Dectin3 Promotes The Development Of Systemic Lupus Erythematosus Through Regulating FoxO1-mediated LOX-1~+M-MDSCs

Systemic lupus erythematosus（SLE）is a typical autoimmune disease involving multiple organs and multiple systems.Recently studies have shown that myeloid-derived suppressor cells（MDSCs）are a group of heterogeneous immature myeloid cells.MDSCs have the function of inhibiting T cells under pathological conditions.Such as tumors,obesity and chronic inflammation.However,we and some researchers have shown that the accumulation of MDSCs is related to SLE progression.However,the mechanisms responsible for the acquisition of pathological activity by MDSCs in SLE remain unclear.Toll-like receptors（TLRs）are a kind of pathogenic pattern recognition receptors,which are mainly expressed in innate immune cells.It is generally believed that the abnormally high expression and activation of TLRs in lupus patients and mouse models is closely associated with the progress of SLE,especially the abnormally activation of TLR7.Studies indicate that C-type lectin-like receptors（CLRs）may be involved in the development of autoimmune diseases.The activation of CLRs induce the expansion of MDSCs.In addition,studies indicated that TLRs and CLRs could synergistically initiate Th cell differentiation/polarization to promote inflammatory diseases progress.CLRs are mainly expressed in myeloid cells,including Dectin-1、Dectin-2、Dectin-3 and Mincle.Interestingly,our pre-experiment found that TLR7agonist R848 promoted the expression of Dectin-3 in MDSCs and the lupus symptoms of Dectin3^-/-mice were relieved compared with the symptoms of WT lupus mice.Therefore,not only TLR7 but Dectin3 pathway play a vital role in regulating MDSCs in SLE.However,the mechanisms of how TLR7 and Dectin3 regulate MDSCs in SLE remain uncertain.Therefore,in this study,we used the lupus model mice as the research object.We explored whether TLR7 regulated MDSCs of SLE associated with Dectin3.Furthermore,we revealed the mechanism of how TLR7 and Dectin3 synergistically regulated MDSCs.The experimental methods and results of this study were as follows:1.TLR7 affects the development of systemic lupus erythematosus and is related to MDSCs by regulating Dectin3 signal:To investigate whether the TLR7 signal and Dectin3 signal have a synergistic effect on the pathogenesis of lupus,we utilized WT and Dectin3^-/-mice to induce the lupus model,including pristane and TLR7 agonist（imiquimod）.We detected the lupus symptoms and immune cells in tissues during different time points.The results indicated that the lupus symptoms of Dectin3^-/-were relieved compared with WT lupus mice from the 3rd months Compared with WT lupus mice,the percentages of immune cells of Dectin3^-/-in PBMC,BM,Kd and SP were significantly reduced,including MDSCs,Th17 cells,Treg cells,activated B cells and activated T cells.In addition,the immunosuppressive activity of MDSCs in Dectin3^-/-lupus mice was stronger than that in WT lupus mice.Similar results were also obtained in a mouse model induced by TLR7 agonist-imiquimod.To determine whether Dectin3 promote lupus progress is dependent on MDSCs,we adoptively transferred MDSCs of Dectin3^-/-lupus mice to imiquimod-induced WT lupus mice.The results showed that the lupus symptoms of WT lupus mice treated with transplanted MDSCs were relieved compared with WT lupus mice treated with PBS.These data indicated that TLR7 promoted lupus progress probably via Dectin3pathway.Meanwhile,Dectin3 promoted lupus development via regulating MDSCs.However,the mechanisms of how Dectin3 regulate the expansion and function of MDSCs in SLE remain unambiguous.2.Dectin3 regulates MDSCs apoptosis and immune function through Syk-AKT1 FoxO1 signal axis:To explore the molecular mechanism of Dectin3 regulating the expansion and function of MDSCs,we sorted and purified the MDSCs of spleen in WT and Dectin3^-/-lupus mice.We performed transcriptome sequencing of MDSCs and found the apoptosis signal was in the top 10.We speculated the significantly reduced percentage of MDSCs in Dectin3^-/-lupus mice probably associated with MDSCs apoptosis.We further detected the apoptpsis of MDSCs in WT and Dectin3^-/-lupus mice and found that pro-apoptosis proteins of Bim and Bax increased in MDSCs of Dectin3^-/-lupus mice compared with WT lupus mice.Meanwhile,the inhibitory apoptosis protein Bcl-2 was decreased in MDSCs of Dectin3^-/-lupus mice.Through the analysis of the full signal transduction network,we found that the FoxO1 gene was located in an important position on the network and the expression of FoxO1 in MDSCs of Dectin3^-/-lupus mice were reduced compared with WT lupus mice,which indicated FoxO1 played a vital role in regulating MDSCs apoptosis.We found that FoxO1 and Akt1 have a targeting relationship through gene co-expression analysis;Syk can regulate the transcriptional expression of Akt1.Dectin3 can activate the downstream innate immune response by activating Syk.We performed a series of interference and overexpression experiments on bone marrow-induced MDSCs of WT and Dectin3^-/-mice in vitro.The results showed that Dectin3 inhibited the apoptosis of MDSCs through the Syk-Akt1-FoxO1 signal axisreduced the immunosuppressive activity of MDSCs and promoted its inflammatory production.MDSCs are a group of highly heterogeneous cells,and different subgroups may perform different functions.Therefore,it is necessary to find out which kind of MDSCs that FoxO1 regulates in lupus.3.Dectin3 promotes LOX-1⁺M-MDSCs to accelerate lupus process through FoxO1:To determine Dectin3 regulate which subgroup of MDSCs to participate in SLE progress,we detected the percentages changes of G-MDSCs and M-MDSCs in spleen,bone marrow and kidney tissues of WT and Dectin3^-/-lupus mice.Compared with WT lupus mice,Dectin3^-/-lupus mice significantly reduced the expansion of M-MDSCs.To determine the mechanism of how Dectin3 regulate M-MDSCs,we sorted the M-MDSCs of WT and Dectin3^-/-lupus mice to perform full-transcriptome gene chip analysis.The results indicated that the surface marker related gene LOX-1 in Dectin3^-/-lupus mice was significantly reduced compared with WT lupus mice.In order to explore whether Dectin3 regulates LOX-1 expression on M-MDSCs via FoxO1 and affects the function of M-MDSCs involved in LOX-1 expression,we performed flow cytometric analysis of MDSCs of WT and Dectin3^-/-lupus mice and found that the expansion of LOX-1⁺M-MDSCs was reduced compared with WT lupus mice.Meanwhile,the immunosuppressive function of LOX-1⁺M-MDSCs was weakened.In order to further reveal the regulatory effect of FoxO1 on the expression of LOX-1 in M-MDSCs,we performed knockdown and overexpression of FoxO1experiments in bone marrow-induced M-MDSCs.The results showed that increased FoxO1 expression in M-MDSCs significantly inhibited the expression of LOX-1.In addition,compared with LOX-1⁺M-MDSCs,LOX-1^-M-MDSCs possessed increased immunosuppressive ability on T cells and decreased pro-inflammatory ability.Furthermore,we treated WT and Dectin3^-/-lupus mice with FoxO1 small interference fragments or recombinant FoxO1 adenovirus in vivo.The results suggested that Dectin3^-/-lupus mice which knocked down of FoxO1 inhibited the apoptosis level of MDSCs,promoted the expansion of LOX-1⁺M-MDSCs and aggravated the lupus symptoms compared with Dectin3^-/-lupus mice.To further confirm that Dectin3 promotes the accumulation of LOX-1⁺M-MDSCs via regulating FoxO1 expression to aggravate SLE progress,we acquired peripheral blood of SLE patients,which were divided into inactive patients and active patients.We performed the peripheral blood samples by flow cytometry analysis and QPCR.The results indicated that the expression of Dectin3 in MDSCs of active group was increased compared with inactive group.In addition,the FoxO1expression was reduced in active group MDSCs compared with inactive group.Meanwhile,we found that the proportion of LOX-1⁺M-MDSCs in active group was higher than that in inactive group.In conclusion:（1）We firstly found that Dectin3 played a vital role in SLE progress via regulating MDSCs,which was related to TLR7 activation.（2）We revealed that Dectin3 regulated the expansion and function of MDSCs via Syk-Akt1-FoxO1 signal axis.（3）We found that Dectin3 promoted the accumulation of LOX-1⁺M-MDSCs via regulating FoxO1,which probably were potential target cells for the exacerbation of the lupus process.