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Thiol/redox metabolomic profiling implicates GSH dysregulation in early experimental graft versus host disease (GVHD).

Suh JH, Kanathezhath B, Shenvi S, Guo H, Zhou A, Tiwana A, Kuypers F, Ames BN, Walters MC - PLoS ONE (2014)

Bottom Line: While most metabolic changes were similar in both groups, plasma glutathione (GSH) was significantly decreased, and GSH disulfide (GSSG) was increased after allogeneic compared to syngeneic recipient and non-transplant controls.Despite a significant rise in oxidative stress, compensatory increase in hepatic GSH synthesis was absent following Allo BMT.Early shifts in hepatic oxidative stress and plasma GSH loss preceded a statistically significant rise in TNF-α.

View Article: PubMed Central - PubMed

Affiliation: Children's Hospital Oakland Research Institute, Oakland, California, United States of America.

ABSTRACT
Graft-versus-host disease (GVHD) is a common complication of allogeneic bone marrow transplantation (BMT). Upregulation of inflammatory cytokines precedes the clinical presentation of GVHD and predicts its severity. In this report, thiol/redox metabolomics was used to identify metabolic perturbations associated with early preclinical (Day+4) and clinical (Day+10) stages of GVHD by comparing effects in Syngeneic (Syn; major histocompatibility complex- identical) and allogeneic transplant recipients (Allo BMT) in experimental models. While most metabolic changes were similar in both groups, plasma glutathione (GSH) was significantly decreased, and GSH disulfide (GSSG) was increased after allogeneic compared to syngeneic recipient and non-transplant controls. The early oxidation of the plasma GSH/GSSG redox couple was also observed irrespective of radiation conditioning treatment and was accompanied by significant rise in hepatic protein oxidative damage and ROS generation. Despite a significant rise in oxidative stress, compensatory increase in hepatic GSH synthesis was absent following Allo BMT. Early shifts in hepatic oxidative stress and plasma GSH loss preceded a statistically significant rise in TNF-α. To identify metabolomic biomarkers of hepatic GVHD injury, plasma metabolite concentrations analyzed at Day+10 were correlated with hepatic organ injury. GSSG (oxidized GSH) and β-alanine, were positively correlated, and plasma GSH cysteinylglycine, and branched chain amino acids were inversely correlated with hepatic injury. Although changes in plasma concentrations of cysteine, cystathionine (GSH precursors) and cysteinylglycine (a GSH catabolite) were not significant by univariate analysis, principal component analysis (PCA) indicated that accumulation of these metabolites after Allo BMT contributed significantly to early GVHD in contrast to Syn BMT. In conclusion, thiol/redox metabolomic profiling implicates that early dysregulation of host hepatic GSH metabolism and oxidative stress in sub-clinical GVHD before elevated TNF-α levels is associated with GVHD pathogenesis. Future studies will probe the mechanisms for these changes and examine the potential of antioxidant intervention strategies to modulate GVHD.

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Plasma metabolome discriminates Allo from Syn BMT and untreated controls at Day+4.Lethally irradiated B6 recipients were transplanted with 5×106 T-cell depleted bone marrow cells and 3×106 CD90+ T-cells from B6 Thy1.1 (Syngeneic) or Balb/C (Allogeneic) donor mice (N = 4 per group). Principal component analysis (PCA) and partial-least squares discriminant analysis (PLS-DA) was performed using plasma metabolite concentrations quantified at Day+4. Panel A shows the PCA scores plot. The different colors and letters signify the five groups in the study: Healthy controls (A; red), Syn Day+4 (B; Green), Syn Day+10 (C; Blue), Allo Day+4 (D; Cyan), and Allo Day+10 (E; Purple). Untreated controls and the BMT groups are separated along the PC1 axis whereas Allo are separated from the Syn group along the PC 3 y-axis. Solid line shows the direction of Allo separation from Syn. Panel B shows the corresponding PCA loading plot for PC1 and PC3 shown in panel A. Total Cysgly, GSH, Cys, Trp and Cysth were variables that contributed the most to the separation of groups identified by the PCA analysis. Panel C shows the PLS-DA scores plot. The group IDs are represented by letters and colors described in Panel A. Panel D shows the heat-map generated from the top 10 metabolites contributing to group discrimination as identified by PLS-DA analysis. Each metabolite is arranged in columns and the individual concentrations within a column are normalized by respective median concentrations. Rows represent different mice and their group ID is shown on the right side of each row. These group IDs are represented by different colors on the left side that correspond to the same color codes in Panels A and C. Concentrations that are two fold above or below the mean are highlighted in amber or in blue, respectively. Dendogram and the 3 nodes (1–3) classified by hierarchical clustering analysis are shown on the left.
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pone-0088868-g005: Plasma metabolome discriminates Allo from Syn BMT and untreated controls at Day+4.Lethally irradiated B6 recipients were transplanted with 5×106 T-cell depleted bone marrow cells and 3×106 CD90+ T-cells from B6 Thy1.1 (Syngeneic) or Balb/C (Allogeneic) donor mice (N = 4 per group). Principal component analysis (PCA) and partial-least squares discriminant analysis (PLS-DA) was performed using plasma metabolite concentrations quantified at Day+4. Panel A shows the PCA scores plot. The different colors and letters signify the five groups in the study: Healthy controls (A; red), Syn Day+4 (B; Green), Syn Day+10 (C; Blue), Allo Day+4 (D; Cyan), and Allo Day+10 (E; Purple). Untreated controls and the BMT groups are separated along the PC1 axis whereas Allo are separated from the Syn group along the PC 3 y-axis. Solid line shows the direction of Allo separation from Syn. Panel B shows the corresponding PCA loading plot for PC1 and PC3 shown in panel A. Total Cysgly, GSH, Cys, Trp and Cysth were variables that contributed the most to the separation of groups identified by the PCA analysis. Panel C shows the PLS-DA scores plot. The group IDs are represented by letters and colors described in Panel A. Panel D shows the heat-map generated from the top 10 metabolites contributing to group discrimination as identified by PLS-DA analysis. Each metabolite is arranged in columns and the individual concentrations within a column are normalized by respective median concentrations. Rows represent different mice and their group ID is shown on the right side of each row. These group IDs are represented by different colors on the left side that correspond to the same color codes in Panels A and C. Concentrations that are two fold above or below the mean are highlighted in amber or in blue, respectively. Dendogram and the 3 nodes (1–3) classified by hierarchical clustering analysis are shown on the left.

Mentions: Supervised Principle Component Analysis (PCA) and unsupervised (PLS-DA) modeling permit identification of subtle metabolic shifts that may not achieve statistical significance in univariate analyses. These techniques were used to examine the metabolomic separation of the Allo and Syn BMT and baseline control groups. The PCA scores plot (Figure 5: Panel A) displays each mouse sample as a point on the plot (Figure5A) and shows the intrinsic segregation patterns of individual samples and group variances. An overview of the PCA score matrix using the first five principal components indicated that the best separation of Allo and Syn BMT mice was achieved when principal components 1 and 3 were used (data not shown). The first PC (PC 1, x-axis), which explained 26% of the variability in the data, separated the controls (A) from the other 4 groups: Syn BMT Day+4 (B) and +10 (C) and Allo BMT Day+4 (D) and +10 (E). The separation of Syn and Allo BMT groups at different time points is seen along the y-axis (PC 3), which is marked by the solid line (Figure 5: Panel A). As shown, Syn D+4 (B) and D+10 (C) had similar metabolomic compositions and closely overlapped with each other. Allo D+4 samples (D) segregated below the two Syn groups with a more significant separation being achieved with Allo D+10 (E) samples.


Thiol/redox metabolomic profiling implicates GSH dysregulation in early experimental graft versus host disease (GVHD).

Suh JH, Kanathezhath B, Shenvi S, Guo H, Zhou A, Tiwana A, Kuypers F, Ames BN, Walters MC - PLoS ONE (2014)

Plasma metabolome discriminates Allo from Syn BMT and untreated controls at Day+4.Lethally irradiated B6 recipients were transplanted with 5×106 T-cell depleted bone marrow cells and 3×106 CD90+ T-cells from B6 Thy1.1 (Syngeneic) or Balb/C (Allogeneic) donor mice (N = 4 per group). Principal component analysis (PCA) and partial-least squares discriminant analysis (PLS-DA) was performed using plasma metabolite concentrations quantified at Day+4. Panel A shows the PCA scores plot. The different colors and letters signify the five groups in the study: Healthy controls (A; red), Syn Day+4 (B; Green), Syn Day+10 (C; Blue), Allo Day+4 (D; Cyan), and Allo Day+10 (E; Purple). Untreated controls and the BMT groups are separated along the PC1 axis whereas Allo are separated from the Syn group along the PC 3 y-axis. Solid line shows the direction of Allo separation from Syn. Panel B shows the corresponding PCA loading plot for PC1 and PC3 shown in panel A. Total Cysgly, GSH, Cys, Trp and Cysth were variables that contributed the most to the separation of groups identified by the PCA analysis. Panel C shows the PLS-DA scores plot. The group IDs are represented by letters and colors described in Panel A. Panel D shows the heat-map generated from the top 10 metabolites contributing to group discrimination as identified by PLS-DA analysis. Each metabolite is arranged in columns and the individual concentrations within a column are normalized by respective median concentrations. Rows represent different mice and their group ID is shown on the right side of each row. These group IDs are represented by different colors on the left side that correspond to the same color codes in Panels A and C. Concentrations that are two fold above or below the mean are highlighted in amber or in blue, respectively. Dendogram and the 3 nodes (1–3) classified by hierarchical clustering analysis are shown on the left.
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Related In: Results  -  Collection

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pone-0088868-g005: Plasma metabolome discriminates Allo from Syn BMT and untreated controls at Day+4.Lethally irradiated B6 recipients were transplanted with 5×106 T-cell depleted bone marrow cells and 3×106 CD90+ T-cells from B6 Thy1.1 (Syngeneic) or Balb/C (Allogeneic) donor mice (N = 4 per group). Principal component analysis (PCA) and partial-least squares discriminant analysis (PLS-DA) was performed using plasma metabolite concentrations quantified at Day+4. Panel A shows the PCA scores plot. The different colors and letters signify the five groups in the study: Healthy controls (A; red), Syn Day+4 (B; Green), Syn Day+10 (C; Blue), Allo Day+4 (D; Cyan), and Allo Day+10 (E; Purple). Untreated controls and the BMT groups are separated along the PC1 axis whereas Allo are separated from the Syn group along the PC 3 y-axis. Solid line shows the direction of Allo separation from Syn. Panel B shows the corresponding PCA loading plot for PC1 and PC3 shown in panel A. Total Cysgly, GSH, Cys, Trp and Cysth were variables that contributed the most to the separation of groups identified by the PCA analysis. Panel C shows the PLS-DA scores plot. The group IDs are represented by letters and colors described in Panel A. Panel D shows the heat-map generated from the top 10 metabolites contributing to group discrimination as identified by PLS-DA analysis. Each metabolite is arranged in columns and the individual concentrations within a column are normalized by respective median concentrations. Rows represent different mice and their group ID is shown on the right side of each row. These group IDs are represented by different colors on the left side that correspond to the same color codes in Panels A and C. Concentrations that are two fold above or below the mean are highlighted in amber or in blue, respectively. Dendogram and the 3 nodes (1–3) classified by hierarchical clustering analysis are shown on the left.
Mentions: Supervised Principle Component Analysis (PCA) and unsupervised (PLS-DA) modeling permit identification of subtle metabolic shifts that may not achieve statistical significance in univariate analyses. These techniques were used to examine the metabolomic separation of the Allo and Syn BMT and baseline control groups. The PCA scores plot (Figure 5: Panel A) displays each mouse sample as a point on the plot (Figure5A) and shows the intrinsic segregation patterns of individual samples and group variances. An overview of the PCA score matrix using the first five principal components indicated that the best separation of Allo and Syn BMT mice was achieved when principal components 1 and 3 were used (data not shown). The first PC (PC 1, x-axis), which explained 26% of the variability in the data, separated the controls (A) from the other 4 groups: Syn BMT Day+4 (B) and +10 (C) and Allo BMT Day+4 (D) and +10 (E). The separation of Syn and Allo BMT groups at different time points is seen along the y-axis (PC 3), which is marked by the solid line (Figure 5: Panel A). As shown, Syn D+4 (B) and D+10 (C) had similar metabolomic compositions and closely overlapped with each other. Allo D+4 samples (D) segregated below the two Syn groups with a more significant separation being achieved with Allo D+10 (E) samples.

Bottom Line: While most metabolic changes were similar in both groups, plasma glutathione (GSH) was significantly decreased, and GSH disulfide (GSSG) was increased after allogeneic compared to syngeneic recipient and non-transplant controls.Despite a significant rise in oxidative stress, compensatory increase in hepatic GSH synthesis was absent following Allo BMT.Early shifts in hepatic oxidative stress and plasma GSH loss preceded a statistically significant rise in TNF-α.

View Article: PubMed Central - PubMed

Affiliation: Children's Hospital Oakland Research Institute, Oakland, California, United States of America.

ABSTRACT
Graft-versus-host disease (GVHD) is a common complication of allogeneic bone marrow transplantation (BMT). Upregulation of inflammatory cytokines precedes the clinical presentation of GVHD and predicts its severity. In this report, thiol/redox metabolomics was used to identify metabolic perturbations associated with early preclinical (Day+4) and clinical (Day+10) stages of GVHD by comparing effects in Syngeneic (Syn; major histocompatibility complex- identical) and allogeneic transplant recipients (Allo BMT) in experimental models. While most metabolic changes were similar in both groups, plasma glutathione (GSH) was significantly decreased, and GSH disulfide (GSSG) was increased after allogeneic compared to syngeneic recipient and non-transplant controls. The early oxidation of the plasma GSH/GSSG redox couple was also observed irrespective of radiation conditioning treatment and was accompanied by significant rise in hepatic protein oxidative damage and ROS generation. Despite a significant rise in oxidative stress, compensatory increase in hepatic GSH synthesis was absent following Allo BMT. Early shifts in hepatic oxidative stress and plasma GSH loss preceded a statistically significant rise in TNF-α. To identify metabolomic biomarkers of hepatic GVHD injury, plasma metabolite concentrations analyzed at Day+10 were correlated with hepatic organ injury. GSSG (oxidized GSH) and β-alanine, were positively correlated, and plasma GSH cysteinylglycine, and branched chain amino acids were inversely correlated with hepatic injury. Although changes in plasma concentrations of cysteine, cystathionine (GSH precursors) and cysteinylglycine (a GSH catabolite) were not significant by univariate analysis, principal component analysis (PCA) indicated that accumulation of these metabolites after Allo BMT contributed significantly to early GVHD in contrast to Syn BMT. In conclusion, thiol/redox metabolomic profiling implicates that early dysregulation of host hepatic GSH metabolism and oxidative stress in sub-clinical GVHD before elevated TNF-α levels is associated with GVHD pathogenesis. Future studies will probe the mechanisms for these changes and examine the potential of antioxidant intervention strategies to modulate GVHD.

Show MeSH
Related in: MedlinePlus