The Differentiation,Function And Their Mechanism Of MDSCs In Systemic Lupus Erythematosus

MDSCs are a heterogeneous group of immature myeloid cells that have the ability to suppress T cell functions.MDSCs are derived from the bone marrow and arise from a delay in maturation during pathologic conditions.In recent years,a vast amount of information has been generated detailing the biology and clinical significance of these cells in various pathologic conditions.Recently,MDSCs have been reported to regulate autoimmunity and control the generation and perpetuation of autoimmune diseases.We now know that MDSCs are involved in a number of different autoimmune disorders,including multiple sclerosis,type 1 diabetes,rheumatoid arthritis(RA),inflammatory bowel disease(IBD)and autoimmune hepatitis.Systemic lupus erythematosus(SLE)is a prototypic autoimmune disease characterized by systemic dysregulation of adaptive and innate immunity and results in auto-antibody production,chronic inflammation and eventually multiple organ damage.Although major advancements have made in the etiology of SLE,the role of MDSCs in SLE development and progression are poorly understood.It is necessary to explore the mechanism of expansion and function of MDSC in SLE and it can provide molecular insights into the mechanism of the pathogenesis of SLE.Non-Hodgkin lymphomas(NHL)have a wide range of histological appearances and clinical features at presentation,which can make diagnosis difficult.There is a close relationship between the pathogenesis and the symptoms of NHL and SLE.In NHL,expansion and function of MDSC has been revealed,but the mechanism remains unclear.It is necessary to explore the mechanism of expansion and function of MDSCs in NHL.It is also important to explore the relationship between SLE and NHL from MDSCs.Thus,we investigated the mechanism of expansion and function of MDSCs in the progress of SLE and NHL.1 CCR1 contributed to accumulation of G-MDSCs in 26 week old MRL/lpr mice.Previous study reported that MDSCs were elevated with pronounced immunosuppressive capacities that specifically target B cells and inducing proportions of MDSCs could suppress lupus nephritis in lupus-prone(NZB � NZW)F1 mice.In addition,recent study demonstrated an impaired expansion and defective function of MDSCs in lupus-prone(NZB � NZW)F1 mice.However,these studies draw a different conclusion on percentages of MDSCs in SLE.As is well known,MDSCs can be further divided into monocytic MDSCs(M-MDSCs)and granulocytic MDSCs(G-MDSCs)in mice respectively.However,the detailed percentage and function of M-MDSCs and G-MDSCs in SLE are poorly understood.In this study,MDSCs from MRL/lpr mice,exhibited a marked elevation in the spleens rather than in bone marrow.Furthermore,splenic G-MDSCs rather than M-MDSCs were expanded in 26 week old MRL/lpr mice.We found CCR1 expression as well as protein level was increased in splenic G-MDSCs from 26 week old MRL/lpr mice.We thus preliminarily determined that CCR1 contributed to accumulation of G-MDSCs.Furthermore,the expression of MCP-1,CCL3,CCL4 and CCL5 was all increased in spleens in 26 week old MRL/lpr mice.To confirm CCR1 contributed to accumulation of G-MDSCs in 26 week old MRL/lpr mice,the adoptive transfer experiment of G-MDSCs was conducted.These data confirmed that CCR1 indeed contributed to accumulation of G-MDSCs in 26 week old mice.2 IFN-y results in the high expression of ROS in splenic G-MDSCs,inflammasome activation results in expression of IL-1? in splenic M-MDSCs.Main factors implicated in MDSCs-mediated immune suppression include expression of arginase(ARG1),inducible NOS(iNOS),TGF-?,IL-10,and COX2.We found significantly increased IL-1? expression in splenic M-MDSCs in 26 week MRL/lpr mice.Gp91phox expression was significantly increased in splenic G-MDSCs in 26 week old MRL/lpr mice.Expectantly,splenic G-MDSCs in 26 week old MRL/lpr mice produced more reactive oxygen species(ROS).To elucidate mechanism that high expression of gp91phox in splenic G-MDSCs,we focused on cytokines that were enriched in SLE pathogenesis,such as IFN-?,TNF?,IL-6 and HIF-1?.IFN-? expression was increased in spleens,and its protein level was also upregulated in serum.Moreover,gp91phox expression and protein level was increased in G-MDSCs with treatment of IFN-y in a dose dependent.IFN-y-treated G-MDSCs also produced more ROS.IL-1? from inflammasome activation by AIM2 and NLRP3 has been implicated in the pathogenesis of SLE.Expressions of AIM2 and TLR2 as well as their protein levels were significantly increased in splenic M-MDSCs from 26 week old MRL/lpr mice.Expression of TLR2,caspase-1 and apoptosis-associated speck-like protein containing a CARD(ASC)was also increased.Furthermore,inflammasome activation was activated evidenced by the increase in caspase 1-p20 in splenic M-MDSCs from 26 week old MRL/lpr mice.These results confirmed that the source of IL-1? from splenic M-MDSCs in 26 week old MRL/lpr mice might be attributed to TLR2 and AIM2 inflammasome activation.3 MDSCs contribute to SLE by regulating differentiation of Th17 and TregsPrevious study revealed that G-MDSCs could impair differentiation of Treg depending on ROS,so we co-cultured naive CD4+T cells with splenic G-MDSCs from 6 and 26 week old MRL/lpr mice.Result showed splenic G-MDSCs from 26 week old MRL/lpr mice impaired Treg differentiation,while inhibiting of ROS using N-acetyl-L-cysteine markedly blocked the suppressive effect of G-MDSCs on Treg differentiation.Previous study in experimental autoimmune encephalitis and arthritis revealed that M-MDSCs could facilitate a Th17 response that is dependent on IL-1?.In light of previous reports of the importance of M-MDSCs in the early Th17 differentiation,we initially co-cultured naive CD4+T cells with splenic M-MDSCs from 6 and 26 week old MRL/lpr mice.Results showed splenic M-MDSCs from 26 week old MRL/lpr mice facilitated Thl7 differentiation compared to those from 6 week old MRL/lpr mice,while neutralizing IL-1? using mAbs blocked the effect of M-MDSCs on Th17 differentiation.Deletion of MDSCs changes the percentage of Th17/Treg cell and attenuates lupus-like syndrome.Adoptive transfer of MDSCs modulates the differentiation of Treg and Th17 cells in MRL/lpr mice.4 MiR-30a is high expressed in SLE and NIH,miR-30a promotes the differentiation and function of MDSCs via SOCS3 in B-cell lymphoma,and these studis reveal that there are same characters in MDSCs between SLE and NHL progress.There is a close relationship between the pathogenesis and the symptoms of NHL and SLE.Previous study reported MDSCs were elevated and promoted the progress of B cell lymphoma.Previous study also reports MDSCs are increased and produces immunosuppressive cytokines,including IL-10 but not nitric oxide(NO)or arginase in B cell lymphoma.As is well known,MDSCs can be further divided into M-MDSCs and G-MDSCs in mice.However,the detailed percentage and function of M-MDSCs and G-MDSCs in B cell lymphoma are poorly understood.In our study,MDSCs exhibited a marked elevation in both spleens and bone marrow in B cell lymphoma mice.Furthermore,both G-MDSCs and M-MDSCs were expanded in spleens and bone marrow in B cell lymphoma mice.To detailedly evaluate function of splenic M-MDSCs and G-MDSCs in B cell lymphoma mice,several functional molecules were measured.We found significantly increased IL-10 and ARG-1 expression in splenic M-MDSCs in B cell lymphoma mice.P47phox and ARG-1 expression was significantly increased in splenic G-MDSCs in B cell lymphoma mice.STAT3 has long been considered to be critical factors in the regulation of MDSCs expansion and activity.Suppressor of cytokine signaling(SOCS)proteins are negative regulators of the JAK2/STAT3 pathway and generally function as tumor suppressors.Previouse study reportes SOCS3 regularates the expansion and function of MDSC.Expression of SOCS3 was decreased in B cell lymphoma.Based on these reports,we conclude SOCS3 may change the function and expansion in B cell lymphoma.MiRNAs have been shown involvment in regulating MDSCs differentiation and function.It has been revealed that miR-30a play an important role in in SLE and NHL patients.Interestingly,miR-30a could target the 3'UTR of the SOCS3 directly and regulate the expression of SOCS3,which can promote the activation of JAK2/STAT3 signaling.The expression of miR-30a in G-MDSCs and M-MDSCs from B cell lymphoma mice is increased,but expression of SOCS3 is decreased.Based on thses results,we conclude that miR-30a may regulate the function and expansion of MDSCs via SOCS3.Over expression of miR-30a increases the induction of MDSC from BM cells,whereas the depletion of miR-30a reduces the induction of MDSCs.To define the function of miR-30a in MDSC function,MDSCs were transfected with miR-30a mimics or inhibitors.Results showed that ARG-1,IL-10 and p47phox were increased after overexpressing miR-30a in MDSCs,whereas depleting miR-30a in MDSCs decreased their expression.We further test the levels of the suppressive factors in G-MDSCs and M-MDSCs after miR-30a mimic and inhibitor transfection.Results showed that expression of ARG-1 and p47phox was increased and decreased after miR-30a overexpressing or depleting in G-MDSCs.Expression of ARG-1 and IL-10 was decreased after miR-30a overexpressing or sedepleting in M-MDSCs.MiR-30a also increased the suppressive capacity of MDSCs after co-culture with T cell.To identify special genes targeted by miR-30a in MDSCs expansion and founction,the Targetscan database was used first.The 3 UTR of SOCS3 was found to harbor one putative target site of miR-30a.Next,to verify whether SOCS3 was the direct target of miR-30a,the lucifferarase experiment was conducted.Compared with the negative control,the over-expression of miR-30a resulted in a 57%reduction of luciferase activity.Then we confirmed miR-30a could target the 3'UTR of the SOCS3 directly and regulate the expression of SOCS3.To determine whether miR-30a exerts any effect on the progression tumor growth,miR-30a agomir or antagomir were directly injected into lymphoma mice by i.v.Upregulation of miR-30a significantly promoted the disease progress,accompanied with reduce percentages of MDSCs in lymphoma mice.However,downregulation of miR-30a show significant protective effects on the disease progression.To examine the function of miR-30a in MDSCs in vivo,we injected miR-30a agomir-or antagomir-modified MDSCs to observe B-cell lymphoma progression.Treated with miR-30a agomir-transfected MDSCs,the incidence of lymphoma in mice was increased and significantly promoted the B-cell lymphoma progression progress.Conversely,treated with miR-30a antagomir-modified MDSCs,the incidence of lymphoma in mice was decreased and significantly prolonged the B-cell lymphoma progression progress.Taken together,we found that G-MDSCs were expanded in MRL/lpr mice,and CCR 1 contributes to accumulation of G-MDSCs during the course of lupus progress,however,M-MDSCs remains similar during the course of lupus progress;IFN-?resulted in the high expression of ROS in splenic G-MDSCs,and splenic G-MDSCs from 26 week old MRL/lpr mice impair differentiation of Tregs via ROS;IL-1? from splenic M-MDSCs was attributed to TLR2 and AIM2 inflammasome activation,and splenic M-MDSCs enhanced Th17 differentiation via IL-1?;MDSCs contribute to SLE by regulating differentiation of Th17 and Tregs;G-MDSCs and M-MDSCs were expanded in B cell lymphoma mice.Expression of ARG-1 and P47phox was increased in G-MDSCs in B cell lymphoma and expression of ARG-1 and IL-10 was increased in M-MDSCs in B cell lymphoma.MiR-30a promotes the differentiation and function of MDSC via SOCS3 in B-cell lymphoma mice.