Olfactory cleft mucus proteins associated with olfactory dysfunction in a cohort without chronic rhinosinusitis.

BACKGROUND: Olfactory dysfunction (OD) is a common problem, affecting up to 20% of the general population. Previous studies identified olfactory cleft mucus proteins associated with OD in chronic rhinosinusitis (CRS) but not in a healthy population. In this study we aimed to identify olfactory cleft mucus proteins associated with olfaction in individuals without sinus disease. METHODS: Subjects free of sinus disease completed medical history questionnaires that collected data regarding demographics, comorbidities, and past exposures. Olfactory testing was performed using Sniffin' Sticks, evaluating threshold, discrimination, and identification. Olfactory cleft mucus (OC) and, in select cases, inferior turbinate mucus (IT) were collected with Leukosorb paper and assays performed for 17 proteins, including growth factors, cytokines/chemokines, cell-cycle regulators, and odorant-binding protein (OBP). RESULTS: Fifty-six subjects were enrolled in the study, with an average age of 47.8 (standard deviation [SD], 17.6) years, including 33 females (58.9%). The average threshold/discrimination/identification (TDI) score was 30.3 (SD, 6.4). In localization studies, OBP concentrations were significantly higher in OC than IT mucus (p = 0.006). Cyclin-dependent kinase inhibitor 2A (CDKN2A/p16INK4a), basic fibroblast growth factor (bFGF), chemokine ligand 2 (CCL2/MCP-1), granulocyte macrophage colony-stimulating factor (GM-CSF), and chemokine ligand 20 (CCL20/MIP-3a) all inversely correlated with overall TDI (all rho ≥ -0.479, p ≤ 0.004). Stem cell factor (SCF) correlated positively with overall TDI (rho = 0.510, p = 0.002). CONCLUSION: Placement of Leukosorb paper is relatively site-specific for olfactory proteins and it is feasible to collect a variety of olfactory cleft proteins that correlate with olfactory function. Further study is required to determine mechanisms of OD in non-CRS subjects.

Dietary vitamin D3 deficiency exacerbates sinonasal inflammation and alters local 25(OH)D3 metabolism.

RATIONALE: Patients with chronic rhinosinusitis with nasal polyps (CRSwNP) have been shown to be vitamin D3 (VD3) deficient, which is associated with more severe disease and increased polyp size. To gain mechanistic insights into these observational studies, we examined the impact of VD3 deficiency on inflammation and VD3 metabolism in an Aspergillus fumigatus (Af) mouse model of chronic rhinosinusitis (Af-CRS). METHODS: Balb/c mice were fed control or VD3 deficient diet for 4 weeks. Mice were then sensitized with intraperitoneal Af, and one week later given Af intranasally every three days for four weeks while being maintained on control or VD3 deficient diet. Airway function, sinonasal immune cell infiltrate and sinonasal VD3 metabolism profiles were then examined. RESULTS: Mice with VD3 deficiency had increased Penh and sRaw values as compared to controls as well as exacerbated changes in sRaw when coupled with Af-CRS. As compared to controls, VD3 deficient and Af-CRS mice had reduced sinonasal 1α-hydroxylase and the active VD3 metabolite, 1,25(OH)2D3. Differential analysis of nasal lavage samples showed that VD3 deficiency alone and in combination with Af-CRS profoundly upregulated eosinophil, neutrophil and lymphocyte numbers. VD3 deficiency exacerbated increases in monocyte-derived dendritic cell (DC) associated with Af-CRS. Conversely, T-regulatory cells were decreased in both Af-CRS mice and VD3 deficient mice, though coupling VD3 deficiency with Af-CRS did not exacerbate CD4 or T-regulatory cells numbers. Lastly, VD3 deficiency had a modifying or exacerbating impact on nasal lavage levels of IFN-γ, IL-6, IL-10 and TNF-α, but had no impact on IL-17A. CONCLUSIONS: VD3 deficiency causes changes in sinonasal immunity, which in many ways mirrors the changes observed in Af-CRS mice, while selectively exacerbating inflammation. Furthermore, both VD3 deficiency and Af-CRS were associated with altered sinonasal VD3 metabolism causing reductions in local levels of the active VD3 metabolite, 1,25(OH)2D3, even with adequate circulating levels.

Impact of tobacco smoke on upper airway dendritic cell accumulation and regulation by sinonasal epithelial cells.

BACKGROUND: In these studies we examined the impact of environmental tobacco smoke (ETS) and active smoking on sinonasal dendritic cell (DC) subsets in controls or patients with chronic rhinosinusitis with nasal polyps (CRSwNP). In subsequent in-vitro investigations, we examined the influence of cigarette smoke extract (CSE) on human sinonasal epithelial cells' (HSNECs) ability to regulate DC functions. METHODS: Sinonasal tissue, blood, and hair were collected from patients undergoing sinus surgery. Smoking status and ETS exposure were determined by hair nicotine. DC subsets were examined by flow cytometric analysis. Monocyte-derived dendritic cells (moDCs) were treated with conditioned medium from non-smoked-exposed HSNECs (NS-HSNECs) or cigarette-smoke-extract-exposed HSNECs (CSE-HSNECs) to assess the impact of CSE exposure on HSNEC regulation of moDC functions. RESULTS: Control subjects who were active smokers displayed increased sinonasal moDC and myeloid dendritic 1 (mDC1) cells and reduced mDC2 cells, whereas, in CRSwNP patients, only moDC and mDC2 cells were altered. ETS was found to increase only moDCs in the CRSwNP patients. In vitro, CSE stimulated HSNEC secretion of the moDC regulatory products chemokine (C-C motif) ligand 20, prostaglandin E2 , and granulocyte-macrophage colony-stimulating factor. CSE exposure also promoted HSNECs to stimulate monocyte and moDC migration. moDCs treated with CSE-HSNEC media stimulated an increase in antigen uptake and expression of CD80 and CD86. Last, CSE-HSNEC-treated moDCs secreted increased levels of interleukin-10, interferon-γ, and thymic stromal lymphopoietin. CONCLUSION: Active smoking, and to a lesser degree ETS, alters the sinonasal composition of DCs. A potential mechanism to account for this is that cigarette smoke stimulates HSNECs to induce moDC migration, maturation, and activation.

Reduced sinonasal levels of 1α-hydroxylase are associated with worse quality of life in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Patients with chronic rhinosinusitis with nasal polyps (CRSwNP) have deficiencies in circulating and sinonasal levels of the inactive form of vitamin D3, 25-hydroxycholecalciferol (25VD3). Moreover, CRSwNP patients have reduced epithelial cell-specific expression of 1α-hydroxylase; the enzyme responsible for the conversion of 25VD3 to its metabolically active form, 1α,25-dihydroxyvitamin D3 (1,25VD3). The objective of this work was to determine the impact of sinonasal 1α-hydroxylase levels combined from all cellular sources on subjective disease severity and to identify variables influencing its expression. METHODS: Blood and sinus tissue explants were collected at the time of surgery from control, chronic rhinosinusitis without nasal polyps (CRSsNP), CRSwNP, and allergic fungal rhinosinusitis (AFRS) patients. 1α-Hydroxylase was measured by immunostaining with flow cytometric analysis. Subjective disease severity was measured by the 22-item Sino-Nasal Outcomes Test (SNOT-22). 1,25VD3 and 25VD3 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Patients with CRSwNP or AFRS have reduced 1α-hydroxylase and 1,25VD3 compared to controls or CRSsNP. Circulating 1,25VD3 levels were the same among all groups. No differences in sinonasal 1α-hydroxylase or 1,25VD3 were found between CRSwNP and AFRS. Gender, age, race, atopy, and systemic 25VD3 had no impact on sinonasal 1α-hydroxylase levels in any group. However, CRSwNP patients with asthma had higher 1α-hydroxylase than those without asthma. Total 1α-hydroxylase levels inversely correlated with SNOT-22 in CRSwNP, but not CRSsNP. CONCLUSION: Patients with CRSwNP and AFRS both have reduced sinonasal 1α-hydroxylase and 1,25VD3 compared to controls or CRSsNP. Reductions in intracellular 1α-hydroxylase combined from all sinonasal cell types were associated with more severe subjective disease severity in CRSwNP.

Superoxide dismutase reduces the inflammatory response to Aspergillus and Alternaria in human sinonasal epithelial cells derived from patients with chronic rhinosinusitis.

BACKGROUND: Aspergillus fumigatus and Alternaria alternata are ubiquitous environmental fungal allergens that can exacerbate airway inflammation and contribute to the disease process in patients with chronic rhinosinusitis (CRS). These antigens have been shown to induce human sinonasal epithelial cells (HSNECs) to promote a proinflammatory response, but what is unclear is a means by which to reduce these effects. Inhaled pathogens can induce HSNECs to produce reactive oxygen species (ROS) that trigger cytokine production. OBJECTIVE: This study aimed to determine whether the free radical scavenger superoxide dismutase (SOD) could reduce HSNEC-derived inflammation, as measured by interleukin (IL)-6 and IL-8 production, in response to Aspergillus or Alternaria exposure. METHODS: Sinus tissue explants were collected at the time of surgery from control patients (n = 7) and patients with CRS with nasal polyps (CRSwNP) (n = 9). HSNECs were cultured from the explants and treated with Aspergillus, Alternaria, and SOD for 24 hours. Cell supernatants and lysates were collected, and IL-6 and IL-8 concentrations were measured using enzyme-linked immunosorbent assay. RESULTS: In control and CRSwNP HSNECs, Aspergillus and Alternaria both increased cytokine production (p < 0.05), as measured by IL-6 and IL-8 concentration. SOD treatment reduced the inflammatory response to fungal antigen exposure from CRSwNP HSNECs but not control HSNECs. In CRSwNP patients, SOD significantly decreased IL-6 and IL-8 production after Alternaria exposure and IL-8 after Aspergillus exposure (p < 0.05). CONCLUSIONS: When HSNECs from CRSwNP patients are treated with SOD concurrently with Aspergillus or Alternaria, SOD treatment decreases the fungal antigen-induced inflammatory response. The ability to attenuate inflammation induced by common fungal allergens with SOD treatment could provide a novel therapeutic or preventative approach for patients with CRS or other allergic inflammatory airway diseases.