Toll-like Receptors In Nasal Inflammation And The Regulation Of Glucocorticoid In Nasal Epithelial Cells

Objective1.To investigate the presence and involvment of Toll-like receptors(TLRs)in the inflammation of chronic rhinosinusitis(CRS).2.To examine the expression of TLRs in the nasal mucosa of patients with persistent allergic rhinitis(PER).3.To evaluate the effect of glucocorticoids(GC)on the TLRs and related cytokines in human nasal epithelial cells.Methods1.A total of 26 CRS patients without nasal polyps(CRSsNP),30 CRS patients with nasal polyps(CRSwNP),and 30 controls were enrolled in this study.Nasal mucosa and polyps were collected and detected for TLR1,TLR2,TLR3,TLR4,TLR5,and TLR6 mRNA,and cytokines mRNA by real-time PCR,and further determined the location of TLR2 and TLR4 in tissues from CRSsNP,CRSwNP and controls by immunohistochemistry.And the protein expression of TLR2 and TLR4 in three groups was confirmed using the Western bolt.The relationship among above variables,Lund-Mackay CT scores,and symptom visual analogue scales(VAS)scores were further analyzed.2.Nasal biopsies were obtained from 21 patients with PER and 21 controls.The mRNA of TLR1,TLR2,TLR3,TLR4,TLR5,TLR6,TLR7,TLR8,and TLR9,and related cytokines were determined by real-time PCR.The localization of TLR2 and TLR4,and quantification of corresponding proteins were determined by the immunohistochemistry and Western bolt.3.Four nasal biopsies were obtained from patients with deviation of nasal septum during the surgery,and nasal epithelial cells were prepared and cultured.After stimulation of LPS,or co-cultured with LPS and hydrocortisone,the TLR2 and TLR4 mRNA,and the release of IL-6,IL-8,IL-10,and IL-12 mRNA were detected using real-time PCR.Results1.Immunoreactivity for TLR2 and TLR4 could be demonstrated in the nasal epithelium,and gland,immune cells and vascular endothelial cells of subepithelial tissue in CRSsNP,CRSwNP and controls(especially in CRSwNP).TLR2,TLR3,TLR4,TLR6,IL-5,IL-6,IL-17,and IFN-a mRNA were significantly higher in the nasal mucosa from CRSwNP patients compared with the controls(P<0.05 or P<0.01),while only IL-5 and IFN-a mRNA were elevated in nasal polyps from the same patients(P<0.05).In CRSsNP patients,IL-5,IL-17,and IFN-a mRNA were elevated compared with the controls(P<0.05),but TLR2,TLR3,TLR4,TLR6,and IL-6 mRNA were decreased than CRSwNP patients which have reached significance(P<0.05).The expression of TLR2 and TLR4 protein were detected in CRSsNP,CRSwNP and controls,and in line with mRNA,the protein in CRSwNP mucosa were higher than those in CRSsNP and the controls(P<0.05).There was no correlation between the TLRs and symptom VAS scores,as well as between the TLRs and Lund-Mackay CT scores.The CT scores was positively correlated with the symptomVAS scores,and especially correlated with the degree of nasal obstruction and anosphrasia.2.TLR2 and TLR4 were demonstrated in the nasal epithelium,and gland,immune cells and vascular endothelial cells of subepithelial tissue in PER patients and controls(especially in PER).TLR2,TLR3,TLR4,TLR5,and TLR7 mRNA were higher in PER patients,but only TLR2 and TLR4 reached statistical significance(P<0.05).IL-5,IL-6,and IL-8 mRNA were elevated in PER patients compared with the controls(P<0.05).The expression of TLR2 and TLR4 protein were detected in both PER patients and controls,and in line with mRNA,the protein in PER mucosa were higher than those in the controls(P<0.05).3.Incubation with 100 ng/ml or 1 ?g/ml LPS could upregulated TLR2 and TLR4 mRNA,thus 100 ng/ml LPS were used for next stimulation.The upregulation of TLR2 and TLR4 mRNA was elevated by pretreatment with low concentration hydrocortisone(1 ?g/ml)and co-cultured with LPS(did not reach statistical significance),but inhibited by moderate(10 ?g/ml)and high concentration(100?g/ml)(P<0.05).No significant differences in IL-12 mRNA were found between with and without hydrocortisone.Conclusions1.The up-regulation of TLR2 and TLR4 in the nasal mucosa of patients with CRSwNP but not CRSsNP supports the idea from the point of inflammation that CRSsNP and CRSwNP may have distinct pathogenesis.2.TLR2 and TLR4 may worsen the symptoms of PER and exacerbate nasal inflammation with the existence of persistent allergen challenge.However,the specific role of TLRs in Th1/Th2 immune balance needs to be explored in future studies.3.GC has both positive and negative effects on TLRs in human nasal epithelial cells,and GC might regulate downstream cytokines via TLRs signaling.And the direct or indirect mechanism needs to be further studied.