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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 infiltration by CD4+, CD8+, and CD11b+ cells in allogeneically transplanted grafts. (A,B,C) Representative stainings for CD4, CD8, and CD11b in syngeneic (DBA/1 into DBA/1) or allogeneic (FVB into DBA/1) heart allografts explanted at day 100 after transplantation. (D) In FVB into DBA/1 transplanted hearts, significantly elevated numbers of CD4+ cells were detected (day 60, P = 0.0007 and day 100, P <0.0001; day 60 versus day 100, P = 0.0009) in comparison to DBA/1 into DBA/1 transplanted grafts. (E) FVB allografts transplanted into DBA/1 recipients showed numbers of CD8+ cells (day 60, P <0.0001 and day 100, P <0.0001; day 60 versus day 100, P = 0.1921) that were significantly higher than in the syngeneic group. (F) Significantly higher levels of CD11b+ cells were found in allogeneic (FVB into DBA/1) grafts (day 60, P = 0.0045 and day 100, P = 0.0065; day 60 versus day 100, P = 0.0124) when compared to DBA/1 to DBA/1 transplanted hearts. The histological score is the mean of 3 HPF (20× magnification); at least five mice per group were analyzed. *P <0.05. HPF, high power field.
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Figure 1: Increased infiltration by CD4+, CD8+, and CD11b+ cells in allogeneically transplanted grafts. (A,B,C) Representative stainings for CD4, CD8, and CD11b in syngeneic (DBA/1 into DBA/1) or allogeneic (FVB into DBA/1) heart allografts explanted at day 100 after transplantation. (D) In FVB into DBA/1 transplanted hearts, significantly elevated numbers of CD4+ cells were detected (day 60, P = 0.0007 and day 100, P <0.0001; day 60 versus day 100, P = 0.0009) in comparison to DBA/1 into DBA/1 transplanted grafts. (E) FVB allografts transplanted into DBA/1 recipients showed numbers of CD8+ cells (day 60, P <0.0001 and day 100, P <0.0001; day 60 versus day 100, P = 0.1921) that were significantly higher than in the syngeneic group. (F) Significantly higher levels of CD11b+ cells were found in allogeneic (FVB into DBA/1) grafts (day 60, P = 0.0045 and day 100, P = 0.0065; day 60 versus day 100, P = 0.0124) when compared to DBA/1 to DBA/1 transplanted hearts. The histological score is the mean of 3 HPF (20× magnification); at least five mice per group were analyzed. *P <0.05. HPF, high power field.

Mentions: To determine the number of graft-infiltrating cells, heart allografts were harvested on day 60 and day 100 after transplantation, and were stained for CD4, CD8, and CD11b. In syngeneic grafts (DBA/1 into DBA/1), low numbers of CD4+ (day 60, 16 ± 3 and day 100, 9 ± 1 cells/HPF), CD8+ (day 60, 15 ± 2 and day 100, 12 ± 3 cells/HPF), and CD11b+ cells (day 60, 6 ± 1 and day 100, 22 ± 5 cells/HPF) were detected (Figure 1A,B,C,D,E,F). Allogeneic heart grafts (FVB into DBA/1) at day 60 after transplantation showed significantly higher cell numbers of CD4+ (63 ± 11 cells/HPF; P = 0.0007), CD8+ (121 ± 11 cells/HPF; P <0.0001), and CD11b+ cells (31 ± 7 cells/HPF; P = 0.0045) compared to grafts in the syngeneic group. The numbers of CD4+ and CD11b+ cells in the FVB into DBA/1 group increased further by day 100 after transplantation (day 60 versus day 100, CD4+P = 0.0009; CD11b+P = 0.0124), whereas the increase in the number of CD8+ cells did not reach statistical significance (P = 0.1921). In comparison to control animals at day 100 after transplantation, the allogeneic group showed significantly higher levels of CD4+ (199 ± 31 cells/HPF; P < 0.0001), CD8+ (149 ± 17 cells/HPF; P <0.0001), and CD11b+ cells (128 ± 34 cells/HPF; P = 0.0065).


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 infiltration by CD4+, CD8+, and CD11b+ cells in allogeneically transplanted grafts. (A,B,C) Representative stainings for CD4, CD8, and CD11b in syngeneic (DBA/1 into DBA/1) or allogeneic (FVB into DBA/1) heart allografts explanted at day 100 after transplantation. (D) In FVB into DBA/1 transplanted hearts, significantly elevated numbers of CD4+ cells were detected (day 60, P = 0.0007 and day 100, P <0.0001; day 60 versus day 100, P = 0.0009) in comparison to DBA/1 into DBA/1 transplanted grafts. (E) FVB allografts transplanted into DBA/1 recipients showed numbers of CD8+ cells (day 60, P <0.0001 and day 100, P <0.0001; day 60 versus day 100, P = 0.1921) that were significantly higher than in the syngeneic group. (F) Significantly higher levels of CD11b+ cells were found in allogeneic (FVB into DBA/1) grafts (day 60, P = 0.0045 and day 100, P = 0.0065; day 60 versus day 100, P = 0.0124) when compared to DBA/1 to DBA/1 transplanted hearts. The histological score is the mean of 3 HPF (20× magnification); at least five mice per group were analyzed. *P <0.05. HPF, high power field.
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Related In: Results  -  Collection

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Figure 1: Increased infiltration by CD4+, CD8+, and CD11b+ cells in allogeneically transplanted grafts. (A,B,C) Representative stainings for CD4, CD8, and CD11b in syngeneic (DBA/1 into DBA/1) or allogeneic (FVB into DBA/1) heart allografts explanted at day 100 after transplantation. (D) In FVB into DBA/1 transplanted hearts, significantly elevated numbers of CD4+ cells were detected (day 60, P = 0.0007 and day 100, P <0.0001; day 60 versus day 100, P = 0.0009) in comparison to DBA/1 into DBA/1 transplanted grafts. (E) FVB allografts transplanted into DBA/1 recipients showed numbers of CD8+ cells (day 60, P <0.0001 and day 100, P <0.0001; day 60 versus day 100, P = 0.1921) that were significantly higher than in the syngeneic group. (F) Significantly higher levels of CD11b+ cells were found in allogeneic (FVB into DBA/1) grafts (day 60, P = 0.0045 and day 100, P = 0.0065; day 60 versus day 100, P = 0.0124) when compared to DBA/1 to DBA/1 transplanted hearts. The histological score is the mean of 3 HPF (20× magnification); at least five mice per group were analyzed. *P <0.05. HPF, high power field.
Mentions: To determine the number of graft-infiltrating cells, heart allografts were harvested on day 60 and day 100 after transplantation, and were stained for CD4, CD8, and CD11b. In syngeneic grafts (DBA/1 into DBA/1), low numbers of CD4+ (day 60, 16 ± 3 and day 100, 9 ± 1 cells/HPF), CD8+ (day 60, 15 ± 2 and day 100, 12 ± 3 cells/HPF), and CD11b+ cells (day 60, 6 ± 1 and day 100, 22 ± 5 cells/HPF) were detected (Figure 1A,B,C,D,E,F). Allogeneic heart grafts (FVB into DBA/1) at day 60 after transplantation showed significantly higher cell numbers of CD4+ (63 ± 11 cells/HPF; P = 0.0007), CD8+ (121 ± 11 cells/HPF; P <0.0001), and CD11b+ cells (31 ± 7 cells/HPF; P = 0.0045) compared to grafts in the syngeneic group. The numbers of CD4+ and CD11b+ cells in the FVB into DBA/1 group increased further by day 100 after transplantation (day 60 versus day 100, CD4+P = 0.0009; CD11b+P = 0.0124), whereas the increase in the number of CD8+ cells did not reach statistical significance (P = 0.1921). In comparison to control animals at day 100 after transplantation, the allogeneic group showed significantly higher levels of CD4+ (199 ± 31 cells/HPF; P < 0.0001), CD8+ (149 ± 17 cells/HPF; P <0.0001), and CD11b+ cells (128 ± 34 cells/HPF; P = 0.0065).

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