Metabolic differences between bronchial epithelium from healthy individuals and patients with asthma and the effect of bronchial thermoplasty

Background: Asthma is a heterogeneous disease with differences in onset, severity, and inflammation. Bronchial epithelial cells (BECs) contribute to asthma pathophysiology. Objective: We determined whether transcriptomes of BECs reflect heterogeneity in inflammation and severity in asthma, and whether this was affected in BECs from patients with severe asthma after their regeneration by bronchial thermoplasty. Methods: RNA sequencing was performed on BECs obtained by bronchoscopy from healthy controls (n = 16), patients with mild asthma (n = 17), patients with moderate asthma (n = 5), and patients with severe asthma (n = 17), as well as on BECs from treated and untreated airways of the latter (also 6 months after bronchial thermoplasty) (n = 23). Lipidome and metabolome analyses were performed on cultured BECs from healthy controls (n = 7); patients with severe asthma (n = 9); and, for comparison, patients with chronic obstructive pulmonary disease (n = 7). Results: Transcriptome analysis of BECs from patients showed a reduced expression of oxidative phosphorylation (OXPHOS) genes, most profoundly in patients with severe asthma but less profoundly and more heterogeneously in patients with mild asthma. Genes related to fatty acid metabolism were significantly upregulated in asthma. Lipidomics revealed enhanced levels of lipid species (phosphatidylcholines, lysophosphatidylcholines. and bis(monoacylglycerol)phosphate), whereas levels of OXPHOS metabolites were reduced in BECs from patients with severe asthma. BECs from patients with mild asthma characterized by hyperresponsive production of mediators implicated in neutrophilic inflammation had decreased expression of OXPHOS genes compared with that in BECs from patients with mild asthma with normoresponsive production. BECs obtained after thermoplasty had significantly increased expression of OXPHOS genes and decreased expression of fatty acid metabolism genes compared with BECs obtained from untreated airways. Conclusion: BECs in patients with asthma are metabolically different from those in healthy individuals. These differences are linked with inflammation and asthma severity, and they can be reversed by bronchial thermoplasty

IL-17 attenuates degradation of ARE-mRNAs by changing the cooperation between AU-binding proteins and microRNA16

Interleukin 17A (IL-17), a mediator implicated in chronic and severe inflammatory diseases, enhances the production of pro-inflammatory mediators by attenuating decay of the encoding mRNAs. The decay of many of these mRNAs depends on proteins (AUBps) that target AU-rich elements in the 3'-untranslated region of mRNAs and facilitate either mRNA decay or stabilization. Here we show that AUBps and the target mRNA assemble in a novel ribonucleoprotein complex in the presence of microRNA16 (miR16), which leads to the degradation of the target mRNA. Notably, IL-17 attenuates miR16 expression and promotes the binding of stabilizing AUBps over that of destabilizing AUBps, reducing mRNA decay. These findings indicate that miR16 independently of a seed sequence, directs the competition between degrading and stabilizing AUBps for target mRNAs. Since AUBps affect expression of about 8% of the human transcriptome and miR16 is ubiquitously expressed, IL-17 may in addition to inflammation affect many other cellular processe

Model of IL-17 mediated regulation of ARE- mRNA expression by AUBps and miR16.

<p>Upon exposure to TNF-α, mRNAs of inflammatory mediators, containing ARE- sequences in the 3′UTR (ARE-mRNA), associate with destabilizing AUBps and miR16. The overall effect is ARE-mRNA degradation. In presence of TNF-α plus IL-17, miR16 association to ARE-mRNAs is limited and additional loading of stabilizing AUBps on the ARE-mRNA takes place, leading to attenuated ARE-mRNA degradation resulting in enhanced and prolonged expression of the encoded protein.</p

KHSRP and TTP down-regulate IL-8 expression by promoting degradation of IL-8 mRNA.

<p><b>a, e</b>). KHSRP (75 kD) (<b>a</b>) and TTP (36 kD) (<b>e</b>) were knocked down in NCI-H292 cells using si-RNA. Scrambled si-RNA (si-con) served as a control for specificity and β-actin (42 kD) was determined to verify equal protein loading. <b>b, f</b>). IL-8 released over 24 h in supernatant from NCI-H292 cells, non-transfected (nt) or transfected with si-con or si-KHSRP (<b>b</b>) or si-TTP (<b>f</b>), and either non-stimulated (ns) or stimulated with IL-17 (100 ng/ml), TNF-α (5 ng/ml) or TNF-α plus IL-17 (5 ng/ml+100 ng/ml, respectively). Data are shown as mean ± SD. 2-way ANOVA analysis was done with Bonferroni's multiple comparison post-test. ***P<0.001, **P<0.01, *P<0.05. <b>c, g</b>). Quantitative PCR (q-PCR) analysis of IL-8 mRNA was done for non-transfected or transfected cells with si-con or si-KHSRP (<b>c</b>) or si-TTP (<b>g</b>), at 2 h after stimulation with TNF-α or TNF-α plus IL-17, or left unstimulated. ***P<0.001, **P<0.01, *P<0.05 (2-way ANOVA, Bonferroni's multiple comparison post-test). The data presented is representative of 3 independent experiments done for each AUBp. mRNA results are presented relative to that of unstimulated, non-transfected cells. <b>d, h</b>). Non-transfected, si-con or si-KHSRP (<b>d</b>) or si-TTP (<b>h</b>) transfected cells were left unstimulated or stimulated with TNF-α or TNF-α plus IL-17 for 2 h. RNA was isolated from cells at 0, 30 and 60 minutes (mins) after blocking gene transcription by actinomycin D (5 µg/ml). % of IL-8 mRNA remaining was calculated on basis of q-PCR. Representative experiment is shown representative of 5 experiments.</p

IL-17 limits miR16 expression and promotes cytoplasmic localization of AUF-1.

<p><b>a</b>) Q-PCR showing time dependent expression of miR16 measured in NCI-H292 cells stimulated with TNF-α or TNF-α plus IL-17. miR16 is presented relative to that of unstimulated, non-transfected cells. <b>b–h</b>) Representative confocal images showing the localization of AUF-1 in resting NCI-H292 cells (<b>b</b>) and in cells stimulated with TNF-α (<b>c, e, g</b>) or TNF-α plus IL-17 (<b>d, f, h</b>) for 1, 2 and 4 h respectively as described in methods. The nucleus is stained with DAPI. White arrows indicate the predominant localization of AUF-1. Two experiments were performed with similar results. <b>i, j</b>) Western Blot analysis of AUF-1 in the cytoplasmic (<b>i</b>) and nuclear (<b>j</b>) fractions of resting cells (ns) or cells stimulated with TNF-α or TNF-α plus IL-17. β-actin and tubulin are markers for the cytoplasmic and nuclear fractions respectively. <b>k, l</b>) Quantitative analysis of AUF-1 normalised to β-actin in cytoplasmic fraction (<b>k</b>) and to tubulin in nuclear fraction (<b>l</b>) obtained from the Western Blot.</p

AUF-1 enhances IL-8 expression by halting IL-8 mRNA degradation.

<p><b>a</b>) AUF-1 (approx. 37 kD) was knocked down in NCI-H292 cells with si-AUF-1. Scrambled si-RNA (si-con) served as a control for specificity and β-actin (42 kD) was determined to verify equal protein loading. <b>b</b>). IL-8 released over 24 h in supernatant from NCI-H292 cells, non-transfected (nt) or transfected with si-con or si-AUF-1 and either non-stimulated (ns) or stimulated with IL-17, TNF-α or TNF-α plus IL-17. Data are shown as mean ± SD. ***P<0.001 (2-way ANOVA, Bonferroni's multiple comparison post-test). <b>c</b>). Q-PCR analysis of IL-8 mRNA was done for non-transfected or transfected cells with si-con or si-AUF-1 at 2 h after stimulation with TNF-α or TNF-α plus IL-17, or left unstimulated. **P<0.01 (2-way ANOVA, Bonferroni's multiple comparison post-test). mRNA results are presented relative to that of unstimulated, non-transfected cells <b>d</b>). Non-transfected, si-con or si-AUF-1 transfected cells were left unstimulated or stimulated with TNF-α or TNF-α plus IL-17 for 2 h. RNA was isolated from cells at 0, 30 and 60 mins after blocking gene transcription by actinomycin D. % of IL-8 mRNA remaining was calculated on basis of q-PCR. Representative experiments are shown for 3 experiments.</p

Neutrophilic inflammation in asthma and defective epithelial translational control

Neutrophilic inflammation in asthma is associated with interleukin (IL)-17A, corticosteroid-insensitivity and bronchodilator-induced forced expiratory volume in 1 s (FEV1) reversibility. IL-17A synergises with tumour necrosis factor (TNF)-α in the production of the neutrophil chemokine CXCL-8 by primary bronchial epithelial cells (PBECs).We hypothesised that local neutrophilic inflammation in asthma correlates with IL-17A and TNF-α-induced CXCL-8 production by PBECs from asthma patients.PBECs from most asthma patients displayed an exaggerated CXCL-8 production in response to TNF-α and IL-17A, but not to TNF-α alone, and which was also insensitive to corticosteroids. This hyperresponsiveness of PBECs strongly correlated with CXCL-8 levels and neutrophil numbers in bronchoalveolar lavage from the corresponding patients, but not with that of eosinophils. In addition, this hyperresponsiveness also correlated with bronchodilator-induced FEV1 % reversibility. At the molecular level, epithelial hyperresponsiveness was associated with failure of the translational repressor T-cell internal antigen-1 related protein (TiAR) to translocate to the cytoplasm to halt CXCL-8 production, as confirmed by TiAR knockdown. This is in line with the finding that hyperresponsive PBECs also produced enhanced levels of other inflammatory mediators.Hyperresponsive PBECs in asthma patients may underlie neutrophilic and corticosteroid-insensitive inflammation and a reduced FEV1, irrespective of eosinophilic inflammation. Normalising cytoplasmic translocation of TiAR is a potential therapeutic target in neutrophilic, corticosteroid-insensitive asthma

IL-8 mRNA and miR16 present in immuno-purified preparations of AUBps.

<p><b>a–c-</b>) Fold change in IL-8 mRNA associated with immuno-purified KHSRP (<b>a</b>), TTP (<b>b</b>) and AUF1 (<b>c</b>) in NCI-H292 cells stimulated for 2 h with TNF-α or TNF-α plus IL-17 as compared to non-stimulated (ns) cells. Statistical analysis was done with 1-way ANOVA, Bonferroni's multiple comparison post-test. ***P<0.001, ** P<0.01. <b>d–f</b>) Fold change in miR16 associated with immuno-purified KHSRP (<b>d</b>), TTP (<b>e</b>) and AUF-1 (<b>f</b>) in NCI-H292 cells stimulated for 2 h with TNF-α or TNF-α plus IL-17 with respect to non-stimulated cells. Representative experiments are shown for 3 experiments.</p