Respiratory Research最新文献

Background: Impaired airway epithelial barrier function is a pathogenic driver in a subset of individuals with asthma. MicroRNAs, small RNAs that function as post-transcriptional regulators of gene expression, may be involved in the regulation of the airway epithelial barrier. This study aimed to determine if microRNAs cause epithelial barrier dysfunction through the targeting of mRNAs involved in maintaining airway epithelial barrier integrity.

Methods: Primary human bronchial epithelial cells cultured at air-liquid interface were stimulated with cytokines reflecting different asthma endotypes. Barrier integrity was assessed by FITC-labelled dextran flux. Differentially expressed epithelial barrier-related genes and microRNAs were identified by next-generation RNA sequencing and qPCR. Results were validated with microRNA pull-down, treatment with microRNA mimics and antagomirs, respectively, and evaluated in airway samples from subjects with asthma.

Results: A combination of IL-17A and TNFα, mimicking Th17 inflammation, was identified as a strong driver of epithelial barrier disruption, as compared to IL-4 + IL-13, IL-6 + sIL-6R, and TGFβ. Several microRNAs induced by IL-17A and TNFα stimulation were predicted to target barrier-related genes, which exhibited decreased expression in the same model. Of these microRNAs, miR-146a-3p and miR-363-3p were consistently induced in multiple donors. MicroRNA pull-down, overexpression, and knockdown experiments indicated a potential role for miR-363-3p interacting with several barrier-related genes, including CLDN8, PCDH1, and PTEN. Bronchial lavage samples demonstrated an increase of miR-363-3p in individuals with asthma compared to healthy controls, as well as a positive correlation between miR-363-3p and the number of airway eosinophils and neutrophils.

Conclusions: Our results support the role of microRNAs as mediators of cytokine-induced airway epithelial barrier dysfunction. Specifically, miR-363-3p appears to contribute to epithelial damage by targeting and suppressing gene expressions of several key barrier components, including CLDN8, PCDH1, and PTEN, suggesting a novel role for this microRNA in Th17-driven airway disease. A better understanding of microRNA networks and their role in asthma pathogenesis may lead to novel biomarkers and therapeutic targets, which are currently needed for individuals with T2-low asthma and in Th17-driven asthma.

Background: Severe respiratory infections have imposed an immense burden on healthcare worldwide, which could lead to fatal outcomes. The dysfunctional immune response impacts disease severity of respiratory infections, yet its underlying mechanisms remain largely obscure.

Methods: We explored the immunologic response underpinning severe respiratory viral infections by performing single-cell transcriptome analysis of peripheral blood mononuclear cells (PBMCs) from 17 patients with coronavirus disease 2019 (COVID-19), including moderate, severe, critical, and convalescent cases. Furthermore, we analyzed the host responses following low- or high-dose vaccination with recombinant protein.

Results: We constructed an immunocyte landscape with 299,527 PBMCs. The significant dysregulation of immune homeostasis in severe respiratory viral infections was characterized by an increased ratio of CD14⁺ monocytes and exhausted CD8⁺ T cells, and reduced Tregs, memory T cells and cDC2 cells. Notably, highly inflamed CD14⁺ monocytes overexpressing interleukin (IL)-related genes emerged as central contributor of inflammatory storms in critical patients. Enhanced inflammatory response in exhausted CD8⁺ T cells, FOS/JUN overexpression in Tregs and Th17 cells, as well as suppressed antigen presentation activity in cDC2 cells were also remarkable features of severe and critical cases. Moreover, the high-dose vaccination with recombinant protein inhibited the inflammatory response of CD14⁺ monocytes, while enriched the memory T/NK cells to boost protective immunity. In mechanism, activated IL-6-JAK-STAT in CD14⁺ monocytes and T cells, coupled with impaired interferon (IFN)-α pathway, could drive excessive inflammatory response.

Conclusions: Our study creates a high-resolution transcriptomic atlas that uncovers distinct immune signatures across the disease severity of respiratory infections, which provides valuable resource for mechanistic exploration and therapeutic strategies development. Furthermore, we demonstrate that high-dose recombinant protein vaccines might mitigate the severity of illness.

Trial registration: This study was prospectively registered at ChiCTR (Registration number: ChiCTR2300067787).

Background: Congenital Pulmonary Airway malformations (CPAM) may lead to malignant degeneration, and therefore many surgeons opt to resect CPAM even in asymptomatic patients. Previously, we identified Kirsten rat sarcoma virus (KRAS) mutations in a subset of CPAM patients, possibly indicating a pre-malignant state. In order to unify treatment strategy in (asymptomatic) patients we focused on KRAS mutations as a potential risk factor for developing malignancy in CPAM.

Methods: Resected lung tissue of CPAM patients was separated in affected region ("cyst") and non-affected region ("control") to subsequently initiate airway organoids. Cyst and control organoids from the same patients with and without KRAS mutations (KRAS^POS vs. KRAS^NEG) (n = 3) where processed for single cell RNA sequencing (scRNA-Seq), and the cellular composition of the organoids was validated by immunofluorescent staining. The role of KRAS was identified by manipulating the expression in the organoids.

Results: ScRNA-Seq data revealed differences in cell proportions between KRAS^POS and KRAS^NEG cyst, and control organoids. The significant differentially expressed genes in the KRAS^POS cyst are comparable to those identified in lung cancer patients with KRAS mutations. Manipulation of KRAS expression showed that KRAS^POS cyst organoids grew larger due to more proliferative cells and that KRAS directly affected the cell cycle.

Conclusions: KRAS^POS cyst organoids show transcriptomic similarities with KRAS mutated lung cancers, show changes in cellular composition and have increased growth and proliferation. These findings support the hypothesis that KRAS mutated CPAM cysts belong to a group of CPAM patients at higher risk of developing a malignancy.

Background: Runt-related transcription factor 2 (Runx2), a transcription factor of the RUNX family, is involved in various inflammatory diseases. However, the role of Runx2 was unclear in chronic obstructive pulmonary disease (COPD).

Methods: Pulmonary Runx2 level was compared in COPD patients and control subjects via a case-control study. Runx2 expression was detected in lung tissues of COPD mice and human bronchial epithelial (BEAS-2B) cells simulated with cigarette smoke extracts (CSE).

Results: Pulmonary Runx2 expression was upregulated, and inversely associated with pulmonary function and positively correlated with inflammatory cytokines in COPD patients. Mechanistically, Runx2 activation facilitated the transcription of CDK8, a co-regulator of nuclear factor-κB (NF-κB), and inflammatory cytokines production. Luciferase report gene assay confirmed that CDK8 was the downstream target gene of Runx2. Further analysis found that CSE inhibited Runx2 ubiquitination and proteasomal degradation. Besides, CSE elevated nicotinamide adenine dinucleotide (NAD⁺) consumes and Sirtuin 3 (Sirt3) depletion. Additionally, Runx2 acetylation was increased in CSE-exposed BEAS-2B cells, lungs tissues from COPD mice and patients. Interestingly, Sirt3 overexpression or supplementation with Nicotinamide Riboside (NR), the precursor of NAD⁺, abolished CSE-induced Runx2 acetylation and Runx2-CDK8 axis activation. In vivo experiment further confirmed NR supplementation evidently mitigated cigarette smoke-induced a COPD-like phenotype in mice.

Conclusions: These results indicated that Sirt3 depletion-induced Runx2 acetylation contributes to CDK8 activation and pulmonary inflammation in the progression of COPD.

Background: The impact of wildfire smoke (WFS) on air quality across the contiguous US has become geographically widespread. However, the effects of WFS exposure on psychometric measures of mental and physical health remain largely unknown.

Objectives: To assess the associations between WFS PM_2.5 and black carbon (BC) exposure and psychometric health measures.

Methods: The St. George's Respiratory Questionnaire (SGRQ) and the 36-Item Short Form Survey (SF-36) were administered to participants in the Lovelace Smokers Cohort in New Mexico to assess psychometric health measures in the past 4 weeks. WFS estimates were calculated against Albuquerque metropolitan area for 7-, 15-, 30-, and 60-d prior to questionnaire completion. The associations between exposure and health measures were assessed using linear models.

Results: Associations were observed for all psychometric measures with WFS PM_2.5 and BC exposures estimated for 7-day prior to questionnaire completion. These associations remained for WFS exposure estimated up to 30-day prior to questionnaire completion for all SGRQ subdomains and physical health measures of SF-36, whereas associations with the mental health component were more transient and primarily evident within one week. Additionally, WFS PM_2.5 exhibited stronger potency than total ambient PM_2.5. Male participants, individuals with less than a college education, and those exposed to woodsmoke demonstrated stronger associations with WFS exposure.

Conclusions: Exposure to WFS was associated with worse SGRQ and SF-36 scores, with notable differences in temporal patterns between mental and physical health measures. Our findings also underscore the importance of source-specific risk assessment for air pollution.

Background: Bronchiectasis is a common feature in idiopathic pulmonary fibrosis (IPF) and rheumatoid arthritis-associated interstitial lung disease (RA-ILD). While these so-called traction bronchiectasis are often considered a secondary phenomenon in fibrosing ILD, their prognostic significance and relationship to respiratory pathogen detection and outcomes remain unclear.

Methods: We conducted a retrospective, single-center cohort study in IPF or fibrosing RA-ILD patients with available high-resolution computed tomography (HRCT) and lower-respiratory tract microbial samples between 2014 and 2024. Bronchiectasis was assessed using the bronchiectasis subscore of the Brody score; fibrosis was quantified by deep-learning-based automated HRCT analysis. Primary outcome was 5-year transplant-free survival; secondary outcomes included isolation of pathogens per CDC criteria, PFT trajectories, bronchiectasis-associated symptoms, and hospitalization. Statistical methods included Cox regression, linear mixed-effects modeling and correlation analysis.

Results: 267 IPF and 56 RA-ILD patients were included. Median modified Brody score was 11.5 (IQR 7-16; max possible range 0-72). Higher Brody scores strongly correlated with fibrotic extent (R = 0.6, P < 0.001). Higher scores had significantly lower baseline FVC and DLCO (P < 0.001), but no differences in PFT trajectories over time. In multivariable Cox regression, higher bronchiectasis scores were independently associated with mortality (HR 1.03 per point [95%CI 1.01-1.06], P = 0.003); fibrosis extent showed similar results (HR 1.02, CI 1.00-1.03, P = 0.017). Pathogens were found at a median of 3 months after baseline in 50.9% (IPF) and 46.4% (RA-ILD), without association with survival, symptoms or Brody scores. Staphylococcus aureus was most common (28.9%); Pseudomonas aeruginosa was rare (1.9%).

Conclusion: In both IPF and RA-ILD, higher bronchiectasis scores were associated with fibrosis extent and mortality, but not classical clinical bronchiectasis features. This supports traction bronchiectasis as a marker of fibrotic remodeling rather than a distinct syndrome.

Trial registration: Not applicable.