| BackgroundAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for many hematologic and genetic diseases. When donor-derived immunocompetent T cells play a part in graft-versus-leukemia/lymphoma (GVL), they may also attack the normal organs, known as graft-versus-host disease (GVHD). GVHD includes acute GVHD and chronic GVHD. Acute GVHD is a major cause of nonrelapse mortality, accounting for over 20% of transplantation related mortality (TRM). The clinical use of immunosuppression, cytotoxic drugs and in vitro/vivo T-depletion can significantly reduce the morbidity and mortality of acute GVHD, but also weaken the GVL effect, which bring out the high recurrence rate after transplantation. Many other new immunomodulators, new strategies such as agents targeting cytokines/chemokines-receptor interaction and novel approaches are currently under investigation in animal models especially mouse models. Mice models of allogeneic transplantation have been widely applied in elucidating the pathogenesis of acute GVHD and new strategies intervention in preclinical studies. The conditioning regimen of most allogeneic transplantation models are based on total body irradiation (TBI) and the reports about transplantation models based on chemotherapy are rare, in contrast to the majority of clinical conditioning regimens are combination chemotherapy.The pathophysiology of acute GVHD is known to be an immunoreaction between the immunocompetent T cells in graft and the host histoincompatible allo-antigens involving the activation of immune cells and pro-inflammatory cytokines, which has been broken down into three phases including the tissue damage-phase responding to the conditioning, the T-cell priming-phase and the target tissue distruction phase. The conditioning pre-transplantation can provide space for the donor stem cells in the hematopoietic compartment, eradicate the residual leukemia/lymphoma cells and provide immunosuppression to prevent graft failure. Meanwhile, the host tissue damage from the conditioning pre-transplantation activates the antigen-presenting cells (APCs) by producing pro-inflammatory cytokines, up-regulated expression of adhesion molecules and then promotes the development of acute GVHD. Some experimental studies have confirmed that the conditioning intensity influences the severity of acute GVHD by regulating the expression of inflammatory cytokines. Moreover, the different gene expression profile in the target organ of acute GVHD liver of TBI or BU-Cy conditioned mice has been reported. Those indicate that the acute GVHD conditioned with chemotherapy and TBI in mice are different. It’s necessary to establish a mouse model based on chemotherapy.Busulfan (BU) combined with cyclophosphamide (Cy) and fludarabine (Flu) combined with busulfan have been the two conditioning regimens most commonly applied into the clinical practice. While in the same transplantation condition is the influence of BU-Cy and Flu-BU on acute GVHD equal? While the incidence and severity of acute and chronic GVHD of the two conditioning regimens in clinical researches are conflicting, the relevant experimental report has not showed yet. We were intended to establish the murine model of allogeneic bone marrow transplantation based on the two conditioning regimens and compare the differences of them, hoping to provide a better experimental animal model for the preclinical study of acute GVHD. In our study we established the mouse model of acute GVHD, in which male BALB/c mice (H-2kd) as recipients were conditioned with BU-Cy/ Flu-BU followed by being transplanted with bone marrow cells and spleen cells from female C57BL/6 mice (H-2Kb). We evaluated the established transplanted models from many sides such as the general situation and acute GVHD clinical grading of GVHD mice, survival, histopathology, chimerism, level of T cells (CD4, CD8 cells) and B cells, cytokine levels. We also compared the differences of acute GVHD induced by the two conditioning regimens, so as to provide a better technology platform for the preclinical study in the pathogenesis and prophylaxis and treatment of acute GVHD.Methods1) As recipients male BALB/c (H-2Kd) were conditioned with different doses of Bu-Cy or Flu-BU randomly and then transplanted with bone marrow cells (2×107)-spleen cells 2×107) from donors (female C57BL/6(H-2Kb)) 48 hours later to determine the most appropriate dose.2) Established acute GVHD murine model. Recipients were divided into 8 groups randomly:Group A:normal feeding BALB/c mice in the same condition. Group B:mice injected with DMSO in the same condition. Group C:mice conditioned with BU-Cy. Group D:mice conditioned with BU-Cy and transplanted with bone marrow cells and spleen cells from syngeneic mice (BALB/c). Group E: mice conditioned with BU-Cy and transplanted with bone marrow cells and spleen cells from allogeneic mice (C57BL/6). Group F:mice conditioned with Flu-BU. Group G:mice conditioned with Flu-BU and transplanted with bone marrow cells and spleen cells from syngeneic mice (BALB/c).Group H:mice conditioned with Flu-BU and and transplanted with bone marrow cells and spleen cells from allogeneic mice (C57BL/6).Conditioning doses:BU 100mg/kg-Cy 200mg/kg or Flu 500mg/kg-BU 100mg/kg. Transplanted cells: 2×107bone marrow cells and 2×107 spleen cells.3) Recorded general situation, survival and the acute GVHD clinical score of transplanted mice. Observed the histopathological changes in target organs of acute GVHD such as liver, spleen, skin and intestine with HE staining and then graded. Cleaved-caspase 3 was detected with immunohistochemistry in liver, skin and intestine of transplanted mice. Chimerism in spleen and bone marrow was studied using flow cytometry. The proportion of CD3+, CD19+, donor-derived CD3+, donor-derived CD19+ in bone marrow and CD4+, CD8+in spleen were analyzed with flow cytometry. Measured the concentration of IFN-y and IL-4 in serum on day+3,+5,+7,+14 after transplantation with ELISA. Compared the differences of the above between allogeneic transplanted mice conditioned with BU-Cy and Flu-BU.Statistic methodsAll data were analyzed using SPSS 19.0. Data are presented as mean ± standard error. The Kaplan-Meier and Log rank test were used for analyzing survival data. Acute GVHD clinical scores, histopathology scores were compared between allogeneic transplanted mice conditioned with BU-Cy or Flu-BU using the nonparametric unpaired Mann-Whitney U test. Concentration of cytokines, proportion of CD3+, CD19+, donor-derived CD3+, donor-derived CD19+, CD4+, CD8+ were tested for normality applying the Shapiro-Wilk. If normality was given, an unpaired t-test (two-sided) was applied. If the data didn’t meet the criteria for normality, the nonparametric unpaired Mann-Whitney U test was applied. Statistical significance for all analyses was established when the P value was less than 0.05.Results1. Allogeneic transplanted mice conditioned with BU100mg/kg-Cy200mg/kg developed typical manifestations of acute GVHD, which led to all death within 30 days (the median survival was 10 days). Meanwhile, allogeneic transplanted mice conditioned with Flu500mg/kg-BU100mg/kg developed typical manifestations of acute GVHD, which led to all death within 45 days (the median survival was 23 days). In consideration of the observing time and uniformity of acute GVHD, we chose them as the appropriate conditioning doses.2. Clinical manifestations such as loss of appetite, weight loss were not observed in mice of group A and group B. Mice of group D and group G lost appetite and weight after conditioning, then the weight rose after transplantation and reached a normal control level on about 22 days after transplantation. Mice in group E continued losing weight within 7 days, then the weight were nearly unchanged during 8-12 days after transplantation and declined again after 12 days. Mice in group E developed typical manifestations of acute GVHD such as mental fatigue, low mobility, diarrhea, hunched posture, fur loss on day+7 after transplantation and died within 30 days. Mice in group H continued losing weight within 8-9 days, then the weight were nearly unchanged during 9-12 days after transplantation and declined again after 12 days. Mice in group H developed typical manifestations of acute GVHD such as mental fatigue, low mobility, diarrhea, hunched posture, fur loss on day+7 after transplantation and died within 45 days. All mice were graded according to the acute GVHD clinical assessment of transplanted models. All scores of mice on day 0 were 0; There was no statistical significance(P=0.606) between the score of mice in group E (2.8±0.20) and the score of mice in group H (3.2±0.49) on day+7 after transplantation; Compared with the score of group H (3.75±0.25、4.0±0.00、4.67±0.33)on day+14, day+21, day+28 respectively, the score of mice in group E (5.5±0.29、6.0±0.00、 7.0±0.00) after transplantation were all higher (P=0.017、P=0.046、P=0.034).3. Mice in group A, group B, group D, group G all developed long-term survival (longer than 45 days).The median survival of mice in group E was 10 days, and they all died within 30 days. The median survival of mice in group H was 23 days, and they all died within 45 days. Compared with group E, group H had a significantly longer survival (2=6.34, P=0.012).4. Histopathological signs including normal structure destruction and inflammatory infiltration were detected in target organs of acute GVHD such as liver, spleen, skin, intestine and lung using the HE staining test in both group E and group H on day +14 after transplantation. Compared with group H (3.67±0.58), the histopathology score in liver of group E (6.67±0.58) was higher (P=0.043). In addition, the histopathology score in intestine of group E (6.67±0.58) was also higher (P=0.046) than group H (4.00±1.00).There was no statistically significant difference (P=1.0) between the score in spleen of group E (2.67±0.58) and group H (2.67±0.58). The histopathology score in skin of group E (3.67±0.58) was significantly lower (P=0.043) than group H (5.67±0.58).5. Cleaved-caspase 3 was detected in both liver and intestine of recipients of group E and group H on day+14 after transplantation, while nearly no cleaved-caspase 3 expressed in skin. Compared with group H, higher cleaved-caspase 3 expression was observed in liver and intestine of group E.6. The chimerism of group E in spleen and bone marrow was 90.30%±4.22%, 95.55%±2.42% respectively, reaching full donor chimerism. The same as group E, group H also reached full donor chimerism with 92.59%±4.050%、98.76%±0.30% in spleen and bone marrow respectively. Those indicated the engraftment of hematopoietic stem cells from donor C57BL/6 (H-2Kb) were successful in recipients BALB/c (H-2Kd). There was no significant difference between them (p=0.44,p=0.12).7. The CD3+ proportion in bone marrow of group E (73.97%±5.37%) and group H (53.37%±3.31%) were all higher (P=0.000、P=0.000) than group A (6.77%±1.02%) on day+14 after transplantation, and significant difference was observed between group E and group H (t=5.66,P=0.005), while the donor-derived CD3+proportion in group H is higher than group E (t=21.8, P=0.000). In contrast to CD3+, the CD19+ proportion in bone marrow of group H (7.47%±1.00%) and group E (15.73%±2.75%) were also significantly lower (P=0.000、P=0.000) compared to group A (68.93%±4.48%) on day+14 after transplantation, and there was also significant difference (t=4.90, P=0.008) between group E and group H, while the donor-derived CD19+ in group E was higher than group H (t=13.46, P=0.000). Furthermore, the quantification of lymphocytes subsets of the spleen revealed a strong decrease (P=0.025、P=0.004) in CD4+ proportion and a strong increase ((P=0.000. P=0.000)) in CD8+ proportion comparing group E, group H with group A (27.60%±3.64%, 7.37%±0.93%). While no significant differences was detected in CD4+(t=1.76, P=0.154) and CD8+(t=1.0,P=0.37) comparing group E(17.02%±3.76%, 28.84%±1.54%)and group H (12.57%±2.25%,27.27%±2.21%).8. The IFN-y concentration on day +5 after transplantation were significantly higher (P=0.000、P=0.002) in group E(1607.78±119.53 pg/ml) and group H (959.04±116.09 pg/ml) compared to group A (20.23±2.01 pg/ml). INF-y concentration all reached the top level on day+5 in both group E and group H, and significant difference (t=7.83, P=0.001) was observed between them; The differences were still significant on day+7 (t=13.16, P=0.001) and day+14 (t=9.92, P=0.002). The IL-4 concentration in group A was 8.60±0.78 pg/ml. The IL-4 concentration reached a lower lever on day+5 after transplatation in group E (5.13±0.09pg/ml) and group H (10.14±0.73pg/ml) with significant difference (t=11.83, P=0.007), then the concentration in group E was still lower (t=3.55, P=0.038) than group H on+7 day after transplatation while there was no significant difference (t=2.68, P=0.075) between them on day+14 after transplatation.ConclusionIn this study, we established acute GVHD models of C57BL/6'BALB/c allogeneic transplanted conditioned with BU-Cy, Flu-BU. The acute GVHD mice conditioned with Flu-BU had longer survival, less severe clinical manifestation and pathological changes in liver and intestine, which was maybe related to lower INF-y concentration, higher IL-4 concentration in serum, meanwhile more donor-derived T cells infiltration and worse impairment of B-cell development in the bone marrow could be seen. While the pathology mechanism of the differences observed between the allogeneic transplanted mice conditioned with different regimens needs further study. |
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