| Background:Autoimmune thyroiditis(AIT),a common organ-specific autoimmune disorder,is the main cause of hypothyroidism.AIT is characterized by the presence of thyroid-specific autoantibodies in the serum,massive infiltration of lymphocytic cells and destruction of the follicular structure within the thyroid.Genetic susceptibility,together with environmental factors,contributes to the breakdown of immune tolerance,but the specific molecular events are not clear yet.Abnormal change in thyroid itself or the immue system could lead to the breakdown of immune tolerance and the initiation of autoimmunity.In recent years,innate immune system is considered as the intersection of genetic predisposition and environmental risk factors.The outcome of innate immune response decides whether it develpes into adaptive immune response mediated autoimmune attacts.Studies on pattern recognition receptors in various autoimmune disorders,such as Toll-like receptor,have provides new perspectives on AIT immunopathogenesis.Inflammasomes are intracellular multi-protein plats composed of NOD-like receptors(NLRs)/AIM-like receptors,the apoptosis-associated speck-like protein that contains a caspase recruitment domain(ASC),and the inflammatory effector pro caspase-1.Activation of inflammasomes leads to self-activation of the cysteine protease caspase-1,which mediates maturation of pro interleukin-1βand pro interleukin-18;this is followed by gasdermin D-mediated programmed inflammatory cell death or pyroptosis,and subsequently,the release of active cytokines.Interleukin-18(IL-18),also known as interferon-γinducing factor,translated and storaged as pro IL-18(24KDa)in cytoplasm,and cleaved into active IL-18(18KDa)by caspase-1,which could significantly contribute to type 1 CD4~+T help cell responses.Pyroptosis is a proinflammatory and programmed cell deathmediated by Gasdermin D,characterized by loss of cell membrane integrity and release of cellular content.Inflammasomes have been linked to a variety of autoimmune disorders,such as systemic lupus erythematosus,multiple sclerosis and type 1 diabetes mellitus.But it is remained unknown whether inflammasomes take part in development and progress of AIT.Therefore,this study aims to investigate the expression signature of several classical inflammasomes—the NLRP1,NLRP3,NLRC4 and AIM2 inflammasomes—in the thyroid tissues and PBMCs from AIT patients and controls,also the related cytokines,IL-18 and IL-1β,were detected.In addition,a human thyroid follicular epithelial cell line(Nthy-ori 3-1)was used to explore the potential pathogenic roles of inflammasome activation,the release of active cytokines and pyroptosis.Methods:Part I:We collected thyroid tissues from 50 patients with AIT and 50 sex-and age-matched controls.Peripheral whole blood and serum samples were collected from another 50 patients with AIT and 50 matched controls.Peripheral blood mononuclear cell(PBMC)was separated by density gradient centrifugation.Serum levels of free T3,free T4,thyrotropin,thyroid peroxidase antibody(TPOAb)and thyroglobulin antibody(TgAb)were measured by electrochemiluminescent immunoassays.Total RNA and protein was extracted from thyroid tissues and PBMCs.The mRNA expression of several inflammasome components(NLRP1,NLRP3,NLRC4,AIM2,ASC and Caspase-1)and downstream cytokines(IL-1βand IL-18)was determined by real-time PCR,and their protein expression was determined by Western-blot.The expression levels of cleaved caspase-1、IL-18 and IL-1βwas measured by Western-blot.Immunohistochemistry was used to localize the expression of NLRP1,NLRP3,NLRC4 and AIM2.Serum concentrations of IL-18 and IL-1βwere assayed with enzyme-linked immunosorbent assay(ELISA).PartⅡ:The Nthy-ori 3-1 thyroid cell line was stimulated with gradient cytokines,TNF-αat 0/125/250/500IU/ml,IFN-γat 0/125/250/500IU/ml,IL-17A at 0/0.01/0.1/1ng/ml and IL-6 at 0/0.01/0.1/1 ng/ml for 24 hours.The expression of NLRP1,NLRP3,NLRC4,AIM2,ASC,Caspase-1,IL-1βand IL-18 m RNA was determined by real-time PCR.The levels of IL-18 and IL-1βin the cell supernatant were measured by enzyme-linked immunosorbent assay(ELISA).After stimulated with 250 IU/ml interferon–γand 1μg/ml poly(d A:dT),ASC specks were examined by confocal immunofluorescence microscopic analysis,the concentration of IL-18 in cell supernatant was measured by ELISA.Cell pyroptosis was examined by flow cytometry with FITC-Annexin V/PI dyeing,the N-terminal domain of gasdermin D was detected by western blot analysis,and lactate dehydrogenase(LDH)was quantified by absorptiometry.The level of IL-18 and LDH in cell supernatant was reevaluated after the use of a Caspase-1inhibitor,z-YVAD-FMK.Results:Part I:Expression of NLRP1,NLRP3,NLRC4,AIM2,ASC,Caspase-1,IL-18,IL-1βmRNA and protein was significantly increased in thyroid tissues from patients with AIT(P<0.01).Enhanced posttranslational maturation of Caspase-1,IL-18 and IL-1βwas observed in the AIT thyroid tissues,which indicates increased activity of inflammasomes.Expression of NLRP1,NLRP3,NLRC4 and AIM2 was localized mainly in thyroid follicular cells adjacent to areas of lymphatic infiltration on thyroid tissue paraffin sections.The thyroid mRNA level of NLRP1 and ASC was correlated to the serum TPOAb and TgAb levels in the AIT group.Expression of NLRP3,NLRC4,pro IL-1βand pro IL-18 mRNA and protein were significantly elevated in PBMCs from patients with AIT compared with those from healthy controls,while no difference was observed in NLRP1,AIM2,ASC and pro Caspase-1 expression.PBMCs from AIT patients expressed higher levels of active Caspase-1 and active IL-18 than controls,but equal level of active IL-1βto controls.Serum IL-18 level was significantly higher in AIT group than control group,while serum IL-1βlevel was not different between the two groups.PartⅡ:Throid cells express visible but low level NLRP1,NLRP3,NLRC4 and AIM2 proteins.Incubating cells with gradient IFN-γ(0,250,500,1000 IU/ml)for 24 hours significantly increased the mRNA levels of NLRP3,AIM2,ASC,Caspase-1 and IL-18(P<0.05),but slightly decreased IL-1βmRNA level.Stimulatting cells with gradient TNF-α(0,125,250,500 IU/ml)for 24 hours elevated the mRNA levels of NLRP3,Caspase-1 and IL-1βin a concentration-dependent manner,and significant increase in NLRP1and AIM2 expression were also observed.In contrast,stimulating thyroid cells with gradient IL-17A or IL-6 hours did not influence the mRNA expression of inflammasome component.The levels of both IL-1βand IL-18 in the supernatant did not differ between cells treated with IFN-γonly,TNF-αonly or IFN-γcombined with TNF-α.Preincubation with 250IU/mlIFN-γfor 24 hours,stimulating cells with poly(dA:dT)for12 hours could activate AIM2 inflammasomes,as macromolecular ASC specks were observed to co-localize with AIM2 surrounding the nucleus by immunofluorescence.Compared with the control group,stimulation with poly(dA:dT)combined with IFN-γsignificantly increased the IL-18 and LDH levels in supernatants,the ratio of Annexin V(+)/PI(+)cells and the expression of Gasdermin D-N terminal domain.Inhibting the activity of Caspase-1 could reduce poly(d A:dT)and IFN-γinduced release of IL-18 and LDH.Conclusions:Compared with the control group,AIT thyroid tissues express higher NLRP1、NLRP3、NLRC4、AIM2 inflammasomes,and higher level of NLRP3 and NLRC4 expression was determined in PBMCs from AIT patients.Inflammasomes contribute to AIT immunopathogenesis by activating cytokines(IL-18 and IL-1β)and mediating cell pyroptosis.Proinflammatory cytokines in AIT thyroids,IFN-γand TNF-α,significantly elevated the expression level of inflammasome componets and enhanced damage-associated molecular patterns induced activation of inflammasmes.Therefore,the current study revealed the NLRP1/NLRP3/NLRC4/AIM2-Caspase-1-IL-18/IL-1βaxis as an important participator in AIT pathogenesis. |
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