CD4+T Cell Research In Patients With Allergic Rhinitis

Allergic rhinitis(AR)is a common immune disease mediated by Th2 cells.It is a major problem affecting the health of the world's population.About 20% of the population is affected by AR,and the incidence of AR is on the rise all over the world.In recent years,the symptoms of allergic rhinitis have been somewhat improved due to the development of treatment programs,but they are still high.A high proportion of AR patients develop rhino-sinusitis,nasal polyps,ocular conjunctivitis,asthma and many other diseases,which greatly affect the quality of life of patients.The main clinical symptoms of allergic rhinitis include runny nose,sneezing,nasal itching and nasal congestion.If allergic rhinitis is not controlled and treated for a long time,it is easy to develop into bronchial asthma.Therefore,the intervention and treatment of allergic rhinitis can play a certain role in the prevention and treatment of asthma,and because of this,allergic rhinitis has become more and more a hot spot in basic and clinical research in otolaryngology.In recent years,there have been a lot of researches on the mechanism of allergic rhinitis,but the specific mechanism is still not very clear.At present,it is believed that T cells are the main lymphocytes causing the occurrence and development of allergic rhinitis,and the immune imbalance of Th1/Th2 is the main factor.Further studies have been conducted on related cytokines,such as IL-4,IL-5,IL-9,IL-13,etc.In the cytological experiments of peripheral blood of patients with allergic rhinitis,we found that the changes of chemokine receptors on the cell surface also play a decisive role in the pathogenesis of the disease,so we conducted further in-depth studies.Objective: The purpose of this study was to study the number and proportion of lymphocyte subsets in peripheral blood of patients with allergic rhinitis,T cell subsets and function in patients with allergic rhinitis,and the changes of T cell chemokine receptors in patients with allergic rhinitis,so as to find out the key factors affecting allergic rhinitis and provide molecular basis for the treatment of the disease.Methods:1.Subjects25 patients with AR and 31 healthy controls(HCs,peripheral blood mononuclear cells were collected from 20 HCs)were recruited from October 2019 to February 2020 from the Otorhinolaryngology clinic in Shengjing Hospital affiliated to China Medical University(CMU).All AR patients visit this hospital for the first time.The screening and classification are based on the following criteria:(1)Age:?18 years old.(2)Clinical symptoms: nasal congestion,nasal itching,runny nose,sneezing(?2 items),duration?1 hour/day.(3)Physical signs: pale and edema of the nasal mucosa,watery nasal discharge.(4)Treatment: No immunotherapy was performed at the first visit,and immune diseases of other systems were excluded.(5)Laboratory tests: allergen detection(+),total serum Ig E(+).(6)Classification: Using the 2016 Allergic Rhinitis and Its Effects on Asthma(ARIA)Guidelines: All AR patients are divided into mild group(1 point)and moderate-severe group(2 points and 3 points)according to the severity of the disease.Ethics approval was obtained from the Ethics Committee of Shengjing hospital of China Medical University,and written informed consent for participation in the study was obtained from all study participants.2.Blood Sample ProcessingFive milliliters of peripheral blood was collected from each study participant with vacutainer tubes containing ethylenediaminetetraacetic acid(EDTA;Becton Dickinson,Plymouth,UK).Peripheral blood mononuclear cells(PBMCs)were obtained by Ficoll-Hypaque density gradient centrifugation,before being cryopreserved in fetal calf serum supplemented with 10% dimethyl sulphoxide,and stored in liquid nitrogen within 48 h of collection.Levels of total Ig E in the plasma were measured in AR patients using Immuno CAP 100(Phadia,Uppsala,Sweden).3.Determination of Lymphocyte Subset CountsLymphocyte subset count(T,B,NK,CD4,and CD8)percentages and absolute numbers were determined with a BD FACSCanto(?) II(Becton Dickinson,USA)flow cytometer,using a sixcolor direct immunofluorescence reagent(BD Multitest IMK kit and BD Multitest 6-color TBNK,Becton Dickinson,USA)with trucount tubes(Becton Dickinson,USA).4.Detection of CD4+T Cell Subset and Receptor ExpressionRegulatory T cells(Treg)were defined as CD4+CD25+CD127low/-cells.Other CD4+T subsets were identified by differential expression of CCR4,CXCR3,and CCR6 as previously reported[35,36]:CXCR3+CCR4-CCR6-(Th1),CXCR3-CCR4+CCR6-(Th2),CXCR3-CCR4+CCR6+(Th17),and CXCR3+CCR4-CCR6+(Th1Th17)(Figure 1).In addition,Th17 cells were defined as the summation of CXCR3-CCR4+CCR6+(Th17)and CXCR3+CCR4-CCR6+(Th1Th17).The fluorochromeconjugated antibodies used for polychromatic flow cytometry analysis were CD3-BV510(Biolegend,USA,catalog no.317332),CD4-APC-Cy7(Biolegend,USA,catalog no.317418),CCR4-APC(Biolegend,USA,catalog no.359404),CXCR3-PE(Biolegend,USA,catalog no.353706),CCR6-PE-Cy7(Biolegend,USA,catalog no.353418),CD25-BV421(Biolegend,USA,catalog no.302630),CD127-FITC(Biolegend,USA,catalog no.351312),PE-Cy7-CCR7(Biolegend,USA,catalog no.353226)and CD62L-FITC(Biolegend,USA,catalog no.304804).A viability dye,7-AAD(Becton Dickinson,USA,catalog no.559925),was used to exclude dead cells.Isotype antibodies were also used as negative controls for every detection to set proper gating for the receptor expression.Cells were analyzed by fluorescence-activated cell sorting(FACS),using the BD LSRII cytometer and Flow Jo software.5.Measurement of Serum Cytokines and ChemokinesSerum was collected from AR patients and HCs,separated by centrifugation(2000 × g for 10 min),and stored in 500 ?L aliquots at-80°C until analysis.Serum concentrations of IL-4,IL-2,IP-10,IL-6,TNF-a,MCP-1,IL-17,IL-10,IFN-g,IL-12,and IL-8 were measured with the LEGENDplex human essential immune response panel(Biolegend,USA,catalog no.740929),as per the manufacturer's instructions.Briefly,50 m L of serum was incubated with antibody-coated capture beads for 2h at room temperature.After washing the beads,25 m L of detection antibodies was added,and incubated with the beads for 1h at room temperature.Next,25 ?L of SA-PE was added directly to each well,and incubated for 30 min at room temperature.After washing away unbound SA-PE,the beads were resuspended in sheath fluid for 5min.Finally,samples were assessed on the BD FACSCanto II(Becton Dickinson,USA),and analyzed with LEGENDplex 8.0 software(Biolegend,USA).6.Statistical AnalysisStatistical calculations were performed using SPSS 17.0 software.Categorical data were described and analyzed by frequency and chisquare(c2)tests.For parametric comparisons,two-tailed Mann-Whitney U or paired Wilcoxon tests were used to assess differences among groups.The Spearman's rank correlation test was used to measure correlations between variables.Unless otherwise stated,p values <0.05 were considered statistically significant.Results:1.There was no significant difference in T.B,and NK cell counts between AR patients and HCs,but a mild increased trend of T cells was found in AR patients.2..There was no significant difference between mild and moderate-severe AR patients in percentage and absolute count of peripheral lymphocyte subsets.3.AR patients had more absolute CD4+T cell counts than HCs.4.There was no significant difference between mild and moderate-severe AR patients in CD4+T cell counts.5.AR patients showed significantly less Th1 and Th17 cells,both in percentage and absolute count.6.Moderate-severe AR patients showed a significant reduction in Th1 and Th17 cells compared to mild AR patients.7.AR patients in this study had increased levels of IFN-??IL-10 and IL-6,which suggests an upregulation of both Th1 and Th17 cytokines.8.AR patients had a significantly reduced expression of CCR4,CCR6,CXCR3,and CD62 L on CD4+T cell.9.CXCR3 was significantly downregulated by both MFI and frequency in AR patients with moderate-severe disease severity than mild patients.Conclusion:While T cells are considered to play a primary role in IgE-mediated atopic diseases,little is known about the systemic variations of T cell subsets from patients with allergic rhinitis(AR).To elucidate the characteristics of peripheral T cells,we analyzed natural killer,B cell,and T cell populations,performed T cell subset construction,and assessed chemokine receptor and associated serum cytokine expression in 25 AR patients and 20 healthy controls.Our results revealed increased levels of CD4+T cells,serum interleukin(IL)-10,IL-6,and interferon(IFN)-?,and reduced Th1 and Th17 subsets,identified by their chemokine receptors,in AR patients.These results suggest a systemic activation of T cell responses in AR.We further demonstrated that AR patients exhibit significantly reduced CD4+T cell CXCR3 expression,especially in patients with moderate-severe disease severity,demonstrating that CXCR3 is a potential key molecule that hinders the Th1/Th2 balance in AR pathology.Overall,systemic T cell activation occurred in AR patients and CXCR3 dramatically decreased in CD4+T cells,which may ultimately be used as a potential disease and/or therapeutic target.