Jiangsu Institute Of Parasitic Diseases Role Of The Atypical Protein Kinase C In Th17 Differentiation And Function

Immunoregulation plays central roles for maintaining the homeostasis of the body.Current hot topics in the immunology,such as autoimmune,allergy and cancer,all related to the imbalance of immunoregulation.Th17 cells,regulatory T cells(Treg),natural killer T cells(NKT),follicular helper(Tfh)and their effector cytokines/chemokines are actively involved in the immunoregulation.Recently,we have revealed,for the first time,that atypical protein kinase C ?/?(PKC?/?)is an important signal transduction molecule regulating Th2 differentiation and function and plays an important role in T cell polarization.Protein kinase C(PKC)is widely present in the tissues and cells of the body and plays key roles in the process of transmembrane signaling.PKC is divided into three groups: classical PKC(c PKC),novel PKC(n PKC)and atypical PKC(a PKC).Classical PKC(c PKC)subfamily consists of four members,PKC?,PKC?I,PKC??and PKC?.Novel PKC(n PKC)subfamily consists of four members,PKC?,PKC?,PKC? and PKC?.Atypical PKC(a PKC)subfamily consists of only two members,PKC? and PKC?/?.PKCs are involved in a variety of physiological and pathological activities.They exert important roles in the pathogenesis of tumor,allergic inflammation,autoimmunity etc.,and are becoming important therapeutic targets.We have reported that PKC?/? plays an important role in Th2 cell differentiation and allergic airway inflammation.However,the impacts of PKC?/? on Th17 cells remain to be elucidated.Th17 cells,a newly defined T helper subset,possess their own differentiation and regulation pathways,independent from Th1 or Th2 cells.They play an important immunoregulatory and effector functions.Th17 cells are preferential producers of IL-17(also called IL-17A),IL-17 F,IL-21,IL-22 and other effector cytokines,thereby inducing a massive tissue reaction owing to the broad distribution of the IL-17 and IL-22 receptors.More and more studies have shown that Th17 cells play an important role in allergic airway inflammation.In this study,we investigated the effects of PKC?/? on Th17 differentiation and function in vitro in cell culture systems and in vivo in murine allergic airway inflammation with the use of conditional PKC?/?-deficient mice.Our data support the notion that PKC?/? emerges as a novel regulator for Th17 cells.Part I.Effect of PKC?/?-deficiency on Th17 differentiation and function in vitro ObjectiveTo investigate the effect of PKC?/?-deficiency on the differentiation and cytokine secretion of Th17 cells in vitro.The CD4+ T cells were isolated from activated T cell-specific conditional PKC?/?-deficient mice and their WT littermates.The effects of PKC?/?-deficiency on the differentiation,cytokine secretion of Th17 cells will be investigated under non-polarizing and Th17 polarizing in vitro culture systems.MethodsNa?ve CD4+ T cells were isolated from spleens of PKC?/?-deficient mice(PKC?/?flx/flx Creoxtuv,KO)and their WT littermates(PKC?/?flx/flx Creoxt uw,WT)by the use of automatic magnetic cell separation technique(Auto MACS Pro).Purified CD4+ T cells were stimulated with anti-CD3/CD28 in vitro by non-polarizing conditions,or cultured under Th17/Treg polarizing conditions.The cytokine levels in the culture supernatants were measured by ELISA(enzyme-linked immunosorbent assay),and the cultured cells were assayed by fluorescence-activated cell sorting(FACS)for intracellular cytokine staining.For the detection of Th2,Th17,Treg subsets,cells were stained with IL-4,IL-17 and CD25/Foxp3,respectively,by FACS.The cytokine levels of IL-4,IL-17 and IL-10 etc.in the culture supernatant were measured by ELISA.ResultsWT and PKC?/?-deficient CD4+ T cells were cultured under non-polarizing conditions with anti-CD3/CD28 stimulation for 2 d;or differentiated under Th17-polarizing conditions for 4 days and restimulated with PMA plus ionomycin.Th17 cells were markedly reduced from PKC?/?-deficient CD4+ T cells under Th17-polarzing conditions by FACS assays.Consistently,IL-17 levels were significantly reduced in the culture supernatants under both non-polarizing and Th17-polarzing conditions by ELISA.ConclusionPKC?/?-deficiency was able to significantly inhibit the Th17 differentiation and cytokine secretion in vitro.Part II.Effect of PKC?/?-deficiency on Th17 function in vivo ObjectiveTo study the effect of conditional PKC?/?-deficiency on Th17 function in the model of murine allergic airway inflammation.MethodsWe established the murine asthma model induced by house dust mite(HDM)with the use of PKC?/?-deficient mice(PKC?/?flx/flx Creoxtuv,KO)and their WT littermates(PKC?/?flx/flx Creoxtu w,WT).To evaluate the infiltrating cells in the airways,the bronchoalveolar lavage(BAL)fluids and lung tissues were collected for Kwik-Diff and H&E stainings,respectively.Cytokines and chemokines,such as IL-4,IL-17,IFN-?,Gob-5,Eotaxin,in the BAL fluids and lung tissues were assayed by ELISA and real-time quantitative PCR,respectively.ResultsWe investigated the impacts of PKC?/?-deficiency on the murine allergic airway hyperresponsiveness induced HDM,which is a Th17 and neutrophils dominant murine asthma model,mimicking severe asthma.One of the major features of asthma is the airway infiltrations of various inflammatory cells,such as CD4+ T cells,macrophages,neutrophils and eosinophils,leading to high airway reactivity and structural changes.We found that the infiltrating cells,Th17 and Th2 effector cytokines in BAL fluids and lungs were significantly reduced in KO mice.Eotaxin and Gob-5,important mediators for airway hyperresponsiveness,were also reduced by the loss of PKC?/?.ConclusionWe successful established the HDM-induced murine asthma model with the use of conditional PKC?/?-deficient mice.Our findings indicated that PKC?/?-deficiency not only inhibited Th17 differentiation and cytokine secretion in vitro,but also significantly downregulated HDM-induced murine allergic airway inflammation.This downregulation was related to the reduced Th17 and Th2 functions in the airways.To our knowledge,it is the first study to reveal the impacts of PKC?/?-Th17 axis in allergic airway inflammation with the use of conditional PKC?/?-deficient mice.More in depth investigations are currently ongoing to elucidate the underlying molecular mechanisms.