WDFY4 Involved In Symptoms Of Systemic Lupus Erythematosus By Modulating B-cell Fate Via Non-canonical

Systemic lupus erythematosus(SLE)is a typical autoimmune disease,mainly characterized by inflammatory response and multi-organ damage as a result of massive production of autoantibodies and subsequent formation of immune complex deposits with autoantigens.The attack of SLE is under influence of various factors,among which genetic factors play a vital role.The concordance rate of identical twins(24-69%)is much higher than that of fraternal twins and siblings(2-5%).The pathogenesis of SLE is very complicated with lack of systematically clarification,and the current research focused on several aspects including T cells,B cells and cytokines.Over-activation of B cells is a predominant feature for SLE patients,who manifests as overproduction of autoantibodies,abnormal antigen presentation and production of various cytokines,such as IL-10,IL-6.In addition,many genes can impact the process of SLE by acting during the differentiation and development ofB cells.The development of Genome Wide Association Study(GWAS)has greatly promoted the genetic study of SLE,which led the research on genetic susceptibility to be a breakthrough point inthe pursuit ofitspathogenesis.At present,more than 80 genes locus have been found to be associated with SLE,including WDFY4,a functional locus that is susceptible to SLE.WDFY4(WDFY family member 4),the fourth member of the WDFY family,is located at 10q11.23.Its genomic sequence contains nearly 300,000 bases and the largest transcript encodes a huge protein of 3,184 amino acids.As reported,WDFY4 is susceptible to SLE in multiple populations based on GWAS studies,and our previous study has identified specific susceptible variant.However,the specific mechanism is still unknown with few function study for WDFY4.Part IConstruction and phenotype analysis of Wdfy4-Cd19 Cre miceIt is known that WDFY4 is associated with susceptibility to SLE,and our previous study showed that WDFY4 is highly expressed in B lymphocytes.Thus,we constructed a mouse model that conditionally knocked out Wdfy4 in B lymphocytes,to explore the role it played in the pathogenetic process of SLE with loxp-cre system.We inserted Loxp sequences on both sides of the third exon of Wdfy4,and obtained Wdfy4flox/+ mice.After,we used the Wdfy4flox/+ mice to hybridize with Cd19-Cre transgenic mice.Under the function of Cd19 recombinase who exclusively expressed in B cells,the third exon of Wdfy4 was removed,leading to the frameshift mutation and subsequent early termination at transitional level,and loss of function for Wdfy4.The Wdfy4flox/flox Cdl9-Cre+/-mice obtained by the final hybridization were Wdfy4 CKO mice,and their littermates of Wdfy4flox/flox mice were control mice.All mice were born in accordance with Mendelian genetic segregation ratio.Next,we analyzed the fundamental phenotypes of Wdfy4 CKO mice,compared with control ones.The number of CD 19+ cells in the spleen of Wdfy4 CKO mice was significantly less than control ones,indicating it played a role in the survival of B lymphocytes.With regards to several subtypes of B lymphocytes,transition type 1(T1),mature type and follicular type B lymphocytes in CKO mice were significantly reduced,while transition type 2(T2)and marginal B lymphocytes appeared as no difference.That means,Wdfy4plays a vital role in the maturation of B lymphocytes.To explore if Wdfy4participate in the early period of B lyymphocytes development,we detected the number of B cells at each stage of development in the bone marrow.And we demonstrated that the number of total B cells remained no change,while Pre-B cells reduced,indicating that the early period of B lymphocytes devlopment was unacted with knockout of Wdfy4,except the transition process of B cells from Pro ones to Pre ones.The main function of B lymphocytes is to secrete antibodies,thus we detected the volume of antibodies secretion in the serum of immunized mice with ELISA.Without treatment of artificial antigen,the level of IgG and IgM showed no difference for Wclfy4 CKO mice compared with WT littermates.Next,we treated the mice with thymus-independent(TI)antigens and thymus-dependent(TD)ones respectively,and detected the level of immunoglobulins intermittently.And we found no difference for the level of LgG and IgM after treatment of NP-Ficoll,a kind of TI antigen.The level of IgG decreased obviously with treatment of high-affinitive antigen NP1-4 and NP20-29,while the level of IgM remained no change with treatment of NP-CGG,a kind of TD antigen.Consistently,the number of B lymphocytes in the bone marrow and spleen reduced apparently,while the number of plasma cells and germinal center B cells also decreased.Thus,we conclude that WDFY4 might play a role in the maturation process of antibody affinity for B cells.In summary,reduced number of B lymphocytes and inhibited function were observed after knockout of Wdfy4 in B lymphocytes.Part ?The function of Wdfy4 in the pathogenesis of SLE with lupus modelExcessive activation of B lymphocytes is one of the most important abnormal characteristics in SLE patients.And many genes who play a role in the differentiation and development of B lymphocytes may participate in the pathogenesis of SLE.Based on previous results,we concluded that Wdfy4 is involved in the development and differentiation of B cells.Then,we aimed to test whether Wdfy4affect the occurrence of SLE through its impact on B lymphocytes.To identify the role of Wdfy4 in the pathogenesis of SLE,we induced the mice to suffer from lupus with injection ofPristane onWdfy4 CKO mice and control ones,and analyzed its phenotypes after 6 months.Mice were sacrificed and observed a number of physiological indexes after 6 mouths with injection of Pristane.First,the weight of spleen and the ratio of spleen weight to body weight were measured to estimate the severity degree of lupus.And the spleens of WT mice were significantly swollen,while the Wdfy4 CKO ones were relieved.Besides,the ratio of spleen weight to body weight of Wdfy4 CKO ones was smaller.In addition,HE staining of the spleens revealed disordered structure and unclear border between the red pulp and white pulp for control mice with treatment of Pristane,while the Wdfy4 CKO mice relieved apparently.That means,the extent of impairament of spleenis greatly relieved the Wdfy4 CKO mice compared with WT littermates after inducement.Generally,aggregation of massive autoantibodies and immune complexes in the kidney will induce lupus-like nephriti.In order to examine the degree of damage for kidneys,we collected the urine for 24h with a metabolic cage,and detected the content of proteinuria by coomassie blue labeling.The level of urine proteins for Wdfy4 CKO was much lower than control ones.Besides,abnormal renal tissue,enlarged glomeruli,thickened glomerular capillary basement membrane,and obvious inflammatory cell infiltration were observed in controls,while Wdfy4 CKO mice was normal.Next,we examined the deposition of renal immune complexes by immunofluorescence staining,indicating that the IgG deposition in the kidneys of Wdfy4 CKO mice was significantly less than that of the control mice.Therefore,the lupus nephritis phenotype in Wdfy4 CKO mice was milder than that of control mice after lupus induction.As for the total level,no difference was seen in the level of IgM,IgG and anti-dsDNA IgM antibodies,while the level of anti-dsDNA IgG was much lower in Wdfy-4-CKOmice than control mice.Taken together,the pathological manifestations of lupus in Wdfy4 CKO mice were significantly reduced after induction with Pristane.Part IIIThe Mechanism of WDFY4 Participating in the Development of SLEBased on previous studies,we demonstrated that Wdfy4might play a role in the pathogenesis of SLE by involving in the development and differentiation of B lymphocytes or affecting its normal function.Then,we endeavored to identify the precise mechanism underlying this process.At present,functional analysis upon Wdfy4was scarce,so we designed to deduce its function from its known structure.The structure of WDFY4 is greatly homologous with WDFY3,which is a autophagy-related protein.Besides,many studies revealed that autophagy is an important role,not only in the development and differentiation of B lymphocytes,but also the pathogenesis of autoimmune diseases.Thus,we focused on the correlation of Wdfy4d and autophagy in B lymphocytes,and even the pathogenesis of SLE.To validate our hypothesis,we constructed a shWDFY4 Raji cell line(human B lymphocytoma cell line)and a shcontrol cell line with lentiviral vectors.First,obvious aggregation of LC3II was observed in shWDFY4 cell line.The behaviour of autophagy is dynamic with complex regulating factors.For example,the accumulation of LC3II might be a result of excessive autophagy;nevertheless,the inhibition of autophagy level by affecting lysosomal digestion also could contribute to the accumulation of LCII.Next,with treatment of CQ(chloroquine),a kind of lysosomal inhibitor,we found that the accumulation of LC3II was further increased.Therefore,we concluded that the level of autophagy of B lymphocytes was increased after knocking down WDFY4.Besides,to further verify the increased level of autophagy,we examined the number of LC3 focuses by IF staining in shWDFY4 HEK293 cell line.And we found apparent increase of LC3 focuses in shWDFY4 cells,indicating the enhancement of autophagy after knocking down WDFY4.In addition,it is worth noting that P62,a classical autophagy substrate,remained no change after knocking down WDFY4,while the expression of PI3KC3 was increased,who function in the process of non-classical autophagy.Therefore,we concluded that WDFY4 might play a role in the non-classical autophagy pathway.Besides,previous studies have demonstrated that non-canonical autophagy could inhibit autoinflammatory and reduce lupus symptoms,which is consistent with our second part of the study.Next,we performed in vivo examination against the above results.We sorted B cells from the spleen of Wdfy4 CKO mice to analyse the level of autophagy.The result of western blot showed obvious accumulation of LC3II and PI3KC3 in CKO mice.Simultaneously,severer aggregation of LC3 focuses was observed in CKO mice.In summary,we conclude that WDFY4plays a role in inhibiting the non-canonical autophagy pathway.