Zhuoya Zhang

Allogeneic mesenchymal stem cells inhibited T follicular helper cell generation in rheumatoid arthritis

T follicular helper (Tfh) cells provide help for antigen-specific B cells. We have previously shown that Tfh cell frequency was increased and associated with auto-antibodies in patients with rheumatoid arthritis (RA), suggesting a possible involvement of Tfh cells in its pathogenesis. Mesenchymal stem cells (MSCs) represent a promising alternative cell therapy for RA by modulating T and B cell activation and proliferation. However, it remains unknown whether MSCs have immunoregulation on Tfh cells. In this paper, we have demonstrated that allogeneic MSCs could suppress Tfh cell differentiation in RA patients partly via the production of indoleamine 2,3-dioxygenase (IDO). IFNγ generated from Tfh cell differentiation system induced IDO expression on MSCs. MSCs transplantation (MSCT) into collagen-induced arthritis (CIA) mice prevented arthritis progression by inhibiting both the number and function of Tfh cells in vivo. These findings reveal a novel suppressive function of MSCs in Tfh cells, which has implication in understanding the underlying mechanisms of the immunotherapeutic effects of MSCs on RA patients.

Mesenchymal stem cells promote CD206 expression and phagocytic activity of macrophages through IL-6 in systemic lupus erythematosus.

Human umbilical cord-derived mesenchymal stem cells (UCMSCs) show therapeutic effects on systemic lupus erythematosus (SLE). Deficiency in functional polarization and phagocytosis in macrophages has been suggested in the pathogenesis of SLE. We found that macrophages from B6.MRL-Fas(lpr) mice exhibited lower level of CD206, the marker for alternatively activated macrophage (AAM, also called M2). In addition, the phagocytic activity of B6.MRL-Fas(lpr) macrophages was also decreased. UCMSC transplantation improved the proportion of CD206(+) macrophages and their phagocytic activity in B6.MRL-Fas(lpr) mice. Importantly, macrophages from SLE patients also showed lower expression of CD206 and reduced phagocytic activity, which were corrected by being co-cultured with UCMSCs in vitro and in SLE patients receiving UCMSC transplantation. Mechanistically, we demonstrated that IL-6 was required for the up-regulation of CD206 expression and phagocytic activity of UCMSC-treated SLE macrophages. Our results indicate that UCMSCs alleviate SLE through promoting CD206 expression and phagocytic activity of macrophages in an IL-6 dependent manner.

Umbilical Cord-Derived Mesenchymal Stem Cells Suppress Autophagy of T Cells in Patients with Systemic Lupus Erythematosus via Transfer of Mitochondria

Aberrant autophagy played an important role in the pathogenesis of autoimmune diseases, especially in systemic lupus erythematosus (SLE). In this study, we showed that T cells from SLE patients had higher autophagic activity than that from healthy controls. A correlation between autophagic activity and apoptotic rate was observed in activated T cells. Moreover, activation of autophagy with rapamycin increased T cell apoptosis, whereas inhibition of autophagy with 3-MA decreased T cell apoptosis. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) could inhibit respiratory mitochondrial biogenesis in activated T cells to downregulate autophagy and consequently decrease T cell apoptosis through mitochondrial transfer and thus may play an important role in SLE treatment.

Serum IFN‐γ Predicts the Therapeutic Effect of Mesenchymal Stem Cells Transplantation in Systemic Lupus Erythematosus Patients

Umbilical cord (UC)‐derived mesenchymal stem cells (MSCs) show immunoregulatory properties on various immune cells and display therapeutic effects on various autoimmune diseases such as systemic lupus erythematosus (SLE). The aim of this study was to investigate the effect of the SLE environment on UC MSCs and to identify a potential serum biomarker to predict the therapeutic effect. UC MSCs were cocultured with peripheral blood mononuclear cells (PBMCs) from active lupus patients, and the proliferation, apoptosis and surface markers of UC MSCs were observed. UC MSC functional molecules were assessed by real‐time polymerase chain reaction, and the signaling pathways were analyzed by Western blot. The clinical effect of MSC transplantation (MSCT) for lupus patients was followed‐up, whereas baseline serum cytokines were analyzed by enzyme‐linked immunosorbent assay. The coculture of PBMC from lupus patients promoted MSC proliferation. Lupus PBMCs were more potent in stimulating UC MSCs to secrete vascular endothelial growth factor (VEGF) and CXCL‐12. Furthermore, lupus PBMCs activated Akt, IκB, and Stat5 signaling pathways in UC MSCs but did not affect Erk1/2 and Smad1/5/8 pathways. Moreover, our clinical study showed that higher baseline levels of IFN‐γ might predict a good response to MSCT in active lupus patients. Baseline IFN‐γ levels may predict clinical response to MSC therapy for active lupus patients, which will help to choose suitable patients for clinical transplantation. Stem Cells Translational Medicine 2017;6:1777–1785

Human Umbilical Cord Mesenchymal Stem Cells Inhibit T Follicular Helper Cell Expansion through the Activation of iNOS in Lupus-Prone B6.MRL-Faslpr Mice

The aberrant generation or activation of T follicular helper (Tfh) cells contributes to the pathogenesis of systemic lupus erythematosus (SLE), yet little is known about how these cells are regulated. In this study, we demonstrated that the frequency of Tfh cells was increased in lupus-prone B6.MRL-Faslpr (B6.lpr) mice and positively correlated to plasma cell proportions and serum total IgG as well as anti-dsDNA antibody levels. Transplantation of mesenchymal stem cells derived from Whartons jelly of human umbilical cords (hUC-MSCs) ameliorated lupus symptoms in B6.lpr mice, along with decreased percentages of Tfh cells. In vitro studies showed that the differentiation and proliferation of Tfh cells were markedly suppressed by hUC-MSCs. The production of inducible nitric oxide synthase (iNOS) was dramatically upregulated in hUC-MSCs when cocultured with CD4+ T cells directly, while adding the specific inhibitor of iNOS into the coculture system significantly reversed the inhibitory effect of hUC-MSCs on Tfh cell generation. Interestingly, the efficacy of hUC-MSCs in inhibiting Tfh cells was impaired in the Transwell system, with the reduction of iNOS in both mRNA and protein levels. Taken together, our findings suggest that hUC-MSCs could effectively inhibit Tfh cell expansion through the activation of iNOS in lupus-prone B6.lpr mice, which is highly dependent on cell-to-cell contacts.

Mesenchymal stem cells upregulate Treg cells via sHLA-G in SLE patients

Background: Soluble human leukocyte antigen‐G (sHLA‐G) is a non‐classical HLA class I molecule, exhibiting strong immunosuppressive properties by inducing the differentiation of T regulatory cells (Treg). Mesenchymal stem cells (MSCs) transplantation alleviates disease progression in systemic lupus erythematosus (SLE) patients. However, the underlying mechanisms are largely unknown. Objectives: To explore whether sHLA‐G is involved in upregulating effects of MSCs on Treg, which contributes to therapeutic effects of MSCs transplantation in SLE. Methods: The serum sHLA‐G levels of SLE patients and healthy controls were detected by ELISA. The percentages of peripheral blood CD4+ILT2+, CD8+ILT2+, CD19+ILT2+ cells and Treg cells were examined by flow cytometry. Ten patients with active SLE, refractory to conventional therapies, were infused with umbilical cord derived MSCs (UC‐MSCs) and serum sHLA‐G was measured 24 h and 1 month after infusion. The mice were divided into three groups: C57BL/6 mice, B6.MRL‐Faslpr mice infused with phosphate buffer saline (PBS), and B6.MRL‐Faslpr mice infused with bone marrow MSCs (BM‐MSCs). Then, the concentrations of serum Qa‐2 were detected. Peripheral blood mononuclear cells (PBMCs) were isolated from SLE patients and co‐cultured with UC‐MSCs for 3 days at different ratios (50:1, 10:1, and 2:1) with or without HLA‐G antibody, and the frequencies of CD4+CD25+Foxp3+ T cells were then determined by flow cytometry. Results: The concentrations of serum sHLA‐G were comparable between SLE patients and healthy controls. However, there was a negative correlation between sHLA‐G levels and SLE disease activity index (SLEDAI) scores in active SLE patients (SLEDAI > 4). We found that serum sHLA‐G levels were negatively correlated with blood urea nitrogen, serum creatinine and 24‐hour urine protein in SLE patients. The sHLA‐G levels were significantly lower in SLE patients with renal involvement than those without renal involvement. The expression of ILT2 on CD4+ T cells from SLE patients decreased significantly compared to that of healthy controls. A positive correlation between the frequencies of Treg and CD4+ILT2+ T cells was found in SLE patients. The levels of sHLA‐G increased 24 h post UC‐MSCs transplantation. The concentrations of Qa‐2 in BM‐MSCs transplanted mice were significantly higher than those of control group. In vitro studies showed that MSCs increased the frequency of Treg cells in SLE patients in a dose‐dependent manner, which was partly abrogated by the anti‐HLA‐G antibody. Conclusions: Our results suggested that MSCs may alleviate SLE through upregulating Treg cells, which was partly dependent on sHLA‐G. HighlightsSerum sHLA‐G levels are correlated with creatinine in SLE patients.ILT2 expression on T and B lymphocytes was downregulated in SLE patients.Serum sHLA‐G and mouse Qa‐2 increased after MSCs transplantation.MSCs upregulated Treg cells via HLA‐G/ILT2.

OP0166 Il-27 Participates in Sjogren's Syndrome by Regulating Lymphocyte Subsets

Background Primary Sjogrens syndrome (pSS) is a chronic, systemic autoimmune disorder characterized by inflammation of exocrine glands and functional impairment of the salivary and lacrimal glands [1]. The pathogenesis of pSS is complicated with many respects remaining elusive. Interleukin-27 (IL-27) is a new member of the IL-12 cytokine family. It is largely secreted by activated antigen-presenting cells, such as macrophages and dendritic cells (DCs). IL-27 participates in multiple autoimmune diseases by regulating T lymphocyte subsets. Recent studies showed that IL-27 was involved in anti-inflammatory functions in SS [2]. However, the underlying mechanism of IL-27 in SS is still unknown. Objectives In the present study, the changes of lymphocyte subsets were studied in Il-27 knock-out and wild-type pSS models and pSS patients for the purpose to explore the specific mechanism of IL-27 in pSS and provide the basis for clinical treatment. Methods The NOD mice, Il-27 knockout (Il-27–/–) mice, Il-27 knockout mice with recombinant IL-27 treatment were studied. The saliva flow rates, weight, weight of submandibular glands and spleens were detected. Murine submandibular glands were fixed and paraffin sections were used for hematoxylin and eosin staining. The lymphocyte subsets of spleens from experimental mice and the peripheral blood from pSS patients were measured by flow-cytometric assay. Results Compared to WT NOD mice, the saliva flow rates decreased significantly, and the indexes of submandibular glands and spleens increased significantly in Il-27–/– mice (Fig 1A-E). The histological results showed that the number of lymphocytic infiltrates in submandibular glands of WT NOD was lower than Il-27–/– mice (Fig 1F). The splenic-Treg cells decreased significantly, but the levels of splenic Th17 and B cells increased significantly in Il-27–/– mice. After IL-27 treatment, the saliva flow rates, submandibular glands, Treg and Th17 cells were partially reversed in Il-27–/– mice (Fig 1G-M). Consistent with above observations, the changes of Terg and Th17 cells in pSS patients showed similar pattern. The Th2, Tfh and plasma cells increased significantly, whilst the B cells decreased significantly in pSS patients (Fig 1N-T). Compared with healthy control, the serum IL-27 decreased in pSS patients. Conclusions In general, these findings indicated that IL-27 deficiency deteriorated the disease symptoms of pSS and resulted in changes of Th17/Treg balance. In addition, pSS patients showed similar lymphocyte imbalance. Our data suggested that IL-27 might play an important role on the development and pathogenesis of pSS through regulating lymphocyte subsets, and targeting IL-27 and Th17/Treg balance may be a new direction of pSS treatment. References Xu J, Wang D, Liu D, et al. Allogeneic mesenchymal stem cell treatment alleviates experimental and clinical Sjogren syndrome. Blood, 2012, 120(15): 3142–3151. Lee BH, Carcamo WC, Chiorini JA, et al. Gene therapy using IL-27 ameliorates Sjögrens syndrome-like autoimmune exocrinopathy. Arthritis Res Ther, 2012, 14(4): R172. Disclosure of Interest None declared

THU0380 Increased Expression of Bruton Tyrosine Kinase in Patients with Lupus Nephritis and its Clinic Significance

Background Systemic lupus erythematosus (SLE) is an autoimmune disease manifested by multiorgan impairment including glomerulonephritis, cutaneous lesions and arthritis. B cells participate in the onset of SLE. As a downstream signaling molecule of B-cell receptor (BCR) signaling pathway, Bruton tyrosine kinase (Btk) is involved in the development, activation and survival of B cells. It is reported that transgenic mice overexpressing Btk specifically in B cells could produce antinuclear antibody and develop a lupus-like symptoms. Objectives The aim of our study was to identify the specific role of Btk in lupus nephritis. Methods The percentages of Btk positive CD19+ B cells from 28 SLE patients and 28 healthy donors were examined by flow cytometry. The correlation between the percentages of Btk positive CD19+ B cells and some lupus related clinical indicators were analyzed. Immunohistochemistry was used to detect the Btk expression in kidney biopsies from 8 lupus nephritis (LN) patients and 8 controls. The levels of Btk expression in glomerulus were detected by image-pro plus analysis, and represented by mean optical density (MOD). Results The frequency of Btk+CD19+ B cells from SLE patients is up-regulated compared with the healthy controls (3.89±0.31 vs 2.61±0.21, p<0.01), significantly correlating with the SLE activity (SLEDAI) (r=0.53), levels of serum anti-dsDNA antibody (r=0.41), levels of serum C3 (r=-0.41), and the amount of 24 hours urine protein (r=0.59). We also found that the frequency of Btk+CD19+ B cells in the patients with lupus nephritis was significantly higher compared with the patients without lupus nephritis (4.71±0.481 vs 3.19±0.30, p<0.05). The levels of Btk expression in glomerulus were markedly increased in LN patients compared with controls (0.04±0.01 vs 0.01±0.00, p<0.001). Conclusions Btk expression is significantly increased in LN, which may be a promising therapeutic target for the molecular therapy of SLE. Disclosure of Interest None declared

Mesenchymal stem cells prevent podocyte injury in lupus-prone B6.MRL-Faslpr mice via polarizing macrophage into an anti-inflammatory phenotype.

BACKGROUND Podocyte injury plays a pathogenic role in the development of lupus nephritis (LN). Mesenchymal stem cells (MSCs) have shown promising therapeutic potential for LN. However, whether MSCs can prevent podocyte injury in LN remains unknown. METHODS Human umbilical cord-derived MSCs (UC-MSCs) were infused into lupus-prone B6.MRL-Faslpr (B6.lpr) mice to investigate the influences of UC-MSCs on podocyte injury in LN. Podocytes and macrophages were co-cultured with UC-MSCs in vitro to study the mechanism by which UC-MSC protect podocytes. We further explored the effects of UC-MSCs on macrophage polarization. RESULTS We found that UC-MSCs promoted the expression of podocyte-specific markers, podocin and synaptopodin, in lupus-prone B6.lpr mice, along with the improvement of lupus renal pathology in terms of reduced IgG and C3 deposition in glomeruli and decreased anti-dsDNA antibody level. Besides, UC-MSC treatment decreased podocyte foot process effacement, as UC-MSCs-treated macrophages led to less podocyte injury in vitro. Interestingly, we further found that UC-MSCs-treated macrophages exhibited an anti-inflammatory phenotype with higher expression of CD206, and lower expression of tumor necrosis factor-α and interleukin-1β. Additionally, UC-MSCs-treated lupus mice showed reduced renal macrophage infiltration and elevated CD206 expression in kidney. CONCLUSIONS Our results demonstrated that UC-MSCs ameliorated LN by preventing podocyte injury possibly through reducing macrophage infiltration and polarizing macrophage into an anti-inflammatory phenotype.

Mesenchymal Stem Cells Control Complement C5 Activation by Factor H in Lupus Nephritis

Lupus nephritis (LN) is one of the most severe complications of systemic lupus erythematosus (SLE) caused by uncontrolled activation of the complement system. Mesenchymal stem cells (MSCs) exhibit clinical efficacy for severe LN in our previous studies, but the underlying mechanisms of MSCs regulating complement activation remain largely unknown. Here we show that significantly elevated C5a and C5b-9 were found in patients with LN, which were notably correlated with proteinuria and different renal pathological indexes of LN. MSCs suppressed systemic and intrarenal activation of C5, increased the plasma levels of factor H (FH), and ameliorated renal disease in lupus mice. Importantly, MSCs transplantation up-regulated the decreased FH in patients with LN. Mechanistically, interferon-α enhanced the secretion of FH by MSCs. These data demonstrate that MSCs inhibit the activation of pathogenic C5 via up-regulation of FH, which improves our understanding of the immunomodulatory mechanisms of MSCs in the treatment of lupus nephritis.