Rosiglitazone prevents graft-versus-host disease (GVHD)

Transplant Immunology 27 (2012) 128–137

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Rosiglitazone prevents graft-versus-host disease (GVHD) Eun-Kee Song a, b, c, d, e, Jun-Mo Yim a, d, e, Joo-Yun Yim a, d, e, Min-Young Song a, b, d, e, Hye-Won Rho a, d, e, Sung Kyun Yim a, b, So Yeon Jeon a, b, Hee Sun Kim f, Ho-Young Yhim a, d, Na-Ri Lee a, b, c, d, e, Jae-Yong Kwak a, b, c, d, e, Myung-Hee Sohn b, c, d, g, Ho Sung Park b, c, d, h, Kyu Yun Jang b, c, d, h, Chang-Yeol Yim a, b, c, d, e,⁎ a

Department of Internal Medicine, Chonbuk National University Medical School, Jeonju, Jeonbuk 561‐180, Republic of Korea Department of Medical Science, Chonbuk National University Graduate School, Jeonju, Jeonbuk 561‐180, Republic of Korea c Institute for Medical Sciences, Chonbuk National University, Jeonju, Jeonbuk 561‐180, Republic of Korea d Research Institute of Clinical Medicine, Chonbuk National University Hospital, Jeonju, Jeonbuk 561‐712, Republic of Korea e Advanced Research Center for Cancer, Chonbuk National University Hospital, Jeonju, Jeonbuk 561‐712, Republic of Korea f Department of Nursing, Woosuk University, Wanju, Jeonbuk 565‐701, Republic of Korea g Department of Nuclear Medicine, Chonbuk National University Medical School, Jeonju, Jeonbuk 561‐180, Republic of Korea h Department of Pathology, Chonbuk National University Medical School, Jeonju, Jeonbuk 561‐180, Republic of Korea b

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Article history: Received 4 May 2012 Received in revised form 5 September 2012 Accepted 6 September 2012 Keywords: GVHD PPARγ PTEN Rosiglitazone T cell

a b s t r a c t The effect of rosiglitazone, an agonist of peroxisome proliferator-activated receptor-γ (PPARγ), was investigated in a mouse parent-to-F1 GVHD model. Rosiglitazone inhibited mixed lymphocyte reactions, inducing enhanced apoptosis in CD4+, CD8+, and B220 + cells, but not in NK1.1+, Mac-1+, CD4 +/CD25 + and CD3 +/NK1.1 + cells. Rosiglitazone administration prevented GVHD in the liver, skin, spleen and intestine. Rosiglitazone inhibited GVHD-induced increases in serum levels of tumor necrosis factor-alpha, interferongamma, interleukin (IL)-6, and IL-12, and the GVHD-induced decreases in transforming growth factor-beta and IL-10. Immunophenotyping of splenic leukocytes demonstrated that while rosiglitazone treatment increased the population percentages of both donor and host CD4+/CD25 + and CD3 +/NK1.1 + cells, the treatment resulted in lower fractions of both donor and host CD8+ cells. Rosiglitazone inhibited the GVHD-induced decreases in the expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN), as well as the GVHD-induced increase in the splenic p-Akt and nuclear factor-kappa B expression. These results indicate that rosiglitazone and PPARγ activation may be useful in protecting the host from GVHD. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is an important treatment modality in the management of a number of malignant and nonmalignant hematopoietic disorders [1]. Unfortunately, the utility of HSCT is limited by transplant-related complications, including graft-versus-host disease (GVHD) [1]. Currently available posttransplantation immunosuppressive therapy is insufficient and GVHD remains a major cause of morbidity and mortality in allogeneic HSCT [1]. The pathophysiology of GVHD is believed to be a multistep process. The initial step involves the development of an inflammatory milieu resulting from damage to host tissues induced by preparative chemotherapy or radiotherapy. Damaged tissues secrete inflammatory ⁎ Corresponding author at: Department of Internal Medicine, Chonbuk National University Medical School, San 2‐20 Geumam-dong, Deokjin-gu, Jeonju, Jeonbuk 561‐180, Republic of Korea. Tel.: +82 63 250 1682; fax: +82 63 254 1609. E-mail address: [email protected] (C.-Y. Yim). 0966-3274/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.trim.2012.09.001

cytokines, including tumor necrosis factor-alpha (TNFα), interferongamma (IFNγ), and interleukin-1 (IL-1) [2–4]. In the second step, antigen-presenting cells from both the recipient and the donor, in concert with inflammatory cytokines, trigger the activation of donorderived T cells, which expand and differentiate into effector T cells [4]. In the third step, activated donor T cells mediate cytotoxicity targeted towards host tissues through Fas–Fas ligand interactions [5,6], perforin-granzyme B [4], and the production of cytokines such as TNFα [6,7]. GVHD predominantly affects the skin, upper and lower gastrointestinal tracts, liver, and occasionally the eyes and oral mucosa [1]. The treatment and prevention of GVHD is based on disruption of this multistep pathophysiologic phenomenon. GVHD has been usually treated with corticosteroid, cyclosporine, tacrolimus, mycophenolate mofetil, antithymocyte globulin and modulating agents of TNFα or IL-2. However, many patients with GVHD remain refractory to these agents and are associated with increased mortality. Thus the exploration of alternative approaches for GVHD treatment is needed. Peroxisome proliferator-activated receptors (PPARs) are transducer proteins that belong to the nuclear receptor superfamily and regulate

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gene expressions in response to ligand binding [8]. Three PPAR isotypes exist: α, β (or δ), and γ. PPARγ is distinct from the other two isotypes in that it is expressed principally in adipose tissue and is an important determinant of adipocyte differentiation and insulin sensitivity [9,10]. PPARγ agonists such as pioglitazone, troglitazone, and rosiglitazone have therefore been used clinically as insulin-sensitizing agents. PPARγ is also reportedly expressed in inflammatory cells, such as macrophages and certain T-cell subtypes [11–13], and PPARγ agonists have been shown to inhibit T-cell proliferative responses [13]. Additionally, PPARγ activation induces various cell types to down-regulate synthesis and release of immunomodulatory cytokines [11,14,15]. PPARγ has been shown to regulate phosphatidylinositol 3-kinase (PI3K) signaling by modulating phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression in inflammatory cells [16]. Recently, studies demonstrate that PPARγ agonists up-regulate PTEN expression, and reduce inflammation and airway hyperresponsiveness in a murine model of allergic asthma [17,18]. The synthetic PPARγ agonist rosiglitazone has been demonstrated to be clinically safe with effective immunomodulating activity [10,12]. These properties led us to investigate the effect of this agent in mouse GVHD. The present study was designed to assess the effect of rosiglitazone on the GVHD process in unirradiated F1 mice that had received parental splenocytes. Since the spleen is a site of hematopoiesis in the mouse, hematopoietic precursors will also be transferred when splenocytes are used as the inoculum of donor cells. When housed in specific pathogen-free conditions, the resulting GVHD mice undergo a nonlethal GVHD course. Various parameters of immune functions were compared in GVHD mice that did and did not receive rosiglitazone therapy. 2. Materials and methods 2.1. Animals Specific pathogen-free C57BL/6J and (C57BL/6J × BALB/c) F1 mice (6–8 weeks-of-age) were obtained from Damul Science Animal Center (Daejon, Korea) and housed at the Chonbuk National University Hospital Animal Care Facility (Jeonbuk, Korea). The mice were maintained under guidelines established by the Chonbuk National University Hospital Animal Care Committee, which also approved all experimental protocols. The mice were age and sex matched at the beginning of each experiment. The experiments that yielded the data presented here were conducted at least three times with highly concordant results. 2.2. Culture conditions Cells were cultured at 37 °C in a humidified 95% air/5% CO2 atmosphere in RPMI 1640 medium supplemented with 5% FBS (HyClone Laboratories, Inc., Logan, UT), 100 U/ml penicillin G (Sigma Chemical Co., St. Louis, MO), 50 μg/ml streptomycin (Sigma), and 2 mM glutamine (Sigma) (working medium) or in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin G, 50 μg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate (Sigma), and 0.1 mM nonessential amino acids (Sigma) (complete medium). Rosiglitazone was added to the cultures when needed at indicated concentrations. Dimethyl sulfoxide (Sigma) was used as a vehicle to dissolve rosiglitazone. Control experiments demonstrated that treating cells with the same concentrations of dimethyl sulfoxide alone had no effect on the experimental results of the present study (data not shown). Mycoplasmal infection was excluded by surveillance cultures of media as previously described [19]. All reagents and media for tissue culture experiments were tested by Limulus amoebocyte lysate assay (detection limit 10 pg/ml; Whittaker Bioproducts, Walkersville, MD) to exclude lipopolysaccharide contamination.

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2.3. Mixed lymphocyte reaction (MLR) and culture of control splenocytes Splenocytes were obtained using gentle teasing of spleens as previously described [20]. MLRs were performed with responder splenocytes from C57BL/6J (H-2b) mice and mitomycin-C-inactivated stimulator splenocytes from BALB/c (H-2d) mice. A total of 5 × 10 5 responder cells and an equal number of stimulator cells were cocultured in complete medium in 96-well microtiter plates at 37 °C in a 5% CO2 incubator for 4 days. Rosiglitazone was added to each well at various concentrations (0–200 μM) upon culture initiation. After 3 days, the cells were pulsed with 0.5 μCi tritiated thymidine ([3H]TdR, 2.0 Ci/mmol, DuPont, Boston, MA) per well and cultured for an additional 18 h. The cells were harvested onto glass fiber filters using a cell harvester (Cambridge Technology, Cambridge, MA). The samples were suspended in Opti‐Fluor® scintillation fluid (Packard Instrument Co., Downers Grove, IL) and a scintillation counter (TRI-CARB 2900TR, Packard) was used to measure the incorporated [ 3H]TdR. Each assay was performed in triplicate and the results are presented as mean±standard deviation (SD) cpm. The cells were harvested from a 4 day-cultured MLR without tritiated thymidine for evaluation of cytolytic activity, immunophenotypic changes, and apoptosis of the responding cells. Control F1 splenocytes were cultured at 2 × 106 cells/ml in 75 cm2 tissue culture flasks in the presence or absence of 50 μM rosiglitazone. Following a 4 day culture, the cells were harvested to evaluate cell recovery, immunophenotypic changes, and apoptosis. 2.4. Induction of GVHD, rosiglitazone administration, and body weight measurement GVHD was induced by transferring splenocytes (1×108 cells in 0.5 ml phosphate buffered saline [PBS]/mouse) isolated from C57BL/6J mice (H-2b) into the tail vein of normal (C57BL/6J×BALB/c) F1 mice (H-2b/d). Rosiglitazone (1 mg/kg, 5 mg/kg, or 10 mg/kg; GlaxoSmithKline Pharmaceuticals, Seoul, Korea) dissolved in distilled water was administered via oral gavage once a day for 15 days following GVHD induction. Distilled water was administered to the untreated GVHD group. Untreated and rosiglitazone alone-treated normal (non-GVHD) F1 mice served as controls. The weights of mice were determined at weekly intervals from the day of donor splenocyte injection. 2.5. Cytotoxicity assay A previously described 4-h 51Cr release assay [21] was used to evaluate the cytolytic activity of donor effector cells (H-2 b) harvested from an MLR targeted towards Meth-A tumor cell (H-2 d) targets. After effector-target cells were co-cultured for 4 h at 37 °C in a 5% CO2 incubator, a 0.1-ml aliquot of cell-free supernatant was removed from each well and a gamma counter was used to quantify the amount of 4-h 51Cr released into the medium (Packard, Downers Grove, IL). Maximum release was assessed by solubilizing an identical aliquot of target cells with 0.5% Triton X-100 (Bio-Rad Laboratories, Richmond, CA). Spontaneous release was assessed in wells containing target cells alone as a control. Cytotoxic activity was expressed as percentage of lysis, which was calculated according to the following formula: Cytotoxicityð% Þ ¼ 100  ðexperimental release−spontaneous releaseÞ =ðmaximum release−spontaneous releaseÞ: Each assay was performed in triplicate and data are presented as mean ± SD at each effector-to-target cell ratio. 2.6. Direct immunofluorescent staining and flow cytometry The following monoclonal antibodies (BD Biosciences, San Diego, CA) were used for flow cytometric analyses: anti-CD4-allophycocyanin (APC), anti-CD8-fluorescein isothiocyanate (FITC), anti-B220-phycoerythrin

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(PE), anti-CD25-PE, anti-Mac-1-FITC, anti-CD3-APC, anti-CD-H-2 d-FITC, anti-CD-H-2 d-PE, anti-CD-H-2b-FITC, and anti-CD-H-2b-PE. Cells were stained as previously described [22]. All cell sorts and analyses were performed on a FACS machine (FACS Calibur E6181, BD Biosciences).

2.7. Detection of apoptosis in immunophenotypic subsets of cells by annexin-V staining Cells were harvested from MLR cultures and stained with fluorophore (PE or APC)-conjugated antibodies specific for CD4, CD8, B220, NK1.1, Mac-1, CD3, or CD25, followed by washing in PBS with 1% bovine serum albumin. The cells were washed in binding buffer and stained with annexin-V-FITC according to the manufacturer's recommendations (BD Biosciences, San Diego, CA). Multi-color flow cytometry was used to analyze triplicate samples (10 4 cells/sample). Data acquisition and analysis were performed with CellQuest software (BD Biosciences). Data are presented as a percentage of apoptotic cells (mean ± SD).

2.8. Cytokine measurements and isolation of blood leukocytes Fifteen days after GVHD induction, blood samples were harvested by retro-orbital punctures and centrifuged at 1500 × g for 10 min to isolate the serum. Sera were frozen and stored at − 7 °C until experimental use. A multiplexed flow cytometric assay (Cytometric Bead Array [CBA]; Mouse Inflammation Kit, BD Biosciences, San Diego, CA) was performed according to the manufacturer's recommendations to measure TNFα, IFNγ, IL-6, and IL-12 levels. Following acquisition of sample data using flow cytometric analysis, BD CBA Analysis Software was used to generate results in tabular format. Results are presented as mean ± SD of triplicate samples. In order to isolate blood leukocytes, harvested bloods were washed twice with working medium and briefly exposed to Tris-buffered 0.16 M ammonium chloride to lyse erythrocytes. After extensive washes, the cells were suspended in working medium.

2.9. Cytosolic and nuclear protein extractions for nuclear factor-kappa B (NF-κB) p65 analysis Cells were homogenized in 2 volumes of buffer A (50 mM Tris– HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 0.5 mM DTT, 5 mM MgCl2, and 1 mM PMSF) containing a protease inhibitor cocktail (Sigma). The homogenates were centrifuged at 1000 × g for 15 min at 4 °C. The supernatants were incubated on ice for 10 min and centrifuged at 100,000 × g for 1 h at 4 °C to obtain cytosolic protein extracts. The pellets were washed twice in buffer A, resuspended in buffer B (1.3 M sucrose, 1.0 mM MgCl2, and 10 mM potassium phosphate buffer, pH 6.8) and centrifuged at 1000 ×g for 15 min. The pellets were suspended in buffer B at a final sucrose concentration of 2.2 M and centrifuged at 100,000 ×g for 1 h. The resulting pellets were washed once with a solution containing 0.25 M sucrose, 0.5 mM MgCl2, and 20 mM Tris–HCl (pH 7.2) and centrifuged at 1000 × g for 10 min. The pellets were then solubilized with a solution containing 50 mM Tris–HCl (pH 7.2), 0.3 M sucrose, 150 mM NaCl, 2 mM EDTA, 20% glycerol, 2% Triton X-100, 2 mM PMSF, and a protease inhibitor cocktail (Sigma). The samples were kept on ice for 1 h with gentle stirring and centrifuged at 12,000 × g for 30 min. The resulting supernatant was considered to contain the soluble nuclear proteins. 2.10. Western blot analysis for PTEN, p-Akt and NF-κB p65 expression All reagents and chemicals for Western blot analysis were purchased from KDR Biotechnology (Seoul, Korea) or Sigma-Aldrich Korea (Seoul, Korea). Spleens were isolated on the 15th day after GVHD induction and homogenized in ice-cold lysis buffer (20 mM HEPES, pH 7.2, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin). After centrifugation, the supernatants were stored at −80 °C until experimental use. A bicinchoninic acid protein assay was used to measure protein concentration of each sample (Pierce, Rockford, IL). Protein concentrations of all samples were equalized. Proteins were separated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Immunobilon-P, Millipore, Billerica, MA), and

Fig. 1. Effect of rosiglitazone on responder cell proliferation, recovery, and cytotoxicity during mixed lymphocyte reaction (MLR). MLR responder splenocytes (C57BL/6J mice) and mitomycin-C-inactivated stimulator splenocytes (BALB/c mice) were cocultured with varying concentrations of rosiglitazone for 4 days. Proliferation rates are represented by [3H] TdR incorporation (A). Parallel cultures were evaluated for cell recovery using trypan blue exclusion (B) and cytotoxicity against Meth-A target cells via a 4-h 51Cr release assay (C). Results are presented as mean ± standard deviation of triplicate samples.

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incubated overnight at 4 °C with primary antibodies specific for PTEN (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-Akt (Cell Signaling Technology, Denver, CO, USA), NF-κB p65 (Santa Cruz Biotechnology, Santa Cruz, CA), or β-actin (BD Biosciences). Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (BD Biosciences). A chemiluminescence reagent (SUPEX, Pohang University of Science and Technology, Pohang, Korea) was used to detect bound antibody. Densitometric scanning (LAS-3000; Fujifilm, Tokyo, Japan) was used to analyze the enhanced chemiluminescence. Blots for β-actin confirmed equal protein loading in all experiments. 2.11. Histologic evaluation After the mice were killed with CO2, the liver, spleen, intestine, and abdominal skin were harvested on the 15th day after GVHD induction. Harvested organs were fixed in 4% paraformaldehyde and embedded in a paraffin block for histologic evaluation. Tissue sections (5 μm) were deparaffinized with xylene and rehydrated with graded ethanol. Sections were stained with hematoxylin and eosin. For evaluation of GVHD, tissue sections were screened by an examiner blinded to the treatment received by each animal. To facilitate semiquantitative assessment of GVHD, grading of GVHD was confined to analysis of the liver. Inflammatory cell infiltrates in the portal tract and around the central veins were graded on a scale of 1 to 5 reflecting Grade 1: normal or minimal perivascular cuffing; Grade 2: perivascular cuffing, 1 to 2 cells in thickness, involving up to 10% of vessels; Grade 3: perivascular cuffing, 1 to 4 cells in thickness, involving 10 to 30% of vessels; Grade 4: perivascular cuffing, 3 to 6 cells in thickness, involving 30% to 50% of vessels; Grade 5: perivascular cuffing, greater than 7 cells in thickness, involving greater than 50% of vessels. 2.12. Statistical analysis Statistical analysis was performed using Student's t-test. p b 0.05 was considered statistically significant. 3. Results 3.1. Effect of rosiglitazone on MLR responding cell proliferation, recovery and cytolytic activity To evaluate the effect of rosiglitazone on allogeneic responses in vitro, we performed MLRs in the presence of increasing concentrations (25–200 μM) of rosiglitazone. MLR cultures incubated without rosiglitazone served as controls. Rosiglitazone at concentrations ≥ 25 μM inhibited the tritiated thymidine incorporation of control MLR cultures in a dose-dependent manner (Fig. 1A). The number of cells recovered from MLR cultures decreased in the presence of rosiglitazone in a dosedependent manner (Fig. 1B). Rosiglitazone also inhibited the cytolytic activity of responding cells (H-2b) against Meth-A tumor cell (H-2d) targets in the MLR cultures in a dose-dependent manner (Fig. 1C). 3.2. Effect of rosiglitazone on immunophenotypic changes and apoptosis of MLR responding cells Immunophenotypes of the responding cells isolated from MLR cultures incubated in the presence of varying concentrations (25–200 μM) of rosiglitazone were evaluated by flow cytometry (Fig. 2A). MLR cultures incubated without rosiglitazone served as controls. When rosiglitazone was administered at 50 μM, 100 μM, or 200 μM, the fractions of CD4+, CD8+, and B220 + cells were significantly reduced. In contrast, rosiglitazone increased the fractions of Mac-1+, CD4+/CD25+, and CD3+/NK1.1+

Fig. 2. Effect of rosiglitazone on fractional changes and apoptosis of immunophenotypic subsets of responder cells during mixed lymphocyte reaction (MLR). MLR responder splenocytes (C57BL/6J mice) and mitomycin-C-inactivated stimulator splenocytes (BALB/c mice) were cocultured with varying concentrations of rosiglitazone. After a 4-day culture, changes in cell subsets were analyzed by direct immunofluorescence staining (A), and apoptosis of immunophenotypic subsets was evaluated by annexinV-FITC immunostaining using dual or triple labeling with phycoerythrin (PE)- and/or allophycocyanin (APC)-labeled monoclonal antibodies specific for cell-surface antigens (B). Samples were analyzed by flow cytometry. Results are presented as mean ± SD of triplicate samples. (* p b 0.05, ** p b 0.01, vs. control [rosiglitazone 0 μM]).

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cells at the same concentrations. Rosiglitazone had no effect on the percentage of NK1.1+ cell populations. These changes in population percentages indicate that rosiglitazone had variable effects on the phenotypic subsets. We postulated that rosiglitazone-induced apoptosis

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selectively occurred in CD4+, CD8+, and B220 + cells, but not in NK1.1+, Mac-1+, CD4+/CD25+, and CD3+/NK1.1+ cells. To test this possibility, responding cells harvested from MLR cultures incubated in the presence of increasing concentrations (25–200 μM) of rosiglitazone were stained with annexin V-FITC and PE- or APCconjugated antibodies to identify the cell type (Fig. 2B). MLR cultures incubated without rosiglitazone served as controls. The highest concentration of rosiglitazone (200 μM) resulted in significantly elevated fractions of annexin V+ cells in each immunophenotypic subset tested. In contrast, both 50 μM and 100 μM of rosiglitazone significantly increased the percentage of annexin V+ cells within CD4+, CD8+, and B220+ cell populations, but no significant increase was detectable among NK1.1+, Mac-1+, CD4+/CD25+, and CD3+/NK1.1+ cells at these lower concentrations. We also evaluated the effect of 50 μM rosiglitazone on the total cell recovery, fractional changes of immunophenotypic subsets, and their apoptosis in normal F1 splenocytes cultured for 4 days without MLR stimulation. No significant differences were observed in all the three evaluated parameters between the control and rosiglitazone-added cultures of splenocytes (Fig. 3A, B and C). 3.3. Effect of rosiglitazone on GVHD in target organs and body weight We then sought to evaluate whether the in vitro inhibitory effect of rosiglitazone on MLR could be translated to the in vivo environment. Specifically, we tested whether rosiglitazone administration improved GVHD in a mouse parent-to-F1 GVHD model. We first evaluated the dose-dependent therapeutic effect of rosiglitazone administration on GVHD severity. Rosiglitazone was administered to three treatment groups (rosiglitazone 1 mg/kg, rosiglitazone 5 mg/kg, and rosiglitazone 10 mg/kg) of mice (4 mice/group) through once-daily oral gavage, for 15 days after GVHD induction. Following 15 days of daily rosiglitazone treatment, GVHD-induced pathologic changes were evaluated microscopically in hematoxylin and eosin-stained liver tissue sections. Untreated and rosiglitazone alone-treated normal (non-GVHD) mice and PBS only-treated GVHD mice served as controls. Rosiglitazone administration improved GVHD in the liver in a dose-dependent trend (data not shown), resulting in maximum improvement when treated with 10 mg/kg rosiglitazone. We therefore chose 10 mg/kg rosiglitazone as the optimal therapeutic dose, and used only four groups [normal control group, rosiglitazone alone-treated normal control group, untreated GVHD group, and rosiglitazone (10 mg/kg)-treated GVHD group] in the following rosiglitazone therapy experiments. The principal target organs of GVHD are the liver, skin, immune system, and intestine [23,24]. We therefore performed histologic evaluations to determine GVHD severity in the liver, skin, spleen, and intestine after 15 days of rosiglitazone (10 mg/kg) therapy (Fig. 4). The liver of normal control mice exhibited no significant pathologic findings. Only a few lymphocytes were detectable in the portal tract. In the untreated GVHD mice, a significant increase in lymphocyte infiltration was evident in the portal tract and around the central veins. In contrast, rosiglitazone treatment resulted in a significant decrease in GVHD-induced lymphocyte infiltration of the liver. We also assessed the effect of rosiglitazone on the GVHD-induced perivascular infiltration of inflammatory cells in

the liver using a semiquantitative scoring system. As shown in Fig. 5A, rosiglitazone treatment significantly improved the GVHD-induced perivascular infiltration of inflammatory cells. Similarly, the microscopic findings of the skin demonstrated a significant increase in perivascular and interstitial lymphocyte infiltration in the untreated GVHD mice compared with the normal control mice. Rosiglitazone therapy caused a decrease in GVHD-induced lymphocyte infiltration of the skin. Microscopic examination of the spleen demonstrated normal white and red pulp regions in the normal control mice. But, in the spleen of the untreated GVHD mice, most of the lymphoid follicles of the white pulp had been replaced with granulomas without necrosis. The red pulp revealed an increase in reticulocytes and megakaryocytes, suggesting increased extramedullary hematopoiesis. Rosiglitazone therapy significantly inhibited GVHD-induced changes, including granuloma formation in the white pulp and the increased extramedullary hematopoiesis in the red pulp. Induction of GVHD also resulted in increased lymphocyte infiltration in the intestine, which was inhibited by rosiglitazone administration. Rosiglitazone alone treatment resulted in no significant histologic changes in the above examined organs of normal control mice. The weights of both control F1 and GVHD mice were determined at weekly intervals after initiation of rosiglitazone therapy. Body weight was not altered in normal F1 control mice by administration of rosiglitazone over a five-week time period (Fig. 5B). Although GVHD mice revealed a trend of weighing less than the normal F1 mice, no significant differences in body weight of GVHD mice that did or did not receive rosiglitazone therapy were noted (Fig. 5C). 3.4. Effect of rosiglitazone on splenic and blood leukocyte subset composition in GVHD mice We then examined whether in vitro rosiglitazone-induced cell population changes in MLR cultures were consistent with the in vivo findings in GVHD mice receiving rosiglitazone therapy. On the 15th day following onset of rosiglitazone treatment, splenocytes and blood leukocytes isolated from the three groups of mice (normal control, untreated GVHD, and 10 mg/kg rosiglitazone-treated GVHD mice) were analyzed to determine the proportion of donor-versus-host phenotypes of lymphocytes and macrophages (Fig. 6). As expected, GVHD induction resulted in expansion of all donor cell types/subtypes analyzed in both splenic and blood leukocytes. In the analysis of GVHD splenocytes (Fig 6A), as compared with normal splenocytes, the fractions of host CD8+, NK1.1+, Mac-1+, and CD3+/NK1.1 + cell populations expanded, but the fractions of host CD4+, B220+, and CD4+/CD25+ cell populations decreased. While the fractions of donor CD4+, NK1.1+, B220+, and Mac-1+ cells remained unchanged in GVHD mice following rosiglitazone treatment, those of donor CD4+/CD25 + and CD3+/NK1.1+ cells were significantly increased. Additionally, rosiglitazone treatment induced a significant decrease in both donor and host CD8+ cells. In contrast, treatment with this agent resulted in larger fractions of host CD4+/CD25+ and CD3+/NK1.1+ cells. The fractions of host CD4+, NK1.1+, B220+, and Mac-1+ cells were not statistically different between untreated and rosiglitazone-treated GVHD mice. The total cell number and donor cell number

Fig. 3. Effect of rosiglitazone on cell recovery, immunophenotypic composition and apoptosis of cultured splenocytes. Splenocytes of (C57BL/6J × BALB/c) F1 mice were cultured in the presence or absence of 50 μM rosiglitazone. After a 4-day culture, cell recovery was measured using trypan blue exclusion (A). Changes in cell subsets were analyzed by direct immunofluorescence staining (B), and apoptosis of immunophenotypic subsets was evaluated by annexin-V-FITC immunostaining using dual or triple labeling with phycoerythrin (PE)- and/or allophycocyanin (APC)-labeled monoclonal antibodies specific for cell-surface antigens (C). Samples were analyzed by flow cytometry. Results are presented as mean ± SD of triplicate samples.

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Fig. 4. Effect of rosiglitazone on graft-versus-host disease (GVHD) severity in target organs. Parental (C57BL/6J mice) splenocytes were injected intravenously into (C57BL/6J×BALB/c) F1 mice. Rosiglitazone (10 mg/kg/day) treatment was begun on the day of the splenocyte injection. After a 15-day rosiglitazone treatment, tissues of target organs were harvested and stained with hematoxylin and eosin. Normal, rosiglitazone alone-treated non-GVHD, and untreated GVHD mice served as controls. Arrows on the intestine micrographs point to infiltrating inflammatory cells. Similar results were obtained in three other separate experiments. Original magnification, ×400 in the liver, skin and intestine, ×100 in the spleen.

recovered per spleen were measured in both untreated GVHD mice and rosiglitazonetreated GVHD mice. Rosiglitazone treatment had not affected the total cell number or donor cell number recovered per spleen, although wide range was observed (Fig. 6B and C). These results indicated that the fractional changes of the immunophenotypic subsets were not due simply to the fractional changes of other subsets. The pattern of fractional changes of blood leukocyte subsets was similar to that of splenic leukocytes except for the fractions of NK1.1+ and CD3+/NK1.1+ cells which were unaffected by rosiglitazone administration in GVHD mice (Fig. 6D). GVHD

induction also unaffected the fractions of blood NK1.1+ and CD3+/NK1.1 + cells in normal F1 mice. 3.5. Effect of rosiglitazone on the inflammatory cytokine levels in the serum Previous studies have demonstrated the important role of inflammatory cytokines in the development of GVHD [1–3,25]. We therefore measured serum levels of TNFα, IFNγ, IL-6, IL-12, TGFβ, and IL-10 in each experimental group [normal control, untreated

Fig. 5. Effect of rosiglitazone administration on pathologic grade of inflammatory cell infiltration in the liver, and body weight. Parental (C57BL/6J mice) splenocytes were injected intravenously into (C57BL/6J × BALB/c) F1 mice. Rosiglitazone (10 mg/kg/day) treatment was begun on the day of the splenocyte injection. After a 15-day rosiglitazone treatment, liver tissues were stained with hematoxylin and eosin, and evaluated for pathologic grade (A). Normal, rosiglitazone alone-treated non-GVHD, and untreated GVHD mice served as controls. Ten mice per group were analyzed. In parallel experiments, body weight was assessed weekly for 5 weeks (B; normal vs. rosiglitazone alone-treated non-GVHD), (C; GVHD vs. rosiglitazone-treated GVHD). Results are presented as mean±SD. (** pb 0.01); rosi, rosiglitazone.

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E.-K. Song et al. / Transplant Immunology 27 (2012) 128–137 GVHD, and rosiglitazone (10 mg/kg)-treated GVHD] on the 15th day after GVHD induction. As shown in Fig. 7, serum levels of TNFα, IFNγ, IL-6, and IL-12 were significantly increased in untreated GVHD mice compared to normal control mice, and these increases were effectively inhibited by treatment with rosiglitazone. In contrast, serum TGFβ and IL-10 levels were decreased in untreated GVHD mice compared to normal mice, and these decreases were also effectively restored by rosiglitazone administration. 3.6. Effect of rosiglitazone on splenic PTEN and P-Akt expression PPARγ has been shown to regulate phosphatidylinositol 3-kinase (PI3K) signaling by modulating phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression in inflammatory cells [26]. We thus performed Western blot analyses to assess PTEN and p-Akt expression in spleens harvested from GVHD mice after 15 days of rosiglitazone administration (Fig. 8). Spleens harvested from normal control and untreated GVHD mice served as controls. GVHD induction resulted in reduced PTEN expression in the normal spleen compared with the normal mice. Reduction in PTEN expression due to GVHD induction was significantly inhibited by rosiglitazone administration. In contrast, GVHD induction increased p-Akt levels and this increase was significantly inhibited by treatment with rosiglitazone. These results indicate that p-Akt expression in GVHD is correlated with PTEN levels. 3.7. Effect of rosiglitazone on splenic NF-κB p65 expression in GVHD mice Previous studies have demonstrated that p-Akt may enhance NF-κB activity, and have implicated NF-κB in the modulation of various immunologic responses [27]. Western blot analysis of NF-κB p65 was thus performed in the spleens harvested from rosiglitazone-treated GVHD mice. Normal and GVHD mice served as controls. GVHD mouse spleens had elevated levels of NF-κB p65 protein in nuclear protein extracts compared to those from the normal mice (Fig. 9A). Treating the mice with rosiglitazone significantly inhibited GVHD-induced increase in nuclear NF-κB p65. Consistent with rosiglitazone-mediated decrease in nuclear NF-κB p65, the levels of cytosolic NF-κB p65 protein were increased in GVHD mice as a result of rosiglitazone treatment (Fig. 9B).

4. Discussion

Fig. 6. Effect of rosiglitazone on graft-versus-host disease (GVHD)-induced immunophenotypic changes of splenic and blood leukocytes. Parental (C57BL/6J mice) splenocytes were injected intravenously into (C57BL/6J × BALB/c) F1 mice. Rosiglitazone (10 mg/kg/day) treatment was begun on the day of the splenocyte injection. After a 15-day rosiglitazone treatment regimen, splenic (A) and blood (D) leukocytes were stained for cell surface antigen expressions by direct immunofluorescence. Normal and untreated GVHD mice served as negative and positive controls, respectively. Results were expressed as mean ± standard deviation of triplicate samples. In parallel experiments, total cell number (B) and donor cell number (C) per spleen were measured in GVHD mice and rosiglitazone-treated GVHD mice (10 mice/group, mean ± standard deviation). (* p b 0.05, ** p b 0.01).

The data shown here demonstrate the novel finding that rosiglitazone, a synthetic PPARγ agonist, inhibits the proliferative response, viable cell recovery, and cytotoxic activity of MLR responding cells. Importantly, the in vitro results were consistent with in vivo data from a murine GVHD model in which rosiglitazone therapy improved signs of GVHD. Addition of rosiglitazone to the MLR cultures resulted in reduced cytolytic activity of the responding cells against Meth-A tumor cells, which present stimulator (host) MHC antigen. These results indicate that rosiglitazone inhibits alloimmunization during MLR and that this agent may be useful as a potential immunosuppressive agent in GVHD. In the cytotoxicity test, the percentage of lysis of target Meth-A cells was relatively low compared to what reported in previous studies employing different target cells [28]. The low target cell lysis may be due to the characteristic relative resistance of Meth-A cells to cytotoxic lymphocyte activity as previously reported [19]. GVHD is a set of immune responses mediated by interactions among a variety of effector cells including T cells, B cells, NK cells, macrophages, dendritic cells, and granulocytes. PPARγ is expressed in these immune cells and affects both cell function and survival [29]. In the present study, addition of rosiglitazone at concentrations of ≥50 μM to MLR cultures decreased the population fractions of CD4+, CD8+, and B220+ cells, but increased the fractions of Mac-1+ cells. Interestingly, CD4+/CD25+ and CD3+/NK1.1+ cell populations, which are known to have immunosuppressive activities during GVHD [30], were also increased following exposure to rosiglitazone at the same concentrations (≥50 μM). The number of cells recovered from MLR cultures decreased in a dose-dependent manner in the presence of rosiglitazone. The change in cell population percentage and decreased cell recovery suggest that rosiglitazone has variable effects on each phenotypic subset. We therefore tested whether CD4+, CD8+, and B220+ cells were more prone to rosiglitazone-induced apoptosis compared to Mac-1+, CD4+/CD25+, and CD3+/NK1.1+ cells. The results demonstrate that the highest concentration of rosiglitazone tested (200 μM) in the

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Fig. 7. Effect of rosiglitazone on serum levels of cytokines in graft-versus-host disease (GVHD) mice. Parental (C57BL/6J mice) splenocytes were injected intravenously into (C57BL/ 6J × BALB/c) F1 mice. Rosiglitazone (10 mg/kg/day) treatment was begun on the day of the splenocyte injection. After a 15-day rosiglitazone treatment regimen, cytokine levels in the serum were measured. Normal and untreated GVHD mice served as negative and positive controls, respectively. Results are expressed as mean ± standard deviation of triplicate samples. (* p b 0.01); rosi, rosiglitazone.

MLR cultures increased the percentage of annexin V+ cells in all cell types tested. This general increase in annexin V+ cells may be due to a toxic effect of rosiglitazone at this high concentration. At lower concentrations (50 μM and 100 μM), rosiglitazone significantly increased the proportion of annexin V+ cells among CD4+, CD8+, and B220+ cells, but not among NK1.1+, Mac-1+, CD4+/CD25+, or CD3+/ NK1.1+ cells. These results demonstrate that CD4+, CD8+, and

Fig. 8. Effect of rosiglitazone on graft-versus-host disease (GVHD)-mediated PTEN and p-Akt protein expression in the spleen. Parental (C57BL/6J mice) splenocytes were injected intravenously into (C57BL/6J×BALB/c) F1 mice. Rosiglitazone (10 mg/kg/day) treatment was begun on the day of the splenocyte injection. After a 15-day rosiglitazone treatment regimen, splenic tissues were harvested and analyzed for PTEN and p-Akt protein expressions by Western blotting. Normal and untreated GVHD mice served as negative and positive controls, respectively. Similar results were obtained in three other separate experiments.

Fig. 9. Effect of rosiglitazone on graft-versus-host disease (GVHD)-mediated NF-κB p65 protein expression in nuclear and cytosolic splenic protein extracts. Parental (C57BL/6J mice) splenocytes were intravenously injected into (C57BL/6J × BALB/c) F1 mice. Rosiglitazone (10 mg/kg/day) treatment was begun on the day of the splenocyte injection. After a 15-day rosiglitazone treatment regimen, splenic tissues were harvested and Western blotting analysis was conducted to assess expression of nuclear and cytosolic NF-κB protein. Normal and untreated GVHD mice served as negative and positive controls, respectively. Similar results were obtained in three other separate experiments.

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B220+ cells are indeed more prone to rosiglitazone-mediated apoptosis compared to Mac-1+, NK 1.1+, CD4+/CD25+, and CD3+/NK1.1+ cells. We then examined whether these in vitro rosiglitazone-induced changes in cell population fraction and numbers also occur in vivo. To address this objective, we analyzed the splenic leukocytes of GVHD mice during rosiglitazone therapy. While the proportions of both donor and host CD4+/CD25+ and CD3/NK1.1+ cells expanded in response to rosiglitazone treatment, both donor and host CD8+ cell populations decreased. Because CD4+/CD25+ and CD3+/NK1.1+ cells are known to suppress GVHD, and CD8 + cells are well-known promoters of GVHD [31–33], the increase in CD4+/CD25+ and CD3+/NK1.1+ cells and the decrease in CD8+ cells may mediate potential effector mechanisms for the rosiglitazone-induced improvement of GVHD. However, although the differences in cellular composition induced by rosiglitazone reached statistically significant values both in vitro and in vivo, they were relatively small. Further evaluation is needed to elucidate whether the rosiglitazone-induced immunophenotypic changes have relevance to the improvement of GVHD. The pattern of fractional changes of blood leukocyte subsets was similar to that of splenic leukocytes except for the fractions of NK1.1 + and CD3+/NK1.1+ cells which were unaffected by rosiglitazone administration in GVHD mice. In the present study, the percentage of donor cells engrafted in the spleen and blood seems relatively lower than what was previously proposed in the similar GVHD model [28]. The differences of experimental conditions including mice, antibodies, and environments may be the source of the changes. Rosiglitazone treatment improved the GVHD-mediated lesions of the liver, skin, spleen, and intestine. The spleen is an important immunologic and hematopoietic organ. In the GVHD model in the present study, granulomatous inflammation severely altered normal architecture of the spleen. Rosiglitazone treatment also significantly reduced inflammation, indicating that this agent may protect the immunologic and hematopoietic functions of the spleen from GVHD-induced injury. We examined the effect of rosiglitazone on GVHD at 15 days after the injection of donor splenocytes. The timing of this protocol was chosen on the basis of previous studies demonstrating that a peak of donor cell engraftment was observed between 2 and 3 weeks after donor splenocyte injection in the GVHD model [34,35]. Induction of GVHD also resulted in increased levels of serum IFNγ, TNFα, IL-6, and IL-12, but decreased levels of serum TGFβ and IL-10. In contrast, rosiglitazone treatment restored GVHD-induced effects on serum cytokine levels close to normal control levels. These results are consistent to a previous report showing that treatment with a PPARγ agonist was associated with reduced expression of Th1 cytokines and elevated expression of Th2 cytokines in a murine model of acute colitis [36]. Researchers have considered acute GVHD to be both Th1- and Th2-mediated on the basis of the predominance of cytotoxic T cell-mediated pathology and of the increased production of Th1 cytokines including IFNγ, TNFα, IL-12, and IL-2 [37]. However, several recent studies suggested that the influence of Th1 and Th2 cytokines in acute and chronic GVHD is not so simple and that timing, amount, and duration of secretion of a particular cytokine may determine the specific effects of that cytokine on GVHD severity [38]. A number of studies have demonstrated that inflammatory mediators attract and activate lymphocytes via signal transduction pathways involving PI3K [39]. PTEN functions primarily as a lipid phosphatase, which regulates crucial signal transduction pathways mediated by phosphatidylinositol-3,4,5-trisphosphate (PIP3) [40]. PTEN has been implicated in regulating cell survival signaling through the PI3K/Akt pathway. PTEN also counteracts PI3K function by dephosphorylating the signal lipid PIP3. PIP3 is produced by PI3K following activation by receptor tyrosine kinases, and activated Ras or G proteins. Production of PIP3 leads to the stimulation of several downstream targets, including serine/threonine protein kinase Akt [41]. PPARγ has been shown to

regulate PI3K/Akt signaling by modulating PTEN expression in inflammatory cells [16]. In mice with GVHD, splenic PTEN expression was down-regulated, but p-Akt expression was up-regulated. Rosiglitazone treatment significantly abrogated the GVHD-induced changes in expression of both PTEN and p-Akt. These results suggest that PTEN and PI3K/Akt are potential signaling molecules regulating the pathogenesis of GVHD and that rosiglitazone may improve GVHD by modulating the expression of PTEN and p-Akt. Previous reports have shown that NF-κB plays a critical role in immune and inflammatory responses [42,43]. Nuclear localized NF-κB protein in splenic tissues is substantially increased in GVHD mice, suggesting that NF-κB is activated. Rosiglitazone administration to GVHD mice significantly reduced nuclear NF-κB levels, while increasing cytosolic NF-κB levels. These results indicate that rosiglitazone inhibits NF-κB activity by preventing translocation of this transcription factor into the nucleus. Inhibitory κB (IκB) has 5–7 conserved domains, each consisting of approximately 30 amino acids and forming a unit that can interact with NF-κB subunits. This interaction prevents NF-κB activation and its translocation to the nucleus [44,45]. Activation of Akt has been shown to enhance degradation of IκB and to cooperate with other factors to induce NF-κB-mediated activation of NF-κB-responsive model promoters [46]. Therefore, we suggest that rosiglitazone inhibits the NF-κB signal transduction pathway by decreasing NF-κB binding to the promoter regions of a large number of genes involved in GVHD-mediated inflammatory responses through the regulation of the PTEN/PI3K/Akt signaling pathway. 5. Conclusions The results presented here demonstrate that rosiglitazone, a synthetic PPARγ agonist, prevents inflammation in GVHD target organs. Furthermore, these results indicate that rosiglitazone and PPARγ activation may be useful in protecting the host from GVHD via the upregulation of PTEN expression. Role of funding source This study was supported by a grant from the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (0620220), and by funds of Chonbuk National University Hospital Research Institute of Clinical Medicine. References [1] Hill GR, Crawford JM, Cooke KR, Brinson YS, Pan L, Ferrara JL. Total body irradiation and acute graft-versus-host disease: the role of gastrointestinal damage and inflammatory cytokines. Blood 1997;90:3204-13. [2] Hill GR, Ferrara JL. The primacy of the gastrointestinal tract as a target organ of acute graft-versus-host disease: rationale for the use of cytokine shields in allogeneic bone marrow transplantation. Blood 2000;95:2754-9. [3] Ferrara JL, Cooke KR, Teshima T. The pathophysiology of acute graft-versus-host disease. Int J Hematol 2003;78:181-7. [4] Schmaltz C, Alpdogan O, Horndasch KJ, Muriglan SJ, Kappel BJ, Teshima T, et al. Differential use of Fas ligand and perforin cytotoxic pathways by donor T cells in graft-versus-host disease and graft-versus-leukemia effect. Blood 2001;97:2886-95. [5] Wasem C, Frutschi C, Arnold D, Vallan C, Lin T, Green DR, et al. Accumulation and activation-induced release of preformed Fas (CD95) ligand during the pathogenesis of experimental graft-versus-host disease. J Immunol 2001;167:2936-41. [6] Socie G, Mary JY, Lemann M, Daneshpouy M, Guardiola P, Meignin V, et al. Prognostic value of apoptotic cells and infiltrating neutrophils in graft-versus-host disease of the gastrointestinal tract in humans: TNF and Fas expression. Blood 2004;103:50-7. [7] Remberger M, Jaksch M, Uzunel M, Mattsson J. Serum levels of cytokines correlate to donor chimerism and acute graft-vs.-host disease after haematopoietic stem cell transplantation. Eur J Haematol 2003;70:384-91. [8] Berger J, Moller DE. The mechanisms of action of PPARs. Annu Rev Med 2002;53: 409-35. [9] Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, et al. The nuclear receptor superfamily: the second decade. Cell 1995;83:835-9. [10] Vamecq J, Latruffe N. Medical significance of peroxisome proliferator-activated receptors. Lancet 1999;354:141-8.

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