A modified graft‐versus‐host‐induced model for systemic sclerosis, with pulmonary fibrosis in Rag2‐deficient mice

Abstract

Systemic sclerosis (SSc) is a connective tissue disease that results in fibrosis in multiple organs. Various animal models for this disease have been developed, both genetic and induced. One of the induced models, sclerodermatous graft‐versus‐host disease (scl‐GvHD), exhibits the main characteristics of SSc, but involves lethal γ‐irradiation of recipients. We sought to develop a modified scl‐GvHD model. Spleen cells from B10.D2 donor mice were transplanted into immunodeficient Rag‐2 recipients on the BALB/c genetic background. Tissue fibrosis was analyzed at 3 and 9 weeks after transplantation. In addition to serum levels of anti‐Scl‐70 autoantibody and cytokines, tissue inflammation, fibrosis, expression of collagen‐I and α‐smooth muscle actin (α‐SMA), infiltration of leukocytes, mRNA expression of transforming growth factor (TGF)‐β, collagen‐I, α‐SMA, tumor necrosis factor (TNF)‐α, and interleukin (IL)‐6, the classical signal pathway of TGF‐β, Smad‐3, and p‐Smad‐3 expression in tissue were analyzed. Skin thickening and increased collagen synthesis, as well as the manifestation of tissue fibrosis, could be detected in skin, kidney, and lung of modified scl‐GvHD mouse model. Increased serum levels of anti‐Scl‐70 autoantibody, IL‐10, and TGF‐β could be detected. Increased CD4 + T cells and F4/80 + macrophage infiltration were found in skin, kidney, and lung. Gene expression of collagen‐I, TGF‐β, α‐SMA, TNF‐α, and IL‐6 was increased in tissue of the scl‐GvHD model. Moreover, TGF‐β expression and Smad‐3 phosphorylation were detected in skin, kidney, and lung of scl‐GvHD mice. Our data show that spleen cells from B10.D2 donor mice transplanted into immunodeficient Rag‐2 recipients could induce typical fibrosis not only of the skin and kidney but also of lung, which was missing from previous scl‐GvHD models. Thus, the modified scl‐GvHD model might be a promising model to explore the immunologic mechanisms of SSc and may be useful for investigation of new therapies for systemic sclerosis.

Combination of 5‐aminolevulinic acid and iron prevents skin fibrosis in murine sclerodermatous graft‐versus‐host disease

“…The number of macrophages and CD4 + T cells had also increased in the lesional skin and ear in early diffuse cutaneous scl‐GvHD. Furthermore, previous histological analysis of the affected skin and ear of the scl‐GvHD mice showed complex inflammatory cell infiltration . Immunohistochemistry revealed CD4 + T cell infiltration in the skin and ear of the scl‐GvHD group, which was not observed in the 5‐ALA/SFC treatment group (Figures and ).…”

“…Immunostaining was performed to detect cutaneous collagen‐I protein and α‐SMA deposition. By weeks 3 and 9 after the spleen cell transplantation, the total cutaneous collagen‐I protein and α‐SMA, which had increased in the untreated scl‐GvHD group, had dramatically decreased in the treatment group as shown by immunostaining (Figures and ). The number of macrophages and CD4 + T cells had also increased in the lesional skin and ear in early diffuse cutaneous scl‐GvHD.…”

“…Sections were viewed under a fluorescence microscope (BX51, OLYMPUS, Tokyo, Japan) with a fluorescein isothiocyanate (FITC) colour filter, and the results were analysed using morphometric software (MetaMorph software; Universal Imaging, Downingtown, PA). Image analysis was performed using ImageJ software (a freely available Java image processing program) as described previously …”

“…To ensure the data were representative and homogenous, at least three images were evaluated for each mouse and four independent microscopic fields were randomly selected for each sample. Image analysis was performed using ImageJ software (a freely available Java image processing program) as described previously …”

“…To study the effects of 5‐ALA in vivo, we constructed an animal model comprehensively mimicking SSc . Traditionally, scl‐GvHD can be induced by transplantation of B10.D2 bone marrow and spleen cells into lethally γ‐irradiated BALB/mice in recipients.…”