ARL5b inhibits human rhinovirus 16 propagation and impairs macrophage-mediated bacterial clearance.

Human rhinovirus is the most frequently isolated virus during severe exacerbations of chronic respiratory diseases, like chronic obstructive pulmonary disease. In this disease, alveolar macrophages display significantly diminished phagocytic functions that could be associated with bacterial superinfections. However, how human rhinovirus affects the functions of macrophages is largely unknown. Macrophages treated with HRV16 demonstrate deficient bacteria-killing activity, impaired phagolysosome biogenesis, and altered intracellular compartments. Using RNA sequencing, we identify the small GTPase ARL5b to be upregulated by the virus in primary human macrophages. Importantly, depletion of ARL5b rescues bacterial clearance and localization of endosomal markers in macrophages upon HRV16 exposure. In permissive cells, depletion of ARL5b increases the secretion of HRV16 virions. Thus, we identify ARL5b as a novel regulator of intracellular trafficking dynamics and phagolysosomal biogenesis in macrophages and as a restriction factor of HRV16 in permissive cells.

Human Lung Conventional Dendritic Cells Orchestrate Lymphoid Neogenesis during Chronic Obstructive Pulmonary Disease.

Rationale: Emerging evidence supports a crucial role for tertiary lymphoid organs (TLOs) in chronic obstructive pulmonary disease (COPD) progression. However, mechanisms of immune cell activation leading to TLOs in COPD remain to be defined.Objectives: To examine the role of lung dendritic cells (DCs) in T follicular helper (Tfh)-cell induction, a T-cell subset critically implicated in lymphoid organ formation, in COPD.Methods: Myeloid cell heterogeneity and phenotype were studied in an unbiased manner via single-cell RNA sequencing on HLA-DR+ cells sorted from human lungs. We measured the in vitro capability of control and COPD lung DC subsets, sorted using a fluorescence-activated cell sorter, to polarize IL-21+CXCL13+ (IL-21-positive and C-X-C chemokine ligand type 13-positive) Tfh-like cells. In situ imaging analysis was performed on Global Initiative for Chronic Obstructive Lung Disease stage IV COPD lungs with TLOs.Measurements and Main Results: Single-cell RNA-sequencing analysis revealed a high degree of heterogeneity among human lung myeloid cells. Among these, conventional dendritic type 2 cells (cDC2s) showed increased induction of IL-21+CXCL13+ Tfh-like cells. Importantly, the capacity to induce IL-21+ Tfh-like cells was higher in cDC2s from patients with COPD than in those from control patients. Increased Tfh-cell induction by COPD cDC2s correlated with increased presence of Tfh-like cells in COPD lungs as compared with those in control lungs, and cDC2s colocalized with Tfh-like cells in TLOs of COPD lungs. Mechanistically, cDC2s exhibited a unique migratory signature and (transcriptional) expression of several pathways and genes related to DC-induced Tfh-cell priming. Importantly, blocking the costimulatory OX40L (OX40 ligand)-OX40 axis reduced Tfh-cell induction by control lung cDC2s.Conclusions: In COPD lungs, we found lung EBI2+ (Epstein-Barr virus-induced gene 2-positive) OX-40L-expressing cDC2s that induced IL-21+ Tfh-like cells, suggesting an involvement of these cells in TLO formation.

Arpin is critical for phagocytosis in macrophages and is targeted by human rhinovirus.

Human rhinovirus is a causative agent of severe exacerbations of chronic obstructive pulmonary disease (COPD). COPD is characterised by an increased number of alveolar macrophages with diminished phagocytic functions, but how rhinovirus infection affects macrophage function is still unknown. Here, we describe that human rhinovirus 16 impairs bacterial uptake and receptor-mediated phagocytosis in macrophages. The stalled phagocytic cups contain accumulated F-actin. Interestingly, we find that human rhinovirus 16 downregulates the expression of Arpin, a negative regulator of the Arp2/3 complex. Importantly, re-expression of the protein rescues defective internalisation in human rhinovirus 16-treated cells, demonstrating that Arpin is a key factor targeted to impair phagocytosis. We further show that Arpin is required for efficient uptake of multiple targets, for F-actin cup formation and for successful phagosome completion in macrophages. Interestingly, Arpin is recruited to sites of membrane extension and phagosome closure. Thus, we identify Arpin as a central actin regulator during phagocytosis that it is targeted by human rhinovirus 16, allowing the virus to perturb bacterial internalisation and phagocytosis in macrophages.

Use of precision cut lung slices as a translational model for the study of lung biology.

Animal models remain invaluable for study of respiratory diseases, however, translation of data generated in genetically homogeneous animals housed in a clean and well-controlled environment does not necessarily provide insight to the human disease situation. In vitro human systems such as air liquid interface (ALI) cultures and organ-on-a-chip models have attempted to bridge the divide between animal models and human patients. However, although 3D in nature, these models struggle to recreate the architecture and complex cellularity of the airways and parenchyma, and therefore cannot mimic the complex cell-cell interactions in the lung. To address this issue, lung slices have emerged as a useful ex vivo tool for studying the respiratory responses to inflammatory stimuli, infection, and novel drug compounds. This review covers the practicality of precision cut lung slice (PCLS) generation and benefits of this ex vivo culture system in modeling human lung biology and disease pathogenesis.

The lung environment controls alveolar macrophage metabolism and responsiveness in type 2 inflammation.

Fine control of macrophage activation is needed to prevent inflammatory disease, particularly at barrier sites such as the lungs. However, the dominant mechanisms that regulate the activation of pulmonary macrophages during inflammation are poorly understood. We found that alveolar macrophages (AlvMs) were much less able to respond to the canonical type 2 cytokine IL-4, which underpins allergic disease and parasitic worm infections, than macrophages from lung tissue or the peritoneal cavity. We found that the hyporesponsiveness of AlvMs to IL-4 depended upon the lung environment but was independent of the host microbiota or the lung extracellular matrix components surfactant protein D (SP-D) and mucin 5b (Muc5b). AlvMs showed severely dysregulated metabolism relative to that of cavity macrophages. After removal from the lungs, AlvMs regained responsiveness to IL-4 in a glycolysis-dependent manner. Thus, impaired glycolysis in the pulmonary niche regulates AlvM responsiveness during type 2 inflammation.

Dual Role For A MEK Inhibitor As A Modulator Of Inflammation And Host Defense Mechanisms With Potential Therapeutic Application In COPD.

Background: Unlike p38 mitogen-activated protein Kinases (MAPK) that has been extensively studied in the context of lung-associated pathologies in COPD, the role of the dual-specificity mitogen-activated protein kinase kinase (MEK1/2) or its downstream signaling molecule extracellular signal-regulated kinases 1/2 (ERK1/2) in COPD is poorly understood. Objectives: The aim of this study was to address whether MEK1/2 pathway activation is linked to COPD and that targeting this pathway can improve lung inflammation through decreased immune-mediated inflammatory responses without compromising bacterial clearance. Methods: Association of MEK1/2 pathway activation to COPD was investigated by immunohistochemistry using lung tissue biopsies from COPD and healthy individuals and through analysis of sputum gene expression data from COPD patients. The anti-inflammatory effect of MEK1/2 inhibition was assessed on cytokine release from lipopolysaccharide-stimulated alveolar macrophages. The effect of MEK1/2 inhibition on bacterial clearance was assessed using Staphylococcus aureus killing assays with RAW 264.7 macrophage cell line and human neutrophils. Results: We report here MEK1/2 pathway activation demonstrated by increased pERK1/2 staining in bronchial epithelium and by the presence of MEK gene activation signature in sputum samples from COPD patients. Inhibition of MEK1/2 resulted in a superior anti-inflammatory effect in human alveolar macrophages in comparison to a p38 inhibitor. Furthermore, MEK1/2 inhibition led to an increase in bacterial killing in human neutrophils and RAW 264.7 cells that was not observed with the p38 inhibitor. Conclusion: Our data demonstrate the activation of MEK1/2 pathway in COPD and highlight a dual function of MEK1/2 inhibition in improving host defense responses whilst also controlling inflammation.

Differential anti-inflammatory effects of budesonide and a p38 MAPK inhibitor AZD7624 on COPD pulmonary cells.

BACKGROUND: The effects of anti-inflammatory drugs in COPD patients may vary between different cell types. The aim of the current study was to assess the anti-inflammatory effects of the corticosteroid budesonide and a p38 MAPK inhibitor (AZD7624) on different cell types obtained from COPD patients and healthy controls. METHODS: Eight healthy smokers, 16 COPD infrequent exacerbators, and 16 frequent COPD exacerbators (≥2 exacerbations in the last year) were recruited for bronchoscopy and blood sampling. The anti-inflammatory effects of budesonide and AZD7624 were assessed on cytokine release from lipopolysaccharide-stimulated alveolar macrophages and peripheral blood mononuclear cells and polyinosinic:polycytidylic acid-stimulated bronchial epithelial cells. RESULTS: The anti-inflammatory effects of budesonide varied greatly within a patient according to the cell type studied. Bronchial epithelial cells showed the lowest sensitivity to budesonide, while peripheral blood mononuclear cells showed the greatest sensitivity. AZD7624 had a greater effect than budesonide on cytokine production from bronchial epithelial cells. Exacerbation frequency did not influence corticosteroid sensitivity. CONCLUSION: We observed variable corticosteroid and p38 MAPK inhibitor anti-inflammatory responses within the same individual depending on the cell type studied. These findings support the use of multiple anti-inflammatory strategies in COPD patients due to differences between cell types.

The development of AZD7624 for prevention of exacerbations in COPD: a randomized controlled trial.

Background: p38 mitogen-activated protein kinase (MAPK) plays a central role in the regulation and activation of pro-inflammatory mediators. COPD patients have increased levels of activated p38 MAPK, which correlate with increased lung function impairment, alveolar wall inflammation, and COPD exacerbations. Objectives: These studies aimed to assess the effect of p38 inhibition with AZD7624 in healthy volunteers and patients with COPD. The principal hypothesis was that decreasing lung inflammation via inhibition of p38α would reduce exacerbations and improve quality of life for COPD patients at high risk for acute exacerbations. Methods: The p38 isoform most relevant to lung inflammation was assessed using an in situ proximity ligation assay in severe COPD patients and donor controls. Volunteers aged 18-55 years were randomized into the lipopolysaccharide (LPS) challenge study, which investigated the effect of a single dose of AZD7624 vs placebo on inflammatory biomarkers. The Proof of Principle study randomized patients aged 40-85 years with a diagnosis of COPD for >1 year to AZD7624 or placebo to assess the effect of p38 inhibition in decreasing the rate of exacerbations. Results: The p38 isoform most relevant to lung inflammation was p38α, and AZD7624 specifically inhibited p38α and p38ß isoforms in human alveolar macrophages. Thirty volunteers were randomized in the LPS challenge study. AZD7624 reduced the increase from baseline in sputum neutrophils and TNF-α by 56.6% and 85.4%, respectively (p<0.001). In the 213 patients randomized into the Proof of Principle study, there was no statistically significant difference between AZD7624 and placebo when comparing the number of days to the first moderate or severe exacerbation or early dropout. Conclusion: Although p38α is upregulated in the lungs of COPD patients, AZD7624, an isoform-specific inhaled p38 MAPK inhibitor, failed to show any benefit in patients with COPD.