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Bortezomib attenuates acute graft-vs.-host disease through interfering with host immature dendritic cells
Experimental Hematology 2011;39:710–720
Bortezomib attenuates acute graft-vs.-host disease through interfering with host immature dendritic cells Yi Taoa, Weiwei Zhangb, Yuan Fanga, Dan Yanga, Liping Wanga, Hong Zhoua, and Jianmin Wanga a
Department of Hematology; bLaboratory Diagnosis, Changhai Hospital, Second Military Medical University, Shanghai, PR China (Received 8 December 2010; revised 16 February 2011; accepted 1 March 2011)
Objective. To explore the conditions under which proteasome inhibitor bortezomib improves acute graft-vs.-host disease (aGVHD) and the mechanism underlying the differential effects of bortezomib on aGVHD. Materials and Methods. Murine aGVHD models (C57BL/6/BALB/c) of different severities were set up by infusing with decreasing doses of donor splenocytes (SC). Bortezomib were administered immediately or 6 days after bone marrow transplantation (BMT). Serum levels of tumor necrosis factorLa (TNF-a) and lipopolysaccharide along with the number of donor TNF-a+ T cells in recipients before intervention were determined. Major histocompatibility complex II expression and interleukin-12 production were analyzed to evaluate the maturation state of host dendritic cells (DCs) before intervention. Phenotypic changes, apoptosis, allogeneic stimulation, and IkBa expression levels in bortezomib-treated mature DCs or immature DCs were analyzed in vitro. Results. Neither early bortezomib (day 0 BMT) administration in a modest (SC 1 3 107) or severe (SC 2 3 107) aGVHD model, nor delayed administration (day +6 BMT) could protect mice form aGVHD. Marked inhibition of aGVHD was observed in a mild aGVHD model (SC 5 3 106) with early intervention. This inhibition correlated with a relatively immature state of host DCs before intervention. Additional in vitro studies showed that, in comparison to mature DCs, bortezomib inhibited phenotypic and functional maturation as well as induced more potent apoptosis in immature DCs through suppression of nuclear factorLkB activity. Conclusions. Manipulating host immature DCs may represent a novel mechanism by which bortezomib improves aGVHD. Ó 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
Allogeneic bone marrow transplantation (BMT) is a potentially curative therapy for malignant hematological diseases. However, acute graft-vs.-host disease (aGVHD) remains a frequent and severe complication that limits its further applications [1]. aGVHD is an inflammatory response dependent on the ability of donor T cells to recognize and react to antigenic disparities present in an immunocompromised host. Antigen-presenting cells (APCs), specifically host dendritic cells (DCs) [2,3], play a central role in the initiation [1,4]. Allogeneic T-cell priming in this context is significantly influenced by the state of DC activation due to ‘‘danger signals’’ during and after conditioning of the recipients [5,6].
Offprint requests to: Jianmin Wang, M.D., Department of Hematology, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai, 200433, PR China; E-mail: [email protected]
Immature DCs (imDCs) are specialized for antigen capture but with low APC ability [7], resulting in less T-cell activation considered to be an important pathway by which tolerance is maintained [8]. Mature DCs (mDCs), upon maturation signals such as pathogen-derived products lipopolysaccharide (LPS) or endogenous inflammatory cytokine tumor necrosis factora (TNF-a) [9,10], migrate to the lymphoid tissue with increased APC ability and T-cell activation potential [11]. Experimental data have suggested that aGVHD can be regulated by qualitatively or quantitatively modulating distinct DC subsets [12,13]. Nuclear factorkB (NF-kB), a widespread transcription factor in virtually all cell types, controls the expression of a number of genes important for immune and inflammatory responses characteristic of aGVHD pathophysiology [14]. Moreover, NF-kB pathway has also been shown to be involved in the maturation and antigen presentation of DCs [15,16].
0301-472X/$ - see front matter. Copyright Ó 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2011.03.001
Y. Tao et al./ Experimental Hematology 2011;39:710–720
Bortezomib (PS-341; Velcade), a reversible proteasome inhibitor, has been demonstrated to inhibit IkB degradation and subsequent NF-kB activation [17]. In addition to antitumor effects [18,19], the ability to inhibit NF-kB pathway makes bortezomib a potentially attractive option for prevention of aGVHD. Evidence from the animal models indicates a potential role for bortezomib in the treatment of aGVHD [14,20]. However, contradictory results of aGVHD aggravation were observed in murine models with delayed bortezomib administration after BMT [21]. To define the possible mechanisms underlying these paradoxical phenomena and to better improve the efficacy of bortezomib in aGVHD treatment clinically, we designed aGVHD murine models (C57BL/6/BALB/c) of different severities by infusing decreasing donor splenocytes (SCs) and chose the different timing of bortezomib administration after BMT. We report here, for the first time, that the beneficial effects of bortezomib on aGVHD, which were observed in a mild aGVHD model with early administration, correlate with a relatively immature state of host DCs before intervention. The aGVHD inhibition might be due to the more potent ability of bortezomib to inhibit maturation and induce apoptosis in imDCs compared to already matured DCs.
Materials and methods Mice and induction of GVHD BALB/c (H2d) and C57BL/6 (B6, H2b) mice between 6 and 8 weeks of age were purchased from the Animal Production Area of the Chinese Scientific Academy (Shanghai). Bone marrow (BM) cells were washed and resuspended in Dulbecco’s modified Eagle’s medium before injection. Red blood cells were removed from SC suspensions by hypotonic lysis using red blood cell buffer (BD Pharmingen, San Diego, CA, USA). Lethally irradiated (8 Gy) BALB/c mice received 1.0 107 B6 BM cells with or without B6 SCs (520 106) intravenously. Mice were then injected intraperitoneally (IP) with phosphate-buffered saline or bortezomib (Millennium Pharmaceuticals, Cambridge, MA, USA) at 0.25 mg/kg at the indicated time phase. All animals were handled in accordance with institutional and governmental directives and were approved by local authorities.
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ents in each group were purified using CD11c microbeads (Miltenyi, Bergisch Gladbach, Germany) according to manufacturer’s recommendations. Purity was generally 85% to 90%. Purified DCs were cultured ex vivo in RPMI-1640 containing 10% fetal bovine serum in 96-well plates at a cell concentration of 2 105 cells/well. After 18 hours, the supernatants were collected for assessing the secretion of interleukin (IL)-12 (p70). Cytokine analysis Host mice were kindly exsanguinated at the indicated time after transplantation, and blood samples were collected and centrifuged to obtain serum. Supernatants from cell cultures were harvested at the indicated time phase. TNF-a and IL-12 levels in serum and supernatants were assayed by enzyme-linked immunosorbent assay kit according to manufacturer’s instructions (eBioscience, San Diego, CA, USA). Flow cytometry For intracellular TNF-a analysis in donor T cells, spleens were collected from mice at 6 hours post-BMT and cell suspensions were restimulated with 50 ng/mL phorbol 12-myristate 13-acetate, 800 ng/mL ionomycin and 1 Brefeldin A (eBioscience, San Diego, CA, USA) for 5 hours. Cells were then surface labeled with phycoerythrin anti-H2b and PeCy5 anti-CD3 (Biolegend, San Diego, CA, USA) followed by fixation and permeabilization using fixation and permeabilization kit (eBioscience). Subsequently, cells were stained intracellularly with fluorescein isothiocyanate (FITC)TNF-a or isotype FITC-labeled rat IgG1 (eBioscience). Labeled cells were measured for three-color analysis on FACScan flow cytometry. For analysis of host splenic DCs, splenocytes were stained with phycoerythrin anti-H2b and FITC anti-CD11c. Mean fluorescence intensity of major histocompatibility class (MHC) II stained in combination with the anti-CD11c antibody was determined on gated CD11chigh DC-enriched cells as described here. For phenotype analysis, BM-DCs, imDCs, or mDCs treated with bortezomib as indicated were stained with specific antibodies (eBioscience). For determination of cell apoptosis, cells were stained with FITC-conjugated Annexin V (eBioscience) and propidium iodide according to manufacturer’s instructions. Flow cytometric experiments were performed on a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Serum lipopolysaccharide determination For determination of endotoxin concentration, the Tachypleus Limulus Amebocyte Lysate assay (Lonza Walkersville, Walkersville, MD, USA) was performed according to manufacturer’s protocol. Absorbance of the assay plate was read at 545 nm. Samples and standards were run in duplicate and the lower limit of detection was 0.10 EU/mL.
Culture of murine DCs and treatment with bortezomib Mouse DCs were prepared from BM progenitors by culturing in 10 ng/mL recombinant murine granulocyte-macrophage colonystimulating factor and 1 ng/mL recombinant murine IL-4 (R&D, Minneapolis, MN, USA) as described previously [23]. The remaining loosely adherent clusters were used on day 6 as imDCs. Then, 5 nM bortezomib was added to imDC on day 6 of culture for 24 hours and subsequently 10 ng/mL LPS or 20 ng/mL TNF-a (R&D) was added during the last 16 hours. mDCs were generated from imDCs stimulated with 10 ng/mL LPS or 20 ng/mL TNF-a for 24 hours. Afterward, mDCs were treated with 5 nM bortezomib for 24 hours.
DCs preparation from recipient spleens Splenocytes were released by teasing and treatment with 1 mg/mL typeⅠcollagenase (Sigma-Aldrich, St Louis, MO, USA). For flow cytometry analysis, DC-enriched cells were obtained as a lowdensity fraction on bovine serum albumin columns as described previously [22]. For functional assays, splenic DCs from recipi-
Mixed lymphocyte reaction Responder CD4þor CD8þT cells (H-2b, 4 105) were cultured in 96-well round-bottom plates with imDCs or mDCs (H-2d, 4 104) that had been treated with bortezomib as indicated and then g-irradiated (30 Gy cyanocobalamin 60Co) before coculture. Responder CD4þor CD8þT cells were obtained by mouse
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CD4þ- or CD8þT-positive selection kit (Stem Cell, Vancouver, Canada). The purity was O95% for each. Control wells consisted of responder CD4þor CD8þT cells only. After 5 days of coculture, T-cell proliferation was evaluated by CCK-8 assay (Dojindo, Kumamoto, Japan) according to manufacturer’s instructions. The value of optical density (OD) in each well was measured with a Thermomax microplate reader according to the absorbance at 450 nm. Stimulation indices were calculated as follows: (OD value of cocultures OD value of control medium) / (OD value of control T OD value of control medium). Supernatants in parallel experiments were collected and stored at 70 C until use for cytokine determination.
Western blotting analysis Western blotting was performed as described [24]. In brief, cell lysates were extracted in sodium dodecyl sulfate sample buffer (6.25 M Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate, 10% glycerol, 2% 2-mercaptoethanol, 0.005% bromophenol blue). Cell lysates were immediately boiled at 100 C for 10 minutes and stored at 20 C for subsequent use. Proteins were separated on a sodium dodecyl sulfate polyacrylamide gel (10%) and electroblotted onto a nitrocellulose membrane. Proteins were visualized by probing the membranes with antiIkBa, antib-actin (Santa Cruz Biotechnology) antibodies. The resulting band intensities were quantitated using an image scanning densitometer (Furi Technology, Shanghai, China).
Statistics Survival data were plotted by the Kaplan-Meier method and analyzed by log-rank test. Group comparisons were performed using the unpaired Student t-test. Data are shown as mean 6 standard deviation for separate experiments. A p value of !0.05 was considered significant.
Results Early bortezomib administration prolonged survival in mice with mild aGVHD Lethally irradiated BALB/c mice underwent transplantation with allogeneic BM and SCs from full MHC major and minor antigen disparate C57BL/6 donors. Because the severity of aGVHD correlates with the number of donor T cells transfused [25], we designed a series of aGVHD murine models with different severity by infusing with decreasing doses of donor SCs (2 107, 1 107, 5 106). Results showed that transplantation of 2 107 SCs along with BM resulted in severe aGVHD with obvious outward manifestations of aGVHD (ie, diarrhea, posture, or grooming changes) observed in the recipients, all of which succumbed within 13 days. Transplantation of 1 107 SCs along with BM led to relatively modest aGVHD and all mice died within 23 days. Results showed that early administration (day 02 post-BMT) of bortezomib imposed no improved effects on survival in mice from each aGVHD model (Fig. 1A, B). In contrast, when the number of donor SCs was further decreased to 5 106 in which all control mice succumbed within 36 days, significant increases in survival were observed in recipients from this mild aGVHD model (SC 5 106), with O70% of mice becoming long-term survivors (Fig. 1C). However, delaying bortezomib administration (day 68 post-BMT) resulted in significantly greater mortality than did phosphate-buffered saline treatment of aGVHD control animals (Fig. 1C). Notably, no morbidity was observed in animals that underwent transplantation with BM and then were treated with bortezomib at any time point post-BMT (Fig. 1C).
Figure 1. Dose response of donor SCs and the timing of administration necessary for different effects of bortezomib on aGVHD. (A, B) Lethally irradiated BALB/c (H2d) recipients of 10 million B6 (H2b) BM and 20 million (A) or 10 million (B) SCs were treated with 0.25 mg/kg per dose of bortezomib (:) or vehicle control (phosphate-buffered saline [PBS]) (-) daily from day 0 through day þ2 post-BMT. (C) Lethally irradiated BALB/c recipients of 10 million B6 BM and 5 million SCs were treated with 0.25 mg/kg per dose of bortezomib daily from day 0 through day þ2 (:) or from day þ6 through day þ8 (6) post-BMT, or treated with vehicle control (PBS) (-). Lethally irradiated BALB/c recipients of 10 million B6 BM were treated with 0.25 mg/kg per dose of bortezomib daily from day 0 through day þ2 () or from day þ6 through day þ8 (+) post-BMT. Data are pooled from two independent experiments. Each experiment consisted of 8 to 10 mice per group.
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More infused donor SCs induced increases in serum LPS levels and numbers of donor TNF-aþ T cells in recipient spleens Both LPS and TNF-a are important stimuli for DCs activation and maturation. Our data showed that as early as 6 hours post-BMT significant increases in serum LPS levels were observed in recipients with 10 million SCs in the modest aGVHD model compared to those with 5 million
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SCs in the mild aGVHD model (Fig. 2A). However, at this time point, there was no significant difference in serum TNF-a levels between these two models (Fig. 2B). Additionally, serum levels of LPS (Fig. 2A) and TNF-a (Fig. 2B) significantly elevated on day 6 post-BMT compared to those on day 0 post-BMT in recipients of each aGVHD model. Flow cytometry analysis showed that the frequency of donor T cells increased in the spleens
Figure 2. Low levels of serum LPS and less numbers of donor TNF-aþ T cells in the spleens observed in mild aGVHD mice. Lethally irradiated BALB/c (H2d) recipients were infused with 10 million B6 (H2b) BM, or 10 million B6 BM þ5 million SCs, or 10 million B6 BM þ10 million SCs. (A) Serum LPS levels were determined in recipients of 10 million B6 BM together with 10 million or 5 million SCs on day 0 (6 hours post-BMT) and day 6 post-BMT. (B) Serum TNF-a levels were determined in recipients of 10 million B6 BM together with 10 million or 5 million SCs on day 0 (6 hours post-BMT) and day 6 post-BMT. (CLE) At 6 hours post-BMT, spleen cells were collected from irradiated BALB/c recipients in each group, reacted with anti-CD3, anti-H2b, and antiTNF-a antibodies, and analyzed by flow cytometry. (C) Representative dot plots. (D) Frequency of donor T cells in recipient spleens. (E) Absolute number of donor TNF-aþ T cells in recipient spleens. *p ! 0.05; **p ! 0.01, data are shown as mean 6 standard deviation from three independent experiments performed in triplicate.
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of recipients with more B6 SCs infused (Fig. 2C, D) at 6 hours post-BMT. With the intracellular staining of TNF-a, the results further demonstrated that the absolute number of donor-derived TNF-aþ T cells significantly increased in the spleens of recipients with 10 million SCs compared to those with 5 million SCs (Fig. 2E).
More infused donor SCs made host splenic DCs more mature Because serum LPS levels and the number of donor TNF-aþ T cells differed in recipients with different doses of donor SCs, we were interested in the fate of host splenic DCs in recipients when more donor SCs were infused. Flow cytometry analysis demonstrated that the infusion of more donor SCs did not change the percentage and the absolute number of host DCs in recipient spleens at 8 hours post-BMT (Fig. 3A, B). To address the activation and maturation state of host splenic DCs, we investigated the phenotype of the splenic CD11chigh population by analyzing the antigen-presenting molecule MHC II expressed on these cells. Results demonstrated that host splenic DCs from irradiated mice with more donor SCs (SC 1 107) expressed increased numbers of MHC II at 8 hours post-BMT (Fig. 3C, D). When splenic DCs were isolated and cultured ex vivo overnight, DCs from irradiated mice with more donor SCs produced significantly higher levels of IL-12 than those from irradiated mice with less SCs did (Fig. 3F), consistent with the increased levels of serum IL-12 detected in these irradiated mice (Fig. 3E). Our data thus reflected that the infusion of more donor SCs made host DCs more mature in vivo.
Bortezomib inhibited imDCs maturation in response to inflammatory signals Subsequently, we undertook ex vivo experiments to determine whether the maturation state of DCs is related to the differential effects of bortezomib on DCs. Stimulation with LPS or TNF-a leads to DCs maturation [9,10], which is accompanied by marked upregulation of the costimulatory molecules CD80, CD86, and CD40, maturation antigen CD83 as well as presentation molecules MHC II [11]. Our results showed that bortezomib prevented imDCs maturation in response to LPS or TNF-a. Namely, imDCs pretreatment with bortezomib prevented upregulation of CD80, CD86, CD40, and CD83 as well as MHC II (Fig. 4B, E). The normal upregulations of integrin CD54 and chemokine receptor CXCR4 molecules in response to these inflammatory signals were slightly reduced by bortezomib. We next examined the effects of bortezomib on DCs at the matured stage (mDCs) induced by LPS or TNF-a. In contrast to pretreated imDCs, no significant difference was found in expression of surface markers on bortezomibtreated (Fig. 4C, F) vs. untreated mDCs (Fig. 4A, D).
Bortezomib induced more potent apoptosis in imDCs compared to already matured DCs A dose-dependent induction of cell apotosis was observed in both imDCs and mDCs after 24 hours treatment of bortezomib. However, apoptosis of imDCs in the presence of LPS was significantly improved after 24 hours of exposure to a low concentration of 5nM bortezomib, as was not observed in already matured DCs. Moreover, our data showed that pretreated imDCs in response to LPS were more susceptible to bortezomib-induced apoptosis than already matured DCs (Fig. 5A). Besides, similar results were obtained when using TNF-a as a maturation stimulus (Fig. 5B). Bortezomib blunted imDCs allostimulatory capacity for CD4þ T cells The most characteristic function featuring DCs is their ability to induce T-cell proliferation. Results demonstrated that pretreatment with bortezomib reduced the capacity of imDCs to induce proliferation in alloreactive CD4þ and CD8þ T cells (Fig. 6A). Given the pivotal role donor T-cellderived TNF-a plays in GVHD [26], we evaluated the TNF-a levels in supernatants from mixed lymphocyte reaction. Here, we found significant decreases in TNF-a levels from alloreactive CD4þ T cells, but not from CD8þ T cells, when cocultured with LPS-primed imDCs pretreated with bortezomib (Fig. 6B). However, bortezomib did not substantially affect mDCs’ capacity to prime allogeneic lymphocyte reaction. Significant increases in IkBa levels in imDCs after bortezomib treatment It was previously reported that bortezomib inhibited activation of the transcription factor NF-kB by preventing proteasomal degradation of IkBa, the NF-kB inhibitor in multiple myeloma [27]. To clarify if NF-kB pathway was affected in DCs, we detected IkBa levels in imDCs and mDCs treated with bortezomib for 12 and 24 hours by immunoblotting. In imDCs, pretreatment with bortezomib increased the expression level of IkBa gradually upon LPS stimulation in a time-dependent fashion, indirectly indicating an inhibition of NF-kB activity. In contrast, expression of IkBa remained unaffected in mDCs after bortezomib treatment (Fig. 7).
Discussion In the present study, we performed dose responses of donor SCs as well as different timing of administration to ascertain the effects of bortezomib on aGVHD. Our data demonstrated that protection from aGVHD by bortezomib was only observed in a mild aGVHD model with less donor SCs infused. These findings contrasted with those of Sun et al. [20], who found that bortezomib treatment could decrease GVHD mortality in an aggressive model of
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Figure 3. More infused donor SCs made host splenic DCs more mature. Lethally irradiated BALB/c (H2d) recipients were infused with 10 million B6 (H2b) BM, or 10 million B6 BM þ5 million SCs, or 10 million B6 BM þ10 million SCs. At 8 hours post-BMT, spleen cells and serum were isolated from irradiated BALB/c recipients in each group. (A) Percentage and (B) absolute number of host splenic DCs. (C) Expression of MHC II on host splenic DCs was analyzed by flow cytometry. Isotype-matched IgG was used as control. (D) The mean fluorescence intensity (MFI) of MHC II on host splenic DCs. (E) IL-12 in the serum from irradiated BALB/c recipients in each group was examined by enzyme-linked immunosorbent assay (ELISA). (F) IL-12 in the supernatants of cultured splenic DCs was examined by ELISA. Splenic DCs were separately purified from irradiated BALB/c recipients in each group at 8 hours post-BMT as described in Materials and Methods. Supernatants were collected from the cultures of DCs overnight. *p ! 0.05; **p ! 0.01, results are presented as mean 6 standard deviation from three independent experiments performed in triplicate.
aGVHD in which all control mice died within 10 days. Although the exact explanation for these disparate results is unclear, certain differences between the studies are noteworthy. BALB/c mice were used as recipients to receive allografts from B6 in our study, while Sun et al. utilized B6 recipients receiving allografts from BALB/c mice. These contrast phenomena can be related to interstrain differences in their genetic background. For example,
Chen et al. found that B6 mice have a substantially lower frequency of CD4þCD25þ T-regulatory (Treg) cells in the peripheral lymphoid tissues, including spleens, than BALB/c mice [28]. It has been clearly established that the immunosuppressive effect of Treg cells could be demonstrated by inhibition of CD4þCD25 T-cell proliferation in vitro. B6 Treg cells are not only lower in number but also less able to suppress syngeneic CD4þCD25
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Figure 4. Effects of bortezomib on phenotypic changes in DCs. imDCs were prepared from mouse BM progenitors by culturing in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 6 days. imDCs were then incubated in the presence of 5 nM bortezomib (B, E) or medium alone (A, D) for 24 hours. LPS (10 ng/mL) or TNF-a (20 ng/mL) was added during the last 16 hours where indicated in the figure. To produce already matured DCs (mDCs), imDCs were stimulated with LPS (10 ng/mL) (C) or TNF-a (20 ng/mL) (F) for 24 hours. mDCs were then washed with phosphate-buffered saline and then cultured in the presence of 5 nM bortezomib for another 24 hours where indicated in the figure. Thereafter, cells were stained with the designated monoclonal antibody and analyzed by flow cytometry. Matched isotype controls are presented as solid histograms. Results are representative of three independent experiments.
T-responder cells than Treg cells from BALB/c mice. In addition, CD4þCD25 T-responder cells from B6 mice were notably more resistant than those from BALB/c mice to inhibition by Treg cells from either mouse strain [28]. Maybe it is one of the reasons why fewer B6 SCs are needed to induce similar severity of aGVHD in BALB/c mice. Besides, it has been shown that activation of donor T cells in the spleen by host APCs is complete by 24 hours after BMT [29]. Thus, in our study, once activated by host mDCs after infusion into BALB/c recipients, B6 T-cell proliferation might be refractory to suppression by Treg cells later on. So the successful intervention phase for aGVHD in this model might be brief, possibly just before donor T-cell activation or even before host DCs maturation. Furthermore, differences in susceptibility to bortezomib between two strains of mice should not be neglected. Compared to B6 mice, BALB/c mice proved to be
more susceptible to bortezomib-induced toxicity, tolerating no more than 5 mg per dose in our study (data not shown). Additionally, consistent with previous reports [14,21], our results also suggest delayed administration of bortezomib accelerated aGVHD morbidity, indicating a narrow therapeutic window for bortezomib intervention. Zhang et al. previously reported that about 5% to 10% of all infused donor T cells were recruited into the spleen by 6 to 24 hours after transplantation [30]. Our results also demonstrated that approximately 12% to 16% of infused B6 T cells reached host spleens by 6 hours post-BMT (data not shown). Importantly, compared with mild aGVHD mice (infused with less donor SCs), more TNF-asecreting donor T cells were found in the spleens of modest aGVHD mice (infused with more donor SCs). It has been reported that during aGVHD pathophysiology, gut epithelial damage induced first by BMT conditioning and subsequently by
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Figure 5. Effects of bortezomib on apoptosis in DCs. imDCs were prepared from mouse BM progenitors by incubating in granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4containing medium. At day 6 of culture, imDCs were stimulated for 24 hours with indicated concentrations of bortezomib and were added LPS (10 ng/mL) or TNF-a (20 ng/mL) during the last 16 hours of incubation. mDCs were obtained from imDCs cocultured with LPS (10 ng/mL) or TNF-a (20 ng/mL) for 24 hours. Afterward, mDCs were stimulated with indicated concentrations of bortezomib for another 24 hours. Bortezomib induces a dose-dependent apoptosis in imDCs and mDCs. (A) Bortezomib-pretreated imDCs (6) in response to LPS were more susceptible to bortezomib-induced apoptosis than already-matured DCs (LPS-induced) (:). (B) Bortezomib-pretreated imDCs (6) in response to TNF-a were more susceptible to bortezomib-induced apoptosis than already-matured DCs (TNF-ainduced) (:). **p ! 0.01, results are shown as mean 6 standard deviation from three independent experiments.
alloreactive donor T cells allows passage of LPS from the gut lumen into the circulation [31]. Thus, it was not surprising that higher serum levels of LPS were observed in irradiated recipients infused with more donor SCs than those with less donor SCs. Based on the notion that TNFa and LPS are strong activators for DCs maturation and the pivotal role host DCs play in aGVHD initiation [4,9], we were interested in the maturation state of host DCs in recipients with different aGVHD severity. As is known, mDCs express high levels of MHC class II [11] and have a great ability to release IL-12 [32]. DCs that produce
IL-12 stimulate na€ıve allogenic CD4þ T cells to develop polarized Th1 responses [33]. Thus, the intensities of MHC II expression and production of IL-12 are good indicators for determining the DC maturation stage. Our results showed that DCs from modest aGVHD mice expressed higher levels of MHC II and secreted larger amounts of IL-12 compared to those from mild aGVHD mice as early as 8 hours post-BMT, suggesting more mature subsets of DCs. In addition, because the percentage and absolute number of host DCs did not differ between the two models, it was unlikely that the distinct effects of bortezomib were
Figure 6. Effects of bortezomib on DCs allostimulatory capacity. imDCs were prepared from mouse BM progenitors by incubating in granulocytemacrophage colony-stimulating factor (GM-CSF) and IL-4–containing medium. At day 6 of culture, cells were stimulated with 5 nM bortezomib or medium alone (phosphate-buffered saline [PBS] control) for 24 hours and were added LPS (10 ng/mL) during the last 16 hours of incubation. To produce already matured DCs (mDCs), imDCs were incubated with LPS for 24 hours to induce mDCs, which were subsequently treated with 5 nM bortezomib for another 24 hours. Thereafter, cells were harvested, washed, irradiated, and used as stimulators for purified splenic CD4þ T cells or CD8þ T cells at the ratio of 1:10 in the absence of bortezomib. (A) Proliferation was measured at day 5 by CCK-8 incorporation. Stimulation indices (SI) were calculated as described in Materials and Methods. (B) Supernatants were collected at day 5 for TNF-a determination. *p ! 0.05; **p ! 0.01, data are presented as mean 6 standard deviation from three independent experiments performed in triplicate.
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Figure 7. Effects of bortezomib on IkBa levels in DCs. imDCs were prepared from mouse BM progenitors by incubating in granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4–containing medium. At day 6 of culture, cells were treated with 5 nM bortezomib or medium alone (phosphate-buffered saline [PBS] control) for 24 hours and were stimulated with LPS (10 ng/mL) during the last 16 hours of incubation. To generate already matured DCs (mDCs), imDCs were incubated with LPS for 24 hours to induce mDCs, which were subsequently treated with 5 nM bortezomib or medium alone for another 24 hours. Immunoblot analysis of imDCs and mDCs after treatment with bortezomib at the indicated time points was performed using antiIkBa monoclonal antibodies (mAb). Immunoblot was probed with antib-actin mAb to compare protein loading within samples. One representative experiment out of three is presented (left). The ratio of band intensities of IkBa to b-actin was obtained as relative IkBa protein level indicated (right). **p ! 0.01, data are shown as mean 6 standard deviation from three separate experiments.
attributed to different ratios between drug doses and target cells. So far, we hypothesized that changes in the levels of pathogenic component LPS and the numbers of TNFasecreting donor T cells after allogeneic BMT resulted in different maturation states of host DCs, which might be responsible for the distinct effects of bortezomib in aGVHD models with different severity. The mechanism by which the proteasome inhibitor affects aGVHD is not definitively known. This study was aimed at defining a possible explanation by examining whether this compound exerts its effects on DCs at different maturation stage. Data on plasma levels of bortezomib in patients with advanced solid tumors have shown peak concentrations ranging between 10 and 100 nM [34]. In addition, the minimum effective dose Sun et al. utilized in a murine study in vitro was 4 nM [20]. Therefore, the concentrations (i.e., 520 nM) we used in the present study match clinically and experimentally effective doses. The present in vitro study demonstrated that bortezomib could inhibit phenotypic maturation of imDCs, providing a basis for the immunosuppressive effects, given the key role of costimulatory signals and antigen presentation for optimal T-cell activation [35]. As expected, DC-primed T-cell response was significantly reduced for pretreated imDCs, which was proven by decreases in allogeneic T-cell proliferation and TNF-a production. These findings are in alignment with similar roles bortezomib played in human imDCs [36,37]. As compared to mDCs, imDCs show more susceptibility to bortezomib-induced inhibition of phenotypic and functional maturation as well as apoptosis, similar to a previous report demonstrating differential effects of bortezomib on human monocyte-derived imDCs and mDCs [38]. Notably, bortezomib inhibited TNF-a secretion
mostly in CD4þ T cells, consistent with previous findings that TNF-a blockade had a highly significant effect (100% survival rate) on CD4þ T-cell, but not on CD8þ T-cellmediated aGVHD [39]. Therefore, in vitro experimental results suggest that bortezomib may improve aGVHD by reducing interactions between host imDCs and donor T cells in secondary lymphoid organs, thus resulting in suppression of T-cellmediated immune responses. Given the differential capacity of bortezomib to interfere with aGVHD in vivo and DCs in vitro, it is possible that under the prerequisite of relatively imDCs, which is the case in early bortezomib administration in mild aGVHD models, bortezomib could protect mice from aGVHD by inhibiting maturation and function of imDCs in response to maturation stimuli. On the other hand, in the case of mature state of host DCs that were observed in modest or even severe aGVHD models, early bortezomib treatment failed to improve aGVHD due to its inability to affect mDCs phenotypically and functionally. Pharmacokinetics analysis showed proteasome function returned to normal by 72 hours after bortezomib treatment. And it has been reported that host APCs, rather than donor APCs, are responsible for the first stage of aGVHD (from day 0 to day 7 post-BMT) [40]. Thus, host DCs were most likely to be targeted by bortezomib during early administration (from day 0 through day þ2) post-BMT in our study. Additionally, our data showed systemic LPS and TNF-a increased by day 6 after BMT, indicating ongoing aGVHD. Systemic inflammation has been demonstrated to induce the disappearance of activated DCs within 24 to 48 hours in the T-cell areas of spleen [11,41]. We observed that the absolute number of host splenic CD11cþ DCs began to decline by 6 hours after
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TBI and by day 6 nearly all host DCs in the spleen were eliminated, consistent with findings from Zhang et al. [30]. Considering the inability of delayed administration of bortezomib to improve aGVHD, it is possible that there are few host DCs left to be affected by bortezomib. Furthermore, the gut as a major GVHD target organ has been demonstrated to be highly sensitive to injury by TNFa [26]. And it has been shown that proteasome inhibition can enhance TNF-ainduced apoptosis in gastric epithelial cells [42], partly explaining an increase in mortality with delayed bortezomib administration in our study. Furthermore, we were interested in the intracellular mechanism specifically responsible for the differential effects of bortezomib on immature vs. mature DCs. Bortezomib has been shown to inhibit NF-kB activity in multiple myeloma cells by blocking IkBa degradation [27]. Additionally, Yoshimura et al. study found that NF-kB was an effective target for blocking DC antigen presentation and inhibiting T-celldependent immune responses [16], suggesting that NF-kB pathway may be differentially regulated by bortezomib in the two subtypes of DCs. This notion was supported by our findings that increased IkBa expression was observed in bortezomib-pretreated imDCs in response to LPS, whereas the levels of IkBa were not affected in mDCs. Degradation of IkBa in the proteasome leads to activation and translocation of released NF-kB to the nucleus [43]. Thus, the increased IkBa protein level in imDCs treated with bortezomib indicated an inhibition of NF-kB activity, while unchanged IkBa expression in mDCs implied unaffected NF-kB activity. Because active protein synthesis was found to represent an upstream prerequisite for DC apoptosis induced by bortezomib [44], it could be speculated that observed changes in imDCs with bortezomib may be due to active NF-kBdependent protein synthesis in imDCs upon maturation stimuli. The immunomodulatory properties of bortezomib we observed in the aGVHD model implied that caution should be taken when applying this compound for aGVHD prevention in a twice-weekly schedule, as in the treatment of multiple myeloma. Monitoring TNF-a, LPS, or the maturation stage of DC in recipients before intervention is possibly beneficial for the positive roles bortezomib played for aGVHD control. In a phase I clinical trial, bortezomib proved effective in ameliorating aGVHD in combination with tacrolimus and methotrexate after reduced-intensity conditioning allogeneic stem cell transplantation [45], implying combination regimens including immunosuppressive agents probably retained immature stage of DC in recipients during bortezomib administration. We should acknowledge that apart from the intervention of host DCs, there are other plausible mechanisms involved in the positive actions of bortezomib in aGVHD. A lowering or dampening of the donor T-cell responses has been demonstrated as one of those [20], although the whole picture of its mechanisms awaits further determination. In conclusion, proteasome inhibitor bortezo-
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mib, as a double-edged sword in aGVHD, could confer protection from aGVHD as a result, in part, of interfering with host DCs at immature stage but not at mature stage. Our data provide new insights into the mechanisms underlying the differential effects of bortezomib on aGVHD, suggesting the importance of manipulating host DCs at an immature stage for therapeutic application.
Acknowledgments This work is supported by grants from the National Natural Science Foundation of China (No. 30871100, 81090110) and Science and Technology Commission of Shanghai Municipality, China (No. 05DZ19327, 08JC1406500) to J.W. The authors would like to thank Lu Gao for technical assistance.
Conflict of interest disclosure No financial interest/relationships with financial interest relating to the topic of this article have been declared.
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Bortezomib attenuates acute graft-vs.-host disease through interfering with host immature dendritic cells Yi Taoa, Weiwei Zhangb, Yuan Fanga, Dan Yanga, Liping Wanga, Hong Zhoua, and Jianmin Wanga a
Department of Hematology; bLaboratory Diagnosis, Changhai Hospital, Second Military Medical University, Shanghai, PR China (Received 8 December 2010; revised 16 February 2011; accepted 1 March 2011)
Objective. To explore the conditions under which proteasome inhibitor bortezomib improves acute graft-vs.-host disease (aGVHD) and the mechanism underlying the differential effects of bortezomib on aGVHD. Materials and Methods. Murine aGVHD models (C57BL/6/BALB/c) of different severities were set up by infusing with decreasing doses of donor splenocytes (SC). Bortezomib were administered immediately or 6 days after bone marrow transplantation (BMT). Serum levels of tumor necrosis factorLa (TNF-a) and lipopolysaccharide along with the number of donor TNF-a+ T cells in recipients before intervention were determined. Major histocompatibility complex II expression and interleukin-12 production were analyzed to evaluate the maturation state of host dendritic cells (DCs) before intervention. Phenotypic changes, apoptosis, allogeneic stimulation, and IkBa expression levels in bortezomib-treated mature DCs or immature DCs were analyzed in vitro. Results. Neither early bortezomib (day 0 BMT) administration in a modest (SC 1 3 107) or severe (SC 2 3 107) aGVHD model, nor delayed administration (day +6 BMT) could protect mice form aGVHD. Marked inhibition of aGVHD was observed in a mild aGVHD model (SC 5 3 106) with early intervention. This inhibition correlated with a relatively immature state of host DCs before intervention. Additional in vitro studies showed that, in comparison to mature DCs, bortezomib inhibited phenotypic and functional maturation as well as induced more potent apoptosis in immature DCs through suppression of nuclear factorLkB activity. Conclusions. Manipulating host immature DCs may represent a novel mechanism by which bortezomib improves aGVHD. Ó 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
Allogeneic bone marrow transplantation (BMT) is a potentially curative therapy for malignant hematological diseases. However, acute graft-vs.-host disease (aGVHD) remains a frequent and severe complication that limits its further applications [1]. aGVHD is an inflammatory response dependent on the ability of donor T cells to recognize and react to antigenic disparities present in an immunocompromised host. Antigen-presenting cells (APCs), specifically host dendritic cells (DCs) [2,3], play a central role in the initiation [1,4]. Allogeneic T-cell priming in this context is significantly influenced by the state of DC activation due to ‘‘danger signals’’ during and after conditioning of the recipients [5,6].
Offprint requests to: Jianmin Wang, M.D., Department of Hematology, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai, 200433, PR China; E-mail: [email protected]
Immature DCs (imDCs) are specialized for antigen capture but with low APC ability [7], resulting in less T-cell activation considered to be an important pathway by which tolerance is maintained [8]. Mature DCs (mDCs), upon maturation signals such as pathogen-derived products lipopolysaccharide (LPS) or endogenous inflammatory cytokine tumor necrosis factora (TNF-a) [9,10], migrate to the lymphoid tissue with increased APC ability and T-cell activation potential [11]. Experimental data have suggested that aGVHD can be regulated by qualitatively or quantitatively modulating distinct DC subsets [12,13]. Nuclear factorkB (NF-kB), a widespread transcription factor in virtually all cell types, controls the expression of a number of genes important for immune and inflammatory responses characteristic of aGVHD pathophysiology [14]. Moreover, NF-kB pathway has also been shown to be involved in the maturation and antigen presentation of DCs [15,16].
0301-472X/$ - see front matter. Copyright Ó 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2011.03.001
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Bortezomib (PS-341; Velcade), a reversible proteasome inhibitor, has been demonstrated to inhibit IkB degradation and subsequent NF-kB activation [17]. In addition to antitumor effects [18,19], the ability to inhibit NF-kB pathway makes bortezomib a potentially attractive option for prevention of aGVHD. Evidence from the animal models indicates a potential role for bortezomib in the treatment of aGVHD [14,20]. However, contradictory results of aGVHD aggravation were observed in murine models with delayed bortezomib administration after BMT [21]. To define the possible mechanisms underlying these paradoxical phenomena and to better improve the efficacy of bortezomib in aGVHD treatment clinically, we designed aGVHD murine models (C57BL/6/BALB/c) of different severities by infusing decreasing donor splenocytes (SCs) and chose the different timing of bortezomib administration after BMT. We report here, for the first time, that the beneficial effects of bortezomib on aGVHD, which were observed in a mild aGVHD model with early administration, correlate with a relatively immature state of host DCs before intervention. The aGVHD inhibition might be due to the more potent ability of bortezomib to inhibit maturation and induce apoptosis in imDCs compared to already matured DCs.
Materials and methods Mice and induction of GVHD BALB/c (H2d) and C57BL/6 (B6, H2b) mice between 6 and 8 weeks of age were purchased from the Animal Production Area of the Chinese Scientific Academy (Shanghai). Bone marrow (BM) cells were washed and resuspended in Dulbecco’s modified Eagle’s medium before injection. Red blood cells were removed from SC suspensions by hypotonic lysis using red blood cell buffer (BD Pharmingen, San Diego, CA, USA). Lethally irradiated (8 Gy) BALB/c mice received 1.0 107 B6 BM cells with or without B6 SCs (520 106) intravenously. Mice were then injected intraperitoneally (IP) with phosphate-buffered saline or bortezomib (Millennium Pharmaceuticals, Cambridge, MA, USA) at 0.25 mg/kg at the indicated time phase. All animals were handled in accordance with institutional and governmental directives and were approved by local authorities.
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ents in each group were purified using CD11c microbeads (Miltenyi, Bergisch Gladbach, Germany) according to manufacturer’s recommendations. Purity was generally 85% to 90%. Purified DCs were cultured ex vivo in RPMI-1640 containing 10% fetal bovine serum in 96-well plates at a cell concentration of 2 105 cells/well. After 18 hours, the supernatants were collected for assessing the secretion of interleukin (IL)-12 (p70). Cytokine analysis Host mice were kindly exsanguinated at the indicated time after transplantation, and blood samples were collected and centrifuged to obtain serum. Supernatants from cell cultures were harvested at the indicated time phase. TNF-a and IL-12 levels in serum and supernatants were assayed by enzyme-linked immunosorbent assay kit according to manufacturer’s instructions (eBioscience, San Diego, CA, USA). Flow cytometry For intracellular TNF-a analysis in donor T cells, spleens were collected from mice at 6 hours post-BMT and cell suspensions were restimulated with 50 ng/mL phorbol 12-myristate 13-acetate, 800 ng/mL ionomycin and 1 Brefeldin A (eBioscience, San Diego, CA, USA) for 5 hours. Cells were then surface labeled with phycoerythrin anti-H2b and PeCy5 anti-CD3 (Biolegend, San Diego, CA, USA) followed by fixation and permeabilization using fixation and permeabilization kit (eBioscience). Subsequently, cells were stained intracellularly with fluorescein isothiocyanate (FITC)TNF-a or isotype FITC-labeled rat IgG1 (eBioscience). Labeled cells were measured for three-color analysis on FACScan flow cytometry. For analysis of host splenic DCs, splenocytes were stained with phycoerythrin anti-H2b and FITC anti-CD11c. Mean fluorescence intensity of major histocompatibility class (MHC) II stained in combination with the anti-CD11c antibody was determined on gated CD11chigh DC-enriched cells as described here. For phenotype analysis, BM-DCs, imDCs, or mDCs treated with bortezomib as indicated were stained with specific antibodies (eBioscience). For determination of cell apoptosis, cells were stained with FITC-conjugated Annexin V (eBioscience) and propidium iodide according to manufacturer’s instructions. Flow cytometric experiments were performed on a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Serum lipopolysaccharide determination For determination of endotoxin concentration, the Tachypleus Limulus Amebocyte Lysate assay (Lonza Walkersville, Walkersville, MD, USA) was performed according to manufacturer’s protocol. Absorbance of the assay plate was read at 545 nm. Samples and standards were run in duplicate and the lower limit of detection was 0.10 EU/mL.
Culture of murine DCs and treatment with bortezomib Mouse DCs were prepared from BM progenitors by culturing in 10 ng/mL recombinant murine granulocyte-macrophage colonystimulating factor and 1 ng/mL recombinant murine IL-4 (R&D, Minneapolis, MN, USA) as described previously [23]. The remaining loosely adherent clusters were used on day 6 as imDCs. Then, 5 nM bortezomib was added to imDC on day 6 of culture for 24 hours and subsequently 10 ng/mL LPS or 20 ng/mL TNF-a (R&D) was added during the last 16 hours. mDCs were generated from imDCs stimulated with 10 ng/mL LPS or 20 ng/mL TNF-a for 24 hours. Afterward, mDCs were treated with 5 nM bortezomib for 24 hours.
DCs preparation from recipient spleens Splenocytes were released by teasing and treatment with 1 mg/mL typeⅠcollagenase (Sigma-Aldrich, St Louis, MO, USA). For flow cytometry analysis, DC-enriched cells were obtained as a lowdensity fraction on bovine serum albumin columns as described previously [22]. For functional assays, splenic DCs from recipi-
Mixed lymphocyte reaction Responder CD4þor CD8þT cells (H-2b, 4 105) were cultured in 96-well round-bottom plates with imDCs or mDCs (H-2d, 4 104) that had been treated with bortezomib as indicated and then g-irradiated (30 Gy cyanocobalamin 60Co) before coculture. Responder CD4þor CD8þT cells were obtained by mouse
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CD4þ- or CD8þT-positive selection kit (Stem Cell, Vancouver, Canada). The purity was O95% for each. Control wells consisted of responder CD4þor CD8þT cells only. After 5 days of coculture, T-cell proliferation was evaluated by CCK-8 assay (Dojindo, Kumamoto, Japan) according to manufacturer’s instructions. The value of optical density (OD) in each well was measured with a Thermomax microplate reader according to the absorbance at 450 nm. Stimulation indices were calculated as follows: (OD value of cocultures OD value of control medium) / (OD value of control T OD value of control medium). Supernatants in parallel experiments were collected and stored at 70 C until use for cytokine determination.
Western blotting analysis Western blotting was performed as described [24]. In brief, cell lysates were extracted in sodium dodecyl sulfate sample buffer (6.25 M Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate, 10% glycerol, 2% 2-mercaptoethanol, 0.005% bromophenol blue). Cell lysates were immediately boiled at 100 C for 10 minutes and stored at 20 C for subsequent use. Proteins were separated on a sodium dodecyl sulfate polyacrylamide gel (10%) and electroblotted onto a nitrocellulose membrane. Proteins were visualized by probing the membranes with antiIkBa, antib-actin (Santa Cruz Biotechnology) antibodies. The resulting band intensities were quantitated using an image scanning densitometer (Furi Technology, Shanghai, China).
Statistics Survival data were plotted by the Kaplan-Meier method and analyzed by log-rank test. Group comparisons were performed using the unpaired Student t-test. Data are shown as mean 6 standard deviation for separate experiments. A p value of !0.05 was considered significant.
Results Early bortezomib administration prolonged survival in mice with mild aGVHD Lethally irradiated BALB/c mice underwent transplantation with allogeneic BM and SCs from full MHC major and minor antigen disparate C57BL/6 donors. Because the severity of aGVHD correlates with the number of donor T cells transfused [25], we designed a series of aGVHD murine models with different severity by infusing with decreasing doses of donor SCs (2 107, 1 107, 5 106). Results showed that transplantation of 2 107 SCs along with BM resulted in severe aGVHD with obvious outward manifestations of aGVHD (ie, diarrhea, posture, or grooming changes) observed in the recipients, all of which succumbed within 13 days. Transplantation of 1 107 SCs along with BM led to relatively modest aGVHD and all mice died within 23 days. Results showed that early administration (day 02 post-BMT) of bortezomib imposed no improved effects on survival in mice from each aGVHD model (Fig. 1A, B). In contrast, when the number of donor SCs was further decreased to 5 106 in which all control mice succumbed within 36 days, significant increases in survival were observed in recipients from this mild aGVHD model (SC 5 106), with O70% of mice becoming long-term survivors (Fig. 1C). However, delaying bortezomib administration (day 68 post-BMT) resulted in significantly greater mortality than did phosphate-buffered saline treatment of aGVHD control animals (Fig. 1C). Notably, no morbidity was observed in animals that underwent transplantation with BM and then were treated with bortezomib at any time point post-BMT (Fig. 1C).
Figure 1. Dose response of donor SCs and the timing of administration necessary for different effects of bortezomib on aGVHD. (A, B) Lethally irradiated BALB/c (H2d) recipients of 10 million B6 (H2b) BM and 20 million (A) or 10 million (B) SCs were treated with 0.25 mg/kg per dose of bortezomib (:) or vehicle control (phosphate-buffered saline [PBS]) (-) daily from day 0 through day þ2 post-BMT. (C) Lethally irradiated BALB/c recipients of 10 million B6 BM and 5 million SCs were treated with 0.25 mg/kg per dose of bortezomib daily from day 0 through day þ2 (:) or from day þ6 through day þ8 (6) post-BMT, or treated with vehicle control (PBS) (-). Lethally irradiated BALB/c recipients of 10 million B6 BM were treated with 0.25 mg/kg per dose of bortezomib daily from day 0 through day þ2 () or from day þ6 through day þ8 (+) post-BMT. Data are pooled from two independent experiments. Each experiment consisted of 8 to 10 mice per group.
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More infused donor SCs induced increases in serum LPS levels and numbers of donor TNF-aþ T cells in recipient spleens Both LPS and TNF-a are important stimuli for DCs activation and maturation. Our data showed that as early as 6 hours post-BMT significant increases in serum LPS levels were observed in recipients with 10 million SCs in the modest aGVHD model compared to those with 5 million
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SCs in the mild aGVHD model (Fig. 2A). However, at this time point, there was no significant difference in serum TNF-a levels between these two models (Fig. 2B). Additionally, serum levels of LPS (Fig. 2A) and TNF-a (Fig. 2B) significantly elevated on day 6 post-BMT compared to those on day 0 post-BMT in recipients of each aGVHD model. Flow cytometry analysis showed that the frequency of donor T cells increased in the spleens
Figure 2. Low levels of serum LPS and less numbers of donor TNF-aþ T cells in the spleens observed in mild aGVHD mice. Lethally irradiated BALB/c (H2d) recipients were infused with 10 million B6 (H2b) BM, or 10 million B6 BM þ5 million SCs, or 10 million B6 BM þ10 million SCs. (A) Serum LPS levels were determined in recipients of 10 million B6 BM together with 10 million or 5 million SCs on day 0 (6 hours post-BMT) and day 6 post-BMT. (B) Serum TNF-a levels were determined in recipients of 10 million B6 BM together with 10 million or 5 million SCs on day 0 (6 hours post-BMT) and day 6 post-BMT. (CLE) At 6 hours post-BMT, spleen cells were collected from irradiated BALB/c recipients in each group, reacted with anti-CD3, anti-H2b, and antiTNF-a antibodies, and analyzed by flow cytometry. (C) Representative dot plots. (D) Frequency of donor T cells in recipient spleens. (E) Absolute number of donor TNF-aþ T cells in recipient spleens. *p ! 0.05; **p ! 0.01, data are shown as mean 6 standard deviation from three independent experiments performed in triplicate.
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of recipients with more B6 SCs infused (Fig. 2C, D) at 6 hours post-BMT. With the intracellular staining of TNF-a, the results further demonstrated that the absolute number of donor-derived TNF-aþ T cells significantly increased in the spleens of recipients with 10 million SCs compared to those with 5 million SCs (Fig. 2E).
More infused donor SCs made host splenic DCs more mature Because serum LPS levels and the number of donor TNF-aþ T cells differed in recipients with different doses of donor SCs, we were interested in the fate of host splenic DCs in recipients when more donor SCs were infused. Flow cytometry analysis demonstrated that the infusion of more donor SCs did not change the percentage and the absolute number of host DCs in recipient spleens at 8 hours post-BMT (Fig. 3A, B). To address the activation and maturation state of host splenic DCs, we investigated the phenotype of the splenic CD11chigh population by analyzing the antigen-presenting molecule MHC II expressed on these cells. Results demonstrated that host splenic DCs from irradiated mice with more donor SCs (SC 1 107) expressed increased numbers of MHC II at 8 hours post-BMT (Fig. 3C, D). When splenic DCs were isolated and cultured ex vivo overnight, DCs from irradiated mice with more donor SCs produced significantly higher levels of IL-12 than those from irradiated mice with less SCs did (Fig. 3F), consistent with the increased levels of serum IL-12 detected in these irradiated mice (Fig. 3E). Our data thus reflected that the infusion of more donor SCs made host DCs more mature in vivo.
Bortezomib inhibited imDCs maturation in response to inflammatory signals Subsequently, we undertook ex vivo experiments to determine whether the maturation state of DCs is related to the differential effects of bortezomib on DCs. Stimulation with LPS or TNF-a leads to DCs maturation [9,10], which is accompanied by marked upregulation of the costimulatory molecules CD80, CD86, and CD40, maturation antigen CD83 as well as presentation molecules MHC II [11]. Our results showed that bortezomib prevented imDCs maturation in response to LPS or TNF-a. Namely, imDCs pretreatment with bortezomib prevented upregulation of CD80, CD86, CD40, and CD83 as well as MHC II (Fig. 4B, E). The normal upregulations of integrin CD54 and chemokine receptor CXCR4 molecules in response to these inflammatory signals were slightly reduced by bortezomib. We next examined the effects of bortezomib on DCs at the matured stage (mDCs) induced by LPS or TNF-a. In contrast to pretreated imDCs, no significant difference was found in expression of surface markers on bortezomibtreated (Fig. 4C, F) vs. untreated mDCs (Fig. 4A, D).
Bortezomib induced more potent apoptosis in imDCs compared to already matured DCs A dose-dependent induction of cell apotosis was observed in both imDCs and mDCs after 24 hours treatment of bortezomib. However, apoptosis of imDCs in the presence of LPS was significantly improved after 24 hours of exposure to a low concentration of 5nM bortezomib, as was not observed in already matured DCs. Moreover, our data showed that pretreated imDCs in response to LPS were more susceptible to bortezomib-induced apoptosis than already matured DCs (Fig. 5A). Besides, similar results were obtained when using TNF-a as a maturation stimulus (Fig. 5B). Bortezomib blunted imDCs allostimulatory capacity for CD4þ T cells The most characteristic function featuring DCs is their ability to induce T-cell proliferation. Results demonstrated that pretreatment with bortezomib reduced the capacity of imDCs to induce proliferation in alloreactive CD4þ and CD8þ T cells (Fig. 6A). Given the pivotal role donor T-cellderived TNF-a plays in GVHD [26], we evaluated the TNF-a levels in supernatants from mixed lymphocyte reaction. Here, we found significant decreases in TNF-a levels from alloreactive CD4þ T cells, but not from CD8þ T cells, when cocultured with LPS-primed imDCs pretreated with bortezomib (Fig. 6B). However, bortezomib did not substantially affect mDCs’ capacity to prime allogeneic lymphocyte reaction. Significant increases in IkBa levels in imDCs after bortezomib treatment It was previously reported that bortezomib inhibited activation of the transcription factor NF-kB by preventing proteasomal degradation of IkBa, the NF-kB inhibitor in multiple myeloma [27]. To clarify if NF-kB pathway was affected in DCs, we detected IkBa levels in imDCs and mDCs treated with bortezomib for 12 and 24 hours by immunoblotting. In imDCs, pretreatment with bortezomib increased the expression level of IkBa gradually upon LPS stimulation in a time-dependent fashion, indirectly indicating an inhibition of NF-kB activity. In contrast, expression of IkBa remained unaffected in mDCs after bortezomib treatment (Fig. 7).
Discussion In the present study, we performed dose responses of donor SCs as well as different timing of administration to ascertain the effects of bortezomib on aGVHD. Our data demonstrated that protection from aGVHD by bortezomib was only observed in a mild aGVHD model with less donor SCs infused. These findings contrasted with those of Sun et al. [20], who found that bortezomib treatment could decrease GVHD mortality in an aggressive model of
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Figure 3. More infused donor SCs made host splenic DCs more mature. Lethally irradiated BALB/c (H2d) recipients were infused with 10 million B6 (H2b) BM, or 10 million B6 BM þ5 million SCs, or 10 million B6 BM þ10 million SCs. At 8 hours post-BMT, spleen cells and serum were isolated from irradiated BALB/c recipients in each group. (A) Percentage and (B) absolute number of host splenic DCs. (C) Expression of MHC II on host splenic DCs was analyzed by flow cytometry. Isotype-matched IgG was used as control. (D) The mean fluorescence intensity (MFI) of MHC II on host splenic DCs. (E) IL-12 in the serum from irradiated BALB/c recipients in each group was examined by enzyme-linked immunosorbent assay (ELISA). (F) IL-12 in the supernatants of cultured splenic DCs was examined by ELISA. Splenic DCs were separately purified from irradiated BALB/c recipients in each group at 8 hours post-BMT as described in Materials and Methods. Supernatants were collected from the cultures of DCs overnight. *p ! 0.05; **p ! 0.01, results are presented as mean 6 standard deviation from three independent experiments performed in triplicate.
aGVHD in which all control mice died within 10 days. Although the exact explanation for these disparate results is unclear, certain differences between the studies are noteworthy. BALB/c mice were used as recipients to receive allografts from B6 in our study, while Sun et al. utilized B6 recipients receiving allografts from BALB/c mice. These contrast phenomena can be related to interstrain differences in their genetic background. For example,
Chen et al. found that B6 mice have a substantially lower frequency of CD4þCD25þ T-regulatory (Treg) cells in the peripheral lymphoid tissues, including spleens, than BALB/c mice [28]. It has been clearly established that the immunosuppressive effect of Treg cells could be demonstrated by inhibition of CD4þCD25 T-cell proliferation in vitro. B6 Treg cells are not only lower in number but also less able to suppress syngeneic CD4þCD25
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Figure 4. Effects of bortezomib on phenotypic changes in DCs. imDCs were prepared from mouse BM progenitors by culturing in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 6 days. imDCs were then incubated in the presence of 5 nM bortezomib (B, E) or medium alone (A, D) for 24 hours. LPS (10 ng/mL) or TNF-a (20 ng/mL) was added during the last 16 hours where indicated in the figure. To produce already matured DCs (mDCs), imDCs were stimulated with LPS (10 ng/mL) (C) or TNF-a (20 ng/mL) (F) for 24 hours. mDCs were then washed with phosphate-buffered saline and then cultured in the presence of 5 nM bortezomib for another 24 hours where indicated in the figure. Thereafter, cells were stained with the designated monoclonal antibody and analyzed by flow cytometry. Matched isotype controls are presented as solid histograms. Results are representative of three independent experiments.
T-responder cells than Treg cells from BALB/c mice. In addition, CD4þCD25 T-responder cells from B6 mice were notably more resistant than those from BALB/c mice to inhibition by Treg cells from either mouse strain [28]. Maybe it is one of the reasons why fewer B6 SCs are needed to induce similar severity of aGVHD in BALB/c mice. Besides, it has been shown that activation of donor T cells in the spleen by host APCs is complete by 24 hours after BMT [29]. Thus, in our study, once activated by host mDCs after infusion into BALB/c recipients, B6 T-cell proliferation might be refractory to suppression by Treg cells later on. So the successful intervention phase for aGVHD in this model might be brief, possibly just before donor T-cell activation or even before host DCs maturation. Furthermore, differences in susceptibility to bortezomib between two strains of mice should not be neglected. Compared to B6 mice, BALB/c mice proved to be
more susceptible to bortezomib-induced toxicity, tolerating no more than 5 mg per dose in our study (data not shown). Additionally, consistent with previous reports [14,21], our results also suggest delayed administration of bortezomib accelerated aGVHD morbidity, indicating a narrow therapeutic window for bortezomib intervention. Zhang et al. previously reported that about 5% to 10% of all infused donor T cells were recruited into the spleen by 6 to 24 hours after transplantation [30]. Our results also demonstrated that approximately 12% to 16% of infused B6 T cells reached host spleens by 6 hours post-BMT (data not shown). Importantly, compared with mild aGVHD mice (infused with less donor SCs), more TNF-asecreting donor T cells were found in the spleens of modest aGVHD mice (infused with more donor SCs). It has been reported that during aGVHD pathophysiology, gut epithelial damage induced first by BMT conditioning and subsequently by
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Figure 5. Effects of bortezomib on apoptosis in DCs. imDCs were prepared from mouse BM progenitors by incubating in granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4containing medium. At day 6 of culture, imDCs were stimulated for 24 hours with indicated concentrations of bortezomib and were added LPS (10 ng/mL) or TNF-a (20 ng/mL) during the last 16 hours of incubation. mDCs were obtained from imDCs cocultured with LPS (10 ng/mL) or TNF-a (20 ng/mL) for 24 hours. Afterward, mDCs were stimulated with indicated concentrations of bortezomib for another 24 hours. Bortezomib induces a dose-dependent apoptosis in imDCs and mDCs. (A) Bortezomib-pretreated imDCs (6) in response to LPS were more susceptible to bortezomib-induced apoptosis than already-matured DCs (LPS-induced) (:). (B) Bortezomib-pretreated imDCs (6) in response to TNF-a were more susceptible to bortezomib-induced apoptosis than already-matured DCs (TNF-ainduced) (:). **p ! 0.01, results are shown as mean 6 standard deviation from three independent experiments.
alloreactive donor T cells allows passage of LPS from the gut lumen into the circulation [31]. Thus, it was not surprising that higher serum levels of LPS were observed in irradiated recipients infused with more donor SCs than those with less donor SCs. Based on the notion that TNFa and LPS are strong activators for DCs maturation and the pivotal role host DCs play in aGVHD initiation [4,9], we were interested in the maturation state of host DCs in recipients with different aGVHD severity. As is known, mDCs express high levels of MHC class II [11] and have a great ability to release IL-12 [32]. DCs that produce
IL-12 stimulate na€ıve allogenic CD4þ T cells to develop polarized Th1 responses [33]. Thus, the intensities of MHC II expression and production of IL-12 are good indicators for determining the DC maturation stage. Our results showed that DCs from modest aGVHD mice expressed higher levels of MHC II and secreted larger amounts of IL-12 compared to those from mild aGVHD mice as early as 8 hours post-BMT, suggesting more mature subsets of DCs. In addition, because the percentage and absolute number of host DCs did not differ between the two models, it was unlikely that the distinct effects of bortezomib were
Figure 6. Effects of bortezomib on DCs allostimulatory capacity. imDCs were prepared from mouse BM progenitors by incubating in granulocytemacrophage colony-stimulating factor (GM-CSF) and IL-4–containing medium. At day 6 of culture, cells were stimulated with 5 nM bortezomib or medium alone (phosphate-buffered saline [PBS] control) for 24 hours and were added LPS (10 ng/mL) during the last 16 hours of incubation. To produce already matured DCs (mDCs), imDCs were incubated with LPS for 24 hours to induce mDCs, which were subsequently treated with 5 nM bortezomib for another 24 hours. Thereafter, cells were harvested, washed, irradiated, and used as stimulators for purified splenic CD4þ T cells or CD8þ T cells at the ratio of 1:10 in the absence of bortezomib. (A) Proliferation was measured at day 5 by CCK-8 incorporation. Stimulation indices (SI) were calculated as described in Materials and Methods. (B) Supernatants were collected at day 5 for TNF-a determination. *p ! 0.05; **p ! 0.01, data are presented as mean 6 standard deviation from three independent experiments performed in triplicate.
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Figure 7. Effects of bortezomib on IkBa levels in DCs. imDCs were prepared from mouse BM progenitors by incubating in granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4–containing medium. At day 6 of culture, cells were treated with 5 nM bortezomib or medium alone (phosphate-buffered saline [PBS] control) for 24 hours and were stimulated with LPS (10 ng/mL) during the last 16 hours of incubation. To generate already matured DCs (mDCs), imDCs were incubated with LPS for 24 hours to induce mDCs, which were subsequently treated with 5 nM bortezomib or medium alone for another 24 hours. Immunoblot analysis of imDCs and mDCs after treatment with bortezomib at the indicated time points was performed using antiIkBa monoclonal antibodies (mAb). Immunoblot was probed with antib-actin mAb to compare protein loading within samples. One representative experiment out of three is presented (left). The ratio of band intensities of IkBa to b-actin was obtained as relative IkBa protein level indicated (right). **p ! 0.01, data are shown as mean 6 standard deviation from three separate experiments.
attributed to different ratios between drug doses and target cells. So far, we hypothesized that changes in the levels of pathogenic component LPS and the numbers of TNFasecreting donor T cells after allogeneic BMT resulted in different maturation states of host DCs, which might be responsible for the distinct effects of bortezomib in aGVHD models with different severity. The mechanism by which the proteasome inhibitor affects aGVHD is not definitively known. This study was aimed at defining a possible explanation by examining whether this compound exerts its effects on DCs at different maturation stage. Data on plasma levels of bortezomib in patients with advanced solid tumors have shown peak concentrations ranging between 10 and 100 nM [34]. In addition, the minimum effective dose Sun et al. utilized in a murine study in vitro was 4 nM [20]. Therefore, the concentrations (i.e., 520 nM) we used in the present study match clinically and experimentally effective doses. The present in vitro study demonstrated that bortezomib could inhibit phenotypic maturation of imDCs, providing a basis for the immunosuppressive effects, given the key role of costimulatory signals and antigen presentation for optimal T-cell activation [35]. As expected, DC-primed T-cell response was significantly reduced for pretreated imDCs, which was proven by decreases in allogeneic T-cell proliferation and TNF-a production. These findings are in alignment with similar roles bortezomib played in human imDCs [36,37]. As compared to mDCs, imDCs show more susceptibility to bortezomib-induced inhibition of phenotypic and functional maturation as well as apoptosis, similar to a previous report demonstrating differential effects of bortezomib on human monocyte-derived imDCs and mDCs [38]. Notably, bortezomib inhibited TNF-a secretion
mostly in CD4þ T cells, consistent with previous findings that TNF-a blockade had a highly significant effect (100% survival rate) on CD4þ T-cell, but not on CD8þ T-cellmediated aGVHD [39]. Therefore, in vitro experimental results suggest that bortezomib may improve aGVHD by reducing interactions between host imDCs and donor T cells in secondary lymphoid organs, thus resulting in suppression of T-cellmediated immune responses. Given the differential capacity of bortezomib to interfere with aGVHD in vivo and DCs in vitro, it is possible that under the prerequisite of relatively imDCs, which is the case in early bortezomib administration in mild aGVHD models, bortezomib could protect mice from aGVHD by inhibiting maturation and function of imDCs in response to maturation stimuli. On the other hand, in the case of mature state of host DCs that were observed in modest or even severe aGVHD models, early bortezomib treatment failed to improve aGVHD due to its inability to affect mDCs phenotypically and functionally. Pharmacokinetics analysis showed proteasome function returned to normal by 72 hours after bortezomib treatment. And it has been reported that host APCs, rather than donor APCs, are responsible for the first stage of aGVHD (from day 0 to day 7 post-BMT) [40]. Thus, host DCs were most likely to be targeted by bortezomib during early administration (from day 0 through day þ2) post-BMT in our study. Additionally, our data showed systemic LPS and TNF-a increased by day 6 after BMT, indicating ongoing aGVHD. Systemic inflammation has been demonstrated to induce the disappearance of activated DCs within 24 to 48 hours in the T-cell areas of spleen [11,41]. We observed that the absolute number of host splenic CD11cþ DCs began to decline by 6 hours after
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TBI and by day 6 nearly all host DCs in the spleen were eliminated, consistent with findings from Zhang et al. [30]. Considering the inability of delayed administration of bortezomib to improve aGVHD, it is possible that there are few host DCs left to be affected by bortezomib. Furthermore, the gut as a major GVHD target organ has been demonstrated to be highly sensitive to injury by TNFa [26]. And it has been shown that proteasome inhibition can enhance TNF-ainduced apoptosis in gastric epithelial cells [42], partly explaining an increase in mortality with delayed bortezomib administration in our study. Furthermore, we were interested in the intracellular mechanism specifically responsible for the differential effects of bortezomib on immature vs. mature DCs. Bortezomib has been shown to inhibit NF-kB activity in multiple myeloma cells by blocking IkBa degradation [27]. Additionally, Yoshimura et al. study found that NF-kB was an effective target for blocking DC antigen presentation and inhibiting T-celldependent immune responses [16], suggesting that NF-kB pathway may be differentially regulated by bortezomib in the two subtypes of DCs. This notion was supported by our findings that increased IkBa expression was observed in bortezomib-pretreated imDCs in response to LPS, whereas the levels of IkBa were not affected in mDCs. Degradation of IkBa in the proteasome leads to activation and translocation of released NF-kB to the nucleus [43]. Thus, the increased IkBa protein level in imDCs treated with bortezomib indicated an inhibition of NF-kB activity, while unchanged IkBa expression in mDCs implied unaffected NF-kB activity. Because active protein synthesis was found to represent an upstream prerequisite for DC apoptosis induced by bortezomib [44], it could be speculated that observed changes in imDCs with bortezomib may be due to active NF-kBdependent protein synthesis in imDCs upon maturation stimuli. The immunomodulatory properties of bortezomib we observed in the aGVHD model implied that caution should be taken when applying this compound for aGVHD prevention in a twice-weekly schedule, as in the treatment of multiple myeloma. Monitoring TNF-a, LPS, or the maturation stage of DC in recipients before intervention is possibly beneficial for the positive roles bortezomib played for aGVHD control. In a phase I clinical trial, bortezomib proved effective in ameliorating aGVHD in combination with tacrolimus and methotrexate after reduced-intensity conditioning allogeneic stem cell transplantation [45], implying combination regimens including immunosuppressive agents probably retained immature stage of DC in recipients during bortezomib administration. We should acknowledge that apart from the intervention of host DCs, there are other plausible mechanisms involved in the positive actions of bortezomib in aGVHD. A lowering or dampening of the donor T-cell responses has been demonstrated as one of those [20], although the whole picture of its mechanisms awaits further determination. In conclusion, proteasome inhibitor bortezo-
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mib, as a double-edged sword in aGVHD, could confer protection from aGVHD as a result, in part, of interfering with host DCs at immature stage but not at mature stage. Our data provide new insights into the mechanisms underlying the differential effects of bortezomib on aGVHD, suggesting the importance of manipulating host DCs at an immature stage for therapeutic application.
Acknowledgments This work is supported by grants from the National Natural Science Foundation of China (No. 30871100, 81090110) and Science and Technology Commission of Shanghai Municipality, China (No. 05DZ19327, 08JC1406500) to J.W. The authors would like to thank Lu Gao for technical assistance.
Conflict of interest disclosure No financial interest/relationships with financial interest relating to the topic of this article have been declared.
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