Arginase-1-dependent Promotion Of Th17 Differentiation And Disease Progression By MDSC In Systemic Lupus Erythematosus

Objective:Myeloid-derived suppressor cells(MDSC) are a heterogeneous population of immature cells derived from myeloid progenitors with immune-suppressive functions. Recently, an increasing number of reports have been published describing the expansion of MDSC in murine models of autoimmune diseases. However, it remains unclear whether the effect of MDSC is beneficial or harmful in autoimmunity. Some studies demonstrated that MDSC serve a beneficial role in autoimmunity by limiting T cell-mediated inflammation, but the others showed that MDSC play a deleterious role by promoting Th17 differention. The role of MDSC in autoimmunity has been studied almost exclusively in mouse models, and it is largely unkown if these cells play a similar role in autoimmune patients. This study is designed to address this question in patients with systemic lupus erythematosus(SLE). By combining the analysis of peripheral blood immune cell populations with in vitro fucntrional assays and humanized mouse models, we aim to understand the role and the underlying mechanisms of MDSC in Th17 differentiation and disease progression in SLE patinets. Methods:(1) We measured the frequencies and numbers of HLADR-CD11b+CD33+ MDSC and their subsets in peripheral blood mononuclear cells(PBMC) of SLE patients by flow cytometry(FCM), and analyzed the correlation of circulating MDSC frequencies with the disease status indicated by SLEDAI scores. M-MDSC and G-MDSC were sorted from SLE patients and healthy controls(HCs), and examined morphologically by hematoxylin and eosin(HE) staining.(2) We measuerd Th17 cell frequencies(i.e., CD4+ T cells producing IL-17A/IL-17F) in PBMCs and IL-17A/IL-17 F levels in sera from SLE patients and HCs by FCM and ELISA, respectively. Pathological changes in renal tissues of SLE patients were evaluated by periodic acid-Schiff(PAS) and HE staining, and by immunofluorescence(IF) analysis of IL-17 A and IL-17 F deposition in the glomeruli.(3) We measured serum Arg-1 activity in SLE patients and in HCs, Arg-1 production by MDSC and their subsets from SLE patients and HCs by FCM and IF analysis. Serum levels of IL-6 in SLE patients and HCs were measured by ELISA. Arg-1 mRNA in MDSC cultured with human IL-6, human IL-17, or media only were measured by qRT-PCR to determine the role of IL-6 and IL-17 in regulating Arg-1 production by MDSC.(4) Na?ve CD4 T cells from HCs were cultured under Th17 differentiation without MDSC, with autologous MDSC, or with autologous MDSC + nor-NOHA(Arg-1 inhibitor), their secretion of Th17 cytokines and expression of the relevant transcription and signaling factors(RORγt、GCN2、phosphorylated eIF2α and mTOR) were determined by ELISA and FCM, respectively.(5) Humanized SLE mice were established by intravenous injection of PBMCs from SLE patients with active disease(SLEDAI≥9; dsDNA≥1:10) into immunodeficient NOD/SCID mice. To determine the role of MDSC and Arg-1 in the disease development, NOD/SCID mice were administered unaltered PBMCs, MDSC-depleted PBMC, or unaltered PBMC plus Arg-1 inhibitor nor-NOHA. The mice were examined for the levels of serum human anti-dsDNA antibodies and urinary proteinuria by ELISA, human IL-17 A mRNA in the spleen and kidney by qRT-PCR, and human IL-17 A and IgG deposition in the kidney by IF staining. Rresults:(1) Circulating MDSC are increased and positively correlated with disease activity in patients with SLE. HE staining of sorted M-MDSC and G-MDSC revealed no detectable difference in morphology between SLE patients and HCs.(2) The number of Th17 cells and serum level of Th17 cytokines are increased in SLE patients. Both IL-17 A and IL-17 F were primarily detected in the glomeruli.(3) Intracellular staining demonstrated a marked increase in Arg-1 production by MDSC from SLE patients compared to those isolated from HCs. Interestingly, G-MDSC produced more Arg-1 compared to M-MDSC in both HCs and SLE patients, and only G-MDSC from the SLE patients showed a significantly increased Arg-1 production. A significant increase in serum Arg-1 activity, which was also positively correlated with the disease activity(SLEDAI), circulating MDSC and serum IL-17 A levels. Real-time quantative polymerase chain reaction(qPCR) revealed a significant increase in Arg-1 mRNA expression in MDSC after treatment with IL-6.(4) MDSC significantly enhanced Th17 cell differentiation in vitro. MDSC-mediated Th17 differentiation was completely diminished in presence of nor-NOHA, a selective Arg-1 inhibitor, suggesting that the effect of MDSC is mediated by their production of Arg-1. MDSC markedly enhanced the expression of RORγt、GCN2、phosphorylated eIF2α(EIF2S1) and mTOR, and MDSC-induced up-regulation was effectively reversed by nor-NOHA. Thus, mTOR and GCN2-eIF2α signaling are likely to be involved in Arg-1–dependent induction of TH17 differentiation by MDSC.(5) Humanized SLE mice established by injection of unaltered PBMCs from SLE patients all developed lupus nephritis-like symptoms within 4-5 weeks, such as the production of human anti-dsDNA antibodies and development of proteinuria. The potential of PBMCs to induce proteinuria in humanized mice correlated positively with the SLEDAI scores of patients from whom PBMCs were obtained. However, mice receiving MDSC-depleted PBMCs from the same patients showed markedly less severe symptom, as shown by significantly lower levels of serum human anti-dsDNA antibodies and proteinuria, indicating that MDSC are essential for the disease pathogenesis in vivo. The disease-promoting role of MDSC is likely to be dependent on Arg-1, as treatment with Arg-1 inhibitor nor-NOHA inhibited the disease progression to a similar extent as MDSC depletion. Compared to mice receiving unaltered PBMCs, mice that received MDSC-depleted PBMCs or unaltered PBMCs plus nor-NOHA showed markedly reduced IL-17 A expression in the spleen and kidney. Histological analysis of kidney tissue revealed reduced IL-17 A and human IgG deposition and reduced mesangial cell proliferation in the glomeruli of the mice receiving MDSC-depleted PBMCs or unaltered PBMCs plus nor-NOHA compared to those injected with unaltered PBMCs. Conclusions:The present study reveals a previously unknown role for MDSC-derived Arg-1 in promoting Th17 cell differentiation in the context of inflammatory condition and autoimmune disease. Our data highlight the importance of the MDSC–T cell interaction in shaping of autoimmune T cell responses and suggest that MDSC may provide a promising target to develop an efficacious treatment for Th17-driven autoimmune disorders such as SLE.