All chronic rhinosinusitis endotype clusters demonstrate improvement in patient-reported and clinical outcome measures after endoscopic sinus surgery.

BACKGROUND: It is unclear whether chronic rhinosinusitis (CRS) endotypes show a differential response to endoscopic sinus surgery (ESS). We explored patient mucous inflammatory cytokine expression and associations with patient-reported and clinically measured post-operative outcome measures. METHODS: Patients with CRS were prospectively recruited between 2016 and 2021 into a national multicenter, observational study. Mucus was collected from the olfactory cleft preoperatively and evaluated for 26 biomarkers using cluster analysis. Patient-reported outcome measures included the 22-item Sino-Nasal Outcome Test (SNOT-22) and Questionnaire of Olfactory Dysfunction (QOD). Additional clinical measures of disease severity included threshold, discrimination, and identification (TDI) scores using "Sniffin' Sticks" testing and Lund-Kennedy endoscopic score (LKES). RESULTS: A total of 115 patients were clustered into type 2 inflammatory, non-type 2 inflammatory, noninflammatory, and two indeterminate clusters based on individual protein levels. Overall, the type 2 inflammatory cluster was found to have the highest mean improvement in both SNOT-22 (-28.3 [standard deviation, ±16.2]) and TDI (6.5 [standard deviation, ±7.9]) scores 6 months after ESS. However, on average, all endotype clusters demonstrated improvement in all outcome measures after ESS without statistically significant between-group differences in SNOT-22 (p = 0.738), QOD (p = 0.306), TDI (p = 0.358), or LKES (p = 0.514) measures. CONCLUSIONS: All CRS endotype clusters responded favorably to surgery and showed improvements in patient-reported and objective outcome measures. Thus, ESS should be considered a more generalized CRS therapy, and benefits appear to not be limited to specific endotypes.

Sinonasal IgG4-related sclerosing disease: A rare entity and challenging diagnosis.

Objectives: To describe the rare presentation, imaging and histological findings, and treatments in patients with IgG4-related disease (IgG4-RD) and diagnostic pitfalls and difficulties. Methods: Cases of sinonasal IgG4-RD were retrieved, and clinicopathological features were reviewed. Results: Seven cases of sinonasal IgG4-RD were identified over an 11-year period, including four males and three females, with an age range of 19-66 years (median 58 years). Patients presented with symptoms related to the mass effect of the lesions or the destructive nature of the disease including fullness, swelling, obstruction, and pain. Serum IgG and IgG4 levels, IgG/IgG4 ratios, storiform fibrosis, obliterative phlebitis, and plasma cell infiltration were seen in varying proportions. Bony erosion and tissue inflammation were present in some cases. Conclusion: Sinonasal IgG4-RD is exceedingly rare among other IgG4-RD and varied in its clinical presentation thus posing as a clinically difficult disease to diagnosis. Proper clinical, pathological, and immunohistopathological analysis is required for accurate diagnosis. Such disease should be considered in all cases of similar presentation to those in this study.Level of Evidence: 4.

Utilization of Nasal Mucus to Investigate the Pathophysiology of Chronic Rhinosinusitis.

BACKGROUND: Nasal mucus is proving to be a useful means by which to study the pathogenesis of chronic rhinosinusitis (CRS). Given the increase in publications examining nasal mucus and the lack of a review on this topic, we will focus on this noninvasive approach to studying CRS. Particular attention will be drawn towards inflammatory cytokines and biomarkers and their influence on disease severity. METHODS: A literature review of papers published in English pertaining to nasal mucus was performed using the PubMed database. The search utilized combinations of the following keywords: sinusitis, polyps, sample collection, nasal mucus, or nasal secretion. Studies solely on acute or bacterial sinusitis, allergic rhinitis, or cystic fibrosis were not included. RESULTS: A wide variety of materials and methods have been used to collect nasal mucus. Numerous assay types have been performed with the most common being ELISA, cytometric bead array, and proteomics. Most studies have focused on examining the levels of Th1/Th2 cytokines along with chemokines associated with type 2 immunity. Other factors identified include growth factors, senescence-associated proteins, complement, and antimicrobial defenses have also been identified. Nasal mucus cytokines have proven useful in cluster analysis and predicting postoperative improvement in Sino-nasal Outcome Test (SNOT-22) scores. One limitation of the use of nasal mucus is that some studies have suggested that nasal mucus does not always reflect the tissue microenvironment. CONCLUSIONS: Nasal mucus represents a critical tool by which to examine the sinonasal microenvironment in a noninvasive manner. Unlike studies of tissue, it can be utilized in both surgically and medically managed patients and avoids the trauma of biopsies. However, studies are still needed to determine the most effective method for nasal mucus collection. Studies should also take care to confirm that nasal mucus markers do, in fact, reflect the levels of the product studied in the tissue.

Role of C3a as a Novel Regulator of 25(OH)D3 to 1α,25-Dihydroxyvitamin D3 Metabolism in Upper Airway Epithelial Cells.

In patients with chronic rhinosinusitis with nasal polyps, primary human sinonasal epithelial cell (HSNEC) 1α-hydroxylase levels are reduced, as is their ability to metabolize 25-hydroxycholecalciferol [25(OH)D3] to its active metabolite, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In this study, we sought to identify the factor responsible for the regulation of HSNEC metabolism of 25(OH)D3, focusing on C3 and C3a. Multiple inhaled irritants trigger the release of complement components, C3 and C3a, leading to suppression of 1α-hydroxylase levels in HSNECs. Recombinant C3a was able to decrease 1α-hydroxylase and impair 25(OH)D3 to 1,25(OH)2D3 metabolism, while addition of a C3a receptor antagonist restored conversion. Conversely, 1,25(OH)2D3 suppressed Aspergillus fumigatus-induced C3 and C3a levels in HSNEC supernatant. Given the ability of 1,25(OH)2D3 to modulate LL37 in other cell types, we examined its regulation in HSNECs and relationship to C3a. 1,25(OH)2D3 stimulated the secretion of LL37, whereas A. fumigatus and C3a suppressed it. Conversely, LL37 reduced the release of C3/C3a by HSNECs. Lastly, oral steroid use and in vitro dexamethasone application both failed to increase 1α-hydroxylase or reduce C3a levels. In summary, in this article, we describe for the first time a novel relationship between complement activation and local vitamin D metabolism in airway epithelial cells. The presence of elevated C3/C3a in patients with asthma and/or chronic rhinosinusitis with nasal polyps may account for their impaired HSNEC 25(OH)D3 to 1,25(OH)2D3 metabolism and explain why they receive limited therapeutic benefit from oral vitamin D3 supplementation.

Dexamethasone Alters Tracheal Aspirate T-Cell Cytokine Production in Ventilated Preterm Infants.

Postnatal corticosteroids improve respiratory status and facilitate respiratory support weaning in preterm infants with bronchopulmonary dysplasia (BPD). Older literature describes characteristic cytokine profiles in tracheal aspirates (TA) of BPD patients which are altered with corticosteroids. Corticosteroids also influence peripheral blood T-cell presence. However, little is known regarding TA T-cell phenotype and cytokine production before or after exogenous corticosteroids. We hypothesized that postnatal dexamethasone alters the TA T-cell cytokine profiles of preterm infants. TA samples were collected from 14 infants born from 23 0/7 to 28 6/7 weeks who were mechanically ventilated for at least 14 days. Samples were collected up to 72 h before a ten-day dexamethasone course and again 1 to 3 calendar days after dexamethasone initiation. The primary outcome was change in T cell populations present in TA and their intracellular cytokine profile after dexamethasone treatment, ascertained via flow cytometry. Following dexamethasone treatment, there were significant decreases in respiratory severity score (RSS), percent CD4+IL-6+ cells, CD8+IL-6+ cells, CXCR3+IL-6+ cells, and CXCR3+IL-2+ cells and total intracellular IFN-γ in TA. RSS significantly correlated with TA percent CD4+IL-6+ cells. To our knowledge, this is the first study demonstrating that dexamethasone reduced T-cell IL-6 and this reduction was associated with improved RSS in pre-term infants with evolving BPD.

Olfactory cleft mucus inflammatory proteins in CRS: a case-control study.

BACKGROUND: Multiple hypotheses are evolving that suggest several, potentially overlapping etiologies for olfactory dysfunction (OD) in chronic rhinosinusitis (CRS). Understanding inflammatory cytokine profiles of the olfactory cleft (OC) and their association with olfactory function is foundational for future clinical care and research. METHODS: This cross-sectional, case-control study evaluates associations among OC mucus inflammatory proteins, psychophysical olfactory testing, and computed tomography (CT) analysis of the OC and sinuses. Normative reference intervals were determined for each protein and odds ratios (ORs) were used to compare proportions of altered expression between CRS without nasal polyposis (CRSsNP) and CRS without nasal polyposis (CRSwNP). RESULTS: Case subjects with CRS (n = 151) and controls (n = 74) were evaluated. A majority of OC proteins tested were found within detectable ranges for cases and controls. The CRS cohort had significantly higher concentrations for 23 of 26 proteins. CRS cases with abnormal levels of C-C motif chemokine ligand 2 (CCL2), CCL3, interleukin 5 (IL5), IL10, and IL13 associated with greater olfactory deficits. The prevalence of elevated IL5 and IL13 in anosmic patients was 64.6% and 62.5%, respectively (p < 0.004). CRS cases with the highest odds of elevated expression in CRSwNP were IL5 (OR = 10.83) and IL13 (OR = 8.36). However, both IL5 and IL13 were still elevated in approximately 14% of CRSsNP patients. The highest magnitude of correlation between the total percent of OC opacification was found to be with IL5 (r = 0.543; p < 0.001), whereas other moderate correlations were noted with immunoglobulin E (IgE), IL10, and IL13. CONCLUSION: This study confirmed that OC inflammatory proteins vary both by disease phenotype and in their association with OD. Type 2 inflammatory mediators are increased in CRS, especially within the CRSwNP group. However, a substantial proportion of CRSsNP also express type 2 inflammatory mediators. Further research is necessary to understand the complex roles OC mucous inflammatory proteins might play in defining endotype and in impacting CRS-related OD. ©2021 ARSAAOA, LLC.

Endotyping chronic rhinosinusitis based on olfactory cleft mucus biomarkers.

BACKGROUND: Although chronic rhinosinusitis (CRS) is considered the most treatable form of olfactory dysfunction, there has been relatively little clinical attention focused on assessing endotypes as they pertain to olfactory loss. OBJECTIVES: The goal of this study was to explore inflammatory endotypes in CRS using an unsupervised cluster analysis of olfactory cleft (OC) biomarkers in a phenotype-free approach. METHODS: Patients with CRS were prospectively recruited and psychophysical olfactory testing, Questionnaire of Olfactory Dysfunction (QOD-NS), and bilateral OC endoscopy were obtained. Mucus was collected from the OC and evaluated for 26 biomarkers using principal component analysis. Cluster analysis was performed using only OC biomarkers and differences in olfactory measures were compared across clusters. RESULTS: A total of 198 subjects (128 with CRS and 70 controls) were evaluated. Evaluation of OC biomarkers indicated 6 principal components, explaining 69.50% of the variance, with type 2, mixed type 1/Th17-cell, growth factor, and neutrophil chemoattractant inflammatory signatures. A total of 10 clusters were identified that differed significantly in frequency of controls, and subjects with CRS with nasal polyps, and subjects with CRS without nasal polyps across the clusters (likelihood ratio test, χ182=178.64; P < .001). Olfactory measures differed significantly across clusters, including olfactory testing, QOD-NS, and OC endoscopy (P < .001 for all). CONCLUSIONS: Clustering based solely on OC biomarkers can organize patients into clinically meaningful endotypes that discriminate between subjects with CRS and controls. Validation studies are necessary to confirm these findings and further refine olfactory endotypes.

Effects of vitamin D supplementation on circulating concentrations of growth factors and immune-mediators in healthy women during pregnancy.

BACKGROUND: For the second aim of the Kellogg Foundation grant, this double-blind RCT investigated the impact of plasma vitamin D metabolite 25-hydroxyvitamin D (25(OH)D) on plasma immune-mediators during pregnancy. We hypothesized that higher 25(OH)D concentrations would associate with reduced pro-inflammatory and increased tolerogenic immune-mediator concentrations. METHODS: Pregnant women enrolled at 10-14 weeks gestation were randomized to 400 or 4400 IU vitamin D3/day. Data on health, safety, circulating 25(OH)D, and 9 immune-mediators were collected at each trimester. Associations between immune-mediators and 25(OH)D at baseline and at second and third trimesters were examined. RESULTS: Baseline TGF-ß and second and third trimesters IFN-γ and IL-2 were associated with baseline 25(OH)D. Baseline immune-mediators were associated with immune-mediators at second and third trimesters for all immune-mediators except IL-5 and IL-10. Race was associated with baseline TGF-ß, VEGF and IL-10 and with IL-10 at second and third trimesters. CONCLUSIONS: Both treatment groups had increased 25(OH)D at second and third trimesters, greatest in the 4400 IU group. Though associations between baseline 25(OH)D and baseline TGF-ß and second and third trimester IFN-γ and IL-2 were noted, vitamin D supplementation throughout pregnancy did not impact immune-mediators at later trimesters. Supplementing with vitamin D before conception conceivably influences immune-mediator responses during pregnancy. IMPACT: In this vitamin D supplementation clinical trial, baseline (first trimester) but not increasing plasma 25(OH)D concentration impacted select plasma immune-mediator profiles in pregnant women. Baseline 25(OH)D was associated with baseline TGF-ß and with IFN-γ and IL-2 at second and third trimesters. Baseline IFN-γ, CRP, TGF-ß, TNF-α, VEGF, IL-2, and IL-4 were associated with concentrations at second and third trimesters for respective immune-mediators; however, 25(OH)D concentration at second and third trimesters were not. Some racial differences existed in immune-mediator concentrations at baseline and at second and third trimesters. This study assesses the impact of vitamin D supplementation on multiple immune-mediators in pregnant women of different racial/ethnic groups using longitudinal data from a relatively large randomized controlled trial. This study found that race was associated with baseline TGF-ß, VEGF, and IL-10 and with IL-10 at second and third trimesters, a novel finding that sheds light where relationships were less well defined. The results of this study suggest that vitamin D supplementation before conception or early in pregnancy, rather than during pregnancy, may be necessary to significantly impact immune-mediator response. This study sets premise for future clinical trials to evaluate the effect of vitamin D supplementation before conception or prior to pregnancy.

Olfactory cleft mucus proteome in chronic rhinosinusitis: a case-control pilot study.

BACKGROUND: Mechanisms of smell loss in chronic rhinosinusitis (CRS) are still unclear and likely multifactorial. Little attention has been given to olfactory cleft (OC) mucus proteins involved in odorant binding and metabolizing enzymes and their potential role in smell loss. METHODS: Mucus from the OC was sampled from patients with CRS (n = 20) and controls (n = 10). Liquid chromatography and mass spectrometry were performed, followed by data processing so that protein groups could be identified, quantified, and compared. Hierarchical clustering and bioinformatic analysis were performed on significantly different proteins to explore for enrichment in known biologic pathways. RESULTS: A total of 2514 proteins were found in OC mucus from all 30 subjects. Significant differences in protein abundance were found between CRS and controls, including both CRSsNP (n = 351 proteins; log2 fold change range: -3.88 to 6.71) and CRSwNP (n = 298 proteins; log2 fold change range: -4.00 to -6.13). Significant differences were found between patients with normosmia and those with dysosmia (n = 183; log2 fold change range: -3.62 to -2.16) and across groups of interest for a number of odorant binding proteins and metabolizing enzymes. CONCLUSION: OC mucous in CRS displays a rich and abundant array of proteins, many of which have been implicated in odorant transport and metabolization in animal studies. Significant differences in the olfactory mucus proteome were seen between CRS subtypes and controls, as well as between those with normal and abnormal olfaction. Further study should confirm these findings and explore the role individual proteins play in odorant transport and metabolization. ©2020 ARSAAOA, LLC.

A Community-Based Study on the Prevalence of Olfactory Dysfunction.

BACKGROUND: Olfactory dysfunction (OD) is common, affecting an estimated 13 million adults in the United States. Prior studies may underestimate OD prevalence due to use of brief smell identification tests or age-adjusted cutoff values, which concede that it is acceptable for older people to have a decreased sense of smell. OBJECTIVE: To determine OD prevalence in the healthy community when the goal and expectation is ideal olfactory function, rather than age-based population norms. Secondary goals were to explore factors associated with OD. METHODS: Subjects without otolaryngic complaints were recruited from the community surrounding the Medical University of South Carolina. Olfactory-specific information was collected, and olfactory function was assessed using the Sniffin' Sticks test (Burghardt, Wedel, Germany) to measure threshold, discrimination, and identification (TDI). OD was defined as a TDI score < 31. Bivariate analysis and linear regression were used to determine factors associated with OD. RESULTS: In total, 176 subjects were included with mean age of 52 years (range: 20-93), 111 (63%) female, and 127 (72%) white. Mean TDI score was 28.8 (6.9) and OD was present in 94 (53%) subjects. Multivariate linear regression revealed that TDI decreased an average of 1 point every 5 years. TDI was also associated with Mini-Mental Status Examination (MMSE) score, asthma, and gastroesophageal reflux disease. Threshold was associated with age, heart problems, and gastroesophageal reflux disease. Discrimination was associated with age and MMSE scores. Identification was associated with age, heart problems, and anxiety. CONCLUSIONS: In a community-based sample, OD affects greater than 50% of subjects. Aging impacts all aspects of olfaction, while the effects of factors such as asthma, MMSE scores, gastroesophageal reflux disease, heart problems, and anxiety may only be evident in specific olfactory subtests.

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.

C3a receptor antagonism as a novel therapeutic target for chronic rhinosinusitis.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory disease with an unknown etiology. Recent studies have implicated the complement system as a potential modulator of disease immunopathology. We performed proteomic pathway enrichment analysis of differentially increased proteins, and found an enrichment of complement cascade pathways in the nasal mucus of individuals with CRSwNP as compared to control subjects. Sinonasal mucus levels of complement 3 (C3) correlated with worse subjective disease severity, whereas no significant difference in systemic C3 levels could be determined in plasma samples. Given that human sinonasal epithelial cells were the predominate sinonasal source of C3 and complement anaphylatoxin 3a (C3a) staining, we focused on their role in in vitro studies. Baseline intracellular C3 levels were higher in CRSwNP cells, and following exposure to Aspergillus fumigatus (Af) extract, they released significantly more C3 and C3a. Inhibition of complement 3a receptor (C3aR) signaling led to a decrease in Af-induced C3 and C3a release, both in vitro and in vivo. Finally, we found in vivo that C3aR deficiency or inhibition significantly reduced inflammation and CRS development in a mouse model of Af-induced CRS. These findings demonstrate that local sinonasal complement activation correlates with subjective disease severity, and that local C3aR antagonism significantly ameliorates Af-induced CRS in a rodent model.

Glucocorticoid receptor isoform expression in peripheral blood mononuclear leukocytes of patients with chronic rhinosinusitis.

BACKGROUND: In several inflammatory disorders, altered peripheral blood mononuclear leukocyte (PBML) glucocorticoid (GC) receptor isoform expression has been associated with GC resistance and disease severity. However, it is unclear if PBML GC receptor isoforms are expressed differentially and are associated with worsened disease severity in chronic rhinosinusitis (CRS). METHODS: PBMLs were isolated from control (n = 8), CRS without nasal polyps (CRSsNP) (n = 8), atopic CRS with nasal polyps (CRSwNP) (n = 8), non-atopic CRSwNP (n = 8), and allergic fungal rhinosinusitis (AFRS) (n = 8) patients. Demographics, atopic status, asthmatic status, 22-item Sino-Nasal Outcome Test (SNOT-22) scores, Lund-Kennedy nasal endoscopy scores, Lund-Mackay sinus computed tomography (CT) scores, Kennedy Osteitis scores, and GC utilization 6 months postoperatively were collected. Intracellular immunostaining was then performed for functional GC receptor α (GCRα) and nonfunctional GC receptor ß (GCRß), followed by flow cytometry analysis of geometric mean fluorescent intensity (MFI) and the percentage of cells expressing each GC receptor isoform. RESULTS: Compared to controls, each CRS subtype had decreased PBML GCRα and GCRα:GCRß MFI expression, but no difference in GCRß expression. Decreasing PBML GCRα in AFRS was associated with increasing Lund-Mackay sinus CT scores (r = -0.880, p =0.004). No significant associations were found between GC receptor isoform expression and other clinical measures. CONCLUSION: CRS patients have reduced functional PBML GCRα expression and decreased GCRα:GCRß compared to controls. Reductions in GCRα in AFRS are associated with worsening Lund-Mackay sinus CT scores. The clinical implications of decreased functional GC receptor expression merits further investigation.

Novel Radiographic Assessment of the Cribriform Plate.

Background The cribriform plate (CP) is a common site of spontaneous cerebrospinal fluid (SCSF) leaks. Radiographic assessment of the anterior and lateral skull base has shown thinner bone in patients with SCSFs; however, prior assessment of the CP has required postmortem cadaver dissection. Objective To develop novel radiographic techniques to assess the anatomy of the CP. Methods Computed tomography (CT) scans were performed on cadaveric specimens. Bone density and anatomy of a predefined volume of interest of the posterior CP were assessed by two independent reviewers. CT assessment of olfactory foramina was also performed and validated using anatomic dissection of cadaver specimens. Results Interclass correlation coefficients (ICCs) for measuring the same volume of each CP was 0.96, confirming reproducible anatomic localization. Cadaver CPs had a mean Hounsfield units of 263, indicating a mix of bone and soft tissue, and ICC was 0.98, confirming reproducible radiographic measurements. Optimal CT estimates of bone composition of CPs averaged 85% (range 76% to 96%) compared to actual anatomic dissection which averaged 84% bone (range 74% to 91%, r = .690, P = .026). Conclusion Our novel, noninvasive CT method for assessing CP anatomy is reproducible and correlates with anatomic dissection assessing bone composition. The clinical implications of anatomic changes in the CP are an area for further study.

The role of regulatory T cells in the regulation of upper airway inflammation.

Allergic rhinitis (AR) and chronic rhinosinusitis with nasal polyps (CRSwNP) are inflammatory diseases of the upper airway, with a similar immunologic profile, characterized by aberrant and persistent type 2 inflammation. One cell population that has been identified as altered in both disease types is regulatory T cell (Treg). Tregs have the capacity to modulate T-effector function and suppress inflammatory cytokine production in a broad range of cell types. Given the ability of Tregs to control inflammation, the role of Tregs in respiratory diseases has attracted much attention. As discussed in this article, alterations in the Treg numbers and function, or both, have been identified in AR and CRSwNP, although much of the data is conflicting. Here, we explored what is known and, in many cases, unknown about the mechanisms by which Tregs differentiate and function, and how these functions can be controlled in the mucosal microenvironment. By gaining a greater understanding of these processes, it may be possible to harness the natural immunosuppressive activity of Tregs to ameliorate the chronic inflammation associated with AR and CRSwNP.

Sinonasal T-cell expression of cytotoxic mediators granzyme B and perforin is reduced in patients with chronic rhinosinusitis.

BACKGROUND: CD8+ T cells and natural killer (NK) cells are cytotoxic cells that use granzyme B (GrB) and perforin. Defective cytotoxic function is known to play a role in dysregulated immune response as seen in chronic sinusitis, also referred to as chronic rhinosinusitis (CRS). However, to our knowledge, in the United States, neither GrB or perforin expression has been reported in patients with CRS. OBJECTIVE: The aim of this study was to investigate sinonasal cytotoxic cells, their mediators, and cell-specific distribution of these mediators in patients with CRS with nasal polyp (CRSwNP) and in patients with CRS without nasal polyp (CRSsNP). METHODS: Blood and sinus tissue samples were taken from patients with CRSsNP (n = 8) and CRSwNP (n = 8) at the time of surgery. Control subjects (n = 8) underwent surgery for cerebrospinal fluid leak repair or to remove non-hormone-secreting pituitary tumors. The cells were analyzed via flow cytometry by using CD8 expression to identify cytotoxic T cells and CD56 expression to identify NK cells. Intracellular GrB and perforin expression were analyzed with flow cytometry. RESULTS: We observed no significant differences in plasma or peripheral blood immune cell numbers or specific levels of GrB or perforin among the groups. In the sinonasal mucosa of the patients with CRSsNP and the patients with CRSwNP, there was a significant decrease in GrB and perforin levels (p < 0.05) despite similar or increased numbers of cytotoxic cells when compared with the controls. The overall decrease in GrB and perforin in the sinonasal mucosa of the patients with CRSsNP and the patients with CRSwNP was due to decreased T cell production. There was no difference in total NK cell count or expression of perforin or GrB among all the groups. CONCLUSION: Total levels of sinonasal GrB and perforin were decreased in the sinonasal mucosa of both the patients with CRSwNP and the patients with CRSsNP compared with the controls, whereas sinonasal CD8+ T cells, (but not NK cells,), intracellular stores of GrB and perforin were reduced in the patients with CRSwNP compared with the controls.

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.

Induction of CFTR gene expression by 1,25(OH)2 vitamin D3, 25OH vitamin D3, and vitamin D3 in cultured human airway epithelial cells and in mouse airways.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which often leads to protein misfolding and no CFTR surface localization. This then leads to chronic airway infections, inflammation, and tissue damage. Although vitamin D has been explored as a therapy to treat CF due to its antimicrobial-inducing and anti-inflammatory properties, the effect of 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on CFTR directly has not been studied. We treated cultured healthy and diseased bronchial epithelial cells (BEC) with 10nM 1α,25(OH)2D3 for 6 and 24h and found that 1α,25(OH)2D3 increases both mRNA and protein CFTR levels using RT-qPCR, flow cytometry and fluorescence immunohistochemistry. Treatment of CF cells with 10nM 1α,25(OH)2D3 led to an increase in both total and surface CFTR expression, suggesting 1α,25(OH)2D3 could be used to increase properly localized CFTR in airway cells. To determine if BEC could convert the more clinically relevant cholecalciferol to 25OHD3, cultured non-CF and CF BECs were treated with a range of cholecalciferol concentrations, and 25OHD3 levels were quantified by ELISA. We found that 25OHD3 levels increased in a concentration-dependent manner. Treatment of BEC with 10µM cholecalciferol led to increases in both CYP24A1 and CFTR mRNA levels, even when added to the apical surface of cells grown in an air-liquid interface, suggesting that topical administration of vitamin D could be used therapeutically. To demonstrate this in vivo, we intranasally delivered 1µM 1α,25(OH)2D3 into mice. After 6h, we observed induction of both Cyp24A1 and CFTR expression in the tracheas of treated mice. The major findings of this study are that vitamin D can be converted to the active form when topically administered to the airway, and this could be used to increase CFTR levels in patients with CF. This could potentially be useful as an adjunctive therapy, together with newly developed CF treatments.