Mediators Of Airway Inflammation Research Articles

DerP1, a house dust mite protease, directly activates airway vagal afferents and induces airway hyperreactivity after chronic exposure

Airway hyperreactivity (AHR) is one of the main symptoms of allergic asthma. Current therapeutics used in the treatment of allergic asthma do not target the AHR. Airway sensory afferents have been shown to be critical in AHR, but the mechanisms remain elusive. We have previously shown that house dust mite (HDM) extract, which contains multiple different proteases, can directly activate these airway sensory afferents in the lung via PAR1 receptors. In this study we identified the most potent protease DerP1 is capable of directly and robustly activating airway sensory vagal afferents. DerP1 caused a significant release of CGRP, a neuropeptide released after nerve terminal activation, from the airways. We observed that DerP1 caused a calcium influx in 65% of TRPV1 positive lung specific neurons. We also observed that in our two-photon ex vivo imaging of the vagal ganglia of PirtCre;R26 ‐ GCaMP6s mice, DerP1 caused a robust activation of approximately 60% of naïve airway-specific C-fibers. Calcium imaging of the lung specific vagal neurons from wild type, TRPA1 KO and TRPV1 KO revealed that the DerP1 mediated activation was absent in TRPV1 KO mice neurons. We then found that mice exposed to DerP1 chronically over a period of 12 days (without an adjuvant) had significant AHR and Th2 mediated airway inflammation. Our data suggests that direct activation of airway vagal C-fiber afferents by DerP1 via TRPV1 chronically leads to airway hyperreactivity and Th2 mediated airway inflammation. Warren Alpert Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

IL-5 antagonism reverses priming and activation of eosinophils in severe eosinophilic asthma

Eosinophils are key effector cells mediating airway inflammation and exacerbation in patients with severe eosinophilic asthma. They are present in increased number and activation state in the airway mucosa and lumen. Interleukin-5 (IL-5) is the key eosinophil growth factor that is thought to play a role in eosinophil priming and activation. However, the mechanism of these effects is still not fully understood. The anti-IL-5 antibody mepolizumab reduces eosinophil counts in the airway modestly but has a large beneficial effect on the frequency of exacerbations of severe eosinophilic asthma, suggesting that reduction in eosinophil priming and activation is of central mechanistic importance. In this study, we used the therapeutic effect of mepolizumab and single cell RNAseq to investigate the mechanism of eosinophil priming and activation by IL-5. We demonstrated that IL-5 is a dominant driver of eosinophil priming and plays multifaceted roles in eosinophil function. It enhances eosinophil responses to other stimulators of migration, survival and activation by activating PI3K, MAPK and p38 signal pathways. It also enhances the pro-fibrotic roles of eosinophils in airway remodeling via TGF-β pathway. These findings provide mechanistic understanding of eosinophil priming in severe eosinophilic asthma and the therapeutic effect of anti-IL-5 approaches in the disease.

Calcium Signaling in Airway Epithelial Cells: Current Understanding and Implications for Inflammatory Airway Disease.

Airway epithelial cells play an indispensable role in protecting the lung from inhaled pathogens and allergens by releasing an array of mediators that orchestrate inflammatory and immune responses when confronted with harmful environmental triggers. While this process is undoubtedly important for containing the effects of various harmful insults, dysregulation of the inflammatory response can cause lung diseases including asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. A key cellular mechanism that underlies the inflammatory responses in the airway is calcium signaling, which stimulates the production and release of chemokines, cytokines, and prostaglandins from the airway epithelium. In this review, we discuss the role of major Ca2+ signaling pathways found in airway epithelial cells and their contributions to airway inflammation, mucociliary clearance, and surfactant production. We highlight the importance of store-operated Ca2+ entry as a major signaling hub in these processes and discuss therapeutic implications of targeting Ca2+ signaling for airway inflammation.

Parkin Promotes Airway Inflammatory Response to Interferon Gamma.

Increased type 2 interferon (i.e., IFN-γ) signaling has been shown to be involved in airway inflammation in a subset of asthma patients who often show high levels of airway neutrophilic inflammation and poor response to corticosteroid treatment. How IFN-γ mediates airway inflammation in a mitochondrial dysfunction setting (e.g., Parkin up-regulation) remains poorly understood. The goal of this study was to determine the role of Parkin, an E3 ubiquitin ligase, in IFN-γ-mediated airway inflammation and the regulation of Parkin by IFN-γ. A mouse model of IFN-γ treatment in wild-type and Parkin knockout mice, and cultured human primary airway epithelial cells with or without Parkin gene deficiency were used. Parkin was found to be necessary for the production of neutrophil chemokines (i.e., LIX and IL-8) and airway neutrophilic inflammation following IFN-γ treatment. Mechanistically, Parkin was induced by IFN-γ treatment both in vivo and in vitro, which was associated with less expression of a Parkin transcriptional repressor Thap11. Overexpression of Thap11 inhibited Parkin expression in IFN-γ-stimulated airway epithelial cells. Our data suggest a novel mechanism by which IFN-γ induces airway neutrophilic inflammation through the Thap11/Parkin axis. Inhibition of Parkin expression or activity may provide a new therapeutic target for the treatment of excessive neutrophilic inflammation in an IFN-γ-high environment.

Mitoquinone ameliorated airway inflammation by stabilizing β-catenin destruction complex in a steroid-insensitive asthma model

Background and purposeMitochondrial dysfunction is an essential part of the pathophysiology of asthma, and potential treatments that target the malfunctioning mitochondria have attracted widespread attention. We have previously demonstrated that aberrant epithelial β-catenin signaling played a crucial role in a toluene diisocyanate (TDI)-induced steroid-insensitive asthma model. The objective of this study was to determine if the mitochondrially targeted antioxidant mitoquinone(MitoQ) regulated the activation of β-catenin in TDI-induced asthma. MethodMice were sensitized and challenged with TDI to generate a steroid-insensitive asthma model. Human bronchial epithelial cells (16HBE) were exposed to TDI-human serum albumin (HSA) and ethidium bromide(EB) to simulate the TDI-induced asthma model and mitochondrial dysfunction. ResultsMitoQ dramatically attenuated TDI-induced AHR, airway inflammation, airway goblet cell metaplasia, and collagen deposition and markedly protected epithelial mitochondrial functions by preserving mass and diminishing the production of reactive oxygen species (ROS). MitoQ administration stabilized β‐catenin destruction complex from disintegration and inhibited the activation of β‐catenin. Similarly, YAP1, an important constituent of β‐catenin destruction complex, was inhibited by Dasatinib, which alleviated airway inflammation and the activation of β‐catenin, and restored mitochondrial mass. In vitro, treating 16HBE cells with EB led to the activation of YAP1 and β-catenin signaling, decreased the expression of glucocorticoid receptors and up-regulated interleukin (IL)-1β, IL6 and IL-8 expression. ConclusionOur results indicated that mitochondria mediates airway inflammation by regulating the stability of the β-catenin destruction complex and MitoQ might be a promising therapeutic approach to improve airway inflammation and severe asthma. Availability of data and materialsThe data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

Molecular Mechanisms Underlying TNFα-Induced Mitochondrial Biogenesis in Human Airway Smooth Muscle.

Proinflammatory cytokines such as TNFα mediate airway inflammation. Previously, we showed that TNFα increases mitochondrial biogenesis in human ASM (hASM) cells, which is associated with increased PGC1α expression. We hypothesized that TNFα induces CREB and ATF1 phosphorylation (pCREBS133 and pATF1S63), which transcriptionally co-activate PGC1α expression. Primary hASM cells were dissociated from bronchiolar tissue obtained from patients undergoing lung resection, cultured (one-three passages), and then differentiated by serum deprivation (48 h). hASM cells from the same patient were divided into two groups: TNFα (20 ng/mL) treated for 6 h and untreated controls. Mitochondria were labeled using MitoTracker green and imaged using 3D confocal microscopy to determine mitochondrial volume density. Mitochondrial biogenesis was assessed based on relative mitochondrial DNA (mtDNA) copy number determined by quantitative real-time PCR (qPCR). Gene and/or protein expression of pCREBS133, pATF1S63, PCG1α, and downstream signaling molecules (NRFs, TFAM) that regulate transcription and replication of the mitochondrial genome, were determined by qPCR and/or Western blot. TNFα increased mitochondrial volume density and mitochondrial biogenesis in hASM cells, which was associated with an increase in pCREBS133, pATF1S63 and PCG1α expression, with downstream transcriptional activation of NRF1, NRF2, and TFAM. We conclude that TNFα increases mitochondrial volume density in hASM cells via a pCREBS133/pATF1S63/PCG1α-mediated pathway.

IGFBP-6 Network in Chronic Inflammatory Airway Diseases and Lung Tumor Progression.

The lung is an accomplished organ for gas exchanges and directly faces the external environment, consequently exposing its large epithelial surface. It is also the putative determinant organ for inducing potent immune responses, holding both innate and adaptive immune cells. The maintenance of lung homeostasis requires a crucial balance between inflammation and anti-inflammation factors, and perturbations of this stability are frequently associated with progressive and fatal respiratory diseases. Several data demonstrate the involvement of the insulin-like growth factor (IGF) system and their binding proteins (IGFBPs) in pulmonary growth, as they are specifically expressed in different lung compartments. As we will discuss extensively in the text, IGFs and IGFBPs are implicated in normal pulmonary development but also in the pathogenesis of various airway diseases and lung tumors. Among the known IGFBPs, IGFBP-6 shows an emerging role as a mediator of airway inflammation and tumor-suppressing activity in different lung tumors. In this review, we assess the current state of IGFBP-6's multiple roles in respiratory diseases, focusing on its function in the inflammation and fibrosis in respiratory tissues, together with its role in controlling different types of lung cancer.

Airway microbiome-immune crosstalk in chronic obstructive pulmonary disease.

Chronic Obstructive Pulmonary Disease (COPD) has significantly contributed to global mortality, with three million deaths reported annually. This impact is expected to increase over the next 40 years, with approximately 5 million people predicted to succumb to COPD-related deaths annually. Immune mechanisms driving disease progression have not been fully elucidated. Airway microbiota have been implicated. However, it is still unclear how changes in the airway microbiome drive persistent immune activation and consequent lung damage. Mechanisms mediating microbiome-immune crosstalk in the airways remain unclear. In this review, we examine how dysbiosis mediates airway inflammation in COPD. We give a detailed account of how airway commensal bacteria interact with the mucosal innate and adaptive immune system to regulate immune responses in healthy or diseased airways. Immune-phenotyping airway microbiota could advance COPD immunotherapeutics and identify key open questions that future research must address to further such translation.

Abstract P045: γδ T cells contribute to an inflammatory and immunosuppressive microenvironment in lung squamous cell carcinoma tumorigenesis

Abstract Lung cancer (LC) is the leading cause of cancer death worldwide. Lung squamous cell carcinoma (LSCC) is the second most prevalent subtype of LC, accounting for approximately 22% of cases. It is strongly associated with smoking, with approximately 90% of cases attributable to cigarette smoke exposure. Despite the strong association with smoking, not all smokers will develop lung cancer. From this, two key questions arise: which patients will develop the lesions preceding LSCC, and among those, which will progress to frank carcinoma? Stratification of at-risk patients for diversion into intervention and prevention strategies, such as increased monitoring and chemical interception, remains an urgent need, and a greater understanding of the factors contributing to progression of LSCC premalignancy is critical in addressing this. Previous evidence suggests a role for the immune system in the development of preinvasive disease, and we hypothesize that immune differences between at-risk smokers may also play a role in which patients progress on to frank LSCC and which do not. Using the N-Nitrosotris-(2-chloroethyl) urea (NTCU) carcinogen-induced murine model of LSCC, we sought to explore the immune contexture of LSCC premalignancy in both NTCU-sensitive and NTCU-resistant mouse strains. We immunophenotyped mouse lungs via flow cytometry and imaging mass cytometry over the course of disease initiation and progression to carcinoma. We found that although the frequency of γδ T cells in control animals were the same between sensitive and resistant mice, in sensitive mice the frequency of γδ T cells and the expression of activation/exhaustion markers increased with histological progression of PMLs, along with a decrease in the frequency of cytotoxic CD8+ T cells. Given that pro-tumor γδ T cells have been established to mediate airway inflammation via recruitment of myeloid cells (e.g., macrophages, neutrophils, and myeloid derived suppressor cells) and to influence maturation/exhaustion of T cells, these findings suggest γδ T cells potentially contribute to the early establishment of an inflammatory and immunosuppressive microenvironment in developing LSCC premalignancy. By better understanding how immune alterations within the precancerous microenvironment contribute to lesion progression, immunomodulatory intervention agents can be developed and deployed in at-risk patients to ameliorate the development of frank lung squamous cell carcinoma and ultimately reduce LC mortality. Citation Format: Táchira Pichardo, Anna Belkina, Jennifer Snyder-Cappione, Jennifer Beane, Sarah Mazzilli. γδ T cells contribute to an inflammatory and immunosuppressive microenvironment in lung squamous cell carcinoma tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P045.

Activities of Periostin and Inteleukin-13 among Asthma Patients in Comparison with Healthy people in Southern India

Background and objectives: Asthma is a chronic inflammatory airway disease which requires biomarkers that reflect refractoriness to conventional therapy with inhalational steroids. Periostin is an extracellular matrix protein that is secreted in the airway epithelium, in response to stimulation by Interleukin -13 (IL-13). IL-13 is a cytokine that mediates airway inflammation following the Type 2 immune response. Both these biomarkers can be used to signify type 2 inflammatory response, which reflects steroid hypo-responsiveness in asthmatics. The objectives of the study were to: 1. Obtain the reference value of serum Periostin and IL-13 levels in healthy south Indian adult population 2. To compare the reference range of Periostin and IL-13 with that of the asthmatics on inhalational corticosteroids. Methodology: The study was carried out among 50 asthmatics and 50 healthy volunteers aged between 25 to 65 years. After procuring the informed consent, the Pulmonary Function test assessment was done to recruit the study subjects. The blood sample was collected for assessment of Serum Periostin and IL-13. Results: The median (IQR) baseline level of Serum Periostin among control group and in asthmatics was 13.2 (8.8-28.1) ng/ml and 16.7 (10.9-20.7) respectively. Also, the median (IQR) baseline level of Serum IL-13 among healthy individuals and among asthmatics was 42.9 (37.8-52.4) pg/ml and 73.5 (60.0-91.1) pg/ml respectively, which was statistically significant. Conclusion: The obtained baseline values of Serum IL-13 and Periostin could be of clinical utility in asthmatics. The validity of the data obtained from this study can be tested out on larger study populations.

The ketone body β-hydroxybutyrate mitigates ILC2-driven airway inflammation by regulating mast cell function.

Ketone bodies are increasingly understood to have regulatory effects on immune cell function, with β-hydroxybutyrate (BHB) exerting a predominantly anti-inflammatory response. Dietary strategies to increase endogenous ketone body availability such as the ketogenic diet (KD) have recently been shown to alleviate inflammation of the respiratory tract. However, the role of BHB has not been addressed. Here, we observe that BHB suppresses group 2 innate lymphoid cell (ILC2)-mediated airway inflammation. Central to this are mast cells, which support ILC2 proliferation through interleukin-2 (IL-2). Suppression of the mast cell/IL-2 axis by BHB attenuates ILC2 proliferation and the ensuing type 2 cytokine response and immunopathology. Mechanistically, BHB directly inhibits mast cell function in part through GPR109A activation. Similar effects are achieved with either the KD or 1,3-butanediol. Our data reveal the protective role of BHB in ILC2-driven airway inflammation, which underscores the potential therapeutic value of ketone body supplementation for the management of asthma.

EphA2 recognizes Dermatophagoides pteronyssinus to mediate airway inflammation in asthma

Most of the asthma with low Th2 is severe steroid-resistant asthma, the exact pathogenesis of which has not yet been fully elucidated. We found that IL-6 and IL-8 were highly expressed in the sputum supernatant of severe asthma and ephrin type-A receptor 2 (EphA2) was highly expressed on bronchial epithelial cells. So, is there a connection between these two phenomena? To clarify this issue, we stimulated bronchial epithelial cells 16HBE with Dermatophagoides pteronyssinus and its compontents LPS, respectively, and detected the activation of EphA2, activation of downstream pathways and secretion of inflammatory cytokines. A mouse asthma model was established, and the therapeutic effects of inhibiting or blocking EphA2 on mouse asthma were investigated. The results showed that D. pteronyssinus and its component LPS phosphorylated EphA2 on 16HBE, activated downstream signaling pathways STAT3 and p38 MAPK, and promoted the secretion of IL-6 and IL-8. After knockout of EphA2 on 16HBE, the activation of inflammatory pathways was attenuated and the secretion of IL-6 and IL-8 was significantly reduced. Inhibition or blockade of EphA2 on mouse airways resulted in a significant reduction in airway hyperresponsiveness and airway inflammation, and a significant decrease in the expression levels of IL-6, IL-17F, IL-1α, IL-1β and TNF in bronchoalveolar lavage fluid and lung tissue. Our study uncovers a novel role for EphA2 expressed on airway epithelial cells in the pathogenesis of asthma; EphA2 recognizes D. pteronyssinus or its component LPS and promotes the secretion of IL-6 and IL-8 by airway epithelial cell, thereby mediating airway inflammation. Thus, it is possible to provide a new molecular therapy for severe asthma.

LncRNA-CCAT1/miR-152-3p is involved in CSE-induced inflammation in HBE cells via regulating ERK signaling pathway

Emerging studies have noted that dysregulated long non-coding RNAs (lncRNAs) are implicated in the pathological processes of chronic obstructive pulmonary disease (COPD). LncRNA colon cancer-associated transcript 1 (CCAT1) plays well-defined roles in the inflammatory progression. The study aims to figure out the effect and regulatory mechanism of CCAT1 in the cigarette smoke induced inflammation in COPD. The results showed that CCAT1 was highly expressed in lung tissues of smokers with COPD compared with never-smokers without COPD. In human bronchial epithelial (HBE) cells, cigarette smoke extract (CSE) treatment led to an increase in CCAT1 expression in a dose- and time- dependent manner. Functional experiments showed that knockdown of CCAT1 amelioratedCSE-inducedinflammation. Mechanistically, CCAT1 directly targeted miR-152-3p, and miR-152-3p overexpression reversed the pro-inflammatory effects of CCAT1 on HBE cells. Subsequently, miR-152-3p was found to regulate ERK signaling pathway. PD98059, an ERK specific inhibitor, reversed miR-152-3p knockdown mediated inflammation in HBE cells. In addition, CCAT1 acted as a sponge for miR-152-3p to positively regulate ERK signaling pathway. Overall, current findings suggest that CCAT1 promoted inflammation by activating ERK signal pathway via sponging miR–152–3p in CSE–treated HBE cells. These results may provide a novel therapeutic target for alleviating cigarette smoke mediated airway inflammation.

Lysophosphatidylserine Induces MUC5AC Production via the Feedforward Regulation of the TACE-EGFR-ERK Pathway in Airway Epithelial Cells in a Receptor-Independent Manner.

Lysophosphatidylserine (LysoPS) is an amphipathic lysophospholipid that mediates a broad spectrum of inflammatory responses through a poorly characterized mechanism. Because LysoPS levels can rise in a variety of pathological conditions, we sought to investigate LysoPS’s potential role in airway epithelial cells that actively participate in lung homeostasis. Here, we report a previously unappreciated function of LysoPS in production of a mucin component, MUC5AC, in the airway epithelial cells. LysoPS stimulated lung epithelial cells to produce MUC5AC via signaling pathways involving TACE, EGFR, and ERK. Specifically, LysoPS- dependent biphasic activation of ERK resulted in TGF-α secretion and strong EGFR phosphorylation leading to MUC5AC production. Collectively, LysoPS induces the expression of MUC5AC via a feedback loop composed of proligand synthesis and its proteolysis by TACE and following autocrine EGFR activation. To our surprise, we were not able to find a role of GPCRs and TLR2, known LyoPS receptors in LysoPS-induced MUC5AC production in airway epithelial cells, suggesting a potential receptor-independent action of LysoPS during inflammation. This study provides new insight into the potential function and mechanism of LysoPS as an emerging lipid mediator in airway inflammation.

CBX4 Regulates Long-Form Thymic Stromal Lymphopoietin-mediated Airway Inflammation through SUMOylation in House Dust Mite-induced Asthma.

Thymic stromal lymphopoietin presents in two distinct isoforms: short-form (sfTSLP) and long-form (lfTSLP). lfTSLP promotes inflammation, whereas sfTSLP inhibits inflammation, in allergic asthma. However, little is known about the regulation of lfTSLP and sfTSLP during allergic attack in the asthma airway epithelium. Here, we report that small ubiquitin-like modifier (SUMOylation) was enhanced in house dust mite-induced allergic asthma airway epithelium. Inhibition of SUMOylation significantly alleviated airway T-helper cell type 2 inflammation and lfTSLP expression. Mechanistically, chromobox 4 (CBX4), a SUMOylation E3 ligase, enhanced lfTSLP mRNA translation, but not sfTSLP, through the RNA-binding protein muscle excess (MEX)-3B. MEX-3B promoted lfTSLP translation by binding the lfTSLP mRNA through its K homology domains. Furthermore, CBX4 regulated MEX-3B transcription in human bronchial epithelial cells through enhancing SUMOylation concentrations of the transcription factor TFII-I. In conclusion, we demonstrate an important mechanism whereby CBX4 promotes MEX-3B transcription through enhancing TFII-I SUMOylation and MEX-3B enhances the expression of lfTSLP through binding to the lfTSLP mRNA and promoting its translation. Our findings uncover a novel target of CBX4 for therapeutic agents for lfTSLP-mediated asthma.

RAGE mediates airway inflammation via the HDAC1 pathway in a toluene diisocyanate-induced murine asthma model

BackgroundExposure to toluene diisocyanate (TDI) is a significant pathogenic factor for asthma. We previously reported that the receptor for advanced glycation end products (RAGE) plays a key role in TDI-induced asthma. Histone deacetylase (HDAC) has been reported to be important in asthmatic pathogenesis. However, its effect on TDI-induced asthma is not known. The aim of this study was to determine the role of RAGE and HDAC in regulating airway inflammation using a TDI-induced murine asthma model.MethodsBALB/c mice were sensitized and challenged with TDI to establish an asthma model. FPS-ZM1 (RAGE inhibitor), JNJ-26482585 and romidepsin (HDAC inhibitors) were administered intraperitoneally before each challenge. In vitro, the human bronchial epithelial cell line 16HBE was stimulated with TDI-human serum albumin (TDI-HSA). RAGE knockdown cells were constructed and evaluated, and MK2006 (AKT inhibitor) was also used in the experiments.ResultsIn TDI-induced asthmatic mice, the expression of RAGE, HDAC1, and p-AKT/t-AKT was upregulated, and these expressions were attenuated by FPS-ZM1. Airway reactivity, Th2 cytokine levels in lymph supernatant, IgE, airway inflammation, and goblet cell metaplasia were significantly increased in the TDI-induced asthmatic mice. These increases were suppressed by JNJ-26482585 and romidepsin. In addition, JNJ-26482585 and romidepsin ameliorated the redistribution of E-cadherin and β-catenin in TDI-induced asthma. In TDI-HSA-stimulated 16HBE cells, knockdown of RAGE attenuated the upregulation of HDAC1 and phospho-AKT (p-AKT). Treatment with the AKT inhibitor MK2006 suppressed TDI-induced HDAC1 expression.ConclusionsThese findings indicate that RAGE modulates HDAC1 expression via the PI3K/AKT pathway, and that inhibition of HDAC prevents TDI-induced airway inflammation.

Role of interleukin 5-induced eosinophils in interleukin 33-triggered airway inflammation in mice.

Interleukin (IL)-5 is essential for allergen induced eosinophilic airway inflammation, but not activation of T helper type 2 (Th2) cells in the lung. Although an excessive Th2 reaction is observed without IL-5 signaling, the mechanisms have remained unknown. To evaluate the negative-feedback mechanism in eosinophilic airway inflammation, we examined IL-33 triggered eosinophilic airway inflammation in IL-5Rα-/- mice. Mice were administered intranasal IL-33 for 3 days. Airway hyperresponsiveness (AHR) was evaluated and bronchoalveolar lavage (BAL) was performed 24 h after the last IL-33 treatment. The number of inflammatory cells and cytokine levels in the BAL fluid (BALF) were analyzed, and histologic examination was performed. Compared with IL-33 treated wild-type (WT) mice, intranasal administration of IL-33 in IL-5Rα-/- mice reduced eosinophilic airway inflammation, AHR, and basement membrane thickening, while we found excessive IL-33 induced IL-5 and IL-13 production in the airway without IL-5 signaling. The numbers of eosinophils with a ringshaped nucleus (resident) and segmented nucleus (inflammatory) were increased in WT mice, but not in IL-5Rα-/- mice following intranasal administration of IL-33, and the numbers of SiglecF-positive eosinophils with (resident) or without (inflammatory) expression of CD62L were also significantly increased by IL-33 treatment in WT mice, but not in IL5Rα-/- mice. The number of ILC2 cells in the BALF was significantly higher in IL-33 treated IL-5Rα-/- mice than in IL-33 treated WT mice. These findings suggest the possibility that IL-5 induced eosinophils contribute to the negative-feedback mechanisms in IL-33 induced ILC2 mediated airway inflammation.

Irg1/itaconate metabolic pathway is a crucial determinant of dendritic cells immune-priming function and contributes to resolute allergen-induced airway inflammation

Itaconate is produced from the mitochondrial TCA cycle enzyme aconitase decarboxylase (encoded by immune responsive gene1; Irg1) that exerts immunomodulatory function in myeloid cells. However, the role of the Irg1/itaconate pathway in dendritic cells (DC)-mediated airway inflammation and adaptive immunity to inhaled allergens, which are the primary antigen-presenting cells in allergic asthma, remains largely unknown. House dust mite (HDM)-challenged Irg1−/− mice displayed increases in eosinophilic airway inflammation, mucous cell metaplasia, and Th2 cytokine production with a mechanism involving impaired mite antigen presentations by DC. Adoptive transfer of HDM-pulsed DC from Irg1-deficient mice into naïve WT mice induced a similar phenotype of elevated type 2 airway inflammation and allergic sensitization. Untargeted metabolite analysis of HDM-pulsed DC revealed itaconate as one of the most abundant polar metabolites that potentially suppress mitochondrial oxidative damage. Furthermore, the immunomodulatory effect of itaconate was translated in vivo, where intranasal administration of 4-octyl itaconate 4-OI following antigen priming attenuated the manifestations of HDM-induced airway disease and Th2 immune response. Taken together, these data demonstrated for the first time a direct regulatory role of the Irg1/itaconate pathway in DC for the development of type 2 airway inflammation and suggest a possible therapeutic target in modulating allergic asthma.

Airway bacterial and fungal microbiome in chronic obstructive pulmonary disease

Little is known about airway mycobiome, and its relationship with bacterial microbiome in chronic obstructive pulmonary disease (COPD). Here we report the first simultaneous characterization of sputum bacterial and fungal microbiome in 84 stable COPD and 29 healthy subjects, using 16S ribosomal DNA and fungal internal transcribed spacer DNA sequencing. Ascomycota predominated over Basidiomycota in fungal microbiome both in COPD patients and healthy controls. Meyerozyma, Candida, Aspergillus and Schizophyllum were most abundant at the genus level. There was a significant inverse correlation between bacterial and fungal microbial diversity, both of which altered in opposite directions in COPD patients versus controls, and in frequent versus non-frequent exacerbators. An enhanced bacterial-fungal ecological interaction was observed in COPD patients, which was characterized by higher proportion of co-occurrence intrakingdom interactions and co-exclusive interkingdom interactions. In COPD, four mutually co-occurring fungal operational taxonomic units (OTUs) in Candida palmioleophila, Aspergillus and Sordariomycetes exhibited co-exclusive relationships with other fungal OTUs, which was specifically present in frequent exacerbators but not in non-frequent exacerbators. The perturbed bacterial-fungal interactions in COPD were associated with increased airway inflammatory mediators such as IL-6 and IL-8. The disruption of airway bacterial-fungal community balance, characterized by the loss of commensal bacterial taxa and enriched pathogenic fungal taxa, is implicated in COPD. The emergence of pathogenic fungi such as Candida and Aspergillus could be a marker for the frequent exacerbator phenotype. The airway mycobiome is an important cofactor mediating pathogenic infection and host inflammation in COPD.

TLR9 mediates the activation of NLRP3 inflammasome and oxidative stress in murine allergic airway inflammation

Toll-like receptor 9 (TLR9) has been reported to mediate airway inflammation, however, the underlying mechanism is poorly understood. In the present study, our objective was to reveal whether TLR9 regulates NLRP3 inflammasome and oxidative stress in murine allergic airway inflammation and Raw264.7 cells. Female wild type(WT)and TLR9−/−mice on C57BL/6 background were used to induce allergic airway inflammation by challenge of OVA, and Raw264.7 cells with or without TLR9 knockdown by small interfering RNA (siRNA) were stimulated by S.aureus. The results demonstrated that deletion of TLR9 effectively attenuated OVA-induced allergic airway inflammation including inflammatory cells infiltration and goblet cell hyperplasia. Meanwhile, OVA-induced protein expression of NLRP3, caspase-1(p20) and mature IL-1β, as well as secretion of IL-1β and IL-18 in wild type mice (WT) was obviously suppressed by TLR9 deficiency. Concomitantly, the expression of oxidative markers 8-OhDG and nitrotyrosine was increased in OVA-challenged WT mice, while TLR9 deficiency significantly inhibited such increase. Similarly, in the in vitro study, we found that knockdown of TLR9 markedly suppressed S.aureus-induced activation of NLRP3 inflammasome and oxidative stress in Raw264.7 cells. Collectively, our findings indicated that TLR9 may mediate allergic airway inflammation via activating NLRP3 inflammasome and oxidative stress.