Green tea epigallo-catechin-galleate ameliorates the development of obliterative airway disease

Abstract

Lung transplantation has the worst outcome compared to all solid organ transplants due to chronic rejection known as obliterative bronchiolitis (OB). Pathogenesis of OB is a complex interplay of alloimmune-dependent and -independent factors, which leads to the development of inflammation, fibrosis, and airway obliteration that have been resistant to therapy. The alloimmune-independent inflammatory pathway has been the recent focus in the pathogenesis of rejection, suggesting that targeting this may offer therapeutic benefits. As a potent anti-inflammatory agent, epigallo-catechin-galleate (EGCG), a green tea catechin, has been very effective in ameliorating inflammation in a variety of diseases, providing the rationale for its use in this study in a murine heterotopic tracheal allograft model of OB. Mice treated with EGCG had reduced inflammation, with significantly less neutrophil and macrophage infiltration and significantly reduced fibrosis. On further investigation into the mechanisms, inflammatory cytokines keratinocyte (KC), interleukin-17 (IL-17), and tumor necrosis factor-α (TNF-α), involved in neutrophil recruitment, were reduced in the EGCG-treated mice. In addition, monocyte chemokine monocyte chemoattractant protein-1 (MCP-1) was significantly reduced by EGCG treatment. Antifibrotic cytokine interferon-γ-inducible protein-10 (IP-10) was increased and profibrotic cytokine transforming growth factor-β (TGF-β) was reduced, further characterizing the antifibrotic effects of EGCG. These findings suggest that EGCG has great potential in ameliorating the development of obliterative airway disease.

FIGURE 1

Less tracheal luminal obliteration in EGCG-treated recipients. Grafts were explanted 2 days (PBS, n = 6; EGCG, n = 6) or 2 weeks (PBS, n = 5; EGCG, n = 6) after transplantation. Comparison of PBS-control and EGCG-treated groups using samples stained with H&E revealed a higher degree of luminal obliteration in the PBS-controls than in the EGCG-treated recipients 2 weeks after transplantation (A) (40× original magnification). Histological grading of H&E-stained samples indicated median obliteration was significantly lower in EGCG-treated mice (B), **P < .01. All error bars indicate SEM. (Color figure available online.)

FIGURE 2

EGCG improves epithelial regeneration. (A and B) Immunohistochemical staining with antibodies against cytokeratin 5 (brown) revealed that tracheas in EGCG-treated group (n = 5) had significantly higher percent of epithelial coverage than those in PBS-controls (n = 6) 2 weeks post transplantation (200× original magnification), **P < .01. All error bars indicate SEM. (Color figure available online.)

FIGURE 3

Reduced neutrophil and macrophage accumulation in EGCG-treated recipients. To assess neutrophil infiltration, 3 to 5 grafts from each treatment group were explanted 2 days, 1 week, or 2 weeks after transplantation. The tracheas in the EGCG-treated group showed significantly decreased numbers of infiltrated polymorphonuclear cells stained with antibodies against Ly-6C/6G (dark brown spots) when compared with PBS-controls 2 weeks after transplantation (A) (200× original magnification). Myeloperoxidase (MPO) activity, an index of neutrophil infiltration, was markedly reduced in EGCG-treated group both 1 week and 2 weeks after transplantation (B), *P < .05. To assess macrophage accumulation, grafts from each treatment group were explanted 2 days (PBS, n = 6; EGCG, n = 6) or 2 weeks (PBS, n = 5; EGCG, n = 6) after transplantation. Comparison of PBS-control and EGCG-treated groups using samples stained with antibodies against Mac-3 (brown) showed more macrophages in PBS-control tracheas than in EGCG-treated tracheas 2 weeks after transplantation (C) (200× original magnification). The number of macrophages per high-power field was significantly less in EGCG-treated group 2 weeks post transplantation (D), **P < .01. All error bars indicate SEM. (Color figure available online.)

FIGURE 4

Analysis of proinflammatory and proangiogenic mediators in transplants. Proinflammatory and proangiogenic mediators in both PBS-control (n = 6 for 2-day and 2-week time points) and EGCG-treated (n = 6 for 2-day and n = 5 for 2-week time points) groups were analyzed. EGCG treatment decreased levels of proinflammatory chemokine KC (A), proinflammatory cytokines IL-17 (B), TNF-α (C), MCP-1 (D), and proangiogenic VEGF (E) 2 days after transplantation, *P < .05. All error bars indicate SEM.

FIGURE 5

Reduced angiogenesis in EGCG-treated recipients. (A) Microphotographs showing vWF-labeled blood vessels (brown) in PBS-control and EGCG-treated groups. (B) Blood vessel number per trachea (n = 5 to 6 for both PBS and EGCG groups at each time point) is also lower in EGCG-treated group 2 weeks post transplantation, *P < .05. All error bars indicate SEM. (Color figure available online.)

FIGURE 6

Less fibrosis in EGCG-treated recipients. (A) Masson’s trichrome staining of collagen deposition in PBS-control and EGCG-treated groups. (B) Hydroxyproline-based quantification of collagen was performed with tracheal transplants from PBS-control (n = 5 to 6 for each time point) and EGCG-treated (n = 5 to 6 for each time point) groups. EGCG-treated mice had less collagen deposition 2 weeks after transplantation. (C) Significant inhibitory effects of EGCG on profibrotic cytokine TGF-β could be demonstrated both at 2 days (PBS, n = 6; EGCG, n = 6) and 1 week (PBS, n = 6; EGCG, n = 6) after transplantation. (D) Although initially lower at 2 days post transplantation (PBS, n = 6; EGCG, n = 5), EGCG had significantly increased antifibrotic cytokine IP-10 production 2 weeks after transplantation (PBS, n = 6; EGCG, n = 6), *P < .05. All error bars indicate SEM. (Color figure available online.)