Airway epithelial cells together with macrophages constitute the first line of defense to interact and respond to external stimuli, thus driving immune responses. This, together with their abundance, makes airway epithelial cells potential major contributors to the inflammation and pathogenesis of asthma
Recently, we identified a defect in translational control in bronchial epithelial cells from patients with asthma that correlated well with neutrophilic responses.
Several earlier reports have shown that both genetic and acquired defects
in airway epithelial cells
can lead to impaired mucociliary clearance
and barrier function.
Apart from genetic defects that may underlie such a defect, airway epithelial cells are continuously exposed to an inflammatory milieu, as a consequence of which epithelial cell biology may change. Even though asthma and COPD are different in etiology and pathophysiology, it is likely that the underlying innate immune mechanisms, especially those directed by bronchial epithelium, may at least in part be shared.
Subjects and design
involved patients with mild asthma and healthy controls. The MATERIAL trial (NTR01520051)
enrolled patients with mild asthma, and the RILCA (Role of Innate Lymphoid Cells in Asthma) study (NL48912.018.14) enrolled patients with moderate asthma and patients with severe asthma from the TASMA (Unravelling Targets of Therapy in Bronchial Thermoplasty in Severe Asthma) (NCT02225392) trial.
The patients with COPD were patients included from the RILCO (Role of Innate Lymphoid Cells in COPD) study (NL53354.018.15). All study protocols were reviewed and approved by the ethical review committee and were in accordance with the Declaration of Helsinki. All study participants provided written informed consent. The collection of BECs in the aforementioned trials and studies was conducted by 1 center, namely, the Department of Respiratory Medicine of the Amsterdam University Medical Center, Amsterdam, The Netherlands. The TASMA trial was conducted at the Department of Pulmonology, University Medical Center Groningen, Groningen, The Netherlands, and Royal Brompton and Imperial College Hospital London, United Kingdom. The baseline characteristics of the patients with asthma and healthy controls who were involved in this study are provided in Table I.
Table IBaseline characteristics of patients with mild, moderate, and severe asthma and healthy controls for transcriptome analyses
Feno, Fraction of exhaled nitric oxide represented in terms of ppb; max, maximum; min, minimum; na, not available; PC20, histamine provocative concentration causing a 20% drop in FEV1.
in the Methods section of the Online Repository (available at www.jacionline.org). Patients with severe asthma (from the TASMA trial) fulfilling the World Health Organization or modified innovative medicines initiative criteria of severe refractory asthma were included.
The RILCA (Role of Innate Lymphoid Cells in Asthma) study and RILCO (Role of Innate Lymphoid Cells in COPD) were 2 explorative studies involving the impact of rhinovirus-16 challenge in patients with moderate asthma and patients with COPD, respectively, and exploring mechanisms driven by innate lymphoid cells. The inclusion and exclusion criteria are mentioned in detail in the Methods section of the Online Repository.
The patients with mild asthma had not received inhaled or systemic corticosteroids or any other treatment other than inhaled short-acting β2-agonists within 2 weeks before the start of the study. The patients with moderate asthma used a dose equivalent of no more than 500 μg of fluticasone propionate per day. The patients with severe asthma had been using an inhaled corticosteroid (ICS) at a dosage of at least 500 μg of fluticasone equivalent per day and a long-acting ß2-agonist at a dosage of at least 100 μg of salmeterol per day or equivalent for the past 6 months, as well as a systemic corticosteroid (≤20 mg of prednisone equivalent per day). The patients with COPD were allowed specific medication, namely, long-acting ß2-agonists and long-acting muscarinic agonists, but no ICSs.
Sampling and RNA isolation
Mucosal brushes collected by brushing the left lower lobe consisted predominantly (>95%) of bronchial epithelial cells. Two brushes were obtained from each participant and then pooled and pelleted by centrifugation at 1240 rpm (using a Rotanta 460S centrifuge) for 10 minutes at 4°C. The pellet was dissolved in 1 mL of TRIzol (Thermofischer Scientific, Paisley, United Kingdom) and stored at –80°C until the RNA was isolated. After all the samples had been obtained, they were thawed at room temperature, and after 200 μL of chloroform was added, they were shaken vigorously for 30 seconds. The samples were kept at room temperature for 10 minutes, after which the phases were separated by centrifugation at 16,000 g for 15 minutes at 4°C. The aqueous phase was concentrated with protocol 5.3 using the NucleoSpin system and an RNA XS extraction kit (Macherey-Nagel, Duren, Germany). The quality and concentration of the samples were assessed by using a fragment analyzer (Advanced Analytical Technologies, Inc, Ankeny, Iowa).
RNA sequencing, analysis, and heat maps
The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (Ipswich, Mass) was used to process the samples. The sample preparation was performed according to the protocol for the NEBNextUltra Directional RNA Library Prep Kit for Illumina (NEB, catalog no. 7420S/L). Briefly, oligo-dT magnetic beads were used to isolate mRNA from total RNA. cDNA synthesis was performed after fragmentation of the mRNA. This was used for ligation with sequencing adapters followed by PCR amplification of the resulting product. The quality and yield after sample preparation were measured with the fragment analyzer. The size of the resulting products was consistent with the expected size distribution of between 300 and 500 bp. To evaluate the quality of the library preparation and kits used, the raw data were sampled and mapped to annotated genomic references. Mapping positions were classified as intragenic, exonic, intergenic, intronic, and rRNA. Clustering and DNA sequencing were performed according to the manufacture’s protocol using the Illumina NextSeq 500. A concentration of 1.6 pM was used as the input. The reads were trimmed for adapter sequences by using Trimmomatic, version 0.30, before the alignment. Presumed adapter sequences were removed from the read when the bases matched a sequence in the adapter sequence set (TruSeq adapters) with 2 or fewer mismatches and an alignment score of at least 12. The reference Homo_sapiens.GRCh37.75 was used to align the reads. The mapping to the reference sequence was done by using a short reader aligner based on Burrows-Wheeler transform. The default of mismatch rate of 2% (3 mismatches in a read of 150 bases) was used. The frequency of the reads mapped on the transcript was determined in terms of counts, which were used as an input for the downstream analysis.
Additionally, reads per kilobase of exon per million reads mapped and fragments per kilobase of exon per million reads mapped values were calculated. Image analysis, base calling, and quality check were performed with Illumina data analysis pipeline RTA, version 2.4.11, and Bcl2fastq, version 17. The read counts were loaded into the DESeq2 software package, which is a statistical package within the R/BioConductor platform that is designed to determine the differentially expressed genes and in which an adjusted P value less than .05 was considered statistically significant. The DEseq2 method uses Benjamini-Hochberg correction as an adjustment for false discovery rates.
For the heat maps, log2 gene expression values were normalized with z scores calculated by using the equation X – (μ/σ), where X is the value of the individual sample, μ is the average of the row, and σ is the SD of the row. The clustering of genes represented in the heat maps was based on k-means clustering. The combination of z scores for all genes is represented below the heat maps in red and blue, with red indicating high expression and blue indicting low expression.
Lipidomics and metabolome analysis
Table IIBaseline characteristics of patients with severe asthma, patients with COPD, and healthy controls for lipidomic and metabolomic analyses
max, Maximum; min, minimum.
The BECs were plated on 6-well plates precoated with PurCol (Advanced Biomatrix, Carlsbad, Calif) and grown until confluence in bronchial epithelial basal medium (Lonza, Basel, Switzerland) supplemented with growth factors (Lonza) and ciprofloxacin (Sigma, St Louis, Mo) at a concentration of 2 μg/mL. The cells were then passaged into T25 flasks (passage 1) until grown confluent. Subsequently, the cells were detached by using trypsin/EDTA (Lonza), and after the addition of trypsin-neutralizing solution, they were pelleted by centrifugation for 7 minutes at 1240 rpm (with a Rotanta 460S centrifuge) at 8°C. The pellet was washed twice with PBS and stored at –80°C until all samples were analyzed in parallel.
The extract of the cell pellet was injected onto a normal phase column (LiChrospher 2 × 250-mm silica-60 column) and a reverse-phase column (Acquity UPLC HSS T3, Milford, Mass). A Q Exactive plus Orbitrap (Thermo Scientific) mass spectrometer was used in the negative and positive electrospray ionization modes. In both ionization modes, mass spectra of the lipid species were obtained by continuous scanning from mass-to-charge ratio 150 to mass-to-charge ratio 2000 with a resolution of 280,000 full width at half maximum. Detailed analysis of the data is provided in the Methods section of the Online Repository.
All subjects provided prior written, informed consent. The design of the TASMA trial has been elaborately described elsewhere.
A total of 23 patients
(6 patients with severe asthma were included in addition to those in the severe asthma group mentioned in Table I) were treated with BT by using the Alair System (Boston Scientific, Boston, Mass) according to the current standard
and sedated by using remifentanil/propofol
or general anaesthesia. Patients were treated with 50 mg of prednisolone 3 days before treatment, on the day of the procedure itself, and 1 day thereafter. During the first procedure, the right lower lobe was treated; during the second procedure, the left lower lobe was treated; and finally, both upper lobes were treated. The right middle lobe remained untreated; therefore, this region could be used to compare the treatment effect. Six months after BT, bronchoscopy was performed. During the bronchoscopy procedure, endobronchial brushes were obtained from the untreated middle lobe and treated left lower lobe airways. The baseline characteristics of the patients with severe asthma used to analyze the effects of BT on bronchial epithelium are provided in Table III; they are no different from those reported originally for all patients.
Table IIIBaseline characteristics of patients with severe asthma who underwent the BT procedure
ACQ, Asthma Control Questionnaire; AQLQ, Asthma Quality of Life Questionnaire; max, maximum; min, minimum; PC20, histamine provocative concentration causing a 20% drop in FEV1 value.
Unbiased transcriptome analysis of BECs from patients with severe asthma compared with those from healthy controls showed a profound reduction in OXPHOS genes belonging to complexes I, III, IV, and V of the electron transport chain, whereas for BECs from patients with mild asthma, this reduction was heterogeneous. Genes related to fatty acid metabolism, however, were significantly upregulated in BECs from all patients with asthma, thus differentiating patients with asthma from healthy controls. This differential expression in bronchial epithelium was validated by lipidomics with enhanced levels of lipid species (PCs, LPCs, and BMPs). The reduction in metabolites in bronchial epithelium of patients with severe asthma was observed trendwise only, with no clear differences. Most interestingly, in BECs from patients with severe asthma who had received BT, a metabolic shift toward that in BECs from healthy individuals was observed. In BECs from patients with COPD also, there was a marked upregulation of lipid profiles compared with that in BECs from healthy controls, and the upregulation was even more pronounced than that in BECs from patients with severe asthma.
and dendritic cells,
which has been linked to inflammation. Another study showed that enhanced glycolysis in lung epithelium is required for IL-1α/β-induced proinflammatory responses.
There could be several reasons for this attenuated OXPHOS gene expression. For example, inflammatory mediators such as TNF-α, can inhibit COX1 by tyrosine phosphorylation, thereby switching from aerobic metabolism to glycolysis.
Interestingly, here we have shown that the attenuated OXPHOS gene expression is observed in BECs that are hyperresponsive, which results in an increased production of proinflammatory mediators, prominently, the neutrophilic chemoattractant CXCL-8, owing to a defective translocation of the translational repressor T-cell internal antigen-1–related protein (TiAR).
Interestingly, TiAR and the closely related T-cell internal antigen-1 (TIA-1) have also been implicated in mitochondrial biogenesis.
In addition, TiAR and TIA-1 knockdown attenuates complex V (ATP synthase) of the OXPHOS pathway.
Therefore, a possible scenario is that a defective TiAR and/or TIA-1 in BECs from patients with asthma leads to the observed metabolic shift and the resulting lipid mediators may contribute to activation of the bronchial epithelium,
which can lead to chronic inflammation in asthma.
Alternatively, but not mutually exclusively, enhanced reactive oxygen species and mitochondrial damage in bronchial epithelium were found to parallel high levels of CXCL-8, IL-6, and IL-1β production.
Further support for a link between mitochondrial dysfunction and neutrophilic inflammation comes from clustering of genes in sputum from patients with asthma, in which case high neutrophilic inflammation was strongly associated with a significant reduction in OXPHOS genes in transcriptome-associated cluster 2 (TAC2),
and ether lipids from neutrophils
downregulate mitochondrial activity.
Finally, BECs from patients with COPD, which is associated with neutrophilic inflammation,
also display a very distinct increase in lipid species, even when compared with that from patients with asthma. Together, these findings provide strong evidence for a prominent reduction in mitochondrial activity in BECs that is linked to neutrophilic inflammation in asthma, and possibly in COPD.
The differences in metabolites between BECs from patients with severe asthma and those from healthy individuals appear to affect purine metabolism, amino acid biosynthesis, and glycolysis. In BECs from patients with COPD, the pathways that may be affected are creatine metabolism and the tricarboxylic acid cycle. This translation of metabolites to metabolic pathways, however, is based merely on the presence of 3 or more significantly different metabolites and should thus be considered with caution. The consequences of these different metabolic pathways on the functioning of BECs remain to be determined, but they likely affect biosynthesis, energy housekeeping, and ultimately inflammatory responses and possibly lung function. In our analysis, 2 patients with severe asthma displayed markedly lower levels of lipid species and had lower FEV1 % reversibility than other the patients did. Despite the low numbers, it would be interesting to explore whether higher lipid metabolism in bronchial epithelium results in higher FEV1 % reversibility in patients with severe asthma.
Consequently, mitochondrial damage in bronchial epithelium of patients with asthma leads to ciliary dysfunction, resulting in poor mucus clearance,
and in COPD this has been linked to exacerbations.
In the current study, the BECs from some patients with asthma from the steroid-naive cohort with mild asthma displayed reduced OXPHOS gene expression. Therefore, at least for this cohort, the reduced expression of OXPHOS genes is independent of corticosteroid use.
However, our results show a significantly increased expression of OXPHOS genes in BECs obtained from treated parts compared with in BECs from untreated parts (middle lobe). Interestingly, imatinib, a protein tyrosine kinase inhibitor, is shown to improve airway hyperresponsiveness in severe refractory asthma,
and it also inhibits the platelet-derived growth factor that induces airway smooth muscle cell proliferation.
In addition, imatinib induces an increase in OXPHOS gene expression in bronchial epithelium, specifically, in the responders to the treatment.
This suggests that reversing metabolic defects in bronchial epithelium from patients with asthma could be beneficial for patients with asthma.
but as the samples were collected on different days, those analyses may have been biased. In the BT trial we collected and analyzed BECs from treated versus nontreated airways in parallel. However, there are some potential limitations to the current study. First, the patients with severe asthma were taking both oral corticosteroids and ICSs, which may have influenced the alteration of metabolic genes in their bronchial epithelium. In some patients with mild steroid-naive asthma, however, there was a reduced expression of OXPHOS and enhanced fatty acid metabolism genes. Second, functional validation by lipidomics and metabolome analysis was done after culturing and expanding the bronchial epithelium. This ex vivo expansion of BECs may affect gene expression
and we cannot exclude the possibility that mitochondrial dysfunction in bronchial epithelium of patients with severe asthma is reversible by culturing BECs ex vivo. However, it also has been demonstrated that cultured bronchial epithelial cells maintain their phenotype ex vivo
and depict asthma severity.
This indicates that bronchial epithelium can retain intrinsic features, even after culturing ex vivo.
Third, because of the high variability of lipid mediators measured in lung epithelial lining fluids,
it is possible that more patients should have been included for functional validation by metabolome analysis. The transcriptome data, however, were substantiated by lipidomics. Fourth, OXPHOS is known to be attenuated by aging,
and both those patients with severe asthma and those with COPD were older than those in the healthy cohort. However, the reduced expression of OXPHOS genes was also observed in younger patients included in the cohort of patients with mild asthma, and thus, it is unlikely that aging underlies the observed differences between patients and healthy controls. In addition, BT apparently resets the metabolic changes in BECs, suggesting that the reduced expression of OXPHOS genes is acquired rather than due to aging. With respect to the latter, we cannot exclude the possibility that regional differences in the airways underlie the observed different bronchial epithelial transcriptomes after BT. Finally, for our study we used submerged cultures, which typically contain undifferentiated BECs as opposed to, for example, BECs grown at air-liquid interface cultures, which are considered more representative of airway epithelial cells. Submerged cultures were chosen because the cells could be analyzed after a shorter culture time span (2 weeks), as opposed to the 6 weeks required for air-liquid interface cultures, and were therefore less likely to lose their phenotype. As our control BECs were also cultured submerged, our findings are genuine, but the consequences of this for differentiated cells need to be established.
these metabolic effects appear to underlie airway inflammation. In this context, it is of interest that BT partially normalizes the metabolic differences, thereby resetting the bronchial epithelium, which may contribute to the response to BT treatment.
Published online: February 05, 2021
Received in revised form:
Supported by the Netherlands Asthma Foundation (currently the Lung Foundation [projects 3.2.10.069, 3.2.07.012, and 3.2.06.031]), GSK ( CRT 114696 ), and Stichting Astma Bestrijding (project 2013_009 ). The explorative trial RILCO (Role of Innate Lymphoid Cells in COPD) was supported in part by Medimmune (Gaithersburg, Md). The TASMA (Unravelling Targets of Therapy in Bronchial Thermoplasty in Severe Asthma) trial is funded by the Netherlands Lung Foundation (grant 5.2.13.064JO ), The Netherlands Organization for Health Research and Development (ZonMw grant 90713477 ), and Boston Scientific Corporation .
Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.
© 2021 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology.