Efficacy of immunomodulatory therapy with all-trans retinoid acid in adult patients with chronic immune thrombocytopenia

Efficacy of immunomodulatory therapy with all-trans retinoid acid in adult patients with chronic immune thrombocytopenia

Thrombosis Research 140 (2016) 73–80 Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromre...

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Thrombosis Research 140 (2016) 73–80

Contents lists available at ScienceDirect

Thrombosis Research journal homepage: www.elsevier.com/locate/thromres

Efficacy of immunomodulatory therapy with all-trans retinoid acid in adult patients with chronic immune thrombocytopenia☆ Lan Dai 1, Ri Zhang 1, Zhaoyue Wang ⁎, Yang He ⁎, Xia Bai, Mingqing Zhu, Ziqiang Yu, Chang-geng Ruan MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006, China; Collaborative Innovation Center of Hematology, Soochow University, 1 Shizi Street, Suzhou 215006, China

a r t i c l e

i n f o

Article history: Received 30 November 2015 Received in revised form 22 January 2016 Accepted 12 February 2016 Available online 15 February 2016 Keywords: Chronic immune thrombocytopenia All-trans retinoid acid Regulatory T cells Immunomodulatory therapy

a b s t r a c t Introduction: Immune thrombocytopenia (ITP) is a common hematologic disorder characterized by isolated thrombocytopenia. In adults, ITP is more likely to be chronic, requiring individualised treatment and management. Corticosteroids and splenectomy are the most common therapy for ITP. However, these routine approaches failed in these patients with chronic ITP. The aim of this study was to evaluate the efficacy of immunomodulatory therapy with all-trans retinoid acid (ATRA) in adult patients with chronic ITP. Materials and methods: ATRA therapy was applied in a total of 35 patients with chronic ITP who failed with standard dose corticosteroids and/or splenectomy. The response ratio and the change of the T cell subsets including Th1, Th2, Th17 and Treg, were evaluated. Results: Complete response and overall response were observed in 10 (28.6%) and 19 patients (54.3%), respectively. Compared with the control group, a significant decreased level of Treg cells, IL-10 and Foxp3 expression were found in ITP patients. ATRA therapy could significantly increase the percentage of Treg cell, IL-10 level and Foxp3 expression. Conclusions: Our findings indicate that ATRA therapy could induce significant changes of Treg cells to induce response in patients with chronic ITP. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Chronic immune thrombocytopenia (ITP), an autoimmune disease, is characterized by reduced platelet counts and a variably increased risk of bleeding. A high risk of death and disease-related or therapy-related complications could be presented in patients with chronic ITP [1,2]. Treatment options of these patients include aggressive immunosuppressant agents, and most recently thrombopoietin (TPO) receptor agonists [3,4]. Moreover, single-agent immunosuppressant drugs such as azathioprine and cyclosporine were also used to treat patients with moderate success [5]; however, dose escalation to achieve treatment efficacy can also cause morbidity. Therefore, other therapeutic options are needed. All-trans retinoic acid (ATRA), a vitamin A metabolite, has been shown to be involved in a wide range of biological processes, including cell proliferation and differentiation [6]. Previous studies have been shown to be effective in treatment of acute promyelocytic leukemia (APL) through differentiation induction and terminal cell division of

☆ Grant support: This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (YX21100214). ⁎ Corresponding authors at: 188 Shizi St., The First Affiliated Hospital of Soochow University, Suzhou 215006, China. E-mail addresses: [email protected] (Z. Wang), [email protected] (Y. He). 1 These two authors contributed equally to this work.

http://dx.doi.org/10.1016/j.thromres.2016.02.013 0049-3848/© 2016 Elsevier Ltd. All rights reserved.

leukemia cells [7–9]. Recent studies revealed that ATRA promotes the development and function of T helper (Th) cells and Foxp3 + Treg cells [10,11]. However, the effectiveness of ATRA in the treatment of chronic ITP remains unknown. Here, we firstly conducted a prospective study to investigate the efficacy of ATRA therapy in the treatment of chronic ITP in adult patients and explore the possible mechanisms. 2. Material and methods 2.1. Patients The study included 35 adults with chronic ITP who received ATRA therapy, who were enrolled in a clinical trial between January 2011 and August 2012 (trial NCT01668615 registered at http://www.clinicaltrials. gov). The study was approved by the institutional review boards of the First Affiliated Hospital of Soochow University, and written informed consent was obtained from all the participants in accordance with the Declaration of Helsinki. ITP diagnosis was according to criteria published in the guideline of American Society of Hematology [12]. Patients in whom treatment with standard dose corticosteroids and/or splenectomy fails, and who require further therapy because of unsafe platelet counts or clinical bleeding were included in this study. ATRA (10 mg, three times daily; Shandong Liangfu Group Pharmaceutical Co. Ltd. China) together

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Table 1 Sequence of primers used in reverse transcription-polymerase chain reaction (RT-PCR). Primer name

Forward primer (5′–3′)

Reverse primer (5′–3′)

T-bet GATA-3 Foxp3 ROR-γ GAPDH

ACTGGAGCACAATCATCTGGG GAAGAGTCCGGAGCTGTAC GTGGCATCATCCGACAAGG GCTGGTTAGGATGTGCCG GGAAGGTGAAGGTCGGAGTC

TTGGGTGCAGTGTGGAAAGGC AGGACGAGAAAGAGTGCC TGTGGAGGAACTCTGGGAAT GGATGCTTTGGCGATGA CGTTCTCAGCCTTGACGGT

2.2. Response criteria Patients were evaluated for response if they completed 4 week infusion. Respondents were classified as complete responders (CR), partial responders (PR), and nonresponders (NR). CR was defined as platelet count maintained ≥ 100 × 109/L for at least 3 months. PR was defined as a platelet N50 × 109/L after treatment or doubling of baseline. NR was defined as platelet count b50 × 109/L. 2.3. Antibodies and reagents

Table 2 Summary of patient demographics and baseline characteristics. Responder Non-responder p (n = 19) (n = 16) value Median (years; range) Sex (n, %) Male Female Platelet count (×109/ml; mean ± SD) Day 0 Day 30 Day 180 Patients with any bleeding (n, %) Before During the treatment Cumulative bleeding events during treatment (n, %) Oral Cutaneous Genitourinary Intracranial Mean duration of disease (month; range) Splenectomy status (n, %) Yes No Immunosuppressant (n, %) Yes No

40 (25–48)

39 (26–50)

NS

8 (42) 11 (58)

7 (44) 9 (56)

NS NS

34 ± 13 106 ± 29 104 ± 20

35 ± 12 36 ± 15 35 ± 18

NS b0.05 b0.05 NS

10 (53) 4 (21) 11

9 (53) 4 (24) 30

b0.05

5 (45) 4 (37) 2 (18) 0 (0) 26 (12–120)

15 (50) 10 (33) 5 (17) 0 (0) 24 (14–118)

NS

2 (11) 17 (89)

1 (7) 15 (93)

NS

9 (47) 10 (53)

10 (60) 6 (40)

NS NS

The following monoclonal antibodies used were from Beckman Coulter (Fullerton, CA): anti-CD4 conjugated to peridin chlorophyll protein (PerCP); anti-CD127 conjugated to phycoerythrin (PE); anti-CD25 conjugated to fluorescein isothiocyanate (FITC); anti-IL-4 FITC; anti-IFN-γ PE; isotype-matched, directly conjugated (FITC, PE and PerCP) control antibodies. Anti-IL-17 PE and related control antibody were purchased from BD Bioscience (San Diego, CA, USA). 2.4. Th1/Th2/Th17/Treg detection Peripheral blood monocytes (PBMCs) in RPMI 1640 were stimulated with 25 ng/ml phorbol 12-myristate 13-acetate, 1 μg/ml ionomycin, and 10 μg/ml Brefeldin A (Sigma, St Louis, MO, USA) at 37 °C for 4 h. After incubation, PBMCs were washed with PBS twice. Then, the cells were stained with anti-CD4 at room temperature (RT) in dark for 15 min. After treatment with permeabilizing solution, cells were stained with anti-IL-17, anti-IL-4 and anti-IFN-γ at RT for 45 min. Moreover, in some settings of experiments, the cells were also stained with anti-CD25 and anti-CD127 at RT for 45 min without permeabilizing treatment. For each tube, at least 10,000 events were collected in a gate created around the viable lymphocyte population. Quadrants were applied to the isotype control dot plots to exclude nonspecific staining. The percentage of CD4+ IL-17+(Th17), CD4+ IL-4+(Th2), CD4+ IFN-γ+(Th1) and CD4+ CD25highCD127intensity/dim (Treg) cell were determined.

NS: not significant.

2.5. Blood sampling and inflammatory cytokine detection with prednisone (10 mg, twice daily; Sinepharm Co. Ltd., Shanghai, China) was administered after discontinuation of previous treatment. The mean treatment duration was 3 months (range: 2–6 months). Peripheral blood were collected from all the patients prior to and 1 month after the treatment.

Peripheral blood was collected from median cubital vein into vaccutainer tubes containing ethylenediaminetetraacetic acid (EDTA)anticoagulant. Serum was obtained by 3000 rpm centrifugation for 10 min and was stored in − 80 °C before further processing.

Fig. 1. Representative dot-plots of regulatory T cell (Treg) cells as evaluated by flow cytometry. Samples from the same patient, in this case a responder, were analyzed before and after all-transretinoic acid (ATRA) therapy. The sample of a control subject was employed for comparison. CD4+ cells were sorted into Treg cells according to the expression of both CD25 and CD127.

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Fig. 2. Flow cytometry analysis of T cell subset in responder. CD4+ cells were sorted into Th1, Th2, Treg and Th17 subsets according to the expression of IFN-γ, IL-4, CD127 and CD25, and IL17. The percentage change of Th1, Th2, Treg and Th17 subsets in ITP patients compared to control were illustrated before ATRA treatment. *p b 0.05.

Fig. 3. Flow cytometry analysis of T cell subset at pre- and post-treatment. CD4+ cells were sorted into Th1, Th2, Treg and Th17 subsets according to the expression of IFN-γ, IL-4, CD127 and CD25, and IL-17. The percentage change of Th1, Th2, Treg and Th17 subsets before and after all-trans-retinoic acid (ATRA) treatment was illustrated. *p b 0.05.

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Fig. 4. The levels of Inflammatory cytokines in responders. Serum samples were collected from patients and the level of inflammatory cytokines were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA). Decreased level of IL-10 and TGF-β were found in ITP patients compared to control before ATRA treatment. *p b 0.05.

Inflammatory cytokines including IFN-γ, IL-4, IL-17, IL-10 and TGF-β, were measured by enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, Grand Island, NY, USA) according to manufacturer's instruction.

gene was assessed simultaneously in all samples as an internal control. Relative gene expression was determined by the 2− ΔΔCT method. Oligonucleotide primers specific for T-bet, GATA-3, Foxp3 and RORγ and GAPDH are listed in Table 1.

2.6. Reverse-transcriptase chain reaction (RT-PCR) 2.7. Statistical analysis Two milliliter peripheral blood was collected from all these ITP patients. Total cellular RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RT-PCR was carried out using a One Step SYBR® PrimeScript™ RT-PCR kit (Takara, Dalian, China) and an iQ5 Real-time PCR Detection system (Bio-Rad, Hercules, CA, USA). Expression of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

All the statistical analyses were performed with SPSS version 17.0 (SPSS Inc. Chicago, IL). Continuous variables and category variables were expressed as mean ± SEM and frequencies, respectively. Student t-test and one way ANOVA test was employed for 2 group and multiple group comparison. p b 0.05 was considered as statistically significant.

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Fig. 5. The levels of Inflammatory cytokines in responders at pre- and post-treatment. Serum samples were collected from patients and the level of inflammatory cytokines were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA). Increased level of IL-10 and TGF-β were found in the responder group after all-trans-retinoic acid (ATRA) treatment. *p b 0.05.

3. Results

3.3. Increased percentage of Treg cells were present in ITP responders after ATRA treatment

3.1. Characteristic of patients The demographic data of 35 chronic ITP patients with ATRA treatment was shown in Table 2. During the treatment, 5 patients were presented with dry mouth symptoms, 2 patients with symptoms of dry skin, and 3 with severe headaches, which were improved after symptomatic treatment.

3.2. Response rate of chronic ITP patients to ATRA Among them, nineteen (54.3%) achieved an overall response defined as platelet count above 50 × 109/L or doubling of baseline, and 10 (28.6%) complete response defined as platelet count above 100 × 109/ L. Among the 19 responders, the mean platelet count was (34 ± 13) × 109/L before ATRA therapy and (106 ± 29) × 109/L after treatment. The mean follow-up time was 14 ± 7 months (range: 3 to 20 months). Relapse was only presented in 2 patients, while other seventeen patients (49%) remained in remission at the last contact.

Before ATRA treatment, we did not observe a significant difference on Th1, Th2 and Th17 cell between chronic ITP patients and control subjects. However, significantly decreased percentage of Treg cells were found in chronic ITP patients compared with control subjects (6.4% ± 1.6% vs. 8.1% ± 1.5%, p b 0.05) (Figs. 1 and 2). ATRA treatment could significantly increase percentage of Treg cells in ITP responders (6.1% ± 1.6% vs. 7.6% ± 2.1%, p b 0.05), while no significant difference was found on percentage of Treg cell in ITP non-responders at preand post-treatment (Fig. 3).

3.4. Increased IL-10 level were present in ITP responders after ATRA treatment Before ATRA treatment, we did not observe a significant difference on the level of IFN-γ, IL-4, IL-17 and TGF-β between chronic ITP patients and control subjects. However, significantly decreased level of IL-10 was found in chronic ITP patients compared with control subjects (vs. p b 0.05) (Fig. 4). ATRA treatment could significantly increase IL-10

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Fig. 6. Expression of T cell subsets transcription factors in responders. T cell expressed transcription factor T-bet, GATA-3, RORγ and Foxp3, representing Th1, Th2, Th17 and Treg. Significantly decreased level of Foxp3 was found in responder compared to control before ATRA treatment. *p b 0.05.

Fig. 7. Expression of T cell subsets transcription factors at pre- and post-treatment. T cell expressed transcription factor T-bet, GATA-3, RORγ and Foxp3, representing Th1, Th2, Th17 and Treg. Significantly increased level of Foxp3 was found in responder after all-trans-retinoic acid (ATRA) treatment. *p b 0.05.

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level in ITP responders (vs. p b 0.05), while no significant difference was found on IL-10 level in ITP non-responders at pre- and post-treatment (Fig. 5). 3.5. Expression of T cell subset transcription factors Since the significant change of T cell subset percentage between ITP patients and control subjects, we further examined the related T cell subset transcription factors. We observed a significantly decreased level of Foxp3 between ITP patients and control subjects while no significant difference was found on GATA-3, ROR-γ, and T-bet (Fig. 6). Furthermore, a significantly increased level of Foxp3 was found between pre- and post-treatment in ITP responders while no significant difference was found in non-responders (Fig. 7). 4. Discussion Several previous studies have shown that ATRA exerts regulatory effects on immune homeostasis and has been used to control and modulate multiple autoimmune diseases, including inflammatory bowel disease, rheumatoid arthritis, type I diabetes and experimental encephalomyelitis [13–16]. However, its effect on chronic ITP is rarely reported. This study involved 35 patients with chronic ITP who failed with prior immunosuppressant drugs and/or splenectomy and received ATRA treatment, of whom 10 (28.6%) and 19 patients (54.3%) achieved complete response and overall response, respectively. Furthermore, we also found a significant decreased level of Treg cells, IL-10 and Foxp3 expression in ITP patients compared to the control subjects, whereas ATRA therapy could significantly increase the percentage of Treg cell, IL-10 level and Foxp3 expression. To the best of our knowledge, our results demonstrated for the first time that ATRA therapy could induce significant changes of Treg cells to induce response in patients with chronic ITP. CD4+ T cells are crucial in achieving a regulated effective immune response to pathogens. Naive CD4+ T cells are activated after interaction with antigen-MHC complex and differentiate into specific subtypes depending mainly on the pattern of cytokine secretion and expression of specific transcription factors [17]. Four major lineages could be differentiated from naïve CD4+ T cells, including Th1, Th2, Th17 and Treg cells [18]. Besides the regulatory effect on CD4+ T cell differentiation [19,20], ATRA could also promote the development and function of Treg cells [21,22]. In present study, we found a decreased level of Treg in chronic ITP patients compared to the healthy control subjects. Moreover, a decreased expression of Foxp3 was also detected in ITP patients. ATRA therapy could elevate the number of Treg cells in these responders, but not in the non-responders. In addition, we did not observe a significant change on the number of Th1, Th2, Th17, their related cytokines as well as related transcription factors after ATRA treatment. These results suggested that Treg development might be the major response cells involved in treatment effect exerted by ATRA, which was consistent with previous studies [23–26]. Furthermore, we also investigate the possible mechanisms involved in the development of Treg cells. Previous study by Van et al. has shown that induced expression of TGF-β and Foxp3 were involved in the effects of ATRA [27]. We therefore examined the serum TGF-β level and peripheral cell Foxp3 expression. We found that a remarkably reduced level of TGF-β and Foxp3 before ATRA treatment, and ATRA treatment could restore the level of TGF-β and Foxp3 in these responders, but not nonresponders. These results suggested that ATRA could induce the Treg facilitated microenvironment by upregulation of TGF-β level. Besides the use of ATRA, low dose of prednisone was also applied in the ITP patients to prevent bleeding episodes. Since the different action mechanisms exerted by ATRA and prednisone, they could be combined used to achieve a synergistic effect. Advancements have been made in the management of chronic ITP during recent years, and immunosuppressant agents were the most

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common drugs used in ITP treatment. However, only a relative small proportion of patients could response to these drugs. ATRA is considered as immunomodulatory agents which exert not only inhibitory effects. Moreover, our results also suggested that development of Tregbase therapy might be useful in ITP treatment. In addition, it should be noted that nearly half of patients exhibited no response to ATRA therapy, which might require further exploration of the no response mechanisms. 5. Conclusions In conclusion, our study shows for the first time that ATRA therapy can induce significant changes of Treg cells to induce response in patients with chronic ITP. The preliminary results of our study suggested that ATRA might be served as a novel therapeutic agent in chronic ITP patients who failed with conventional treatment. In addition, further investigation of these changes will be necessary in order to fully understand the effects of ATRA therapy on the immune system both in ITP and other autoimmune disorders. Conflicts of interest None. Acknowledgements None. References [1] R. McMillan, C. Durette, Long-term outcomes in adults with chronic ITP after splenectomy failure, Blood 104 (2004) 956–960. [2] J.E. Portielje, R.G. Westendorp, H.C. Kluin-Nelemans, A. Brand, Morbidity and mortality in adults with idiopathic thrombocytopenic purpura, Blood 97 (2001) 2549–2554. [3] J.B. Bussel, D. Provan, T. Shamsi, G. Cheng, B. Psaila, L. Kovaleva, et al., Effect of eltrombopag on platelet counts and bleeding during treatment of chronic idiopathic thrombocytopenic purpura: a randomised, double-blind, placebo-controlled trial, Lancet 373 (2009) 641–648. [4] D.J. Kuter, J.B. Bussel, R.M. Lyons, V. Pullarkat, T.B. Gernsheimer, F.M. Senecal, et al., Efficacy of romiplostim in patients with chronic immune thrombocytopenic purpura: a double-blind randomised controlled trial, Lancet 371 (2008) 395–403. [5] S.K. Vesely, J.J. Perdue, M.A. Rizvi, D.R. Terrell, J.N. George, Management of adult patients with persistent idiopathic thrombocytopenic purpura following splenectomy: a systematic review, Ann. Intern. Med. 140 (2004) 112–120. [6] Z.M. Liu, K.P. Wang, J. Ma, Zheng S. Guo, The role of all-trans retinoic acid in the biology of Foxp3+ regulatory T cells, Cell. Mol. Immunol. 12 (2015) 553–557. [7] T.R. Breitman, S.J. Collins, B.R. Keene, Terminal differentiation of human promyelocytic leukemic cells in primary culture in response to retinoic acid, Blood 57 (1981) 1000–1004. [8] M.E. Huang, Y.C. Ye, S.R. Chen, J.C. Zhao, L.J. Gu, J.R. Cai, et al., All-trans retinoic acid with or without low dose cytosine arabinoside in acute promyelocytic leukemia. Report of 6 cases, Chin. Med. J. 100 (1987) 949–953. [9] M.E. Huang, Y.C. Ye, S.R. Chen, J.R. Chai, J.X. Lu, L. Zhoa, et al., Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia, Blood 72 (1988) 567–572. [10] J.A. Hill, J.A. Hall, C.M. Sun, Q. Cai, N. Ghyselinck, P. Chambon, et al., Retinoic acid enhances Foxp3 induction indirectly by relieving inhibition from CD4+ CD44hi cells, Immunity 29 (2008) 758–770. [11] D. Mucida, K. Pino-Lagos, G. Kim, E. Nowak, M.J. Benson, M. Kronenberg, et al., Retinoic acid can directly promote TGF-beta-mediated Foxp3(+) Treg cell conversion of naive T cells, Immunity 30 (2009) 471–472 (author reply 2–3). [12] C. Neunert, W. Lim, M. Crowther, A. Cohen, L. Solberg Jr., M.A. Crowther, The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia, Blood 117 (2011) 4190–4207. [13] M. Osanai, N. Nishikiori, M. Murata, H. Chiba, T. Kojima, N. Sawada, Cellular retinoic acid bioavailability determines epithelial integrity: role of retinoic acid receptor alpha agonists in colitis, Mol. Pharmacol. 71 (2007) 250–258. [14] N. Miyagawa, T. Homma, H. Kagechika, K. Shudo, H. Nagai, Effect of synthetic retinoid, TAC-101, on experimental autoimmune disease, Pharmacology 67 (2003) 21–31. [15] S.J. Zunino, D.H. Storms, C.B. Stephensen, Diets rich in polyphenols and vitamin A inhibit the development of type I autoimmune diabetes in nonobese diabetic mice, J. Nutr. 137 (2007) 1216–1221. [16] M.K. Racke, D. Burnett, S.H. Pak, P.S. Albert, B. Cannella, C.S. Raine, et al., Retinoid treatment of experimental allergic encephalomyelitis. IL-4 production correlates with improved disease course, J. Immunol. 154 (1995) 450–458.

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