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Differentiation between acute skin rejection in allotransplantation and T-cell mediated skin inflammation based on gene expression analysis.

Wolfram D, Morandi EM, Eberhart N, Hautz T, Hackl H, Zelger B, Riede G, Wachter T, Dubrac S, Ploner C, Pierer G, Schneeberger S - Biomed Res Int (2015)

Bottom Line: Advances in microsurgical techniques and immunosuppressive medication have rendered transplantation of vascularized composite allografts possible, when autologous tissue is neither available nor sufficient for reconstruction.Gene expression levels varied significantly across skin types and regions, indicating localization specific mechanism of leukocyte migration and infiltration.Expression of Il12b, Il17a, and Il1b gene expression levels differed significantly between rejection and inflammation, independent of the skin type.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria.

ABSTRACT
Advances in microsurgical techniques and immunosuppressive medication have rendered transplantation of vascularized composite allografts possible, when autologous tissue is neither available nor sufficient for reconstruction. However, skin rejection and side effects of long-term immunosuppression still remain a major hurdle for wide adoption of this excellent reconstructive technique. Histopathologic changes during acute skin rejection in vascular composite allotransplantation often mimic inflammatory skin disorders and are hard to distinguish. Hence, the identification of diagnostic and therapeutic markers specific for skin rejection is of particular clinical need. Here we present novel markers allowing for early differentiation between rejection in hind limb allotransplantation and contact hypersensitivity. Assessment of Ccl7, Il18, and Il1b expression is most indicative of distinguishing skin rejection from skin inflammatory disorders. Gene expression levels varied significantly across skin types and regions, indicating localization specific mechanism of leukocyte migration and infiltration. Expression of Il12b, Il17a, and Il1b gene expression levels differed significantly between rejection and inflammation, independent of the skin type. In synopsis of the RNA expression profile and previously assessed protein expression, the Il1 family appears as a promising option for accurate skin rejection diagnosis and, as a following step, for development of novel rejection treatments.

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Heat map visualizing mean log2-fold changes of cytokine/chemokine mRNA levels in two different inflammatory models (DTH, CHS) and two different skin types of allograft rejection at POD 5 in rat hind limb transplantation. Relative expressions are normalized to the mean of the controls from the respective group. Greater levels of inflammatory mediators (log2-fold changes > 0) are indicated in red and smaller levels (log2-fold changes < 0) are indicated in blue according to the legend. Similar profiles of mediators across all conditions are grouped by average linkage hierarchical clustering as indicated by the dendrogram (tree) at the left.
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fig5: Heat map visualizing mean log2-fold changes of cytokine/chemokine mRNA levels in two different inflammatory models (DTH, CHS) and two different skin types of allograft rejection at POD 5 in rat hind limb transplantation. Relative expressions are normalized to the mean of the controls from the respective group. Greater levels of inflammatory mediators (log2-fold changes > 0) are indicated in red and smaller levels (log2-fold changes < 0) are indicated in blue according to the legend. Similar profiles of mediators across all conditions are grouped by average linkage hierarchical clustering as indicated by the dendrogram (tree) at the left.

Mentions: Furthermore we aimed to clarify whether there are differences in cytokine gene expression patterns between skin types and if they have an impact on the distinction between rejection and inflammation. To address these questions, samples from both inflammatory models were analyzed separately, the DTH model on the pinna (hairy skin type) and the CHS model on the planta pedis (nonhairy skin type), and compared with hairy thigh skin and the nonhairy footpad skin from allografts. The relative expression levels of the 17 cytokines/chemokines (ΔCT) were then normalized to the mean within the respective controls and isografts (ΔΔCT≈ log2-fold changes). In Figure 5, a heat map is showing the mean of all log2-fold changes within each of the four groups for all genes studied. A distinct expression pattern differentiating the two anatomical sites in rejection (REJ) as well as inflammation (INF) was observed suggesting differential relative cytokine gene expression in the examined skin types. The results of this analysis indicate that for some genes the magnitude of the differences in relative expression between the skin types exceeds those between rejection and inflammation. To account for the anatomical location in the separation analysis we performed a two-way ANOVA analysis with skin type as one variable and rejection/inflammation model as the other variable and considered their interdependence. A high mutual dependence between these variables was observed for Il12a, Il2, and Tnf (interaction P < 0.001) (Table 2). Since these cytokines were also highly significantly differentially expressed between the two skin types this indicates that the differences between rejection and inflammation are systematically biased by the anatomical location. Il12b, Il17a, and Il1b gene expression levels significantly differ between the rejection and inflammatory disease models with only moderate interaction and were independent from skin types (Figure 6 and Table 2). To test for differences in the profile of these three genes between the individual groups a Tukey HSD post hoc procedure was performed. As shown in Figure 6 the expression levels of these three genes differ significantly on the footpad between DTH inflammation model and the rejection group (Il12b, adjusted P = 0,0022; Il1b, adjusted P = 7.8 × 10−4; Il17a, adjusted P = 0,0017). Moreover, Il12b distinguished contact hypersensitivity from rejection in the thigh skin (adjusted P = 0,012).


Differentiation between acute skin rejection in allotransplantation and T-cell mediated skin inflammation based on gene expression analysis.

Wolfram D, Morandi EM, Eberhart N, Hautz T, Hackl H, Zelger B, Riede G, Wachter T, Dubrac S, Ploner C, Pierer G, Schneeberger S - Biomed Res Int (2015)

Heat map visualizing mean log2-fold changes of cytokine/chemokine mRNA levels in two different inflammatory models (DTH, CHS) and two different skin types of allograft rejection at POD 5 in rat hind limb transplantation. Relative expressions are normalized to the mean of the controls from the respective group. Greater levels of inflammatory mediators (log2-fold changes > 0) are indicated in red and smaller levels (log2-fold changes < 0) are indicated in blue according to the legend. Similar profiles of mediators across all conditions are grouped by average linkage hierarchical clustering as indicated by the dendrogram (tree) at the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4338383&req=5

fig5: Heat map visualizing mean log2-fold changes of cytokine/chemokine mRNA levels in two different inflammatory models (DTH, CHS) and two different skin types of allograft rejection at POD 5 in rat hind limb transplantation. Relative expressions are normalized to the mean of the controls from the respective group. Greater levels of inflammatory mediators (log2-fold changes > 0) are indicated in red and smaller levels (log2-fold changes < 0) are indicated in blue according to the legend. Similar profiles of mediators across all conditions are grouped by average linkage hierarchical clustering as indicated by the dendrogram (tree) at the left.
Mentions: Furthermore we aimed to clarify whether there are differences in cytokine gene expression patterns between skin types and if they have an impact on the distinction between rejection and inflammation. To address these questions, samples from both inflammatory models were analyzed separately, the DTH model on the pinna (hairy skin type) and the CHS model on the planta pedis (nonhairy skin type), and compared with hairy thigh skin and the nonhairy footpad skin from allografts. The relative expression levels of the 17 cytokines/chemokines (ΔCT) were then normalized to the mean within the respective controls and isografts (ΔΔCT≈ log2-fold changes). In Figure 5, a heat map is showing the mean of all log2-fold changes within each of the four groups for all genes studied. A distinct expression pattern differentiating the two anatomical sites in rejection (REJ) as well as inflammation (INF) was observed suggesting differential relative cytokine gene expression in the examined skin types. The results of this analysis indicate that for some genes the magnitude of the differences in relative expression between the skin types exceeds those between rejection and inflammation. To account for the anatomical location in the separation analysis we performed a two-way ANOVA analysis with skin type as one variable and rejection/inflammation model as the other variable and considered their interdependence. A high mutual dependence between these variables was observed for Il12a, Il2, and Tnf (interaction P < 0.001) (Table 2). Since these cytokines were also highly significantly differentially expressed between the two skin types this indicates that the differences between rejection and inflammation are systematically biased by the anatomical location. Il12b, Il17a, and Il1b gene expression levels significantly differ between the rejection and inflammatory disease models with only moderate interaction and were independent from skin types (Figure 6 and Table 2). To test for differences in the profile of these three genes between the individual groups a Tukey HSD post hoc procedure was performed. As shown in Figure 6 the expression levels of these three genes differ significantly on the footpad between DTH inflammation model and the rejection group (Il12b, adjusted P = 0,0022; Il1b, adjusted P = 7.8 × 10−4; Il17a, adjusted P = 0,0017). Moreover, Il12b distinguished contact hypersensitivity from rejection in the thigh skin (adjusted P = 0,012).

Bottom Line: Advances in microsurgical techniques and immunosuppressive medication have rendered transplantation of vascularized composite allografts possible, when autologous tissue is neither available nor sufficient for reconstruction.Gene expression levels varied significantly across skin types and regions, indicating localization specific mechanism of leukocyte migration and infiltration.Expression of Il12b, Il17a, and Il1b gene expression levels differed significantly between rejection and inflammation, independent of the skin type.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria.

ABSTRACT
Advances in microsurgical techniques and immunosuppressive medication have rendered transplantation of vascularized composite allografts possible, when autologous tissue is neither available nor sufficient for reconstruction. However, skin rejection and side effects of long-term immunosuppression still remain a major hurdle for wide adoption of this excellent reconstructive technique. Histopathologic changes during acute skin rejection in vascular composite allotransplantation often mimic inflammatory skin disorders and are hard to distinguish. Hence, the identification of diagnostic and therapeutic markers specific for skin rejection is of particular clinical need. Here we present novel markers allowing for early differentiation between rejection in hind limb allotransplantation and contact hypersensitivity. Assessment of Ccl7, Il18, and Il1b expression is most indicative of distinguishing skin rejection from skin inflammatory disorders. Gene expression levels varied significantly across skin types and regions, indicating localization specific mechanism of leukocyte migration and infiltration. Expression of Il12b, Il17a, and Il1b gene expression levels differed significantly between rejection and inflammation, independent of the skin type. In synopsis of the RNA expression profile and previously assessed protein expression, the Il1 family appears as a promising option for accurate skin rejection diagnosis and, as a following step, for development of novel rejection treatments.

Show MeSH
Related in: MedlinePlus