| As a transcription factor,Aire plays important roles in the maintenance of self tolerance.A single gene mutation in Aire results in autoimmune polyglandular syndrome type I（APSI）,which shares major characteristics with many autoimmune diseases,such as the involvement of endocrine glands and chronic mucosal candida infection.It is well known that autoimmune diseases are caused by the combined effects of autoreactive T cells and autoreactive B cells.Multiple studies have already confirmed that Aire is primarily expressed in medullary thymic epithelial cells（mTECs）,and it prevents the development of autoimmune diseases through the clearance of autoreactive T cells.In addition,Aire can also be expressed in dendritic cells（DCs）,our previous studies revealed that the transplantation of the Aire overexpressing mouse DC cell line DC2.4 educated T lymphocytes into streptozotocin-induced type 1 diabetes mellitus model mice could delay disease progression,suggesting that Aire expressed in peripheral DCs might be involved in the maintenance of peripheral immune tolerance.However,the exact mechanism underlying this effect and whether it is caused by inhibiting the activation of autoreactive B lymphocytes are still not clear.It has been shown that T follicular helper（Tfh）cells are an important subpopulation that facilitates the activation of autoreactive B cells.An abnormal level of Tfh cells has been closely associated with the development of autoimmune diseases.Tfh cells located in lymphoid follicles,and their characteristic transcription factor is B cell lymphoma 6（BCL-6）,which is essential for the differentiation of Tfh.Tfh express high levels of various biomarker molecules,including C-X-C chemokine receptor type 5（CXCR5）and programmed death 1（PD-1）.Tfh cell differentiation is a multistage,multifactorial process,and it starts at initial DCs priming of a na?ve CD4+T cell.IL-6,secreted by DCs,interacts with its corresponding receptor to induce BCL-6 expression,which is a key transcription factor involved in the induction of Tfh differentiation.IL-6 deficiency in DCs results in poor Tfh cell differentiation.Inducible costimulator molecule ligand（ICOSL）is primarily expressed in DCs,B cells,and other antigen-presenting cells（APCs）,and it belongs to the B7 family.ICOS,the ligand for ICOSL,is only expressed in activated T cells and belongs to the CD28 family.The ICOSL-ICOS interaction can regulate humoral immunity and germinal center reaction.This signaling pathway is very important for mediating the production of Tfh cells in mice.Blocking ICOSL in DCs can reduce the expression levels of CXCR5 and ICOS in CD4+T cells.Tfh cell development has been reported to be inhibited in ICOS deficient mice and humans.It has been shown that spleen DCs derived from Aire deficient mice are able to more strongly induce Tfh cell differentiation than those from wild-type mice.However,whether Aire influences Tfh cell differentiation through the regulation of ICOSL and IL-6 expression in DCs is still not clear.Based on the above background,in order to clarify the effect of Aire on the expression of IL-6 and ICOSL in DCs and its role in inducing Tfh differentiation,we performed the following studies.1.We observed the number of Tfh cells in the lymph nodes and spleen,the expression of ICOSL and IL-6 in DCs in Aire knockout mice infected with the FM1 strain of the H1N1 influenza virus and immunized with OVA.We try to investigate the relationships between Aire and both ICOSL expression and IL-6 secretion in DCs and changes in the number of Tfh cells.2.We observed the effects of bone marrow DCs（BMDCs）from Aire knockout mice(Aire-/--BMDCs)on Tfh cell differentiation in vitro,and we examined the expression levels of ICOSL and IL-6 inAire-/--BMDCs to study the role of Aire in Tfh cell differentiation and the possible mechanisms.I.The effects of Aire on Tfh cell differentiation in vivo1.Establishment of H1N1 infection mice model to observe the effect of Aire on Tfh differentiation（1）Establishment of a mouse model infected with the H1N1 influenza virusTo observe the effects of Aire on Tfh cell differentiation in vivo,Aire knockout mice and wild-type mice were infected with the FM1 strain of the H1N1 influenza virus by the intranasal infection.Using normal saline as control group.To confirm whether the H1N1 infected mouse model was established successfully,peripheral blood from model mice was collected on day 8 for the detection of H1N1-IgG using ELISA.The results showed that,the serum H1N1-IgG concentrations of model mice were between 4-10μg/ml after H1N1 infection,and the concentrations in the Aire knockout mice were significantly higher than those in wildtype mice.In addition,HE staining was performed on lung tissues from model mice on day 8 after virus infection.The results showed that model mice exhibited obvious inflammatory cells infiltration,and the infiltration was more serious in the Aire knockout mice than it in wildtype mice.These results indicated that the H1N1 infected mouse model was established successfully and that the degree of antibody response in the Aire knockout mice was significantly higher than that in the control group.（2）Tfh cell differentiation in Aire knockout miceTo further investigate the reason for the increase of anti-H1N1 antibodies observed in Aire knockout mice,the number of Tfh cells that facilitated the production of antibodies by B cells was examined in vivo.The numbers of Tfh cells in the spleens,SLNs,and MLNs from model mice were detected on day 8 after H1N1infection using FACS.The results showed that the numbers of Tfh cells in the spleens and lymph nodes from knockout mice were higher than those in wildtype mice,suggesting that Aire might inhibit Tfh cell differentiation.This may be used to explain the increased antibodies in Aire knockout mice after H1N1 infection.（3）ICOSL and IL-6 expression in Aire knockout miceTo make it clear whether Aire can influence key molecules involved in Tfh differentiation,including the costimulator molecule,ICOSL and the cytokine,IL-6.ICOSL expression levels in DCs from the spleens,SLNs,and MLNs of mice model were detected on day 8 after H1N1 infection using FACS.IL-6 expression in DCs from the spleens was detected using FACS.The data showed that ICOSL in DCs from the spleens and lymph nodes of knockout mice were significantly upregulated compared with that in wildtype mice,and IL-6 in splenic DCs of knockout mice were significantly increased than those in the wildtype mice,suggesting that Aire might inhibit ICOSL and IL-6 expression in vivo.This may be the possible cause of Aire’s influence on Tfh differentiation.2.Establishment of OVA immunized mice model to observe the effect of Aire on Tfh differentiation（1）Establishment of a mouse model immunized with OVAIn order to further clarify the effect of Aire on Tfh differentiation under the condition of non-pathogen infectionin vivo,in addition to applying H1N1 infected mouse model,we also established OVA immunized mice model by subcutaneous injection of OVA and aluminium hydroxide（adjuvant）and strengthening immunity on the 15th day,using normal saline as control group.To confirm whether the model was established successfully,peripheral blood from model mice was collected on day 22for the detection of OVA antibody level using ELISA.The results showed that,the titer of anti-OVA antibody is greater than 1:3,200,and the titer in the Aire knockout mice were higher than that in wildtype mice.The above results indicated that the OVA immunized mice model was established successfully and that the antibody response level in the Aire knockout mice was higher than that in the control group.（2）Tfh cell differentiation in Aire knockout miceTo further investigate the reasons for the increase of anti-OVA antibody potency in Aire knockout mice,the numbers of Tfh cells in the spleens,SLNs,and MLNs from model mice were detected on day 22 after OVA immunization using FACS.The results showed that the numbers of Tfh cells in the spleens and lymph nodes from knockout mice were higher than those in wildtype mice,suggesting that Aire might inhibit Tfh cell differentiation in non-infectious bodies,presumably,this may be the cause of elevated antibody potency in Aire knockout after OVA immunization.（3）ICOSL and IL-6 expression in Aire knockout miceTo make it clear whether Aire can influence key molecules involved in Tfh differentiation,including the costimulator molecule,ICOSL and the cytokine,IL-6 in OVA immuned mice.ICOSL expression levels in DCs from the spleens,SLNs,and MLNs of mice model were detected on day 22 after OVA immunization using FACS,and IL-6 expressionlevels in splenic DCs were detected using FACS.The results showed that ICOSL in DCs from the spleens and lymph nodes of knockout mice were significantly increased compared with that in wildtype mice,and IL-6 in splenic DCs of knockout mice were significantly increased than those in wildtype mice,suggesting that Aire might inhibit ICOSL and IL-6 expression in vivo.Ⅱ.The effects of Aire-/--BMDCs on Tfh cell differentiation in vitroThe above results showed that Aire was related to ICOSL expression in DCs and IL-6 production and to the numbers of Tfhcells inAire knockout mice.However,it was not clear whether a direct association existed.Therefore,we further observed the effects of Aire-/--BMDCs on Tfh cell differentiation in vitro,and examined the expression levels of ICOSL and IL-6 in BMDCs derived from Aire knockout mice to investigate the effects of Aire on Tfh cell differentiation and the possible underlying mechanisms.1.The effects of Aire-/--BMDCs on Tfh cell differentiationIsolation of mouse bone marrow cells was performed,and immature BMDCs were obtained after induction for 6 days,Its purity could reach 82.7%.To observe the effects of Aire-/--BMDCs on Tfh cell differentiation both at a resting and stimulated state,two types of stimuli were selected:（1）LPS,one of the major cell wall components of Gram-negative bacteria that present important antigens to DCs after infection;and（2）poly（I:C）,a double-stranded RNA analogue and a virus infection associated molecular pattern.poly（I:C）can be recognized by toll-like receptor 3（TLR3）to induce nuclear factor kappa B（NF-κB）activation and cytokine production.We performed na?ve CD4+T cell sorting using magnetic beads.The purity of cells detected using FACS could reach 96.1%.Resting BMDCs,stimulated BMDCs with 1μg/ml LPS for 24 h or 100μg/ml poly（I:C）for 24 h were co-cultured with na?ve CD4+T cells for 72 h.The results showed that after co-culture of Aire knockout BMDCs and na?ve CD4+T cells,the number of Tfh was increased compared with that of control group,suggesting that Aire might inhibit Tfh cell differentiation under normal circumstances.2.The mechanisms of Aire-/--BMDCs influencing Tfh cell differentiation（1）Expression of ICOSL and IL-6 in Aire-/--BMDCsDCs could promote Tfh cell differentiation through ICOSL and IL-6.Our study showed that the numbers of Tfh cells in the spleens and lymph nodes of Aire knockout mice and the expression levels of ICOSL in DCs and IL-6 levels in serum all increased compared to those of the control group in vivo.The above results showed that Aire-/--BMDCs promoted Tfh cell differentiation in vitro.Therefore,we speculated that Aire might influence Tfh cell differentiation through the two above mentioned molecules,ICOSL and IL-6.We observed the effect of Aire on the expression of ICOSL and IL-6 in BMDCs.The RT-qPCR results showed that the ICOSL mRNA expression level in Aire-/--BMDCs significantly increased after LPS stimulation for 6 h compared to that in the control group.In addition,after poly（I:C）stimulation for 24 h,IL-6 mRNA expressionin Aire-/--BMDCs also significantly increased compared to that in the control.At the protein level,the detection of ICOSL in DCs using FACS and the detection of IL-6 in the cell supernatant using ELISA also showed the same trend.These results indicated that Aire might inhibit ICOSL and IL-6 expression in BMDCs.（2）The effects of Aire on Tfh cell differentiation through the regulation of ICOSL and IL-6 expression in BMDCsTo further confirm that Aire knockout DCs promoted Tfh cell differentiation through ICOSL and IL-6,supernatants from Aire-/--BMDCs were blocked using ICOSL or IL-6 antibodies,and then Aire-/--BMDCs were co-cultured with na?veCD4+T cells to observe the differentiation of Tfhcells.The results showed that,after blocking with anti-ICOSL or anti-IL-6 antibodies,the numbers of Tfh cells in the Aire knockout and control groups were both reduced,and there was no difference between these two groups.These results indicated that Aire inhibited Tfh cell differentiation through the downregulation of ICOSL and IL-6 expression in BMDCs.（3）The mechanisms of Aire influencing ICOSL and IL-6 expression on BMDCsLPS and poly（I:C）can be combined with TLR4 and TLR3 respectively to activate the transcription of ICOSL and IL-6 via NF-κB classical signaling pathway in BMDCs.Therefore,in order to explore the molecular mechanism of Aire affecting the expression of ICOSL and IL-6 in DCs,we use the Western Blot to detect the changes of IκBαand p65,which are the key molecules in the NF-κB classical pathway,in Aire-/--BMDCs after the stimulation by the above two stimuli,and the changes of TICAM after the stimulation of poly（I:C）.The results showed that the levels of phosphorylated IκBαand p65 in the BMDCs of Aire knockout mice were higher than those in the control group,and the level of TICAM was significantly increased than that in the control group after the stimulation of poly（I:C）.The results suggested that Aire is likely to influence the expression of ICOSL and IL-6 in DCs by regulating the relevant molecules in the NF-κB classical pathway.Through the above research,the following conclusions are obtained:1.Aire can inhibit the differentiation of Tfh,and decrease the antibody secretion by B lymphocytes in vivo.2.Aire can regulate NF-κB classical pathway and inhibit the expression of ICOSL and IL-6 to inhibit the differentiation of Tfh,which may eventually inhibit B cell activation and thus play a role in maintaining peripheral tolerance.In this study,the effects and mechanisms of Aire on Tfh differentiation were observed with Aire knockout mice as the research object.The results show that Aire can affect the NF-κB classical pathway of ICOSL and IL-6 expression regulation in DCs,and Aire can inhibit the differentiation of Tfh by inhibiting the expression of IL-6 and ICOSL.This study provides a new way to fully understand the role and mechanisms of Aire in peripheral immune tolerance.In addition,this study provides experimental basis for the treatment of various autoimmune diseases through the regulation of Tfh cell differentiation and the production of autoantibodies using Aire as the target. |
PDF Full Text Request