Repeated COVID-19 mRNA-based vaccination contributes to SARS-CoV-2 neutralizing antibody responses in the mucosa.

To prevent infection by respiratory viruses and consequently limit virus circulation, vaccines need to promote mucosal immunity. The extent to which the currently used messenger RNA (mRNA)-based COVID-19 vaccines induce mucosal immunity remains poorly characterized. We evaluated mucosal neutralizing antibody responses in a cohort of 183 individuals. Participants were sampled at several time points after primary adenovirus vector-based or mRNA-based COVID-19 vaccination and after mRNA-based booster vaccinations. Our findings revealed that repeated vaccination with mRNA boosters promoted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies in nasal secretions. Nasal and serum neutralizing antibody titers of both IgG and IgA isotypes correlated to one another. We investigated the source of these mucosal antibodies in a mouse model wherein mice received repeated mRNA vaccines for SARS-CoV-2. These experiments indicated that neutralizing antibody-producing cells reside in the spleen and bone marrow, whereas no proof of tissue homing to the respiratory mucosa was observed, despite the detection of mucosal antibodies. Serum transfer experiments confirmed that circulating antibodies were able to migrate to the respiratory mucosa. Collectively, these results demonstrate that, especially upon repeated vaccination, the currently used COVID-19 mRNA vaccines can elicit mucosal neutralizing antibodies and that vaccination might also stimulate mucosal immunity induced by previous SARS-CoV-2 infection. Moreover, migration of circulating antibodies to the respiratory mucosa might be a main mechanism. These findings advance our understanding of mRNA vaccine-induced immunity and have implications for the design of vaccine strategies to combat respiratory infections.

The multi-omics single-cell landscape of sinus mucosa in uncontrolled severe chronic rhinosinusitis with nasal polyps.

Uncontrolled severe chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with elevated levels of type 2 inflammatory cytokines and raised immunoglobulin concentrations in nasal polyp tissue. By using single-cell RNA sequencing, transcriptomics, surface proteomics, and T cell and B cell receptor sequencing, we found the predominant cell types in nasal polyps were shifted from epithelial and mesenchymal cells to inflammatory cells compared to nasal mucosa from healthy controls. Broad expansions of CD4 T effector memory cells, CD4 tissue-resident memory T cells, CD8 T effector memory cells and all subtypes of B cells in nasal polyp tissues. The T and B cell receptor repertoires were skewed in NP. This study highlights the deviated immune response and remodeling mechanisms that contribute to the pathogenesis of uncontrolled severe CRSwNP. CLINICAL IMPLICATIONS: We identified differences in the cellular compositions, transcriptomes, proteomes, and deviations in the immune profiles of T cell and B cell receptors as well as alterations in the intercellular communications in uncontrolled severe CRSwNP patients versus healthy controls, which might help to define potential therapeutic targets in the future.

A substantial neutrophilic inflammation as regular part of severe type 2 chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is generally associated with severe type 2 immune reactions in the white population. However, recent findings suggest an additional role for neutrophils in severe type 2 inflammation. OBJECTIVE: This study aimed to characterize the neutrophilic inflammation in CRSwNP and its relation to eosinophilic inflammation in severe type 2 immune reactions. METHODS: The presence and activation of neutrophils and eosinophils was analyzed in CRS without NP and CRSwNP by measuring cell and activation markers via immunohistochemistry, immunofluorescence, Luminex assay, ELISA, UniCAP, fluorescence-activated cell sorting, and PCR. Differential neutrophil migration was assessed via Boyden-chamber assay and neutrophil survival was analyzed via flow cytometry. RESULTS: Both CRS without NP and CRSwNP displayed variable degrees of eosinophilic and neutrophilic inflammation, with a profound neutrophilic infiltration and activation in type 2 CRSwNP, associated with eosinophil extracellular traps cell death and Charcot-Leyden crystals, but independent of IL-17. Neutrophil extracellular traps cell death in CRSwNP was associated with bacterial colonization, however, neutrophils were less prone to undergo neutrophil extracellular traps cell death in the tissue of patients with severe type 2 CRSwNP. Neutrophils did not show increased migration nor survival in the CRSwNP environment in vitro. CONCLUSIONS: This study demonstrated a severe neutrophilic inflammation associated with severe eosinophilic type 2 inflammatory CRSwNP, the role of which needs further study.

Unravelling the expression of interleukin-9 in chronic rhinosinusitis: A possible role for Staphylococcus aureus.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a Th2 biased inflammation, associated with nasal colonization of Staphylococcus (S.) aureus. Interleukin (IL)-9 is a pro-inflammatory Th2 cytokine with a pivotal role in asthma, allergy and chronic obstructive pulmonary disease (COPD), but is less studied in CRSwNP. We aimed to characterize the expression and cellular source of IL-9 and examined S. aureus as potential local trigger in CRSwNP. We showed increased numbers of interleukin-9 producing neutrophils and mononuclear cells in the tissue of CRSwNP patients. This interleukin-9 production was stimulated by S. aureus and its enterotoxin B in vitro. These findings underline the contribution of S. aureus and define IL-9 as another relevant cytokine in type 2 CRSwNP.

Mouse Strain-Dependent Difference Toward the Staphylococcus aureus Allergen Serine Protease-Like Protein D Reveals a Novel Regulator of IL-33.

Staphylococcus aureus (S. aureus) can secrete a broad range of virulence factors, among which staphylococcal serine protease-like proteins (Spls) have been identified as bacterial allergens. The S. aureus allergen serine protease-like protein D (SplD) induces allergic asthma in C57BL/6J mice through the IL-33/ST2 signaling axis. Analysis of C57BL/6J, C57BL/6N, CBA, DBA/2, and BALB/c mice treated with intratracheal applications of SplD allowed us to identify a frameshift mutation in the serine (or cysteine) peptidase inhibitor, clade A, and member 3I (Serpina3i) causing a truncated form of SERPINA3I in BALB/c, CBA, and DBA/2 mice. IL-33 is a key mediator of SplD-induced immunity and can be processed by proteases leading to its activation or degradation. Full-length SERPINA3I inhibits IL-33 degradation in vivo in the lungs of SplD-treated BALB/c mice and in vitro by direct inhibition of mMCP-4. Collectively, our results establish SERPINA3I as a regulator of IL-33 in the lungs following exposure to the bacterial allergen SplD, and that the asthma phenotypes of mouse strains may be strongly influenced by the observed frameshift mutation in Serpina3i. The analysis of this protease-serpin interaction network might help to identify predictive biomarkers for type-2 biased airway disease in individuals colonized by S. aureus.

Chronic rhinosinusitis: assessment of changes in nociceptive neurons.

BACKGROUND: Pain is a major symptom of chronic rhinosinusitis (CRS). It is mainly associated with CRS without nasal polyps (CRSsNP) and has a major impact in the decision to move on to surgery. Patients with CRS with nasal polyps (CRSwNP) are characterized by trigeminal hypoesthesia and suffer from less pain. The aim of this study was to investigate whether CRS induces alterations in the peripheral nociceptive neurons, mainly focusing on quantitative changes. METHODS: Sinus mucosa and inferior turbinate (IT) samples were obtained from patients with CRS, and IT tissue of healthy patients served as controls. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed for neuronal markers including CNTNAP2, FAM19A1, GFRA2, NEFH, NTRK1, PLXNC1, RET, SCN10A, SCN11A, TRPV1, and PGP 9.5; enzyme-linked immunosorbent assay (ELISA) was performed for KCNK18, SCN10A, MRGPRD, and MAP2. For PGP 9.5, immunohistochemistry was additionally used to analyze tissue slides. RESULTS: We included 35 patients with CRSsNP, 47 patients with CRSwNP, and 18 control patients. No differences in expression of the neuronal markers were observed between CRSsNP, CRSwNP, and controls. SCN10A was the only marker exclusively expressed on nociceptive neurons in sinus tissue. No histological difference in nerve fibers was observed between sinus mucosa of both phenotypes. CONCLUSION: Our results indicate that the nociceptive nerve density in CRSwNP is not lower than in CRSsNP, as was assumed previously. The nociceptive neurons in sinonasal mucosa cannot be classified into subtypes due to the lack of specificity of the respective marker genes. Our findings question the generally accepted claim that nasal polyp tissue does not contain any nerves.

Type 2 inflammation in chronic rhinosinusitis without nasal polyps: Another relevant endotype.

BACKGROUND: Chronic rhinosinusitis without nasal polyps (CRSsNP) is mainly considered a type 1 mediated disease. The role and clinical significance of type 2 immune responses in CRSsNP have not been addressed sufficiently; a recent cluster analysis for CRS described the existence of a subgroup of patients with CRSsNP with a type 2 inflammation. OBJECTIVE: We aimed to characterize the underlying type 2 immune response and its clinical significance in patients with CRSsNP. METHODS: A total of 240 patients with CRSsNP were endotyped and subdivided on the basis of expression of marker cytokines. Clinical data such as recurrence, comorbid asthma and allergy, and numbers of blood eosinophils and neutrophils were collected from all patients. A selection of 15 patients was further characterized for the presence of eosinophils, neutrophils, Charcot-Leyden crystals, and eosinophil extracellular traps in the mucosae. RESULTS: A type 2 immune response with increased levels of IL-4, IL-5, eosinophil cationic protein, IgE, and Staphylococcus aureus enterotoxin-specific IgE was observed in 49% of patients with CRSsNP. Those patients showed increased numbers of blood and tissue eosinophils, and they displayed a considerable eosinophilic inflammation associated with eosinophil extracellular trap cell death and Charcot-Leyden crystals. A significantly increased prevalence of recurrence and asthma was observed in patients with type 2 CRSsNP compared with in patients with non-type 2 CRSsNP. However, only 4 of 117 patients with type 2 CRSsNP developed nasal polyps within 12 years. CONCLUSION: This study shows that type 2 immune responses in CRSsNP follow similar patterns but are less pronounced than in chronic rhinosinusitis with nasal polyps. Also CRSsNP with a moderate type 2 immune response showed a considerable eosinophilic inflammation with clinical impact.

Staphylococcus aureus Induces a Mucosal Type 2 Immune Response via Epithelial Cell-derived Cytokines.

RATIONALE: Chronic rhinosinusitis with nasal polyps is characterized by a T-helper cell type 2-skewed upper airway inflammation. Mucosal Staphylococcus aureus colonization is found in the majority of patients with nasal polyps. S. aureus is known to induce type 2 cytokine release via enterotoxins. OBJECTIVES: To investigate the impact of non-enterotoxin-producing S. aureus on type 2 cytokine release. METHODS: TSLP (thymic stromal lymphopoietin), IL-33, and type 2 cytokines were assessed in a human mucosal tissue model upon S. aureus infection. MEASUREMENTS AND MAIN RESULTS: S. aureus exposure increased the expression of IL-33, TSLP, IL-5, and IL-13 in nasal polyp tissue, accompanied by elevated expression levels of TSLP and IL-33 receptors, predominantly on CD3+ T cells. S. aureus infection led to the release of TSLP, but not IL-33, IL-5, or IL-13, from healthy inferior turbinate tissue. In contrast, S. epidermidis did not induce any epithelial cell-derived cytokines in nasal polyp or healthy tissue. S. aureus infection also increased the release of IL-33 and TSLP in BEAS-2B epithelial cells, accompanied by activation of NF-κB (nuclear factor κB) pathways. Incubation with CU-CPT22, a specific Toll-like receptor 2 antagonist, significantly reduced the S. aureus-induced release of TSLP and IL-33, and the activity of the NF-κB signal in BEAS-2B cells. CONCLUSIONS: This study demonstrates for the first time that S. aureus can directly induce epithelial cell-derived cytokine release via binding to Toll-like receptor 2, and may thereby propagate type 2 cytokine expression in nasal polyp tissue.

IL-21 Is Increased in Nasal Polyposis and after Stimulation with Staphylococcus aureus Enterotoxin B.

BACKGROUND: Chronic rhinosinusitis with nasal polyposis (CRSwNP) is an inflammatory disease associated with lymphoid aggregates and local IgE production related to Staphylococcus aureus enterotoxins. T-follicular helper cells and their effector cytokine interleukin (IL)-21 play an important role in germinal center proliferation. METHODS: IL-21 was determined on the mRNA level by qPCR in nasal tissue of 3 groups of patients: control (n = 17), chronic rhinosinusitis without nasal polyposis (CRSsNP; n = 23), and CRSwNP (n = 35). The expression of IL-21 by CD4+ T cells was analyzed in tissue at baseline and after 24-h stimulation of tissue fragments with S. aureus enterotoxin B (SEB) using flow cytometry. Finally, human nasal IL-21+CXCR5+CD4+ T cells were isolated and coincubated with human blood naive B cells to investigate their functionality. RESULTS: IL-21 mRNA expression was increased in the CRSwNP group (p < 0.05) compared to the control group, and B-cell lymphoma-6 and B-lymphocyte-induced maturation protein-1 were upregulated in CRSwNP versus CRSsNP. Furthermore, SEB was able to increase IL-21 mRNA expression significantly (p < 0.01) in nasal polyps. Flow cytometry revealed that the source of IL-21 was predominantly CD4+ T cells and that IL-21+CD4+ T cells were significantly increased in polyp tissue and further increased after SEB stimulation. Finally, tissue CXCR5+CD4+ T cells derived from nasal polyp tissue were able to induce maturation of human naive B cells. CONCLUSIONS: IL-21- and IL-21-producing CD4+ T cells were increased in CRSwNP. In addition, SEB induced an increase in IL-21 and IL-21+CD4+ T cells, suggesting that S. aureus can modulate the function of Tfh cells in nasal polyps. We speculate that T-follicular helper cells and IL-21 are important in the pathophysiology of CRSwNP.

Extracellular eosinophilic traps in association with Staphylococcus aureus at the site of epithelial barrier defects in patients with severe airway inflammation.

BACKGROUND: Chronic rhinosinusitis with nasal polyps is characterized by TH2-biased eosinophilic inflammation. Eosinophils have been shown to generate so-called extracellular eosinophilic traps (EETs) under similar pathologic conditions. OBJECTIVE: Our aim was to investigate a possible link between EET formation and the presence of Staphylococcus aureus, an organism frequently colonizing the upper airways, at the human mucosal site of the disease. METHODS: Tissue slides were investigated for the presence of EETs and S aureus by using immunofluorescent staining and the PNA-Fish assay, respectively. An ex vivo human mucosal disease tissue model was used for artificial infection with S aureus. Cell markers were analyzed by using immunohistochemistry, the Luminex Multiplex assay, ELISA, PCR, and immunoblotting and linked to the presence of EETs. RESULTS: About 8.8% ± 4.8% of the infiltrating eosinophils exhibited EETs in patients' nasal polyp tissues. Formation of EETs was associated with increased IL-5 (P < .05) and periostin (P < .05) tissue levels and colonization with S aureus (P < .05). By using an ex vivo human mucosal disease tissue model, EET formation was induced (4.2 ± 0.9-fold) on exposure to S aureus but not Staphylococcus epidermidis. Eosinophils were shown to migrate (P < .01) toward S aureus and entrap the bacteria both inside and outside the mucosal tissue. Blocking NAPDH oxidase activity led to a complete inhibition (P < .05) of EET formation by S aureus. CONCLUSION: Eosinophils are likely to be specifically recruited to S aureus and possibly other microorganisms and form EETs at sites of airway epithelial damage to protect the host from infections in patients with chronic rhinosinusitis with nasal polyps.

Inflammatory endotypes of chronic rhinosinusitis based on cluster analysis of biomarkers.

BACKGROUND: Current phenotyping of chronic rhinosinusitis (CRS) into chronic rhinosinusitis with nasal polyps (CRSwNP) and chronic rhinosinusitis without nasal polyps (CRSsNP) might not adequately reflect the pathophysiologic diversity within patients with CRS. OBJECTIVE: We sought to identify inflammatory endotypes of CRS. Therefore we aimed to cluster patients with CRS based solely on immune markers in a phenotype-free approach. Secondarily, we aimed to match clusters to phenotypes. METHODS: In this multicenter case-control study patients with CRS and control subjects underwent surgery, and tissue was analyzed for IL-5, IFN-γ, IL-17A, TNF-α, IL-22, IL-1ß, IL-6, IL-8, eosinophilic cationic protein, myeloperoxidase, TGF-ß1, IgE, Staphylococcus aureus enterotoxin-specific IgE, and albumin. We used partition-based clustering. RESULTS: Clustering of 173 cases resulted in 10 clusters, of which 4 clusters with low or undetectable IL-5, eosinophilic cationic protein, IgE, and albumin concentrations, and 6 clusters with high concentrations of those markers. The group of IL-5-negative clusters, 3 clusters clinically resembled a predominant chronic rhinosinusitis without nasal polyps (CRSsNP) phenotype without increased asthma prevalence, and 1 cluster had a TH17 profile and had mixed CRSsNP/CRSwNP. The IL-5-positive clusters were divided into a group with moderate IL-5 concentrations, a mixed CRSsNP/CRSwNP and increased asthma phenotype, and a group with high IL-5 levels, an almost exclusive nasal polyp phenotype with strongly increased asthma prevalence. In the latter group, 2 clusters demonstrated the highest concentrations of IgE and asthma prevalence, with all samples expressing Staphylococcus aureus enterotoxin-specific IgE. CONCLUSION: Distinct CRS clusters with diverse inflammatory mechanisms largely correlated with phenotypes and further differentiated them and provided a more accurate description of the inflammatory mechanisms involved than phenotype information only.

Systemic expression of inflammatory mediators in patients with chronic rhinosinusitis and nasal polyps with and without Aspirin Exacerbated Respiratory Disease.

BACKGROUND: Systemic reactions are related to the pathogenesis of Aspirin Exacerbated Respiratory Disease (AERD). With this work we wanted to study the changes in the systemic levels of inflammatory mediators in both baseline and after oral aspirin challenge in patients with and without AERD. METHODS: Patients with nasal polyposis and asthma with AERD (n=20) and without (n=18) were orally challenged with aspirin in a single-blind placebo controlled study. Serum samples and urine were collected before and 6h after placebo and aspirin oral challenges. Serum levels of inflammatory mediators were assayed by using the Luminex technology and ELISA. The concentrations of 9-alpha, 11-beta prostaglandin F2, and leukotriene E4 (uLTE4) were measured in urine samples by ELISA. The expression of T-cell surface markers was analyzed in peripheral blood mononuclear cells isolated before and after the challenges. RESULTS: AERD patients showed significantly higher baseline levels of s-IL-5R-alpha, uLTE4 and percentage of CD4(+)CD25(+)CD127(pos) and CD4(+)CD45RA(-)CD45RO(+) but decreased levels of TGF-ß1 and number of CD4(+)CD25(+)CD127(neg) cells. Aspirin challenge induced the release of uLTE4, IL-6 and increased the number of CD4(+)CD45RA(-)CD45RO(+) memory T-cells only in AERD patients but failed to reduce the levels of sCD40L as observed in non-AERD subjects. Further, IL-8 and sIL-5R-alpha levels directly correlated with the PD20ASA and the effects of aspirin on IL-6 and number of memory T-cells was more pronounced in subjects showing more strong reaction (bronchial and nasal). CONCLUSIONS: AERD patients have a differential baseline inflammatory pattern that supports the role inflammation as underlying mechanism of the disease. Systemic response to oral aspirin challenge was related to an increase in serum IL-6 and the number of circulating memory T-cells in AERD patients.

The expression of dendritic cell subsets in severe chronic rhinosinusitis with nasal polyps is altered.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized as a Th2-driven disease. Activated dendritic cells (DCs) are the main T-cell activators; their role in the chronic inflammatory process of nasal polyposis is still unclear. METHODS: The regulation of DC subsets was analyzed in nasal polyp tissue from CRSwNP patients and compared to inferior turbinate tissue from healthy subjects. Tissue localization and expression of both plasmacytoid and myeloid DCs were assayed by means of immunohistochemistry and flow cytometry. Plasmacytoid DCs were also assayed by PCR, and tissue homogenates were assayed for various inflammatory markers. RESULTS: The number of plasmacytoid (pDCs) and myeloid (mDCs) dendritic cells was significantly increased in nasal polyp tissue when compared to non-inflamed nasal mucosa. The number of pDCs, but not mDCs, was down-regulated in more severe cases (nasal polyps with asthma) and varied with the cytokine milieu. The amount of pDCs was significantly decreased in IL5+IFNγ - nasal polyp tissue compared to tissues with high IFNγ levels (IL5+IFNγ+). Furthermore, levels of indoleamine 2,3-dioxygenase were increased in nasal polyp compared to inferior turbinate tissue and correlated negatively with the number of pDCs. CONCLUSIONS: There is an altered balance of pDC and mDC numbers in nasal polyp tissue. pDCs seem to be more susceptible to an inflammatory cytokine milieu and may play a crucial role in disease severity.

Inflammatory patterns in upper airway disease in the same geographical area may change over time.

BACKGROUND: Inflammatory patterns of nasal polyps (NPs) may vary. Changes over time have not been investigated so far. This study was designed to evaluate the inflammatory patterns of NPs in Thailand at two time points 12 years apart, explore differences in Staphylococcus aureus (SA) mucosal carriage rates over time, and the latter's relationship with the inflammatory patterns. METHODS: Formalin-fixed nasal tissue was obtained from 89 (47 in 1999 and 42 in 2011) patients suffering from chronic rhinosinusitis with NPs (CRSwNPs). Tissues were evaluated for eosinophils, neutrophils, IgE(+) cells, IgE and macrophage mannose receptors, interleukin (IL)-5 and IL-17 cytokine profile, and the presence of SA, using automated immunohistochemistry and peptide nucleic acid-fluorescence in situ hybridization. RESULTS: We found a significant increase in the absolute values of eosinophils and IgE(+) cells in the 2011 CRSwNP tissue series compared with 1999 and a significant but smaller increase in neutrophils. Semiquantitative evaluation revealed significantly higher mean values of positive cells for all studied inflammatory markers in the 2011 group of patients, except for the high-affinity IgE receptor. This "eosinophilic shift" of inflammation was accompanied by higher SA carriage, as well as higher frequencies of SA invasion (54.8% versus 10.6%; p < 0.001) in the 2011 compared with 1999 subjects. Patients with asthma were more likely to have higher SA carriage rates compared with nonasthmatic patients. CONCLUSION: There was a shift from predominantly neutrophilic to eosinophilic CRSwNPs in Thai patients within 12 years, with an increase in various inflammatory markers including IgE, which is associated with an increase in intramucosal presence of SA.

Omalizumab is effective in allergic and nonallergic patients with nasal polyps and asthma.

BACKGROUND: Adult patients with nasal polyps often have comorbid asthma, adding to the serious effect on the quality of life of these patients. Nasal polyps and asthma might represent a therapeutic challenge; inflammation in both diseases shares many features, such as airway eosinophilia, local IgE formation, and a T(H)2 cytokine profile. Omalizumab is a human anti-IgE mAb with proved efficacy in patients with severe allergic asthma. Omalizumab could be a treatment option for patients with nasal polyps and asthma. OBJECTIVE: The goal of this study was to investigate the clinical efficacy of omalizumab in patients with nasal polyps and comorbid asthma. METHODS: A randomized, double-blind, placebo-controlled study of allergic and nonallergic patients with nasal polyps and comorbid asthma (n = 24) was conducted. Subjects received 4 to 8 (subcutaneous) doses of omalizumab (n = 16) or placebo (n = 8). The primary end point was reduction in total nasal endoscopic polyp scores after 16 weeks. Secondary end points included a change in sinus computed tomographic scans, nasal and asthma symptoms, results of validated questionnaires (Short-Form Health Questionnaire, 31-item Rhinosinusitis Outcome Measuring Instrument, and Asthma Quality of Life Questionnaire), and serum/nasal secretion biomarker levels. RESULTS: There was a significant decrease in total nasal endoscopic polyp scores after 16 weeks in the omalizumab-treated group (-2.67, P = .001), which was confirmed by means of computed tomographic scanning (Lund-Mackay score). Omalizumab had a beneficial effect on airway symptoms (nasal congestion, anterior rhinorrhea, loss of sense of smell, wheezing, and dyspnea) and on quality-of-life scores, irrespective of the presence of allergy. CONCLUSION: Omalizumab demonstrated clinical efficacy in the treatment of nasal polyps with comorbid asthma, supporting the importance and functionality of local IgE formation in the airways.

RAGE processing in chronic airway conditions: involvement of Staphylococcus aureus and ECP.

BACKGROUND: The receptor for advanced glycation end products (RAGE) is a multiligand receptor that exists as a membrane-bound (mRAGE) form and a soluble (sRAGE) form. RAGE is reported to play a role in diverse pathologies including lower airway conditions, but the exact mechanism of action remains poorly understood. In the upper airways, the involvement of RAGE remains completely unexplored. OBJECTIVE: To investigate the involvement of RAGE in the human upper airway conditions chronic rhinosinusitis without nasal polyps (CRSsNP) and chronic rhinosinusitis with nasal polyps (CRSwNP). METHODS: Protein levels of sRAGE, mRAGE, IL-5, and eosinophil cationic protein (ECP) were quantitatively assessed in inflamed tissue of CRSsNP and CRSwNP patients. Nasal tissue of subjects without disease served as control. Ex vivo human sinonasal tissue stimulation assays were used to assess the effect of Staphylococcus aureus and ECP on sRAGE processing. RESULTS: sRAGE protein levels were higher in CRSsNP tissue, whereas mRAGE protein levels were lower than in controls. In CRSwNP patients, both tissue sRAGE and mRAGE protein levels were reduced. Low tissue sRAGE protein concentrations were associated with high IL-5 and ECP protein levels. In vitro, S aureus induced the release of sRAGE from the tissue, while ECP was shown to be implicated in the breakdown of free sRAGE. CONCLUSIONS: We demonstrate for the first time that RAGE protein is highly expressed in human upper airways under normal physiology and that it is subject to differential processing in CRSsNP and CRSwNP, identifying S aureus and ECP as novel and crucial players in this process.

Mepolizumab, a humanized anti-IL-5 mAb, as a treatment option for severe nasal polyposis.

BACKGROUND: Approximately 85% of nasal polyps (NPs) in white subjects are characterized by prominent eosinophilia. IL-5 is the key driver of eosinophilic differentiation and survival. OBJECTIVE: We sought to investigate the therapeutic potential of inhibiting IL-5 with a humanized mAb as treatment for severe nasal polyposis. METHODS: Thirty patients with severe nasal polyposis (grade 3 or 4 or recurrent after surgery) refractory to corticosteroid therapy were randomized in a double-blind fashion to receive either 2 single intravenous injections (28 days apart) of 750 mg of mepolizumab (n = 20) or placebo (n = 10). Change from baseline in NP score was assessed monthly until 1 month after the last dose (week 8). Computed tomographic scans were also performed at week 8. RESULTS: Twelve of 20 patients receiving mepolizumab had a significantly improved NP score and computed tomographic scan score compared with 1 of 10 patients receiving placebo at week 8 versus baseline. CONCLUSION: Mepolizumab achieved a statistically significant reduction in NP size for at least 1 month after dosing in 12 of 20 patients. IL-5 inhibition is a potential novel therapeutic approach in patients with severe eosinophilic nasal polyposis.

Different regulation of cigarette smoke induced inflammation in upper versus lower airways.

BACKGROUND: Cigarette smoke (CS) is known to initiate a cascade of mediator release and accumulation of immune and inflammatory cells in the lower airways. We investigated and compared the effects of CS on upper and lower airways, in a mouse model of subacute and chronic CS exposure. METHODS: C57BL/6 mice were whole-body exposed to mainstream CS or air, for 2, 4 and 24 weeks. Bronchoalveolar lavage fluid (BAL) was obtained and tissue cryosections from nasal turbinates were stained for neutrophils and T cells. Furthermore, we evaluated GCP-2, KC, MCP-1, MIP-3alpha, RORc, IL-17, FoxP3, and TGF-beta1 in nasal turbinates and lungs by RT-PCR. RESULTS: In both upper and lower airways, subacute CS-exposure induced the expression of GCP-2, MCP-1, MIP-3alpha and resulted in a neutrophilic influx. However, after chronic CS-exposure, there was a significant downregulation of inflammation in the upper airways, while on the contrary, lower airway inflammation remained present. Whereas nasal FoxP3 mRNA levels already increased after 2 weeks, lung FoxP3 mRNA increased only after 4 weeks, suggesting that mechanisms to suppress inflammation occur earlier and are more efficient in nose than in lungs. CONCLUSIONS: Altogether, these data demonstrate that CS induced inflammation may be differently regulated in the upper versus lower airways in mice. Furthermore, these data may help to identify new therapeutic targets in this disease model.

TGF-beta signaling and collagen deposition in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis is an inflammatory disease with distinct cytokine and remodeling patterns. OBJECTIVE: The objective was to analyze the presence of TGF-beta isoforms, receptors, intracellular signaling, and collagen deposition in chronic rhinosinusitis. METHODS: Sinonasal mucosal samples obtained from chronic rhinosinusitis with nasal polyps (CRSwNP; n = 13), chronic rhinosinusitis without nasal polyps (CRSsNP; n = 13), and controls (n = 10) were analyzed for TGF-beta isoforms 1 and 2 by means of ELISA and IHC, and for TGF-beta R1, 2, and 3 by RT-PCR and IHC. As downstream proteins, phospho-Smad 2 (pSmad 2) and collagen were analyzed by performing immunostaining and picrosirius red staining, respectively. RESULTS: TGF-beta 1 and 2 protein concentrations, TGF-beta receptor (R) I and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly higher in CRSsNP versus controls. In CRSwNP, TGF-beta 1 protein concentration, TGF-beta RII and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly lower versus controls. Only TGF-beta 2 protein was found higher in CRSwNP versus controls. CONCLUSION: A high TGF-beta 1 protein expression, increased TGF-beta RI expression, and a high number of pSmad 2-positive cells all indicate an enhanced TGF-beta signaling in CRSsNP, whereas a low TGF-beta 1 protein concentration, a decreased expression of TGF-beta RII, and a low number of pSmad 2-positive cells in CRSwNP indicate a low level of TGF-beta signaling in CRSwNP. These findings are compatible with the remodeling patterns observed, reflected by a lack of collagen in CRSwNP, and excessive collagen production with thickening of the collagen fibers in the extracellular matrix in CRSsNP.