Epithelial Mesenchymal Transition in Human Ocular Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD) of the ocular surface and lacrimal gland is a vision-threatening condition that occurs after allogeneic bone marrow transplantation. In this study, we used immunohistochemistry and electron microscopy to investigate whether epithelial mesenchymal transition (EMT) contributed to the pathogenesis of ocular cGVHD. We detected down-regulation of E-cadherin and translocation of β-catenin from the intercellular junction to the cytoplasm and nucleus of cGVHD conjunctival basal epithelia and lacrimal gland myoepithelia. Notable findings included expression of Snail, an inducer of EMT, in the nucleus of ocular cGVHD epithelia. The fibrosis markers heat shock protein 47, α-smooth muscle actin, and fibroblast specific protein-1 were overexpressed in ocular cGVHD epithelia. In addition, p63, a marker of conjunctival basal epithelia, was observed in the nuclei of subconjunctival cells beneath disrupted basal lamina. Disrupted basal lamina and the presence of altered collagen bundles were observed in the cytoplasm and beneath cGVHD epithelia. In contrast, these observations were rarely observed in the normal conjunctiva and in Sjögren’s syndrome lacrimal gland epithelia. These findings together indicate that ocular cGVHD epithelia gain the mesenchymal phenotype and the capacity to migrate into the subepithelial stroma. Our findings suggest that EMT may be partially responsible for the conjunctival and lacrimal gland fibrosis found in patients with cGVHD.Allogeneic hematopoietic stem cell transplantation is a potentially curative treatment for hematological malignancies. However, chronic graft-versus-host disease (cGVHD) is a major complication after allogeneic hematopoietic stem cell transplantation (HSCT), which has hampered the success of HSCT.¹ Although numerous advances have been made for treating acute GVHD, the pathogenesis of cGVHD remains largely unknown and effective therapy has not been established. We previously studied the pathogenesis of cGVHD by focusing on the ocular surface and lacrimal gland and found that excessive fibrosis and a subset of fibroblasts contribute to the pathogenesis of ocular cGVHD.^2,3Recently, several studies have reported that epithelial-mesenchymal transition (EMT) contributes to various fibrotic diseases of the kidney,⁴ lung,⁵ and liver.⁶ For example, 40% of fibroblasts in kidney fibrosis arise from epithelial cells via local EMT triggered by inflammatory stress.⁷ EMT is involved in embryonic development, organ fibrosis, and also cancer metastasis.⁸ EMT is characterized by the loss of apical/basal cell polarity and loss of cell-to-cell adhesions, followed by the acquisition of a mesenchymal phenotype, ie, migration and invasion ability and expression of mesenchymal markers.EMT is triggered by various stimuli including irradiation,⁹ hypoxia,¹⁰ reactive oxygen species,¹¹ inflammatory cytokines such as transforming growth factor-β and fibroblast growth factor,⁸ disruption of basal lamina, and exposure of cytoplasm to extracellular matrix.¹² These triggers of EMT also participate in conjunction with the pathogenesis of cGVHD after HSCT. In a clinical setting, total body irradiation before HSCT and migrating inflammatory cells after HSCT generate substantial proinflammatory cytokines.¹ This “cytokine storm” then acts on T cells in the graft, prompting them to attack host antigens.¹³ In addition, reactive oxygen species-mediated organ injury was reported in bone marrow transplant recipients.¹⁴ The purpose of this study was to elucidate whether EMT is involved in mucosal and exocrine gland cGVHD.