Correlative Studies Of AhR Expression And Th17Response And The Influence Of AhR Ligand Intervention In Patients With Chronic Rhinosinusitis With Nasal Polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common clinical disease ofotolaryngology department, which is characterized by chronic inflammation of the nasalcavity and sinusitis mucosa and the appearance of nasal polyps (NPs) that develop in thenasal cavity; these NPs are visible to physicians during nasal endoscopy. According to thecurrent epidemiological survey, CRSwNP is a major public health concern andbrings serious economic burden to person because of its high prevalence worldwide(2%–5%), which affects quality of life, sleep, and work performance. The level ofdiagnosis and treatment of CRSwNP has been greatly promoted, however, the therapeuticeffect of CRSwNP is still cannot make all patients satisfied completely, and the rate ofrecurrence is also high. In the past decades, otolaryngologists and researchers had carriedout extensive and in-depth research on the Etiology, aetiopathogenesis and treatment ofCRSwNP. Currently; several studies have proved that the chronic inflammatory reactionof mucosa was caused by single or multiple factors such as anatomical variation,infection, atopic constitution, and abnormality of immunoregulation. Moreover, apredominant Th17population is the marker of CRswNP in Chinese patients.Recent studies have focused on the role of AhR, a ligand-activated transcriptionfactor, in modulating immune reactions, which are involved in the pathogenesis ofallergic asthma, atopic dermatitis, experimental autoimmune encephalomyelitis, andrheumatoid arthritis. Animal studies have yielded increasing evidence that AhRpossesses strong immunomodulation potential by controlling the differentiation of T lymphocytes and dendritic cells (DCs). In particular, after activation by its ligands, suchas2-(1H-indole-3-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), AhR couldsuppresse Th17cell proliferation. Therefore, we hypothesize that AhR may play a keyrole in the formation of chronic inflammation patterns in Chinese patients withCRSwNP.Our experiments attempt not only to explore whether AhR involved in thepathogenesis of CRSwNP and the correlative studies of AhR expression and th17response, but also analyze physiological action of AhR-DC-Th17signal pathwaythrough intervening the immune cells which from atopic and non-atopic CRSwNPpatients and controls, in order to find out a new target in thetreatment of CRSwNP.PART ONE. RELATIVITY ANALYSIS OF AHR AND TH17RESPONSE IN PATIENTS WITH NASAL POLYPOSISObject: In order to further elucidate the pathogenesis and find new targets fortreatment of CRSwNP, the aim of the present study was to investigate whether AhR,which regulates Th17cell differentiation, played a role in the pathogenesis of CRSwNPby evaluating AhR expression in nasal polyps (NPs) and peripheral blood mononuclearcells (PBMCs) obtained from CRSwNP patients.Methods: A total of48CRSwNP patients (atopic,24; non-atopic,24) and13cerebrospinal rhinorrhea patients (controls) were recruitted for this study. To explore therole of AhR in CRSwNP, we analyzed the expression of AhR (RT-qPCR and Westernblot), retinoid-related orphan receptor C (RORC; RT-qPCR), interleukin (IL)-17andIL-10(ELIASA), and the differentiation of Th17cells (FCM).Results: The expression of AhR was reduced in CRSwNP, and the expression ofAhR was lower in the atopic group than in the non-atopic group. However, there was avery low level of Th17and its associated factors (RORC, IL-17) in the control groupcompared to the two CRSwNP groups. In particular, the polarization of Th17cells in atopic CRSwNP patients was increased compared with non-atopic individuals. Therewere significantly negative correlations between AhR mRNA levels and Th17/IL-17inPBMCs, as well as between AhR mRNA and RORC mRNA/IL-17levels in NPs. Positivecorrelations were found between Th17and IL-17in PBMCs and between RORC mRNAand IL-17in NPs. Positive correlations were also found between AhR mRNA in NPs andPBMCs, RORC mRNA in NPs and Th17in PBMCs, and IL-17in NPS and PBMCs.Conclusions: The decreased expression of AhR and overexpression of Th17inNPs and PBMCs from CRSwNP patients, which May play an important role in thepathogenesis of CRSwNP. Moreover, the lower AhR expression and more intense Th17response in atopic group demonstrate that atopic constitution may aggravate Th17response by reducing AhR expression.PART TWO. AHR LIGAND INTERVENED AND REGULATEDTH17RESPONSE IN PBMCS OF CRSWNP PATIENTSObject: In this section, ITE acts as an intervention factor, and we observe the effectsof ITE on the expression of AhR and Th17and the related cytokines IL-17and IL-10so asto further understand the pathogenesis of CRSwNP. Based on the previous experiment,we aim to demonstrate that ITE treatment may suppress the inflammatory responsethrough attenuating Th17responses in CRSwNP patients in vitro.Methods: Heparinized peripheral blood was obtained from eighteen CRSwNPpatients (atopic,9; non-atopic,9) and9controls. PBMCs were obtained by standardFicoll-Hypaque density-gradient centrifugation, and then intervened by ITE. Weanalyzed the expression of AhR in PBMCs by western blot, and detected the level ofTh17cells by FCM. The levels of IL-10and IL-17in the supernatant of CulturedPBMCs were investigated by ELISA.Results: AhR expression and IL-10production in ITE-treated PBMCs wasmarkedly higher than that in untreated PBMCs, the Th17cells differentiation and IL-17 prodction in ITE-treated PBMCs was significantly lower than that in untreated PBMCs inatopic and non-atopic CRSwNP patients and controlsConclusions: ITE intervention in PBMCs promoted AhR expression, andattenuated Th17response, which showed that AhR is a negative regulator of Th17response in CRSwNP. ITE, may serve to suppress the inflammatory response associatedwith CRSwNP through AhR–Th17signaling pathway.PART THREE. AN ARYL HYDROCARBON RECEPTOR LIGANDACTS ON DENDRITIC CELLS AND T CELLS TO SUPPRESS THETH17RESPONSE IN CRSWNP PATIENTSObject: In order to find new targets for AhR in atopic and non-atopic CRswNPpatients. We aim to detect the expression of AhR and related cytokins in DCs and CD4+T cell from atopic CRswNP patients, non-atopic CRswNP patients, and controls.Methods: Eighteen CRSwNP patients (atopic,9; non-atopic,9) and9controlswere included in this study. The expression of AhR,(interleukin) IL-1β, IL-6, and IL-10in DCs and AhR, IL-10, and IL-17in CD4+T cells were measured using RT-qPCR,Western Bolt, and ELISA.Results: We found significantly decreased AhR Expression is observed in DCs, butnot in CD4+T Cells from CRSwNP Patients. AhR expression in DCs was markedlylower in atopic patients than in non-atopic patients. We also found significantlyincreased levels of IL-6and IL-1β in the supernatants of DCs and IL-17in thesupernatants of CD4+T cells from atopic and non-atopic patients. IL-6, IL-1β and IL-17levels were markedly higher in atopic patients than in non-atopic patients. But weobserved significantly decreased IL-10levels in supernatants of DCs and CD4+T cellsfrom atopic and non-atopic patients. In addition, IL-10levels were signifcantly lower inthe atopic group than in the non-atopic group.Conclusions: Our work demonstrated that the deficiency of AhR may be the target of Th17response. The activation of AhR in DCs may play a critical role in regulatinganti-inflammatory response. Moreover, the deficiency of AhR is more severe in atopicgroup than in non-atopic group, this data suggested that atopy may exacerbate Th17response via AhR signaling.PART FOUR. AN ARYL HYDROCARBON RECEPTOR LIGANDACTS ON DENDRITIC CELLS AND T CELLS TO SUPPRESS THETH17RESPONSE IN CRSWNP PATIENTSObject: To analyze the effect of AhR–DC–CD4+T cell axis and AhR–CD4+T cellaxis on Th17response by investigating the action of ITE on DCs and CD4+T cells fromatopic and non-atopic CRswNP patients.Methods: Eighteen CRSwNP patients (atopic,10; non-atopic,10) and12controlswere included in this study. The expression of (interleukin) IL-1β, IL-6, and IL-10inDCs and IL-10, and IL-17, and the presence of Th17cells in CD4+T cells and aDC-CD4+T cell co-culture system were measured using mRNA or protein detectionmethods before and after treatment with ITE.Results: We showed that ITE significantly induced cell secretion of IL-10andinhibited IL-1β and IL-6production in DCs, and promoted IL-10production andsuppressed IL-17expression in CD4+T cells in vitro. It also suppressed the expansion ofTh17cells in vitro.Conclusions: Our work demonstrated that ITE acts on DCs and CD4+T cells toinhibit the Th17response that suppresses CRSwNP; the AhR–DC–Th17axis may be animportant pathway in the treatment of CRSwNP. ITE, a non-toxic AhR ligand,attenuated the Th17response; thus, it appears to be a promising therapeutic candidatefor suppressing the inflammatory responses associated with CRSwNP.