The Mechanisms Of GVHD-induced Pulmonary Fibrosis

Background and objective:Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for many individuals with malignant and non malignant conditions. Unfortunately, a number of complications limit its successful outcomes. Diffuse lung injury occurs in 25-60% of transplant recipients and can account for half of the non-relapse mortality. Lung injury following allo-HSCT can be classified as infectious or noninfectious in nature, and can be either early occuring within 100 days or late-onset occuring over 100 days post-transplantation. Recently, idiopathic pneumonia syndrome (IPS), bronchiolitis obliterans (BO) and bronchiolitis obliterans organizing pneumonia (BOOP) are now recognized as part of a spectrum of post-HSCT lung diseases. Within the past decade, significant progress has been made in elucidating the mechanisms underlying the noninfectious lung injury post-transplantation. Yet the pathophysiology of these disorders is not fully understood. Emerging evidences indicate that there exists a strong association between acute graft-versus-host disease (aGVHD) and IPS, and BO or BOOP is pathognomonic of chronic GVHD (cGVHD). Recent prevalence estimates suggest that noninfectious lung injury is likely underdiagnosed, and when severe noninfectious lung injury does occur, current treatments have been largely ineffective. The lack of good therapeutic options may be related to the paucity of knowledge on the pathogenesis. Currently, research on the pathogenesis of aGVHD mainly focuses on the immunologic cells and various cytokines and chemokines. What happened after the target cells being attacked is largely unknown. Clinical investigation found that those patients with lung injury in the early stage of post-HSCT were vulnerable to develop late onset non-infectious pulmonary complications.Whether early or late lung injury after transplantation, their common pathological feature is the fibrosis of lung tissue.In the non-transplantation patients, the lung fibrosis may be due to viral or bacterial infection, smoking, radioaction, toxic compounds, environment exposure to allergens, or collagen angiopathy and so on. The pathophysiologic process of lung fibrosis is very complicated and its pathogenesis is still unclear. It is largely accepted that the lung damage induces a cascade of cellular responses and molecular processes, trying to control the damage, to fight the infection, and to repair the damaged tissue. The repair process goes away and the immune and repair response overreact, leading to inflammation, uncontrolled apoptosis, improper tissue remodeling, and excessive extracellular matrix secretion. Lung fibrosis includes three pathological stage:alveolitis, alveolar epithelial cell injury and fibrosis repair after the damage, and finally characterizes by progressive scarring of the lung parenchyma, leading to areas of inflammation,excessive collagen deposition and fibroblast proliferation. Clinical investigation found that the first stage of lung fibrosis is characterised by alternating with regions of alveolar epithelial cell injury and hyperplasia, basal membrane disruption and capillary leakage. Many matrix metalloproteinase, cytokines, growth factors, are involved in this complex network of regulation. During the initial injury phase, activated alveolar epithelial cells and recruited inflammatory cells (eg, macrophages, neutrophils) release potent fibrogenic growth factors that perpetuate the cycle of injury, failed repair, and fibrosis. These growth factors, particularly transforming growth factor β(TGF-β), are recognized as one of the most important profibrogenic cytokines.TGF-β via (1) triggering the epithelial-mesenchymal transition (EMT) signaling pathway and leading to the fibrogenesis; (2) involving in injury and apoptosis of alveolar epithelial cells, and in activation, invasion,or apoptosis resistance of fibroblasts and myofibroblasts; (3) stimulating the fibroblasts to synthesize and secrete ECM; (4)inducing inflammatory cell to release the profibrogenic cytokines, such as tumour necrosis factor α(TNF-α), platelet-derived growth factor (PDGF), interleukin 1(IL-1) and interleukin 8(IL-8). On the contrary, interferon γ (IFN-γ) has anti-fibrosis effect, it can (1) down-regulate the TGF-β expression to inhibit the proliferation and migration of fibroblasts; (2) directly inhibit synthesis and secretion of collagen by fibroblasts and myofibroblasts; (3) act on alveolar macrophage indirectly prompting myofibroblasts apoptosis; (4) act on interferon induced protein 10 (IP-10) inhibiting angiogenesis and result in lung fibrosis amelioration. In most instances, these processes are well controlled leading to proper healing without any pathological problems. In some cases, activated alveolar epithelial cells and recruited macrophages release potent TGF-β, leading to the imbalance of the TGF-β and IFN-γ, then trigger the downstream signal pathway that activates the myofibroblasts ultimately resulting in the increased deposition of extracellular matrix.In the allo-HSCT recipients, the pathological hallmark of the GVHD-induced noninfectious diffuse lung injury is fibrosis, especially the fibrosis of alveolar and bronchioles. The pathophysiology of these disorders is not fully understood yet. Whether GVHD-induced noninfectious diffuse lung injury has the similar pathological physiology as the usual pulmonary fibrosis is largely unknown. In this study, we established a murine model of aGVHD-induced lung injury to explore the effect and molecular mechanism of microenvironmental changes on lung epithelial cell biology behavior, and to clarify whether EMT is involved in airway remodeling of GVHD-induced lung injury. These studies will supply theoretical basis in preventing and treating aGVHD-induced diffuse lung injury and pulmonary fibrosis in patients underwent allo-HSCT.Methods and materials:1) Establishment of an aGVHD-induced lung injury animal model:BALB/c mice received 8 Gy total body irradiation, and administrated by an intravenous injection of 10×106 C57BL/6 spleen cells and 5×106 C57BL/6 bone marrow cells as GVHD group, and only injected bone marrow cells as control group. Survival and clinical GVHD severity were assessed in BALB/c recipeints on every other day post transtplantation. Peripheral blood from recipeints was collected for chimerism detection. Formalin-preserved of the right lobe of the lungs were embedded in paraffin, and 5-μm-thick sections were stained with hematoxylin and eosin for histologic examination, and stained with masson for collgen analysis.2) Analysis of T lymphocyte subpopulation distribution:Peripheral blood mononuclear cells, broncho-alveolar lavage fluid cells and lung tissue lymphoid cells from individual mice at different time point(7d,14d,21d,28d) after HSCT were assessed by flow cytometry and the acquired data were further analyzed using BD-FACSDiva Software.3) Detection of cytokine expressions in peripheral blood, broncho-alveolar lavage fluid and lung tissue:Concentrations of IFN-γ, TNF-α, and TGF-β were measured in the serum and broncho-alveolar lavage fluid by enzyme-linked immunosorbent assay, performed according to the manufacturer’s protocol. Real-time polymerase chain reaction was used for detecting cytokine expressions of lung tissue.4) Research on the regulation of EMT signaling pathway:Immunofluorescent labeling of epithelial marker E-cadherin and mesenchymal marker Vimentin were performed with multiple steps of antibody staining on lung tissue from patients with GVHD-induced lung injury. And samples were visualized with an Olympus FV10i-W Intelligent laser scanning confocal microscope. The mRNA and protein expressions of E-cadherin and Vimentin in GVHD mice lung tissue, were detected by RT-PCR and western blotting. The related transcription factor and key proteins of epithelial to mesenchymal transition signalling pathways were detected at the same time.Statistical analysisSPSS 13.0 software was used for statistical analysis. Results were described as mean ± standard deviation (x±s). Measurement data was analyzed by independent-samples T test, p<0.05 was considered as significant difference. Survival was analyzed by Kaplan-Meier method, and group differences were evaluated according to the log-rank test. Figures were showed with the use of GraphPad Prism5 software.Results1) We established an aGVHD-induced lung injury animal model of C57BL/6â†'>BALB/c mice. The chimerism of donor marker H2Kb exceeded 98% on 28 days post-transplantation, which indicated that the engraftment was succeeded. In the GVHD group, the manifestations of aGVHD, such as weight loss, hunching, activity and diarrhea, can be seen easily. The survival between the two groups was significant difference(χ2=32.514, P<0.001). The clinical score of GVHD group was much higher than that of the bone marrow control group. Lung tissue was evaluated using a semiquantitative scoring system that incorporates both the severity and extent of histopathology. Compared with the BMT control, inflammatory cells infiltrate and interstitial thickening were easily seen in the GVHD lungs. In Masson staining, we can find collagen deposition around the airway. All these results suggest diffuse lung injury accompanied by fibrosis occur in the GVHD mice.2) In the aGVHD-induced lung injury mice, CD4+ lymphocyte infiltration appeared in the early stage of transplantation, and then swithched to CD8+ lymphocytes infiltration in the late stage post-HSCT. The CD4+ lymphocytes of blood increased on 7 days and 14 days(P<0.05), and then gradually decreased on 21 days and 28 days after transplantation while the CD8+ lymphocytes were not significant difference between the two groups(P>0.05). The peak of CD4+ lymphocytes in broncho-alveolar lavage fluid was on 7 days, whereas the peak of CD8+ lymphocytes was on 28 days after transplantation.The CD4+ lymphocytes of lung tissue increased on 7 days,14 days and 21 days post-HSCT in GVHD group (P<0.05), and the CD8+ lymphocytes in GVHD lung tissue were significantly higher as compared to the the control group at all time points after transplantation (P<0.05).3) There was a discrepancy expression of cytokines in serum, broncho-alveolar lavage fluid and lung tissue between the two groups. Compared with BMT control group, the expression of serum IFN-γ in GVHD mice was lower on 28 days after allo-HSCT (P<0.05). High levels of TNF-α in serum was seen on 7 days,14 days and 21 days post-HSCT in GVHD group(P<0.05). The serum TGF-β level had no significant difference between two groups (P>0.05). The peak of IFN-y in broncho-alveolar lavage fluid was on 7 days and the peak of TNF-a was on 14 days after transplantation.While the TGF-β level in broncho-alveolar lavage fluid increased gradually, and was significant higher than BMT mice on 7 days,14 days and 21 days post-HSCT (P<0.05). The variation tendency of TGF-β in lung tissue was similar to that in broncho-alveolar lavage fluid. The expressions of IFN-γ and TNF-α in lung tissue increased in early stage and dropped in late stage of allo-HSCT.4) Epithelial to mesenchymal transition involved in the airway remodeling of GVHD-induced lung injury. Epithelial cell marker E-cadherin and mesenchymal cell marker Vimentin were found coexpression on the epithelial cells of the patients with GVHD-induced lung injury. Futher researches on the animal model showed that GVHD mice have lower levels of E-cadherin and increased levels of mesenchymal Vimentin, suggesting epithelial cells undergoing EMT. Interestingly, these changes were in a time dependent manner.In addition, an elevated expression of EMT-related transcription factor Slug and key proteinsβ-catenin and Claudin-1 was found in GVHD mice.Conclusions1) An aGVHD-induced lung injury murine model in allo-HSCT was successfully established. By observing the pathological changes and collagen deposition, lung tissue fibrosis was confirmed in aGVHD mice.2) The CD4+ lymphocytes appeared in the early stage while CD8+ lymphocytes elevated in the late stage post-HSCT. The swithched lymphocyte infiltration contributed to the differential expression of cytokines.3) The imbalance expression of cytokines in lung tissue might be the "switch" that initiates the pathogenetic cascade of fibrosis in aGVHD-induced lung injury.4) EMT plays an important role in airway remodeling during aGVHD-induced lung injury. One of the reasons for lung fibrosis may be associated with the EMT, in which the alveolar epithelial cells undergo transition to fibroblasts that was attributed to the microenvironment changes in lung tissue.