Donor-But Not Recipient-Derived Cells Produce Collagen-1 in Chronically Rejected Cardiac Allografts

Abstract

Fibrosis is a prominent feature of chronic allograft rejection, caused by an excessive production of matrix proteins, including collagen-1. Several cell types produce collagen-1, including mesenchymal fibroblasts and cells of hematopoietic origin. Here, we sought to determine whether tissue-resident donor-derived cells or allograft-infiltrating recipient-derived cells are responsible for allograft fibrosis, and whether hematopoietic cells contribute to collagen production. A fully MHC-mismatched mouse heterotopic heart transplantation model was used, with transient depletion of CD4⁺ T cells to prevent acute rejection. Collagen-1 was selectively knocked out in recipients or donors. In addition, collagen-1 was specifically deleted in hematopoietic cells. Tissue-resident macrophages were depleted using anti-CSF1R antibody. Allograft fibrosis and inflammation were quantified 20 days post-transplantation. Selective collagen-1 knock-out in recipients or donors showed that tissue-resident cells from donor hearts, but not infiltrating recipient-derived cells, are responsible for production of collagen-1 in allografts. Cell-type-specific knock-out experiments showed that hematopoietic tissue-resident cells in donor hearts substantially contributed to graft fibrosis. Tissue resident macrophages, however, were not responsible for collagen-production, as their deletion worsened allograft fibrosis. Donor-derived cells including those of hematopoietic origin determine allograft fibrosis, making them attractive targets for organ preconditioning to improve long-term transplantation outcomes.

Keywords: allograft fibrosis; chronic rejection; collagen-1; fibrocytes; transplantation.

Figure 1

Mice with a heterozygous ubiquitous deficiency of collagen-1 used as recipients or donors of cardiac allografts - Quantification of fibrosis. C57BL/6 mice with a heterozygous ubiquitous deficiency of collagen-1 (Ubicre.col^wt/fl) or the appropriate control (UbiCre.col^wt/wt) were used either as recipients or donors of cardiac allografts. Recipients were depleted of CD4⁺ T cells and allografts were analyzed on day 20-post heart transplantation (HTx). (A) Collagen-1 positive (collagen-1⁺, col1⁺) area (%) and representative images (collagen-1 in red). (B) Fibrotic area (%) and representative Masson–Trichrome (MT) stainings (fibrosis in blue). (C) Fibronectin positive (Fibronectin⁺) area (%) and representative images (fibronectin in red). In each of the two donor groups one sample is missing because not enough tissue was left for fibronectin detection. Scale bar = 50µm; *p ≤ 0.05; **p < 0.01; ns, not significant. Data are mean +/- SEM and were compared using Mann-Whitney U test.

Figure 2

Mice with a heterozygous ubiquitous deficiency of collagen-1 used as recipients or donors of cardiac allografts - Gene expression analysis. Transplantation was performed as described in . Allografts were harvested on day 20 and gene expression was analyzed by RT-PCR. (A–D) Expression of col1a1, IL-6, TGF-β and Arg-1 within the allografts.*p ≤ 0.05; **p < 0.01; ns, not significant. Data are mean +/- SEM and were compared using Mann-Whitney U test.

Figure 3

Mice with a heterozygous ubiquitous deficiency of collagen-1 used as recipients or donors of cardiac allografts - Quantification of inflammation. Transplantation was performed as described in . Allografts were harvested on day 20. (A, B) Quantification of allograft-infiltrating CD3⁺ T cells and MAC-2⁺ cells by immunohistology. Representative images show CD3 and MAC-2 staining in brown. (C–E) Quantification of allograft-infiltrating CD8⁺ T cells, CCR2⁺ monocytes and neutrophils by flow cytometry. Scale bar = 50µm; *p ≤ 0.05; **p < 0.01; ns, not significant. Data are mean +/- SEM and were compared using Mann-Whitney U test.

Figure 4

Mice with a selective deficiency of collagen-1 in hematopoietic cells used as recipients or donors of cardiac allografts – Quantification of fibrosis. C57BL/6 mice with a selective homozygous deficiency of collagen-1 (col1) in hematopoietic cells (CD45^wt/cre.col^fl/fl) or appropriate controls (B6.CD45^wt/cre.col1^wt/wt and B6.CD45^wt/wt.col1^fl/fl) were used either as recipients or donors of cardiac allografts. Recipients were depleted of CD4⁺ T cells and allografts analyzed at day 20. (A) Collagen-1 positive (collagen-1⁺) area (%) and representative images (collagen-1 in red). (B) Fibrotic area (%) and representative Masson–Trichrome stainings (fibrosis in blue). (C) Fibronectin positive (fibronectin⁺) area (%) and representative images (fibronectin in red). Scale bar = 50µm; *p ≤ 0.05; **p < 0.01; ns, not significant. Data are mean +/- SEM and were compared using Mann-Whitney U test.

Figure 5

Mice with a selective deficiency of collagen-1 in hematopoietic cells used as recipients or donors of cardiac allografts – Gene expression analysis. Transplantation was performed as described in . Allografts were harvested on day 20. (A–D) Expression of col1a1, IL-6, TGF-β and Arg-1 within the allografts. *p ≤ 0.05; **p < 0.01; ns, not significant. Data are mean +/- SEM and were compared using Mann-Whitney U test.

Figure 6

Mice with a selective deficiency of collagen-1 in hematopoietic cells used as recipients or donors of cardiac allografts – Quantification of fibrosis. C57BL/6 mice with a selective homozygous deficiency of collagen-1 in hematopoietic cells (VavCre.col^fl/fl) or appropriate controls (VavCre.col^wt/wt and col ^fl/fl) were used as either recipients or donors of cardiac allografts. Recipient were depleted of CD4⁺ T cells and allografts analyzed on day 20. (A) Collagen-1 positive (collagen-1⁺) area (%) and representative images (collagen-1 in red). (B) Fibrotic area (%) and representative Masson–Trichrome stainings (fibrosis in blue). (C) Fibronectin positive (fibronectin⁺) area (%) and representative images (fibronectin in red). Scale bar = 50µm; *p ≤ 0.05; **p < 0.01; ns, not significant. Data are mean +/- SEM and were compared using Mann-Whitney U test.

Figure 7

Mice with a selective deficiency of collagen-1 in hematopoietic cells used as recipients or donors of cardiac allografts – Gene expression analysis. Heart transplantation was performed as described in . Allografts were harvested on day 20. (A-D) Expression of col1a1, IL-6, TGF-β and Arg-1 within the allografts. *p ≤ 0.05; **p < 0.01; ns, not significant. Data are mean +/- SEM and were compared using Mann-Whitney U test.