Prevention of allergic reactions during oxaliplatin desensitization through inhibition of Bruton tyrosine kinase.

BACKGROUND: Acute infusion reactions to oxaliplatin, a chemotherapeutic used to treat gastrointestinal cancers, are observed in about 20% of patients. Rapid drug desensitization (RDD) protocols often allow the continuation of oxaliplatin in patients with no alternative options. Breakthrough symptoms, including anaphylaxis, can still occur during RDD. OBJECTIVE: Our aim was to evaluate whether pretreatment with acalabrutinib, a Bruton tyrosine kinase inhibitor, can prevent anaphylaxis during RDD in a patient sensitized to oxaliplatin. METHODS: A 52-year-old male with locally advanced gastric carcinoma developed anaphylaxis during his fifth cycle of oxaliplatin. As he required 6 additional cycles to complete his curative-intent treatment regimen, he underwent RDD to oxaliplatin but still developed severe acute reactions. The risks and benefits of adding acalabrutinib before and during RDD were reviewed, and the patient elected to proceed. RESULTS: With acalabrutinib taken before and during the RDD, the patient was able to tolerate oxaliplatin RDD without complication. Consistent with its mechanism of action, acalabrutinib completely blocked the patient's positive skin prick response to oxaliplatin. Acalabrutinib did not alter the percentage of circulating basophils (1.24% vs 0.98%) before the RDD but did protect against basopenia (0.74% vs 0.09%) after the RDD. Acalabrutinib was associated with a drastic reduction in the ability of basophils to upregulate CD63 in vitro following incubation with oxaliplatin (0.11% vs 2.38%) or polyclonal anti-human IgE antibody (0.08% vs 44.2%). CONCLUSIONS: Five doses of acalabrutinib, 100 mg, orally twice daily starting during the evening 2 days before and continuing through RDD allowed a sensitized patient to receive oxaliplatin successfully and safely.

Evidence that oncostatin M synergizes with IL-4 signaling to induce TSLP expression in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Oncostatin M (OSM) may promote type 2 inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP) by inducing thymic stromal lymphopoietin (TSLP). OBJECTIVE: We sought to study the impact of OSM on TSLP synthesis and release from nasal epithelial cells (NECs). METHODS: OSM receptors, IL-4 receptors (IL-4R), and TSLP were evaluated in mucosal tissue and primary NECs from patients with CRSwNP by quantitative PCR and immunofluorescence. Air-liquid interface-cultured NECs were stimulated with cytokines, including OSM, and quantitative PCR, ELISA, Western blot, and flow cytometry were used to assess the expression of OSM receptors, IL-4R, and TSLP. RESULTS: Increased levels of OSM receptor ß chain (OSMRß), IL-4Rα, and TSLP were observed in nasal polyp tissues and primary epithelial cells from nasal polyps of patients with CRSwNP compared with control tissues or cells from control subjects. The level of expression of OSMRß in tissue was correlated with levels of both IL-4Rα and TSLP. OSM stimulation of NECs increased the expression of OSMRß and IL-4Rα. Stimulation with IL-4 plus OSM augmented the production of TSLP; the response was suppressed by a signal transducer and activator of transcription 6 inhibitor. Stimulation of NECs with IL-4 plus OSM increased the expression of proprotein convertase subtilisin/kexin 3, an enzyme that truncates and activates TSLP. CONCLUSIONS: OSM increases the expression of IL-4Rα and synergizes with IL-4 to induce the synthesis and release of TSLP in NECs. Because the combination of IL-4 and OSM also augmented the expression of proprotein convertase subtilisin/kexin 3, these results suggest that OSM can induce both synthesis and posttranslational processing/activation of TSLP, promoting type 2 inflammation.

Epigenetic responses to rhinovirus exposure in airway epithelial cells are correlated with key transcriptional pathways in chronic rhinosinusitis.

BACKGROUND: Viruses may drive immune mechanisms responsible for chronic rhinosinusitis with nasal polyposis (CRSwNP), but little is known about the underlying molecular mechanisms. OBJECTIVES: To identify epigenetic and transcriptional responses to a common upper respiratory pathogen, rhinovirus (RV), that are specific to patients with CRSwNP using a primary sinonasal epithelial cell culture model. METHODS: Airway epithelial cells were collected at surgery from patients with CRSwNP (cases) and from controls without sinus disease, cultured, and then exposed to RV or vehicle for 48 h. Differential gene expression and DNA methylation (DNAm) between cases and controls in response to RV were determined using linear mixed models. Weighted gene co-expression analysis (WGCNA) was used to identify (a) co-regulated gene expression and DNAm signatures, and (b) genes, pathways, and regulatory mechanisms specific to CRSwNP. RESULTS: We identified 5585 differential transcriptional and 261 DNAm responses (FDR <0.10) to RV between CRSwNP cases and controls. These differential responses formed three co-expression/co-methylation modules that were related to CRSwNP and three that were related to RV (Bonferroni corrected p < .01). Most (95%) of the differentially methylated CpGs (DMCs) were in modules related to CRSwNP, whereas the differentially expressed genes (DEGs) were more equally distributed between the CRSwNP- and RV-related modules. Genes in the CRSwNP-related modules were enriched in known CRS and/or viral response immune pathways. CONCLUSION: RV activates specific epigenetic programs and correlated transcriptional networks in the sinonasal epithelium of individuals with CRSwNP. These novel observations suggest epigenetic signatures specific to patients with CRSwNP modulate response to viral pathogens at the mucosal environmental interface. Determining how viral response pathways are involved in epithelial inflammation in CRSwNP could lead to therapeutic targets for this burdensome airway disorder.

Studies on activation and regulation of the coagulation cascade in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Increased activation of the coagulation cascade and diminished fibrinolysis combine to promote fibrin deposition and polyp formation in chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP). More information is needed concerning mechanisms of coagulation in CRSwNP. OBJECTIVE: We investigated the mechanisms as well as the initiation and regulation of coagulation cascade activation in CRS. METHODS: Samples were collected from 135 subjects with CRSwNP, 80 subjects with chronic CRS without nasal polyps (NP), and 65 control subjects. The levels of activated factor X (FXa), prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex, tissue factor (TF), and TF pathway inhibitor (TFPI) were monitored in CRS by real-time PCR, ELISA, immunohistochemistry, or immunofluorescence. Heteromeric complexes of TF with activated factor VII (FVII) and TF with activated FVII and FXa were assessed by coimmunoprecipitation and Western blotting. RESULTS: Increased levels of FXa, F1+2, and thrombin-antithrombin complex were detected in NP tissue compared to uncinate tissue from CRS and control subjects. Although free TF protein levels were not increased in NP, immunoprecipitation of TF in NP tissue revealed increased complexes of TF with FVII. Local expression of FVII was detected in sinonasal mucosa, and the ratio of TFPI to FXa was lower in NP tissue. CONCLUSION: The coagulation cascade is associated with NP compared to control and uncinate tissue from CRS patients, and TF and FVII are produced locally in sinonasal mucosa in patients. TF and FVII can activate the extrinsic coagulation pathway, suggesting that this pathway may activate fibrin deposition in CRSwNP. Reduced formation of the complex of FXa and TFPI in NP may reduce natural suppression of the extrinsic coagulation pathway in CRSwNP.

Retinoic acid promotes fibrinolysis and may regulate polyp formation.

BACKGROUND: Patients with aspirin-exacerbated respiratory disease (AERD) regularly exhibit severe nasal polyposis. Studies suggest that chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by excessive fibrin deposition associated with a profound decrease in epithelial tissue plasminogen activator (tPA). Retinoids, including vitamin A and its active metabolite retinoic acid (RA), are necessary for maintaining epithelial function and well-known inducers of tPA in endothelial cells. OBJECTIVES: This study sought to determine whether endogenous retinoids are involved in NP pathophysiology and disease severity in patients with CRSwNP and AERD. METHODS: NP tissue was collected from patients with AERD or CRSwNP, and concentrations of retinoids and fibrinolysis markers were measured using ELISA. Normal human bronchial epithelial cells were stimulated alone or in combination with RA and IL-13 for 24 hours. RESULTS: This study observed lower retinoid levels in nasal polyps of patients with AERD than those with CRSwNP or healthy controls (P < .01). Levels of the fibrin-breakdown product d-dimer were the lowest in AERD polyps (P < .01), which is consistent with lower tPA expression (P < .01). In vitro, all-trans RA upregulated tPA levels in normal human bronchial epithelial cells by 15-fold and reversed the IL-13-induced attenuation of tPA expression in cultured cells (P < .01). CONCLUSIONS: RA, a potent inducer of epithelial tPA in vitro, is reduced in tissue from patients with AERD, a finding that may potentially contribute to decreased levels of tPA and fibrinolysis in AERD. RA can induce tPA in epithelial cells and can reverse IL-13-induced tPA suppression in vitro, suggesting the potential utility of RA in treating patients with CRSwNP and/or AERD.

Studies of the role of basophils in aspirin-exacerbated respiratory disease pathogenesis.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by the triad of chronic rhinosinusitis with nasal polyps (CRSwNP), asthma, and intolerance to cyclooxygenase-1 enzyme inhibitors. The underlying mechanisms contributing to AERD pathogenesis are not fully understood, but AERD is characterized by an enhanced type 2 inflammatory phenotype. Basophils are potent type 2 effector cells, but their involvement in AERD pathophysiology remains unclear. OBJECTIVE: We sought to characterize the systemic and local basophil responses in patients with AERD compared with patients with CRSwNP. METHODS: Sinonasal tissues including inferior turbinate and/or nasal polyps (NPs) and peripheral blood were collected from controls, patients with AERD, and patients with CRSwNP. Expression of cell surface (CD45, FcεRI, CD203c), activation (CD63), and intracellular (2D7) markers associated with basophils was characterized using flow cytometry. Clinical data including Lund-Mackay scores and pulmonary function were obtained. RESULTS: The mean number of basophils (CD45+CD203c+FcεRI+CD117-) detected in AERD NPs (147 ± 28 cells/mg tissue) was significantly elevated compared with that detected in CRSwNP NPs (69 ± 20 cells/mg tissue; P = .01). The number of circulating basophils was significantly elevated in patients with AERD (P = .04). Basophils in NPs had significantly higher CD203c and CD63 mean fluorescence intensity compared with blood in both conditions (P < .01). Basophils from AERD NPs had lower expression of the granule content marker 2D7 compared with those from matched blood (P < .01) or NPs of patients with CRSwNP (P = .06), suggesting ongoing degranulation. Basophil 2D7 mean fluorescence intensity significantly correlated with pulmonary function (r = 0.62; P = .02) and inversely correlated with sinonasal inflammation (r = -0.56; P = .004). CONCLUSIONS: Increased basophil numbers and extent of ongoing degranulation in NPs of patients with AERD compared with patients with CRSwNP may contribute to the exaggerated disease pathogenesis and severity unique to AERD.

Activation of the 15-lipoxygenase pathway in aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), and an intolerance of medications that inhibit cyclooxygenase-1. Patients with AERD have more severe upper and lower respiratory tract disease than do aspirin-tolerant patients with CRSwNP. A dysregulation in arachidonic acid metabolism is thought to contribute to the enhanced sinonasal inflammation in AERD. OBJECTIVE: Our aim was to utilize an unbiased approach investigating arachidonic acid metabolic pathways in AERD. METHODS: Single-cell RNA sequencing (10× Genomics, Pleasanton, Calif) was utilized to compare the transcriptional profile of nasal polyp (NP) cells from patients with AERD and patients with CRSwNP and map differences in the expression of select genes among identified cell types. Findings were confirmed by traditional real-time PCR. Lipid mediators in sinonasal tissue were measured by mass spectrometry. Localization of various proteins within NPs was assessed by immunofluorescence. RESULTS: The gene encoding for 15-lipooxygenase (15-LO), ALOX15, was significantly elevated in NPs of patients with AERD compared to NPs of patients with CRSwNP (P < .05) or controls (P < .001). ALOX15 was predominantly expressed by epithelial cells. Expression levels significantly correlated with radiographic sinus disease severity (r = 0.56; P < .001) and were associated with asthma. The level of 15-oxo-eicosatetraenoic acid (15-Oxo-ETE), a downstream product of 15-LO, was significantly elevated in NPs from patients with CRSwNP (27.93 pg/mg of tissue) and NPs from patients with AERD (61.03 pg/mg of tissue) compared to inferior turbinate tissue from controls (7.17 pg/mg of tissue [P < .001]). Hydroxyprostaglandin dehydrogenase, an enzyme required for 15-Oxo-ETE synthesis, was predominantly expressed in mast cells and localized near 15-LO+ epithelium in NPs from patients with AERD. CONCLUSIONS: Epithelial and mast cell interactions, leading to the synthesis of 15-Oxo-ETE, may contribute to the dysregulation of arachidonic acid metabolism via the 15-LO pathway and to the enhanced sinonasal disease severity observed in AERD.

Increased thrombin-activatable fibrinolysis inhibitor levels in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis (CRS) is a heterogeneous chronic inflammatory disease subdivided based on the presence or absence of nasal polyps (NPs). Histologic features of chronic rhinosinusitis with nasal polyps (CRSwNP) include inflammatory cell infiltration and excessive fibrin deposition in NPs. Thrombin-activatable fibrinolysis inhibitor (TAFI) is an enzyme that plays an antifibrinolytic role in the body. The significance of TAFI has been documented in patients with chronic inflammatory diseases, including chronic lung disease; however, it has not been evaluated in the pathogenesis of NPs. OBJECTIVE: The objective of this study was to evaluate the potential role of TAFI in the pathogenesis of NPs. METHODS: Nasal lavage fluid was collected from control subjects and patients with CRS. We measured levels of thrombin/anti-thrombin complex (TATc) and TAFI protein using an ELISA. RESULTS: TATc levels in nasal lavage fluid were significantly increased in patients with CRSwNP and patients with chronic rhinosinusitis without nasal polyps (CRSsNP) compared with control subjects, and TAFI levels in nasal lavage fluid were also significantly increased in patients with CRSwNP compared with those in control subjects and patients with CRSsNP. There was a significant correlation between TATc and TAFI levels in nasal lavage fluid. Interestingly, patients with CRS and asthma showed increased TATc and TAFI levels in nasal lavage fluid compared with those in patients with CRS without asthma, especially patients with CRSwNP. CONCLUSIONS: Increased TATc and TAFI levels in nasal passages of patients with CRSwNP might participate in fibrin deposition in NPs and might play a role in the pathogenesis of CRSwNP and asthma.

rBPI21 (Opebacan) Promotes Rapid Trilineage Hematopoietic Recovery in a Murine Model of High-Dose Total Body Irradiation.

The complexity of providing adequate care after radiation exposure has drawn increasing attention. While most therapeutic development has focused on improving survival at lethal radiation doses, acute hematopoietic syndrome (AHS) occurs at substantially lower exposures. Thus, it is likely that a large proportion of such a radiation-exposed population will manifest AHS of variable degree and that the medical and socioeconomic costs of AHS will accrue. Here, we examined the potential of rBPI21 (opebacan), used without supportive care, to accelerate hematopoietic recovery after radiation where expected survival was substantial (42-75%) at 30 days). rBPI21 administration was associated with accelerated recovery of hematopoietic precursors and normal marrow cellularity, with increases in megakaryocyte numbers particularly marked. This translated into attaining normal trilineage peripheral blood counts 2-3 weeks earlier than controls. Elevations of hematopoietic growth factors observed in plasma and the marrow microenvironment suggest the mechanism is likely multifactorial and not confined to known endotoxin-neutralizing and cytokine down-modulating activities of rBPI21 . These observations deserve further exploration in radiation models and other settings where inadequate hematopoiesis is a prominent feature. These experiments also model the potential of therapeutics to limit the allocation of scarce resources after catastrophic exposures as an endpoint independent of lethality mitigation. This article is protected by copyright. All rights reserved.

Microparticles in nasal lavage fluids in chronic rhinosinusitis: Potential biomarkers for diagnosis of aspirin-exacerbated respiratory disease.

BACKGROUND: Microparticles (MPs) are submicron-sized shed membrane vesicles released from activated or injured cells and are detectable by flow cytometry. MP levels have been used as biomarkers to evaluate cell injury or activation in patients with pathological conditions. OBJECTIVE: We sought to compare MP types and levels in nasal lavage fluids (NLFs) from controls and patients with chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), and aspirin-exacerbated respiratory disease (AERD). METHODS: We collected NLFs from patients with CRSsNP (n = 33), CRSwNP (n = 45), and AERD (n = 31) and control (n = 24) subjects. Standardized flow cytometry methods were used to characterize the following MP types: endothelial MPs, epithelial MPs (epithelial cell adhesion molecule [EpCAM](+)MPs, E-cadherin(+)MPs), platelet MPs (CD31(+)CD41(+)MPs), eosinophil MPs (EGF-like module-containing mucin-like hormone receptor-like 1[EMR1](+)MPs), mast cell MPs (high-affinity IgE receptor [FcεRI](+)c-kit(+)MPs), and basophil MPs (CD203c(+)c-kit(-)MPs). Basophil activation was evaluated by the mean fluorescence intensity of CD203c on basophil MPs. RESULTS: Activated mast cell MPs (CD137(+) FcεRI(+)c-kit(+)MPs) were significantly increased in NLFs of controls compared with NLFs of patients with CRSsNP (2.3-fold; P < .02), CRSwNP (2.3-fold; P < .03), and AERD (7.4-fold; P < .0001). Platelet MPs (3.5-fold; P < .01) and basophil MPs (2.5-fold; P < .05) were increased only in patients with AERD. Mean fluorescence intensity of CD203c on MPs was increased in patients with CRSwNP (P < .002) and AERD (P < .0001), but not in patients with CRSsNP. EpCAM(+)MPs in patients with CRSwNP were no different from control (P = .91) and lower than those in patients with CRSsNP (P < .02) and AERD (P < .002). CONCLUSIONS: Based on released MPs, mast cells, platelets, and basophils were more highly activated in patients with AERD than in patients with CRS. Epithelial injury was lower in patients with CRSwNP than in patients with CRSsNP and AERD. MP analysis may help identify phenotypes of CRS, and in distinguishing AERD from CRSwNP.

Classical complement pathway activation in the nasal tissue of patients with chronic rhinosinusitis.

BACKGROUND: Complement plays a major role in inflammatory diseases, but its involvement and mechanisms of activation in patients with chronic rhinosinusitis (CRS) are not known. OBJECTIVES: After earlier studies discovering autoantibodies in patients with CRS, we sought to investigate the nature, extent, and location of complement activation in nasal tissue of patients with CRS. Specifically, we were interested in whether antibody-mediated activation through the classical pathway was a major mechanism for complement activation in patients with CRS. METHODS: Nasal tissue was obtained from patients with CRS and control subjects. Tissue homogenates were analyzed for complement activation products (ELISA-C5b-9, C4d, activated C1, and C5a) and major complement-fixing antibodies (Luminex). Tissue sections were stained for C5b-9, C4d, and laminin. Antibodies were purified with protein A/G columns from nasal polyps (NP), matching patient serum, and control serum and assayed for basement membrane binding by means of ELISA. RESULTS: C5b-9 levels were significantly increased in NP tissue compared with uncinate tissue (UT) of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and those with chronic rhinosinusitis without nasal polyps (CRSsNP; P < .01). Similarly, C4d levels were increased in NPs compared with UT of patients with CRSwNP, patients with CRSsNP, and control subjects (P < .05). Activated C1 levels were also increased in NP tissue compared with UT of patients with CRSsNP and control subjects (P < .05) and correlated with levels of C5a (P < .01), local immunoglobulins (especially IgM, P < .0001), and anti-double-stranded DNA IgG (P < .05). Immunofluorescence showed that C5b-9 and C4d deposition occurred linearly along the epithelial basement membrane. NP tissue extracts had significantly more anti-basement membrane antibodies than sera from patients with CRSwNP and control subjects (P < .0001). CONCLUSION: Levels of C5b-9, C4d, and activated C1 were significantly increased locally in NP tissue. C5b-9 and C4d were almost universally deposited linearly along the basement membrane of NP tissue. Furthermore, activated C1 levels were best correlated with local immunoglobulin and C5a levels. Together, these data suggest that the classical pathway plays a major role in complement activation in patients with CRS.

Neutrophils are a major source of the epithelial barrier disrupting cytokine oncostatin M in patients with mucosal airways disease.

BACKGROUND: We have previously shown that oncostatin M (OSM) levels are increased in nasal polyps (NPs) of patients with chronic rhinosinusitis (CRS), as well as in bronchoalveolar lavage fluid, after segmental allergen challenge in allergic asthmatic patients. We also showed in vitro that physiologic levels of OSM impair barrier function in differentiated airway epithelium. OBJECTIVE: We sought to determine which hematopoietic or resident cell type or types were the source of the OSM expressed in patients with mucosal airways disease. METHODS: Paraffin-embedded NP sections were stained with fluorescence-labeled specific antibodies against OSM, GM-CSF, and hematopoietic cell-specific markers. Live cells were isolated from NPs and matched blood samples for flow cytometric analysis. Neutrophils were isolated from whole blood and cultured with the known OSM inducers GM-CSF and follistatin-like 1, and OSM levels were measured in the supernatants. Bronchial biopsy sections from control subjects, patients with moderate asthma, and patients with severe asthma were stained for OSM and neutrophil elastase. RESULTS: OSM staining was observed in NPs, showed colocalization with neutrophil elastase (n = 10), and did not colocalize with markers for eosinophils, macrophages, T cells, or B cells (n = 3-5). Flow cytometric analysis of NPs (n = 9) showed that 5.1% ± 2% of CD45+ cells were OSM+, and of the OSM+ cells, 56% ± 7% were CD16+Siglec-8-, indicating neutrophil lineage. Only 0.6 ± 0.4% of CD45+ events from matched blood samples (n = 5) were OSM+, suggesting that increased OSM levels in patients with CRS was locally stimulated and produced. A majority of OSM+ neutrophils expressed arginase 1 (72.5% ± 12%), suggesting an N2 phenotype. GM-CSF levels were increased in NPs compared with those in control tissue and were sufficient to induce OSM production (P < .001) in peripheral blood neutrophils in vitro. OSM+ neutrophils were also observed at increased levels in biopsy specimens from patients with severe asthma. Additionally, OSM protein levels were increased in induced sputum from asthmatic patients compared with that from control subjects (P < .05). CONCLUSIONS: Neutrophils are a major source of OSM-producing cells in patients with CRS and severe asthma.

Proton pump inhibitors decrease eotaxin-3/CCL26 expression in patients with chronic rhinosinusitis with nasal polyps: Possible role of the nongastric H,K-ATPase.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is often characterized by tissue eosinophilia that is associated with poor prognosis. Recent findings that proton pump inhibitors (PPIs) directly modulate the expression of eotaxin-3, an eosinophil chemoattractant, in patients with eosinophilic diseases suggest therapeutic potential for PPIs in those with CRSwNP. OBJECTIVE: We assessed the effect of type 2 mediators, particularly IL-13 and eotaxin-3, on tissue eosinophilia and disease severity in patients with chronic rhinosinusitis (CRS). Further investigation focused on PPI suppression of eotaxin-3 expression in vivo and in vitro, with exploration of underlying mechanisms. METHODS: Type 2 mediator levels in nasal tissues and secretions were measured by using a multiplex immunoassay. Eotaxin-3 and other chemokines expressed in IL-13-stimulated human sinonasal epithelial cells (HNECs) and BEAS-2B cells with or without PPIs were assessed by using ELISA, Western blotting, real-time PCR, and intracellular pH imaging. RESULTS: Nasal tissues and secretions from patients with CRSwNP had increased IL-13, eotaxin-2, and eotaxin-3 levels, and these were positively correlated with tissue eosinophil cationic protein levels and radiographic scores in patients with CRS (P < .05). IL-13 stimulation of HNECs and BEAS-2B cells dominantly induced eotaxin-3 expression, which was significantly inhibited by PPIs (P < .05). Patients with CRS taking PPIs also showed lower in vivo eotaxin-3 levels compared with those without PPIs (P < .05). Using intracellular pH imaging and altering extracellular K+, we found that IL-13 enhanced H+,K+-exchange, which was blocked by PPIs and the mechanistically unrelated H,K-ATPase inhibitor, SCH-28080. Furthermore, knockdown of ATP12A (gene for the nongastric H,K-ATPase) significantly attenuated IL-13-induced eotaxin-3 expression in HNECs. PPIs also had effects on accelerating IL-13-induced eotaxin-3 mRNA decay. CONCLUSION: Our results demonstrated that PPIs reduce IL-13-induced eotaxin-3 expression by airway epithelial cells. Furthermore, mechanistic studies suggest that the nongastric H,K-ATPase is necessary for IL-13-mediated epithelial responses, and its inhibitors, including PPIs, might be of therapeutic value in patients with CRSwNP by reducing epithelial production of eotaxin-3.

Evidence for altered levels of IgD in the nasal airway mucosa of patients with chronic rhinosinusitis.

BACKGROUND: IgD is an enigmatic antibody isotype best known when coexpressed with IgM on naive B cells. However, increased soluble IgD (sIgD) levels and increased IgD+IgM- B-cell populations have been described in the human upper respiratory mucosa. OBJECTIVE: We assessed whether levels of sIgD and IgD+ B cell counts are altered in nasal tissue from patients with chronic rhinosinusitis (CRS). We further characterized IgD+ B-cell populations and explored clinical and local inflammatory factors associated with tissue sIgD levels. METHODS: sIgD levels were measured by means of ELISA in nasal tissues, nasal lavage fluid, sera, and supernatants of dissociated nasal tissues. IgD+ cells were identified by using immunofluorescence and flow cytometry. Inflammatory mediator levels in tissues were assessed by using real-time PCR and multiplex immunoassays. Bacterial cultures from the middle meatus were performed. Underlying medical history and medicine use were obtained from medical records. RESULTS: sIgD levels and numbers of IgD+ cells were significantly increased in uncinate tissue (UT) of patients with chronic rhinosinusitis without nasal polyps (CRSsNP) compared with that of control subjects (4-fold, P < .05). IgD+ cells were densely scattered in the periglandular regions of UT from patients with CRSsNP. We also found that IgD+CD19+CD38bright plasmablast numbers were significantly increased in tissues from patients with CRSsNP compared with control tissues (P < .05). Among numerous factors tested, IL-2 levels were increased in UT from patients with CRSsNP and were positively correlated with tissue IgD levels. Additionally, supernatants of IL-2-stimulated dissociated tissue from patients with CRSsNP had significantly increased sIgD levels compared with those in IL-2-stimulated dissociated control tissue ex vivo (P < .05). Tissue from patients with CRS with preoperative antibiotic use or those with pathogenic bacteria showed higher IgD levels compared with tissue from patients without these variables (P < .05). CONCLUSION: sIgD levels and IgD+CD19+CD38bright plasmablast counts were increased in nasal tissue of patients with CRSsNP. IgD levels were associated with increased IL-2 levels and the presence of pathogenic bacteria. These findings suggest that IgD might contribute to enhancement mucosal immunity or inflammation or respond to bacterial infections in patients with CRS, especially CRSsNP.

Potential Involvement of the Epidermal Growth Factor Receptor Ligand Epiregulin and Matrix Metalloproteinase-1 in Pathogenesis of Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is a heterogeneous chronic inflammatory disease of the nose and paranasal sinuses that presents without or with nasal polyps (CRSwNP). Notable features of CRSwNP are the frequent presence of type 2 allergic inflammation and high prevalence of Staphylococcus aureus (SA) colonization. As inflammation persists, sinus tissue undergoes epithelial damage and repair along with polyp growth, despite active medical management. Because one feature of damaged tissue is enhancement of growth factor signaling, we evaluated the presence of epidermal growth factor receptor (EGFR) ligands and matrix metalloproteinases (MMPs) in CRS. The objectives of this study were to analyze the expression of EGFR ligands and MMPs in patients with CRS and to investigate the possible role of SA on epithelial activation. Sinonasal tissues were collected during surgery from control subjects and patients with CRS. Tissues were processed as described previously for analysis of mRNA (RT-PCR) and proteins (ELISA) for the majority of EGFR ligands within the tissue extracts. CRS tissue was used for evaluation of the distribution of epiregulin (EREG), an EGFR ligand, and MMP-1 by immunohistochemistry. In parallel studies, expression of these genes and proteins was analyzed in cultured primary airway epithelial cells. Elevated expression of EREG and MMP-1 mRNA and protein was observed in uncinate and polyp tissue from patients with CRSwNP. Immunohistochemistry study of clinical samples revealed that airway epithelial cells expressed both of these proteins. Cultured primary human airway epithelial cells expressed MMP-1, and MMP-1 was further induced by stimulation with EREG or heat-killed SA (HKSA). The induction of MMP-1 by HKSA was blocked by an antibody against EREG, suggesting that endogenous EREG induces MMP-1 after stimulation with HKSA. EREG and MMP-1 were found to be elevated in nasal polyp and uncinate tissues in patients with CRSwNP. Elevated expression of EREG and MMP-1 may be related to polyp formation in CRS, and colonization of SA might further enhance this process.

Role of Aspergillus fumigatus in Triggering Protease-Activated Receptor-2 in Airway Epithelial Cells and Skewing the Cells toward a T-helper 2 Bias.

Aspergillus fumigatus (AF) infection and sensitization are common and promote Th2 disease in individuals with asthma. Innate immune responses of bronchial epithelial cells are now known to play a key role in determination of T cell responses upon encounter with inhaled pathogens. We have recently shown that extracts of AF suppress JAK-STAT signaling in epithelial cells and thus may promote Th2 bias. To elucidate the impact of AF on human bronchial epithelial cells, we tested the hypothesis that AF can modulate the response of airway epithelial cells to favor a Th2 response and explored the molecular mechanism of the effect. Primary normal human bronchial epithelial (NHBE) cells were treated with AF extract or fractionated AF extract before stimulation with poly I:C or infection with human rhinovirus serotype 16 (HRV16). Expression of CXCL10 mRNA (real-time RT-PCR) and protein (ELISA) were measured as markers of IFN-mediated epithelial Th1-biased responses. Western blot was performed to evaluate expression of IFN regulatory factor-3 (IRF-3), NF-κB, and tyrosine-protein phosphatase nonreceptor type 11 (PTPN11), which are other markers of Th1 skewing. Knockdown experiments for protease-activated receptor-2 (PAR-2) and PTPN11 were performed to analyze the role of PAR-2 in the mechanism of suppression by AF. AF and a high-molecular-weight fraction of AF extract (HMW-AF; > 50 kD) profoundly suppressed poly I:C- and HRV16-induced expression of both CXCL10 mRNA and protein from NHBE cells via a mechanism that relied upon PAR-2 activation. Both AF extract and a specific PAR-2 activator (AC-55541) suppressed the poly I:C activation of phospho-IRF-3 without affecting activation of NF-κB. Furthermore, HMW-AF extract enhanced the expression of PTPN11, a phosphatase known to inhibit IFN signaling, and concurrently suppressed poly I:C-induced expression of both CXCL10 mRNA and protein from NHBE cells. These results show that exposure of bronchial epithelial cells to AF extract suppressed poly I:C and HRV16 signaling via a mechanism shown to involve activation of PAR-2 and PTPN11. This action of AF may promote viral disease exacerbations and may skew epithelial cells to promote Th2 inflammation in allergic airway disorders mediated or exacerbated by AF, such as asthma and chronic rhinosinusitis.

Cytokines in Chronic Rhinosinusitis. Role in Eosinophilia and Aspirin-exacerbated Respiratory Disease.

RATIONALE: The mechanisms that underlie the pathogenesis of chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), and aspirin-exacerbated respiratory disease (AERD) are not clear. OBJECTIVES: To first evaluate the inflammatory profiles of CRSsNP and CRSwNP tissues and then to investigate whether clinical differences observed between CRSwNP and AERD are in part secondary to differences in inflammatory mediator expression within nasal polyp (NP) tissues. METHODS: Expression levels of numerous inflammatory mediators were determined by quantitative real-time polymerase chain reaction, ELISA, and multiplex immunoassay. MEASUREMENTS AND MAIN RESULTS: CRSwNP NP had increased levels of type 2 mediators, including IL-5 (P < 0.001), IL-13 (P < 0.001), eotaxin-2 (P < 0.001), and monocyte chemoattractant protein (MCP)-4 (P < 0.01), compared with sinonasal tissue from subjects with CRSsNP and control subjects. Expression of IFN-γ messenger RNA or protein was low and not different among the chronic rhinosinusitis subtypes examined. Compared with CRSwNP, AERD NP had elevated protein levels of eosinophil cationic protein (ECP) (P < 0.001), granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.01), and MCP-1 (P = 0.01), as well as decreased gene expression of tissue plasminogen activator (tPA) (P = 0.02). Despite the higher eosinophilia in AERD, there was no associated increase in type 2 mediator protein levels observed. CONCLUSIONS: CRSwNP was characterized by a predominant type 2 inflammatory environment, whereas CRSsNP did not reflect a classic type 1 milieu, as has been suggested previously. AERD can be distinguished from CRSwNP by elevated ECP levels, but this enhanced eosinophilia is not associated with elevations in traditional type 2 inflammatory mediators associated with eosinophil proliferation and recruitment. However, other factors, including GM-CSF, MCP-1, and tPA, may be important contributors to AERD pathogenesis.

Oncostatin M promotes mucosal epithelial barrier dysfunction, and its expression is increased in patients with eosinophilic mucosal disease.

BACKGROUND: Epithelial barrier dysfunction is thought to play a role in many mucosal diseases, including asthma, chronic rhinosinusitis (CRS), and eosinophilic esophagitis. OBJECTIVE: The objective of this study was to investigate the role of oncostatin M (OSM) in epithelial barrier dysfunction in human mucosal disease. METHODS: OSM expression was measured in tissue extracts, nasal secretions, and bronchoalveolar lavage fluid. The effects of OSM stimulation on barrier function of normal human bronchial epithelial cells and nasal epithelial cells cultured at the air-liquid interface were assessed by using transepithelial electrical resistance and fluorescein isothiocyanate-dextran flux. Dual-color immunofluorescence was used to evaluate the integrity of tight junction structures in cultured epithelial cells. RESULTS: Analysis of samples from patients with CRS showed that OSM mRNA and protein levels were highly increased in nasal polyps compared with those seen in control uncinate tissue (P < .05). OSM levels were also increased in bronchoalveolar lavage fluid of allergic asthmatic patients after segmental allergen challenge and in esophageal biopsy specimens from patients with eosinophilic esophagitis. OSM stimulation of air-liquid interface cultures resulted in reduced barrier function, as measured by decreased transepithelial electrical resistance and increased fluorescein isothiocyanate-dextran flux (P < .05). Alterations in barrier function by OSM were reversible, and the viability of epithelial cells was unaffected. OSM levels in lysates of nasal polyps and uncinate tissue positively correlated with levels of α2-macroglobulin, a marker of epithelial leak, in localized nasal secretions (r = 0.4855, P < .05). CONCLUSIONS: These results suggest that OSM might play a role in epithelial barrier dysfunction in patients with CRS and other mucosal diseases.

Involvement of Toll-like receptor 2 and epidermal growth factor receptor signaling in epithelial expression of airway remodeling factors.

Staphylococcus aureus (SA) colonization and infection is common, and may promote allergic or inflammatory airway diseases, such as asthma, cystic fibrosis, and chronic rhinosinusitis by interacting with airway epithelial cells. Airway epithelial cells not only comprise a physical barrier, but also play key roles in immune, inflammatory, repair, and remodeling responses upon encounters with pathogens. To elucidate the impact of SA on epithelial-mediated remodeling of allergic airways, we tested the hypothesis that SA can enhance the remodeling process. Normal human bronchial epithelial (NHBE) cells were stimulated with heat-killed SA (HKSA) or transforming growth factor (TGF) α. Cell extracts were collected to measure mRNA (real-time RT-PCR) and signaling molecules (Western blot); supernatants were collected to measure protein (ELISA) after 24 hours of stimulation. Epidermal growth factor receptor (EGFR) signaling inhibition experiments were performed using a specific EGFR kinase inhibitor (AG1478) and TGF-α was blocked with an anti-TGF-α antibody. HKSA induced both mRNA and protein for TGF-α and matrix metalloproteinase (MMP) 1 from NHBE cells by a Toll-like receptor 2-dependent mechanism. Recombinant human TGF-α also induced mRNA and protein for MMP-1 from NHBE cells; anti-TGF-α antibody inhibited HKSA-induced MMP-1, suggesting that endogenous TGF-α mediates the MMP-1 induction by HKSA. HKSA-induced MMP-1 expression was suppressed when a specific EGFR kinase inhibitor was added, suggesting that EGFR signaling was mediating the HKSA-induced MMP-1 release. Exposure or colonization by SA in the airway may enhance the remodeling of tissue through a TGF-α-dependent induction of MMP-1 expression, and may thereby promote remodeling in airway diseases in which SA is implicated, such as asthma and chronic rhinosinusitis.

Suppression of epithelial signal transducer and activator of transcription 1 activation by extracts of Aspergillus fumigatus.

Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-ß or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ-induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-ß and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-ß or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-ß and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-ß. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-ß, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6 hours. Our results show that AF extracts suppressed expression of inflammatory cytokines in association with inhibition of the IFN-ß signaling pathway and suppression of the formation of pSTAT1.