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IL-13 signaling via IL-13Rα2 triggers TGF-β1-dependent allograft fibrosis.

Brunner SM, Schiechl G, Kesselring R, Martin M, Balam S, Schlitt HJ, Geissler EK, Fichtner-Feigl S - Transplant Res (2013)

Bottom Line: Graft-infiltrating cells were isolated and analyzed by flow cytometry.The allogeneic grafts were infiltrated by significantly increased numbers of CD4+ (P <0.0001), CD8+ (P <0.0001), and CD11b+ cells (P = 0.0065) by day 100.The expression of IL-13 and IL-13Rα2 resulted in significantly increased TGF-β1 levels (P <0.0001), higher numbers of CD11bhighGr1intermediateTGF-β1+ cells, and elevated cardiac collagen deposition (P = 0.0094).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, University Medical Center Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg 93053, Germany. stefan.brunner@ukr.de.

ABSTRACT

Background: Allograft fibrosis still remains a critical problem in transplantation, including heart transplantation. The IL-13/TGF-β1 interaction has previously been identified as a key pathway orchestrating fibrosis in different inflammatory immune disorders. Here we investigate if this pathway is also responsible for allograft fibrosis and if interference with the IL-13/TGF-β1 interaction prevents allograft fibrosis.

Methods: FVB or control DBA/1 donor hearts were transplanted heterotopically into DBA/1 recipient mice and hearts were explanted at day 60 and 100 post-transplantation. Cardiac tissue was examined by Masson's trichrome staining and immunohistochemistry for CD4, CD8, CD11b, IL-13, Fas ligand, matrix metalloproteinase (MMP)-1, MMP-13, β2-microglobulin, and Gremlin-1. Graft-infiltrating cells were isolated and analyzed by flow cytometry. IL-13 and TGF-β1 levels were determined by enzyme-linked immunosorbent assay (ELISA) and the amount of collagen was quantified using a Sircol assay; IL-13Rα2 expression was detected by Western blotting. In some experiments IL-13/ TGF-β1 signaling was blocked with specific IL-13Rα2 siRNA. Additionally, a PCR array of RNA isolated from the allografts was performed to analyze expression of multiple genes involved in fibrosis.

Results: Both groups survived long-term (>100 days). The allogeneic grafts were infiltrated by significantly increased numbers of CD4+ (P <0.0001), CD8+ (P <0.0001), and CD11b+ cells (P = 0.0065) by day 100. Furthermore, elevated IL-13 levels (P = 0.0003) and numbers of infiltrating IL-13+ cells (P = 0.0037), together with an expression of IL-13Rα2, were detected only within allografts. The expression of IL-13 and IL-13Rα2 resulted in significantly increased TGF-β1 levels (P <0.0001), higher numbers of CD11bhighGr1intermediateTGF-β1+ cells, and elevated cardiac collagen deposition (P = 0.0094). The allograft fibrosis found in these experiments was accompanied by upregulation of multiple profibrotic genes, which was confirmed by immunohistochemical stainings of allograft tissue. Blockage of the IL-13/TGF-β1 interaction by IL-13Rα2 siRNA led to lower numbers of CD11bhighGr1intermediateTGF-β1+, CD4+, CD8+, and CD11b+ cells, and prevented collagen deposition (P = 0.0018) within these allografts.

Conclusions: IL-13 signaling via IL-13Rα2 induces TGF-β1 and causes allograft fibrosis in a murine model of chronic transplant rejection. Blockage of this IL-13/TGF-β1 interaction by IL-13Rα2 siRNA prevents cardiac allograft fibrosis. Thus, IL-13Rα2 may be exploitable as a future target to reduce allograft fibrosis in organ transplantation.

No MeSH data available.


Related in: MedlinePlus

Activation of IL-13/TGF-β1 pathway in allogeneically transplanted grafts. (A) ELISA of supernants of cultured allograft-infiltrating cells detected significantly elevated IL-13 levels in allografts (day 60, P = 0.0031 and day 100, P = 0.0003)compared to syngrafts or FVB control hearts (P = 0.0055 and P = 0.0042). (B) Immunohistochemistry showed significantly higher and over time increasing numbers of IL-13+ cells in FVB hearts transplanted into DBA/1 recipients (day 60, P = 0.0228; day 100, P = 0.0037) relative to syngeneic animals (day 60 versus day 100, P = 0.0083). (C) Representative immunohistochemical stainings showed more IL-13+ cells in allografts ( FVB into DBA/1) compared to controls (DBA/1 into DBA/1; day 100). (D) Western blot analysis revealed expression of IL-13Rα2 only in allograft-infiltrating cells isolated from allogeneically transplanted hearts (FVB into DBA/1) in contrast to cells isolated from FVB controls or syngrafts (DBA/1 into DBA/1) without IL-13Rα2 expression. (E) Measurement of TGF-β1 by ELISA detected significantly elevated TGF-β1 levels in cells isolated from DBA/1 mice grafted with FVB hearts at day 60 (88 ± 5 versus 46 ± 5 pg/mL; P = 0.0010) and at day 100 (133 ± 6 versus 42 ± 7 pg/mL; P <0.0001) in comparison to syngrafts,and FVB control hearts (40 ± 8 pg/mL; P = 0.0048 and P = 0.0009, respectively). (F) Flow cytometry of graft-infiltrating cells extracted from allografts showed a higher percentage of CD11bhighGr1intermediateTGF-β1+ cells (7.3%) than in the syngeneic controls (1.2%; day 100). (G) In the flow cytometric analysis (pre-gated for CD45) these CD11bhigh cells were the only source of TGF-β1 production. The histological score is the mean of 3 HPF (20x magnification); at least five mice per group were analyzed. *P <0.05. ELISA, enzyme-linked immunosorbent assay; HPF, high power field; IL-13, interleukin 13; TGF-β1, transforming growth factor beta 1.
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Figure 2: Activation of IL-13/TGF-β1 pathway in allogeneically transplanted grafts. (A) ELISA of supernants of cultured allograft-infiltrating cells detected significantly elevated IL-13 levels in allografts (day 60, P = 0.0031 and day 100, P = 0.0003)compared to syngrafts or FVB control hearts (P = 0.0055 and P = 0.0042). (B) Immunohistochemistry showed significantly higher and over time increasing numbers of IL-13+ cells in FVB hearts transplanted into DBA/1 recipients (day 60, P = 0.0228; day 100, P = 0.0037) relative to syngeneic animals (day 60 versus day 100, P = 0.0083). (C) Representative immunohistochemical stainings showed more IL-13+ cells in allografts ( FVB into DBA/1) compared to controls (DBA/1 into DBA/1; day 100). (D) Western blot analysis revealed expression of IL-13Rα2 only in allograft-infiltrating cells isolated from allogeneically transplanted hearts (FVB into DBA/1) in contrast to cells isolated from FVB controls or syngrafts (DBA/1 into DBA/1) without IL-13Rα2 expression. (E) Measurement of TGF-β1 by ELISA detected significantly elevated TGF-β1 levels in cells isolated from DBA/1 mice grafted with FVB hearts at day 60 (88 ± 5 versus 46 ± 5 pg/mL; P = 0.0010) and at day 100 (133 ± 6 versus 42 ± 7 pg/mL; P <0.0001) in comparison to syngrafts,and FVB control hearts (40 ± 8 pg/mL; P = 0.0048 and P = 0.0009, respectively). (F) Flow cytometry of graft-infiltrating cells extracted from allografts showed a higher percentage of CD11bhighGr1intermediateTGF-β1+ cells (7.3%) than in the syngeneic controls (1.2%; day 100). (G) In the flow cytometric analysis (pre-gated for CD45) these CD11bhigh cells were the only source of TGF-β1 production. The histological score is the mean of 3 HPF (20x magnification); at least five mice per group were analyzed. *P <0.05. ELISA, enzyme-linked immunosorbent assay; HPF, high power field; IL-13, interleukin 13; TGF-β1, transforming growth factor beta 1.

Mentions: To examine if TGF-β1 stimulated by IL-13 signaling is elevated in mice receiving allogeneic transplants, IL-13 levels were measured by ELISA in supernatants of cultured allograft-infiltrating cells. Syngeneic DBA/1 heart grafts showed similar IL-13 concentrations at day 60 (108 ± 13 pg/mL) and at day 100 (112 ± 12 pg/mL) (P = 0.8415). FVB allografts placed in DBA/1 recipients showed significantly elevated IL-13 levels at day 60 (187 ± 10 pg/mL; P = 0.0031) and at day 100 after transplantation (303 ± 23 pg/mL; P = 0.0003) in comparison to allogeneic grafts at the same respective time points (Figure 2A). Additionally, immunohistochemical staining for IL-13 in FVB allografts transplanted into DBA/1 mice showed significantly increased numbers of IL-13+ cells/HPF at day 60 (16 ± 4 versus 6 ± 2 cells/HPF; P = 0.0228) and at day 100 (96 ± 26 versus 7 ± 2 cells/HPF; P = 0.0037), relative to the syngeneic controls (day 60 versus day 100; P = 0.0083; Figure 2B,C). Western blot analyses of lysates from allograft-infiltrating cells indicated detectable expression of IL-13Rα2 only in the allogeneic FVB to DBA/1 mice, both at day 60 and at day 100 after heart transplantation (Figure 2D).


IL-13 signaling via IL-13Rα2 triggers TGF-β1-dependent allograft fibrosis.

Brunner SM, Schiechl G, Kesselring R, Martin M, Balam S, Schlitt HJ, Geissler EK, Fichtner-Feigl S - Transplant Res (2013)

Activation of IL-13/TGF-β1 pathway in allogeneically transplanted grafts. (A) ELISA of supernants of cultured allograft-infiltrating cells detected significantly elevated IL-13 levels in allografts (day 60, P = 0.0031 and day 100, P = 0.0003)compared to syngrafts or FVB control hearts (P = 0.0055 and P = 0.0042). (B) Immunohistochemistry showed significantly higher and over time increasing numbers of IL-13+ cells in FVB hearts transplanted into DBA/1 recipients (day 60, P = 0.0228; day 100, P = 0.0037) relative to syngeneic animals (day 60 versus day 100, P = 0.0083). (C) Representative immunohistochemical stainings showed more IL-13+ cells in allografts ( FVB into DBA/1) compared to controls (DBA/1 into DBA/1; day 100). (D) Western blot analysis revealed expression of IL-13Rα2 only in allograft-infiltrating cells isolated from allogeneically transplanted hearts (FVB into DBA/1) in contrast to cells isolated from FVB controls or syngrafts (DBA/1 into DBA/1) without IL-13Rα2 expression. (E) Measurement of TGF-β1 by ELISA detected significantly elevated TGF-β1 levels in cells isolated from DBA/1 mice grafted with FVB hearts at day 60 (88 ± 5 versus 46 ± 5 pg/mL; P = 0.0010) and at day 100 (133 ± 6 versus 42 ± 7 pg/mL; P <0.0001) in comparison to syngrafts,and FVB control hearts (40 ± 8 pg/mL; P = 0.0048 and P = 0.0009, respectively). (F) Flow cytometry of graft-infiltrating cells extracted from allografts showed a higher percentage of CD11bhighGr1intermediateTGF-β1+ cells (7.3%) than in the syngeneic controls (1.2%; day 100). (G) In the flow cytometric analysis (pre-gated for CD45) these CD11bhigh cells were the only source of TGF-β1 production. The histological score is the mean of 3 HPF (20x magnification); at least five mice per group were analyzed. *P <0.05. ELISA, enzyme-linked immunosorbent assay; HPF, high power field; IL-13, interleukin 13; TGF-β1, transforming growth factor beta 1.
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Figure 2: Activation of IL-13/TGF-β1 pathway in allogeneically transplanted grafts. (A) ELISA of supernants of cultured allograft-infiltrating cells detected significantly elevated IL-13 levels in allografts (day 60, P = 0.0031 and day 100, P = 0.0003)compared to syngrafts or FVB control hearts (P = 0.0055 and P = 0.0042). (B) Immunohistochemistry showed significantly higher and over time increasing numbers of IL-13+ cells in FVB hearts transplanted into DBA/1 recipients (day 60, P = 0.0228; day 100, P = 0.0037) relative to syngeneic animals (day 60 versus day 100, P = 0.0083). (C) Representative immunohistochemical stainings showed more IL-13+ cells in allografts ( FVB into DBA/1) compared to controls (DBA/1 into DBA/1; day 100). (D) Western blot analysis revealed expression of IL-13Rα2 only in allograft-infiltrating cells isolated from allogeneically transplanted hearts (FVB into DBA/1) in contrast to cells isolated from FVB controls or syngrafts (DBA/1 into DBA/1) without IL-13Rα2 expression. (E) Measurement of TGF-β1 by ELISA detected significantly elevated TGF-β1 levels in cells isolated from DBA/1 mice grafted with FVB hearts at day 60 (88 ± 5 versus 46 ± 5 pg/mL; P = 0.0010) and at day 100 (133 ± 6 versus 42 ± 7 pg/mL; P <0.0001) in comparison to syngrafts,and FVB control hearts (40 ± 8 pg/mL; P = 0.0048 and P = 0.0009, respectively). (F) Flow cytometry of graft-infiltrating cells extracted from allografts showed a higher percentage of CD11bhighGr1intermediateTGF-β1+ cells (7.3%) than in the syngeneic controls (1.2%; day 100). (G) In the flow cytometric analysis (pre-gated for CD45) these CD11bhigh cells were the only source of TGF-β1 production. The histological score is the mean of 3 HPF (20x magnification); at least five mice per group were analyzed. *P <0.05. ELISA, enzyme-linked immunosorbent assay; HPF, high power field; IL-13, interleukin 13; TGF-β1, transforming growth factor beta 1.
Mentions: To examine if TGF-β1 stimulated by IL-13 signaling is elevated in mice receiving allogeneic transplants, IL-13 levels were measured by ELISA in supernatants of cultured allograft-infiltrating cells. Syngeneic DBA/1 heart grafts showed similar IL-13 concentrations at day 60 (108 ± 13 pg/mL) and at day 100 (112 ± 12 pg/mL) (P = 0.8415). FVB allografts placed in DBA/1 recipients showed significantly elevated IL-13 levels at day 60 (187 ± 10 pg/mL; P = 0.0031) and at day 100 after transplantation (303 ± 23 pg/mL; P = 0.0003) in comparison to allogeneic grafts at the same respective time points (Figure 2A). Additionally, immunohistochemical staining for IL-13 in FVB allografts transplanted into DBA/1 mice showed significantly increased numbers of IL-13+ cells/HPF at day 60 (16 ± 4 versus 6 ± 2 cells/HPF; P = 0.0228) and at day 100 (96 ± 26 versus 7 ± 2 cells/HPF; P = 0.0037), relative to the syngeneic controls (day 60 versus day 100; P = 0.0083; Figure 2B,C). Western blot analyses of lysates from allograft-infiltrating cells indicated detectable expression of IL-13Rα2 only in the allogeneic FVB to DBA/1 mice, both at day 60 and at day 100 after heart transplantation (Figure 2D).

Bottom Line: Graft-infiltrating cells were isolated and analyzed by flow cytometry.The allogeneic grafts were infiltrated by significantly increased numbers of CD4+ (P <0.0001), CD8+ (P <0.0001), and CD11b+ cells (P = 0.0065) by day 100.The expression of IL-13 and IL-13Rα2 resulted in significantly increased TGF-β1 levels (P <0.0001), higher numbers of CD11bhighGr1intermediateTGF-β1+ cells, and elevated cardiac collagen deposition (P = 0.0094).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, University Medical Center Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg 93053, Germany. stefan.brunner@ukr.de.

ABSTRACT

Background: Allograft fibrosis still remains a critical problem in transplantation, including heart transplantation. The IL-13/TGF-β1 interaction has previously been identified as a key pathway orchestrating fibrosis in different inflammatory immune disorders. Here we investigate if this pathway is also responsible for allograft fibrosis and if interference with the IL-13/TGF-β1 interaction prevents allograft fibrosis.

Methods: FVB or control DBA/1 donor hearts were transplanted heterotopically into DBA/1 recipient mice and hearts were explanted at day 60 and 100 post-transplantation. Cardiac tissue was examined by Masson's trichrome staining and immunohistochemistry for CD4, CD8, CD11b, IL-13, Fas ligand, matrix metalloproteinase (MMP)-1, MMP-13, β2-microglobulin, and Gremlin-1. Graft-infiltrating cells were isolated and analyzed by flow cytometry. IL-13 and TGF-β1 levels were determined by enzyme-linked immunosorbent assay (ELISA) and the amount of collagen was quantified using a Sircol assay; IL-13Rα2 expression was detected by Western blotting. In some experiments IL-13/ TGF-β1 signaling was blocked with specific IL-13Rα2 siRNA. Additionally, a PCR array of RNA isolated from the allografts was performed to analyze expression of multiple genes involved in fibrosis.

Results: Both groups survived long-term (>100 days). The allogeneic grafts were infiltrated by significantly increased numbers of CD4+ (P <0.0001), CD8+ (P <0.0001), and CD11b+ cells (P = 0.0065) by day 100. Furthermore, elevated IL-13 levels (P = 0.0003) and numbers of infiltrating IL-13+ cells (P = 0.0037), together with an expression of IL-13Rα2, were detected only within allografts. The expression of IL-13 and IL-13Rα2 resulted in significantly increased TGF-β1 levels (P <0.0001), higher numbers of CD11bhighGr1intermediateTGF-β1+ cells, and elevated cardiac collagen deposition (P = 0.0094). The allograft fibrosis found in these experiments was accompanied by upregulation of multiple profibrotic genes, which was confirmed by immunohistochemical stainings of allograft tissue. Blockage of the IL-13/TGF-β1 interaction by IL-13Rα2 siRNA led to lower numbers of CD11bhighGr1intermediateTGF-β1+, CD4+, CD8+, and CD11b+ cells, and prevented collagen deposition (P = 0.0018) within these allografts.

Conclusions: IL-13 signaling via IL-13Rα2 induces TGF-β1 and causes allograft fibrosis in a murine model of chronic transplant rejection. Blockage of this IL-13/TGF-β1 interaction by IL-13Rα2 siRNA prevents cardiac allograft fibrosis. Thus, IL-13Rα2 may be exploitable as a future target to reduce allograft fibrosis in organ transplantation.

No MeSH data available.


Related in: MedlinePlus