C Gao

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 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.

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 …

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

THU0176 Efficiency and safety of rapamycin combined with low-dose IL-2 treatment compared with methotrexate in patients with rheumatoid arthritis

Background The molecular target rapamycin (mTOR) signaling can regulate between effector and regulatory T cell lineage commitment [1]. Rapamycin, the inhibitor of mTOR, has appeared to be a new therapy for several autoimmune diseases, such as systemic lupus erythematosus [2]. Objectives To evaluate whether rapamycin is beneficial in patients with Rheumatoid Arthritis (RA), and compared with Methotrexate in efficiency and safety. Methods Fifty-eight DMARDs-naive RA patients were enrolled, thirty-eight were treated with Rapamycin (0.5 mg every 2 days, combined with IL-2 50WIU per day for 5 days), the others with Methotrexate (10mg per week) taken as control. Clinical improvement and immunological assessments were performed at baseline, 1 and 12 weeks. Treatment group assessed CD4+ T cell subsets by flow cytometry at baseline, 1 and 12 weeks. Results We enrolled 58 patients. At baseline, patients had a mean DAS28 of 3.34 (0.81). Rapamycin group and Methotrexate group included 38 and 20 patients, respectively, with no significant differences in baseline characteristics. At 1 week, the mean DAS28 after Rapamycin treatment (2.43 [0.77]) and Methotrexate (2.25 [0.86]) was not significantly different (P=0.43). Same as ESR (24.74 [24.53], 21.76 [24.27], P=0.66). The dose of glucocorticoid during hospitalization of rapamycin treatment group (720.8 [554.3]) was lower than Methotrexate (1202.3 [943.1], P=0.042). The length of hospital stay of Rapamycin (14.5 [3.9]) was lower than Methotrexate (21.0 [3.8], P<0.001). Rapamycin administration resulted in an increase in the absolute counts of Treg cells from a median of 36.82 cell/ul (at week 0) to 99.80 cell/ul (at week 1) (P<0.001). The ratios of Th17/Treg cells showed a reduction from a median of 0.16 to 0.09, and the difference was significant (P=0.047). At 12 week, 5 patients treated with Rapamycin dropped out because of non-compliance. the mean DAS28 was not significantly different (2.36 [0.97], 2.16 [0.86], P=0.51). The same as the daily dose of glucocorticoid (10.21 [32.3], 9.16 [40.1], P=0.804). The absolute counts of Treg cells increased from a median of 36.82 cell/ul (at baseline) to 43.26 cell/ul after Rapamycin administration (P=0.028). The ratios of Th17/Treg had no significant difference from a median of 0.16 at baseline to 0.12 at week 12 (P=0.937). Liver enzyme elevations occurred on 2 patients after Methotrexate therapy for 1 week. However, there were no serious adverse events observed during the 12-week period of rapamycin treatment. Conclusions Rapamycin combined with the low-dose IL-2 appears to be a safe and effective therapy for RA, by a rapid increase of circulating Treg cells and a correction of the ratio of Th17/Treg cells, which has gotten a same response compared with Methotrexate. References Zheng Y. The mTOR kinase differentially regulates effector and regulatory T cell lineage commitment.[J]. Immunity, 2009, 30(6):832–844. Fernandez D, Bonilla E, Mirza N, et al. Rapamycin Reduces Disease Activity and Normalizes T Cell Activation–Induced Calcium Fluxing in Patients With Systemic Lupus Erythematosus[J]. Arthritis & Rheumatology, 2006, 54(9):2983–2988. Disclosure of Interest None declared

OP0132 Low-dose INTERLEUKIN-2 selectively restore regulatory T cell numbers in patients with behcet's disease

Background The lack of CD4+CD25+Foxp3+ T regulatory cell (Treg) has been associated with human systemic autoimmune diseases, such as Behcets disease (BD). IL-2, an essential growth and survival factor for Treg cells.However, the significance of Treg cells in the pathogenesis and the effect of low dose of IL-2 on BD are remain to investigate. Objectives The lack of CD4+CD25+Foxp3+ T regulatory cell (Treg) has been associated with human systemic autoimmune diseases, such as Behcets disease (BD). IL-2, an essential growth and survival factor for Treg cells.However, the significance of Treg cells in the pathogenesis and the effect of low dose of IL-2 on BD are remain to investigate. Methods Eighty patients with BD and seventy healthy donors were enrolled. CD4+T cell subsets in peripheral blood mononuclear cells from these people were measured by multicolour flow cytometry. Twenty-six patients were treated daily with subcutaneous injections of 0.5 million IU of human IL-2 for five consecutive days, CD4+T cell subsets were analysed before and after treatment by flow cytometry. Results Compared to health control,the absolute counts of circulating Treg cells were significantanty decreased in patients with BD (median:29.93 cell/uL VS median:33.16 cell/uL; p=0.039) and it is negative correlation with disease activity. While the ratios of Th17/Treg in patients with BD (median:0.29;n=80,p=0.034) were significantly higher than those of health control (median:0.2;n=70).No diffrernce in the absolute counts of circulating Th17 cells (CD4+IL-17+) between patients with BD and health control. Treatment of patients with BD with a low-dose of IL-2 regimen selectively increased the absolute counts of Treg cells, from a median of 18.97cell/uL to 74.68 cell/uL (at 5 days) (p=0.000). No significant difference was observed in the absolute counts of circulating Th17, Th1 and Th2 cells after IL-2 treatment. Conclusions Th17/Treg cells may play a role in the pathogenesis of Patients with BD, low-dose of IL-2 proposes a selective biological treatment strategy by restoring immune tolerance. Disclosure of Interest None declared

OP0043 The number of circulating regulatory t cells is reduced and low-dose il-2 selectively stimulates its proliferation in patients with systemic lupus erythematosus

Background The imbalance of T help 17 cells (Th17)/regulatory T cells (Tregs) is considered to be a pivotal cause of autoimmune diseases1, including systemic lupus erythematosus (SLE). However, previous reports1,2 describing the respective changes of Tregs and Th17 cells in SLE patients were controversial because a few samples or diverse markers were used to identify Tregs with little consensus. Objectives To clarify the status of Tregs and Th17 in SLE, we investigated the frequencies of Tregs and Th17 cells on a large scale and whether those defects can be corrected by the supplementation of low-dose human recombinant interleukin-2 (IL-2). Methods Two hundred and thirty-five SLE patients (219 women and 16 men), with mean age of 37.80±14.00 years, were enrolled. The disease activity using European League Against Rheumatism (EULAR) criteria was judged for SLE patients with erythrocyte sedimentation rate (ESR) and SLEDAI scores. The frequencies of CD3+CD4+FOXP3+Treg cells and Th17 cells in peripheral blood from these patients were measured by flow cytometry. And low-dose IL-2 was used among 127 patients at a dosage of fifty WIU every day for five days. Immunological and clinical assessments were performed again at the end of IL-2 treatment. Ninety healthy volunteers, matched for patients age and gender, were also included for the estimation of CD4+ T cell subsets. Results As compared to healthy controls (median of Treg cells: 33.09 cells/ul), the frequencies of circulating CD4+CD25+FOXP3+Treg cells were significantly decreased in SLE patients (median: 15.49 cells/ul, P<0.001). The median ratios of Th17/Tregs cells in patients were greatly higher than those of healthy volunteers [0.42 (0.19, 0.88) vs. 0.21 (0.15, 0.34), P<0.001]. There was not significantly different in circulating Th17 cell between two groups. Moreover, CD4+CD25+FOXP3+Treg cells were negatively correlated with ESR and SLEDAI score (r=-0.198, P=0.01; r=-0.25, P=0.002).While no obvious correlation was seen between Th17 cells and SLEDAI score. After IL-2 therapy in SLE, there was a four-fold increase in circulating CD4+CD25+FOXP3+Treg cells [43.73 (24.08,74.22) vs. 11.95 (7.51,20.34),P<0.001], whereas Th17 cells were increased slightly. The ratio of Th17/Tregs was decreased significantly in patients with IL-2 treatment [0.19 (0.09,0.41) vs. 0.52 (0.23,0.95),P<0.001], tended to balance and had no difference with healthy individual (P=0.275). Conclusions With the increase of disease activity,CD4+CD25+FOXP3+Treg cells were gradually reducing, while Th17 cells did not show a significant change, indicating that the reduction of Tregs but not the elevation of Th17 cells may be the major reason for imbalance of Th17/Tregs. It is speculated that SLE is an autoimmune disease triggered by the defect of immunotolerance. More importantly, low-dose IL-2 selectively modulated the abundance of Tregs, which effectively induced autoimmune tolerance and further improved clinical symptoms. References Ji Y, Xue Y, Hejian Z, et al.Recovery of the immune balance betweenTh17 and regulatory T cells as a treatment for systemic lupus erythematosus[J]. Rheumatology 2011;50:1366–1372. Steinmetz OM, Turner JE, Paust HJ et al. CXCR3 mediates renal Th1 and Th17 immune response in murine lupus nephritis[J]. J Immunol 2009;183:4693–704. 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

SAT0213 Low dose il-2 restores decreased absolute number of regulatory t cells and imbalance between th17 and regulatory t cells in patients with rheumatoid arthritis

Background A therapeutic revolution in the past decade is still a considerable unmet need in the treatment of rheumatoid arthritis (RA). On the other hand, dysfunction of regulatory T cells (Tregs) has been considered to be a pivotal cause of RA and correction of this dysfunction to be a potential RA therapy1. However, abnormalities of Tregs in patients with RA were reported controversially in previous studies2, in which only proportion was measured and Tregs were defined using different protein markers. Objectives In this study, we measured both absolute numbers and proportions of CD4+CD25+Foxp3+ Tregs in peripheral blood of RA patients and investigated the effects of low-dose recombinant human IL-2 (rhIL-2) on Tregs and CD4+ effector T cell subsets in patients with RA. 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 population, were examined by flow cytometry in 342 patients with RA with different 28-joint Disease Activities (DAS28), including 75 who had never received disease-modifying antirheumatic drugs (DMARD) and 151 who were receiving or had received DMARD. Among these patients, 112 consented at enrollment to receive rhIL-2 treatment. Before and after treatment, the Th17 and Treg cells in peripheral blood were analyzed by flow cytometry. Results The absolute count of Treg cells in patients with RA was significantly low compared with that of healthy controls (P<0.05), but the absolute count of Th17 cell was no different between RA and healthy controls. After the course of rhIL-2 treatment, there was a significant increase in the absolute count of Th17 and Treg cells in the CD4+ T cell population (P<0.01), but Th17/Treg was significantly low after the treatment. Conclusions The data suggest that, besides propotion, the decrease of Treg cell number, defined as the CD4+CD25+FOXp3+population, may contribute to the pathogenesis of RA. Over the treatment of rhIL-2, there was a more significant increase in the absolute count of Treg cells than that of Th17, and consequently restore the balance of Th17/Treg. References Miyara M, Ito Y, Sakaguchi S. TREG-cell therapies for autoimmune rheumatic diseases. Nature reviews Rheumatology 2014;10:543–51. Morita T, Shima Y, Wing JB, Sakaguchi S, Ogata A, Kumanogoh A. The Proportion of Regulatory T Cells in Patients with Rheumatoid Arthritis: A Meta-Analysis. PloS one 2016;11:e0162306. Disclosure of Interest None declared