Bleomycin-Treated Chimeric Thy1-Deficient Mice with Thy1-Deficient Myofibroblasts and Thy-Positive Lymphocytes Resolve Inflammation without Affecting the Fibrotic Response

Abstract

Lung fibrosis is characterized by abnormal accumulation of fibroblasts in the interstitium of the alveolar space. Two populations of myofibroblasts, distinguished by Thy1 expression, are detected in human and murine lungs. Accumulation of Thy1-negative (Thy1−) myofibroblasts was shown in the lungs of humans with idiopathic pulmonary fibrosis (IPF) and of bleomycin-treated mice. We aimed to identify genetic changes in lung myofibroblasts following Thy1 crosslinking and assess the impact of specific lung myofibroblast Thy1-deficiency, in vivo, in bleomycin-injured mouse lungs. Thy1 increased in mouse lung lymphocytes following bleomycin injury but decreased in myofibroblasts when fibrosis was at the highest point (14 days), as assessed by immunohistochemistry. Using gene chip analysis, we detected that myofibroblast Thy1 crosslinking mediates downregulation of genes promoting cell proliferation, survival, and differentiation, and reduces production of extracellular matrix (ECM) components, while concurrently mediating the upregulation of genes known to foster inflammation and immunological functions. Chimeric Thy1-deficient mice with Thy1+ lymphocytes and Thy1− myofibroblasts showed fibrosis similar to wild-type mice and an increased number of CD4/CD25 regulatory T cells, with a concomitant decrease in inflammation. Lung myofibroblasts downregulate Thy1 expression to increase their proliferation but to diminish the in vivo inflammatory milieu. Inflammation is not essential for evolution of fibrosis as was previously stated.

A Profibrotic Phenotype in Naïve and in Fibrotic Lung Myofibroblasts Is Governed by Modulations in Thy-1 Expression and Activation

“…In addition, as we have shown in our previous publication [ 15 ], gene chip analysis revealed that Thy1 activation as detailed by Cohen et al [ 46 ] downregulates expression of some genes that have a role in myofibroblast differentiation and actin cytoskeleton regulation. Three genes are of special interest here.…”

“…Thy1 expression is associated either with a low or a high rate of cell growth in different cell types [ 16 , 25 , 26 , 28 – 31 ]. Since we found an increase in the proportion of Thy1 − myofibroblasts among the total myofibroblast population at day 14 following bleomycin IT [ 15 ], we assessed whether the high rate of lung myofibroblast proliferation is associated with an absence of Thy1 expression. To this end, myofibroblasts were isolated from the lungs of Thy1-deficient and WT-untreated mice.…”

“…As we have previously shown [ 15 ], gene chip analysis revealed that Thy1 activation downregulated the expression of several genes that have a role in lung fibroblast proliferation [ 46 ], including genes affecting the cell cycle, as well as genes involved in signaling in the MAPK [ 47 ], insulin [ 48 , 49 ], and TGF β [ 50 , 51 ] pathways. In this study, we validated these gene chip results by real-time RT-PCR.…”

“…Myofibroblasts are thought to be primarily responsible for increased deposition of collagen within the lung [ 55 , 64 ]. We have previously shown that Thy1 activation downregulates genes promoting fibroblast cell survival and differentiation [ 15 ], functions that are considered to be profibrotic. In this case, Thy1 may also serve as a protector against fibroblast overactivity, as shown by others who report an inverse correlation between Thy1 expression on fibroblasts and the evolution of lung fibrosis [ 16 , 65 ].…”

“…Using gene chip analysis, we found that myofibroblast Thy1 cross-linking mediates downregulation of genes promoting cell proliferation, survival, and differentiation and reduces production of extracellular matrix (ECM) components, while concurrently mediating the upregulation of genes known to foster inflammation and immunological functions [ 15 ].…”