Rapamycin blocks fibrocyte migration and attenuates bronchiolitis obliterans in a murine model

Abstract

Background: Fibrocytes are integral in the development of fibroproliferative disease. The CXCL12/CXCR4 chemokine axis has been shown to play a central role in fibrocyte migration and the development of bronchiolitis obliterans (BO) after lung transplantation. Inhibition of the mammalian target of rapamycin (mTOR) pathway with rapamycin has been shown to decrease expression of both CXCR4 and its receptor agonist CXCL12. Thus, we hypothesized that rapamycin treatment would decrease fibrocyte trafficking into tracheal allografts and prevent BO.

Methods: A total alloantigenic mismatch murine heterotopic tracheal transplant (HTT) model of BO was used. Animals were either treated with rapamycin or dimethyl sulfoxide (DMSO) for 14 days after tracheal transplantation. Fibrocyte levels were assessed by flow cytometry, and allograft neutrophil, CD3(+) T-cell, macrophage, and smooth muscle actin (SMA) levels were assessed by immunohistochemistry. Tracheal luminal obliteration was assessed on hematoxylin and eosin (H&E) stains.

Results: Compared with DMSO-treated controls, rapamycin-treated mice showed a significant decrease in fibrocyte levels in tracheal allografts. Fibrocyte levels in recipient blood showed a similar pattern, although it was not statistically significant. Furthermore, animals treated with rapamycin showed a significant decrease in tracheal allograft luminal obliteration compared with controls. Based on immunohistochemical analyses, populations of α-SMA-positive (α-SMA(+)) cells, neutrophils, CD3(+) T cells, and macrophages were all decreased in rapamycin-treated allografts versus DMSO controls.

Conclusions: Rapamycin effectively reduces recruitment of fibrocytes into tracheal allografts and mitigates development of tracheal luminal fibrosis. Further studies are needed to determine the cellular and molecular mechanisms that mediate the protective effect of rapamycin against BO.

Copyright © 2013 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Representative immunohistochemical stains of tracheal grafts for SMA at Day 14 post transplant. DMSO controls demonstrate positive SMA staining (Fast-Red) within the tracheal lumen, indicative of the developing fibrosis. Rapamycin-treated tracheas show no SMA staining within the lumen. Magnification of images is 40×. BF = bright field. Fast-Red = SMA stain. DAPI = nuclear stain. Merged = Fast-Red + DAPI together.

Figure 2

Rapamycin treatment reduces luminal obliteration of tracheal allografts at Day 28 post transplant. (A) Images of representative H&E stains of tracheal allografts. DMSO-treated show complete luminal obliteration with fibrosis; rapamycin-treated shows no luminal obliteration (pink substance within lumen is mucus). (B) Bar graph of percent luminal obliteration in each treatment group. n = 8 tracheas per group. Data shown are mean ± standard error of the mean.