Andrew P. Lane

Altered expression of genes associated with innate immunity and inflammation in recalcitrant rhinosinusitis with polyps.

Background The role of the innate immune system in the pathophysiology of chronic rhinosinusitis (CRS) is poorly understood. In this study, we compared sinonasal expression of toll-like receptors (TLRs), complement components, serum amyloid A, and inflammatory genes (chemokines and cytokines) in control subjects and patients undergoing sinus surgery for CRS. Methods Eleven control subjects and 30 subjects with CRS unresponsive to medical management were enrolled prospectively before undergoing endoscopic sinus surgery. Ethmoid mucosal specimens were obtained surgically and processed for RNA extraction. Real-time polymerase chain reaction was used to quantitate the level of expression of messenger RNA (mRNA) for TLR, acute phase proteins, and cytokine genes. Subjects were followed for a minimum of 6 months postoperatively with nasal endoscopy to assess for recurrence of polyps. Results mRNA for all target genes was detected in the ethmoid mucosa of both control and CRS subjects. The level of gene expression was normalized to the housekeeping genes 18s RNA and glyceraldehyde-3-phosphate dehydrogenase. As compared with controls, CRS was associated with significantly higher expression of TLR2 and the inflammatory genes macrophage-inflammatory protein a, RANTES, and granulocyte-macrophage colony-stimulating factor. Patients with early recurrence of polyps after surgery had significantly decreased expression of TLR2, 9, and serum amyloid A and increased expression of macrophage-inflammatory protein a compared with surgery-responsive patients. Conclusion This study shows the increased levels of expression of TLR2 and a variety of inflammatory genes in sinonasal mucosa of CRS patients compared with controls. Whether these differences play a role in pathogenesis or are merely manifestations of disease activity is worthy of investigation.

Treatment-recalcitrant chronic rhinosinusitis with polyps is associated with altered epithelial cell expression of interleukin-33.

Background Abnormalities in host mucosal immunity exist in chronic rhinosinusitis with nasal polyps (CRSwNPs), but it is unclear whether this is a cause or an effect of the eosinophilic inflammation and frequent microbial colonization that characterizes the disease. Sinonasal epithelial cells (SNECs) are critical participants in healthy antimicrobial innate immune defense. They also can promote Th2 inflammation with various mediators, including interleukin (IL)-33, which induces T helper cells to produce Th2 cytokines. Methods CRSwNP SNECs were obtained during sinus surgery and stored. Patients were subsequently classified as either treatment responsive or treatment recalcitrant, based on long-term outcomes of medical and surgical therapy. Epithelial cells from these patients were grown in air–liquid interface (ALI) culture and treated with IL-13, as well as the bacteria-associated molecule, CpG. Expression of IL-33 mRNA was determined by real-time polymerase chain reaction. Results Recalcitrant CRSwNP epithelial cells had increased baseline expression of IL-33 compared with responsive CRSwNPs, which was further increased by 24-hour exposure to CpG. Treatment-responsive epithelial cells were not induced by CpG to express IL-33. Prolonged treatment with IL-13 during differentiation at the ALI diminished the baseline expression of IL-33 and prevented the subsequent induction of IL-33 by CpG. Conclusion Mucosal innate immunity likely plays an important role in CRSwNP pathogenesis. A definitive link between infectious triggers and the development of Th2 inflammation has been elusive. We have found constitutive IL-33 expression by SNECs in recalcitrant CRSwNPs, which can be further induced by a bacteria-associated molecular pattern. Dysregulated epithelial cell immune interactions between host and environment may contribute to Th2 inflammation in CRSwNPs.

Th2 cytokines associated with chronic rhinosinusitis with polyps down-regulate the antimicrobial immune function of human sinonasal epithelial cells

Background Chronic rhinosinusitis with nasal polyps (CRSwNPs) is a disorder characterized by persistent eosinophilic Th2 inflammation and frequent sinonasal microbial colonization. It has been postulated that an abnormal mucosal immune response underlies disease pathogenesis. The relationship between Th2 inflammatory cytokines and the innate immune function of sinonasal epithelial cells (SNECs) has not been explored. Methods Human SNECs (HSNECs) isolated from control subjects and patients with CRS were assessed for expression of antimicrobial innate immune genes and proinflammatory cytokine genes by real-time polymerase chain reaction, ELISA, and flow cytometry. A model of the Th2 inflammatory environment was created by exposure of primary HSNEC to the Th2 cytokine interleukin (IL)-4 or IL-13 for 36 hours, with subsequent assessment of innate immune gene expression. Results HSNEC obtained from CRSwNP patients displayed decreased expression of multiple antimicrobial innate immune markers, including toll-like receptor 9, human beta-defensin 2, and surfactant protein A. Baseline expression of these genes by normal and CRS HSNEC in culture is significantly down-regulated after incubation with IL-4 or IL-13. Conclusion Expression of multiple innate immune genes by HSNEC is reduced in CRSwNP. One mechanism appears to be a direct effect of the leukocyte-derived Th2 cytokines present in the sinonasal mucosa in CRSwNP. Impaired mucosal innate immunity may contribute to microbial colonization and abnormal immune responses associated with CRSwNP.

Sinonasal epithelial cell expression of toll-like receptor 9 is decreased in chronic rhinosinusitis with polyps

Background Innate immune recognition of pathogens by sinonasal epithelial cells may play an important role in the pathogenesis of chronic rhinosinusitis (CRS). Previous studies have indicated that toll-like receptor (TLR) mRNA is present in sinonasal mucosa, and levels of TLR9 expression are decreased in recalcitrant CRS with nasal polyps (CRSwNP). However, the cellular source and function of TLR9 in the sinonasal epithelium is not known. In this study, primary epithelial cell cultures were analyzed from control subjects and CRSwNP patients to determine the presence and function of TLR9 protein. Methods Primary epithelial cell cultures were established from 5 controls and 10 CRSwNP patients undergoing sinus surgery. Flow cytometry was used to confirm purity of epithelial cells and to assess expression of TLR9 protein. Epithelial cells were stimulated with TLR9 agonist, and mRNA was analyzed by real-time PCR for expression of human β-defensin (HBD) 2 and interleukin (IL)-8. Results Flow cytometry showed TLR9 protein in 100% of epithelial cells from controls and CRSwNP patients. The level of expression was 50% lower in CRS patients than in controls. Stimulation of epithelial cells with TLR9 agonist produced a 1.5- to 9-fold increase in HBD-2 and IL-8 mRNA expression. Conclusion Functional TLR9 protein is expressed by normal and diseased sinonasal epithelial cells. The level of TLR9 expression is decreased in CRSwNP patients, consistent with the previous finding of decreased TLR9 mRNA in whole sinonasal tissue. These findings suggest that impaired innate immune responses to pathogens via TLR9 on sinonasal epithelial cells may represent a critical mechanism in chronic inflammatory sinus disease.

Innate immunity of the sinonasal cavity and its role in chronic rhinosinusitis

Chronic rhinosinusitis (CRS) is one of the most common health problems in the United States. Medical therapy and surgery are successful in treating the majority of patients with sinusitis; however, CRS patients recalcitrant to traditional therapy are increasingly prevalent. Although traditionally this illness could be explained by sinus ostial obstruction and persistent bacterial infection, the rhinologic literature over the years has suggested a significant underlying inflammatory component. Adaptive immune components, including lymphocytes and their associated cytokines, have been the subject of most research in chronic nasal inflammation. A recent appreciation of the importance of the innate immune system is leading to new areas of investigation regarding the pathogenesis of CRS. This review will outline our current knowledge of sinonasal innate immunity, the role of innate immunity in the pathogenesis of CRS, and potential therapeutic targets in the innate immune system.

Serum amyloid A, properdin, complement 3, and toll-like receptors are expressed locally in human sinonasal tissue

Background There is a growing appreciation of the role that nasal mucosa plays in innate immunity. In this study, the expression of pattern recognition receptors known as toll-like receptors (TLRs) and the effector molecules complement factor 3 (C3), properdin, and serum amyloid A (SAA) were examined in human sinonasal mucosa obtained from control subjects and patients with chronic rhinosinusitis (CRS). Methods Sinonasal mucosal specimens were obtained from 20 patients with CRS and 5 control subjects. Messenger RNA (mRNA) was isolated and tested using Taqman real-time polymerase chain reaction with primer and probe sets for C3, complement factor P, and SAA. Standard polymerase chain reaction was performed for the 10 known TLRs. Immunohistochemistry was performed on the microscopic sections using antibodies against C3 Results Analysis of the sinonasal sample mRNA revealed expression of all 10 TLRs in both CRS samples and in control specimens. Expression of the three effector proteins was detected also, with the levels of mRNA for C3 generally greater than SAA and properdin in CRS patients. No significant differences were found in TLR or innate immune protein expression in normal controls. Immunohistochemical analysis of sinonasal mucosal specimens established C3 staining ranging from 20 to 85% of the epithelium present. Conclusion These studies indicate that sinonasal mucosa expresses genes involved in innate immunity including the TLRs and proteins involved in complement activation. We hypothesize that local production of complement and acute phase proteins by airway epithelium on stimulation of innate immune receptors may play an important role in host defense in the airway and, potentially, in the pathogenesis of CRS.

International Consensus Statement on Allergy and Rhinology: Rhinosinusitis

Isam Alobid, MD, PhD1, Nithin D. Adappa, MD2, Henry P. Barham, MD3, Thiago Bezerra, MD4, Nadieska Caballero, MD5, Eugene G. Chang, MD6, Gaurav Chawdhary, MD7, Philip Chen, MD8, John P. Dahl, MD, PhD9, Anthony Del Signore, MD10, Carrie Flanagan, MD11, Daniel N. Frank, PhD12, Kai Fruth, MD, PhD13, Anne Getz, MD14, Samuel Greig, MD15, Elisa A. Illing, MD16, David W. Jang, MD17, Yong Gi Jung, MD18, Sammy Khalili, MD, MSc19, Cristobal Langdon, MD20, Kent Lam, MD21, Stella Lee, MD22, Seth Lieberman, MD23, Patricia Loftus, MD24, Luis Macias‐Valle, MD25, R. Peter Manes, MD26, Jill Mazza, MD27, Leandra Mfuna, MD28, David Morrissey, MD29, Sue Jean Mun, MD30, Jonathan B. Overdevest, MD, PhD31, Jayant M. Pinto, MD32, Jain Ravi, MD33, Douglas Reh, MD34, Peta L. Sacks, MD35, Michael H. Saste, MD36, John Schneider, MD, MA37, Ahmad R. Sedaghat, MD, PhD38, Zachary M. Soler, MD39, Neville Teo, MD40, Kota Wada, MD41, Kevin Welch, MD42, Troy D. Woodard, MD43, Alan Workman44, Yi Chen Zhao, MD45, David Zopf, MD46

A Genetic Model of Chronic Rhinosinusitis-Associated Olfactory Inflammation Reveals Reversible Functional Impairment and Dramatic Neuroepithelial Reorganization

Inflammatory sinus and nasal disease is a common cause of human olfactory loss. To explore the mechanisms underlying rhinosinusitis-associated olfactory loss, we have generated a transgenic mouse model of olfactory inflammation, in which tumor necrosis factor α (TNF-α) expression is induced in a temporally controlled manner specifically within the olfactory epithelium (OE). Like the human disease, TNF-α expression leads to a progressive infiltration of inflammatory cells into the OE. Using this model, we have defined specific phases of the pathologic process. An initial loss of sensation without significant disruption is observed, followed by a striking reorganization of the sensory neuroepithelium. An inflamed and disrupted state is sustained chronically by continued induction of cytokine expression. After prolonged maintenance in a deficient state, there is a dramatic recovery of function and a normal histologic appearance when TNF-α expression is extinguished. Although obstruction of airflow is also a contributing factor in human rhinosinusitis, this in vivo model demonstrates for the first time that direct effects of inflammation on OE structure and function are important mechanisms of olfactory dysfunction. These features mimic essential aspects of chronic rhinosinusitis-associated olfactory loss, and illuminate underlying cellular and molecular aspects of the disease. This manipulable model also serves as a platform for developing novel therapeutic interventions.

Use of Computed Tomography in the Assessment of Mandibular Invasion in Carcinoma of the Retromolar Trigone

Carcinomas originating in the retromolar trigone (RMT) are uncommon and characterized by early spread. Determination of mandibular invasion is significant for planning therapy and determining prognosis. For oral cavity cancers in general, CT is reasonably accurate in assessing bone invasion. However, there is a paucity of information specifically addressing the value of CT in the RMT. In this study, the records of patients with biopsy-proven RMT carcinomas treated between 1984 and 1998 were reviewed with attention to preoperative CT scans and histopathologic findings during surgery. Half of the patients who were treated with primary resection had mandibular invasion. Bone invasion was not identified radiographically in 27% of patients with preoperative CT scans. The sensitivity of CT for bone involvement in RMT cancers was 50%, with a negative predictive value of 61.1%. The positive predictive value was 91.1%. These findings suggest that CT is a useful, but potentially inaccurate, predictor of bone invasion in the RMT.

Medical therapy reduces microbiota diversity and evenness in surgically recalcitrant chronic rhinosinusitis

Chronic rhinosinusitis (CRS) is a highly prevalent and heterogeneous condition frequently treated with antibiotics and corticosteroid therapy. However, the effect of medical therapy on sinus microbiota remains unknown.