X Li

Combination with Methotrexate and Cyclophosphamide Attenuated Maturation of Dendritic Cells: Inducing Treg Skewing and Th17 Suppression In Vivo

Immune disorder is considered the main pathogenesis of autoimmune diseases, such as rheumatoid arthritis (RA). The balance of the two special subsets of CD4+T cells, T helper cell 17 (Th17), and Regulator T cell (Treg) is the key factor of maintaining a normal immune response. Dendritic cells (DCs), which are the most powerful antigen-presenting cells, play an important role in regulating the balance of Th17 and Treg. The combination of disease modifying antirheumatic drugs (DMARDs) is an important strategy of RA therapy. In this study, we investigated the effect of MTX and CTX on DC maturation in ovalbumin (OVA) immunized mice. Th17 inflammatory response is stronger, while the level of DCs maturity is higher. In contrast, the immunosuppression of Treg is stronger. We found that MTX combined with CTX significantly inhibited the DCs maturity and downregulated the antigen presenting capacity of DCs. As a result, it reestablished a balance of Th17 and Treg. Our study adds a novel mechanism and therapeutic target of MTX combined with CTX for autoimmune disease treatment.

High-Performance Liquid Chromatography (HPLC) Quantification of Liposome-Delivered Doxorubicin in Arthritic Joints of Collagen-Induced Arthritis Rats

Background Neoangiogenesis occurring in inflamed articular synovium in early rheumatoid arthritis (RA) is characterized by enhanced vascular permeability that allows nanoparticle agents, including liposomes, to deliver encapsulated drugs to arthritic joints and subsequently improve therapeutic efficacy and reduce adverse effects. However, the targeting distribution of liposomes in arthritic joints during RA has not been quantitatively demonstrated. We performed this study to evaluate the targeting distribution of PEGylated doxorubicin liposomes in the arthritic joints of collagen-induced arthritis (CIA) rats by high-performance liquid chromatography (HPLC). Material/Methods Two doxorubicin formulations were administered to CIA rats via tail intravenous injection at a single dose (50 mg/m2). CIA rats were sacrificed and the tissues of the inflamed ankle joints were collected. The content of doxorubicin in the arthritic joints was analyzed by a validated and reproducible HPLC method. A two-way ANOVA for 2×5 factorial design was used for statistical analysis. Results The developed HPLC method was sensitive, precise, and reproducible. The method was successfully applied to quantify doxorubicin content in arthritic tissues. At each time point (6, 12, 24, 48, and 72 h), doxorubicin content in the arthritic joints of the doxorubicin liposome group (DOX-LIP group) was higher than in the free doxorubicin group (DOX group) (P<0.05). In the DOX-LIP group, doxorubicin levels in the arthritic joints increased gradually and significantly in the interval of 6–72 h post-administration. Conclusions PEGylated doxorubicin liposomes were targeted to, accumulated, and retained in the arthritic joints of CIA rats. The present study indicates that liposome encapsulation increases the therapeutic efficacy of antirheumatic drugs, presenting a promising therapeutic strategy for RA.

Methotrexate, combined with cyclophosphamide attenuates murine collagen induced arthritis by modulating the expression level of Breg and DCs

To explore the mechanism of methotrexate (MTX) and its combination with cyclophosphamide (CTX) in collagen-induced arthritis (CIA), we investigated the levels of several immune cells and cytokines in mice with different treatments. CIA was induced in DBA/1 mice at the age of 7 weeks by primary immunization with 100μl emulsion containing 2mg/ml bovine type II collagen which was mixed with complete Freunds adjuvant (CFA). The booster immunization was performed with 50-100μl emulsion containing 2mg/ml bovine type II collagen (CII) mixed with incomplete Freunds adjuvant (IFA). MTX, CTX or both were administrated after the booster immunization. Therapeutic effect was evaluated by arthritic scores, X-rays and assessment of histopathological joint destruction. The expression of TNF-α, IL-6, IL-23, IL-10 were also measured. The frequencies of different immune cell subsets in the lymph node, spleen and bone marrow were determined by flow cytometry analysis. Our results showed that CTX and MTX treatment attenuated the severity of arthritis of CIA mice and reduced the levels of several cytokines. CTX and MTX treated mice showed a lower frequency of B cells in bone marrow. Also, when treated the CIA mice with MTX, alone or together with CTX, the lymph nodes and spleen exhibited a decrease in regulatory B cells (Breg) and dendritic cells (DCs). Notably, the combination of MTX and CTX had a more pronounced effect. By measuring the levels of different immune cells those participated in the development of rheumatoid arthritis (RA), our experiment may help to evaluate the therapeutic effects and prognosis of arthritic diseases.

Combination of 4-hydroperoxy cyclophosphamide and methotrexate inhibits IL-6/sIL-6R-induced RANKL expression in fibroblast-like synoviocytes via suppression of the JAK2/STAT3 and p38MAPK signaling pathway

&NA; Although conventional combination therapy is effective for most patients with rheumatoid arthritis (RA), many still do not respond to current therapies. Therefore, novel combination regimens that better target cellular processes involved in RA pathogenesis are required. Preliminary studies have demonstrated the beneficial effects of a combination of cyclophosphamide (CTX) and methotrexate (MTX) in models of RA. Using western blotting, real‐time polymerase chain reaction, enzyme‐linked immunosorbent assays, and immunofluorescent staining, we demonstrated that the combination of 4‐hydroperoxy CTX (4‐H‐CTX) and MTX inhibited the expression of receptor activator of nuclear factor‐&kgr;B ligand (RANKL) in fibroblast‐like synoviocytes (FLS) treated with the interleukin (IL)‐6/soluble IL‐6 receptor (sIL‐6R) complex. To elucidate the mechanisms underlying this effect, we treated RA‐FLS with the JAK2/STAT3 inhibitor AG490 or p38MAPK inhibitor SB203580. The results showed that IL‐6/sIL‐6R‐induced RANKL upregulation required phosphorylation‐mediated activation of STAT3 and p38 signaling, and that 4‐H‐CTX and/or MTX inhibited RANKL expression in IL‐6/sIL‐6R‐stimulated FLS by suppressing JAK2/STAT3 and p38MAPK signaling. This study demonstrated for the first time the inhibitory effects of 4‐H‐CTX and MTX on RANKL expression in IL‐6/sIL‐6R‐stimulated FLS via suppression of STAT3 and p38MAPK phosphorylation. These results identify promising therapeutic agents that might have clinical applications in patients with RA who are at high risk of bone erosion or do not respond well to conventional therapy. HighlightsIL‐6/sIL‐6R‐induced RANKL expression is regulated by JAK2/STAT3 and p38MAPK pathway.4‐H‐CTX and MTX inhibit RANKL expression by suppressing STAT3 and p38MAPK signaling.CTX‐MTX combination therapy is promising for rheumatoid arthritis.

Methotrexate Combined with 4-Hydroperoxycyclophosphamide Downregulates Multidrug-Resistance P-Glycoprotein Expression Induced by Methotrexate in Rheumatoid Arthritis Fibroblast-Like Synoviocytes via the JAK2/STAT3 Pathway

Objective Rheumatoid arthritis (RA) multidrug resistance is associated with P-glycoprotein (P-gp) overexpression. We investigated the effects of methotrexate (MTX) alone and combined with 4-hydroperoxycyclophosphamide (4-HC) on P-gp expression in fibroblast-like synoviocytes (FLSs) from patients with RA and examined the signaling pathway involved. Methods RA-FLSs were treated with MTX, MTXu2009+u20094-HC, AG490u2009+u2009MTX, or AG490u2009+u2009MTXu2009+u20094-HC for 72u2009h. Proliferation inhibition rates were determined by MTT assay; P-gp expression was measured by flow cytometry and real-time polymerase chain reaction (RT-PCR); JAK2 and STAT3 were measured by RT-PCR and cell-based ELISA to assess STAT3 signaling. Results MTX alone significantly induced P-gp expression and mRNA production in RA-FLSs. P-gp expression and mRNA levels were lower in the MTXu2009+u20094-HC group than in the MTX-alone group. In contrast to MTX, MTXu2009+u20094-HC reduced the STAT3 phosphorylation and downregulated JAK2 and STAT3 mRNA production. Inhibition of constitutively active STAT3 accompanied by 4-HC suppressed P-gp levels in RA-FLSs. The MTT assays revealed no significant differences in proliferation inhibition rates among groups. Conclusions The increased anti-P-gp effect of MTXu2009+u20094-HC versus MTX alone in RA-FLSs was mediated via inhibition of the JAK2/STAT3 pathway and may have helped reverse MDR in refractory RA patients with high-P-gp levels.

The Proportion of Regulatory T Cells in Patients with Systemic Lupus Erythematosus: A Meta-Analysis

Background Accumulating evidence indicates that a deficiency in or dysfunction of regulatory T cells (Tregs) is involved in the pathogenesis of systemic lupus erythematosus (SLE). As different markers have been used to identify Tregs, recent studies on the proportions of Tregs in SLE patients have generated controversial results. To clarify the status of Tregs in such patients, we determined the proportions of Tregs present during development of the disease, with special consideration of controversial cellular markers. Methods We identified studies reporting the proportions of Tregs in SLE patients by searching relevant databases through March 2018. Using the PRISMA guidelines, we performed a random effects meta-analysis of the frequencies of Tregs defined in different ways. Inconsistency was evaluated using the I-squared index (I 2), and publication bias was assessed by examining funnel plot asymmetry using the Begger and Egger tests. Results Forty-four studies involving 2779 participants were included in the meta-analysis. No significant difference in the proportions of Tregs was evident between 1772 patients and 1007 controls [−0.191, (−0.552, 0.362), p = 0.613, I 2 = 95.7%]. We next conducted subanalyses based on individual definitions of Tregs. When the Treg definition included “FOXP3-positive” cells, the proportions did not differ between SLE patients and controls [−0.042, (−0.548, 0.632), p = 0.889, I 2 = 96.6%]; this was the case when Tregs were defined as either “CD25low/−FOXP3+” or “CD25high/+FOXP3+” cells. SLE patients had lower proportions of Tregs that were “single CD25-positive” [−1.428, (−1.982, −0.873), p < 0.001, I 2 = 93.4%] and “CD127-negative” [−1.093, (−2.002, −0.183), p = 0.018, I 2 = 92.6%] compared to controls. Tregs defined as “CD25bright,” “CD25bright/highCD127low/−,” and “CD25highCD127low/−FOXP3+” did not differ in proportion between SLE patients and controls. Conclusions The Treg proportions varied by the cellular identification method used. The proportions of Tregs that were accurately identified and functionally validated fell among patients with SLE. Stricter definitions of Tregs are necessary when evaluating the status of such patients.

Short-term and low-dose IL-2 therapy restores the Th17/Treg balance in the peripheral blood of patients with primary Sjögren’s syndrome

Based on evidence that low-dose interleukin-2 (IL-2) increases CD4+CD25+FOXP3+ Treg (CD4Treg) cells in patients with graft-versus-host disease, induction of autoimmune tolerance has been proposed as a treatment.1 However, evidence-based guidelines for the management ofxa0primary Sjogren’s syndrome (pSS) are lacking.2 Using a modified method of flow cytometry, we aimed to revaluate the exact levels of Th17 and CD4Treg cells in the peripheral blood (PB) of patients with pSS and explore the effects of short-term and low-dose IL-2.3 4nnA total of 190 patients with pSS (169, treated with immunosuppressants; 21, new-onset (sampled as treated or new pSS patients below)) consented at enrolment to donate PB samples for comprehensive immunophenotyping (see online supplementary table S1xa0and figure S1). In the study, BD Trucount tubes were used to determine the absolute counts of total CD4+ T cells in the PB, and then, the absolute number of CD4+ T subsets, such as CD4Treg cells, were calculated.xa0Detailed methods and statistical analysis is in online version (see online supplementary text).xa0nn### Supplementary file 11nn[annrheumdis-2018-213036-SP11.docx]nnAmong the CD4+ T subsets in the PB of the healthy control …

AB0321 Negative correlation of the absolute number of cd4 +cd25+foxp3+regulatory t cells to the levels of rheumatoid factor in peripheral blood of new onset patients with rheumatoid arthritis

Background Rheumatoid arthritis (RA) is a progressive immune-mediated disease that can culminate in joint destruction and early mortality. High levels of serum RF are associated with a worse prognosis in RA. The role of is not fully understood. Recently, Our studies have found that the absolute number of peripheral CD4+ CD25+Foxp3+ regulatory T (CD4+Treg) cells decreased in RA patients. Interestingly, regulatory T cell epitopes (Tregitopes) in IgG have been reported as the main component of intravenous immunoglobulin therapy (IVIG) and provide one explanation for the expansion and activation of Treg cells following IVIg treatment. We hypothesise that RF joins with IgG to form large molecular complexes, which interrupts the production of Tregitopes in antigen presenting cells as a cause of reduction of CD4+Treg cells. Objectives The aim of this study is to investigate whether the absolute number of CD4+Treg cells is associated with the titers of auto-antibodies in blood of new-onset diagnosed patients with RA. Methods A total of 57 new-onset diagnosed patients with RA were enrolled and healthy donors as control. The absolute number of peripheral CD4+Tregs cells was detected by multicolor flow cytometry combined with an internal microsphere counting standard. 46 of new-onset cases were tested the levels of RF and anti-CCP with ELISA method. IIF method was used to detect APF and AKA. Low titers group and high titers group around with the value 1:80. All data was analysed by SPSS 22.0. Results The absolute number of CD4+Treg cells in peripheral blood of new-onset patients with RA was significantly lower than in healthy controls [25.1 (16.01, 40.75) vs 33.0567 (22.9, 43.18), p<0.05]. Interestingly, the reduction of peripheral CD4+Treg cells was negatively correlated to RF titers (correlation coefficient −0.488, p<0.01) but not to other auto-antibodies against CCP, APF and AKA. The absolute number of CD4+Tregs in high titers RF group was lower than in low titers RF group [20.5 (14.0,40.0 vs 34.0 (29.7,44.6),p<0.05]. There was statistically significant difference in two titers groups.Abstract AB0321 – Table 1 Spearman correlation analysis of absolute number of CD4+ Treg cells and autoantibodies titers in 46 new-onset RA patients RF AKA APF a-CCP CD4+Treg −0.488** −0.126 −0.328 0.104 **p<0.01Abstract AB0321 – Table 2 The absolute number of CD4+Treg cells Contrast with different titers group RF n M (P 25, P 75) Z P Low titer group 13 34.0 (29.7,44.6) −2.127 0.033 High titer group 33 20.5 (14.0,40.0) Abstract AB0321 – Figure 1 The absolute number of CD4+ Treg cells were reduced in peripheral blood of the all enrolled new-onset RA patients (n=57) (A). The reduction of peripheral CD4+ Treg cells from new-onset patients were negatively correlated with the levels of RF tested in these subjects (B). There was statistically significant difference in two titers groups of RF (C). Conclusions The absolute number of CD4+Treg cells in peripheral blood of new-onset patients with RA was significantly decreased compared with that in health controls. Furthermore, the reduction of peripheral CD4+Treg cells was negatively correlated to the titers of RF, suggesting that RF contributes to the reduction of CD4 +Tregs cells. The correlation of decreased CD4+Treg and RF may be involved in the pathogenesis of poor prognosis in RA. Reference [1] Cousens LP, Najafian N, Mingozzi F, Elyaman W, Mazer B, Moise L, Messitt TJ, Su Y, Sayegh M, High K, Khoury SJ, Scott DW, De Groot AS. In vitro and in vivo studies of IgG-derived Treg epitopes (Tregitopes): a promising new tool for tolerance induction and treatment of autoimmunity. J Clin Immunol2013Jan;33(Suppl 1):S43–9. doi:10.1007/s10875-012-9762-4 [Epub 2012 Sep 2. Review]. Disclosure of Interest None declared

AB0535 Effect of metformin on the absolute number of cd4+ t cell subsets in patients with primary sjogren’s syndrome

Background Primary Sjogren’s syndrome is a chronic inflammatory autoimmune disease characterised by the infiltration of lymphocytes into exocrine glands such as the salivary gland and lacrimal gland. Although its etiology and pathogenesis is unclear at present, we consider immune dysfunction plays a significant role in the process. A lot of studies have confirmed that the formation of Treg and Th17 cells interact between each other, and their balance can affect the immune response results, notably reflected in various autoimmune and inflammatory diseases, including primary Sjogren’s syndrome. However, there are few studies on the absolute number of CD4 +T cells in peripheral blood of patients with primary Sjogren’s syndrome. In addition, metformin can affect the balance of Th17/Treg cells through the AMPK-mTOR pathway. Objectives To explore whether metformin can affect the balance of Th17/Treg cells in peripheral blood of patients with primary Sjogren’s syndrome, and then be applied in the treatment of pSS patients. Methods The number of Treg cells{28.74 (21.22,38.68) vs 34.05 (30.14,42.31),P=0.023}significantly increased after the treatment. At the same time, there was a significantly decrease in the ratio of h17/Treg cells{0.25 (0.08,0.44) vs 0.18 (0.04,0.32),P=0.014},]. Besides, after the treatment the absolute number of Th17 cells were increased, but it was not statistically significant{4.5 (3.64,14.23) vs 7.87 (2.37,19.89),P=0.835}. In addition, the clinical symptoms of the metformin group were obviously improved, while the dosage of prednisone, leflunomide or hydroxychloroquine reduced significantly. Results The number of Treg cells{28.74 (21.22,38.68) vs 34.05 (30.14,42.31),P=0.023}significantly increased after the treatment. At the same time, there was a significantly decrease in the ratio of h17/Treg cells{0.25 (0.08,0.44) vs 0.18 (0.04,0.32),P=0.014},]. Besides, after the treatment the absolute number of Th17 cells were increased, but it was not statistically significant{4.5 (3.64,14.23) vs 7.87 (2.37,19.89),P=0.835}. In addition, the clinical symptoms of the metformin group were obviously improved, while the dosage of prednisone, leflunomide or hydroxychloroquine reduced significantly.Abstract AB0535 – Figure 1 Conclusions Metformin can increase the absolute number of Treg cells and decrease the ratio of Th17/Treg cells in the peripheral blood of patients with pSS, while reducing the use of hormones and DMARDs drugs. And it may be one of the mechanisms adopted in the treatment for pSS. References [1] Matsui K, Sano H. T Helper 17 Cells in Primary Sjogren’s syndrome [J]. Journal of Clinical Medicine2017;6(7):65. [2] Saito M, Otsuka K, Ushio A, et al. Unique Phenotypes and Functions of Follicular Helper T Cell and Regulatory T Cell in Sjogren’s Syndrome. [J] Curr Rheumatol Rev2017;13(999). [3] Lee SY, Moon SJ, Kim EK, et al. Metformin Suppresses Systemic Autoimmunity in Roquin (san/san) Mice through Inhibiting B Cell Differentiation into Plasma Cells via Regulation of AMPK/mTOR/STAT3[J]. Journal of Immunology2017;198(7):2661. Disclosure of Interest None declared

SAT0459 Low dose IL-2 restores imbalance between TH17 and regulatory T cells in patients with psoriatic arthritis

Background Psoriasis arthritis is one of chronic, relapsing, inflammatory autoimmune disorders with skin lesions and joint damage. A therapeutic revolution of psoriatic arthritis (PsA) is still a considerable unmet need in the past decades. It has been well known that the imbalance of Th17 cells and regulatory T cells (Tregs) may be a pivotal cause of PsA. Correction of this dysfunction can be a potential therapy of PsA. Objectives In this study, we measured and compared both absolute numbers and proportions of CD4+CD17+ Th17 cells and CD4+CD25+Foxp3+ Treg cells in peripheral blood of PsA patients and healthy controls to explore the immunopathogenesis of PsA; on the other hand, the effects of low-dose recombinant human IL-2 (rhIL-2) on Th17 and Treg cells were investigated in patients with PsA. Methods Both absolute numbers and proportions of Treg and Th17 cells in peripheral blood, defined as the CD4+CD25+Foxp3+T or CD4+IL-17+ T cell populations, were examined by flow cytometry in 40 healthy controls and 77 patients with PsA, including 39 patients who had never received disease-modifying antirheumatic drugs (DMARDs) and 38 patients who were receiving or had received DMARDs. Among these patients, 20 patients consented at enrollment to receive rhIL-2 treatment. Before and after treatment (50WIU/d for 5 days, IH), Th17 and Treg cells in peripheral blood were analyzed by flow cytometry. Results The absolute count of Th17 cells in patients with PsA was very significantly higher than that of healthy controls (P<0.01), but the proportions of Th17 cell were not seen difference between PsA and healthy controls (P>0.05). In contrast with treated-PsA patients, the absolute count of Th17 cells was significant higher in untreated-PsA patients (P<0.05). After the course of rhIL-2 treatment, there was a significant increase in the absolute count of Treg cells (P<0.05), but no diference in the absolute count of Th17 cells, Th17/Treg was significantly lower and went back to nomal. Conclusions The results suggest that, not the proportion, but the decrease in the absolute count of Th17 cells, defined as the CD4+CD17+ populations, contributes to the pathogenesis of PsA. After the treatment of rhIL-2, there was a more significant increase in the absolute count of Treg cells than that of Th17, and consequently the balance of Th17/Treg was restored to normal, leading to the development of new therapies. References Karczewski J, Dobrowolska A, Rychlewskahańczewska A, et al. New insights into the role of T cells in pathogenesis of psoriasis and psoriatic arthritis[J]. Autoimmunity, 2016:1.DOI:10.3109/08916934.2016.1166214. Yoo I S, Lee J H, Song S T, et al. T-helper 17 cells: the driving force of psoriasis and psoriatic arthritis.[J]. International Journal of Rheumatic Diseases, 2012, 15(6):531–537. DOI:10.1111/j.1756–185X.2012.01813.x. Szodoray P, Nakken B, Barath S, et al. Altered Th17 cells and Th17/regulatory T-cell ratios indicate the subsequent conversion from undifferentiated connective tissue disease to definitive systemic autoimmune disorders[J]. Human Immunology, 2013, 74(12):1510–8. DOI:10.1016/j.humimm.2013.08.003. Raychaudhuri S P. Role of IL-17 in psoriasis and psoriatic arthritis.[J]. Clinical Reviews in Allergy & Immunology, 2013, 44(2):183–193. DOI: 10.1007/s12016–012–8307–1. Disclosure of Interest None declared