Study Of The Immunoregulation Of Purinoceptor P2Y₆ On DC And NK Cell And The Underlying Mechanisms

Immune response is an extraordinary complicated and strictly regulated process,which divided into innate immune response and adoptive immune response.On one hand,the body can timely remove infected or damaged cells by activating the immune response.On the other hand,the body can also control the immune response timely and moderately through some negative regulation mechanisms to prevent the excessive activation of the immune response and maintain immunity tolerance and steady state.DC and NK cells,as important immune cells in immune response,play a key role in activating immune response and maintaining immune tolerance.Extracellular nucleotides are released during pathgen infection,inflammatory response or tumor.On one hand,these extracellular nucleotides can be used as a dangerous signal to induce immune cells to migrate to the site of inflammation.On the other hand,they can also activate innate immune response and adoptive immune response though act on their receptor in the immune cells.The high expression of the purine receptor P2Y6 on DC and NK cells was found by consulting the database,and the expression of P2Y6 receptor was significantly reduced after DC and NK cells activation in this study.These results suggest that P2Y6 receptor may have important regulatory functions on DC and NK cells.Therefore,this study explores the function of P2Y6 receptor in immune response from the perspective of DC and NK cells,respectively,which provided potential targets for the treatment of autoimmune disease or tumor.Part ? Study on the effect of purinoceptor P2Y6 on the development and function of DC and the underlying mechanismsDendritic cells(DCs),as a professional antigen presenting cells(APCs),which have strong antigen-engulfing ability in the immune system.After antigen capture or stimulated by some factors,DCs differentiate into mature DCs.DC maturation is characterized by reduced phagocytic and endocytic activity,enhanced capacity of migration,enhanced expression of surface marker CD80,CD86 and MHC? molecules,enhanced antigen presentation and cytokine production activity,and increased ability to stimulate T and B cells.DC is the pivotal point in the initiation,regulation and maintenance of the immune response.The excessive activation of DCs can lead to the dysfunction of T cells,B cells or the whole immune response.DCs play a central role in activating immune response and maintaining immune tolerance.Furthermore,DCs also play an indispensable bridge between innate immune responses and adaptive immune responses.However,the mechanism for controlling the maturation and inducing T cell differentiation of DCs remain unclear.DCs express various kinds of pathogen recognition receptor(PRR)including Toll-like receptors(TLRs),which can sense the invading pathogens or recognize microbial components and subsequently trigger the innate immune response against pathogens.A large number of clinical data have proved that excessive activation of DC plays an important role in the development of autoimmune diseases(AID)such as multiple sclerosis(MS)and systemic lupus erythematosus(SLE),etc.Therefore,finding a negative regulation mechanism of DCs,which were over activation by TLRs,has an important theoretical significance for further exploring the regulatory mechanism of immune recognition and immune response,and also provides new ideas or targets for the treatment of some autoimmune diseases.The G protein coupled receptor(GPCR),also called the seven trans-membrane receptor,is the largest family of molecules involved in signal transmission.About 4%of the gene encoding protein in the human genome is the GPCR family,which plays an important role in introducing extracellular signal into the intracellular.GPCRs are extremely abundant in the body and play an important physiological role in various systems.They are therefore used as target sites for the treatment of various diseases such as cardiovascular system,nervous system,immune system,metabolism and endocrine system disease and tumor.The P2Y6 receptor is an extracellular nucleotide receptor,which located on chromosome 11,encoding 328 amino acid residues.It belongs to the GPCR and plays a vital role in modulating cellular responses to inflammation through autocrine or paracrine actions of uracil nucleotides.Recent studies suggest a role of P2Y6 receptor in innate immune responses against bacterial and viral intervention.Some groups demonstrated that P2Y6 receptor activation triggers chemokine release from DCs and monocytes,thus promoting inflammatory cells migrate to the inflammation or infection site and P2Y6 receptor deficiency increases the activation of effector T cells.In addition,the activation of P2Y6 receptor by endogenous agonist UDP triggers microglial phagocytosis.Similarly,blocking P2Y6 receptor signaling prevents neuronal loss and death by activated microglia phagocytosis of neurons.Our previous studies found that the expression level of P2Y6 receptor decreased if DCs were activated,suggesting that P2Y6 may play a role in regulating the function of DCs.On the basis of these observations,we investigated how P2Y6 receptor negatively regulated the function of DCs and subsequently prevented the pathgenesis of autoimmune diseases in this study.1.P2Y6 receptor significantly suppresses DC maturationIn order to examine whether P2Y6 repector controls DCs differentiation and function,we first analyzed mRNA in BMDCs after LPS stimulation by using transcription microarray technique.We observed the expression of P2Y6 in activated DCs by LPS stimulation was significantly downregulated.We also analyzed CD 11c+cells from spleens of wild-type mice after LPS and Poly(I:C)stimulation and found the level of P2Y6 was also downregulated.In order to investigate the maturation state of DCs in wild type and P2Y6-/-mice spleens,we detected the expression of surface moleculars such as CD40,CD86 and MHC II after LPS stimulation,the data showed that higher expression level of surface molecular CD40 and CD86 was observed in DCs from spleen of P2Y6-/-mice after in vitro lipopolysaccharides(LPS)stimulation.Howerer,no significant elevation of MHC-II was observed after LPS stimulation inDCs from spleen of P2Y6-/-mice.We next investigated the maturation and function of DCs in P2Y6-/-BMDCs.We found that P2Y6-/-BMDCs had higher expression of CD40 and CD86 as compared to wild type BMDCs,but no significant difference was detected in MHC II expression between P2Y6-/-and wild type BMDCs.Of note,we found the expression of CD40 and CD86 were markedly reduced in BMDCs stimulated with UDP,and no significant difference was detected in MHC ? expression.2.P2Y6 receptor significantly inhibits the antigen presentation of DCsWe first analyzed the antigen uptake of BMDCs and found P2Y6 receptor deficiency did not affect the uptake of antigens by BMDCs.However,we found P2Y6-/-DCs pulsed by OVA peptides markedly reduced the capacity to induce proliferation of OT-? T cell in vitro.In addition,we further used the contact hypersensitivity model to investigate whether P2Y6 deficiency also regulated the antigen presenting function of DCs in vivo.The results showed that P2Y6-/-DCs could induce stronger CHS response compared to wild type DCs.Accordingly,histological analysis also revealed significantly increased thickness of ear was detected in the mice injected by P2Y6-/-DCs.The data from both in-vitro and in-vivo analysis supported our hypothesis that P2Y6 receptor deficiency increased the antigen presenting function of DCs after antigen uptake.3.P2Y6 receptor changes the release of cytokines by mature DCsThe synthesis and release of cytokines with important modulatory function on inflammation and T cell differentiation by mature DCs are a major attribute.Therefore,we investigated whether P2Y6 receptor affected the secretion of cytokines from DCs at RNA and protein levels,respectively.The data showed that P2Y6-/-DCs produced more IL-12 and IL-23 at the RNA and protein levels.To further verify this result,we also used UDP,a ligand of the P2Y6 receptor and the results showed that UDP can inhibit the expression of IL-12p35,IL-12p40 and IL-23pl9 in DCs.4.P2Y6 receptor inhibits DC-mediated differentiation of Thl and Thl7cellsStudies have showed that IL-12 supports differentiation of Thl cells,while IL-23 plays an important role in Th17 cells defferentiation.Therefore,we cultured CD4+T cells with the supernatants from P2Y6-/-DCs or wild type DCs,the data showed that more Thl and Thl7 cells were induced if the supernantants from P2Y6-/-DCs were used.In addition,the supernatants from DCs treated by the agonist of P2Y6 receptor could induce less Thl and Thl7 cells compared with the supernatants from the untreated DCs.5.P2Y6 receptor does not affect T cell differentiation directlyNext,we tested whether P2Y6 receptor deficiency affect the T cell development and differentiation during steady state.P2Y6 deletion didn't affect the proporation of CD3,CD4 and CD8 T cells in the spleen of P2Y6-/-mice.Likewise,the proporation of splenic GD11b+ cells and B cells were similar between P2Y6-/-and wild type animals.To further investigate whether P2Y6 receptor deficiency has an effect on T cell subset differentiation,we detected the frequency of Thl and Th17 cells in the spleen of wild type and P2Y6-/-mice.No significant differences were detected between wild type and P2Y6-/-mice.In addition,the frequency and total number of Treg cells in spleen of P2Y6-/-mice remained equal to wild type mice.Thus,P2Y6 receptor deficiency has no effect on T cells activation and differentiation in steady state.To further verify this result,we induced T cell subsets differentiation with the different cytokines in vitro.The results show that the proporation of Thl,Thl7 and Treg cells was similar with or P2Y6 receptor.These data strongly supported that P2Y6 receptor deficiency does not affect T cell differentiation directly.6.P2Y6 receptor inhibits LPS-induced NF-?B activationThe above data suggested that P2Y6 receptor inhibits LPS-triggered DCs maturation,antigen presentiation and IL-12 and IL-23 production,which potently suppresses Thl and Th17 cells generation.In order to investigate the molecular mechanism through which P2Y6 receptor suppresses the LPS-triggered activation of DCs,we examined TLR signaling pathways in P2Y6-/-BMDCs and the downstream signaling pathway of TLR such as MAPK and NF-?B.Upon stimulation via LPS,the levels of p-ERK,p-JNK and p-p38 had not been changed,whereas the phosphorylation of NF-?B p65 in P2Y6-/-DCs was increased after LPS stimulation.In addition,P2Y6-/-DCs had more phosphorylation of kinases IKK?/? and the inhibitory NF-?B chaperone IKBa and more degradation of IKBa than wild type DCs.On the contrary,we also used the agonist of P2Y6 receptor and found the phosphorylation of NF-?B p65 was noticeably decreased after LPS stimulation and the level of p-ERK,p-JNK and p-p38 was not significantly altered.We concluded from above data that P2Y6 receptor selectively inhibited the LPS-triggered activation of NF-?B,but did not affect MAPK signaling pathway.7.P2Y6 receptor inhibits the development of EAEStudies have shown that Thl and Th17 cells play an important role in the autoimmune disease such as EAE.In order to prove whether P2Y6 receptor plays a role in the development of autoimmune diseases by inhibiting the DC ? IL-12/IL-23 ?Thl/Th17 cells pathway,we established EAE model by co-immunization of MOG(35-55)polypeptides and PTX and analyzed the function of the P2Y6 receptor in EAE model,as well as the relevant cellular and molecular mechanisms.The results showed that P2Y6-deficient EAE mice had higher clinical scores,more infiltration of inflammatory cells and more serious demyelination in the spinal cord.The proportion of Thl and Th17 cells in the spleen of P2Y6-deficient mice increased.The secretion of IL-12 and IL-23 in splenic DCs from P2Y6-deficient mice also increased.The above results showed that the inhibition of IL-12 and IL-23 production from DCs by P2Y6 receptor leaded to the suppression of Thl and Th17 differenriation and eventually contributed to the prevention of experimental autoimmune encephalomyelitis.To sum up,the following conclusions are drawn:Firstly,the expression of P2Y6 receptor is down regulated after DCs maturation and activation.Secondly,P2Y6 receptor inhibits the maturation and the antigen presentation as well as the production of IL-12 and IL-23 of DCs by suppressing LPS-induced NF-?B signaling pathway activation.Finally,P2Y6 receptors inhibits the occurrence of autoimmune diseases by suppressing DCs function.Part ? Study on the effect of purinoceptor P2Y6 on the maturation and function of NK cell and the underlying mechanismsThe natural immune response plays a crucial role in the defense against pathogen infection and tumor.NK cell is an important immune cell and plays a key role in mediating immune response and immune tolerance.NK cell can kill tumor cells and virus-infected cells by secreting some cytokines and cytotoxic granules.The development and activation of NK cells are highly regulated by the immune system,but the regulatory mechanism remains unclear.The development of NK cells is extremely complex.NK cells originate from common lymphoid precursor(CLP),experiencing NKp cell stage,iNK cell stage and mNK cell stage.The maturation and activation of NK cells are characterized by enhanced production of cytokines and cytotoxic granules as well as exhibited significantly stronger cytotoxicity.A large number of clinical data have proved that excessive activation of NK cell plays a key role in the development of autoimmune diseases(AID)such as Multiple sclerosis(MS)and Systemic lupus erythematosus(SLE),etc.Although much is known about how NK cells are activated to eliminate infected cells and tumor,the detailed mechanisms for the negative regulation of NK activation remain unclear.Therefore,the study on the negative regulation of NK cell development and activation can provide a significant theoretical basis for further exploration of immune tolerance and immunoregulatory mechanism and provide potential targets for the treatment of autoimmune diseases and tumors caused by the disordered NK cells.P2Y6 receptor is a member of the extracellular nucleotide receptor family,which belongs to GPCR.There are very few studies about how GPCRs regulate the function of NK cells.Recently,the studies show that GPR56(ADGRG1)is an inhibitory receptor of NK cells,and ATP can inhibit NK cell migration and cytotoxic via activating P2Y11 receptor.Our previous data showed that the expression of P2Y6 was downregulated in activated NK cells,which suggested that P2Y6 maybe played the role in the function of NK cells.Threrefore,the study was carried out on how P2Y6 receptor negatively regulated NK cell activation and tumor-killing function from the perspective of development,maturation,activation and cytokines production of NK cells and the molecular mechanism.1.P2Y6 receptor significantly inhibits the maturation of NK cellsFirstly,we analyzed the expression of P2Y6 receptor in activated NK cells with different activators.In vivo and in vitro experiments showed that the expression of P2Y6 receptor in splenic NK cells was significantly downregulated after Poly(I:C)stimulation.In order to investigate whether other activators can also affect the expression of P2Y6 receptors,we used IL-15 to stimulate the spleen cells.The results showed that the expression of P2Y6 receptor after IL-15 stimulation in NK cells was also significantly downregulated.In addition,the human NK cell line NK-92 cells were stimulated by Poly(I:C),IL-15 and IL-2,respectively.The results showed that the expression of P2Y6 receptors on NK-92 cells was also downregulated.Furthermore,the human peripheral blood NK cells were isolated and stimulated by IL-15.The results showed that the expression of P2Y6 receptor was also downregulated.In order to study whether P2Y6 deficiency affects the immune system of mice,we analyzed the proportion of immune cells in the P2Y6-deficient mice.The results showed that there was no significant change in the T,B and CD11b+ monocyte cells in the spleen of P2Y6-deficient mice.We also detected the proportion of total NK cells in the bone marrow,lymph nodes,liver and lungs of the P2Y6-deficient mice and found no significant changes.P2Y6 receptor can not affect the proportion of total NK cells in mice,so we investigate whether P2Y6 receptor affects the maturation and development of NK cells.The results showed that the percentage of NKp cells decreased and the proportion of iNK cells increased significantly in P2Y6-deficient mice compared to wild type mice.These results indicated that P2Y6 receptor inhibits the development of NKp cells into iNK cells.In order to further study the effects of P2Y6 receptor in NK cell maturation,we analyzed NK cell subsets in bone marrow,lymph node,spleen,liver and lung from WT and P2Y6-deficient mice.The results showed that the proportion of CD11b+ CD27+ NK cells,CD11b+ CD27+ NK cells and CD11b+ CD27+ NK cells in bone marrow and lymph node was similar in WT and P2Y6-deficient mice mice.However,the proportion of CD11b+CD27-NK cells in the spleen,liver and lung significantly increased in P2Y6-deficient mice.2.P2Y6 receptor suppresses the effector function of NK cellsFirstly,we studied the secretion of cytokine and cytotoxic granules and the expression of inhibitory and activator receptor in the steady state of NK cells.The results showed that the IFN-y and granzyume B(Gzm B)secreted by splenic NK cells did not change significantly in WT and P2Y6-deficient mice.We also detected the expression of activating(NKG2D)and inhibitory(NKG2A and KLRG1)receptor on the surface of NK cells and found the same results.These results showed that P2Y6 receptor deficiency did not affect the biological functions of NK cells in the steady state.In order to further investigate whether P2Y6 receptor affects the IFN-y secretion in activation NK cells,the mouse spleen cells were stimulated by Poly(I:C),IL-15,the combination of IL-12 and IL-15,or PMA,respectively.The results showed that the production of IFN-y significant increased in P2Y6-1-NK cells after the stimulation with Poly(I:C),IL-15,the combination of IL-12 and IL-15,or PMA.In addition,NK-92 cells or human peripheral blood NK cells was stimulated by the P2Y6 receptor agonist UDP or UDP analogues 5-OMe-UDP.The results showed that UDP or 5-OMe-UDP inhibited the secretion of IFN-? in NK cells.In order to further investigate how the P2Y6 receptor affects the function of NK cells,we designed a cytotoxic experiment in vitro and tumor metastasis model in vivo.The results of the in vitro experiment showed that the killing effect of P2Y6-/-NK cells was higher than that of the wild type NK cells.In vivo tumor metastasis model experiment showed that the numbers of lung nodules in mice injected with P2Y6-/-NK cells were significantly lower than those of mice injected with WT NK cells,and the lung weight was also significantly lower than that of mice injected with WT NK cells.3.P2Y6 receptor inhibits the maturation and function of NK cells by suppressing the expression of T-betIn order to further investigate the mechanism of P2Y6 receptor affecting the maturation of NK cells,we detected the expression of transcription factors T-bet and Eomes in NK cells and their subsets in spleen of WT and P2Y6-deficient mice.The results showed that the expression of T-bet in P2Y6-/-NK cells was significantly higher than that of WT NK cells,but there was no obvious change in the expression of Eomes.Similarly,the results for the detection of T-bet in splenic NK cell subsets showed that the higher expression of T-bet in CD11b-CD27+ NK cells,CD llb+ CD27+ NK cells and CD11b+ CD27-NK cells from P2Y6-deficient mice was detected.However,there was no significant difference in the expression of Eomes.We found that the expression of T-bet in P2Y6-/-NK cells was significantly higher than that of WT NK cells after IL-15 stimulation.In order to further verify this result,we also used P2Y6 receptor agonist UDP or 5-OMe-UDP to stimulate mouse NK cells,human NK cell lines NK-92 or human peripheral blood NK cells.The results showed that the expression of T-bet significantly decreased after UDP or 5-OMe-UDP stimulation.4.P2Y6 receptor inhibits NK cells activation induced by IL-15 via suppressing the mTOR signaling pathwayIn order to investigate the mechanism by which P2Y6 receptor deficiency increases the expression of T-bet in NK cells,and then leads to the maturation and activation of NK cells,we detected the downstream signaling pathways of IL-15 firstly.The results showed that there was no significant difference in the level of p-Foxol between WT and P2Y6-/-NK cells.Furthermore,there was no significant difference in Foxol expression between total NK cells and NK cells in different mature stages.We also detected the expression of IL-15 receptor CD122 and CD132 on the WT and P2Y6-/-NK cells.The results showed that there was no significant change in both of them.Then,we detected the experession of CD71 and CD98 which was associated with mTOR signaling pathway,the data showed that the expression of CD71 and CD98 on P2Y6-/-NK cells after IL-15 stimulation was significantly higherthan that of WT NK cells.Finally,we further investigated the activation of mTOR signaling pathway.The results showed that the phosphorylation level of Akt and S6 protein after IL-15 stimulation in WT NK cells was significantly lower than that in P2Y6-/-NK cells,but the level of 4EBP1 phosphorylation did not change significantly.To sum up,the following conclusions are drawn:Firstly,the expression of P2Y6 receptor can be reduced after NK cells activation.Secondly,the P2Y6 receptor can inhibit the maturation and activation of NK cells by suppressing the expression of T-bet.Thirdly,IL-15 can induce T-bet expression in NK cells via activating mTOR signaling pathway,but P2Y6 receptor can inhibit the expression of T-bet by suppressing the level of p-Akt and pS6 in NK cells,thus leading to the activation and maturation of NK cells and eventually inhibiting the NK cytotoxic function.This study has proved the negative regulation function of P2Y6 receptor on the maturation and activation of NK cells,and elucidated its molecular mechanism,which provided potential targets for the treatment of NK cell-related diseases.