Lung Slices Research Articles

Ex vivo tissue perturbations coupled to single-cell RNA-seq reveal multilineage cell circuit dynamics in human lung fibrogenesis.

Pulmonary fibrosis develops as a consequence of failed regeneration after injury. Analyzing mechanisms of regeneration and fibrogenesis directly in human tissue has been hampered by the lack of organotypic models and analytical techniques. In this work, we coupled ex vivo cytokine and drug perturbations of human precision-cut lung slices (hPCLS) with single-cell RNA sequencing and induced a multilineage circuit of fibrogenic cell states in hPCLS. We showed that these cell states were highly similar to the in vivo cell circuit in a multicohort lung cell atlas from patients with pulmonary fibrosis. Using micro-CT-staged patient tissues, we characterized the appearance and interaction of myofibroblasts, an ectopic endothelial cell state, and basaloid epithelial cells in the thickened alveolar septum of early-stage lung fibrosis. Induction of these states in the hPCLS model provided evidence that the basaloid cell state was derived from alveolar type 2 cells, whereas the ectopic endothelial cell state emerged from capillary cell plasticity. Cell-cell communication routes in patients were largely conserved in hPCLS, and antifibrotic drug treatments showed highly cell type-specific effects. Our work provides an experimental framework for perturbational single-cell genomics directly in human lung tissue that enables analysis of tissue homeostasis, regeneration, and pathology. We further demonstrate that hPCLS offer an avenue for scalable, high-resolution drug testing to accelerate antifibrotic drug development and translation.

Evaluating Cytotoxicity and Membrane Integrity in Lung Slices via Lactate Dehydrogenase Assay

Evaluating Cytotoxicity and Membrane Integrity in Lung Slices via Lactate Dehydrogenase Assay

Evaluating Cytotoxicity and Membrane Integrity in Lung Slices via Lactate Dehydrogenase Assay

Evaluating Cytotoxicity and Membrane Integrity in Lung Slices via Lactate Dehydrogenase Assay

Agarose-Based Preparation of Human Precision-Cut Lung Slices

Agarose-Based Preparation of Human Precision-Cut Lung Slices

An Assay to Measure Chemically-Induced Cytotoxicity in Human Precision-Cut Lung Slices

An Assay to Measure Chemically-Induced Cytotoxicity in Human Precision-Cut Lung Slices

Agarose-Based Preparation of Human Precision-Cut Lung Slices

Agarose-Based Preparation of Human Precision-Cut Lung Slices

An Assay to Measure Chemically-Induced Cytotoxicity in Human Precision-Cut Lung Slices

An Assay to Measure Chemically-Induced Cytotoxicity in Human Precision-Cut Lung Slices

Hydrogel-Embedded Precision-Cut Lung Slices Model Lung Cancer Premalignancy Ex Vivo.

Lung cancer is the leading global cause of cancer-related deaths. Although smoking cessation is the best prevention, 50% of lung cancer diagnoses occur in people who have quit smoking. Research into treatment options for high-risk patients is constrained to rodent models, which are time-consuming, expensive, and require large cohorts. Embedding precision-cut lung slices (PCLS) within an engineered hydrogel and exposing this tissue to vinyl carbamate, a carcinogen from cigarette smoke, creates an in vitro model of lung cancer premalignancy. Hydrogel formulations are selected to promote early lung cancer cellular phenotypes and extend PCLS viability to six weeks. Hydrogel-embedded PCLS are exposed to vinyl carbamate, which induces adenocarcinoma in mice. Analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content after six weeks reveals that vinyl carbamate induces premalignant lesions with a mixed adenoma/squamous phenotype. Putative chemoprevention agents diffuse through the hydrogel and induce tissue-level changes. The design parameters selected using murine tissue are validated with hydrogel-embedded human PCLS and results show increased proliferation and premalignant lesion gene expression patterns. This tissue-engineered model of human lung cancer premalignancy is the foundation for more sophisticated ex vivo models that enable the study of carcinogenesis and chemoprevention strategies.

Direct assessment of nitrative stress in lipid environments: Applications of a designer lipid-based biosensor for peroxynitrite

Lipid membranes and lipid-rich organelles are targets of peroxynitrite (ONOO-), a highly reactive species generated under nitrative stress. We report a membrane-localized phospholipid (DPPC-TC-ONOO-) that allows the detection of ONOO- in diverse lipid environments: biomimetic vesicles, mammalian cell compartments, and within the lung lining. DPPC-TC-ONOO- and POPC self-assemble to membrane vesicles that fluorogenically and selectively respond to ONOO-. DPPC-TC-ONOO-, delivered through lipid nanoparticles, allowed for ONOO- detection in the endoplasmic reticulum upon cytokine-induced nitrative stress in live mammalian cells. It also responded to ONOO- within lung tissue murine models upon acute lung injury. We observed nitrative stress around bronchioles in precision cut lung slices exposed to nitrogen mustard and in pulmonary macrophages following intratracheal bleomycin challenge. Results showed that DPPC-TC-ONOO- functions specifically toward iNOS, a key enzyme modulating nitrative stress, and offers significant advantages over its hydrophilic analog in terms of localization and signal generation.

Caffeic Acid Phenethyl Ester (CAPE), a natural polyphenol to increase the therapeutic window for lung adenocarcinomas

Background and purposeLung cancers are highly resistant to radiotherapy, necessitating the use of high doses, which leads to radiation toxicities such as radiation pneumonitis and fibrosis. Caffeic Acid Phenethyl Ester (CAPE) has been suggested to have anti-proliferative and pro-apoptotic effects in tumour cells, while radioprotective anti-inflammatory and anti-oxidant effects in the normal tissue. We investigated the radiosensitizing and radioprotective effects of CAPE in lung cancer cell lines and normal tissue in vitro and ex vivo, respectively. Materials and methodsThe cytotoxic and radiosensitizing effects of CAPE in lung cancer were investigated using viability and clonogenic survival assays. The radioprotective effects of CAPE were assessed in vitro and ex vivo using precision cut lung slices (PCLS). Potential underlying molecular mechanisms of CAPE focusing on cell cycle, cell metabolism, mitochondrial function and pro-inflammatory markers were investigated. ResultsTreatment with CAPE decreased cell viability in a dose-dependent manner (IC50 57.6 ± 16.6 μM). Clonogenic survival assays showed significant radiosensitization by CAPE in lung adenocarcinoma lines (p < 0.05), while no differences were found in non-adenocarcinoma lines (p ≥ 0.13). Cell cycle analysis showed an increased S-phase (p < 0.05) after incubation with CAPE in the majority of cell lines. Metabolic profiling showed that CAPE shifted cellular respiration towards glycolysis (p < 0.01), together with mitochondrial membrane depolarization (p < 0.01). CAPE induced a decrease in NF-κB activity in adenocarcinomas and decreased pro-inflammatory gene expression in PCLS. ConclusionThe combination of CAPE and radiotherapy may be a potentially effective approach to increase the therapeutic window in lung cancer patients.

Exogenous hydrogen sulfide attenuates hyperoxia effects on neonatal mouse airways.

Supplemental O2 remains a necessary intervention for many premature infants (<34 wk gestation). Even moderate hyperoxia (<60% O2) poses a risk for subsequent airway disease, thereby predisposing premature infants to pediatric asthma involving chronic inflammation, airway hyperresponsiveness (AHR), airway remodeling, and airflow obstruction. Moderate hyperoxia promotes AHR via effects on airway smooth muscle (ASM), a cell type that also contributes to impaired bronchodilation and remodeling (proliferation, altered extracellular matrix). Understanding mechanisms by which O2 initiates long-term airway changes in prematurity is critical for therapeutic advancements for wheezing disorders and asthma in babies and children. Immature or dysfunctional antioxidant systems in the underdeveloped lungs of premature infants thereby heightens susceptibility to oxidative stress from O2. The novel gasotransmitter hydrogen sulfide (H2S) is involved in antioxidant defense and has vasodilatory effects with oxidative stress. We previously showed that exogenous H2S exhibits bronchodilatory effects in human developing airway in the context of hyperoxia exposure. Here, we proposed that exogenous H2S would attenuate effects of O2 on airway contractility, thickness, and remodeling in mice exposed to hyperoxia during the neonatal period. Using functional [flexiVent; precision-cut lung slices (PCLS)] and structural (histology; immunofluorescence) analyses, we show that H2S donors mitigate the effects of O2 on developing airway structure and function, with moderate O2 and H2S effects on developing mouse airways showing a sex difference. Our study demonstrates the potential applicability of low-dose H2S toward alleviating the detrimental effects of hyperoxia on the premature lung.NEW & NOTEWORTHY Chronic airway disease is a short- and long-term consequence of premature birth. Understanding effects of O2 exposure during the perinatal period is key to identify targetable mechanisms that initiate and sustain adverse airway changes. Our findings show a beneficial effect of exogenous H2S on developing mouse airway structure and function with notable sex differences. H2S donors alleviate effects of O2 on airway hyperreactivity, contractility, airway smooth muscle thickness, and extracellular matrix deposition.

Abstract 12185: Hypoxia Exacerbates Flow-Induced Vascular Remodeling via CXCL12/CXCR4 Pathway

Intro: Patients with congenital heart disease are at risk of developing pulmonary arterial hypertension (PAH-CHD) due to increased blood flow and exposure to hypoxia (Hx). We aimed to establish a“two-hit” mouse model of pulmonary hypertension (PH) combining increased blood flow via left pneumonectomy (LP) and Hx ( Fig 1a ). Our objective was to investigate the pathogenesis of flow-induced vascular remodeling and elucidate the underlying mechanism mediated by smooth muscle cells (SMCs) through the CXCL12/CXCR4 pathway. Hypothesis: We hypothesize SMCs contribute to flow-induced vascular remodeling by upregulating the Cxcl12/Cxcr4 pathway. Methods: Mice underwent LP followed by 3 weeks of Hx(LP/Hx). PH was evaluated by measuring right ventricular systolic pressure (RVSP) and Fulton Index. Whole lung lobes and precision cut lung slices underwent tissue clearing, immunofluorescence staining and advanced microscopy. SMCs were isolated using Dynabeads and Cxcl12 signaling measured by RT-qPCR. Tissue samples from patients with PAH-CHD (ventricular septal defect) were stained for CXCL12. Results: LP/Hx mice demonstrated severe PH compared to Hx controls, as evidenced by increased RVSP (41 vs 33mm Hg), right ventricle hypertrophy (FI: 40 vs 32%) and RV dysfunction ( Fig 1b ). SMCs from LP/Hx mice exhibited an 8.6-fold increase in Cxcl12 expression compared to controls, with Hx and LP alone showing 5-fold and 2-fold increases, respectively. Advanced microscopy revealed excessive remodeling of distal arterioles in LP/Hx mice ( Fig 1c ). Furthermore, CXCL12 expression was upregulated in SMCs of patients with PAH-CHD compared to controls. Conclusion: Our study, using a novel mouse model combining increased blood flow and hypoxia, demonstrated that upregulation of Cxcl12 in SMCs and ECs contributes to flow-induced vascular remodeling. This model has the potential to serve as a pre-clinical model for studying the pathology of PAH-CHD and exploring therapeutic interventions.

Inflammation plays a critical role in damage to the bronchiolar epithelium induced by Trueperella pyogenes in vitro and in vivo.

Trueperella pyogenes can cause severe pulmonary disease in swine, but the mechanism of pathogenesis is not well defined. T. pyogenes-induced damage to porcine bronchial epithelial cells (PBECs), porcine precision-cut lung slices (PCLS), and respiratory epithelium of mice remains unknown. In this study, we used T. pyogenes 20121 to infect PBECs in air-liquid interface conditions and porcine PCLS. T. pyogenes could adhere to, colonize, and induce cytotoxic effect on PBECs and the luminal surface of bronchi in PCLS, which damaged the bronchiolar epithelium. Moreover, bronchiolar epithelial cells showed extensive degeneration in the lungs of infected mice. Furthermore, western blot showed that the NOD-like receptor (NLR)/C-terminal caspase recruitment domain (ASC)/caspase-1 axis and nuclear factor-kappa B pathway were involved in inflammation in PCLS and lungs of mice, which also confirms that porcine PCLS provide a platform to analyze the pulmonary immune response. Meanwhile, the levels of p-c-Jun N-terminal kinase, p-extracellular signal-regulated kinase, and p-protein kinase B (AKT) were increased significantly, which indicated the mitogen-activated protein kinase and Akt pathways were also involved in inflammation in T. pyogenes-infected mice. In addition, we used T. pyogenes 20121 to infect tumor necrosis factor-alpha (tnf-α-/-) mice, and the results indicated that apoptosis and injury in respiratory epithelium of infected tnf-α-/- mice were alleviated. Thus, the pro-inflammatory cytokine TNF-α played a role in apoptosis and the respiratory epithelium injury in mouse lungs. Collectively, our study provides insight into the inflammatory injury induced by T. pyogenes and suggests that blocking NLR may be a potential therapeutic strategy against T. pyogenes infection.

Neuropeptide stimulation of physiological and immunological responses in precision-cut lung slices.

Organotypic lung slices, sometimes known as precision-cut lung slices (PCLS), provide an environment in which numerous cell types and interactions can be maintained outside the body (ex vivo). PCLS were maintained exvivo for up to a week and demonstrated health via the presence of neurons, maintenance of tissue morphology, synthesis of mucopolysaccharides, and minimal cell death. Multiple phenotypes of neuronal fibers were present in lung slices with varied size, caliber, and neurotransmitter immunoreactivity. Of the neuropeptides present in fibers, calcitonin gene-related peptide (CGRP) was the most prevalent. Exposing PCLS to recombinant CGRP resulted in the proliferation and dispersion of CD19+ B cells in slices taken selectively from females. The number of granules containing immunoreactive (ir) surfactant protein C (SPC), which are representative of alveolar type 2 cells, increased in slices from females within 24 h of exposure to CGRP. Additionally, ir-SPC granule size increased in slices from males and females across 48 h of exposure to CGRP. Exposure of PCLS to exogenous CGRP did not alter the number of solitary pulmonary neuroendocrine cells (PNEC) but did result in neuroendocrine bodies that had significantly more cells. Neuronal fiber numbers were unchanged based on ir-peripherin; however, ir-CGRP became non-detectable in fibers while unchanged in PNECs. The effects of exogenous CGRP provide insight into innate immune and neuroendocrine responses in the lungs that may be partially regulated by neural fibers. The sex-dependent nature of these changes may point to the basis for sex-selective outcomes among respiratory diseases.

Targeting IL-11 system as a treatment of pulmonary arterial hypertension

IL-11 is linked to fibrotic diseases, but its role in pulmonary hypertension is unclear. We examined IL-11's involvement in idiopathic pulmonary arterial hypertension (iPAH). Using samples from control (n = 20) and iPAH (n = 6) subjects, we assessed IL-11 and IL-11Rα expression and localization through RT-qPCR, ELISA, immunohistochemistry, and immunofluorescence. A monocrotaline-induced PAH model helped evaluate the impact of siRNA-IL-11 on pulmonary artery remodeling and PH. The effects of recombinant human IL-11 and IL-11Rα on human pulmonary artery smooth muscle cell (HPASMC) proliferation, pulmonary artery endothelial cell (HPAEC) mesenchymal transition, monocyte interactions, endothelial tube formation, and precision cut lung slice (PCLS) pulmonary artery remodeling and contraction were evaluated. IL-11 and IL-11Rα were over-expressed in pulmonary arteries (3.2-fold and 75-fold respectively) and serum (1.5-fold and 2-fold respectively) of patients with iPAH. Therapeutic transient transfection with siRNA targeting IL-11 resulted in a significant reduction in pulmonary artery remodeling (by 98%), right heart hypertrophy (by 66%), and pulmonary hypertension (by 58%) in rats exposed to monocrotaline treatment. rhIL-11 and soluble rhIL-11Rα induce HPASMC proliferation and HPAEC to monocyte interactions, mesenchymal transition, and tube formation. Neutralizing monoclonal IL-11 and IL-11Rα antibodies inhibited TGFβ1 and EDN-1 induced HPAEC to mesenchymal transition and HPASMC proliferation. In 3D PCLS, rhIL-11 and soluble rhIL-11Rα do not promote pulmonary artery contraction but sensitize PCLS pulmonary artery contraction induced by EDN-1.In summary, IL-11 and IL-11Rα are more highly expressed in the pulmonary arteries of iPAH patients and contribute to pulmonary artery remodeling and the development of PH.

A method for TAT-Cre recombinase-mediated floxed allele modification in ex vivo tissue slices.

Precision-cut lung slices (PCLS) are used for a variety of applications. However, methods to manipulate genes in PCLS are currently limited. We developed a new method, TAT-Cre recombinase-mediated floxed allele modification in tissue slices (TReATS), to induce highly effective and temporally controlled gene deletion or activation in ex vivo PCLS. Treatment of PCLS from Rosa26-flox-stop-flox-EYFP mice with cell-permeant TAT-Cre recombinase induced ubiquitous EYFP protein expression, indicating successful Cre-mediated excision of the upstream loxP-flanked stop sequence. Quantitative real-time PCR confirmed induction of EYFP. We successfully replicated the TReATS method in PCLS from Vangl2flox/flox mice, leading to the deletion of loxP-flanked exon 4 of the Vangl2 gene. Cre-treated Vangl2flox/flox PCLS exhibited cytoskeletal abnormalities, a known phenotype caused by VANGL2 dysfunction. We report a new method that bypasses conventional Cre-Lox breeding, allowing rapid and highly effective gene manipulation in ex vivo tissue models.

Luminescent reporter cells enable the identification of broad-spectrum antivirals against emerging viruses.

The emerging viruses SARS-CoV-2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS-CoV-2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P), respectively. We report improved luciferase-based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high-throughput manner. We identified 23 FDA-approved small molecules, among them monensin which displayed broad activity against furin and SKI-1/S1P. Monensin inhibited arenaviruses and SARS-CoV-2 in a dose-dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS-CoV-2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS-CoV-2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad-spectrum antivirals with therapeutic potential to combat current and future emerging viruses.

Amitriptyline inhibits bronchoconstriction and directly promotes dilatation of the airways

IntroductionThe standard therapy for bronchial asthma consists of combinations of acute (short-acting ß2-sympathomimetics) and, depending on the severity of disease, additional long-term treatment (including inhaled glucocorticoids, long-acting ß2-sympathomimetics, anticholinergics, anti-IL-4R antibodies). The antidepressant amitriptyline has been identified as a relevant down-regulator of immunological TH2-phenotype in asthma, acting—at least partially—through inhibition of acid sphingomyelinase (ASM), an enzyme involved in sphingolipid metabolism. Here, we investigated the non-immunological role of amitriptyline on acute bronchoconstriction, a main feature of airway hyperresponsiveness in asthmatic disease.MethodsAfter stimulation of precision cut lung slices (PCLS) from mice (wildtype and ASM-knockout), rats, guinea pigs and human lungs with mediators of bronchoconstriction (endogenous and exogenous acetylcholine, methacholine, serotonin, endothelin, histamine, thromboxane-receptor agonist U46619 and leukotriene LTD4, airway area was monitored in the absence of or with rising concentrations of amitriptyline. Airway dilatation was also investigated in rat PCLS by prior contraction induced by methacholine. As bronchodilators for maximal relaxation, we used IBMX (PDE inhibitor) and salbutamol (ß2-adrenergic agonist) and compared these effects with the impact of amitriptyline treatment. Isolated perfused lungs (IPL) of wildtype mice were treated with amitriptyline, administered via the vascular system (perfusate) or intratracheally as an inhalation. To this end, amitriptyline was nebulized via pariboy in-vivo and mice were ventilated with the flexiVent setup immediately after inhalation of amitriptyline with monitoring of lung function.ResultsOur results show amitriptyline to be a potential inhibitor of bronchoconstriction, induced by exogenous or endogenous (EFS) acetylcholine, serotonin and histamine, in PCLS from various species. The effects of endothelin, thromboxane and leukotrienes could not be blocked. In acute bronchoconstriction, amitriptyline seems to act ASM-independent, because ASM-deficiency (Smdp1−/−) did not change the effect of acetylcholine on airway contraction. Systemic as well as inhaled amitriptyline ameliorated the resistance of IPL after acetylcholine provocation. With the flexiVent setup, we demonstrated that the acetylcholine-induced rise in central and tissue resistance was much more marked in untreated animals than in amitriptyline-treated ones. Additionally, we provide clear evidence that amitriptyline dilatates pre-contracted airways as effectively as a combination of typical bronchodilators such as IBMX and salbutamol.ConclusionAmitriptyline is a drug of high potential, which inhibits acute bronchoconstriction and induces bronchodilatation in pre-contracted airways. It could be one of the first therapeutic agents in asthmatic disease to have powerful effects on the TH2-allergic phenotype and on acute airway hyperresponsiveness with bronchoconstriction, especially when inhaled.

Modelling Acid-Induced Lung Damage in Precision-Cut Lung Slices: An Ex Vivo Animal Model

Background: Donor lungs are often discarded, with gastric aspiration accounting for ~9% of lungs unsuitable for transplantation. To increase the donor pool, it is important to understand the pathophysiology of aspiration-induced lung damage (AILD) and to assess its treatment. Methods: Precision-cut lung slices (PCLS) were prepared from murine lungs and exposed to acid—pH 1.5 to 5.5—for 15 min. We also investigated whether acid-exposed slices (pH 3.5) could affect unexposed slices. In addition, we investigated whether dexamethasone (0.5 or 1 μM) could mitigate and treat the damage in each group. In each experiment (n = 3), we analyzed cell viability (ATP/protein content) and markers of inflammation (IL-1β, IL-6, TNF-𝛼, TRAIL). Results: PCLS subjected to pH 1.5–3.5 had a significantly reduced amount of ATP, albeit no increase in inflammation markers. There was no interaction of secretions from acid-exposed slices on unexposed slices. Dexamethasone had no beneficial effects in either group. Conclusion: Direct exposure to acid in the PCLS leads to a decrease in cell viability. Acid-exposed slices had no effect on the cell viability of unexposed slices. Treatment with dexamethasone offered no mitigation. More studies have to be performed to elucidate the pathophysiology of AILD and the possible treatment of aspiration-induced injury.

The RNA binding proteins ZFP36L1 and ZFP36L2 are dysregulated in airway epithelium in human and a murine model of asthma.

Introduction: Asthma is the most common chronic inflammatory disease of the airways. The airway epithelium is a key driver of the disease, and numerous studies have established genome-wide differences in mRNA expression between health and asthma. However, the underlying molecular mechanisms for such differences remain poorly understood. The human TTP family is comprised of ZFP36, ZFP36L1 and ZFP36L2, and has essential roles in immune regulation by determining the stability and translation of myriad mRNAs encoding for inflammatory mediators. We investigated the expression and possible role of the tristetraprolin (TTP) family of RNA binding proteins (RBPs), poorly understood in asthma. Methods: We analysed the levels of ZFP36, ZFP36L1 and ZFP36L2 mRNA in several publicly available asthma datasets, including single cell RNA-sequencing. We also interrogated the expression of known targets of these RBPs in asthma. We assessed the lung mRNA expression and cellular localization of Zfp36l1 and Zfp36l2 in precision cut lung slices in murine asthma models. Finally, we determined the expression in airway epithelium of ZFP36L1 and ZFP36L2 in human bronchial biopsies and performed rescue experiments in primary bronchial epithelium from patients with severe asthma. Results: We found ZFP36L1 and ZFP36L2 mRNA levels significantly downregulated in the airway epithelium of patients with very severe asthma in different cohorts (5 healthy vs. 8 severe asthma; 36 moderate asthma vs. 37 severe asthma on inhaled steroids vs. 26 severe asthma on oral corticoids). Integrating several datasets allowed us to infer that mRNAs potentially targeted by these RBPs are increased in severe asthma. Zfp36l1 was downregulated in the lung of a mouse model of asthma, and immunostaining of ex vivo lung slices with a dual antibody demonstrated that Zfp36l1/l2 nuclear localization was increased in the airway epithelium of an acute asthma mouse model, which was further enhanced in a chronic model. Immunostaining of human bronchial biopsies showed that airway epithelial cell staining of ZFP36L1 was decreased in severe asthma as compared with mild, while ZFP36L2 was upregulated. Restoring the levels of ZFP36L1 and ZFP36L2 in primary bronchial epithelial cells from patients with severe asthma decreased the mRNA expression of IL6, IL8 and CSF2. Discussion: We propose that the dysregulation of ZFP36L1/L2 levels as well as their subcellular mislocalization contributes to changes in mRNA expression and cytoplasmic fate in asthma.