Activation of the PGE2-EP2 pathway as a potential drug target for treating eosinophilic rhinosinusitis.

Current treatments of eosinophilic chronic rhinosinusitis (ECRS) involve corticosteroids with various adverse effects and costly therapies such as dupilumab, highlighting the need for improved treatments. However, because of the lack of a proper mouse ECRS model that recapitulates human ECRS, molecular mechanisms underlying this disease are incompletely understood. ECRS is often associated with aspirin-induced asthma, suggesting that dysregulation of lipid mediators in the nasal mucosa may underlie ECRS pathology. We herein found that the expression of microsomal PGE synthase-1 (encoded by PTGES) was significantly lower in the nasal mucosa of ECRS patients than that of non-ECRS subjects. Histological, transcriptional, and lipidomics analyses of Ptges-deficient mice revealed that defective PGE2 biosynthesis facilitated eosinophil recruitment into the nasal mucosa, elevated expression of type-2 cytokines and chemokines, and increased pro-allergic and decreased anti-allergic lipid mediators following challenges with Aspergillus protease and ovalbumin. A nasal spray containing agonists for the PGE2 receptor EP2 or EP4, including omidenepag isopropyl that has been clinically used for treatment of glaucoma, markedly reduced intranasal eosinophil infiltration in Ptges-deficient mice. These results suggest that the present model using Ptges-deficient mice is more relevant to human ECRS than are previously reported models and that eosinophilic inflammation in the nasal mucosa can be efficiently blocked by activation of the PGE2-EP2 pathway. Furthermore, our findings suggest that drug repositioning of omidenepag isopropyl may be useful for treatment of patients with ECRS.

NRF2 is a spatiotemporal metabolic hub essential for the polyfunctionality of Th2 cells.

Upon encountering allergens, CD4+ T cells differentiate into IL-4-producing Th2 cells in lymph nodes, which later transform into polyfunctional Th2 cells producing IL-5 and IL-13 in inflamed tissues. However, the precise mechanism underlying their polyfunctionality remains elusive. In this study, we elucidate the pivotal role of NRF2 in polyfunctional Th2 cells in murine models of allergic asthma and in human Th2 cells. We found that an increase in reactive oxygen species (ROS) in immune cells infiltrating the lungs is necessary for the development of eosinophilic asthma and polyfunctional Th2 cells in vivo. Deletion of the ROS sensor NRF2 specifically in T cells, but not in dendritic cells, significantly abolished eosinophilia and polyfunctional Th2 cells in the airway. Mechanistically, NRF2 intrinsic to T cells is essential for inducing optimal oxidative phosphorylation and glycolysis capacity, thereby driving Th2 cell polyfunctionality independently of IL-33, partially by inducing PPARγ. Treatment with an NRF2 inhibitor leads to a substantial decrease in polyfunctional Th2 cells and subsequent eosinophilia in mice and a reduction in the production of Th2 cytokines from peripheral blood mononuclear cells in asthmatic patients. These findings highlight the critical role of Nrf2 as a spatial and temporal metabolic hub that is essential for polyfunctional Th2 cells, suggesting potential therapeutic implications for allergic diseases.

Butyrate inhibits type 2 inflammation in eosinophilic chronic rhinosinusitis.

Butyrate and other Short-chain fatty acids (SCFAs) are microbial metabolites from Bacteroides and Clostridium species that may suppress type 2 inflammation. However, the mechanisms of SCFAs in the nasal sinuses are not fully understood. We aimed to clarify the in vitro and in vivo roles of SCFAs in eosinophilic chronic rhinosinusitis (ECRS) pathophysiology. We investigated whether SCFAs induced changes in type 2 cytokines, IgE, and apoptosis and the roles of GPR41, GPR43, and histone deacetylase. Analysis of the control subjects demonstrated that butyrate of SCFAs effectively inhibited type 2 cytokine production in PBMCs, ILC2s, and CD4+ T cells and IgE production in CD19+ B cells. In annexin V analysis, butyrate also induced late apoptosis of PBMCs. The butyrate-induced inhibition of type 2 cytokines appeared involved in histone deacetylase inhibition but not in GPR41 or GPR43. In an analysis of ECRS in humans, butyrate inhibited type 2 cytokine production in PBMCs and nasal polyp-derived cells. The butyrate concentration in nasal lavage fluid was significantly decreased in ECRS patients compared to controls and non-ECRS patients. Our findings confirm that butyrate can inhibit type 2 inflammation and may be a potential therapeutic target for ECRS.

Increased SERPINB2 potentiates 15LO1 expression via STAT6 signalling in epithelial cells in eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: SERPINB2, a biomarker of Type-2 (T2) inflammatory processes, has been described in the context of asthma. Chronic rhinosinusitis with nasal polyps (CRSwNP) is also correlated with T2 inflammation and elevated 15LO1 induced by IL-4/13 in nasal epithelial cells. The aim of this study was to evaluate the expression and location of SERPINB2 in nasal epithelial cells (NECs) and determine whether SERPINB2 regulates 15LO1 and downstream T2 markers in NECs via STAT6 signalling. METHODS: SERPINB2 gene expression in bulk and single-cell RNAseq database was analysed by bioinformatics analysis. SERPINB2, 15LO1 and other T2 markers were evaluated from CRSwNP and HCs NECs. The colocalization of SERPINB2 and 15LO1 was evaluated by immunofluorescence. Fresh NECs were cultured at an air-liquid interface with or without IL-13, SERPINB2 Dicer-substrate short interfering RNAs (DsiRNAs) transfection, exogenous SERPINB2, 15-HETE recombinant protein and pSTAT6 inhibitors. 15LO1, 15-HETE and downstream T2 markers were analysed by qRT-PCR, western blot and ELISA. RESULTS: SERPINB2 expression was increased in eosinophilic nasal polyps compared with that in noneosinophilic nasal polyps and control tissues and positively correlated with 15LO1 and other downstream T2 markers. SERPINB2 was predominantly expressed by epithelial cells in NP tissue and was colocalized with 15LO1. In primary NECs in vitro, SERPINB2 expression was induced by IL-13. Knockdown or overexpression SERPINB2 decreased or enhanced expression of 15LO1 and 15-HETE in NECs, respectively, in a STAT6-dependent manner. SERPINB2 siRNA also inhibited the expression of the 15LO1 downstream genes, such as CCL26, POSTN and NOS2. STAT6 inhibition similarly decreased SERPINB2-induced 15LO1. CONCLUSIONS: SERPINB2 is increased in NP epithelial cells of eosinophilic CRSwNP (eCRSwNP) and contributes to T2 inflammation via STAT6 signalling. SERPINB2 could be considered a novel therapeutic target for eCRSwNP.

Eosinophils in Oral Disease: A Narrative Review.

The prevalence of diseases characterised by eosinophilia is on the rise, emphasising the importance of understanding the role of eosinophils in these conditions. Eosinophils are a subset of granulocytes that contribute to the body's defence against bacterial, viral, and parasitic infections, but they are also implicated in haemostatic processes, including immunoregulation and allergic reactions. They contain cytoplasmic granules which can be selectively mobilised and secrete specific proteins, including chemokines, cytokines, enzymes, extracellular matrix, and growth factors. There are multiple biological and emerging functions of these specialised immune cells, including cancer surveillance, tissue remodelling and development. Several oral diseases, including oral cancer, are associated with either tissue or blood eosinophilia; however, their exact mechanism of action in the pathogenesis of these diseases remains unclear. This review presents a comprehensive synopsis of the most recent literature for both clinicians and scientists in relation to eosinophils and oral diseases and reveals a significant knowledge gap in this area of research.

Functional 2D Nanoplatforms Alleviate Eosinophilic Chronic Rhinosinusitis by Modulating Eosinophil Extracellular Trap Formation.

The therapeutic outcomes of patients with eosinophilic chronic rhinosinusitis (ECRS) remain unsatisfactory, largely because the underlying mechanisms of eosinophilic inflammation are uncertain. Here, it is shown that the nasal secretions of ECRS patients have high eosinophil extracellular trap (EET) and cell-free DNA (cfDNA) levels. Moreover, the cfDNA induced EET formation by activating toll-like receptor 9 (TLR9) signaling. After demonstrating that DNase I reduced eosinophilic inflammation by modulating EET formation, linear polyglycerol-amine (LPGA)-coated TiS2 nanosheets (TLPGA) as functional 2D nanoplatforms with low cytotoxicity, mild protein adsorption, and increased degradation rate is developed. Due to the more flexible linear architecture, TLPGA exhibited higher cfDNA affinity than the TiS2 nanosheets coated with dendritic polyglycerol-amine (TDPGA). TLPGA reduced cfDNA levels in the nasal secretions of ECRS patients while suppressing cfDNA-induced TLR9 activation and EET formation in vitro. TLPGA displayed exceptional biocompatibility, preferential nasal localization, and potent inflammation modulation in mice with eosinophilic inflammation. These results highlight the pivotal feature of the linear molecular architecture and 2D sheet-like nanostructure in the development of anti-inflammation nanoplatforms, which can be exploited for ECRS treatment.

Retinoid orphan receptor gamma t (rorγt) promotes inflammatory eosinophilia but is dispensable for innate immune-mediated colitis.

Inflammatory bowel diseases (IBD) result from uncontrolled inflammation in the intestinal mucosa leading to damage and loss of function. Both innate and adaptive immunity contribute to the inflammation of IBD and innate and adaptive immune cells reciprocally activate each other in a forward feedback loop. In order to better understand innate immune contributions to IBD, we developed a model of spontaneous 100% penetrant, early onset colitis that occurs in the absence of adaptive immunity by crossing villin-TNFAIP3 mice to RAG1-/- mice (TRAG mice). This model is driven by microbes and features increased levels of innate lymphoid cells in the intestinal mucosa. To investigate the role of type 3 innate lymphoid cells (ILC3) in the innate colitis of TRAG mice, we crossed them to retinoid orphan receptor gamma t deficient (Rorγt-/-) mice. Rorγt-/- x TRAG mice exhibited markedly reduced eosinophilia in the colonic mucosa, but colitis persisted in these mice. Colitis in Rorγt-/- x TRAG mice was characterized by increased infiltration of the intestinal mucosa by neutrophils, inflammatory monocytes, macrophages and other innate cells. RNA and cellular profiles of Rorγt-/- x TRAG mice were consistent with a lack of ILC3 and ILC3 derived cytokines, reduced antimicrobial factors, increased activation oof epithelial repair processes and reduced activation of epithelial cell STAT3. The colitis in Rorγt-/- x TRAG mice was ameliorated by antibiotic treatment indicating that microbes contribute to the ILC3-independent colitis of these mice. Together, these gene expression and cell signaling signatures reflect the double-edged sword of ILC3 in the intestine, inducing both proinflammatory and antimicrobial protective responses. Thus, Rorγt promotes eosinophilia but Rorγt and Rorγt-dependent ILC3 are dispensable for the innate colitis in TRAG mice.

Myeloid Bmal1 deletion suppresses the house dust mite-induced chronic lung allergy.

Asthma is the chronic pulmonary inflammatory response that could lead to respiratory failure when allergic reactions exacerbate. It is featured by type 2 immunity with eosinophilic inflammation, mucus, and IgE production, and Th2 cytokine secretion upon repeated challenge of allergens. The symptom severity of asthma displays an apparent circadian rhythm with aggravated airway resistance in the early morning in patients. Bmal1 is the core regulator of the circadian clock, while the regulatory role of Bmal1 in asthma remains unclear. Here, we investigate whether the myeloid Bmal1 is involved in the pathogenesis of house dust mite (HDM)-induced lung allergy. We found that knockdown of Bmal1 in macrophages suppressed the time-of-day variance of the eosinophil infiltration in the alveolar spaces in chronic asthmatic mice. This was accompanied by decreased bronchial mucus production, collagen deposition, and HDM-specific IgE production. However, the suppression effects of myeloid Bmal1 deletion did not alter the allergic responses in short-term exposure to HDM. The transcriptome profile of alveolar macrophages (AMs) showed that Bmal1-deficient AMs have enhanced phagocytosis and reduced production of allergy-mediating prostanoids thromboxane A2 and prostaglandin F2α synthesis. The attenuated thromboxane A2 and prostaglandin F2α may lead to less induction of the eosinophil chemokine Ccl11 expression in bronchial epithelial cells. In summary, our study demonstrates that Bmal1 ablation in macrophages attenuates eosinophilic inflammation in HDM-induced chronic lung allergy, which involves enhanced phagocytosis and reduced prostanoid secretion.

Airway hyperresponsiveness correlates with airway TSLP in asthma independent of eosinophilic inflammation.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is released from the airway epithelium in response to various environmental triggers, inducing a type-2 inflammatory response, and is associated with airway inflammation, airway hyperresponsiveness (AHR), and exacerbations. TSLP may also induce AHR via a direct effect on airway smooth muscle and mast cells, independently of type-2 inflammation, although association between airway TSLP and AHR across asthma phenotypes has been described sparsely. OBJECTIVES: This study sought to investigate the association between AHR and levels of TSLP in serum, sputum, and bronchoalveolar lavage in patients with asthma with and without type-2 inflammation. METHODS: A novel ultrasensitive assay was used to measure levels of TSLP in patients with asthma (serum, n = 182; sputum, n = 81; bronchoalveolar lavage, n = 85) and healthy controls (serum, n = 47). The distribution and association among airway and systemic TSLP, measures of AHR, type-2 inflammation, and severity of disease were assessed. RESULTS: TSLP in sputum was associated with AHR independently of levels of eosinophils and fractional exhaled nitric oxide (ρ = 0.49, P = .005). Serum TSLP was higher in both eosinophil-high and eosinophil-low asthma compared to healthy controls: geometric mean: 1600 fg/mL (95% CI: 1468-1744 fg/mL) and 1294 fg/mL (95% CI: 1167-1435 fg/mL) versus 846 fg/mL (95% CI: 661-1082 fg/mL), but did not correlate with the level of AHR. Increasing age, male sex, and eosinophils in blood were associated with higher levels of TSLP in serum, whereas lung function, inhaled corticosteroid dose, and symptom score were not. CONCLUSIONS: The association between TSLP in sputum and AHR to mannitol irrespective of markers of type-2 inflammation further supports a role of TSLP in AHR that is partially independent of eosinophilic inflammation.

Dupilumab-induced eosinophilia in patients with diffuse type 2 chronic rhinosinusitis.

BACKGROUND: Dupilumab, a monoclonal anti-IL-4Rα antibody, is approved for several type 2 mediated inflammatory diseases like asthma, atopic dermatitis, and diffuse type 2 chronic rhinosinusitis (CRS). Clinical studies had reported a transient increase in blood eosinophils during dupilumab therapy. This study aimed to assess the impact of elevated blood eosinophils on clinical outcome and to investigate the cause of high blood eosinophil levels under dupilumab therapy. METHODS: Patients suffering from diffuse type 2 CRS treated with dupilumab were examined on days 0, 28, 90, and 180 after therapy start. Sino-Nasal-Outcome-Test Score (SNOT-22), Total Nasal Polyp Score (TNPS), and blood samples were collected. Cytokine measurements and proteomics analysis were conducted. Flow cytometry analysis measured receptor expression on eosinophils. RESULTS: Sixty-eighty patients were included. Baseline eosinophilia ≥0.3G/L was observed in 63.2% of patients, and in 30.9% of patients, eosinophils increased by ≥0.5G/L under dupilumab. Subjects with eosinophilia ≥0.3G/L at baseline had the best SNOT-22 mean change compared to no eosinophilia. Eosinophil elevation during dupilumab therapy had no impact on clinical scores. The eosinophil adhesion molecule VCAM-1 decreased significantly during therapy in all patients. The chemokine receptor CXCR4 was significantly down- and IL-4 upregulated in subjects with eosinophil increase. CONCLUSION: Our findings suggest that increased eosinophils in type 2 CRS are associated with a good clinical response to dupilumab. Patients with elevated IL-4 at baseline developed dupilumab-induced transient eosinophilia. We identified the downregulation of VCAM-1 and surface markers CD49d and CXCR4 on eosinophils as possible explanations of dupilumab-induced eosinophilia.

Blood and Sputum Eosinophils of COPD Patients Are Differently Polarized than in Asthma.

Different eosinophil subpopulations have been identified in asthma and other eosinophilic disorders. However, there is a paucity of data on eosinophil subpopulations in patients with chronic obstructive pulmonary disease (COPD). The aim of this study was to compare eosinophil phenotypes in blood and induced sputum in patients with COPD, asthma and controls. Stable patients with mild-to-moderate COPD (n = 15) and asthma (n = 14) with documented blood eosinophilia ≥100 cells/µL in the year prior to the study and the control group (n = 11) were included to the study. The blood and sputum eosinophil phenotypes were analyzed by flow cytometry. IL-5, IL-13, CCL5 and eotaxin-3 levels were measured in the induced sputum. The marker expression on blood eosinophils was similar among control, asthma and COPD groups. The expressions of CD125, CD193, CD14 and CD62L were higher on blood than on sputum eosinophils in all three groups. We found increased levels of CD193+ and CD66b+ sputum eosinophils from COPD patients, and an elevated level of CD11b+ sputum eosinophils in asthma compared to COPD patients. The results of our study suggest that the profile of marker expression on COPD sputum eosinophils differed from other groups, suggesting a distinct phenotype of eosinophils of COPD patients than in asthma or healthy subjects.

Involvement of CD40-CD40L and ICOS-ICOSL in the development of chronic rhinosinusitis by targeting eosinophils.

Background: Chronic rhinosinusitis (CRS), whose prevalence and pathogenesis are age-related, is characterized by nasal tissue eosinophil infiltration. CD40-CD40 ligand (CD40L) pathway involves in the eosinophil-mediated inflammation, and inducible co-stimulator (ICOS)-ICOS ligand (ICOSL) signal can strengthen CD40-CD40L interaction. Whether CD40-CD40L and ICOS-ICOSL have a role in the development of CRS remains unknown. Objectives: The aim of this study is to investigate the association of CD40-CD40L and ICOS-ICOSL expression with CRS and underlying mechanisms. Methods: Immunohistology detected the expression of CD40, CD40L, ICOS, and ICOSL. Immunofluorescence was performed to evaluate the co-localizations of CD40 or ICOSL with eosinophils. Correlations between CD40-CD40L and ICOS-ICOSL as well as clinical parameters were analyzed. Flow cytometry was used to explore the activation of eosinophils by CD69 expression and the CD40 and ICOSL expression on eosinophils. Results: Compared with the non-eCRS subset, ECRS (eosinophilic CRS) subset showed significantly increased CD40, ICOS, and ICOSL expression. The CD40, CD40L, ICOS, and ICOSL expressions were all positively correlated with eosinophil infiltration in nasal tissues. CD40 and ICOSL were mainly expressed on eosinophils. ICOS expression was significantly correlated with the expression of CD40-CD40L, whereas ICOSL expression was correlated with CD40 expression. ICOS-ICOSL expression positively correlated with blood eosinophils count and disease severity. rhCD40L and rhICOS significantly enhanced the activation of eosinophils from patients with ECRS. Tumor necrosis factor-α (TNF-α) and interleukin-5 (IL-5) obviously upregulated CD40 expression on eosinophils, which was significantly inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor. Conclusions: Increased CD40-CD40L and ICOS-ICOSL expressions in nasal tissues are linked to eosinophils infiltration and disease severity of CRS. CD40-CD40L and ICOS-ICOSL signals enhance eosinophils activation of ECRS. TNF-α and IL-5 regulate eosinophils function by increasing CD40 expression partly via p38 MAPK activation in patients with CRS.

Nafamostat has anti-asthmatic effects associated with suppressed pro-inflammatory gene expression, eosinophil infiltration and airway hyperreactivity.

Introduction: Asthma is characterized by an imbalance between proteases and their inhibitors. Hence, an attractive therapeutic option could be to interfere with asthma-associated proteases. Here we exploited this option by assessing the impact of nafamostat, a serine protease inhibitor known to neutralize mast cell tryptase. Methods: Nafamostat was administered in a mouse model for asthma based on sensitization by house dust mite (HDM) extract, followed by the assessment of effects on airway hyperreactivity, inflammatory parameters and gene expression. Results: We show that nafamostat efficiently suppressed the airway hyperreactivity in HDM-sensitized mice. This was accompanied by reduced infiltration of eosinophils and lymphocytes to the airways, and by lower levels of pro-inflammatory compounds within the airway lumen. Further, nafamostat had a dampening impact on goblet cell hyperplasia and smooth muscle layer thickening in the lungs of HDM-sensitized animals. To obtain deeper insight into the underlying mechanisms, a transcriptomic analysis was conducted. This revealed, as expected, that the HDM sensitization caused an upregulated expression of numerous pro-inflammatory genes. Further, the transcriptomic analysis showed that nafamostat suppressed the levels of multiple pro-inflammatory genes, with a particular impact on genes related to asthma. Discussion: Taken together, this study provides extensive insight into the ameliorating effect of nafamostat on experimental asthma, and our findings can thereby provide a basis for the further evaluation of nafamostat as a potential therapeutic agent in human asthma.

CD109 on Dendritic Cells Regulates Airway Hyperreactivity and Eosinophilic Airway Inflammation.

Asthma is a chronic airway inflammatory disease characterized by airway hyperreactivity (AHR) and eosinophilic airway inflammation. Dendritic cells (DCs) are essential for the development of asthma via presenting allergens, causing T-helper cell type 2 (Th2) skewing and eosinophil inflammation. Recent studies have revealed that CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is involved in the pathogenesis of inflammatory diseases such as rheumatoid arthritis and psoriasis. However, no study has addressed the role of CD109 in asthma. This study sought to address the role of CD109 on DCs in the development of AHR and allergic inflammation. CD109-deficient mice (CD109-/-) were sensitized with house dust mite or ovalbumin and compared with wild-type mice for induction of AHR and allergic inflammation. CD109-deficient mice had reduced AHR and eosinophilic inflammation together with lower Th2 cytokine expression compared with wild-type mice. Interestingly, CD109 expression was induced in lung conventional DC2s (cDC2s), but not lung cDC1s, upon allergic challenge. Lung cDC2s from CD109-/- mice had a poor ability to induce cytokine production in ex vivo DC-T cell cocultures with high expression of RUNX3 (runt-related transcription factor 3), resulting in suppression of Th2 differentiation. Adoptive transfer of bone marrow-derived CD109-/- DCs loaded with house dust mite failed to develop AHR and eosinophilic inflammation. Finally, administration of monoclonal anti-CD109 antibody reduced airway eosinophils and significantly decreased AHR. Our results suggest the involvement of CD109 in asthma pathogenesis. CD109 is a novel therapeutic target for asthma.

Exploring proteomic plasma biomarkers in eosinophilic and neutrophilic asthma.

BACKGROUND: Few biomarkers identify eosinophilic and neutrophilic asthma beyond cell concentrations in blood or sputum. Finding novel biomarkers for asthma endotypes could give insight about disease mechanisms and guide tailored treatment. Our aim was to investigate clinical characteristics and inflammation-related plasma proteins in relation to blood eosinophil and neutrophil concentrations in subjects with and without asthma. METHODS: We included 24-26-year-old subjects (n = 2063) from the Swedish population-based cohort BAMSE. Subjects with asthma (n = 239) and without asthma (n = 1824) were subdivided based on blood eosinophil and neutrophil concentrations (cut-offs 0.3 × 109 /L and 5.0 × 109 /L, respectively). We measured the levels of 92 plasma proteins using Olink Proseek Multiplex Inflammation Panel Assay. Group statistics tests were used to analyse the data, as well as adjusted multiple logistic regression models. RESULTS: Among subjects with asthma, 21.8% had eosinophilic asthma and 20.5% neutrophilic asthma. Eosinophilic asthma, but not neutrophilic asthma, was associated with a distinct clinical phenotype with, for example, higher proportions of eczema and sensitization. Most plasma proteins that associated with high eosinophil and/or neutrophil blood concentrations in subjects with asthma showed similar associations in subjects without asthma. However, out of these proteins, MMP10 levels were associated with eosinophilic asthma and were significantly higher as compared to controls with high eosinophilic concentration, while CCL4 levels associated with high neutrophil concentration only in subjects with asthma. CONCLUSIONS: Eosinophilic asthma was associated with a clear clinical phenotype. With our definitions, we identified MMP10 as a possible plasma biomarker for eosinophilic asthma and CCL4 was linked to neutrophilic asthma. These proteins should be evaluated further in clinical settings and using sputum granulocytes to define the asthma endotypes.

Elevated CXCL14 in Induced Sputum Was Associated with Eosinophilic Inflammation and Airway Obstruction in Patients with Asthma.

INTRODUCTION: CXCL14 involved in inflammatory processes was upregulated in the asthma expression profile datasets in our pilot study. However, the expression of CXCL14 in induced sputum and its potential clinical role in asthma were poorly reported. OBJECTIVE: We sought to detect CXCL14 expression in airway epithelium and induced sputum cells of asthma and explore its potential clinical implications. METHODS: The expression of CXCL14 in asthma was analyzed using R software based on multiple microarray datasets, including GSE43696, GSE63142, GSE67940, and GSE76262. Subsequent verification of the CXCL14 expression pattern in induced sputum and bronchial epithelium cells was performed by qRT-PCR and ELISA. Besides, the correlations between CXCL14 and eosinophilic inflammation indicators (FeNO, EOS#, and IgE), Th2 signature genes (SERPINB2, POSTN, and CLCA1), inflammatory cytokines (IL-4, IL-5, IL-10, IL-13, IL-25, IL-33, TSLP, IL-8, IL-17A, IFN-γ, and IL-2), and airway obstruction indicators (pulmonary function and mucin secretion) were further explored. RESULTS: The expression of CXCL14 in epithelium and sputum cells was upregulated in asthma and positively correlated with clinical eosinophilic indicators. The protein levels of CXCL14 were positively associated with Th2 signature genes (SERPINB2, POSTN, and CLCA1) and Th2 cytokines (IL-4, IL-5, IL-10, IL-13, IL-25, IL-33, and TSLP). Increased expression of CXCL14 was also observed in BEAS-2B cells stimulated by the cytokine IL-4. Furthermore, the expression of CXCL14 was positively correlated with MUC5AC secretion and negatively associated with pulmonary function. CONCLUSIONS: Upregulated CXCL14 in asthma was positively correlated with inflammatory indicators and negatively correlated with pulmonary function, which indicated that upregulated CXCL14 might act as a pathogenic gene through involvement in Th2 inflammation in asthma.

Sweet taste receptor agonists attenuate macrophage IL-1ß expression and eosinophilic inflammation linked to autophagy deficiency in myeloid cells.

BACKGROUND: Eosinophilic inflammation is a hallmark of refractory chronic rhinosinusitis (CRS) and considered a major therapeutic target. Autophagy deficiency in myeloid cells plays a causal role in eosinophilic CRS (ECRS) via macrophage IL-1ß overproduction, thereby suggesting autophagy regulation as a potential therapeutic modality. Trehalose is a disaccharide sugar with known pro-autophagy activity and effective in alleviating diverse inflammatory diseases. We sought to investigate the therapeutic potential of autophagy-enhancing agent, trehalose, or related sugar compounds, and the underlying mechanism focusing on macrophage IL-1ß production in ECRS pathogenesis. METHODS: We investigated the therapeutic effects of trehalose and saccharin on macrophage IL-1ß production and eosinophilia in the mouse model of ECRS with myeloid cell-specific autophagy-related gene 7 (Atg7) deletion. The mechanisms underlying their anti-inflammatory effects were assessed using specific inhibitor, genetic knockdown or knockout, and overexpression of cognate receptors. RESULTS: Unexpectedly, trehalose significantly attenuated eosinophilia and disease pathogenesis in ECRS mice caused by autophagy deficiency in myeloid cells. This autophagy-independent effect was associated with reduced macrophage IL-1ß expression. Various sugars recapitulated the anti-inflammatory effect of trehalose, and saccharin was particularly effective amongst other sugars. The mechanistic study revealed an involvement of sweet taste receptor (STR), especially T1R3, in alleviating macrophage IL-1ß production and eosinophilia in CRS, which was supported by genetic depletion of T1R3 or overexpression of T1R2/T1R3 in macrophages and treatment with the T1R3 antagonist gurmarin. CONCLUSION: Our results revealed a previously unappreciated anti-inflammatory effect of STR agonists, particularly trehalose and saccharin, and may provide an alternative strategy to autophagy modulation in the ECRS treatment.

Rheology predicts sputum eosinophilia in patients with muco-obstructive lung diseases.

BACKGROUND: Mucus is known to play a pathogenic role in muco-obstructive lung diseases, but little is known about the determinants of mucus rheology. The purpose of this study is to determine which sputum components influence sputum rheology in patients with muco-obstructive lung diseases. METHODS: We performed a cross sectional prospective cohort study. Spontaneous sputum was collected from consecutive patients with muco-obstructive lung diseases. Sputum rheology was assessed using the Rheomuco® rheometer (Rheonova, Grenoble); the elastic modulus G', viscous modulus G″, and the critical stress threshold σc were recorded. Key quantitative and qualitative biological sputum components were determined by cytology, nucleic acid amplification tests and mass spectrometry. RESULTS: 48 patients were included from January to August 2019. Among them, 10 had asthma, 14 COPD and 24 non-CF bronchiectasis (NCFB). The critical stress threshold σc predicted a sputum eosinophilia superior to 1.25% with 89.19% accuracy (AUC = 0.8762). G' and G″ are positively correlated with MUC5AC protein concentration ((rho = 0.361; P = .013) and (rho = 0.335; P = .021), respectively). σc was positively correlated with sputum eosinophilia (rho = 0.394; P = .012), MUC5B (rho = 0.552; P < .001) and total protein (rho = 0.490; P < .001) concentrations. G' and G″ were significantly higher in asthma patients (G' = 14.49[7.18-25.26]Pa, G'' = 3.0[2.16-5.38]Pa) compared to COPD (G' = 5.01[2.94-6.48]Pa, P = .010; G'' = 1.45[1.16-1.94]Pa, P = .006) and to NCFB (G' = 4.99[1.49-10.49]Pa, P = .003; G'' = 1.46[0.71-2.47]Pa, P = .002). CONCLUSION: In muco-obstructive lung diseases, rheology predicts sputum eosinophilia and is correlated with mucin concentrations, regardless of the underlying disease. CLINICAL TRIAL REGISTRATION: (registrar, website, and registration number), where applicable NCT04081740.

Phosphatidylinositol 3-Kinase (PI3K) Orchestrates Aspergillus fumigatus-Induced Eosinophil Activation Independently of Canonical Toll-Like Receptor (TLR)/C-Type-Lectin Receptor (CLR) Signaling.

Eosinophilia is associated with various persisting inflammatory diseases and often coincides with chronic fungal infections or fungal allergy as in the case of allergic bronchopulmonary aspergillosis (ABPA). Here, we show that intranasal administration of live Aspergillus fumigatus conidia causes fatal lung damage in eosinophilic interleukin-5 (IL-5)-transgenic mice. To further investigate the activation of eosinophils by A. fumigatus, we established a coculture system of mouse bone marrow-derived eosinophils (BMDE) with different A. fumigatus morphotypes and analyzed the secretion of cytokines, chemokines, and eicosanoids. A. fumigatus-stimulated BMDE upregulated expression of CD11b and downregulated CD62L and CCR3. They further secreted several proinflammatory mediators, including IL-4, IL-13, IL-18, macrophage inflammatory protein-1α (MIP-1α)/CC chemokine ligand 3 (CCL3), MIP-1ß/CCL4, and thromboxane. This effect required direct interaction and adherence between eosinophils and A. fumigatus, as A. fumigatus culture supernatants or A. fumigatus mutant strains with impaired adhesion elicited a rather poor eosinophil response. Unexpectedly, canonical Toll-like receptor (TLR) or C-type-lectin receptor (CLR) signaling was largely dispensable, as the absence of MYD88, TRIF, or caspase recruitment domain-containing protein 9 (CARD9) resulted in only minor alterations. However, transcriptome analysis indicated a role for the PI3K-AKT-mTOR pathway in A. fumigatus-induced eosinophil activation. Correspondingly, we could show that phosphatidylinositol 3-kinase (PI3K) inhibitors successfully prevent A. fumigatus-induced eosinophil activation. The PI3K pathway in eosinophils may therefore serve as a potential drug target to interfere with undesired eosinophil activation in fungus-elicited eosinophilic disorders. IMPORTANCE Allergic bronchopulmonary aspergillosis (ABPA) is caused by the fungus Aspergillus fumigatus, afflicts about five million patients globally, and is still a noncurable disease. ABPA is associated with pronounced lung eosinophilia. Activated eosinophils enhance the inflammatory response not only by degranulation of toxic proteins but also by secretion of small effector molecules. Receptors and signaling pathways involved in activation of eosinophils by A. fumigatus are currently unknown. Here, we show that A. fumigatus-elicited activation of eosinophils requires direct cell-cell contact and results in modulation of cell surface markers and rapid secretion of cytokines, chemokines, and lipid mediators. Unexpectedly, this activation occurred independently of canonical Toll-like receptor or C-type lectin receptor signaling. However, transcriptome analysis indicated a role for the PI3K-AKT-mTOR pathway, and PI3K inhibitors successfully prevented A. fumigatus-induced eosinophil activation. The PI3K pathway may therefore serve as a potential drug target to interfere with undesired eosinophil activation in fungus-elicited eosinophilic disorders.

Age related non-type 2 inflammation and its association with treatment outcome in patients with chronic rhinosinusitis with nasal polyp in Korea.

This study aimed to investigate the effect of age in patients with chronic rhinosinusitis with nasal polyp (CRSwNP). 269 patients were divided into eosinophilic and non-eosinophilic groups based on tissue eosinophilia, defined by eosinophils accounting for more than 20% of the total inflammatory cells. Patients were then further divided into younger and older groups based on the age of 35 years. Clinical characteristics including blood eosinophil, Lund Mackay score, and modified Lund-Kennedy (mLK) scores were compared. Levels of 14 cytokines from nasal tissues of an additional 78 patients were analyzed. Tissue eosinophilia was significantly associated with age and the proportion of non-eosinophilic CRSwNP was significantly higher in younger patients as compared to older patients (79.2% vs 56.6%). There was no difference in clinical characteristics and cytokine levels between the younger and older patients with eosinophilic CRSwNP. In contrast, in patients with non-eosinophilic CRSwNP, younger patients had significantly lower preoperative blood eosinophils and higher mLK scores at three and six months, postoperatively, compared to older patients. Alpha-1 antitrypsin and IL-5 levels were significantly lower in younger patients than in older patients in non-eosinophilic CRSwNP. This study suggests a potential association between age, non-type 2 inflammation and treatment outcome in CRSwNP.