Mechanisms and biomarkers of inflammatory endotypes in chronic rhinosinusitis without nasal polyps.

BACKGROUND: Chronic rhinosinusitis (CRS) without nasal polyps (CRSsNP) is a common disease that is characterized by multiple inflammatory endotypes. However, the molecular mechanisms in CRSsNP are poorly understood compared with those of polypoid CRS. OBJECTIVE: Our aim was to identify mechanisms and biomarkers associated with inflammatory endotypes underpinning CRSsNP. METHODS: Ethmoid tissues and nasal lavage fluids (NLFs) were obtained from control patients and patients with CRS. The gene expression profiles were determined by microarray analysis and quantitative RT-PCR, and expression of proteins was measured by ELISA and Luminex analysis. RESULTS: Microarray found that compared with their levels of expression in control tissue, the levels of expression of 126, 241, and 545 genes were more than 3-fold and significantly elevated in CRSsNP with type 1 (T1) endotype, type 2 (T2) endotype, and type 3 (T3) endotype, respectively. Selected identified genes were confirmed by RT-PCR. Gene set enrichment analysis suggested that T1 CRSsNP was associated with IFN-γ signaling and antiviral immunity controlled by T cells (TH1 and CD8+), natural killer cells, and antigen-presenting cells; T2 CRSsNP was associated with STAT6 signaling and IgE-mediated activation controlled by eosinophils, mast cells, TH2 cells, group 2 innate lymphoid cells, and antigen-presenting cells; and T3 CRSsNP was associated with IL-17 signaling, acute inflammatory response, complement-mediated inflammation, and infection controlled by neutrophils, TH17 cells, B cells, and antigen-presenting cells. The results suggest that T1 (CXCL9 and CXCL10), T2 (eosinophilic proteins and CCL26), and T3 (CSF3) endotypic biomarkers in NLF may be able to distinguish tissue endotypes in CRSsNP. CONCLUSIONS: Inflammatory endotypes in CRSsNP were controlled by different molecular mechanisms. NLF biomarker assays may allow for more precise and personalized medical treatments in CRS.

Role of tissue eosinophils in chronic rhinosinusitis-associated olfactory loss.

BACKGROUND: Olfactory dysfunction is 1 of the hallmark symptoms of chronic rhinosinusitis (CRS). Eosinophilic inflammation has been implicated as a potential causative factor. However, prior studies have been limited by retrospective study designs, concomitant use of systemic corticosteroids, and other confounding factors. METHODS: CRS and healthy non-CRS control subjects undergoing endoscopic sinus or skull-base surgery were prospectively enrolled and completed olfactory testing utilizing the 40-item Smell Identification Test (SIT) immediately prior to surgery. Histopathological evaluation of tissue excised from the ethmoid bulla was performed by a pathologist in a blinded fashion. Disease severity and patient-reported outcomes were measured via the Lund-Mackay computed tomography (CT) grading system and 22-item Sino-Nasal Outcome Test (SNOT-22), respectively. The associations between olfactory function, tissue eosinophilia, and disease severity were analyzed using Spearman rank order correlation and multiple linear regression. RESULTS: Twenty-seven (27) subjects with CRS without nasal polyps (CRSsNP), 32 subjects with CRS with nasal polyps (CRSwNP), and 10 healthy non-CRS controls were enrolled. CRSwNP was associated with higher mean tissue eosinophil counts (71.6 vs 28.1 eosinophils/high-power field [HPF], p < 0.05) and lower age/sex-adjusted SIT scores (-17.4 vs -6.2, p < 0.001) when compared to CRSsNP. SIT scores were strongly negatively correlated with tissue eosinophil counts in CRSwNP (r = -0.60, p = 0.0003), but not CRSsNP (r = 0.16, p = 0.42). The correlation between olfactory function and tissue eosinophilia in CRSwNP persisted after adjusting for disease severity. CONCLUSION: Tissue eosinophilia is associated with olfactory loss in CRSwNP, independent of disease severity. These results suggest a possible role for eosinophils or eosinophil-associated cytokines in CRS-associated olfactory loss.

Radiographic Evidence of Sinonasal Inflammation in Asthma-Chronic Obstructive Pulmonary Disease Overlap Syndrome: An Underrecognized Association.

BACKGROUND: Sinonasal inflammation on both clinical examinations and imaging significantly impacts both asthma and chronic obstructive pulmonary disease (COPD). OBJECTIVE: The objective of this study was to examine the association between sinonasal inflammation and asthma-COPD overlap syndrome (ACOS). METHODS: A total of 112 patients with a ratio of forced expiratory volume in 1 s to forced vital capacity of less than 70% were enrolled. COPD, asthma, and ACOS were clinically diagnosed according to the 2014 Global Initiative for Asthma and Global Initiative for Chronic Obstructive Lung Disease guidelines. Sinonasal inflammatory condition was evaluated using sinus computed tomography, and its severity was assessed according to the Lund-Mackay staging (LMS) system. Ethmoid sinus-dominant shadow was defined as the presence of greater LMS scores for the anterior and posterior ethmoid sinuses than for the maxillary sinus. RESULTS: COPD, asthma, and ACOS were diagnosed in 55 (49.1%), 39 (34.8%), and 18 patients (16.1%), respectively. The frequency of radiographic evidence of sinonasal inflammation in patients with COPD, asthma, ACOS was 60.0%, 94.9%, and 72.2%, respectively. Patients with ACOS and COPD had only mild radiographic evidence of sinonasal inflammation (LMS score, 1-7), whereas moderate (LMS score, 8-11) and severe (LMS score, ≥12) radiographic evidence of sinonasal inflammation were detected only in patients with asthma. Furthermore, the frequency of ethmoid sinus-dominant shadow was significantly higher in patients with asthma than in those with COPD and ACOS. CONCLUSIONS: Radiographic evidence of sinonasal inflammation was a common comorbidity in ACOS. Future studies are required to examine the role of sinonasal inflammation in ACOS.

Enhanced expressions of lysozyme, SLPI and glycoprotein 340 in biofilm-associated chronic rhinosinusitis.

Lysozyme, secretory leukocyte proteinase inhibitor (SLPI) and glycoprotein 340 (gp340) are important effectors of the innate immune system in sinonasal mucosa. Bacterial biofilms (BBF) are highly organized bacterial communities resistant to host defense systems. The aim of this study was to investigate the expression of lysozyme, SLPI and gp340 in sinus mucosa from chronic rhinosinusitis (CRS) patients with different BBF status. In this prospective cohort study, 63 CRS patients undergoing endoscopic sinus surgery and 20 controls were enrolled and their mucosal samples from ethmoid sinus were obtained. Biofilms were examined by confocal scanning laser microscopy (CSLM), and the expressions of lysozyme, SLPI and gp340 in mRNA and protein levels were detected using reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry and Western blot assay, respectively. As a result, 35/63 (55.6%) of the patients were BBF positive in the CRS group and none in controls. Both mRNA and protein levels of lysozyme, SLPI and gp340 in patients with CRS were significantly higher than those in controls. When sub-classified according to BBF status, the CRS patients with BBF revealed the significantly enhanced mRNA and protein levels of lysozyme, SLPI and gp340. In conclusion, our study demonstrates that lysozyme, SLPI and gp340 are constitutively expressed in sinus mucosa and their up-regulated expressions on both the mRNA and protein levels are associated with BBF in CRS patients. These findings may offer an insight into the interaction between BBF and the innate immune system.

Increased exhaled nitric oxide and its oxidation metabolism in eosinophilic chronic rhinosinusitis.

OBJECTIVE: Monitoring of fractional concentrations of exhaled nitric oxide (FeNO) has become a reliable marker of inflammation in human nose and paranasal sinuses. However, it is still unknown to what extent nasal NO levels contribute to the pathology of chronic rhinosinusitis (CRS). In the present study, we aimed to examine FeNO levels and the underlying mechanism of NO production and metabolism in patients with eosinophilic chronic rhinosinusitis (ECRS) and non-ECRS. METHODS: Thirty-three untreated ECRS patients, 16 non-ECRS patients, and 38 normal subjects were enrolled in this cross-sectional study of FeNO levels. Oral and nasal FeNO levels were measured before treatment using an electrochemical NO analyzer (NObreath(®)) with a nose adaptor. The mRNA expression of three nitric oxide synthase (NOS) isoforms, interleukin-5 (IL-5), and transforming growth factor-beta (TGF-ß) in the ethmoid sinus mucosa and nasal polyps were analyzed by real-time PCR. Immunohistological localization of inducible NOS (iNOS) and nitrotyrosine (NT), a marker for oxidized NO metabolites, was also examined. RESULTS: ECRS patients showed significantly higher oral FeNO levels compared to non-ECRS patients and normal subjects (mean values, 47.6, 13.5, and 15.3ppb, respectively). Nasal FeNO levels of the non-ECRS patients (30.5ppb) were significantly lower than those of the ECRS patients (53.9ppb) and normal subjects (45.5ppb). Positive correlations existed between the blood eosinophil percentage and FeNO levels in ECRS patients. Histologically, ECRS patients showed higher eosinophil accumulation in the ethmoid mucosa than non-ECRS patients (103.1 vs. 16.3cells/HPF). Real-time PCR analysis showed significant upregulation of iNOS and IL-5 mRNA expression in the ethmoid mucosa of the ECRS patients compared to those of non-ECRS patients. Positive iNOS immunoreactivity was observed in ciliated epithelial cells, submucosal glands and associated inflammatory cells in both groups. NT immunoreactivity was detected in the epithelium and around inflammatory cells. Intense NT staining was co-localized with eosinophil accumulation and ECRS patients showed significantly higher rates of NT-positive cells than non-ECRS patients. CONCLUSION: A combination of oral and nasal FeNO measurement is a valid marker for the classification and definition of different CRS subtypes in Japan. Higher levels of oral and nasal FeNO in ECRS patients may reflect the persistence of eosinophilic inflammation in sinus mucosa with concomitant iNOS upregulation and accompanying deposition of oxidized NO metabolites.

Dysregulated survivin expression in nasal polyps of individuals with aspirin exacerbated respiratory disease.

BACKGROUND: A derailed balance of cell proliferation and apoptosis is presumed to result in cell hyperplasia as a typical feature of nasal polyps. Survivin, a protein of the inhibitors of the apoptosis family is proposed to promote polyp formation. However, studies concerning survivin expression in chronic rhinosinusitis (CRS) with nasal polyps are rare and the specificity of the survivin expression in nasal polyps from individuals with aspirin-exacerbated respiratory disease (AERD) has not been investigated. METHODS: Immunohistochemical survivin expression analysis was performed. Samples were taken from the ethmoid sinus of individuals with CRS with nasal polyps with and without AERD during sinus surgery and control specimens of the inferior turbinate from individuals without CRS. Cell cultures were stimulated with recombinant vascular endothelial growth factor (VEGF(165)) and the resulting survivin expression was analyzed by Western blot. RESULTS: The survivin expression of 61 specimens was analyzed by quantitative immunohistochemistry and a potential VEGF-dependant stimulation of survivin in cell cultures was investigated. The survivin expression in nasal polyps from individuals with AERD was increased compared with the controls (median, 1194 versus 927 arbitrary units [A.U.]; p = 0.054). Western blot analysis revealed in vitro a VEGF-dependant regulation of survivin in nasal polyps from individuals without AERD, but not in those with AERD (p = 0.05). CONCLUSION: Enhanced survivin expression might result in decreased apoptosis and cellular hyperplasia as a part of the largely unknown pathophysiology of nasal polyp formation. Furthermore, we hypothesize a pathological, VEGF-independent constitutive survivin expression in nasal polyps of individuals with AERD.

Migration of regulatory T cells toward airway epithelial cells is impaired in chronic rhinosinusitis with nasal polyposis.

The pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP) is still unclear. To evaluate the role of regulatory T cells (Treg) in the pathogenesis of nasal polyposis, we tested migration potential of Treg purified from subjects with CRSwNP, CRS without NP and controls. The nasal tissue expressions of FOXP3 were analyzed by means of RT-PCR and double immunohistochemistry. Chemotaxis assays were used to evaluate the migration potential of Treg onto bronchial epithelial cells and primary nasal epithelial cells, and toward chemokines. FOXP3(+)CD3(+) cells frequency and FOXP3 transcript expression in nasal tissue, and migration potentials of Treg toward airway epithelial cells and CCL1 were significantly lower in CRSwNP compared with other groups (P<0.05). These results indicate that migration potential of Treg is decreased in CRSwNP subjects, and this may be one of the reasons why tissue infiltration of Treg was decreased as seen in the immunohistochemistry of nasal polyps from CRSwNP subjects.

Detection of natural killer T cells in the sinus mucosa from asthmatics with chronic sinusitis.

BACKGROUND: Chronic sinusitis (CS) with asthma generally exhibits a high degree of sinus tissue eosinophilia and recurrence often occurs even after surgical therapy. However, the cause has not yet been fully clarified. AIMS OF THE STUDY: To elucidate the pathogenesis of this refractory disease, we examined the infiltration of natural killer T (NKT) and type 1 helper T (Th1)/type 2 helper T (Th2) cells, and the cytokine expression in the sinus mucosa. METHODS: Sinus mucosal specimens were obtained surgically from 16 CS patients with nasal polyps. The NKT cells, Th1/Th2 cells and the expression of IL-4, IL-5, IL-13 and IFN-gamma were examined by a polymerase chain reaction or flow cytometry. Nasal mucosal specimens from six other patients with allergic rhinitis (AR) were examined in a similar manner. RESULTS: The NKT cells were detected to varying degrees in the sinus mucosa from asthmatic CS patients, but neither in the nonasthmatics nor in the nasal mucosa from the patients with AR. The Th2 cells and Th2 cytokines were expressed at significantly higher levels in the sinus mucosa from the CS patients with asthma in comparison to those without asthma. However, the Th1 cell infiltration and IFN-gamma expression were not different between these groups. CONCLUSION: Natural killer T cells may, therefore, play important roles in the enhanced Th2 cytokine expression and increased infiltration of Th2 cells and eosinophils observed in the sinus mucosa from asthmatic CS patients through MHC-independent mechanisms.

An intracranial aspergilloma with low signal on T2-weighted images corresponding to iron accumulation.

We present a case of cerebral aspergillosis in an immunocompetent patient. The MRI signal characteristics were compared with the histologic findings. Irregular low-signal zones were demonstrated between the wall of the abscess and the central necrosis on T2-weighted images; the pathology specimen revealed concentrated iron in these transitional zones but no hemosiderin. Iron is an essential element for the growth of fungal hyphae. The low-signal zones may represent the areas where there was active proliferation of aspergillus, and the unique location of the low signal may be a helpful imaging characteristic for the diagnosis of an aspergillus abscess.

Positive identification in situ of mRNA expression of IL-6, and IL-12, and the chemotactic cytokine RANTES in patients with chronic sinusitis and polypoid disease. Clinical relevance and relation to allergy.

Interleukins 6 (IL-6) and 12 (IL-12), and the chemoattractant chemokine RANTES were studied in ethmoidal mucosa, using reverse transcriptase polymerase chain reaction. The 49 patients had chronic sinusitis or nasal/paranasal polyposis, and some also allergy. To the best of our knowledge, this is the first study that demonstrates RANTES and IL-12 on mRNA level in human sinonasal mucosa in situ. mRNA for IL-6, IL-12 and RANTES were detected in 2, 8 and 6 patients with chronic sinusitis, respectively, and in mucosa from patients with polyposis a positive expression was observed in 4, 14 and 10 cases. There were no statistically significant differences. Analysing the entire group of 49 patients, disregarding type of mucosal disease, the number of patients with positive RANTES was significantly higher than that for IL-6. Similarly, IL-12 positivity was more frequently expressed than IL-6. mRNA for IL-6 was expressed in only 2 of the allergic patients. The cytokine production studied thus seems to be unrelated to the clinically defined entities. There is thus a local production in human diseased sinonasal mucosa of RANTES, as well as of IL-6 and IL-12. The local production of RANTES is an important prerequisite for recruitment and migration of inflammatory cells into the tissue. IL-12 is a co-stimulator of antigen-specific responses of established T helper 1 (Th1) clones, and regulates the responsiveness of the clones to a number of T cell growth factors. The study supports a shift towards Th1 cells in these disease entities.