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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

Increased collagen deposition in allogeneically transplanted grafts. (A) Representative Masson’s trichrome stainings showed increased levels of collagen (blue color) in allogeneically (FVB into DBA/1) compared to syngeneically (DBA/1 into DBA/1) transplanted hearts explanted at day 60 and day 100 after transplantation (5× and 20× magnification). (B) Analysis by Sircol assay detected significantly higher amounts of collagen in FVB hearts placed into DBA/1 recipients at day 60 (P = 0.0342) and at day 100 after transplantation (P = 0.0022) compared to the DBA/1 to DBA/1 mice and also to FVB control hearts (P = 0.0094). At least five mice per group were analyzed. *P < 0.05.
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Figure 3: Increased collagen deposition in allogeneically transplanted grafts. (A) Representative Masson’s trichrome stainings showed increased levels of collagen (blue color) in allogeneically (FVB into DBA/1) compared to syngeneically (DBA/1 into DBA/1) transplanted hearts explanted at day 60 and day 100 after transplantation (5× and 20× magnification). (B) Analysis by Sircol assay detected significantly higher amounts of collagen in FVB hearts placed into DBA/1 recipients at day 60 (P = 0.0342) and at day 100 after transplantation (P = 0.0022) compared to the DBA/1 to DBA/1 mice and also to FVB control hearts (P = 0.0094). At least five mice per group were analyzed. *P < 0.05.

Mentions: To prove that FVB hearts transplanted in DBA/1 mice develop fibrosis, Masson’s trichrome staining was performed. In these stainings, a strong collagen deposition was found in the allogeneic grafts at day 60, with a further increase in collagen deposition by day 100 after heart transplantation. No such fibrotic collagen deposition was observed in the syngeneic control mice (Figure 3A).


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)

Increased collagen deposition in allogeneically transplanted grafts. (A) Representative Masson’s trichrome stainings showed increased levels of collagen (blue color) in allogeneically (FVB into DBA/1) compared to syngeneically (DBA/1 into DBA/1) transplanted hearts explanted at day 60 and day 100 after transplantation (5× and 20× magnification). (B) Analysis by Sircol assay detected significantly higher amounts of collagen in FVB hearts placed into DBA/1 recipients at day 60 (P = 0.0342) and at day 100 after transplantation (P = 0.0022) compared to the DBA/1 to DBA/1 mice and also to FVB control hearts (P = 0.0094). At least five mice per group were analyzed. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016099&req=5

Figure 3: Increased collagen deposition in allogeneically transplanted grafts. (A) Representative Masson’s trichrome stainings showed increased levels of collagen (blue color) in allogeneically (FVB into DBA/1) compared to syngeneically (DBA/1 into DBA/1) transplanted hearts explanted at day 60 and day 100 after transplantation (5× and 20× magnification). (B) Analysis by Sircol assay detected significantly higher amounts of collagen in FVB hearts placed into DBA/1 recipients at day 60 (P = 0.0342) and at day 100 after transplantation (P = 0.0022) compared to the DBA/1 to DBA/1 mice and also to FVB control hearts (P = 0.0094). At least five mice per group were analyzed. *P < 0.05.
Mentions: To prove that FVB hearts transplanted in DBA/1 mice develop fibrosis, Masson’s trichrome staining was performed. In these stainings, a strong collagen deposition was found in the allogeneic grafts at day 60, with a further increase in collagen deposition by day 100 after heart transplantation. No such fibrotic collagen deposition was observed in the syngeneic control mice (Figure 3A).

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