NLRP3 inhibition attenuates the allergic rhinitis symptoms in a mouse model.

BACKGROUND: Recent human and animal studies have demonstrated that Nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is closely involved in the development of allergic diseases. OBJECTIVE: To identify the mechanism underlying the activation of NLRP3 inflammasome signaling pathway in an ovalbumin (OVA)-induced allergic rhinitis (AR) mice model and to validate the effect of a specific inhibitor of the NLRP3, MCC950. METHODS: Mice were divided into three groups and each group consisted of ten mice (saline group, the negative control group; OVA group, the OVA-induced AR model group; and OVA+MCC group, treated with 10 mg/kg MCC950). MCC950 was administered intraperitoneally every second day. Multiple parameters of AR, including NLRP3, caspase-1, interleukin (IL)-1ß, and IL-18 were evaluated by using ELISA, RT-qPCR, histopathology, and immunohistochemistry. RESULTS: The mRNA and protein levels of NLRP3, caspase-1, IL-1ß and IL-18 were upregulated in the OVA group compared with those of the saline group. MCC950 significantly inhibited the mRNA and protein levels of NLRP3, caspase-1, IL-1ß and IL-18 in nasal tissue. Further, AR symptoms and eosinophil count were normalized after MCC950 treatment. However, OVA-specific IgE was not restored in the OVA+MCC group. CONCLUSION: NLRP3 inflammasome signaling pathway may be an alternative pathway to induce AR symptoms in OVA-induced AR model. MCC950 is a specific inhibitor of NLRP3 cascade, which attenuates AR symptoms regardless of IgE.

Bone morphogenetic protein-2 as a novel biomarker for refractory chronic rhinosinusitis with nasal polyps.

BACKGROUND: Bone morphogenetic proteins (BMPs), which are members of the TGF-ß superfamily, regulate bone remodeling by stimulating osteoblasts and osteoclasts. Although the association between osteitis and poor surgical outcomes is well known in patients with chronic rhinosinusitis (CRS), BMPs have not been fully investigated as potential biomarkers for the prognosis of CRS. OBJECTIVE: Our aim was to investigate the role of BMPs in osteitis in patients with CRS with nasal polyps (NPs) (CRSwNPs), as well as associations between BMPs and inflammatory markers in sinonasal tissues from patients with CRSwNP. METHODS: We investigated the expression of 6 BMPs (BMP-2, BMP-4, BMP-6, BMP-7, BMP-9, and BMP-10) and their cellular origins in NPs of human subjects by using immunohistochemistry and ELISA of NP tissues. Exploratory factor analysis was performed to identify associations between BMPs and inflammatory markers. Air-liquid interface cell culture of human nasal epithelial cells was performed to evaluate the induction of the epithelial-mesenchymal transition by BMPs. RESULTS: Of the 6 BMPs studied, BMP-2 and BMP-7 were associated with refractoriness. Only BMP-2 concentrations were higher in patients with severe osteitis and advanced disease extent according to the computed tomography findings. Eosinophils and some macrophages were identified as cellular sources of BMP-2 in immunofluorescence analysis. An in vitro experiment revealed that BMP-2 induced epithelial-mesenchymal transition in air-liquid interface-cultured human nasal epithelial cells, particularly in a TH2 milieu. CONCLUSION: BMP-2 could reflect the pathophysiology of mucosa and bone remodeling and may be a novel biomarker for refractory CRSwNP.

Superantigen-related TH2 CD4+ T cells in nonasthmatic chronic rhinosinusitis with nasal polyps.

BACKGROUND: Staphylococcus aureus enterotoxin (SAE) superantigens are detected in nasal polyps (NPs), and SAE-specific IgE predicts asthma comorbidity in patients with NPs. However, roles of SAE superantigens and superantigen-related T-cell responses remain to be elucidated in nonasthmatic patients. OBJECTIVE: We investigated the presence of SAEs and SAE-related T-cell receptor (TCR) Vß (TCRVß) in nonasthmatic NPs, the phenotypes and functions of SAE-related T cells, and the clinical implication of SAE-related T-cell expansion. METHODS: Sinonasal tissue samples were obtained from patients with nonasthmatic chronic rhinosinusitis (CRS) with NPs (CRSwNP), patients with CRS without NPs (CRSsNP), and control subjects. SAE genes were detected by PCR, and the TCRVß distribution and T-cell phenotypes were examined by flow cytometry. RESULTS: Various SAE genes were detected not only in NPs but also in sinonasal mucosa from patients with CRSsNP and from controls. The S aureus enterotoxin I (SEI) gene was detected in all NPs. The fraction of SEI-responsive TCRVß+ (TCRVß1+ and Vß5.1+) CD4+ T cells was significantly increased only in NPs and the ethmoidal mucosa of patients with CRSwNP, indicating superantigen-induced expansion. The expanded TCRVß5.1+ CD4+ T cells expressed proliferation marker Ki-67 and the TH2 transcription factor GATA3. Furthermore, TCRVß5.1+ CD4+ T cells in NPs highly expressed TH2 markers, including IL-17RB, thymic stromal lymphoprotein receptor, and chemoattractant receptor-homologous molecule expressed on TH2 cells, with a potent TH2 cytokine-producing ability. Moreover, the expansion of TCRVß1+ or Vß5.1+ CD4+ T cells was associated with the Lund-Mackay computed tomography score, indicating disease extent. CONCLUSION: In nonasthmatic patients with CRSwNP, superantigen-related expansion of CD4+ T cells with TH2 differentiation was associated with the disease extent.

Tumour necrosis factor alpha and interleukin-5 inhibit olfactory regeneration via apoptosis of olfactory sphere cells in mice models of allergic rhinitis.

BACKGROUND: Olfactory dysfunction is frequently experienced by patients with allergic rhinitis. It is thought to result from structural and functional changes occurring in the olfactory mucosa caused by inflammation. However, the current understanding of the pathophysiology of olfactory dysfunction in allergic rhinitis remains unclear. OBJECTIVE: To investigate the mechanism by which the olfactory neural cells are damaged in allergic rhinitis. METHODS: Olfactory sphere cells (OSCs) were established after dissociation and serial cultures of cells from the mouse olfactory mucosa. Viability and proliferation of OSCs were compared between control and allergic rhinitis mice models, and olfactory stem cell markers were analysed in vivo. To elucidate which cytokines have an inhibitory effect on OSCs, viability and apoptotic markers of OSCs were investigated. RESULTS: Olfactory sphere cells were successfully isolated from the olfactory mucosa of mice, and these cells expressed markers of neural stem cells. To investigate the neural differentiation, we performed the immunocytochemical staining and found significantly elevated expressions of Tuji1, GFAP and O4 on OSCs. On the comparison of the characteristics of OSCs between control and allergic rhinitis model, we detected significantly fewer neurospheres, reduced clonogenic capacity and decreased expression of olfactory neural stem cell markers in allergic rhinitis model. When OSCs were treated with several major allergic cytokines were treated on OSCs, only TNF-α showed an inhibitory effect on OSCs. Interestingly, IL-5 had an inhibitory effect on the viability of OSCs in combination with TNF-α, whereas IL-5 alone does not have an effect. Moreover, TNF-α combined with IL-5 significantly increased the apoptotic expression, compared with TNF-α or IL-5 alone. Additionally, allergic rhinitis mice models showed the increased apoptotic expression. CONCLUSION AND CLINICAL RELEVANCE: Allergic rhinitis mice models showed lower expression of OSCs, and TNF-α combined with IL-5 had an apoptotic effect on OSCs. Therefore, these cytokines may be therapeutic targets for olfactory dysfunction in patients with allergic rhinitis.

Chronic Rhinosinusitis without Nasal Polyps in Asian Patients Shows Mixed Inflammatory Patterns and Neutrophil-Related Disease Severity.

Chronic rhinosinusitis (CRS) shows heterogeneous immunologic features. Western studies revealed that CRS without nasal polyps (CRSsNP) showed a predominantly type 1 immune response and CRS with nasal polyps (CRSwNP) was characterized by type 2 immune response; however, the detailed immunologic profile of CRSsNP in Asian patients has not been thoroughly investigated. Therefore, we investigated the inflammatory endotypes of CRSsNP in Asian patients. Patients with CRSsNP (N = 57), patients with CRSwNP (N = 13), and a control group (N = 10), who underwent endoscopic sinus surgery, were enrolled; uncinate process (UP) tissues were harvested from all patients. Homogenates were prepared from the UP of each group, and immunologic profiles were analyzed, including major cytokines (32 inflammatory mediators). When comparing the UPs between groups, CRSsNP patients showed higher levels of Th2 cytokines (IL-4 and IL-13), eosinophilic chemokines (CCL-11 and CCL-24), ECP, and total IgE expression than control subjects. In addition, several neutrophilic markers (IL-1α, IL-6, IL-8, CXCL-1, CXCL-2, and MPO), IL-17A, IL-22, and TNF-α were dominant in CRSsNP patients. Among these inflammatory mediators, IL-17A showed higher expression levels in CRSsNP patients than in the control group and CRSwNP patients. However, IFN-γ expression was not significantly elevated in CRSsNP patients. The levels of neutrophil-associated cytokines were well correlated with each other; of which, CXCL2, IL-8, and MMP-9/TIMP-1 levels were significantly correlated with disease extent (r = 0.338, r = 0.317, and r = 0.424, respectively). However, the levels of eosinophil-associated cytokines showed little correlation with each other and were not correlated with disease extent. Our study revealed that Asian CRSsNP patients showed a mixed (types 2 and 17) immune response, but neutrophil-related markers were dominant and associated with disease extent. Knowledge of this immunologic feature may help clinicians make better individual treatment decisions for Asian CRSsNP patients.

The role of interleukin-33 in chronic rhinosinusitis.

RATIONALE: Interleukin (IL)-33, a new member of the IL-1 family, is constitutively expressed in epithelial tissues and lymphoid organs and plays an important role in the pathogenesis of allergic disease. However, the role of IL-33 in chronic rhinosinusitis with nasal polyps (CRSwNP) remains unclear. OBJECTIVE: To investigate the role of IL-33 in the pathophysiology of CRSwNP. METHODS: We investigated IL-33 expression and its cellular origins in the nasal polyps (NPs) of human subjects by immunohistochemistry (IHC), quantitative reverse transcription PCR (qRT-PCR), and multiplex cytokine assays. Correlations between IL-33 expression and other inflammatory markers were also explored. To investigate the role of IL-33 in CRSwNP, anti-IL-33 antibody was used in a murine model of CRS. RESULTS: Uncinate process tissues from control (19), CRSsNP (61), CRSwNP (69) and NP tissues (71) were used in this study. Increased expression of IL-33 mRNA and protein in patients with CRSwNP compared with controls was observed. The concentration of IL-33 protein in CRSwNP was positively correlated with the number of neutrophils and the expression of several Th1 and Th17 inflammatory markers, including interferon (IFN)-γ, IL-1ß, tumour necrosis factor (TNF)-α, IL-17A, IL-22, and various markers for neutrophil recruitment. However, protein levels of IL-5 and quantity of eosinophils were inversely correlated with levels of IL-33. The expression of tissue inhibitor of metalloproteinase (TIMP)-1 was negatively correlated with IL-33 protein levels, while the expression of matrix metalloproteinase (MMP)-2 and MMP-9 was positively correlated with IL-33 protein levels. In animal studies, IL-33 expression was upregulated in the CRSwNP group compared with controls. Anti-IL-33 treatment reduced the thickness of oedematous mucosa, subepithelial collagen deposition, and infiltration of neutrophils, but infiltration of eosinophils was not reduced. This treatment also inhibited the expression of neutrophilic inflammatory cytokines, but not IL-4. In addition, the expression of intracellular adhesion molecule 1, vascular adhesion molecule 1 and CXCL-2 in the nasal mucosa was suppressed in mice treated with anti-IL-33 antibody. CONCLUSIONS: Our data suggest a role for IL-33 in the pathogenesis of CRSwNP via neutrophil recruitment. Therefore, anti-IL-33 may provide a new treatment strategy to target infiltrating neutrophils in CRSwNP.

IL-25 Could Be Involved in the Development of Allergic Rhinitis Sensitized to House Dust Mite.

BACKGROUND AND PURPOSE: When house dust mite (HDM), a common allergen, comes into the mucosal membrane, it may stimulate innate immunity. However, the precise role of interleukin- (IL-) 25 in the development of HDM-induced nasal allergic inflammation is still unclear. Therefore, we investigated the role of IL-25 in allergic rhinitis (AR) patients sensitized to HDM. METHODS: To confirm the production of IL-25 in human nasal epithelial cells (HNECs), we stimulated HNECs. IL-25 expression in the nasal mucosa from control, non-AR (NAR) patients, and HDM-sensitized AR patients was assessed using immunohistochemistry, and quantitative reverse transcription PCR. Correlations between IL-25 and other inflammatory markers were explored. RESULTS: An in vitro study showed significantly elevated concentrations of IL-25 in the HNEC samples with highest doses of HDM. Nasal tissues from AR patients sensitized to HDM showed significantly higher IL-25 expression, compared to those from the control or NAR patients. Moreover, the expression of IL-25 in nasal tissues from AR patients sensitized to HDM was positively associated with Th2 markers, such as ECP and GATA3. CONCLUSIONS: IL-25 expression increased with high-dose HDM stimulation and was related to Th2 markers. Therefore, IL-25 neutralization might offer a new strategy for treating patients with HDM-sensitized AR.

IL-25 as a novel therapeutic target in nasal polyps of patients with chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps (NPs) in Western populations is associated with TH2 cytokine polarization. IL-25, an IL-17 family cytokine, was recently reported to induce TH2-type immune responses and to contribute to several allergic diseases, such as atopic dermatitis and asthma. However, the role of IL-25 in Asian patients with nasal polyposis remains unclear. OBJECTIVE: We sought to determine the role of IL-25 in Asian patients with nasal polyposis and CRS. METHODS: We investigated IL-25 expression and its cellular origins in NPs of human subjects using immunohistochemistry (IHC), quantitative RT-PCR, and ELISA of NP tissues. Correlations between IL-25 expression and expression of other inflammatory markers in NP tissues were also explored. Anti-IL-25 neutralizing antibody was administered in an ovalbumin- and staphylococcal enterotoxin B-induced murine NP model to confirm the function of IL-25 during nasal polypogenesis. RESULTS: IL-25 expression was upregulated in NP mucosa from patients with CRS with NPs compared with uncinate process tissue from control subjects and those with CRS without NPs. Overexpression of epithelial IL-25 was confirmed by using IHC, and double IHC staining showed that tryptase-positive cells were one of the main sources of IL-25 among immune cells. Furthermore, IL-17 receptor B levels were also increased in immune cells of patients with NPs compared with those in control subjects. In NPs IL-25 mRNA expression positively correlated with the expression of several inflammatory markers, including T-box transcription factor, RAR-related orphan receptor C, GATA3, eosinophil cationic protein, TGF-ß1, and TGF-ß2. IL-25 was more abundant in the murine NP model compared with control mice, and similar correlations between IL-25 and inflammatory markers were observed in murine models. Anti-IL-25 treatment reduced the number of polyps, mucosal edema thickness, collagen deposition, and infiltration of inflammatory cells, such as eosinophils and neutrophils. This treatment also inhibited expression of local inflammatory cytokines, such as IL-4 and IFN-γ. Furthermore, expression of CCL11, CXCL2, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 in the nasal mucosa was suppressed in the anti-IL-25-treated group. CONCLUSION: Our results suggest that IL-25 secreted from the sinonasal epithelia and infiltrating mast cells plays a crucial role in the pathogenesis of CRS with NPs in Asian patients. In addition, our results suggest the novel possibility of treating nasal polyposis with anti-IL-25 therapy.

Neutrophil Extracellular Traps as a Biomarker in Refractory Non-Type 2 CRSwNP.

PURPOSE: Chronic rhinosinusitis (CRS) is classified into type 2 (T2) and non-T2 inflammation. T2 CRS presents as a severe form, CRS with nasal polyps (CRSwNP), which often occurs with asthma as a comorbidity worldwide. Some cases of non-T2 CRS show nasal polyposis and refractoriness, mainly in Asian countries. However, its mechanism remains elusive. To investigate a biomarker for the refractoriness of non-T2 CRSwNP via RNA sequencing. METHODS: RNA sequencing by using nasal polyps (NPs) and ethmoidal mucosa (EM) from CRS subjects and uncinate tissues from controls was performed, and differentially expressed genes (DEGs) were analyzed (cutoffs: expression change > 2-fold, P < 0.01). Immunofluorescence staining and enzyme-linked immunosorbent assay were performed. RESULTS: We identified DEGs among T2-NP, non-T2-NP, T2-EM, non-T2-EM, and controls (NP vs. controls: 1,877 genes, EM vs. controls: 1,124 genes, T2-NP vs. controls: 1,790 genes, non-T2-NP vs. controls: 2,012 genes, T2-EM vs. controls: 740 genes, non-T2-EM vs. controls: 1,553 genes). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that neutrophil extracellular trap (NET) formation, systemic lupus erythematosus, and the phagosome were enriched in non-T2-NP vs. controls and non-T2-EM vs. controls. Immunofluorescence staining confirmed that NETs were elevated in non-T2-NP. Cytokine analysis demonstrated that NETs were significantly related to the refractoriness in non-T2-NPs. CONCLUSIONS: This study demonstrated DEGs between T2 and non-T2 inflammation. These results suggest that NETs may contribute to the refractoriness in non-T2-NPs and have a promise as a therapeutic strategy for patients with refractory non-T2-NP.

Endotype and Phenotype Related Postoperative Effects of Bacterial Lysate in Chronic Rhinosinusitis.

OBJECTIVES: Chronic rhinosinusitis (CRS) endotypes have demonstrated clinical value in guiding treatment decisions. Bacterial lysates are immunomodulators that have shown beneficial effects in various respiratory inflammatory diseases. This study aimed to evaluate the effect of postoperative bacterial lysate therapy on different CRS endotypes. METHODS: Patients diagnosed with CRS who underwent endoscopic sinus surgery were recruited. Bacterial lysates were administered postoperatively for 10 days per month for 3 months to the experimental group comprising patients with a history of frequent upper respiratory infections without adverse reactions. The remaining participants were allocated to the control group. The results of the postoperative 3-, 6-, and 12-month assessments, including the modified Lund-Kennedy (mLK) endoscopic and Sinonasal Outcome Test (SNOT) 22 scores, for the groups were compared. The tissue samples obtained from the participants were evaluated to detect the presence of relevant inflammatory mediators. RESULTS: Among the 92 participants, 47 started bacterial lysate therapy 2 weeks after the surgery. The tissue cytokine profiles and clinical parameters, such as the disease severity and blood eosinophil percentage, of the bacterial lysate and control groups were comparable before treatment. The mLK endoscopic and SNOT-22 scores did not differ after 3, 6, and 12 months of follow-up. The subgroup analysis revealed that the bacterial lysate group had significantly lower mLK endoscopic scores than the control group for CRS without nasal polyps, while there was a tendency toward significance for the interleukin (IL)-5 negative group after 6 months. CONCLUSION: Postoperative bacterial lysate therapy has some beneficial effects on the endoscopic findings of patients with CRS without nasal polyps or those who are negative for IL-5.

Receptor activator of nuclear factor κB ligand is a biomarker for osteitis of chronic rhinosinusitis.

BACKGROUND: Evidence of osteitis is frequently observed in patients with chronic rhinosinusitis (CRS), especially in recalcitrant cases. However, studies focusing on biological markers of osteitis are limited and it remains unclear whether osteitis is associated with different phenotypes of CRS. This study aimed to analyze the expression and assess the roles of receptor activator of nuclear factor κB ligand (RANKL) in patients with CRS and osteitis. METHODS: CRS patients with nasal polyps (CRSwNP, n = 63), CRS patients without nasal polyps (CRSsNP, n = 8), and control subjects (n = 12) were enrolled. Histologic phenotypes, clinical information, and computed tomography (CT) scores were investigated. The Global Osteitis Scoring Scale (GOSS) and RANKL, a molecular marker of bone remodeling, were analyzed in each type of CRS. CRS mouse models were treated with anti-RANKL. RESULTS: GOSS values were significantly higher in all CRS patients than in the control group. The GOSS value in non-eosinophilic CRSwNP was higher than in eosinophilic CRSwNP. RANKL was upregulated whereas decoy receptor osteoprotegerin (OPG) was downregulated in CRS. RANKL messenger RNA (mRNA) and protein levels were positively correlated with GOSS. RANKL/OPG was increased in recurrent cases compared with primary cases. Multiple inflammatory mediators were positively correlated with the protein level of RANKL in CRS tissues. In the mouse CRSwNP model, anti-RANKL treatment abrogated mucosal inflammation and bone remodeling. CONCLUSION: RANKL expression is associated with clinical osteitis and disease severity in CRSwNP. These findings shed light on the importance of RANKL as a potential biomarker of CRS and a key player in CRS pathogenesis.

Elastase-Positive Neutrophils Are Associated With Refractoriness of Chronic Rhinosinusitis With Nasal Polyps in an Asian Population.

PURPOSE: Various immune cells, including eosinophils and neutrophils, are known to contribute to the development of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the current understanding of the role of neutrophils in the development of CRSwNP still remains unclear. Therefore, we investigated risk factors for refractoriness of CRSwNP in an Asian population. METHODS: Protein levels of 17 neutrophil-related mediators in nasal polyps (NPs) were determined by multiplex immunoassay, and exploratory factor analysis using principal component analysis was performed. Immunofluorescence analysis was conducted to detect human neutrophil elastase (HNE) or myeloperoxidase (MPO)-positive cells. Tissue eosinophilic nasal polyp (ENP) and tissue neutrophilia (Neuhigh) were defined as greater than 70 eosinophils and 20 HNE-positive cells, otherwise was classified into non-eosinophilic nasal polyp (NENP) and absence of tissue neutrophilia (Neulow). RESULTS: In terms of disease control status, NENP-Neulow patients showed the higher rate of disease control than NENP-Neuhigh and ENP-Neuhigh patients. Linear by linear association demonstrated the trend in refractoriness from NENP-Neulow to NENP-Neuhigh or ENP-Neulow to ENP-Neuhigh. When multiple logistic regression was performed, tissue neutrophilia (hazard ratio, 4.38; 95% confidence interval, 1.76-10.85) was found as the strongest risk factor for CRSwNP refractoriness. Additionally, exploratory factor analysis revealed that interleukin (IL)-18, interferon-γ, IL-1Ra, tumor necrosis factor-α, oncostatin M, and MPO were associated with good disease control status, whereas IL-36α and IL-1α were associated with refractory disease control status. In subgroup analysis, HNE-positive cells and IL-36α were significantly upregulated in the refractory group (P = 0.0132 and P = 0.0395, respectively), whereas MPO and IL-18 showed higher expression in the controlled group (P = 0.0002 and P = 0.0009, respectively). Moreover, immunofluorescence analysis revealed that IL-36R⁺HNE⁺-double positive cells were significantly increased in the refractory group compared to the control group. We also found that the ratio of HNE-positive cells to α1 anti-trypsin was increased in the refractory group. CONCLUSIONS: Tissue neutrophilia had an influence on treatment outcomes in the Asian CRSwNP patients. HNE-positive cells and IL-36α may be biomarkers for predicting refractoriness in Asians with CRSwNP. Additionally, imbalances in HNE and α1 anti-trypsin may be associated with pathophysiology of neutrophilic chronic rhinosinusitis.

Enhanced Type 2 Immune Reactions by Increased IL-22/IL-22Ra1 Signaling in Chronic Rhinosinusitis With Nasal Polyps.

PURPOSE: Recent studies have revealed the pathogenic role of interleukin (IL)-22 in atopic dermatitis and asthma. However, little is known about the role of IL-22 in the pathophysiology of chronic rhinosinusitis with nasal polyps. We aimed to investigate the expression of IL-22 and its pathogenic function in type 2 immune reactions of nasal polyps (NP). METHODS: Protein levels of inflammatory mediators were determined by multiplex immunoassay, and principal component analysis (PCA) was performed. Immunofluorescence analysis and mast cell culture were used to determine the cellular sources of IL-22. Normal human bronchial epithelial (NHBE) cells were stimulated using IL-22 in combination with diverse cytokines, and thymic stromal lymphopoietin (TSLP) was measured. RESULTS: IL-22 expression was not up-regulated in NP compared with control tissues, but IL-22-high NP revealed distinct features characterized by type 2 inflammatory cytokines such as chemokine (C-C motif) ligand (CCL)-11, CCL-24, and IL-5 on the PCA. Additionally, IL-22 positively correlated with type 2 immune mediators and the disease severity in NP. For the localization of the cellular sources of IL-22 in eosinophilic NP, it was expressed in cells mostly composed of eosinophil peroxidase-positive cells and partially of tryptase-positive cells. The human mast cell line, LAD2 cells, released IL-22 mediated by immunoglobulin E. Moreover, IL-22 receptor subunit alpha-1 (IL-22Ra1) expression was significantly increased in NP. IL-22Ra1 was up-regulated with poly(I:C) stimulation in NHBE cells. Furthermore, TSLP production was enhanced when stimulated with a combination of IL-13, poly(I:C), and IL-22. Treatment with anti-IL-22Ra1 also inhibited IL-22-induced enhancement of TSLP production. CONCLUSION: IL-22 was associated with type 2 inflammatory reactions in NP. The IL-22/IL-22Ra1 axis was enhanced and might be involved in type 2 inflammatory reactions via TSLP production in NP.

Comparison Between Signature Cytokines of Nasal Tissues in Subtypes of Chronic Rhinosinusitis.

PURPOSE: Endotype in chronic rhinosinusitis (CRS) has been established in the last decade. However, the exact immunologic profile of CRS still has controversy because it has a considerable immunologic heterogeneity. Therefore, we investigated various inflammatory mediators according to different nasal tissues in chronic rhinosinusitis and compared them within the same subject. METHODS: We collected uncinate process mucosa (UP) and nasal polyp (NP) tissues from controls, CRS without NP (CRSsNP) and CRS with NP (CRSwNP). Expression levels of 28 inflammatory mediators including T helper (Th) 1, Th2, Th17, proinflammatory cytokines and remodeling markers were determined by multiplex immunoassay and were analyzed using paired tests as well as principal component analysis (PCA) to investigate endotype in each subtype of CRS. RESULTS: Signature inflammatory mediators are interleukin (IL)-5, C-C motif chemokine ligand (CCL)-24, monocyte chemoattractant protein (MCP)-4, and vascular cell adhesion molecule (VCAM)-1 in eosinophilic NP, whereas IL-17A, IL-1ß, and matrix metallopeptidase (MMP)-9 were detected as signature inflammatory markers in non-eosinophilic NP. Despite differences in inflammatory cytokine profile between eosinophilic and non-eosinophilic NP, the common upregulation of IL-5, CCL-11, IL-23, IL-2Rα, VCAM-1, MMP-3 and MMP-9 were shown in NP compared to UP within the same subject. In the PCA, we observed that Th2 immune response was helpful in discriminating between nasal tissues in subtypes of CRS and that there was a partial overlap between non-eosinophilic NP and eosinophilic NP in terms of Th2 mediators. CONCLUSIONS: Commonly upregulated mediators in NP were Th2-associated, compared with UP regardless of CRS subtypes, whereas signature markers were distinct in each NP subtype. These findings imply that Th2 inflammatory responses may play a role in the development of NP regardless of CRSwNP subtypes.

Immunological Characteristics in Refractory Chronic Rhinosinusitis with Nasal Polyps Undergoing Revision Surgeries.

PURPOSE: Despite medical and surgical treatments, some cases of nasal polyps (NP) exhibit recidivism. However, the endotype of refractory chronic rhinosinusitis with NP (CRSwNP) remains unclear. Therefore, the objective of this study was to characterize the immunological profile of refractory CRSwNP. METHODS: The control (n =23), primary NP group (pNP, n =70) and refractory NP group (rNP, n =86) were enrolled in this study. Patients who underwent revision surgeries due to failed maximal medical treatment after primary surgery were defined as the rNP group. A total of 18 inflammatory markers were investigated in nasal tissues using multiplex cytokine assay or enzyme-linked immunosorbent assay. RESULTS: The clinical characteristics of rNP included more extensive disease and worse clinical course after surgery. Additionally, rNP subjects showed higher infection rate (mucopurulence and culture-positive rate), more frequent use of antibiotics and suffered from symptomatic bacterial infection, increased asthma morbidity compared to pNP. Cytokine profile analysis showed that levels of Th17-associated mediators (myeloperoxidase, interleukin (IL)-8, IL-17A and IL-23), B-cell activating factor (BAFF) and Th1 cytokine (interferon-γ) were up-regulated in rNP compared to controls and pNP. Human neutrophil elastase-positive cells were also enhanced in rNP compared with pNP. Upregulation of Th17/Th1mediators and BAFF were observed in rNP, regardless of tissue eosinophilia or asthmatic comorbidity. Interestingly, eosinophilic markers, such as eosinophil cationic protein and C-C motif chemokine ligand 24, were up-regulated in asthmatic rNP compared to pNP and controls. Levels of anti-dsDNA immunoglobulin (Ig) G and IgA were up-regulated in rNP and highest in asthmatic eosinophilic rNP among subtypes of rNP. CONCLUSIONS: Our results suggest that Th17/Th1-associated mediators and BAFF may play a role and be a potential therapeutic target in refractory CRSwNP. Additionally, eosinophilic markers and autoantibodies may contribute to refractoriness in asthmatic rNP.

Prolonged allergen exposure is associated with increased thymic stromal lymphopoietin expression and Th2-skewing in mouse models of chronic rhinosinusitis.

OBJECTIVES/HYPOTHESIS: Chronic rhinosinusitis (CRS) is characterized by a dysfunctional host-environment interaction at the nasal mucosa. Contributions of host susceptibility factors such as atopy and aspirin sensitivity to CRS pathophysiology are well established. However, clinical studies on the effects of environmental factors are limited. This study investigates the histological and immunological effects of allergen exposure duration in animal models. STUDY DESIGN: Animal study. METHODS: A murine model for CRS with nasal polypoid lesions was induced by instilling ovalbumin/staphylococcal enterotoxin B (SEB) into murine nasal cavities for 12 (short term) or 24 weeks (long term). Histopathological changes were observed. Interleukin (IL)-4, IL-17A, IL-10, and interferon (INF)-γ levels from nasal lavage fluid were measured using enzyme-linked immunosorbent assay. Gene expressions of IL-25, thymic stromal lymphopoietin (TSLP), IL-4, IL-5, INF-γ, C-C motif chemokine ligand (CCL)-11, CCL-24, C-X-C motif chemokine ligand (CXCL)-1, CXCL-2, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, matrix metallopeptidase (MMP)-7, and tissue inhibitor of metalloproteinase (TIMP)-3 were analyzed from the nasal mucosa. RESULTS: Long-term CRS models exhibited increased polypoid lesions, edematous mucosal thickness, and eosinophil infiltration compared with short-term models and showed a higher IL-10 level but lower IFN-γ and IL-17A protein levels. Moreover, CCL-24 and MMP-7 gene expressions increased whereas TIMP-3 expression decreased in long-term models compared to controls and short-term models. IL-25 and TSLP expressions were upregulated at mRNA and protein levels in short-term and long-term CRS models, respectively. Furthermore, TSLP mRNA expression was positively associated with IL-5 (r = 0.8754) and inversely correlated to IFN-γ (r = -0.7212) in CRS models. CONCLUSIONS: Prolonged allergen exposure in ovalbumin/SEB-induced CRS models maintains Th2 inflammation and reduces Th1 inflammation, which was associated with upregulation of TSLP. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E265-E272, 2016.

Age-Related Decline of Neutrophilic Inflammation Is Associated with Better Postoperative Prognosis in Non-eosinophilic Nasal Polyps.

BACKGROUND: Innate and adaptive immune responses change with increasing age and affect the course of diseases. Previous study investigated immunologic alteration in Western nasal polyps (NP) which is mostly eosinophilic. However, there are no reports regarding age-related immune changes of non-eosinophilic NP (NE-NP) which is a predominant subtype in Asian population. METHODS: A total of 153 subjects, including 20 with control, 63 with chronic rhinosinusitis (CRS) without NP (CRSsNP), and 70 with CRS with NP were enrolled. Age-related changes in computed tomography (CT), cytokines and clinical information were investigated. Tissue samples were analyzed for protein levels of IL-5, IL-17A, IL-23, interferon (IFN)-γ, CCL-11, and CXCL-8, using Luminex immunoassay and for mRNA expression levels of interleukin (IL)-5, IL-17A, IL-23p19, IFN-γ, CCL-11, CXCL-1, CXCL-2, CXCL-8, and CXCR2 by quantitative RT-PCR. Immunohistochemistry (IHC) was performed for the number of inflammatory cells. RESULTS: We observed that Lund-Mackay CT scores decreased with age in NE-NP. The number of human neutrophil elastase-positive cells and myeloperoxidase gene expression decreased in older patients with NE-NP, but not in control subjects, CRSsNP, and E-NP. Neutrophil-associated cytokines including IL-17A and IL-23, were negatively correlated with age in NE-NP at the protein and mRNA levels. Additionally, the expression of CXCR2, a receptor for CXCL-1 and CXCL-2, was decreased with age in NE-NP. However, there were no age-related changes in blood neutrophil count, and neutrophil-recruiting chemokines such as CXCL-1, CXCL-2, and CXCL-8. Elderly NE-NP patients showed better endoscopic scores at 12 months after surgery compared with the non-elderly. CONCLUSION: Age-related decline in neutrophil inflammation may favorably affect postoperative results in elderly patients with NE-NP.

Non-Eosinophilic Nasal Polyps Shows Increased Epithelial Proliferation and Localized Disease Pattern in the Early Stage.

BACKGROUND: Non-eosinophilic nasal polyps (NPs) show less inflammatory changes and are less commonly associated with lower airway inflammatory disorders such as asthma, compared with eosinophilic NPs. However, the development of non-eosinophilic NPs which is a predominant subtype in Asian population still remains unclear. METHODS: A total of 81 patients (45 with non-eosinophilic NPs and 36 with eosinophilic NPs) were enrolled. Clinical information and computed tomography (CT), endoscopic, and histological findings were investigated. Tissue samples were analyzed for total IgE levels and for mRNA expression levels of interleukin (IL)-4, IL-5, IL-13, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17A, IL-22, IL-23p19, transforming growth factor (TGF)-ß1, TGF-ß2, TGF-ß3, and periostin. Immunostaining assessment of Ki-67 as a proliferation marker was performed. RESULTS: We found that epithelial in-growing patterns such as pseudocysts were more frequently observed in histological and endoscopic evaluations of non-eosinophilic NPs, which was linked to increase epithelial staining of Ki-67, a proliferating marker. Eosinophilic NPs were characterized by high infiltration of inflammatory cells, compared with non-eosinophilic NPs. To investigate the developmental course of each subtype, CT was analyzed according to CT scores and subtypes. Non-eosinophilic NPs showed more localized pattern and maxillary sinus involvement, but lesser olfactory involvement in early stage whereas eosinophilic NPs were characterized by diffuse ethmoidal and olfactory involvement. In addition, high ethmoidal/maxillary (E/M) CT scores, indicating ethmoidal dominant involvement, were one of surrogate markers for eosinophilic NP. E/M CT scores was positively correlated with levels of TH2 inflammatory markers, including IL-4, IL-5, periostin mRNA expression and total IgE levels in NPs, whereas levels of the TH1 cytokine, IFN- γ were inversely correlated. Moreover, if the combinatorial algorithm meet the three of the four markers, including IL-5 (<2.379), periostin (<3.889), IFN-γ (>0.316), and E/M ratio (<2.167), non-eosinophilic CRSwNP are diagnosed with a sensitivity of 84.4% and a specificity of 84.8%. CONCLUSION: Histologic, immunologic and clinical data suggest that non-eosinophilic NPs showed enhanced epithelial alteration and more localized maxillary involvement. Combination of cutoff value on IL-5, periostin, IFN-γ, and E/M scores may be one of surrogate markers for non-eosinophil NP subtype.

Upregulation of SOX2, NOTCH1, and ID1 in supratentorial primitive neuroectodermal tumors: a distinct differentiation pattern from that of medulloblastomas.

OBJECT: Supratentorial primitive neuroectodermal tumor (PNET) and medulloblastoma are highly malignant embryonal brain tumors. They share morphological similarities, but differ in their differentiation patterns and global gene expression. The authors compared the expression of specific genes involved in neuroglial differentiation in supratentorial PNETs and medulloblastomas to define the distinct characters of these tumors. METHODS: The mRNA expression of 8 genes (SOX2, NOTCH1, ID1, ASCL-1, NEUROD1, NEUROG1, NEUROG2, and NRG1) was evaluated in 25 embryonal tumors (12 supratentorial PNETs and 13 medulloblastomas) by quantitative real-time polymerase chain reaction. The expression levels of the transcripts of these genes were compared between the tumor groups. Activation of the JAK/STAT3 pathway was assessed by immunoblotting. Relative expression levels of STAT3 and phosphorylated STAT3 proteins were compared. RESULTS: Supratentorial PNETs expressed significantly higher levels of SOX2, NOTCH1, ID1, and ASCL-1 transcripts, whereas the transcription of proneural basic helix-loop-helix factors, NEUROD1, NEUROG1 (significantly), and NEUROG2 (not significantly) was upregulated in medulloblastomas. The proportion of phosphorylated STAT3alpha relative to STAT3alpha was significantly greater in supratentorial PNETs than in medulloblastomas, indicating activation of the JAK/STAT3 pathway in supratentorial PNETs. CONCLUSIONS: These results indicate that supratentorial PNET predominantly has glial features and medulloblastoma largely follows a neuronal differentiation pattern. These divergent differentiation patterns may be related to the location and origin of each tumor.