The Role Of IL-17A In Contributing To The Impaired Suppressive Function Of Tregs In Psoriasis

Background:Psoriasis is a common autoimmune chronic disease of dermatology.The typical clinical manifestations of psoriasis are erythema,plaque and scales with a clear boundary.Pathological characters are psoriatic hyperplasia,dermal papillary capillaries,and dermal lymphocyte infiltration.Many factors such as genetics,environmental factors,drugs,and mental stress induce psoriasis.T cells and other immune cells play an important role in the pathogenesis of psoriasis,forming an inflammatory loop,leading to chronic inflammation of psoriasis.The over-activated of Th17 cells produce interleukin 17A(IL-17A)and other cytokines,which is proved to be the central link in the pathogenesis of psoriasis.Biological agents targeting IL-23/IL-17 pathways have achieved satisfactory results in psoriasis treatment,with improved clinical manifestations,pathological and immunohistochemical manifestations.Regulatory T cells(Treg cells),a group of T cell subsets with immunosuppression function,are crucial in maintaining T cell homeostasis and preventing autoimmune disease.When the immunosuppressive function of Treg cells is insufficient or defective,the effector T cells will be abnormally active,leading to a variety of autoimmune diseases.Previous studies by our research group and other scholars have proved that the suppression function of Treg cells from psoriasis patients decreased,but the mechanism is still unclear.Whether the psoriasis-related inflammatory factors represented by IL-17 A directly act on Treg cells to interfere with their immunosuppressive function needs further research.Objective:To discuss the effect and underlying mechanism of IL-17 A on the suppressive function of Treg cells.Explore the effect of IL-17 A on Treg cells in the pathogenesis of psoriasis.To evaluate the restoration of the suppressive function of Treg cells in psoriasis during anti-IL-17 A treatment.Methods:1.The effect of IL-17 A on the suppression function of Treg cells: Collect PBMC from healthy control and psoriasis patients,then use magnetic beads or flow cytometry to sort out Treg cells and Teff cells.Teff cells were co-cultured with Teff cells with different concentrations of IL-17 A.CFSE signal was detected by flow cytometry after 5 days.Then calculate the proliferation rate of Teff cells and the Treg suppression rate.Treg was sorted and cultured with IL-17 Ato detect the effect of IL-17 A on Treg proliferation and apoptosis.2.Effect of IL-17 A on the expression of transcription factors and the secretion of inflammatory factors and inhibitors of Treg cells: Collect PBMC from healthy people and use magnetic beads or flow cytometry to sort out Treg cells.Treg cells were cultured with IL-17 A stimulation for 48 hours.RNA-sequencing and RT-PCR were performed to detect the gene expression level.The release of inhibitory cytokines,including IL-10 and TGF-β,was assessed by flow cytometry.3.Effect of IL-17 A on pathway activation in Treg cells: Collect PBMC from healthy people and use magnetic beads or flow cytometry to sort out Treg cells.Treg cells were cultured with IL-17 A stimulation for 30 minutes.The changes of NF-κB,MAPK and ERK pathways after IL-17 A treatment were detected by flow cytometry and immunofluorescence.Use NF-κB inhibitor and add IL-17 A stimulation to detect the influence of IL-17 A on activation NF-κB pathway related to cytokines secretion.IL-17 A signaling activation was analyzed by flow cytometry and immunofluorescence.4.Analysis of changes in Treg cells function of psoriasis patients after receiving anti-IL-17 A monoclonal antibody treatment: Recruit 3 patients with psoriasis and record their clinical manifestation and PASI score before and after secukinumab treatment.Collect the PBMC of psoriasis patients before and after secukinumab treatment,and co-culture sorted-Treg and Teff.Then detect the proliferation rate of Teff cells and calculate the Treg suppression rate.Detect the proportion of Treg in PBMC and the expression of various inhibitors of Treg by flow cytometry.Blood samples were collected from three psoriasis patients before and after anti-IL-17 A treatment to evaluate the change of suppressive function and inhibiting factors in Treg cells.Results:1.The suppression function of Treg cells in psoriasis patients is impaired: Flow cytometry results showed that compared with Treg cells derived from healthy control,the immunosuppressive function of Treg cells on Teff from psoriasis patients is impaired.Treg gene expression profile of psoriasis patients and healthy control showed that the upregulated genes in Treg cells from psoriasis patients were enriched in the IL-17 pathway.2.The up-regulated genes of Treg from psoriasis enriched in the IL-17 pathway:Analyzing the gene expression profile of Treg from psoriasis patients with and normal control,it was found that the differential genes which abnormally high expression in Tregs from psoriasis patients were mainly enriched in the IL-17 pathway.3.IL-17 A blocks the inhibitory effect of Treg cells on Teff cells by directly modulating the function of Treg cells: Flow cytometry results showed that IL-17 A does not affect the proliferation and apoptosis of Treg cells;IL-17 A does not affect the proliferation of Teff cells.IL-17 A inhibits the suppression function of Treg cells and act directly on Treg cells.4.IL-17 A inhibits suppression function of regulatory T cells on effector T cells by inhibiting the secretion of TGF-β by regulatory T cells: RNA sequencing results showed that TGF-β expression of Treg cells was down-regulated after IL-17 A treatment.RT-PCR results showed that IL-17 A inhibited the expression of TGF-β and promoted the expression of IFN-γ in Treg cells.Flow cytometry results showed that IL-17 A inhibited the secretion of TGF-β and promoted the secretion of IFN-γ in Treg cells.5.IL-17 A phosphorylates NF-κB to promote the production of the inflammatory cytokine IFN-γ: The immunofluorescence results showed that IL-17 A promotes IFN-γsecretion in Treg cells and activated the NF-κB signaling pathway in Treg cells.Nuclear localized phosphorylated pp65 and fluorescence intensity in Treg cells were increased following IL-17 A stimulation.However,neither the fluorescence intensity nor the nuclear localization of pp38 and p ERK was increased upon IL-17 A stimulation.The flow cytometry results revealed that the number of pp65-positive Tregs was significantly increased upon IL-17 A stimulation.Inhibition of the NF-κB pathway blocked IL-17A-induced upregulation of IFN-γ by Treg cells.6.Anti-IL-17 A treatment with secukinumab in psoriasis patients restores the suppressive function of Treg cells via the induction of TGF-β: The lesion improved and the mean PASI score of psoriasis patients was decreased after 12 weeks of Secukinumab treatment.The suppressive ratio of Tregs from psoriasis to autoreactive Teff cells increased after four weeks of secukinumab treatment,indicating restoration to a level of suppressive function similar to that observed in healthy controls.And TGF-β secretion increased in Treg cells of psoriasis after 4 weeks of secukinumab treatment.Conclusion:The lack of immunosuppressive function of Treg cells from psoriasis patients is closely related to the high expression of IL-17 A in the body.IL-17 A acts on Treg cells to interfere with their immunosuppressive functions from two aspects: on the one hand,IL-17 A directly inhibits the production of its main inhibitor TGF-β,making it unable to exert its inhibitory effect on effector T cells;on the other hand,it can also activate the NF-κB signaling pathway of Treg cells,phosphorylated p65 translocating to the nucleus,to mediate the secretion of IFN-γ and other inflammatory cytokines and play a pro-inflammatory effect.Our research proved that under the action of IL-17 A,Treg cells not only lose their immunosuppressive function to effector T cells but also promote inflammation,which is an important new finding in the pathogenesis of psoriasis.The use of IL-17 A monoclonal antibody to treat psoriasis can reverse the interference effect of IL-17 A on Treg cells and restore its immunosuppressive function.It is a supplement to the therapeutic mechanism of this treatment strategy,and it also provides new ideas for long-term psoriasis management.