patients with COPD stage II-IV compared with (non)-smoking controls, and levels were associated with higher neutrophil numbers in bronchial biopsies of patients with COPD. MiR-223-3p expression was also increased in lungs and bronchoalveolar lavage of cigarette smoke (CS)-exposed mice. CS-induced neutrophil and monocyte lung infiltration was stronger in miR-223-deficient mice on acute (5 days) exposure, but attenuated on subchronic (4 wk) exposure. Additionally, miR-223 deficiency attenuated acute and subchronic CS-induced lung infiltration of dendritic cells and T lymphocytes. Finally, in vitro overexpression of miR-223-3p in non-COPD airway epithelial cells suppressed C-X-C motif chemokine ligand 8 (CXCL8) and granulocyte monocyte-colony stimulation factor (GM-CSF) secretion and gene expression of the proinflammatory transcription factor TRAF6. Importantly, this suppressive effect of miR-223-3p was compromised in COPD-derived cultures. In conclusion, we demonstrate that miR-223-3p is increased in lungs of patients with COPD and CS-exposed mice and is associated with neutrophilic inflammation. In vivo data indicate that miR-223 acts as negative regulator of acute CS-induced neutrophilic and monocytic inflammation. In vitro data suggest that miR-223-3p does so by suppressing proinflammatory airway epithelial responses, which is less effective in COPD epithelium.

Neutrophils Affect IL-33 Processing in Response to the Respiratory Allergen Alternaria alternata.

Future precision medicine requires further clarifying the mechanisms of inflammation in the severe endotypes of chronic airway diseases such as asthma and chronic rhinosinusitis (CRS). The presence of neutrophils in the airways is often associated with severe airway inflammation, while their precise contribution to the severe inflammation is largely unknown. We aimed to study the role of neutrophils in BALB/c and C57BL/6 mice exposed to Alternaria alternata (Alt). The mice were exposed to Alt extract for twelve hours or ten days to induce allergic airway inflammation. C57BL/6 mice exposed to Alt responded with eosinophilic infiltration and the characteristic IL-5 upregulation. In contrast, the inflammatory response to Alt extract in BALB/c mice was characterized by a neutrophilic response, high levels of G-CSF, and elastase in the lungs. The lack of neutrophils affected the processing of IL-33 in BALB/c mice, as was demonstrated by depletion of neutrophils through intraperitoneal injections of anti-Ly6G antibody. Our data identifies the key role of neutrophils in airway inflammation through IL-33 cleavage in the Alt-induced airway inflammation in mice, which could potentially underline the different endotypes in human disease.

Quantification and role of innate lymphoid cell subsets in Chronic Obstructive Pulmonary Disease.

OBJECTIVES: Innate lymphoid cells (ILCs) secrete cytokines, such as IFN-γ, IL-13 and IL-17, which are linked to chronic obstructive pulmonary disease (COPD). Here, we investigated the role of pulmonary ILCs in COPD pathogenesis. METHODS: Lung ILC subsets in COPD and control subjects were quantified using flow cytometry and associated with clinical parameters. Tissue localisation of ILC and T-cell subsets was determined by immunohistochemistry. Mice were exposed to air or cigarette smoke (CS) for 1, 4 or 24 weeks to investigate whether pulmonary ILC numbers and activation are altered and whether they contribute to CS-induced innate inflammatory responses. RESULTS: Quantification of lung ILC subsets demonstrated that ILC1 frequency in the total ILC population was elevated in COPD and was associated with smoking and severity of respiratory symptoms (COPD Assessment Test [CAT] score). All three ILC subsets localised near lymphoid aggregates in COPD. In the COPD mouse model, CS exposure in C57BL/6J mice increased ILC numbers at all time points, with relative increases in ILC1 in bronchoalveolar lavage (BAL) fluid. Importantly, CS exposure induced increases in neutrophils, monocytes and dendritic cells that remained elevated in Rag2/Il2rg-deficient mice that lack adaptive immune cells and ILCs. However, CS-induced CXCL1, IL-6, TNF-α and IFN-γ levels were reduced by ILC deficiency. CONCLUSION: The ILC1 subset is increased in COPD patients and correlates with smoking and severity of respiratory symptoms. ILCs also increase upon CS exposure in C57BL/6J mice. In the absence of adaptive immunity, ILCs contribute to CS-induced pro-inflammatory mediator release, but are redundant in CS-induced innate inflammation.

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.

Spotlight on microRNAs in allergy and asthma.

In past 10 years, microRNAs (miRNAs) have gained scientific attention due to their importance in the pathophysiology of allergic diseases and their potential as biomarkers in liquid biopsies. They act as master post-transcriptional regulators that control most cellular processes. As one miRNA can target several mRNAs, often within the same pathway, dysregulated expression of miRNAs may alter particular cellular responses and contribute, or lead, to the development of various diseases. In this review, we give an overview of the current research on miRNAs in allergic diseases, including atopic dermatitis, allergic rhinitis, and asthma. Specifically, we discuss how individual miRNAs function in the regulation of immune responses in epithelial cells and specialized immune cells in response to different environmental factors and respiratory viruses. In addition, we review insights obtained from experiments with murine models of allergic airway and skin inflammation and offer an overview of studies focusing on miRNA discovery using profiling techniques and bioinformatic modeling of the network effect of multiple miRNAs. In conclusion, we highlight the importance of research into miRNA function in allergy and asthma to improve our knowledge of the molecular mechanisms involved in the pathogenesis of this heterogeneous group of diseases.

Charting Extracellular Transcriptomes in The Human Biofluid RNA Atlas.

Extracellular RNAs present in biofluids have emerged as potential biomarkers for disease. Where most studies focus on blood-derived fluids, other biofluids may be more informative. We present an atlas of messenger, circular, and small RNA transcriptomes of a comprehensive collection of 20 human biofluids. By means of synthetic spike-in controls, we compare RNA content across biofluids, revealing a 10,000-fold difference in concentration. The circular RNA fraction is increased in most biofluids compared to tissues. Each biofluid transcriptome is enriched for RNA molecules derived from specific tissues and cell types. Our atlas enables an informed selection of the most relevant biofluid to monitor particular diseases. To verify the biomarker potential in these biofluids, four validation cohorts representing a broad spectrum of diseases were profiled, revealing numerous differential RNAs between case and control subjects. Spike-normalized data are publicly available in the R2 web portal for further exploration.

Targeting neutrophils in asthma: A therapeutic opportunity?

Suppression of airway inflammation with inhaled corticosteroids has been the key therapeutic approach for asthma for many years. Identification of inflammatory phenotypes in asthma has moreover led to important breakthroughs, e.g. with specific targeting of the IL-5 pathway as add-on treatment in difficult-to-treat eosinophilic asthma. However, the impact of interfering with the neutrophilic component in asthma is less documented and understood. This review provides an overview of established and recent insights with regard to the role of neutrophils in asthma, focusing on research in humans. We will describe the main drivers of neutrophilic responses in asthma, the heterogeneity in neutrophils and how they could contribute to asthma pathogenesis. Moreover we will describe findings from clinical trials, in which neutrophilic inflammation was targeted. It is clear that neutrophils are important actors in asthma development and play a role in exacerbations. However, more research is required to fully understand how modulation of neutrophil activity could lead to a significant benefit in asthma patients with airway neutrophilia.

Lyophilization and nebulization of pulmonary surfactant-coated nanogels for siRNA inhalation therapy.

RNA interference (RNAi) enables highly specific silencing of potential target genes for treatment of pulmonary pathologies. The intracellular RNAi pathway can be activated by cytosolic delivery of small interfering RNA (siRNA), inducing sequence-specific gene knockdown on the post-transcriptional level. Although siRNA drugs hold many advantages over currently applied therapies, their clinical translation is hampered by inefficient delivery across cellular membranes. We previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel core (nanogel) coated with Curosurf®, a clinically used pulmonary surfactant (PS). The latter enhances both particle stability as well as intracellular siRNA delivery, which was shown to be governed by the PS-associated surfactant protein B (SP-B). Despite having a proven in vitro and in vivo siRNA delivery potential when prepared ex novo, clinical translation of this liquid nanoparticle suspension requires the identification of a long-term preservation strategy that maintains nanoparticle stability and potency. In addition, to achieve optimal pulmonary deposition of the nanocomposite, its compatibility with state-of-the-art pulmonary administration techniques should be evaluated. Here, we demonstrate that PS-coated nanogels can be lyophilized, reconstituted and subsequently nebulized via a vibrating mesh nebulizer. The particles retain their physicochemical integrity and their ability to deliver siRNA in a human lung epithelial cell line. The latter result suggests that the functional integrity of SP-B in the PS coat towards siRNA delivery might be preserved as well. Of note, successful lyophilization was achieved without the need for stabilizing lyo- or cryoprotectants. Our results demonstrate that PS-coated siRNA-loaded nanogels can be lyophilized, which offers the prospect of long-term storage. In addition, the formulation was demonstrated to be suitable for local administration with a state-of-the-art nebulizer for human use upon reconstitution. Hence, the data presented in this study represent an important step towards clinical application of such nanocomposites for treatment of pulmonary disease.

Translational research into the effects of cigarette smoke on inflammatory mediators and epithelial TRPV1 in Crohn's disease.

Crohn's disease is a pathological condition of the gastro-intestinal tract, causing severe transmural inflammation in the ileum and/or colon. Cigarette smoking is one of the best known environmental risk factors for the development of Crohn's disease. Nevertheless, very little is known about the effect of prolonged cigarette smoke exposure on inflammatory modulators in the gut. We examined the effect of cigarette smoke on cytokine profiles in the healthy and inflamed gut of human subjects and in the trinitrobenzene sulphonic acid mouse model, which mimics distal Crohn-like colitis. In addition, the effect of cigarette smoke on epithelial expression of transient receptor potential channels and their concurrent increase with cigarette smoke-augmented cytokine production was investigated. Active smoking was associated with increased IL-8 transcription in ileum of controls (p < 0,001; n = 18-20/group). In the ileum, TRPV1 mRNA levels were decreased in never smoking Crohn's disease patients compared to healthy subjects (p <0,001; n = 20/group). In the colon, TRPV1 mRNA levels were decreased (p = 0,046) in smoking healthy controls (n = 20/group). Likewise, healthy mice chronically exposed to cigarette smoke (n = 10/group) showed elevated ileal Cxcl2 (p = 0,0075) and colonic Kc mRNA levels (p = 0,0186), whereas TRPV1 mRNA and protein levels were elevated in the ileum (p = 0,0315). Although cigarette smoke exposure prior to trinitrobenzene sulphonic acid administration did not alter disease activity, increased pro-inflammatory cytokine production was observed in the distal colon (Kc: p = 0,0273; Cxcl2: p = 0,104; Il1-ß: p = 0,0796), in parallel with the increase of Trpv1 mRNA (p < 0,001). We infer that CS affects pro-inflammatory cytokine expression in healthy and inflamed gut, and that the simultaneous modulation of TRPV1 may point to a potential involvement of TRPV1 in cigarette smoke-induced production of inflammatory mediators.