Expression Of Toll-like Receptors In Mast Cells In Chronic Rhinosinusitis With Nasal Polyps And Its Association With Type 2 Inflammatory Response

Objective:Chronic rhinosinusitis(CRS)is divided into CRS without nasal polyps(CRSsNP)and CRS with nasal polyps(CRSwNP).CRSwNP is characterized by moderate to severe T helper type 2(Th2)-mediated inflammation,mast cell activation,and increased IgE concentration.The patients with CRSwNP often suffer from other diseases,such as asthma and aspirin intolerance.Although the role of eosinophils in nasal polyps has been well understood,the function of mast cells and their activation in CRSwNP development remains unclear,because activated mast cells can also secrete type 2 inflammatory factors.At present,the inflammation response in response to microbiota has been taken into account to contribute to the pathogenesis of CRSwNP.Proposed theories suggest that the activation of the innate and adaptive immunity defense system of the airway epithelium plays important roles in the development of CRSwNP.Microbiota are recognized by pattern-recognition receptors(PRRs).Toll-like receptors(TLRs)recognize structurally conserved pathogen-related molecular patterns originated from microbes,which lead to the activation of immune cell responses.Mast cells have long been considered as the main effector cells of allergy.Recent studies have revealed that mast cells also play a key role in regulating innate and adaptive immunity through TLRs.The signal adaptor MyD88 has been defined as an intracellular TLR signaling pathway,which promotes early activation of nuclear factor(NF)-?B,followed by the release of inflammation-related cytokines,such as IL-4,IL-5,and IL-13.Therefore,TLRs might affect the inflammatory response in CRSwNP through the activation of mast cells.However,it remains unknown whether the expression of TLRs in mast cells of CRSwNP patients is different from that in normal controls.This study aimed to investigate the expression of toll-like receptors(TLRs)in mast cells isolated from the uncinate process mucosa of patients with chronic rhinosinusitis with nasal polyps(CRSwNP)and normal controls.Then the effect of TLRs on TLR-dependent signaling pathway MyD88/NF-?B,and on IL-5 level in activated mast cells was assessed.Methods:In this study,35 patients who were admitted to the First Affiliated Hospital of China Medical University from June 2019 to December 2019 for their first operation were included.Inclusion criteria included patients aged 18 to 65 with CRSwNP who had no history of sinus surgery.The diagnosis of CRSwNP was made according to the EPOS criteria.The patients diagnosed with maxillary sinus cysts were served as controls.Exclusion criteria included the presence of an isolated anterochoanal polyp,unilateral nasal polyps,allergic fungal sinusitis,or any other systemic diseases including Crohn's disease,primary immunodeficiency,mast cell disorders,hematologic disorders,malignancy,organ transplantation or previous nasal surgery.Fresh uncinate process(UP)sinonasal mucosa was collected from 13 control subjects and 22 CRSwNP subjects.Clinical data were collected including age,gender,Lund-Kennedy score,modified Lund-Mackay CT score,total VAS score,peripheral blood eosinophils(%),peripheral blood basophils(%),inhaled allergen detection,whether accompanied by asthma and tissue eosinophils.Isolation and quantification of viable TLRs+mast cells from uncinate process(UP)mucosa by flow cytometryFresh MUCOSA tissues obtained during functional endoscopic sinus surgery(n=35)was transported on ice in balance salt solution.Briefly,fresh MUCOSA specimens were washed twice in PBS,minced with a scalpel,and then digested with 0.25%trypsin for 50 min at 37?.Single-cell suspension was passed over a 70-?m cell strainer,washed in Hank's balanced salt solution(HBSS),and resuspended in freezing PBS.To avoid nonspecific staining,single-cell suspension was incubated with human FC block at room temperature for 10 min prior to incubation with antibodies.Next,antibodies including surface markers of mast cells(CD117,Fc?Rla,CD203c),TLR2,TLR4,TLR5,TLR3,TLR7 were dropped according to the manufacturer instructions.To detect the intracellular proteins,the mast cells were fixed and permeated using the Cytofix/Cytoperm Soln Kit.Viable mast cells expressing surface or intracellular TLRs were identified using flow cytometry.Unstained and isotype controls were included in each analysis.Human mast cell cultureHuman mast cells HMC-1 were seeded in a 75 cm culture flask and cultured with RPMI 1640 medium,10%bovine serum albumin and 1%penicillin/streptomycin in a CO2 incubator.The culture medium was replaced every 2 days and the cells were kept at a density of 105 cells/ml.Construction of activated human mast cells(HMC-1)secreting IL-5 of Type 2 inflammatory response cytokines.Subculture(0.25%trypsin)was performed when cell density was above 90%.The subcultured cells were seeded into 96-well plates.HMC-1 mast cells at logarithmic growth stage were taken and divided into the following four groups:(1)blank control group;(2)group of 50 ng/ml PMA and 500 ng/ml lonomycin;To inhibit the expression of TLR2,4,and 5,HMC-1 cells were pretreated with Sparstolonin B(TLR2,4 inhibitor,100 ?g/mL)and Th1020(TLR5 inhibitor,0.78 ?M).(3)After pretreatment with 100?g/mL Sparstolonin B for 24 hours,50ng/mL PMA+500ng/mL Ionomycin(PMA+Lonomycin+Sparstolonin B group)was added;(4)After pretreatment with 0.78?M TH1020 for 24h,50ng/ml phobarate+500ng/ml ionomycin(PMA+Lonomycin+TH1020 group)was added.After 6 hours of cell culture,the supernatant of the cells was collected and placed in a 15mL centrifuge tube for 9min(800 RPM/min).The expression of IL-5 was detected by ELISA kit.Detection of cytokine IL-5The supernatant of HMC-1 cells with various treatments was collected.The level of IL-5 in the supernatant was assessed using the Human IL-5 Quantikine ELISA Kit according to manufacturer instructions.The mRNA expression levels of TLR2,TLR4 and TLR5 were detected by Quantitative Real-Time Polymerase Chain Reaction(qRT-PCR)Total RNA was extracted from HMC-1 cells using a Total RNA Extraction Kit.Then cDNA was synthesized using a PrimeScriptTM RT reagent kit.The mRNA expression levels of TLR2,TLR4,TLR5 were quantified using 2×SYBR Green PCR Mastermix.Relative expression normalized to GAPDH was calculated using the 2-??CT method.TLRs,MyD88 and NF-?B protein expression levels were detected by Western blottingTotal protein was extracted according to the kit instructions,and protein quantification was performed by BCA method.20 ?L protein sample added to 5xSDS buffer solution was boiled for 5 min for protein denaturation.After sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE),the protein was transferred to PVDF membrane,sealed with 5%skimmed milk powder for 1 h,and the primary antibodies against TLR2,TLR4,TLR5,MyD88,NF-?B p65,phospho-NF-?B p65 and GAPDH were incubated overnight at 4?.Then the membrane was washed three times with TBST for every 5 min.The secondary antibody(goat anti-rabbit IgG-HRP was incubated at room temperature for 2 h,followed by washing with TBST for three times.ECL was used for visualization of protein bands.Results:This study recruited a total of 35 participators divided into the following groups:CRSwNP(n=22)and controls(n=13).The subjects'clinical data are summarized in this study.There was no age and gender difference between the two groups.However,CRSwNP patients presented a higher Lund-Kennedy score,modified Lund-Mackay CT score,total VAS score,peripheral blood eosinophils(%),peripheral blood basophils(%),inhaled allergen detection,accompanied by asthma and tissue eosinophils,as compared with controls.Comparison of total mast cell%between CRSwNP group and Control groupThe TLRs+mast cells in fresh MUCOSA tissues were identified by flow cytometry.The percentage of total viable mast cells present in CRSwNP group and Control group was 2.60%±1.55%and 1.72%±0.77%,respectively.Thus,the percentage of total mast cells present in CRSwNP group was more than that in control group(P=0.001).Overexpression of TLR2,TLR4,and TLR5 on the surface of mast cells in CRSwNPNext,we assessed TLR2,TLR4,and TLR5 levels in the surface of mast cells isolated from the fresh UP mucosa tissues.Our results showed that the percentage of TLR2,TLR4,and TLR5 positive mast cells in patients with CRSwNP and controls were as follows:TLR2(94.85%±2.17%,88.24%±5.75%),TLR4(94.85%±2.15%,83.48%±3.72%),and TLR5(95.87%±1.50%,85.53%±6.84%).The above findings indicated that TLR2,TLR4,and TLR5 expression on the surface of mast cells in CRSwNP patients was higher than that in controls.Decreased expression of intracellular TLR3 and TLR7 in the mast cells in CRSwNPTo determine the intracellular TLR3 and TLR7 expression,mast cells isolated from the fresh UP mucosa tissues were permeated using the Cytofix/Cytoperm Soln Kit.As detected by flow cytometry,the percentage of TLR3 and TLR7 positive mast cells from CRSwNP and control groups was as follows:TLR3(49.09%±8.43%,74.60%±5.34%)and TLR7(41.94%±8.41%,71.46%±7.33%).These findings demonstrated that the intracellular TLR3 and TLR7 expression in mast cells in CRSwNP patients was lower as compared with the control group.Correlation between TLRs and clinical symptom severityAs assessed by Spearman's rho correlation test,the percentage of TLR2+,TLR4+and TLR5+ mast cells was positively correlated with Lund-Mackay CT score,Lund-Kennedy score,and concomitant asthma,whereas there was a negative correlation between TiR3+,TLR7+ mast cells and,und-Mackay CT score,Lund-Kennedy score,and concomitant asthma.Expression of TLR2,TLR4,and TLR5 in activated HMC-1 cellsThe expression of TLRs was further validated in activated human mast cell line HMC-1.As presented,a significant up-regulation of TLR2,TLR4,and TLR5 mRNA was found in HMC-1 cells after challenge with PMA and lonomycin.Consistently,the protein levels of TLR2,TLR4,and TLR5 were enhanced in activated HMC-1 cells.Effect of TLRs on IL-5 secretion in activated HMC-1 cellsThe ELISA results showed that the release of the cytokine IL-5 was enhanced in HMC-1 cells after stimulation with PMA and lonomycin.Whereas treatment with TLR2,4 inhibitor sparstolonin B or TRL5 inhibitor TH1020 could counteract PMA and lonomycin-mediated IL-5 secretion in HMC-1 cells.Effect of TLRs on MyD88/NF-KB pathway in activated HMC-1 cellsTo determine the activation of TLR downstream MyD88/NF-?B pathway,Western blotting was carried out.As presented,stimulation with PMA and lonomycin led to increased expression of MyD88,p-NF-?B p65 and NF-?B p65 in HMC-1 cells.However,inhibition of TLR2,4 by sparstolonin B or inhibition of TLR5 by TH1020 abolished the above changes.Therefore,TLRs-mediated MyD88/NF-?B pathway activation could contribute to the inflammatory response in CRSwNP.Conclusion:Our results indicate the differential expression of TLRs in mast cells in CRSwNP and its correlation with the severity of clinical symptoms,the activation of TLR downstream MyD88/NF-?B pathway in mast cells and the secretion of cytokines IL-5 which suggest that TLRs might contribute to Type2 inflammation in CRSwNP via activating mast cells.This study provides a new direction for further study on the therapeutic targets of chronic rhinosinusitis with nasal polyps.