Adventitial Stromal Cells Define Group 2 Innate Lymphoid Cell Tissue Niches.

Type 2 lymphocytes promote both physiologic tissue remodeling and allergic pathology, yet their physical tissue niches are poorly described. Here, we used quantitative imaging to define the tissue niches of group 2 innate lymphoid cells (ILC2s), which are critical instigators of type 2 immunity. We identified a dominant adventitial niche around lung bronchi and larger vessels in multiple tissues, where ILC2s localized with subsets of dendritic and regulatory T cells. However, ILC2s were most intimately associated with adventitial stromal cells (ASCs), a mesenchymal fibroblast-like subset that expresses interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP). In vitro, ASCs produced TSLP that supported ILC2 accumulation and activation. ILC2s and IL-13 drove reciprocal ASC expansion and IL-33 expression. During helminth infection, ASC depletion impaired lung ILC2 and Th2 cell accumulation and function, which are in part dependent on ASC-derived IL-33. These data indicate that adventitial niches are conserved sites where ASCs regulate type 2 lymphocyte expansion and function.

Local and systemic responses to SARS-CoV-2 infection in children and adults.

It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children.

Inhaled Fine Particles Induce Alveolar Macrophage Death and Interleukin-1α Release to Promote Inducible Bronchus-Associated Lymphoid Tissue Formation.

Particulate pollution is thought to function as an adjuvant that can induce allergic responses. However, the exact cell types and immunological factors that initiate the lung-specific immune responses are unclear. We found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) and caused inducible bronchus-associated lymphoid tissue (iBALT) formation in the lung. IL-1α release continued for up to 2 weeks after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1α release and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, which coincided with subsequent immunoglobulin E responses, and IL-1-receptor-deficient mice failed to induce iBALT formation. Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation.

Bronchial epithelia from adults and children: SARS-CoV-2 spread via syncytia formation and type III interferon infectivity restriction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.

Bronchial epithelial DNA methyltransferase 3b dampens pulmonary immune responses during Pseudomonas aeruginosa infection.

DNA methyltransferase (Dnmt)3b mediates de novo DNA methylation and modulation of Dnmt3b in respiratory epithelial cells has been shown to affect the expression of multiple genes. Respiratory epithelial cells provide a first line of defense against pulmonary pathogens and play a crucial role in the immune response during pneumonia caused by Pseudomonas (P.) aeruginosa, a gram-negative bacterium that expresses flagellin as an important virulence factor. We here sought to determine the role of Dntm3b in respiratory epithelial cells in immune responses elicited by P. aeruginosa. DNMT3B expression was reduced in human bronchial epithelial (BEAS-2B) cells as well as in primary human and mouse bronchial epithelial cells grown in air liquid interface upon exposure to P. aeruginosa (PAK). Dnmt3b deficient human bronchial epithelial (BEAS-2B) cells produced more CXCL1, CXCL8 and CCL20 than control cells when stimulated with PAK, flagellin-deficient PAK (PAKflic) or flagellin. Dnmt3b deficiency reduced DNA methylation at exon 1 of CXCL1 and enhanced NF-ĸB p65 binding to the CXCL1 promoter. Mice with bronchial epithelial Dntm3b deficiency showed increased Cxcl1 mRNA expression in bronchial epithelium and CXCL1 protein release in the airways during pneumonia caused by PAK, which was associated with enhanced neutrophil recruitment and accelerated bacterial clearance; bronchial epithelial Dnmt3b deficiency did not modify responses during pneumonia caused by PAKflic or Klebsiella pneumoniae (an un-flagellated gram-negative bacterium). Dnmt3b deficiency in type II alveolar epithelial cells did not affect mouse pulmonary defense against PAK infection. These results suggest that bronchial epithelial Dnmt3b impairs host defense during Pseudomonas induced pneumonia, at least in part, by dampening mucosal responses to flagellin.

The development of inducible bronchus-associated lymphoid tissue depends on IL-17.

Ectopic or tertiary lymphoid tissues, such as inducible bronchus-associated lymphoid tissue (iBALT), form in nonlymphoid organs after local infection or inflammation. However, the initial events that promote this process remain unknown. Here we show that iBALT formed in mouse lungs as a consequence of pulmonary inflammation during the neonatal period. Although we found CD4(+)CD3(-) lymphoid tissue-inducer cells (LTi cells) in neonatal lungs, particularly after inflammation, iBALT was formed in mice that lacked LTi cells. Instead, we found that interleukin 17 (IL-17) produced by CD4(+) T cells was essential for the formation of iBALT. IL-17 acted by promoting lymphotoxin-α-independent expression of the chemokine CXCL13, which was important for follicle formation. Our results suggest that IL-17-producing T cells are critical for the development of ectopic lymphoid tissues.

Dual role for CXCR3 and CCR5 in asthmatic type 1 inflammation.

BACKGROUND: Many patients with severe asthma (SA) fail to respond to type 2 inflammation-targeted therapies. We previously identified a cohort of subjects with SA expressing type 1 inflammation manifesting with IFN-γ expression and variable type 2 responses. OBJECTIVE: We investigated the role of the chemotactic receptors C-X-C chemokine receptor 3 (CXCR3) and C-C chemokine receptor 5 (CCR5) in establishing type 1 inflammation in SA. METHODS: Bronchoalveolar lavage microarray data from the Severe Asthma Research Program I/II were analyzed for pathway expression and paired with clinical parameters. Wild-type, Cxcr3-/-, and Ccr5-/- mice were exposed to a type 1-high SA model with analysis of whole lung gene expression and histology. Wild-type and Cxcr3-/- mice were treated with a US Food and Drug Administration-approved CCR5 inhibitor (maraviroc) with assessment of airway resistance, inflammatory cell recruitment by flow cytometry, whole lung gene expression, and histology. RESULTS: A cohort of subjects with increased IFN-γ expression showed higher asthma severity. IFN-γ expression was correlated with CXCR3 and CCR5 expression, but in Cxcr3-/- and Ccr5-/- mice type 1 inflammation was preserved in a murine SA model, most likely owing to compensation by the other pathway. Incorporation of maraviroc into the experimental model blunted airway hyperreactivity despite only mild effects on lung inflammation. CONCLUSIONS: IFNG expression in asthmatic airways was strongly correlated with expression of both the chemokine receptors CXCR3 and CCR5. Although these pathways provide redundancy for establishing type 1 lung inflammation, inhibition of the CCL5/CCR5 pathway with maraviroc provided unique benefits in reducing airway hyperreactivity. Targeting this pathway may be a novel approach for improving lung function in individuals with type 1-high asthma.

Mapping atopic dermatitis and anti-IL-22 response signatures to type 2-low severe neutrophilic asthma.

BACKGROUND: Transcriptomic changes in patients who respond clinically to biological therapies may identify responses in other tissues or diseases. OBJECTIVE: We sought to determine whether a disease signature identified in atopic dermatitis (AD) is seen in adults with severe asthma and whether a transcriptomic signature for patients with AD who respond clinically to anti-IL-22 (fezakinumab [FZ]) is enriched in severe asthma. METHODS: An AD disease signature was obtained from analysis of differentially expressed genes between AD lesional and nonlesional skin biopsies. Differentially expressed genes from lesional skin from therapeutic superresponders before and after 12 weeks of FZ treatment defined the FZ-response signature. Gene set variation analysis was used to produce enrichment scores of AD and FZ-response signatures in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes asthma cohort. RESULTS: The AD disease signature (112 upregulated genes) encompassing inflammatory, T-cell, TH2, and TH17/TH22 pathways was enriched in the blood and sputum of patients with asthma with increasing severity. Patients with asthma with sputum neutrophilia and mixed granulocyte phenotypes were the most enriched (P < .05). The FZ-response signature (296 downregulated genes) was enriched in asthmatic blood (P < .05) and particularly in neutrophilic and mixed granulocytic sputum (P < .05). These data were confirmed in sputum of the Airway Disease Endotyping for Personalized Therapeutics cohort. IL-22 mRNA across tissues did not correlate with FZ-response enrichment scores, but this response signature correlated with TH22/IL-22 pathways. CONCLUSIONS: The FZ-response signature in AD identifies severe neutrophilic asthmatic patients as potential responders to FZ therapy. This approach will help identify patients for future asthma clinical trials of drugs used successfully in other chronic diseases.

Hypersensitivity may be involved in severe COVID-19.

BACKGROUND: Deaths attributed to Coronavirus Disease 2019 (COVID-19) are mainly due to severe hypoxemic respiratory failure. Although the inflammatory storm has been considered the main pathogenesis of severe COVID-19, hypersensitivity may be another important mechanism involved in severe cases, which have a perfect response to corticosteroids (CS). METHOD: We detected the serum level of anti-SARS-CoV-2-spike S1 protein-specific IgE (SP-IgE) and anti-SARS-CoV-2 nucleocapsid protein-specific IgE (NP-IgE) in COVID-19. Correlation of levels of specific IgE and clinical severity were analysed. Pulmonary function test and bronchial provocation test were conducted in early convalescence of COVID-19. We also obtained histological samples via endoscopy to detect the evidence of mast cell activation. RESULT: The levels of serum SP-IgE and NP-IgE were significantly higher in severe cases, and were correlated with the total lung severity scores (TLSS) and the PaO2 /FiO2 ratio. Nucleocapsid protein could be detected in both airway and intestinal tissues, which was stained positive together with activated mast cells, binded with IgE. Airway hyperresponsiveness (AHR) exists in the early convalescence of COVID-19. After the application of CS in severe COVID-19, SP-IgE and NP-IgE decreased, but maintained at a high level. CONCLUSION: Hypersensitivity may be involved in severe COVID-19.

Isolation, identification, and characterization of the human airway ligand for the eosinophil and mast cell immunoinhibitory receptor Siglec-8.

BACKGROUND: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. OBJECTIVE: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. METHODS: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. RESULTS: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. CONCLUSION: A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.

Mitochondria are involved in bronchial smooth muscle remodeling in severe preschool wheezers.

BACKGROUND: Bronchial remodeling is a key feature of asthma that is already present in preschoolers with wheezing. Moreover, bronchial smooth muscle (BSM) remodeling at preschool age is predictive of asthma at school age. However, the mechanism responsible for BSM remodeling in preschoolers with wheezing remains totally unknown. In contrast, in adult asthma, BSM remodeling has been associated with an increase in BSM cell proliferation related to increased mitochondrial mass and biogenesis triggered by an altered calcium homeostasis. Indeed, BSM cell proliferation was decreased in vitro by the calcium channel blocker gallopamil. OBJECTIVE: Our aim was to investigate the mechanisms involved in BSM cell proliferation in preschoolers with severe wheezing, with special attention to the role of mitochondria and calcium signaling. METHODS: Bronchial tissue samples obtained from 12 preschool controls without wheezing and 10 preschoolers with severe wheezing were used to measure BSM mass and establish primary BSM cell cultures. BSM cell proliferation was assessed by manual counting and flow cytometry, ATP content was assessed by bioluminescence, mitochondrial respiration was assessed by using either the Seahorse or Oroboros technique, mitochondrial mass and biogenesis were assessed by immunoblotting, and calcium response to carbachol was assessed by confocal microscopy. The effect of gallopamil was also evaluated. RESULTS: BSM mass, cell proliferation, ATP content, mitochondrial respiration, mass and biogenesis, and calcium response were all increased in preschoolers with severe wheezing compared with in the controls. Gallopamil significantly decreased BSM mitochondrial biogenesis and mass, as well as cell proliferation. CONCLUSION: Mitochondria are key players in BSM cell proliferation in preschoolers with severe wheezing and could represent a potential target to treat BSM remodeling at an early stage of the disease.

C5aR inhibition of nonimmune cells suppresses inflammation and maintains epithelial integrity in SARS-CoV-2-infected primary human airway epithelia.

BACKGROUND: Excessive inflammation triggered by a hitherto undescribed mechanism is a hallmark of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and is associated with enhanced pathogenicity and mortality. OBJECTIVE: Complement hyperactivation promotes lung injury and was observed in patients suffering from Middle East respiratory syndrome-related coronavirus, SARS-CoV-1, and SARS-CoV-2 infections. Therefore, we investigated the very first interactions of primary human airway epithelial cells on exposure to SARS-CoV-2 in terms of complement component 3 (C3)-mediated effects. METHODS: For this, we used highly differentiated primary human 3-dimensional tissue models infected with SARS-CoV-2 patient isolates. On infection, viral load, viral infectivity, intracellular complement activation, inflammatory mechanisms, and tissue destruction were analyzed by real-time RT-PCR, high content screening, plaque assays, luminex analyses, and transepithelial electrical resistance measurements. RESULTS: Here, we show that primary normal human bronchial and small airway epithelial cells respond to SARS-CoV-2 infection by an inflated local C3 mobilization. SARS-CoV-2 infection resulted in exaggerated intracellular complement activation and destruction of the epithelial integrity in monolayer cultures of primary human airway cells and highly differentiated, pseudostratified, mucus-producing, ciliated respiratory tissue models. SARS-CoV-2-infected 3-dimensional cultures secreted significantly higher levels of C3a and the proinflammatory cytokines IL-6, monocyte chemoattractant protein 1, IL-1α, and RANTES. CONCLUSIONS: Crucially, we illustrate here for the first time that targeting the anaphylotoxin receptors C3a receptor and C5a receptor in nonimmune respiratory cells can prevent intrinsic lung inflammation and tissue damage. This opens up the exciting possibility in the treatment of COVID-19.

Involvement of the Innate Immune System in the Pathogenesis of Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a common, socially significant disease characterized by progressive airflow limitation due to chronic inflammation in the bronchi. Although the causes of COPD are considered to be known, the pathogenesis of the disease continues to be a relevant topic of study. Mechanisms of the innate immune system are involved in various links in the pathogenesis of COPD, leading to persistence of chronic inflammation in the bronchi, their bacterial colonization and disruption of lung structure and function. Bronchial epithelial cells, neutrophils, macrophages and other cells are involved in the development and progression of the disease, demonstrating multiple compromised immune mechanisms.

IL-17 regulates DC migration to the peribronchial LNs and allergen presentation in experimental allergic asthma.

IL-17 is associated with different phenotypes of asthma, however, it is not fully elucidated how it influences induction and maintenance of asthma and allergy. In order to determine the role of IL-17 in development of allergic asthma, we used IL-17A/F double KO (IL-17A/F KO) and WT mice with or without neutralization of IL-17 in an experimental allergic asthma model and analyzed airway hyperresponsiveness, lung inflammation, T helper cell polarization, and DCs influx and activation. We report that the absence of IL-17 reduced influx of DCs into lungs and lung draining LNs. Compared to WT mice, IL-17A/F KO mice or WT mice after neutralization of IL-17A showed reduced airway hyperresponsiveness, eosinophilia, mucus hypersecretion, and IgE levels. DCs from draining LNs of allergen-challenged IL-17A/F KO mice showed a reduction in expression of migratory and costimulatory molecules CCR7, CCR2, MHC-II, and CD40 compared to WT DCs. Moreover, in vivo stimulation of adoptively transferred antigen-specific cells was attenuated in lung-draining LNs in the absence of IL-17. Thus, we report that IL-17 enhances airway DC activation, migration, and function. Consequently, lack of IL-17 leads to reduced antigen-specific T cell priming and impaired development of experimental allergic asthma.

Type I and Type III Interferons Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures.

The newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. In this study, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral, surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by proinflammatory cytokines and chemokine induction, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and CXCL8, and identified NF-κB and ATF-4 as key drivers of this proinflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pretreatment and posttreatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients.IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.

Role of iBALT in Respiratory Immunity.

Pulmonary respiration inevitably exposes the mucosal surface of the lung to potentially noxious stimuli, including pathogens, allergens, and particulates, each of which can trigger pulmonary damage and inflammation. As inflammation resolves, B and T lymphocytes often aggregate around large bronchi to form inducible Bronchus-Associated Lymphoid Tissue (iBALT). iBALT formation can be initiated by a diverse array of molecular pathways that converge on the activation and differentiation of chemokine-expressing stromal cells that serve as the scaffolding for iBALT and facilitate the recruitment, retention, and organization of leukocytes. Like conventional lymphoid organs, iBALT recruits naïve lymphocytes from the blood, exposes them to local antigens, in this case from the airways, and supports their activation and differentiation into effector cells. The activity of iBALT is demonstrably beneficial for the clearance of respiratory pathogens; however, it is less clear whether it dampens or exacerbates inflammatory responses to non-infectious agents. Here, we review the evidence regarding the role of iBALT in pulmonary immunity and propose that the final outcome depends on the context of the disease.

Friend or Foe: The Protective and Pathological Roles of Inducible Bronchus-Associated Lymphoid Tissue in Pulmonary Diseases.

Inducible bronchus-associated lymphoid tissue (iBALT) is a tertiary lymphoid structure that resembles secondary lymphoid organs. iBALT is induced in the lung in response to Ag exposure. In some cases, such as infection with Mycobacterium tuberculosis, the formation of iBALT structure is indicative of an effective protective immune response. However, with persistent exposure to Ags during chronic inflammation, allergy, or autoimmune diseases, iBALT may be associated with exacerbation of inflammation. iBALT is characterized by well-organized T and B areas enmeshed with conventional dendritic cells, follicular dendritic cells, and stromal cells, usually located surrounding airways or blood vessels. Several of the molecular signals and cellular contributors that mediate formation of iBALT structures have been recently identified. This review will outline the recent findings associated with the formation and maintenance of iBALT and their contributions toward a protective or pathogenic function in pulmonary disease outcome.

Rhinovirus-Induced SIRT-1 via TLR2 Regulates Subsequent Type I and Type III IFN Responses in Airway Epithelial Cells.

IFN responses to viral infection are necessary to establish intrinsic antiviral state, but if unchecked can lead to heightened inflammation. Recently, we showed that TLR2 activation contributes to limitation of rhinovirus (RV)-induced IFN response in the airway epithelial cells. We also demonstrated that compared with normal airway epithelial cells, those from patients with chronic obstructive pulmonary disease (COPD) show higher IFN responses to RV, but the underlying mechanisms are not known. Initially, RV-induced IFN responses depend on dsRNA receptor activation and then are amplified via IFN-stimulated activation of JAK/STAT signaling. In this study, we show that in normal cells, TLR2 limits RV-induced IFN responses by attenuating STAT1 and STAT2 phosphorylation and this was associated with TLR2-dependent SIRT-1 expression. Further, inhibition of SIRT-1 enhanced RV-induced IFN responses, and this was accompanied by increased STAT1/STAT2 phosphorylation, indicating that TLR2 may limit RV-induced IFN responses via SIRT-1. COPD airway epithelial cells showed attenuated IL-8 responses to TLR2 agonist despite expressing TLR2 similar to normal, indicating dysregulation in TLR2 signaling pathway. Unlike normal, COPD cells failed to show RV-induced TLR2-dependent SIRT-1 expression. Pretreatment with quercetin, which increases SIRT-1 expression, normalized RV-induced IFN levels in COPD airway epithelial cells. Inhibition of SIRT-1 in quercetin-pretreated COPD cells abolished the normalizing effects of quercetin on RV-induced IFN expression in these cells, confirming that quercetin exerts its effect via SIRT-1. In summary, we show that TLR2 is required for limiting RV-induced IFNs, and this pathway is dysregulated in COPD airway epithelial cells, leading to exaggerated IFN production.

Activation of NLRP3 inflammasome by lymphocytic microparticles via TLR4 pathway contributes to airway inflammation.

The presence of elevated T lymphocytic microparticles (TLMPs) during respiratory illness is associated with airway and lung inflammation and epithelial injuries. Although inflammasome and IL-1ß signaling are crucial in airway inflammation, little was known about their regulatory mechanism. We hypothesized that TLMPs trigger inflammasome activation and IL-1ß production in bronchial and alveolar epithelial cells to induce airway and lung inflammation. In this study, TLMPs induced IL-1ß and IL-18 secretion through NLRP3 inflammasome activation and upregulated TLR4 mRNA and protein expression in alveolar (A549) and human airway epithelial (16HBE) cells. Pretreatment with CLI-095, a specific inhibitor of TLR4 signaling, dramatically diminished the TLMP-induced release of IL-1ß and IL-18 by inhibiting the formation of NLRP3/ASC/pro-caspase-1 inflammasome in a dose-dependent manner. The TLMP-induced autophagy inhibition in epithelial cells was dependent on the PI3K/Akt signaling pathway, which significantly increased NLRP3 expression and enhanced TLMP-induced inflammation. TLR4, IL-1ß, and IL-18 proteins harbored in TLMPs were nonessential for the pro-inflammatory effect. In conclusion, TLMPs induce bronchial and alveolar epithelial cell secretion of IL-1ß and IL-18 cytokines by activating the TLR4 and PI3K/Akt signaling pathways and inhibiting autophagy. These effects lead to NLRP3 inflammasome formation and accumulation. TLMPs may be regarded as deleterious markers of airway and lung damage in respiratory diseases.

LncRNA loc105377478 promotes NPs-Nd2O3-induced inflammation in human bronchial epithelial cells through the ADIPOR1/NF-κB axis.

With the wide application of neodymium oxide nanoparticles (NPs-Nd2O3) in various fields, their health hazards have aroused public concern in recent years. However, data regarding the cytotoxicity of NPs-Nd2O3 is limited. In this study, we investigated the function and mechanism of long-chain non-coding RNAs (lncRNAs) in NPs-Nd2O3-induced airway inflammation. Treatment with NPs-Nd2O3 induced an inflammatory response in human bronchial epithelial cells (16HBE) by upregulating the expression of interleukin-6 (IL-6) and interleukin-8 (IL-8). The levels of LDH and intracellular ROS in the cells treated by various doses of NPs-Nd2O3 also increased significantly. After treatment with 10 µg/ml NPs-Nd2O3, RNA microarray and real-time quantitative polymerase chain reaction (qRT-PCR) showed a significant upregulation of lncRNA loc105377478. Functional experiments suggested lncRNA loc105377478 enhanced the expression of IL-6, IL-8 and ROS in NPs-Nd2O3-treated 16HBE cells, and it was further demonstrated that lncRNA loc105377478 promoted the activation of NF-κB by negatively regulating ADIPOR1 expression. Moreover, the expression of IL-6 and IL-8 in NPs-Nd2O3-treated 16HBE cells was regulated by lncRNA loc105377478, which was mediated by the NF-κB signaling pathway. In conclusion, lncRNA loc105377478 promotes NF-κB activation by negatively regulating ADIPOR1 expression, thereby upregulating the expression of IL-6 and IL-8 in 16HBE cells treated with NPs-Nd2O3.