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, 2019, 9015292
eCollection

Treatment With Apocynin Limits the Development of Acute Graft-versus-Host Disease in Mice

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Treatment With Apocynin Limits the Development of Acute Graft-versus-Host Disease in Mice

Barbara Maximino Rezende et al. J Immunol Res.

Abstract

Graft-versus-host disease (GVHD) is the most serious complication limiting the clinical utility of allogeneic hematopoietic stem cell transplantation (HSCT), in which lymphocytes of donors (graft) are activated in response to the host antigen. This disease is associated with increased inflammatory response through the release of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species (ROS). In this study, we have evaluated the role of ROS in GVHD pathogenesis by treatment of recipient mice with apocynin (apo), an inhibitor of intracellular translocation of cytosolic components of NADPH oxidase complex. The pharmacological blockade of NADPH oxidase resulted in prolonged survival and reduced GVHD clinical score. This reduction in GVHD was associated with reduced levels of ROS and TBARS in target organs of GVHD in apocynin-treated mice at the onset of the mortality phase. These results correlated with reduced intestinal and liver injuries and decreased levels of proinflammatory cytokines and chemokines. Mechanistically, pharmacological blockade of the NADPH oxidase was associated with inhibition of recruitment and accumulation of leukocytes in the target organs. Additionally, the chimerism remained unaffected after treatment with apocynin. Our study demonstrates that ROS plays an important role in mediating GVHD, suggesting that strategies aimed at blocking ROS production may be useful as an adjuvant therapy in patients subjected to bone marrow transplantation.

Conflict of interest statement

The authors declare that there is no conflict of interest regarding the publication of this paper.

Figures

Figure 1
Figure 1
Apocynin treatment is associated with reduced mortality and improvement in GVHD clinical signs. GVHD was induced by the adoptive transfer of 107 BM cells + 3 × 107 splenocytes from C57BL/6 mouse donors to B6D2F1 mice. Mice that received syngeneic (B6D2F1) BM cells and splenocytes did not develop disease and were considered the control group. After GVHD induction, recipient mice were treated with apocynin (3 mg/kg, 24 h/24 h, intraperitoneally) or vehicle 30 min before transplantation until the experimental endpoint. The mice were evaluated every 2 d for survival (a) and clinical scoring (b). The results are shown as means ± SEM, and the numbers of animals were as follows: control group (●, n = 5), vehicle group (▲, n = 6), and apo group (■, n = 6). ∗ and #: p < 0.05 compared with the control and vehicle groups, respectively.
Figure 2
Figure 2
Apocynin treatment reduces reactive oxygen species and lipid peroxidation in GVHD target organs. GVHD was induced by the adoptive transfer of 107 BM cells + 3 × 107 splenocytes from C57BL/6 mouse donors to B6D2F1 mice. Mice that received syngeneic (B6D2F1) BM cells and splenocytes did not develop disease and were considered the control group. After GVHD induction, recipient mice were treated with apocynin (3 mg/kg, 24 h/24 h, intraperitoneally) or vehicle 30 min before transplantation until the experimental endpoint. At the onset of mortality, mice were killed, and the levels of reactive oxygen species were evaluated in the (a) spleen and (b) liver by DCF-DA analysis. The lipid peroxidation was also evaluated by TBARS in the (c) liver and (d) jejunum-ileum. Results are presented as the mean ± SEM (n = 4‐7); ∗ and #: p < 0.05 when comparing to the control and vehicle groups, respectively.
Figure 3
Figure 3
Apocynin treatment reduces hepatic and intestinal injuries related to GVHD. GVHD was induced by the adoptive transfer of 107 BM cells + 3 × 107 splenocytes from C57BL/6 mouse donors to B6D2F1 mice. Mice that received syngeneic (B6D2F1) BM cells and splenocytes did not develop disease and were considered the control group. After GVHD induction, recipient mice were treated with apocynin (3 mg/kg, 24 h/24 h, intraperitoneally) or vehicle 30 min before transplantation until the experimental endpoint. At the onset of mortality, the mice were killed, and the (a, c–e) liver and (b, f–h) jejunum-ileum tissues were sampled for histopathologic analysis. (c–e) Histological aspects of H&E-stained liver sections from the control, vehicle group, and apo group, respectively. (f–h) Histologic aspects of hematoxylin and eosin- (H&E-) stained small intestine sections from the control, vehicle group, and apo group, respectively. Scale bar: 50 μm for all panels. Results are presented as the mean ± SEM (n = 4‐6); ∗ and #: p < 0.05 when comparing to the control and vehicle groups, respectively.
Figure 4
Figure 4
Apocynin treatment reduces the concentration of cytokines and chemokines in the liver of mice subjected to GVHD. GVHD was induced by the adoptive transfer of 107 BM cells + 3 × 107 splenocytes from C57BL/6 mouse donors to B6D2F1 mice. Mice that received syngeneic (B6D2F1) BM cells and splenocytes did not develop disease and were considered the control group. After GVHD induction, recipient mice were treated with apocynin (3 mg/kg, 24 h/24 h, intraperitoneally) or vehicle 30 min before transplantation until the experimental endpoint. At the onset of mortality, mice were killed, and the concentrations of (a) TNF, (b) IFN-γ, (c) IL-17, (d) CCL2, (e) CCL3, and (f) CCL5 in the hepatic homogenates were evaluated by ELISA. Results are presented as the mean ± SEM (n = 6‐10); ∗ and #: p < 0.05 when comparing to the control and vehicle groups, respectively.
Figure 5
Figure 5
Apocynin treatment reduces the concentration of cytokines and chemokines in the jejunum-ileum of mice subjected to GVHD. GVHD was induced by the adoptive transfer of 107 BM cells + 3 × 107 splenocytes from C57BL/6 mouse donors to B6D2F1 mice. Mice that received syngeneic (B6D2F1) BM cells and splenocytes did not develop disease and were considered the control group. After GVHD induction, recipient mice were treated with apocynin (3 mg/kg, 24 h/24 h, intraperitoneally) or vehicle 30 min before transplantation until the experimental endpoint. At the onset of mortality, mice were killed, and the concentrations of (a) TNF, (b) IFN-γ, (c) IL-17, (d) CCL2, (e) CCL3, and (f) CCL5 in the intestinal homogenates were evaluated by ELISA. Results are presented as the mean ± SEM (n = 6‐10); ∗ and #: p < 0.05 when comparing to the control and vehicle groups, respectively.
Figure 6
Figure 6
Apocynin treatment reduces leukocyte recruitment into GVHD target organs. GVHD was induced by the adoptive transfer of 107 BM cells + 3 × 107 splenocytes from C57BL/6 mouse donors to B6D2F1 mice. Mice that received syngeneic (B6D2F1) BM cells and splenocytes did not develop disease and were considered the control group. After GVHD induction, recipient mice were treated with apocynin (3 mg/kg, 24 h/24 h, intraperitoneally) or vehicle 30 min before transplantation until the experimental endpoint. The leukocyte recruitment was evaluated on day 13 after transplantation. The mice were anesthetized, and intestinal venules (±40 μm) were selected to count the numbers of rolling and adherent leukocytes by intravital microscopy. (a) The number of rolling cells/minute; (b) the number of adherent cells/100 μm. Results are presented as the mean ± SEM (n = 4). Macrophages were quantified in the (c) liver and (d) jejunum-ileum by enzymatic methods (NAG assay). Results are presented as the mean ± SEM (n = 6‐9). The percentage of (e) hepatic LT CD4+ was evaluated by flow cytometry. Results are presented as the mean ± SEM (n = 4). (f) Representative dot plot of the flow cytometry analysis. ∗ and #: p < 0.05 when comparing to the control and vehicle groups, respectively.
Figure 7
Figure 7
Apocynin treatment did not interfere with chimerism. GVHD was induced by the adoptive transfer of 107 BM cells + 3 × 107 splenocytes from C57BL/6 mouse donors to B6D2F1 mice. Mice that received syngeneic (B6D2F1) BM cells and splenocytes did not develop disease and were considered the control group. After GVHD induction, recipient mice were treated with apocynin (3 mg/kg, 24 h/24 h, intraperitoneally) or vehicle 30 min before transplantation until the experimental endpoint. At day 13 after transplant, the mice were killed and the percentage of H2d+H2b+ cells (marker for B6D2F1 cells) and H2b+ cells (marker for C57BL/6 cells) in the spleen and BM was evaluated by flow cytometry. (a) Frequency of H2b+ in the spleen. (b) Frequency of H2b+H2d+ in the spleen. (c) Frequency of H2b+ in the BM. (d) Frequency of H2b+H2d+ in the BM. (e) Representative dot plot of the flow cytometry analysis in the spleen. (f) Representative dot plot of the flow cytometry analysis in the BM. Results are presented as the mean ± SEM (n = 4); ∗ and #: p < 0.05 when comparing to the control and vehicle groups, respectively.
Figure 8
Figure 8
Summary of GVHD protection induced by apocynin treatment. Apocynin treatment controls oxidative stress, which in turn results in lower liver and intestinal damage, decreased levels of proinflammatory cytokines and chemokines, reduced leukocyte rolling and adhesion, reduced recruitment of macrophages and CD4 lymphocytes to GVHD target organs, and improvement of survival.

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