Validation and shortcomings of the most common mouse model of chronic rhinosinusitis with nasal polyps

BACKGROUND: Chronic rhinosinusitis (CRS) is a highly prevalent airway disease worldwide. Whereas eosinophilic CRS with nasal polyps (eCRSwNP) represents its most severe phenotype, pathogenic mechanisms remain poorly understood despite a wide spectrum of in vitro and in vivo experimental models. A mouse model of experimental ovalbumin (OVA)-induced airway allergy with coadministration of Staphylococcus aureus enterotoxin B (SEB) has been widely used to study eosinophilic eCRSwNP. This study revisits the features of this model and its suitability for studying eCRS. METHODOLOGY: We implemented the most used eCRSwNP mouse model based on OVA+SEB intranasal challenges. Readouts including inflammatory features by (immuno)histology of the sinonasal epithelium (NP formation, eosinophils, epithelial and basement membrane thickness, fibrosis, goblet cells, Charcot-Leyden crystals (CLC)-like, tight junctions) and IgE production by enzyme-linked immunosorbent assay (ELISA), were compared to features of the corresponding human disease. RESULTS: The OVA+SEB model induced eosinophilic inflammation of upper and lower airways, with epithelial and basement membrane thickening, goblet cell hyperplasia and subepithelial fibrosis in the sinuses, along increased IgE production. Except local IgE in nasal lavage (NL), which was only increased in OVA+SEB group, all other features did not differ between OVA and OVA+SEB groups. Macro- or microscopic NP were not detected. CONCLUSIONS: With the notable exception of local IgE production, the addition of SEB did not induce additional inflammatory or structural change in the sinuses from mice exposed to and challenged with OVA. This model might represent a model for severe upper airway allergy rather than a specific model of human eCRSwNP.

[Potential therapeutic implication of focal adhesion kinase in small-cell lung cancer]. / Implication thérapeutique potentielle de la kinase de l'adhérence focale dans le cancer pulmonaire à petites cellules.

The molecular steps leading to small cell lung cancer (SCLC) development and progression are still poorly understood, resulting in the absence of targeted therapy and an extremely poor prognosis. Activation of Focal Adhesion Kinase (FAK) plays a key role in the invasive behavior of this cancer in vitro. Our hypothesis is that FAK could be a therapeutic target in SCLC. Our work aims to describe a mouse model to study the role of FAK and the antitumoral potential of its inhibition in SCLC in vivo.

[Take-home messages from the COPD 2021 biennial of the French Society of Respiratory Diseases. Understanding to so as to better innovate]. / Principaux messages de la première Biennale BPCO 2021 de la SPLF. Mieux comprendre pour innover.

INTRODUCTION: The first COPD biennial organized by the French Society of Respiratory Diseases (SPLF) took place on 17 December 2021. STATE OF THE ART: The objective of the biennial was to discuss current knowledge regarding COPD pathophysiology, current treatments, research development, and future therapeutic approaches. PERSPECTIVES: The different lecturers laid emphasis on the complexity of pathophysiologic mechanisms including bronchial, bronchiolar and parenchymal alterations, and also dwelt on the role of microbiota composition in COPD pathenogenesis. They pointed out that addition to inhaled treatments, ventilatory support and endoscopic approaches have been increasingly optimized. The development of new therapeutic pathways such as biotherapy and cell therapy (stem cells…) call for further exploration. CONCLUSIONS: The dynamism of COPD research was repeatedly underlined, and needs to be further reinforced, the objective being to "understand so as to better innovate" so as to develop effective new strategies for treatment and management of COPD.

[Potential implication of the IgA-pIgR system in idiopathic pulmonary fibrosis]. / Implications potentielles du système IgA-pIgR dans la fibrose pulmonaire idiopathique.

Idiopathic pulmonary fibrosis (IPF) is a lethal respiratory disease characterized by the excessive deposition of extracellular matrix in the alveolar zones. The bronchiolar epithelium has been implicated in the development of this disease and is capable of secreting IgA into the airway lumen thanks to its expression of the polymeric immunoglobulin receptor. Several elements indicate a dysregulation of this system, such as raised serum IgA levels in IPF patients and the pro-fibrotic effect of IgA on several key cell types. Our work aims at studying the underlying mechanisms so as to better understand the role of IgA mucosal immunity in this disease.

Chronic oral corticosteroids use and persistent eosinophilia in severe asthmatics from the Belgian severe asthma registry.

BACKGROUND: Severe asthma (SA) may require frequent courses or chronic use of oral corticosteroids (OCS), inducing many known side effects and complications. Therefore, it is important to identify risk factors of chronic use of OCS in SA, considering the heterogeneity of clinical and inflammatory asthma phenotypes. Another aim of the present analysis is to characterize a subpopulation of severe asthmatics, in whom blood eosinophil counts (BEC) remain elevated despite chronic OCS treatment. METHODS: In a cross-sectional analysis of 982 SA patients enrolled in the Belgian Severe Asthma Registry (BSAR) between March 2009 and February 2019, we investigated the characteristics of the OCS treated patients with special attention to their inflammatory profile. RESULTS: At enrollment, 211 (21%) SA patients were taking maintenance OCS (median dose: 8 [IQR: 5-10]) mg prednisone equivalent). BEC was high (> 400/mm3) in 44% of the OCS treated population. Multivariable logistic regression analysis showed that risk factors for chronic use of OCS in SA were late-onset asthma (i.e. age of onset > 40 yr), frequent exacerbations (i.e. ≥2 exacerbations in the previous year) and non-atopic asthma. Late-onset asthma was also a predictor for persistently high BEC in OCS treated SA patients. CONCLUSION: These data showed a significant association between a persistently high BEC and late-onset asthma in OCS treated SA patients. Whether it is poor compliance to treatment or corticosteroid insensitivity the reasons for this association warrants further investigation.

FcεRI expression and IgE binding by dendritic cells and basophils in allergic rhinitis and upon allergen immunotherapy.

BACKGROUND: In humans, both basophils and dendritic cells (DCs) express the high-affinity IgE receptor (FcεRI). OBJECTIVE: To gain more insight into the relation between serum IgE levels and FcεRI expression and IgE binding by DCs and basophils in house dust mite (HDM) allergy and during subcutaneous immunotherapy (SCIT). METHODS: We measured FcεRI, IgE and HDM allergen on DCs (conventional type 2 DCs, cDC2s; plasmacytoid dendritic cells, pDCs) and basophils by flow cytometry in 22 non-allergic vs 52 allergic subjects and upon HDM SCIT in 28 allergic subjects. IgE levels were measured in serum. RESULTS: Serum IgE correlated differentially with FcεRI expression and IgE binding depending on cell type and allergic status. In non-allergic subjects, FcεRI/IgE surface densities increased with serum IgE to a significantly stronger degree on basophils compared to cDC2s. By contrast, in allergic subjects FcεRI/IgE surface densities increased with serum IgE to a slightly stronger degree on cDC2s compared to basophils. In addition, the data set suggests sequential loading of IgE onto FcεRI expressed by these cells (basophils>cDC2s>pDCs). Finally, HDM SCIT induced a temporary increase in serum IgE, which was paralleled by a peak in FcεRI and IgE on DCs, but not on basophils. CONCLUSIONS & CLINICAL RELEVANCE: This study provides a comprehensive insight into the relation between serum IgE and FcεRI/IgE on basophils and DC subsets. The novel finding that HDM SCIT induces a temporary increase in FcεRI expression on DCs, but not on basophils, can be an incentive for future research on the potential tolerogenic role of IgE/FcεRI signalling in DCs in the setting of allergen immunotherapy.

The epithelial barrier and immunoglobulin A system in allergy.

Airway and intestinal epithelial layers represent first-line physical barriers, playing a key role in mucosal immunity. Barrier dysfunction, characterized by alterations such as disruption of cell-cell apical junctions and aberrant epithelial responses, probably constitutes early and key events for chronic immune responses to environmental antigens in the skin and in the gut. For instance, barrier dysfunction drives Th2 responses in atopic disorders or eosinophilic esophagitis. Such epithelial impairment is also a salient feature of allergic asthma and growing evidence indicates that barrier alterations probably play a driving role in this disease. IgA has been identified as the most abundant immunoglobulin in mucosa, where it acts as an active barrier through immune exclusion of inhaled or ingested antigens or pathogens. Historically, it has been thought to represent the serum factor underlying reaginic activity before IgE was discovered. Despite several studies about regulation and major functions of IgA at mucosal surfaces, its role in allergy remains largely unclear. This review aims at summarizing findings about epithelial functions and IgA biology that are relevant to allergy, and to integrate the emerging concepts and the recent developments in mucosal immunology, and how these could translate to clinical observations in allergy.

Chronic inflammatory airway diseases: the central role of the epithelium revisited.

The respiratory epithelium plays a critical role for the maintenance of airway integrity and defense against inhaled particles. Physical barrier provided by apical junctions and mucociliary clearance clears inhaled pathogens, allergens or toxics, to prevent continuous stimulation of adaptive immune responses. The "chemical barrier", consisting of several anti-microbial factors such as lysozyme and lactoferrin, constitutes another protective mechanism of the mucosae against external aggressions before adaptive immune response starts. The reconstruction of damaged respiratory epithelium is crucial to restore this barrier. This review examines the role of the airway epithelium through recent advances in health and chronic inflammatory diseases in the lower conducting airways (in asthma and chronic obstructive pulmonary disease). Better understanding of normal and altered epithelial functions continuously provides new insights into the physiopathology of chronic airway diseases and should help to identify new epithelial-targeted therapies.

The role of allergen components for the diagnosis of latex-induced occupational asthma.

BACKGROUND: Recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergen components have been developed to assess the patients' allergen sensitization profile and to improve the diagnosis of NRL allergy. OBJECTIVE: To examine whether the determination of specific IgE (sIgE) reactivity to a panel of recombinant allergen components would be helpful for diagnosing NRL-induced occupational asthma (OA) in predicting the outcome of a specific inhalation test. METHODS: sIgE levels to NRL extract and 12 recombinant NRL allergen components were assessed in 82 subjects with OA ascertained by a positive specific inhalation challenge (SIC) with NRL gloves and in 25 symptomatic subjects with a negative challenge. RESULTS: The sensitivity, specificity, positive predictive value, and negative predictive value of a NRL-sIgE level ≥0.35 kUA /l as compared to the result of SICs were 94%, 48%, 86%, and 71%, respectively. The positive predictive value increased above 95% when increasing the cutoff value to 5.41 kUA /l. Subjects with a positive SIC showed a significantly higher rate of sIgE reactivity to rHev b 5, 6.01, 6.02, and 11 than those with a negative SIC. A sIgE sum score against rHev b 5 plus 6.01/6.02 ≥ 1.46 kUA /l provided a positive predictive value >95% with a higher sensitivity (79%) and diagnostic efficiency (Youden index: 0.67) as compared with a NRL-sIgE ≥5.41 kUA /l (49% and 0.41, respectively). CONCLUSION: In suspected OA, high levels of sIgE against rHev b 5 combined with rHev b 6.01 or 6.02 are the most efficient predictors of a bronchial response to NRL.

[Bacterial endotoxin as a trigger for eosinophilic asthma]. / Une injection d'endotoxine bactérienne comme facteur déclenchant d'un asthme éosinophilique.

INTRODUCTION: Lipopolysaccharide is a component of the outer membrane of gram-negative bacteria. It plays an important role in asthma as an adjuvant to allergens in activating the airway epithelium. CASE REPORT: Following treatment of a cutaneous mycosis by injection of endotoxin from Salmonella (Pyrogenalum), a 49-year-old non-smoking man developed an acute bronchitis followed by persistent cough, sometimes productive of sputum. Clinical examination was unremarkable. Lung function tests showed airway obstruction, FEV1 54% predicted, partly reversible with salbutamol, and exhaled NO was increased to 73.5 ppb. There was a moderate blood eosinophilia ranging from 540 to 890 per mm(3) (7.4 to 9.6%). Lung CT scan showed no parenchymal or bronchial abnormalities, and ENT examination showed nasal polyposis. CONCLUSION: Besides its critical adjuvant role in the development of asthma when inhaled, this case suggests that endotoxin can also provoke asthma when administered systemically.

Dendritic cells revisited in human allergic rhinitis and asthma.

The role of dendritic cells (DCs) in airway allergy has been studied for 15 years; recent data has highlighted the cross talk with airway epithelial cells and environmental factors (allergens, virus) during the inception and exacerbation of allergic asthma. Although murine models have provided key information, it remains uncertain to what extent these basic mechanisms take place in human allergic disease, notably with regard to different clinical phenotypes. In the present review, we discuss new evidence regarding mechanisms of DC regulation in the mouse which could be important in human asthma. Finally, after discussing the effects of current therapies on DC biology, we focus on pathways that could represent targets for future therapies.

Raised immunoglobulin A and circulating T follicular helper cells are linked to the development of food allergy in paediatric liver transplant patients.

BACKGROUND: Post-transplant food allergy (LTFA) is increasingly observed after paediatric liver transplantation (LT). Although the immunopathology of LTFA remains unclear, immunoglobulin (Ig) E seems to be implicated. OBJECTIVE: To study humoral and cellular immunity in paediatric LT patients in search for factors associated with LTFA, and compare with healthy controls (HC) and non-transplant food-allergic children (FA). METHODS: We studied serum Ig levels in 29 LTFA, 43 non-food-allergic LT patients (LTnoFA), 21 FA patients and 36 HC. Serum-specific IgA and IgE against common food allergens in LTFA, IgA1 , IgA2 and joining-chain-containing polymeric IgA (pIgA) were measured. Peripheral blood mononuclear cells were analysed by flow cytometry for B and T cell populations of interest. RESULTS: Serum IgA and specific IgA were higher in LTFA compared to LTnoFA. LTFA patients had the highest proportion of circulating T follicular helper cells (cTfh). The percentage of cTfh correlated positively with serum IgA. Unique in LTFA was also the significant increase in serum markers of mucosal IgA and the decrease in the Th17 subset of CXCR5(-) CD4(+) cells compared to HC. Both LT patients exhibited a rise in IgA(+) memory B cells and plasmablasts compared to HC and FA. CONCLUSIONS: LT has an impact on humoral immunity, remarkably in those patients developing FA. The increase in serum markers of mucosal IgA, food allergen-specific IgA and cTfh cells observed in LTFA, point towards a disturbance in intestinal immune homoeostasis in this patient group.

Heterogeneity of phenotypes in severe asthmatics. The Belgian Severe Asthma Registry (BSAR).

UNLABELLED: The Belgian severe asthma registry is a web-based registry encompassing demographic, clinical, functional and inflammatory data of severe asthmatics (SA), aiming at improving awareness, knowledge on its natural history and subphenotypes, and offering tools to optimize care of this asthma population. METHODS: The cross-sectional analyses of this registry included 350 SA as defined by the ATS (2000) from 9 Belgian centres, with at least one year follow up. RESULTS: Mean age was 55 ± 14 yrs. SA were more frequently female (57%) and atopic (70%). Late-onset asthma (≥40 yr) was observed in 31% of SA. Current smokers represented 12% while 31% were ex-smokers. In addition to high doses ICS + LABA, 65% of patients were receiving LTRA, 27% anti-IgE and 24% maintenance oral corticosteroids (8 mg (Interquartile range-IQR:4-8) methylprednisolone). Despite impaired airflow (median FEV1:67%; IQR: 52-81) only 65% had a post-bronchodilator FEV1/FVC ratio <70%. The median blood eosinophil count was 240/mm³. The median FENO was 26 ppb (IQR: 15-43) and 22% of SA had FENO ≥ 50 ppb. Induced sputum was successful in 86 patients. Eosinophilic asthma (sputum Eos ≥ 3%) was the predominant phenotype (55%) while neutrophilic (sputum Neu ≥ 76%) and paucigranulocytic asthma accounted for 22% and 17% respectively. Comorbidities included rhinitis and chronic rhinosinusitis (49%), nasal polyposis (19%), oesophageal reflux (36%), overweight and obesity (47%) and depression (19%). In addition, 8% had aspirin-induced asthma and 3% ABPA. Asthma was not well-controlled in 83% according to ACT < 20 and 77% with ACQ > 1.5. CONCLUSION: In this cohort of patients with severe asthma, the majority displayed indices of persistent airflow limitation and eosinophilic inflammation despite high-dose corticosteroids, suggesting potential for eosinophil-targeted biotherapies.

Features of mesenchymal transition in the airway epithelium from chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) defines a group of disorders characterized by persistent inflammation of the sinonasal tract. Epithelial changes and structural remodelling are present, but whether epithelial differentiation is altered remains uncertain. METHODS: To evaluate the differentiation state of the sinonasal epithelium in CRS, sinonasal biopsies from patients with CRS with nasal polyps (CRSwNP) or CRS without nasal polyps (CRSsNP), or with allergic rhinitis (AR), as compared to controls, were processed by immunohistochemistry and RT-qPCR for terminal differentiation (E-cadherin, high molecular weight cytokeratins (Hmw CK) and CK5, vimentin) and lineage differentiation (ß-tubulin IV+ ciliated cells, MUC5AC+ goblet cells, p63 + basal cells). Findings were correlated with subepithelial fibrosis and clinical CT score. RESULTS: Expression of E-cadherin was decreased at protein and mRNA levels in CRSwNP and CRSsNP, as compared to controls. Staining for Hmw CKs was also reduced in CRSwNP and CRSsNP, and CK5 mRNA was decreased in CRSwNP. These features were not due to changes in lineage specification, but associated with increases in vimentin-expressing epithelial cells. In addition, vimentin expression correlated with the basement membrane thickening and with CT score, as well as with tissue eosinophils. CONCLUSION: Features of epithelial dedifferentiation towards a mesenchymal phenotype are observed in CRSwNP and CRSsNP and correlate with airway fibrosis and inflammation.

Myeloid dendritic cells are primed in allergic asthma for thymic stromal lymphopoietin-mediated induction of Th2 and Th9 responses.

BACKGROUND: Type 1 myeloid dendritic cells (mDCs) contribute to inception of allergic asthma (AA) and are regulated by epithelial-derived cytokines. OBJECTIVES: To evaluate whether mDCs from AA patients are primed for thymic stromal lymphopoietin (TSLP)-driven responses. METHODS: mDCs from 18 AA patients and 15 controls were purified using immunomagnetic sorting. Cells were pulsed with TSLP or Dermatophagoides pteronyssinus (Der p) allergen, before FACS phenotyping and co-culture with allogeneic CD4+ T cells. Bronchial biopsies from 15 AA patients and four controls were immunostained for CD1c and TSLP receptor (TSLPR). RESULTS: Allergic asthma patients had a higher proportion of TSLPR+ mDCs, in blood and bronchial mucosa. When compared to mDCs from controls, both TSLP- and Der p-pulsed blood mDCs from AA patients induced increased polarization of CD4+ T cells into Th2 cells (IL-5, IL-13, and GATA3+), while only TSLP-mDCs promoted Th9 cells (IL-9 and PU.1+ /IRF4+). In addition, OX40L was induced upon TSLP stimulation and was required for the induction of Th2, but not Th9, cells. In contrast, development of Th9 cells in this model depended on TGF-ß1. CONCLUSIONS: Our data indicate overlapping but partially distinct effects of TSLP and Der p allergen pathways, showing that DCs are primed in human asthma for TSLP-driven induction of both Th2 and Th9 cells. This novel TSLP/mDC/Th9 axis operates through a distinct, OX40L-independent pathway. These data further highlight the TSLP pathway as a relevant target in human asthma.

Impaired ICOSL in human myeloid dendritic cells promotes Th2 responses in patients with allergic rhinitis and asthma.

BACKGROUND: Myeloid dendritic cells (mDCs) and costimulatory molecules such as ICOSL/B7H2 play a pivotal role in murine experimental asthma, while little is known in human allergic disease. The aim of this study was to characterize the phenotype and ICOSL expression of mDCs from allergic rhinitis patients (AR) and their functional correlates on mDC regulation of T cell responses. METHODS: Human blood myeloid, CD1c(+) DCs were isolated from AR or healthy controls. Expression of costimulatory molecules inducible costimulatory ligand (ICOSL) and programmed death ligand 1 (PD-L1) was analysed in blood mDCs by flow cytometry and in nasal tissue biopsies by dual immunostaining. Blood mDCs were cocultured with (allogeneic) CD4(+) T cells before immunoassays for cytokine responses. RESULTS: mDCs from AR patients expressed a lower level of ICOSL, in both blood and nasal tissue. mDCs from AR were constitutively primed to induce Th2 cytokines and TNF in allogeneic CD4(+) T cells, while no difference was observed for IFN-γ or IL-10. Production of IL-10 and IL-12 did not differ between AR and control mDCs. Blockade of ICOSL in control DCs up-regulated IL-13 but not IFN-γ in cocultures with T cells, while PD-L1 blockade up-regulated both IL-13 and IFN-γ. CONCLUSIONS: Our data show that mDCs from patients with AR display impaired expression of ICOSL, and this defect licenses mDCs to promote aberrant IL-13- and IL-5-producing Th2 cell responses.

Downregulation of polymeric immunoglobulin receptor and secretory IgA antibodies in eosinophilic upper airway diseases.

BACKGROUND: Immunoglobulin (Ig) A represents a first-line defence mechanism in the airways, but little is known regarding its implication in upper airway disorders. This study aimed to address the hypothesis that polymeric Ig receptor (pIgR)-mediated secretory IgA immunity could be impaired in chronic upper airway diseases. METHODS: Nasal and ethmoidal biopsies, as well as nasal secretions, were collected from patients with chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) or without nasal polyps (CRSsNP), allergic rhinitis (AR) and controls, and assayed for IgA1/IgA2 synthesis, pIgR expression, production of secretory component (SC), IgA and relevant IgA antibodies, and correlated with local eosinophils and inflammatory features (IL-12, IL-13 and ECP). RESULTS: pIgR expression was decreased in the ethmoidal mucosa in patients with CRSwNP (P = 0.003) and in AR (P = 0.006). This pIgR defect was associated with reduced levels of SC (P = 0.007) and IgA antibodies to Staphylococcus aureus enterotoxin B (SAEB) (P = 0.003) in nasal secretions from patients with CRSwNP, and with increased IgA deposition in subepithelial areas. pIgR downregulation was selectively observed in patients with tissue eosinophilia, whilst no clear relation to smoking history was observed. CONCLUSION: Epithelial pIgR expression is decreased in patients with CRSwNP and AR and results in decreased SC and IgA antibodies to certain bacterial antigens (SAEB) in nasal secretions of patients with CRSwNP in parallel to subepithelial accumulation of IgA. This defect in mucosal immunity is associated with eosinophilic, Th2-related inflammation.