Ryan M. Mulligan

Vitamin D3 correlates inversely with systemic dendritic cell numbers and bone erosion in chronic rhinosinusitis with nasal polyps and allergic fungal rhinosinusitis

Vitamin D3 (VD3) is a steroid hormone that regulates bone health and numerous aspects of immune function and may play a role in respiratory health. We hypothesized that T helper type 2 (Th2) disorders, chronic rhinosinusitis with nasal polyps (CRSwNP) and allergic fungal rhinosinusitis (AFRS) would have VD3 deficiencies, resulting in increased mature dendritic cells (DCs) and bone erosion. We conducted a retrospective study examining VD3 levels in patients with AFRS (n = 14), CRSwNP (n = 9), chronic rhinosinusitis without nasal polyps (CRSsNP) (n = 20) and cerebrospinal fluid leak repair (non‐diseased controls) (n = 14) at time of surgery. Circulating immune cell levels were determined by immunostaining and flow cytometric analysis. Plasma VD3 and immune regulatory factors (granulocyte–macrophage colony‐stimulating factor and prostaglandin E2) were measured by enzyme‐linked immunosorbent assay. It was observed that CRSwNP and AFRS demonstrated increased circulating DCs, while chronic rhinosinusitis without nasal polyps displayed increased circulating macrophages. CRSwNP and AFRS were to found to have insufficient levels of VD3 which correlated inversely with circulating numbers of mature DCs, DC regulatory factors and bone erosion. CRSsNP displayed no change in circulating DC numbers or VD3 status compared to control, but did display increased numbers of circulating macrophages that was independent of VD3 status. Lastly, VD3 deficiency was associated with more severe bone erosion. Taken together, these results suggest support a role for VD3 as a key player in the immunopathology of CRSwNP and AFRS.

Increased presence of dendritic cells and dendritic cell chemokines in the sinus mucosa of chronic rhinosinusitis with nasal polyps and allergic fungal rhinosinusitis

The aim of this study was to determine if there is a link between local dendritic cells (DCs) and various subtypes of chronic rhinosinusitis (CRS): CRS with nasal polyposis (CRSwNP), CRS without nasal polyposis (CRSsNP), and allergic fungal rhinosinusitis (AFRS). Once DC presence was established we considered possible mechanisms for DC recruitment to the sinuses.

Antigen-specific IgE in sinus mucosa of allergic fungal rhinosinusitis patients.

Background Local tissue production of antigen-specific immunoglobulin E (IgE) has been shown in patients with allergic rhinitis and in patients with chronic rhinosinusitis (CRS) with nasal polyps. In allergic fungal rhinosinusitis (AFRS), specific IgE has been established in nasal lavage fluid and eosinophilic mucin. In this study, local production of antigen-specific IgE within sinus mucosa of AFRS patients was evaluated. Methods Sinus mucosa homogenates from 11 AFRS patients, 8 patients with CRS without nasal polyps (CRSsNP), and 9 nonrhinosinusitis control patients were assessed for IgE localization by immunohistochemistry. AFRS and control tissue homogenates were also evaluated for antigen-specific IgE to 14 common antigens by ImmunoCAP testing (Phadia AB, Portage, MI). Results There was a significant increase in IgE staining in AFRS sinus epithelium and subepithelium compared with controls and with patients with CRSsNP (p ≤ 0.012 for all group differences). AFRS patients showed increased IgE staining in the subepithelium when compared with epithelium (p < 0.001). AFRS sinus tissue had significantly more IgE measured by ImmunoCAP when compared with control sinus tissue for 7 of 14 specific antigens (p < 0.05) and for total IgE (p = 0.004). Antigens with a significant difference on ImmunoCAP included Cladosporium, Aspergillus, Timothy grass, red maple, cockroach, ragweed, and cocklebur. Conclusion AFRS patients showed significantly more IgE in sinus mucosa tissue specimens, with increased IgE in subepithelial sites when compared with epithelium. The increased expression of antigen-specific IgE is not limited to fungal antigens. These findings support the role of type I hypersensitivity and local manifestations of allergy in AFRS patients.

Vitamin D3 deficiency increases sinus mucosa dendritic cells in pediatric chronic rhinosinusitis with nasal polyps.

Objective Dendritic cells are professional antigen presenting cells, capable of initiating Th1 or Th2 responses, and have been implicated in the pathogenesis of a number of diseases, including sinusitis. Vitamin D3 is a steroid hormone that acts on dendritic cells in a manner similar to corticosteroids. Investigators examined whether children with allergic fungal rhinosinusitis (AFRS) or chronic rhinosinusitis with nasal polyposis (CRSwNP) were vitamin D3 deficient and the relationship of vitamin D3 deficiency to dendritic cell infiltrate in the sinus mucosa. Setting Tertiary care university hospital. Study Design Retrospective, controlled study using samples collected from pediatric patients seen from August 2009 to July 2011. Subjects and Methods Plasma levels of 25-hydroxy vitamin D3 were measured by enzyme-linked immunosorbent assay in children (≤18 years old) with AFRS, CRSwNP, or CRS without nasal polyposis (CRSsNP) and in controls undergoing surgery for adenotonsillar hypertrophy. Vitamin D3 levels were confirmed using clinical diagnostic methods for those with CRSwNP or AFRS. Tissue samples were immunohistochemically stained for the dendritic cell marker CD209 and the costimulatory molecules CD80 and CD86. Results There was no difference in mean vitamin D3 levels between control and CRSsNP, whereas mean CRSwNP and AFRS levels were both well below the minimum recommended level of 30 ng/mL and significantly lower than control and CRSsNP levels. CD209+ dendritic cells inversely correlated with vitamin D3 but not costimulatory molecule expression. Conclusions These studies identify that children with CRSwNP or AFRS are vitamin D3 deficient, which may be linked to increased dendritic cell infiltrate. These results suggest a role for vitamin D3 as a key player in the immunopathology of pediatric CRSwNP.

Local production of antigen-specific IgE in different anatomic subsites of allergic fungal rhinosinusitis patients

Objective: Local production of antigen-specific IgE in allergic fungal rhinosinusitis (AFRS) is likely integral to the expression of allergy. This study examines if there are anatomic variations in local IgE expression or if variations among fungal and nonfungal IgE exist. Study Design: Cross-sectional study. Setting: Tertiary medical center. Subjects and Methods: Specimens from 11 AFRS, 8 chronic rhinosinusitis without nasal polyps (CRSsNP), and 9 control patients underwent immunohistochemical localization for IgE and evaluation for antigen-specific IgE by ImmunoCAP testing. Results: Inferior turbinate (IT) epithelium had greater IgE staining in AFRS than control (P = 0.013) and CRSsNP (P = 0.002). A significant difference was also found at the IT subepithelial level for AFRS compared with controls (P = 0.001) and CRSsNP (P < 0.001). Within AFRS, IgE staining was increased in the subepithelium compared to epithelium (P = 0.003). ImmunoCAP analysis on IT tissue from AFRS and controls demonstrated increased antigen-specific IgE for 5 of 14 antigens (P < 0.05) and total IgE (P < 0.001). There were no significant anatomic differences between IT and sinus IgE staining. Conclusion: More fungal and nonfungal IgE is expressed in IT and sinus tissues of AFRS patients, as compared with control and CRSsNP patients.

Cigarette smoke extract stimulates interleukin-8 production in human airway epithelium and is attenuated by superoxide dismutase in vitro.

Background Cigarette smoke exposure (CSE) results in extensive inflammation in the upper and lower airways. Reactive oxygen species, such as superoxide, have been shown to be potent mediators of this inflammation. Methods Mucosal biopsy specimens were collected from patients undergoing sinonasal surgery and were used as a source of primary epithelial cells. Human sinonasal epithelial (HSNE) cells and were isolated from sinus tissue, maintained in culture, and ultimately treated with varying concentrations of CSE with or without free superoxide dismutase (SOD). Supernatants and cell lysates were examined for the proinflammatory cytokine interleukin (IL)-8. Similar experiments were performed using normal human bronchial epithelial (NHBE) cell lines. Results CSE induces both secretion and intracellular production of the proinflammatory cytokine IL-8 by HSNE cells in a dose-dependent manner. Furthermore, this up-regulation can be suppressed by SOD. CSE induces secretion of IL-8 in NHBEs that is also suppressed by SOD. Conclusion Inflammation in the airway after CSE can be blocked by SOD in this in vitro model. The ability to attenuate CSE-induced inflammation with SOD could provide a therapeutic/preventative approach for individuals with cigarette smoke exposure.

Alterations in Gene Expression of Complement Components in Chronic Rhinosinusitis

Background The complement cascade forms part of the initial innate response to pathogens in the airway. Complement activation is important in the maintenance of host homeostasis, but excessive and uncontrolled activation may lead to inflammation and disease. The role of the complement pathway in the innate response in chronic rhinosinusitis (CRS) is poorly characterized Methods Sinus mucosa biopsy specimens from the anterior ethmoid or uncinate process of patients with allergic fungal rhinosinusitis (AFRS), CRS without NPs (CRS–NPs), and controls were harvested and gene and protein expression of C3, factor B (fB), C5, and C7 complement proteins were analyzed using quantitative polymerase chain reaction and immunohistochemical techniques. Results fB, C3, and C5 gene expression were increased in both AFRS and CRS—NPs compared with controls (p < 0.05). Transcriptional activity for the terminal pathway protein C7 was not significantly increased when compared with controls, with C7 levels actually reduced in AFRS patients when compared with controls. Immunohistochemistry studies showed the presence of C3 and fB on the mucosal surface and in submucosa of both AFRS and CRS—NPs, but not normal controls. Terminal pathway protein C9 was not found in our specimens. Conclusions Both AFRS and CRS—NPs display up-regulation of the complement pathway, in particular, the alternative pathway (fB) and common pathways (C3 and C5). Enhanced innate responses as shown by alterations in complement components may play a pivotal role in the inflammatory response noted in CRS and provide potential therapeutic targets in the future.

Cigarette Smoke Inhibits Dynamic Ciliary Beat Frequency in Pediatric Adenoid Explants

Objective. Environmental tobacco smoke exposure in children increases the incidence of upper respiratory infections, chronic sinusitis, and chronic otitis media. This study investigated the effects of ex vivo and in vitro smoke exposure on dynamic ciliary beat frequency (CBF) in pediatric adenoid explants. Study Design. Blinded and controlled prospective study. Setting. Tertiary care pediatric hospital. Subjects and Methods. Fifty-five children undergoing adenoidectomy for obstructive sleep apnea and adenotonsillar hypertrophy were enrolled in this study. Adenoids were surgically removed using currettage. Hair was collected for nicotine analysis. Tissue was sectioned into 1-mm strips and allowed to equilibrate in DMEM/F12 with 2% fetal bovine serum for 24 hours. Cilia-bearing explant tissues were treated with either DMEM/F12 media, 5% cigarette smoke extract (CSE), or 10% CSE for 24 hours. Cilia were then stimulated using either isoproterenol (10−9 M) or methacholine (10−6M), and CBF was serially recorded using the Sisson-Ammons Video Analysis (SAVA) software. Results. Children with hair nicotine levels ≥1 ng/mg consistent with secondhand smoke exposure display blunted dynamic CBF response ex vivo. Explants incubated with CSE in vitro demonstrate significant impairment of isoproterenol and methacholine-induced CBF. Conclusion. CBF of adenoid explants increases when stimulated with isoproterenol and methacholine. Ex vivo and in vitro smoke exposure blunted ciliostimulation of CBF in adenoid explants. Smoke exposure impairs ciliary function in the pediatric airway and could potentially contribute to disorders such as chronic rhinosinusitis and chronic otitis media.

Primary human sinonasal epithelial cell culture model for topical drug delivery in patients with chronic rhinosinusitis with nasal polyposis.

Objectives  The primary human sinonasal epithelial cell culture (HSNEC) allows for in‐vitro modelling of mucosal responses to topical therapy. Cultures grown from healthy donors may underestimate changes in individuals with chronic sinonasal disease thereby yielding inaccurate results with respect to this large patient population. The purpose of this study was to analyse HSNECs derived from patients with chronic rhinosinusitis with nasal polyposis (CRSwNP) to determine whether expected disease dependent variables salient to topical drug delivery persist in culture.

Human sinonasal epithelial cells direct dendritic function and T-cell T helper 1/T helper 2 skewing following Aspergillus exposure.

In lower airway disease such as asthma, epithelial cells have been shown to be potent regulators of dendritic cell (DC) functions. However, it is unclear how human sinonasal epithelial cells (HSNECs) from patients with sinusitis regulate DC functions. Therefore, in these studies we investigated the ability of Aspergillus fumigatus exposed HSNECs to regulate DC antigen uptake, maturation, and direction of T‐cell T helper 1 (Th1)/Th2 skewing.