A Critical Role for the CXCL3/CXCL5/CXCR2 Neutrophilic Chemotactic Axis in the Regulation of Type 2 Responses in a Model of Rhinoviral-Induced Asthma Exacerbation.

Rhinovirus (RV) infections in asthmatic patients are often associated with asthma exacerbation, characterized by worsened airways hyperreactivity and increased immune cell infiltration to the airways. The C-X-C chemokines, CXCL3 and CXCL5, regulate neutrophil trafficking to the lung via CXCR2, and their expression in the asthmatic lung is associated with steroid-insensitive type 2 inflammatory signatures. Currently, the role of CXCL3 and CXCL5 in regulating neutrophilic and type 2 responses in viral-induced asthma exacerbation is unknown. Inhibition of CXCL3 or CXCL5 with silencing RNAs in a mouse model of RV-induced exacerbation of asthma attenuated the accumulation of CXCR2+ neutrophils, eosinophils, and innate lymphoid cells in the lung and decreased production of type 2 regulatory factors IL-25, IL-33, IL-5, IL-13, CCL11, and CCL24. Suppression of inflammation was associated with decreased airways hyperreactivity, mucus hypersecretion, and collagen deposition. Similar results were obtained by employing RC-3095, which has been shown to bind to CXCR2, or by depletion of neutrophils. Our data demonstrate that CXCL3 and CXCL5 may be critical in the perpetuation of RV-induced exacerbation of asthma through the recruitment of CXCR2-positive neutrophils and by promoting type 2 inflammation. Targeting the CXCL3/CXCL5/CXCR2 axis may provide a new therapeutic approach to attenuating RV-induced exacerbations of asthma.

Single-cell transcriptomic analysis reveals key immune cell phenotypes in the lungs of patients with asthma exacerbation.

BACKGROUND: Asthma exacerbations are associated with heightened asthma symptoms, which can result in hospitalization in severe cases. However, the molecular immunologic processes that determine the course of an exacerbation remain poorly understood, impeding the progression of development of effective therapies. OBJECTIVE: Our aim was to identify candidate genes that are strongly associated with asthma exacerbation at a cellular level. METHODS: Subjects with asthma exacerbation and healthy control subjects were recruited, and bronchoalveolar lavage fluid was isolated from these subjects via bronchoscopy. Cells were isolated through fluorescence-activated cell sorting, and single-cell RNA sequencing was performed on enriched cell populations. RESULTS: We showed that the levels of monocytes, CD8+ T cells, and macrophages are significantly elevated in the bronchoalveolar lavage fluid of patients. A set of cytokines and intracellular transduction regulators are associated with asthma exacerbations and are shared across multiple cell clusters, forming a complicated molecular framework. An additional group of core exacerbation-associated modules is activated, including eukaryotic initiation factor 2 signaling, ephrin receptor signaling, and C-X-C chemokine receptor type 4 signaling in the subpopulations of CD8+ T cells (C1-a) and monocyte clusters (C7 clusters), which are associated with infection. CONCLUSION: Our study identified a significant number of severe asthma-associated genes that are differentially expressed by multiple cell clusters.

IL-17A is a common and critical driver of impaired lung function and immunopathology induced by influenza virus, rhinovirus and respiratory syncytial virus.

BACKGROUND AND OBJECTIVE: Influenza virus (FLU), rhinovirus (RV) and respiratory syncytial virus (RSV) are the most common acute respiratory infections worldwide. Infection can cause severe health outcomes, while therapeutic options are limited, primarily relieving symptoms without attenuating the development of lesions or impaired lung function. We therefore examined the inflammatory response to these infections with the intent to identify common components that are critical drivers of immunopathogenesis and thus represent potential therapeutic targets. METHODS: BALB/c mice were infected with FLU, RV or RSV, and lung function, airway inflammation and immunohistopathology were measured over a 10-day period. Anti-IL-17A mAb was administered to determine the impact of attenuating this cytokine's function on the development and severity of disease. RESULTS: All three viruses induced severe airway constriction and inflammation at 2 days post-infection (dpi). However, only FLU induced prolonged inflammation till 10 dpi. Increased IL-17A expression was correlated with the alterations in lung function and its persistence. Neutralization of IL-17A did not affect the viral replication but led to the resolution of airway hyperresponsiveness. Furthermore, anti-IL-17A treatment resulted in reduced infiltration of neutrophils (in RV- and FLU-infected mice at 2 dpi) and lymphocytes (in RSV-infected mice at 2 dpi and FLU-infected mice at 10 dpi), and attenuated the severity of immunopathology. CONCLUSION: IL-17A is a common pathogenic molecule regulating disease induced by three prevalent respiratory viruses. Targeting the IL-17A pathway may provide a unified approach to the treatment of these respiratory infections alleviating both inflammation-induced lesions and difficulties in breathing.

GSTO1-1 is an upstream suppressor of M2 macrophage skewing and HIF-1α-induced eosinophilic airway inflammation.

BACKGROUND: Glutathione S-transferases omega class 1 (GSTO1-1) is a unique member of the GST family regulating cellular redox metabolism and innate immunity through the promotion of LPS/TLR4/NLRP3 signalling in macrophages. House dust mite (HDM) triggers asthma by promoting type 2 responses and allergic inflammation via the TLR4 pathway. Although linked to asthma, the role of GSTO1-1 in facilitating type 2 responses and/or HDM-driven allergic inflammation is unknown. OBJECTIVE: To determine the role of GSTO1-1 in regulating HDM-induced allergic inflammation in a preclinical model of asthma. METHODS: Wild-type and GSTO1-1-deficient mice were sensitized and aeroallergen challenged with HDM to induce allergic inflammation and subsequently hallmark pathophysiological features characterized. RESULTS: By contrast to HDM-challenged WT mice, exposed GSTO1-1-deficient mice had increased numbers of eosinophils and macrophages and elevated levels of eotaxin-1 and -2 in their lungs. M1 macrophage-associated factors, such as IL-1ß and IL-6, were decreased in GSTO1-1-deficient mice. Conversely, M2 macrophage factors such as Arg-1 and Ym1 were up-regulated. HIF-1α expression was found to be higher in the absence of GSTO1-1 and correlated with the up-regulation of M2 macrophage markers. Furthermore, HIF-1α was shown to bind and activate the eotaxin-2 promotor. Hypoxic conditions induced significant increases in the levels of eotaxin-1 and -2 in GSTO1-deficient BMDMs, providing a potential link between inflammation-induced hypoxia and the regulation of M2 responses in the lung. Collectively, our results suggest that GSTO1-1 deficiency promotes M2-type responses and increased levels of nuclear HIF-1α, which regulates eotaxin (s)-induced eosinophilia and increased disease severity. CONCLUSION & CLINICAL IMPLICATION: We propose that GSTO1-1 is a novel negative regulator of TLR4-regulated M2 responses acting as an anti-inflammatory pathway. The discovery of a novel HIF-1α-induced eotaxin pathway identifies an unknown connection between hypoxia and the regulation of the severity of allergic inflammation in asthma.

TRAIL deficiency and PP2A activation with salmeterol ameliorates egg allergen-driven eosinophilic esophagitis.

Food antigens are common inflammatory triggers in pediatric eosinophilic esophagitis (EoE). TNF-related apoptosis-inducing ligand (TRAIL) promotes eosinophilic inflammation through the upregulation of the E3 ubiquitin ligase Midline (MID)-1 and subsequent downregulation of protein phosphatase 2A (PP2A), but the role of this pathway in EoE that is experimentally induced by repeated food antigen challenges has not been investigated. Esophageal mucosal biopsies were collected from children with EoE and controls and assessed for TRAIL and MID-1 protein and mRNA transcript levels. Wild-type and TRAIL-deficient (Tnfsf10-/-) mice were administered subcutaneous ovalbumin (OVA) followed by oral OVA challenges. In separate experiments, OVA-challenged mice were intraperitoneally administered salmeterol or dexamethasone. Esophageal biopsies from children with EoE revealed increased levels of TRAIL and MID-1 and reduced PP2A activation compared with controls. Tnfsf10-/- mice were largely protected from esophageal fibrosis, eosinophilic inflammation, and the upregulation of TSLP, IL-5, IL-13, and CCL11 when compared with wild-type mice. Salmeterol administration to wild-type mice with experimental EoE restored PP2A activity and also prevented esophageal eosinophilia, inflammatory cytokine expression, and remodeling, which was comparable to the treatment effect of dexamethasone. TRAIL and PP2A regulate inflammation and fibrosis in experimental EoE, which can be therapeutically modulated by salmeterol.

TNF-related apoptosis-inducing ligand (TRAIL) regulates midline-1, thymic stromal lymphopoietin, inflammation, and remodeling in experimental eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is an inflammatory disorder of the esophagus defined by eosinophil infiltration and tissue remodeling with resulting symptoms of esophageal dysfunction. TNF-related apoptosis-inducing ligand (TRAIL) promotes inflammation through upregulation of the E3 ubiquitin-ligase midline-1 (MID1), which binds to and deactivates the catalytic subunit of protein phosphatase 2Ac, resulting in increased nuclear factor κB activation. OBJECTIVE: We sought to elucidate the role of TRAIL in EoE. METHODS: We used Aspergillus fumigatus to induce EoE in TRAIL-sufficient (wild-type) and TRAIL-deficient (TRAIL(-/-)) mice and targeted MID1 in the esophagus with small interfering RNA. We also treated mice with recombinant thymic stromal lymphopoietin (TSLP) and TRAIL. RESULTS: TRAIL deficiency and MID1 silencing with small interfering RNA reduced esophageal eosinophil and mast cell numbers and protected against esophageal circumference enlargement, muscularis externa thickening, and collagen deposition. MID1 expression and nuclear factor κB activation were reduced in TRAIL(-/-) mice, whereas protein phosphatase 2Ac levels were increased compared with those seen in wild-type control mice. This was associated with reduced expression of CCL24, CCL11, CCL20, IL-5, IL-13, IL-25, TGFB, and TSLP. Treatment with TSLP reconstituted hallmark features of EoE in TRAIL(-/-) mice and recombinant TRAIL induced esophageal TSLP expression in vivo in the absence of allergen. Post hoc analysis of gene array data demonstrated significant upregulation of TRAIL and MID1 in a cohort of children with EoE compared with that seen in controls. CONCLUSION: TRAIL regulates MID1 and TSLP, inflammation, fibrosis, smooth muscle hypertrophy, and expression of inflammatory effector chemokines and cytokines in experimental EoE.

A unique role for IL-13 in inducing esophageal eosinophilia through MID-1 and STAT6.

Introduction: Eosinophilic esophagitis (EoE) is associated with allergen-driven inflammation of the esophagus and an upregulated Th2 cytokine signature. Recombinant interleukin (IL)-13 (rIL-13) administration to mice induces some of the hallmark features of EoE, including increased eotaxin expression and eosinophil recruitment. Inflammation in EoE has previously been shown to depend on the expression of TRAIL and MID-1, which reduced protein phosphatase 2A (PP2A) activity. The relationship between IL-13 and TRAIL signalling in esophageal eosinophilia is currently unknown. Objective: To investigate the interaction between IL-13-driven eosinophil infiltration and TRAIL or MID-1 in the esophagus. Method: We administered rIL-13 to wild type (WT), TRAIL-deficient (Tnsf10-/-) or STAT6-deficient (STAT6-/-) mice and targeted MID-1 with small interfering RNA. Results: rIL-13 administration to mice increased TRAIL and MID-1 expression in the esophagus while reducing PP2A activity. TRAIL deficient, but not STAT6 deficient mice demonstrated increased MID-1 expression and PP2A reduction upon IL-13 challenge which correlated with eosinophil infiltration into the esophagus. Silencing MID-1 expression with siRNA completely ablated IL-13 induced eosinophil infiltration of the esophagus, restored PP2A activity, and reduced eotaxin-1 expression. Conclusion: IL-13-driven eosinophil infiltration of the esophagus induced eosinophilia and eotaxin-1 expression in a STAT6-dependent and MID-1-dependent manner. This study highlights a novel mechanism employed by IL-13 to perpetuate eosinophil infiltration.

Inhibition of γ-glutamyl transferase suppresses airway hyperresponsiveness and airway inflammation in a mouse model of steroid resistant asthma exacerbation.

Introduction: Despite recent advances, there are limited treatments available for acute asthma exacerbations. Here, we investigated the therapeutic potential of GGsTop, a γ-glutamyl transferase inhibitor, on the disease with a murine model of asthma exacerbation. Methods: GGsTop was administered to mice that received lipopolysaccharide (LPS) and ovalbumin (OVA) challenges. Airway hyperresponsiveness (AHR), lung histology, mucus hypersecretion, and collagen deposition were analyzed to evaluate the hallmark features of asthma exacerbation. The level of proinflammatory cytokines and glutathione were determined with/without GGsTop. The transcription profiles were also examined. Results: GGsTop attenuates hallmark features of the disease with a murine model of LPS and OVA driven asthma exacerbation. Airway hyperresponsiveness (AHR), mucus hypersecretion, collagen deposition, and expression of inflammatory cytokines were dramatically inhibited by GGsTop treatment. Additionally, GGsTop restored the level of glutathione. Using RNA-sequencing and pathway analysis, we demonstrated that the activation of LPS/NFκB signaling pathway in airway was downregulated by GGsTop. Interestingly, further analysis revealed that GGsTop significantly inhibited not only IFNγ responses but also the expression of glucocorticoid-associated molecules, implicating that GGsTop profoundly attenuates inflammatory pathways. Conclusions: Our study suggests that GGsTop is a viable treatment for asthma exacerbation by broadly inhibiting the activation of multiple inflammatory pathways.

Single-cell RNA transcriptomic analysis identifies Creb5 and CD11b-DCs as regulator of asthma exacerbations.

Immune responses that result in asthma exacerbation are associated with allergen or viral exposure. Identification of common immune factors will be beneficial for the development of uniformed targeted therapy. We employed a House Dust Mite (HDM) mouse model of asthma and challenged allergic HDM mice with allergens (HDM, cockroach extract (CRE)) or respiratory syncytial virus (RSV). Purified lung immune cells underwent high-dimensional single-cell RNA deep sequencing (scRNA-seq) to generate an RNA transcriptome. Gene silencing with siRNA was employed to confirm the efficacy of scRNA-seq analysis. scRNA-seq UMAP analysis portrayed an array of cell markers within individual immune clusters. SCENIC R analysis showed an increase in regulon number and activity in CD11b- DC cells. Analysis of conserved regulon factors further identified Creb5 as a shared regulon between the exacerbation groups. Creb5 siRNAs attenuated HDM, CRE or RSV-induced asthma exacerbation. scRNA-seq multidimensional analysis of immune clusters identified gene pathways that were conserved between the exacerbation groups. We propose that these analyses provide a strong framework that could be used to identify specific therapeutic targets in multifaceted pathologies.

miR-122 promotes virus-induced lung disease by targeting SOCS1.

Virus-induced respiratory tract infections are a major health burden in childhood, and available treatments are supportive rather than disease modifying. Rhinoviruses (RVs), the cause of approximately 80% of common colds, are detected in nearly half of all infants with bronchiolitis and the majority of children with an asthma exacerbation. Bronchiolitis in early life is a strong risk factor for the development of asthma. Here, we found that RV infection induced the expression of miRNA 122 (miR-122) in mouse lungs and in human airway epithelial cells. In vivo inhibition specifically in the lung reduced neutrophilic inflammation and CXCL2 expression, boosted innate IFN responses, and ameliorated airway hyperreactivity in the absence and in the presence of allergic lung inflammation. Inhibition of miR-122 in the lung increased the levels of suppressor of cytokine signaling 1 (SOCS1), which is an in vitro-validated target of miR-122. Importantly, gene silencing of SOCS1 in vivo completely reversed the protective effects of miR-122 inhibition on RV-induced lung disease. Higher miR-122 expression in nasopharyngeal aspirates was associated with a longer time on oxygen therapy and a higher rate of treatment failure in 87 infants hospitalized with moderately severe bronchiolitis. These results suggest that miR-122 promotes RV-induced lung disease via suppression of its target SOCS1 in vivo. Higher miR-122 expression was associated with worse clinical outcomes, highlighting the potential use of anti-miR-122 oligonucleotides, successfully trialed for treatment of hepatitis C, as potential therapeutics for RV-induced bronchiolitis and asthma exacerbations.

In vivo targeting of miR-223 in experimental eosinophilic oesophagitis.

OBJECTIVES: Eosinophilic oesophagitis (EoE) is characterised by oesophageal inflammation, fibrosis and dysfunction. Micro (mi)-RNAs interfere with pro-inflammatory and pro-fibrotic transcriptional programs, and miR-223 was upregulated in oesophageal mucosal biopsy specimens from EoE patients. The therapeutic potential of modulating miR-223 expression in vivo has not been determined. We aimed to elucidate the relevance of oesophageal miR-223 expression in an in vivo model of EoE by inhibiting miR-223 tissue expression. METHODS: The expression of miR-223 and the validated miR-223 target insulin-like growth factor receptor 1 (IGF1R) protein was determined in our paediatric cohort of EoE patients. A murine model of Aspergillus fumigatus-induced EoE was employed, and oesophagi were assessed for miR-233, IGF1R, T lymphocyte type 2 (T2) cytokine expression and eosinophil infiltration. Mice were treated with antagomirs targeting miR-223 or resveratrol targeting its upstream regulator Midline-1(MID-1). RESULTS: There was an inverse relationship between an increased expression of miR-223 and a decreased IGF1R protein concentration in biopsy specimens from EoE patients. TNF-related apoptosis-inducing ligand deficiency, MID-1 inhibition and resveratrol treatment suppressed miR-223 expression. Furthermore, inhibition of miR-223 and treatment with resveratrol in the oesophagus resulted in an amelioration of EoE hallmark features including eosinophilic infiltration, oesophageal circumference and a reduction in T2 cytokine expression. CONCLUSION: miR-223 has a key role in the perpetuation of EoE hallmark features downstream of TNF-related apoptosis-inducing ligand and MID-1 in an experimental model. These studies highlight a potentially critical role of miRNA function in EoE aetiology. miR-223 expression in the oesophagus may be therapeutically modulated by resveratrol, providing a potential new therapeutic option to be explored in EoE patients for this increasingly prevalent condition.

A Selective α7 Nicotinic Acetylcholine Receptor Agonist, PNU-282987, Attenuates ILC2s Activation and Alternaria-Induced Airway Inflammation.

Background: The anti-inflammatory effect of an α7nAChR agonist, PNU-282987, has previously been explored in the context of inflammatory disease. However, the effects of PNU-282987 on type 2 innate lymphoid cells (ILC2s)-mediated allergic airway inflammation has not yet been established. Aims: To determine the effects of PNU-282987 on the function of ILC2s in the context of IL-33- or Alternaria Alternata (AA)- induced airway inflammation. Methods: PNU-282987 was administered to mice that received recombinant IL-33 or AA intranasal challenges. Lung histological analysis and flow cytometry were performed to determine airway inflammation and the infiltration and activation of ILC2s. The previously published α7nAChR agonist GTS-21 was employed as a comparable reagent. ILC2s were isolated from murine lung tissue and cultured in vitro in the presence of IL-33, IL-2, and IL-7 with/without either PNU-282987 or GTS-21. The expression of the transcription factors GATA3, IKK, and NF-κB were also determined. Results: PNU-282987 and GTS-21 significantly reduced goblet cell hyperplasia in the airway, eosinophil infiltration, and ILC2s numbers in BALF, following IL-33 or AA challenge. In vitro IL-33 stimulation of isolated lung ILC2s showed a reduction of GATA3 and Ki67 in response to PNU-282987 or GTS-21 treatments. There was a significant reduction in IKK and NF-κB phosphorylation in the PNU-282987-treated group when compared to the GTS-21-treated ILC2s. Conclusion: PNU-282987 inhibits ILC2-associated airway inflammation, where its effects were comparable to that of GTS-21.