树突状细胞在硼替佐米对急性移植物抗宿主病的不同效应中起重要作用
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摘要
异基因造血干细胞移植是治愈血液系统恶性疾病的根本治愈方法。但是,其严重并发症移植物抗宿主病(GVHD)限制了造血干细胞移植的广泛应用。众所周知,急性GVHD (aGVHD)是一炎性反应过程,主要由供体T淋巴细胞攻击受体组织造成。抗原递呈细胞,尤其是宿主树突状细胞(DC)在aGVHD的启动中发挥重要作用。异源性供体T淋巴细胞的活化在很大程度上取决于预处理时或预处理后受体体内DC的活化状态。未成熟DC (imDCs)主要功能是抗原摄取,但是抗原递呈能力比较弱,因此活化T淋巴细胞能力比较弱。而在病原体来源的细菌脂多糖(LPS)或内源性炎症因子肿瘤坏死因子-α(TNF-α)刺激下,imDCs分化为成熟DC(mDCs),迁移到淋巴组织中发挥效应。mDCs具有较强的抗原递呈能力和活化T淋巴细胞功能。NF-κB是广泛分布在几乎所有细胞中的转录因子,它能控制与免疫及炎症反应相关的基因表达,而这些基因与aGVHD病理过程密切相关。值得注意的是,刺激DC成熟的信号也能活化NF-κB,提示我们NF-κB信号通路可能参与DC成熟过程。
硼替佐米(PS-341;Velcade)是一个可逆性蛋白酶体抑制剂,它有很多生物学功能,其中包括阻断NF-κB活化。硼替佐米阻断NF-κB的能力,使其在aGVHD防治中具有潜在价值。动物模型和部分临床资料的结果显示,硼替佐米能减轻aGVHD。但是,在动物模型中发现,骨髓移植后如果延迟给予硼替佐米反而加重aGVHD。
因此,为明确究竟在什么条件下,硼替佐米能改善aGVHD从而扩大其临床应用价值,我们通过建立了一系列不同严重程度的小鼠aGVHD模型的方法,并选择移植后不同时间给予硼替佐米,观察硼替佐米对aGVHD的影响及涉及机制。以下是主要研究方法和结果：
一.早期硼替佐米干预能缓解轻微aGVHD模型中小鼠aGVHD表现
方法：通过给致死性照射后的BALB/c小鼠输注递减剂量的供体C57BL/6小鼠脾脏细胞(SC)2×107、1×107、5×106,分别建立严重、中等严重和轻微程度小鼠aGVHD模型。在不同模型中,硼替佐米选择在移植后d0-2或d6-8腹腔注射,通过观察移植后小鼠生存时间、体重变化、靶器官病理损害、供体T淋巴细胞增殖、血清炎性因子表达等,分析硼替佐米对小鼠aGVHD影响。
结果：
(一)硼替佐米延长轻微aGVHD模型中小鼠生存时间和恢复小鼠体重硼替佐米在移植后早期干预(移植后d0-2),并不能改善严重(SC 2×107)及中等严重(SC1×107) aGVHD模型中小鼠生存时间(P＞0.05)。然而,当供体SC减少至5×106以进一步减轻aGVHD程度时,硼替佐米显著延长轻微aGVHD模型(SC 5×106)中小鼠生存时间,移植后d52仍有70%小鼠存活(P=0.016)。而且,移植后d26小鼠体重开始逐渐恢复。但是,即使在这种轻微aGVHD模型中,延迟硼替佐米干预(移植后d6-8)反而增加aGVHD小鼠死亡(平均生存天数：aGVHD组26天,干预组7天)。然而,仅接受骨髓和硼替佐米(移植后d0-2或d6-8干预)的小鼠与单纯接受骨髓的小鼠一样,都能长期存活。
(二)硼替佐米改善aGVHD小鼠小肠的病理损害病理分析通过H&E染色表明,硼替佐米干预明显改善aGVHD小鼠小肠粘膜的病理损害。显微镜下病理结构显示,aGVHD小鼠小肠粘膜层绒毛结构紊乱,坏死脱落,隐窝细胞增生,粘膜和粘膜下层明显水肿、充血,伴大量淋巴细胞浸润；硼替佐米干预后急性GVHD小鼠小肠粘膜各层结构基本完整,绒毛轻度脱落,隐窝细胞无明显增生,粘膜和粘膜下层无明显淋巴细胞浸润。
(三)硼替佐米降低aGVHD小鼠体内供体T淋巴细胞增殖硼替佐米对于供体T淋巴细胞的嵌合率无影响,因为硼替佐米干预后aGVHD小鼠与对照aGVHD小鼠相比,均在移植后d+15外周血中供体T淋巴细胞＞90%,在移植后d+30供体T淋巴细胞＞99%。结果显示,当移植后d3,CD4+T淋巴细胞数：aGVHD组(14.20±1.32)×105,干预组(6.26±0.67)×105；CD8+T淋巴细胞数：aGVHD组(7.82±0.72)×105,干预组(1.44±0.15)×105。当移植后d5,CD4+T淋巴细胞数：aGVHD组(21.25±1.77)×105,干预组(3.44±0.22)×105；CD8+T淋巴细胞数：aGVHD组(60.29±5.02)×105,干预组(13.06±0.85)×105。当移植后d7,CD4+T淋巴细胞数：aGVHD组(47.82±2.81)×105,干预组(15.69±1.95)×105；CD8+T淋巴细胞数：aGVHD组(56.72±3.34)×105,干预组(19.03±2.36)×105。以上结果表明,在移植后d+3、+5和+7,硼替佐米干预明显降低aGVHD小鼠体内供体CD4+或CD8+T淋巴细胞数目(P＜0.01)。
(四)硼替佐米降低aGVHD小鼠体内Th1炎症因子水平,而升高Th2炎症因子水平移植后d7,TNF-α水平：aGVHD组(65.83±9.63)pg/ml,干预组(27.41±4.79)pg／ml；IFN-γ水平：aGVHD组(665.54±36.79)pg／ml,干预组(468.52±27.42)pg／ml；IL-4水平：aGVHD组(178.01±11.66)pg／ml,,干预组(225.36±14.61)pg／ml；IL-10水平：aGVHD组(151.42±10.22)pg／ml,,干预组(220.65±33.80)pg／ml；以上结果表明,硼替佐米干预明显降低aGVHD小鼠体内Th1炎症因子TNF-α和IFN-γ水平(P＜0.01),明显升高aGVHD小鼠体内Th2炎症因子IL-4和IL-10水平(P＜0.05)。
二.硼替佐米依赖的aGVHD改善与干预前低水平的TNF-α、LPS以及相对不成熟的宿主DC状态有关
方法：在中等严重或轻微程度小鼠aGVHD模型中(SC 1×107,5×106),通过酶联免疫法检测(ELISA)硼替佐米干预前血清和组织中TNF-α水平,及鲎试剂法检测血清中LPS水平,流式细胞术检测脾脏中表达TNF-α的供体T淋巴细胞数目和宿主树突状细胞(DC)表面MHCⅡ类分子表达。
结果：
(一)硼替佐米干预前血清LPS水平分析无论在轻微aGVHD模型中或是中等严重aGVHD模型中,移植后d6受体小鼠血清LPS水平(SC 5×106组,2.53±0.24 EU／ml:SC 1×107组,8.14±1.17 EU／ml)都明显高于移植后d0(SC 5×106组,0.68±0.03 EU／ml;SC 1×107组,1.13±0.04 EU／ml)(P<0.01).另外,移植后d0,中等严重aGVHD模型中小鼠血清LPS水平高于轻微aGVHD模型中小鼠(P＜0.05)。
(二)硼替佐米干预前血清TNF-α水平分析无论在轻微aGVHD模型中或是中等严重aGVHD模型中,移植后d6受体小鼠血清TNF-α水平(SC 5×106组,54.85±10.51 pg／ml；SC 1×107组,76.55±7.18pg／m1)都明显高于移植后d0(SC 5×106组,10.42±2.22 pg／m1；SC 1×107组,12.54±3.17 pg／m1)(P<0.01).但是,移植后d0,轻微aGVHD模型和中等严重aGVHD模型中小鼠血清TNF-α水平无明显差别(P＞0.05)。
(三)硼替佐米干预前组织TNF-α水平分析移植后d2,中等严重aGVHD模型中小鼠小肠组织中TNF-α水平(603.34±59.05pg/ml)明显高于轻微aGVHD模型中小鼠(399.63±26.19 pg/ml) (P＜0.01)。
(四)硼替佐米干预前表达TNF-α的供体T淋巴细胞数目移植后12小时,中等严重aGVHD模型中受体小鼠脾细胞中表达TNF-α供体T淋巴细胞数目(87.48±12.63)×104明显高于轻微aGVHD模型中小鼠(33.05±3.41)×104(P＜0.05)。
(五)硼替佐米干预前宿主DCs状态受体小鼠脾细胞中CD11chigh/H-2b-群细胞被认为是宿主脾脏DCs。移植后18小时,中等严重aGVHD模型中受体小鼠宿主脾脏DCs表面MHCⅡ分子平均荧光强度(MFI 477.5±37.43)明显高于轻微aGVHD模型中小鼠(MFI276.37±39.62) (P<0.01)。
三.硼替佐米体外能抑制未成熟DC (imDCs)表型和功能成熟,但是对已成熟DC (mDCs)无明显作用
方法：骨髓前体细胞在mGM-CSF和mIL-4条件下分化成imDCs, TNF-α或LPS将imDCs诱导成mDCs。通过流式细胞术,观察硼替佐米对不同成熟状态DC成熟表型的影响；通过Annexin V-FITC和PI双标记,观察硼替佐米对不同成熟状态DC凋亡的影响；通过混合淋巴细胞反应(MLR),观察硼替佐米对不同成熟状态DC刺激异基因T淋巴细胞能力及调节性T细胞(Tregs)的影响；通过Western-blot,观察硼替佐米对不同成熟状态DC中IκBα表达水平的影响。
结果：
(一)硼替佐米对DCs表型影响硼替佐米阻断LPS或TNF-α.诱导的imDCs成熟。即硼替佐米能阻断CD80、CD86、CD40、CD83和MHC-Ⅱ上调表达,轻度下调粘附分子CD54和趋化因子受体CXCR4表达。然而,硼替佐米对于mDCs表面这些成熟表型表达下调作用不明显。
(二)硼替佐米对DCs凋亡影响硼替佐米以剂量依赖方式诱导imDCs和mDCs凋亡。但是,当以LPS为成熟刺激剂时,与mDCs比较, imDCs对硼替佐米诱导的凋亡更加敏感。(0nM,imDC14.30±0.98, mDC13.33±0.78; 5nM, imDC 23.24±2.00, mDC16.31±1.55; 10nM, imDC31.83±2.10, mDC22.67±1.66; 20nM, imDC 39.87±2.21, mDC 27.33±2.49) (P<0.01)。当以TNF-α为成熟刺激剂时,观察到相似的结果(OnM, imDC14.29±1.51, mDC14.63±1.42; 5nM, imDC23.51±2.10, mDC 16.39±1.69; 10nM, imDC 34.43±2.57, mDC 25.23±2.30; 20nM, imDC 41.58±3.52, mDC28.74±2.57) (P<0.01)。
(三)硼替佐米对DCs刺激同种异基因T细胞能力影响低浓度硼替佐米(5nM)明显降低imDCs刺激同种异基因CD4+T(刺激指数SI：干预组20.12±1.45,对照组35.4±1.07)(P＜0.01)和CD8+T淋巴细胞增殖(刺激指数：干预组7.35±1.67,对照组18.19±0.40)(P＜0.05)。而且,混合淋巴细胞反应中,CD4+T淋巴细胞分泌的TNF-α水平也明显降低(干预组290.46±57.79,对照组867.23±110.30)(P＜0.01),但是CD8+T淋巴细胞分泌的TNF-α水平没有明显变化(干预组145.04±34.43,对照组154.85±29.95)(P＞0.05)。而且,硼替佐米预处理后的imDC使CD4+T淋巴细胞中Tregs比例显著增加(imDC组22.00±3.75%,mDC组11.89±1.89%)(P＜0.05)。但是,低浓度硼替佐米对于已经成熟的mDCs刺激T淋巴细胞的增殖能力和分泌TNF-α水平没有明显变化。
(四)硼替佐米对DCs中NF-κB活性影响当以LPS为成熟刺激剂,硼替佐米以时间依赖方式逐渐增加imDCs中1κB-α表达(IκB-α/β-actin:12h干预组26.71±3.08,对照组10.64±1.36；24h干预组35.28±3.69,对照组6.04±1.29)(P＜0.01),间接表明其抑制NF-κB活性。然而,mDCs中IκB-α表达在硼替佐米作用后没有明显变化(IκB-α/β-actin:12h干预组11.02±1.47,对照组11.07±1.79；24h干预组9.58±1.47,对照组10.48±2.52)(P＞0.05)。
结论：蛋白酶体抑制剂硼替佐米在aGVHD防治中是一把双刃剑。早期硼替佐米干预能改善轻微aGVHD,可能是通过干预受体小鼠体内的imDC,而非已经成熟的mDC。本研究对硼替佐米干预的免疫学和药理学有了崭新认识,同时也提示在实验和临床应用方面,imDC可作为硼替佐米调控的靶细胞。
Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for many malignant and non-malignant hematological diseases. However, graft-versus-host disease (GVHD) remains a lethal complication that limits its further applications. Acute GVHD (aGVHD) is a inflammatory response of donor T cells against host tissues. Antigen Presenting Cells (APCs), specifically host DCs, play a central role in the initiation of aGVHD. 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. Immature DCs (imDCs) are specialized for antigen capture but with low APC ability, resulting in less T cell activation. Whereas, mature DCs (mDCs) upon maturation signals such as pathogen-derived products LPS or endogenous inflammatory cytokine TNF-α, migrate to the lymphoid tissue with increased APC ability and T-cell activation potential. NF-κB, a widespread transcription factor in virtually all cell types, controls the expressions of a number of genes important for immune and inflammatory responses characteristic of GVHD pathophysiology. Interestingly, signals that induce DC maturation are also strong activators of NF-κB, suggesting that NF-κB may be involved in DC maturation.
Bortezomib (PS-341; Velcade), a reversible proteasome inhibitor, has been demonstrated to exert numerous biological effects including the blockade of NF-κB activation. The ability to inhibit NF-κB pathway make bortezomib a potentially attractive option for the prevention of GVHD. Evidence from the animal models as well as clinical data indicates a potential role for bortezomib in the treatment of GVHD. However, contradictory results of GVHD aggravation were observed in murine models with delayed bortezomib administration after bone marrow transplantation (BMT).
Thus, to specify the conditions under which bortezomib improves GVHD, we set up aGVHD murine models of different severity fulfilled by infusion of decreasing donor splenocytes (SCs) and chose the different timing of bortezomib administration. The following are the main methods we performed and the major results we obtained in the experiments.
Ⅰ. Early bortezomib administration protected mice form aGVHD in a mild aGVHD model
Methods:Murine aGVHD models with different severity were set up by infusing lethally irradiated BALB/c with decreasing doses of donor C57BL/6 SCs (2×107,1×107,5×106). In each model, bortezomib was administered from dayO to day2 or from day6 to day8 post-BMT. We observed the effects of bortezomib on aGVHD by means of survival, weight changes, pathological impairment of target organs, donor T cell proliferation and levels of proinflamatory cytokines.
Results:
1. Bortezomib prolonged survival and improved weight loss in mice with mild aGVHD Early bortezomib administration (day0-2 post-BMT) imposed no effects on survival improvement in either an aggressive model (SC 2×107) or a modest model (SC 1×107) of aGVHD. In contrast, when the number of donor SCs was decreased to further reduce the severity of the GVHD (SC 5×106), significant (P=0.016) increases in survival were observed, with more than 70% of mice becoming long-term survivors. Bortezomib also improved weight loss of aGVHD mice from day26 post-BMT. But, even in this mild model, delaying the administration of bortezomib (day6-8 post-BMT) resulted in significantly greater mortality than did PBS treatment of GVHD control animals (Median survival:26 days for aGVHD group,7days for intervention group). Notably, animals that underwent transplantation with BM alone and then were treated with bortezomib at early or later time post-BMT had long-term survival similar to those of untreated control mice.
2. Bortezomib improved pathological changes in the small intestine of aGVHD mice Histologic analysis show reduced damage to the small intestines of bortezomib-treated animals compared with PBS-treated GVHD animals. Microscopic examination revealed obvious villous blunting and fusion, crypt-cell hyperplasia and apoptosis, along with inflammatory infiltrates in the small intestine from PBS-treated GVHD control mice. While, animals administered with bortezomib had modest small intestine injury with only mild villous blunting and few inflammatory infiltrates.
3. Bortezomib resulted in reduced donor T cell proliferation in aGVHD mice Mice receiving donor BM and SCs with or without bortezomib had>90% donor T cells at day 15 and>99% donor T cells at day 30 post-BMT, suggesting that donor T cell chimerism was not adversely affected. Our results showed, at day 3 post-BMT, CD4+ T cell number was (14.20±1.32)×105 for aGVHD group, (6.26±0.67)×105 for intervention group. CD8+ T cell number was (7.82±0.72)×105 for aGVHD group, (1.44±0.15)×105 for intervention group. At day 5 post-BMT, CD4+ T cell number was (21.25±1.77)×105 for aGVHD group, (3.44±0.22)×105 for intervention group. CD8+ T cell number was (60.29±5.02)×105 for aGVHD group, (13.06±0.85)xl05 for intervention group. At day 7 post-BMT, CD4+ T cell number was (47.82±2.81)×105 for aGVHD group, (15.69±1.95)×105 for intervention group. CD8+T cell number was (56.72±3.34)×105 for aGVHD group, (19.03±2.36)×105 for intervention group. Thus, bortezomib treatment resulted in significantly less donor CD4+ and CD8+ T cell proliferation at day+3,+5 and +7 post-BMT (P<0.01).
4. Bortezomib induced decreases in Thl proinflamatory cytokines and increases in Th2 proinflamatory cytokines in aGVHD mice On day 7 post-BMT, TNF-αlevel was (65.83±9.63) pg/ml for aGVHD group, (27.41±4.79)pg/ml for intervention group. IFN-γlevel was (665.54±36.79) pg/ml for aGVHD group, (468.52±27.42) pg/ml for intervention group. IL-4 level was (178.01±11.66) pg/ml for aGVHD group, (225.36±14.61) pg/ml for intervention group. IL-10 level was (151.42±10.22) pg/ml for aGVHD group, (220.65±33.80) pg/ml for intervention group. Thus, serum levels of the TNF-α, IFN-γwere both significantly (P<0.01) decreased and serum levels of the IL-4, IL-10 were both significantly (P<0.05) increased in the recipients receiving SCs and bortezomib treatment compared with recipients receiving SCs and PBS.
Ⅱ. Improved aGVHD by bortezomib correlates with low levels of TNF-αand LPS as well as immature state of host DCs before administration
Methods:In murine aGVHD models with different severity (SC 1×107,5×106) before bortezomib administration, TNF-αlevels in the sera and tissues were assayed by ELISA kit. Determination of serum LPS levels was performed by Tachypleus Limulus Amebocyte Lysate (LAL) assay. Flow cytometry was used to determine the number of splenic donor T cells expressing TNF-αand analyse MHCⅡexpression on host DCs.
Results:
1. Analysis of serum LPS level before bortezomib intervention Increased serum LPS levels were observed in recipients on day+6 post-BMT (2.53±0.24 EU/ml for SC 5×106 group,8.14±1.17 EU/ml for SC 1×107 group) compared with those on d0 post-BMT (0.68±0.03 EU/ml for SC 5×106 group, 1.13±0.04 EU/ml for SC 1×107 group) in each model (P<0.01). Besides, on d0 post-BMT, significantly higher serum LPS levels were observed in recipients with 1×107 donor SCs compared to those with 5×106 SC (P<0.05).
2. Analysis of serum TNF-αlevel before bortezomib intervention Increased serum TNF-αlevels were observed in recipients on day+6 post-BMT (54.85±10.51 pg/ml for SC 5×106group,76.55±7.18pg/ml for SC 1×107group) compared with those on d0 post-BMT (10.42±2.22 pg/ml for SC 5×106 group, 12.54±3.17 pg/ml for SC 1×107group) in each model (P<0.01). However, there was no difference in serum TNF-αlevel at d0 between the two models (P>0.05)
3. Analysis of localized TNF-αlevel before bortezomib intervention Significant increases in localized TNF-a in the small intestine were observed in recipients with 1×107donor SCs (603.34±59.05 pg/ml) compared with those with 5×106 SCs (399.63±26.19 pg/ml) on day+2 post-BMT(P<0.01).
4. Analysis of the number of splenic donor T cells expressing TNF-αbefore bortezomib intervention Significant increases in the number of splenic donor T cells expressing TNF-αwere observed in recipients with 1×107 donor SCs (87.48±12.63)×104 compared with those with 5×106 SCs (33.05±3.41)×104 at 12 hours post-BMT (P<0.05).
5. Analysis of the state of host DCs before bortezomib intervention CDllchigh/H-2b- population of DCs was gated as host splenic DCs. The mean fluorescence intensity (MFI) of MHCⅡin host splenic DCs from recipients with 1×107 donor SCs (MFI 477.5±37.43) was significantly higher than that from recipients with 5×106 SC (MFI 276.37±39.62) at 18 hours post-BMT (P<0.01).
Ⅲ. Bortezomib inhibited the phenotypic and functional maturation of imDCs rather than already mDCs in vitro
Methods:imDCs were prepared from mouse BM progenitors by culturing in the presence of GM-CSF and IL-4. To produce already matured DCs (mDCs), imDCs were stimulated with LPS or TNF-a. To observe the effects of bortezomib on DCs with different maturation stage, we performed flow cytometry to assay phenotypes, applied Annexin V-FITC/PI to analyze apoptosis, conducted mixed lymphocyte reaction (MLR) to assay allostimulatory capacity of DCs and impacts on regulatory T cells (Tregs), used western-blot to study the expression of IκBαin DCs.
Results:
1. Effects of bortezomib on phenotypic changes in DCs Bortezomib prevented imDCs maturation in response to LPS or TNF-α. Namely, imDCs pretreatment with the proteasome inhibitor prevented the up-regulation CD80, CD86, CD40 and CD83 as well as MHC-Ⅱ. The normal up-regulation of integrin CD54 and chemokine receptor CXCR4 molecules in response to these inflammatory signals was slightly reduced by the proteasome inhibitor. In contrast to pre-treated imDCs, no significant difference was found regarding the expression of surface markers on bortezomib treated versus untreated mDCs.
2. Effects of bortezomib on apoptosis in DCs A dose-dependent induction of cell apoptosis in both imDCs in response to LPS and already mDCs (LPS-induced) was observed with bortezomib treatment. However, pre-treated imDCs in response to LPS were more susceptible to bortezomib-induced apoptosis than already matured DCs (LPS-induced) (OnM, imDC14.30±0.98, mDC13.33±0.78; 5nM, imDC 23.24±2.00, mDC16.31±1.55;10nM, imDC 31.83±2.10, mDC22.67±1.66; 20nM, imDC 39.87±2.21, mDC 27.33±2.49) (P <0.01). Similar results were obtained when using TNF-a as a maturation stimulus (OnM, imDC14.29±1.51, mDC 14.63±1.42; 5nM, imDC 23.51±2.10, mDC 16.39±1.69;10nM, imDC 34.43±2.57, mDC 25.23±2.30; 20nM, imDC 41.58±3.52, mDC28.74±2.57) (P＜0.01)
3. Effects of bortezomib on DCs allostimulatory capacity 5nM bortezomib reduced the capacity of imDCs to induce proliferation in alloreactive CD4+ T cells (Stimulation indices were 20.12±1.45 for intervention group,35.44±1.07 for control group) (P<0.01) and CD8+ T cells (Stimulation indices were 7.35±1.67 for intervention group,18.19±0.40 for control group) (P< 0.05). Additionally, significant decreases in TNF-αin alloreactive CD4+ T cells (290.46±57.79 for intervention group,867.23±110.30 for control group) (P<0.01), but not in CD8+ T cells (145.04±34.43 for intervention group,154.85±29.95 for control group) (P>0.05), when co-cultured with LPS-primed imDCs pretreated with bortezomib. Further, significant increases were observed in Tregs in CD4+ T cells when cocultured with bortezomib-pretreated imDCs (22.00±3.75% for imDCs group, 11.89±1.89% for mDCs group) (P<0.05). However, bortezomib with the low concentration does not substantially affect mDCs capacity to prime allogeneic lymphocyte proliferation and TNF-a production.
4. Effects of bortezomib on NF-κB activity in DCs In imDCs, the pretreatment with bortezomib increased the expression level of IκB-αgradually upon LPS stimulation in a time dependent fashion (The ratio of IκB-αtoβ-actin at 12h was 26.71±3.08 for intervention group,10.64±1.36 for control group. The ratio at 24h was 35.28±3.69 for intervention group,6.04±1.29 for control group.) (P<0.01), indirectly indicating an inhibition of NF-κB activity. In contrast, the expression of IκB-αremained unaffected in mDCs after bortezomib treatment (The ratio of IκB-αtoβ-actin at 12h was 11.02±1.47 for intervention group, 11.07±1.79 for control group. The ratio at 24h was 9.58±1.47 for intervention group, 10.48±2.52 for control group.) (P>0.05).
In conclusion, proteasome inhibition bortezomib, as a double-edged sword in GVHD, could confer protection from GVHD, in part, due to interfering with DCs at immature stage but not at matured stage. These data provide new insights into the immunopharmacology of bortezomib and suggest a novel approach to the manipulation of DCs for therapeutic and experimental application.
硼替佐米(PS-341;Velcade)是一个可逆性蛋白酶体抑制剂,它有很多生物学功能,其中包括阻断NF-κB活化。硼替佐米阻断NF-κB的能力,使其在aGVHD防治中具有潜在价值。动物模型和部分临床资料的结果显示,硼替佐米能减轻aGVHD。但是,在动物模型中发现,骨髓移植后如果延迟给予硼替佐米反而加重aGVHD。
因此,为明确究竟在什么条件下,硼替佐米能改善aGVHD从而扩大其临床应用价值,我们通过建立了一系列不同严重程度的小鼠aGVHD模型的方法,并选择移植后不同时间给予硼替佐米,观察硼替佐米对aGVHD的影响及涉及机制。以下是主要研究方法和结果：
一.早期硼替佐米干预能缓解轻微aGVHD模型中小鼠aGVHD表现
方法：通过给致死性照射后的BALB/c小鼠输注递减剂量的供体C57BL/6小鼠脾脏细胞(SC)2×107、1×107、5×106,分别建立严重、中等严重和轻微程度小鼠aGVHD模型。在不同模型中,硼替佐米选择在移植后d0-2或d6-8腹腔注射,通过观察移植后小鼠生存时间、体重变化、靶器官病理损害、供体T淋巴细胞增殖、血清炎性因子表达等,分析硼替佐米对小鼠aGVHD影响。
结果：
(一)硼替佐米延长轻微aGVHD模型中小鼠生存时间和恢复小鼠体重硼替佐米在移植后早期干预(移植后d0-2),并不能改善严重(SC 2×107)及中等严重(SC1×107) aGVHD模型中小鼠生存时间(P＞0.05)。然而,当供体SC减少至5×106以进一步减轻aGVHD程度时,硼替佐米显著延长轻微aGVHD模型(SC 5×106)中小鼠生存时间,移植后d52仍有70%小鼠存活(P=0.016)。而且,移植后d26小鼠体重开始逐渐恢复。但是,即使在这种轻微aGVHD模型中,延迟硼替佐米干预(移植后d6-8)反而增加aGVHD小鼠死亡(平均生存天数：aGVHD组26天,干预组7天)。然而,仅接受骨髓和硼替佐米(移植后d0-2或d6-8干预)的小鼠与单纯接受骨髓的小鼠一样,都能长期存活。
(二)硼替佐米改善aGVHD小鼠小肠的病理损害病理分析通过H&E染色表明,硼替佐米干预明显改善aGVHD小鼠小肠粘膜的病理损害。显微镜下病理结构显示,aGVHD小鼠小肠粘膜层绒毛结构紊乱,坏死脱落,隐窝细胞增生,粘膜和粘膜下层明显水肿、充血,伴大量淋巴细胞浸润；硼替佐米干预后急性GVHD小鼠小肠粘膜各层结构基本完整,绒毛轻度脱落,隐窝细胞无明显增生,粘膜和粘膜下层无明显淋巴细胞浸润。
(三)硼替佐米降低aGVHD小鼠体内供体T淋巴细胞增殖硼替佐米对于供体T淋巴细胞的嵌合率无影响,因为硼替佐米干预后aGVHD小鼠与对照aGVHD小鼠相比,均在移植后d+15外周血中供体T淋巴细胞＞90%,在移植后d+30供体T淋巴细胞＞99%。结果显示,当移植后d3,CD4+T淋巴细胞数：aGVHD组(14.20±1.32)×105,干预组(6.26±0.67)×105；CD8+T淋巴细胞数：aGVHD组(7.82±0.72)×105,干预组(1.44±0.15)×105。当移植后d5,CD4+T淋巴细胞数：aGVHD组(21.25±1.77)×105,干预组(3.44±0.22)×105；CD8+T淋巴细胞数：aGVHD组(60.29±5.02)×105,干预组(13.06±0.85)×105。当移植后d7,CD4+T淋巴细胞数：aGVHD组(47.82±2.81)×105,干预组(15.69±1.95)×105；CD8+T淋巴细胞数：aGVHD组(56.72±3.34)×105,干预组(19.03±2.36)×105。以上结果表明,在移植后d+3、+5和+7,硼替佐米干预明显降低aGVHD小鼠体内供体CD4+或CD8+T淋巴细胞数目(P＜0.01)。
(四)硼替佐米降低aGVHD小鼠体内Th1炎症因子水平,而升高Th2炎症因子水平移植后d7,TNF-α水平：aGVHD组(65.83±9.63)pg/ml,干预组(27.41±4.79)pg／ml；IFN-γ水平：aGVHD组(665.54±36.79)pg／ml,干预组(468.52±27.42)pg／ml；IL-4水平：aGVHD组(178.01±11.66)pg／ml,,干预组(225.36±14.61)pg／ml；IL-10水平：aGVHD组(151.42±10.22)pg／ml,,干预组(220.65±33.80)pg／ml；以上结果表明,硼替佐米干预明显降低aGVHD小鼠体内Th1炎症因子TNF-α和IFN-γ水平(P＜0.01),明显升高aGVHD小鼠体内Th2炎症因子IL-4和IL-10水平(P＜0.05)。
二.硼替佐米依赖的aGVHD改善与干预前低水平的TNF-α、LPS以及相对不成熟的宿主DC状态有关
方法：在中等严重或轻微程度小鼠aGVHD模型中(SC 1×107,5×106),通过酶联免疫法检测(ELISA)硼替佐米干预前血清和组织中TNF-α水平,及鲎试剂法检测血清中LPS水平,流式细胞术检测脾脏中表达TNF-α的供体T淋巴细胞数目和宿主树突状细胞(DC)表面MHCⅡ类分子表达。
结果：
(一)硼替佐米干预前血清LPS水平分析无论在轻微aGVHD模型中或是中等严重aGVHD模型中,移植后d6受体小鼠血清LPS水平(SC 5×106组,2.53±0.24 EU／ml:SC 1×107组,8.14±1.17 EU／ml)都明显高于移植后d0(SC 5×106组,0.68±0.03 EU／ml;SC 1×107组,1.13±0.04 EU／ml)(P<0.01).另外,移植后d0,中等严重aGVHD模型中小鼠血清LPS水平高于轻微aGVHD模型中小鼠(P＜0.05)。
(二)硼替佐米干预前血清TNF-α水平分析无论在轻微aGVHD模型中或是中等严重aGVHD模型中,移植后d6受体小鼠血清TNF-α水平(SC 5×106组,54.85±10.51 pg／ml；SC 1×107组,76.55±7.18pg／m1)都明显高于移植后d0(SC 5×106组,10.42±2.22 pg／m1；SC 1×107组,12.54±3.17 pg／m1)(P<0.01).但是,移植后d0,轻微aGVHD模型和中等严重aGVHD模型中小鼠血清TNF-α水平无明显差别(P＞0.05)。
(三)硼替佐米干预前组织TNF-α水平分析移植后d2,中等严重aGVHD模型中小鼠小肠组织中TNF-α水平(603.34±59.05pg/ml)明显高于轻微aGVHD模型中小鼠(399.63±26.19 pg/ml) (P＜0.01)。
(四)硼替佐米干预前表达TNF-α的供体T淋巴细胞数目移植后12小时,中等严重aGVHD模型中受体小鼠脾细胞中表达TNF-α供体T淋巴细胞数目(87.48±12.63)×104明显高于轻微aGVHD模型中小鼠(33.05±3.41)×104(P＜0.05)。
(五)硼替佐米干预前宿主DCs状态受体小鼠脾细胞中CD11chigh/H-2b-群细胞被认为是宿主脾脏DCs。移植后18小时,中等严重aGVHD模型中受体小鼠宿主脾脏DCs表面MHCⅡ分子平均荧光强度(MFI 477.5±37.43)明显高于轻微aGVHD模型中小鼠(MFI276.37±39.62) (P<0.01)。
三.硼替佐米体外能抑制未成熟DC (imDCs)表型和功能成熟,但是对已成熟DC (mDCs)无明显作用
方法：骨髓前体细胞在mGM-CSF和mIL-4条件下分化成imDCs, TNF-α或LPS将imDCs诱导成mDCs。通过流式细胞术,观察硼替佐米对不同成熟状态DC成熟表型的影响；通过Annexin V-FITC和PI双标记,观察硼替佐米对不同成熟状态DC凋亡的影响；通过混合淋巴细胞反应(MLR),观察硼替佐米对不同成熟状态DC刺激异基因T淋巴细胞能力及调节性T细胞(Tregs)的影响；通过Western-blot,观察硼替佐米对不同成熟状态DC中IκBα表达水平的影响。
结果：
(一)硼替佐米对DCs表型影响硼替佐米阻断LPS或TNF-α.诱导的imDCs成熟。即硼替佐米能阻断CD80、CD86、CD40、CD83和MHC-Ⅱ上调表达,轻度下调粘附分子CD54和趋化因子受体CXCR4表达。然而,硼替佐米对于mDCs表面这些成熟表型表达下调作用不明显。
(二)硼替佐米对DCs凋亡影响硼替佐米以剂量依赖方式诱导imDCs和mDCs凋亡。但是,当以LPS为成熟刺激剂时,与mDCs比较, imDCs对硼替佐米诱导的凋亡更加敏感。(0nM,imDC14.30±0.98, mDC13.33±0.78; 5nM, imDC 23.24±2.00, mDC16.31±1.55; 10nM, imDC31.83±2.10, mDC22.67±1.66; 20nM, imDC 39.87±2.21, mDC 27.33±2.49) (P<0.01)。当以TNF-α为成熟刺激剂时,观察到相似的结果(OnM, imDC14.29±1.51, mDC14.63±1.42; 5nM, imDC23.51±2.10, mDC 16.39±1.69; 10nM, imDC 34.43±2.57, mDC 25.23±2.30; 20nM, imDC 41.58±3.52, mDC28.74±2.57) (P<0.01)。
(三)硼替佐米对DCs刺激同种异基因T细胞能力影响低浓度硼替佐米(5nM)明显降低imDCs刺激同种异基因CD4+T(刺激指数SI：干预组20.12±1.45,对照组35.4±1.07)(P＜0.01)和CD8+T淋巴细胞增殖(刺激指数：干预组7.35±1.67,对照组18.19±0.40)(P＜0.05)。而且,混合淋巴细胞反应中,CD4+T淋巴细胞分泌的TNF-α水平也明显降低(干预组290.46±57.79,对照组867.23±110.30)(P＜0.01),但是CD8+T淋巴细胞分泌的TNF-α水平没有明显变化(干预组145.04±34.43,对照组154.85±29.95)(P＞0.05)。而且,硼替佐米预处理后的imDC使CD4+T淋巴细胞中Tregs比例显著增加(imDC组22.00±3.75%,mDC组11.89±1.89%)(P＜0.05)。但是,低浓度硼替佐米对于已经成熟的mDCs刺激T淋巴细胞的增殖能力和分泌TNF-α水平没有明显变化。
(四)硼替佐米对DCs中NF-κB活性影响当以LPS为成熟刺激剂,硼替佐米以时间依赖方式逐渐增加imDCs中1κB-α表达(IκB-α/β-actin:12h干预组26.71±3.08,对照组10.64±1.36；24h干预组35.28±3.69,对照组6.04±1.29)(P＜0.01),间接表明其抑制NF-κB活性。然而,mDCs中IκB-α表达在硼替佐米作用后没有明显变化(IκB-α/β-actin:12h干预组11.02±1.47,对照组11.07±1.79；24h干预组9.58±1.47,对照组10.48±2.52)(P＞0.05)。
结论：蛋白酶体抑制剂硼替佐米在aGVHD防治中是一把双刃剑。早期硼替佐米干预能改善轻微aGVHD,可能是通过干预受体小鼠体内的imDC,而非已经成熟的mDC。本研究对硼替佐米干预的免疫学和药理学有了崭新认识,同时也提示在实验和临床应用方面,imDC可作为硼替佐米调控的靶细胞。
Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for many malignant and non-malignant hematological diseases. However, graft-versus-host disease (GVHD) remains a lethal complication that limits its further applications. Acute GVHD (aGVHD) is a inflammatory response of donor T cells against host tissues. Antigen Presenting Cells (APCs), specifically host DCs, play a central role in the initiation of aGVHD. 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. Immature DCs (imDCs) are specialized for antigen capture but with low APC ability, resulting in less T cell activation. Whereas, mature DCs (mDCs) upon maturation signals such as pathogen-derived products LPS or endogenous inflammatory cytokine TNF-α, migrate to the lymphoid tissue with increased APC ability and T-cell activation potential. NF-κB, a widespread transcription factor in virtually all cell types, controls the expressions of a number of genes important for immune and inflammatory responses characteristic of GVHD pathophysiology. Interestingly, signals that induce DC maturation are also strong activators of NF-κB, suggesting that NF-κB may be involved in DC maturation.
Bortezomib (PS-341; Velcade), a reversible proteasome inhibitor, has been demonstrated to exert numerous biological effects including the blockade of NF-κB activation. The ability to inhibit NF-κB pathway make bortezomib a potentially attractive option for the prevention of GVHD. Evidence from the animal models as well as clinical data indicates a potential role for bortezomib in the treatment of GVHD. However, contradictory results of GVHD aggravation were observed in murine models with delayed bortezomib administration after bone marrow transplantation (BMT).
Thus, to specify the conditions under which bortezomib improves GVHD, we set up aGVHD murine models of different severity fulfilled by infusion of decreasing donor splenocytes (SCs) and chose the different timing of bortezomib administration. The following are the main methods we performed and the major results we obtained in the experiments.
Ⅰ. Early bortezomib administration protected mice form aGVHD in a mild aGVHD model
Methods:Murine aGVHD models with different severity were set up by infusing lethally irradiated BALB/c with decreasing doses of donor C57BL/6 SCs (2×107,1×107,5×106). In each model, bortezomib was administered from dayO to day2 or from day6 to day8 post-BMT. We observed the effects of bortezomib on aGVHD by means of survival, weight changes, pathological impairment of target organs, donor T cell proliferation and levels of proinflamatory cytokines.
Results:
1. Bortezomib prolonged survival and improved weight loss in mice with mild aGVHD Early bortezomib administration (day0-2 post-BMT) imposed no effects on survival improvement in either an aggressive model (SC 2×107) or a modest model (SC 1×107) of aGVHD. In contrast, when the number of donor SCs was decreased to further reduce the severity of the GVHD (SC 5×106), significant (P=0.016) increases in survival were observed, with more than 70% of mice becoming long-term survivors. Bortezomib also improved weight loss of aGVHD mice from day26 post-BMT. But, even in this mild model, delaying the administration of bortezomib (day6-8 post-BMT) resulted in significantly greater mortality than did PBS treatment of GVHD control animals (Median survival:26 days for aGVHD group,7days for intervention group). Notably, animals that underwent transplantation with BM alone and then were treated with bortezomib at early or later time post-BMT had long-term survival similar to those of untreated control mice.
2. Bortezomib improved pathological changes in the small intestine of aGVHD mice Histologic analysis show reduced damage to the small intestines of bortezomib-treated animals compared with PBS-treated GVHD animals. Microscopic examination revealed obvious villous blunting and fusion, crypt-cell hyperplasia and apoptosis, along with inflammatory infiltrates in the small intestine from PBS-treated GVHD control mice. While, animals administered with bortezomib had modest small intestine injury with only mild villous blunting and few inflammatory infiltrates.
3. Bortezomib resulted in reduced donor T cell proliferation in aGVHD mice Mice receiving donor BM and SCs with or without bortezomib had>90% donor T cells at day 15 and>99% donor T cells at day 30 post-BMT, suggesting that donor T cell chimerism was not adversely affected. Our results showed, at day 3 post-BMT, CD4+ T cell number was (14.20±1.32)×105 for aGVHD group, (6.26±0.67)×105 for intervention group. CD8+ T cell number was (7.82±0.72)×105 for aGVHD group, (1.44±0.15)×105 for intervention group. At day 5 post-BMT, CD4+ T cell number was (21.25±1.77)×105 for aGVHD group, (3.44±0.22)×105 for intervention group. CD8+ T cell number was (60.29±5.02)×105 for aGVHD group, (13.06±0.85)xl05 for intervention group. At day 7 post-BMT, CD4+ T cell number was (47.82±2.81)×105 for aGVHD group, (15.69±1.95)×105 for intervention group. CD8+T cell number was (56.72±3.34)×105 for aGVHD group, (19.03±2.36)×105 for intervention group. Thus, bortezomib treatment resulted in significantly less donor CD4+ and CD8+ T cell proliferation at day+3,+5 and +7 post-BMT (P<0.01).
4. Bortezomib induced decreases in Thl proinflamatory cytokines and increases in Th2 proinflamatory cytokines in aGVHD mice On day 7 post-BMT, TNF-αlevel was (65.83±9.63) pg/ml for aGVHD group, (27.41±4.79)pg/ml for intervention group. IFN-γlevel was (665.54±36.79) pg/ml for aGVHD group, (468.52±27.42) pg/ml for intervention group. IL-4 level was (178.01±11.66) pg/ml for aGVHD group, (225.36±14.61) pg/ml for intervention group. IL-10 level was (151.42±10.22) pg/ml for aGVHD group, (220.65±33.80) pg/ml for intervention group. Thus, serum levels of the TNF-α, IFN-γwere both significantly (P<0.01) decreased and serum levels of the IL-4, IL-10 were both significantly (P<0.05) increased in the recipients receiving SCs and bortezomib treatment compared with recipients receiving SCs and PBS.
Ⅱ. Improved aGVHD by bortezomib correlates with low levels of TNF-αand LPS as well as immature state of host DCs before administration
Methods:In murine aGVHD models with different severity (SC 1×107,5×106) before bortezomib administration, TNF-αlevels in the sera and tissues were assayed by ELISA kit. Determination of serum LPS levels was performed by Tachypleus Limulus Amebocyte Lysate (LAL) assay. Flow cytometry was used to determine the number of splenic donor T cells expressing TNF-αand analyse MHCⅡexpression on host DCs.
Results:
1. Analysis of serum LPS level before bortezomib intervention Increased serum LPS levels were observed in recipients on day+6 post-BMT (2.53±0.24 EU/ml for SC 5×106 group,8.14±1.17 EU/ml for SC 1×107 group) compared with those on d0 post-BMT (0.68±0.03 EU/ml for SC 5×106 group, 1.13±0.04 EU/ml for SC 1×107 group) in each model (P<0.01). Besides, on d0 post-BMT, significantly higher serum LPS levels were observed in recipients with 1×107 donor SCs compared to those with 5×106 SC (P<0.05).
2. Analysis of serum TNF-αlevel before bortezomib intervention Increased serum TNF-αlevels were observed in recipients on day+6 post-BMT (54.85±10.51 pg/ml for SC 5×106group,76.55±7.18pg/ml for SC 1×107group) compared with those on d0 post-BMT (10.42±2.22 pg/ml for SC 5×106 group, 12.54±3.17 pg/ml for SC 1×107group) in each model (P<0.01). However, there was no difference in serum TNF-αlevel at d0 between the two models (P>0.05)
3. Analysis of localized TNF-αlevel before bortezomib intervention Significant increases in localized TNF-a in the small intestine were observed in recipients with 1×107donor SCs (603.34±59.05 pg/ml) compared with those with 5×106 SCs (399.63±26.19 pg/ml) on day+2 post-BMT(P<0.01).
4. Analysis of the number of splenic donor T cells expressing TNF-αbefore bortezomib intervention Significant increases in the number of splenic donor T cells expressing TNF-αwere observed in recipients with 1×107 donor SCs (87.48±12.63)×104 compared with those with 5×106 SCs (33.05±3.41)×104 at 12 hours post-BMT (P<0.05).
5. Analysis of the state of host DCs before bortezomib intervention CDllchigh/H-2b- population of DCs was gated as host splenic DCs. The mean fluorescence intensity (MFI) of MHCⅡin host splenic DCs from recipients with 1×107 donor SCs (MFI 477.5±37.43) was significantly higher than that from recipients with 5×106 SC (MFI 276.37±39.62) at 18 hours post-BMT (P<0.01).
Ⅲ. Bortezomib inhibited the phenotypic and functional maturation of imDCs rather than already mDCs in vitro
Methods:imDCs were prepared from mouse BM progenitors by culturing in the presence of GM-CSF and IL-4. To produce already matured DCs (mDCs), imDCs were stimulated with LPS or TNF-a. To observe the effects of bortezomib on DCs with different maturation stage, we performed flow cytometry to assay phenotypes, applied Annexin V-FITC/PI to analyze apoptosis, conducted mixed lymphocyte reaction (MLR) to assay allostimulatory capacity of DCs and impacts on regulatory T cells (Tregs), used western-blot to study the expression of IκBαin DCs.
Results:
1. Effects of bortezomib on phenotypic changes in DCs Bortezomib prevented imDCs maturation in response to LPS or TNF-α. Namely, imDCs pretreatment with the proteasome inhibitor prevented the up-regulation CD80, CD86, CD40 and CD83 as well as MHC-Ⅱ. The normal up-regulation of integrin CD54 and chemokine receptor CXCR4 molecules in response to these inflammatory signals was slightly reduced by the proteasome inhibitor. In contrast to pre-treated imDCs, no significant difference was found regarding the expression of surface markers on bortezomib treated versus untreated mDCs.
2. Effects of bortezomib on apoptosis in DCs A dose-dependent induction of cell apoptosis in both imDCs in response to LPS and already mDCs (LPS-induced) was observed with bortezomib treatment. However, pre-treated imDCs in response to LPS were more susceptible to bortezomib-induced apoptosis than already matured DCs (LPS-induced) (OnM, imDC14.30±0.98, mDC13.33±0.78; 5nM, imDC 23.24±2.00, mDC16.31±1.55;10nM, imDC 31.83±2.10, mDC22.67±1.66; 20nM, imDC 39.87±2.21, mDC 27.33±2.49) (P <0.01). Similar results were obtained when using TNF-a as a maturation stimulus (OnM, imDC14.29±1.51, mDC 14.63±1.42; 5nM, imDC 23.51±2.10, mDC 16.39±1.69;10nM, imDC 34.43±2.57, mDC 25.23±2.30; 20nM, imDC 41.58±3.52, mDC28.74±2.57) (P＜0.01)
3. Effects of bortezomib on DCs allostimulatory capacity 5nM bortezomib reduced the capacity of imDCs to induce proliferation in alloreactive CD4+ T cells (Stimulation indices were 20.12±1.45 for intervention group,35.44±1.07 for control group) (P<0.01) and CD8+ T cells (Stimulation indices were 7.35±1.67 for intervention group,18.19±0.40 for control group) (P< 0.05). Additionally, significant decreases in TNF-αin alloreactive CD4+ T cells (290.46±57.79 for intervention group,867.23±110.30 for control group) (P<0.01), but not in CD8+ T cells (145.04±34.43 for intervention group,154.85±29.95 for control group) (P>0.05), when co-cultured with LPS-primed imDCs pretreated with bortezomib. Further, significant increases were observed in Tregs in CD4+ T cells when cocultured with bortezomib-pretreated imDCs (22.00±3.75% for imDCs group, 11.89±1.89% for mDCs group) (P<0.05). However, bortezomib with the low concentration does not substantially affect mDCs capacity to prime allogeneic lymphocyte proliferation and TNF-a production.
4. Effects of bortezomib on NF-κB activity in DCs In imDCs, the pretreatment with bortezomib increased the expression level of IκB-αgradually upon LPS stimulation in a time dependent fashion (The ratio of IκB-αtoβ-actin at 12h was 26.71±3.08 for intervention group,10.64±1.36 for control group. The ratio at 24h was 35.28±3.69 for intervention group,6.04±1.29 for control group.) (P<0.01), indirectly indicating an inhibition of NF-κB activity. In contrast, the expression of IκB-αremained unaffected in mDCs after bortezomib treatment (The ratio of IκB-αtoβ-actin at 12h was 11.02±1.47 for intervention group, 11.07±1.79 for control group. The ratio at 24h was 9.58±1.47 for intervention group, 10.48±2.52 for control group.) (P>0.05).
In conclusion, proteasome inhibition bortezomib, as a double-edged sword in GVHD, could confer protection from GVHD, in part, due to interfering with DCs at immature stage but not at matured stage. These data provide new insights into the immunopharmacology of bortezomib and suggest a novel approach to the manipulation of DCs for therapeutic and experimental application.
引文
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