The Aberrant Regulatory T Cell Response In Primary Nephrotic Syndrome Or Cow’s Milk Protein Allergy Context And Impaired Early Activation Of B Cells Caused By DOCK8 Deficiency

Background:Primary Nephrotic Syndrome(PNS)is a common primary glomerular disease in children,which is also one of the important reasons that cause end-stage renal disease.Due to the complexity and heterogeneity of the etiology,the current understanding of the pathogenesis of PNS is still limited.Relevant research suggested that immune dysfunction was important pathogenesis of PNS.Among them,the disorders of T cell responses have attracted more and more attention and were considered to be one of the core factors in the occurrence and development of PNS.Regulatory T cell(Regulatory T cell,Treg)is one of the subgroups of CD4~+T cells.,which is marked by the expression of Foxp3.It has strong immunosuppressive ability and plays a central role in the establishment and maintenance of immune tolerance and homeostasis.Treg levels in the peripheral blood of children with PNS in acute phase were down-regulated,suggesting that abnormal Treg responses are involved in the development of PNS.Under a variety of autoimmune,immune disorders and inflammatory conditions,there were instability,plasticity and dysfunction of Treg cells.However,there is still a lack of relevant research in nephrotic syndrome.Therefore,the purpose of this study is to determine whether Treg cells have instability,plasticity and dysfunction under PNS conditions,and to analyze the underlying mechanisms that lead to the above abnormalities.Methods:to investigate this issue,we collected the peripheral blood samples from children with newly-diagnosed PNS during the acute phase alongside age-matched healthy controls(HCs),and conducted a series of ex vivo and in vitro experiments to analyze Treg cell-associated immune phenotypes(including stability,plasticity and dysfunction)in detail.We also attempted to figure out potential molecular machineries involved in the aberrant Treg cell responses using gene expression profiling coupled with specific pharmacological inhibitions.Results:(1)The stability of PNS-Treg was significantly impaired,which was manifested as a significant increase in the proportion of CD25~+Foxp3~-cells,a significant decrease in the expression of Foxp3,and a loss of Foxp3 expression during the proliferation of PNS-Treg cells;(2)PNS-Treg cells showed significant plasticity,which was manifested by a significant increase in the level of Th1-like Treg cells.In the process of proliferation,with the down-regulation of Foxp3 expression,PNS-Treg cells significantly transformed into Th1-like Treg cells secreting IFN-γ,and even completely transdifferentiated into Th1 cells;(3)PNS-Treg cells exhibited significant dysfunction,which was manifested as a significantly weakened ability to inhibit the proliferation of responder T cells in vitro;(4)These abnormal responses of PNS-Treg cells was closely related to the exhaustion induced by over-activation;(5)The TCR signal and its downstream PI3K-AKT and MAPK signal pathways are significantly enriched in PNS-Treg cells after activation in vitro;(6)As a common regulatory target downstream of PI3K-AKT and MAPK signaling pathways,mTORC1 activation are significantly up-regulated in PNS-Treg cells activated in vitro;(7)The expression levels of m TORC1 downstream transcription factors,c-Myc and SREBPs were significantly up-regulated in PNS-Treg cells activated in vitro;(8)The specific inhibitors of m TORC1,c-Myc,and SREBPs can significantly restore the aberrant responses of PNS-Treg in vitro.(9)Gene expression profile analysis suggested that a variety of biosynthetic and bioenergetic pathways are significantly enriched in PNS-Treg cells activated in vitro.Conclusion:Under the pathological conditions of PNS,Treg cells show abnormalities in function,stability,and plasticity,which are closely related to the exhaustion state induced by over-activation.The abnormal activation of m TORC1 mediated by TCR plays an indispensable role in this process.At the same time,this process may be accompanied by abnormally active biosynthesis and bioenergy production.Objevtice: To investigate the influences of CMPA(particularly non-IgE-mediated CMPA)-associated microbial dysbiosis on Treg cell-mediated intestinal immune tolerance and homeostasis,the fecal microbiota from infant donors with cow’milk protein-induced FPIAP(CM-FPIAP)were transplanted into germ-free mice,then examined the changes of intestinal microbioal components and immune microenvironment in the recipient mice.Methods: Fecal samples collected from CM-FPIAP infants(I-FPIAP group)and healthy control infants(I-HC group)were separately transplanted into germ-free mice(M-FPIAP group and M-HC group).At two weeks post fecal microbiota transplantation,the gut microbiome of the recipient mice was analyzed by 16 S r RNA gene sequencing,and the intestinal immunological alterations associated with the Treg cell compartment and intestinal immune homeostasis were detected.The specific gut microbial phylotypes that were potentially responsible for the disruption of intestinal immune homeostasis were also analyzed.Results: 1.After two weeks of transplantation,the main characteristics of the gut microbiome in infant donors could be stably maintained in the recipient mice.2.Mice colonized with the gut microbiome from infants with CM-FPIAP showed significant deficiencies among the levels of total Treg cells,PTreg cells and ROR-γt+ Treg cells,and there were no significant differences in the proliferation and apoptosis of Treg cells.3.The expression of ICOS and CTLA4 in colon Treg cells of M-FPIAP group was significantly decreased,the activation level of Treg cells was significantly down-regulated,the level of IL-10 was significantly decreased,and the inhibitory ability on the proliferation of effector T cells in vitro was significantly weakened.4.These recipient mice showed disrupted intestinal immune homeostasis,which was characterized by an overactivated Th2 biased immune response in the intestine.4.Two genera that might potentially contribute to this disruption.Conclusion: 1.As the main clinical type of non-IgE mediated CMPA,microbiota dysbiosis in CM-FPIAP can significantly damage the development and function of intestinal Treg cells,and then affects intestinal immune tolerance and homeostasis.2.Raoultella and Clostridium Sensu Stricto may have a key effect on the disruption of development and function of colonic Treg cells.Objective: We preliminarily explored the effect of Dock8 deficiency on key events in early activation of B cells and the activation of memory B cells using samples from Dock8 patients and Dock8 knockout mice,in order to deepen the understanding of the pathogenesis of abnormal humoral immunity in patients with Dock8 deficiency.Methods: We purified B cells from peripheral blood of Dock8 patients and spleen of Dock8 knockout mice.Using the soluble antigen-phosphorylation flow cytometry system and membrane-associated antigen-TIRFm system,we analyzed the effects of Dock8 deficiency on early activation-related signal transduction,BCR dynamics,and cell morphology in total B cells or memory B cells.By flow cytometry,we analyzed the effect of Dock8 deficiency on the phenotypic transition of na(?)ve B cell induced by in vitro activation.We also analyzed the effect of Dock8 deficiency on the expression of co-receptor CD19 and Dock8 downstream effector WASP.Results: In Dock8 knockout mice,the phosphorylation level of key signal molecules(CD19,Btk)in early activation of B cells and the overall level of BCR signal-related protein tyrosine phosphorylation,(p Y)are significantly reduced.After activation in vitro,the phosphorylation level of WASP was significantly down-regulated in Dock8 knockout B cells.The transcription and protein levels of WASP in B cells of Dock8 knockout mice were significantly down-regulated.After activation in vitro,p CD19,p Btk,p Y,p WASP and F-actin levels were significantly down-regulated in B cells from Dock8 patients.TIRFm results showed that,after membrane-associated antigen activation,the early activation of memory B cells from Dock8 patients was also significantly impaired,which was manifested as: BCR clusters and B cell expansion in the contact area between memory B cells and membrane-associated antigens were restricted.The recruitment of BCR signaling molecules(p Y,p Btk)are impaired.After activation with soluble antigen,the phosphorylation levels of Erk and Btk in memory B cells from Dock8 patients were significantly down-regulated.The phenotypic transition of naive B cells to memory B cells induced by activation in vitro was significantly restricted in Dock8 patients;the expression of CD19 in peripheral memory B cells of Dock8 patients was significantly down-regulated.Conclusion: Dock8 deficiency will significantly impair the early activation of total B cells and memory B cells;The above effects may be achieved by inhibiting the expression and activation of CD19 and WASP.