Effects Of IRHOM2 On Pulmonary Fibrosis In Mice By Modulation Of TNF-α

Idiopathic pulmonary fibrosis (IPF) is characterized by diffuse interstitial fibrosis, but the mechanism is still unclear. Pulmonary fibrosis has no lasting option for therapy other than transplantation for end-stage. The mechanism of pulmonary fibrosis might be related to severe inflammation and aberrant wound healing.Pulmonary fibrosis is an important step after lung injury. There are three distinct repair steps after lung injury, including an inflammatory phase, a granulation tissue formation and deposition of extracellular matrix phase(ECM), and a final remodeling phase where normal tissue architecture is restored. After tissue damage, a variety of cytokines and chemokines amplify the inflammatory response, trigger fibroblast proliferation and recruitment to the injury site. Then the fibroblast transform into a-smooth muscle actin(a-SMA) expressing myofibroblasts that secrete ECM components in order to promote wound healing and tissue remodeling. Studies have demonstrated that Type â…¡ alveolar epithelial cell in the wound repair process can be found in the IPF patients and animal models, with upregulation of fibrosis markers, such as a-smooth muscle actin and collagen-â… . Epithelial cells cytoskeleton reorganization has morphological changes into spindle, and eventually transform into mesenchymal cells. Excessive deposition of extracellular matrix proteins can lead to fibrosis.TNF-a is a proinflammatory cytokine that plays an important role in the pathological process of pulmonary fibrosis. Earlier studies have found TNF-a involved in lung wound healing, overexpressed after lung injury, and associated with progressive pulmonary fibrosis. TNF-a can upregulate the expressions of collagen-â…  and a-SMA by stimulating deposition of extracellular matrix. Further research found that TNF-a modulated transcription of target genes by transmembrane receptor kinase, then induced proliferation and differentiation of myofibroblast.Inactive rhomboid-like protein2(IRHOM2) belongs to the rhomboid-like protein family, which acts as key regulators in cellular signal transduction, for example, RHBDD3 suppresses activation of TLR3 pathway of NK cell. Freeman found a role of IRHOM2 as an essential factor for the modulation of anti-pathogen defense and sleep. Macrophages secrete TNF-a after invasion, which is shed from the plasma membrane by the ADAM family metalloprotease TACE (TNF-a converting enzyme). IRHOM2 is required in the ER (endoplasmic reticulum) for the maturation of TACE, and acts as a type of cargo receptor to assist the trafficking of TACE to the cell surface. Inhibiting the expression of IRHOM2 could induce dysfunction of TACE. In addition, studies have shown that mice lacking Rhbdf2(encode IRHOM2) were protected from inflammatory arthritis, showing less joint swelling and lower clinical scores, and reflecting less synovial inflammation and cartilage erosion.Our research was aimed to explore the role of IRHOM2/TACE/TNF-a signaling pathway in modulation of pulmonary fibrosis in vivo and in vitro, which would provide new theoretical supports for clinical intervention.Part 1. The effect of pulmonary fibrosis on expressions of IRHOM2 and TNF-a in lung tissue and cells in miceThe effect of pulmonary fibrosis on expressions of IRHOM2 and TNF-a in lung tissueObjective:To study the effect of pulmonary fibrosis on expressions of IRHOM2 and TNF-a in lung tissue in mice.Method:Male C57BL/6 mice were randomly divided into control(Con) and pulmonary fibrosis group (Fb). Bleomycin(BLM,4mg/kg) was given to Fb mice on Day 1, while Con mice were treated with saline (2ml/kg). Lung tissue injury was evaluated by arterial blood gas analysis, histology, expression of hydroxyproline and wet/dry ratio(W/D ratio) on Day 14. Bronchoalveolar lavage fluid(BALF) was isolated for analysis of cytokine levels using ELISA. Immunohistochemtry was used to evaluate levels of collagen-I, a-SMA and fibronectin (FN). The expressions of a-SMA, collagen-I and IRHOM2 were detected by Western Blot.Results:Compared with Con group, Fb mice induced lung pulmonary fibrosis by lower PaO2, marked recruitment of interstitial edema,neutrophils, alveolar haemorrhage, severe pulmonary lesions, and more cytokine in BALF. The expression of a-SMA and collagen-I is increased while IRHOM2 declined in lung tissue of Fb mice.Conclusion:Pulmonary fibrosis was induced by BLM with increased level of TNF-a in BALF and upregulated a-SMA and collagen-I, but decreased expression of IRHOM2.The effect of pulmonary fibrosis on expressions of IRHOM2 and TNF-a in type II alveolar epithelial cell(AT-â…¡) and alveolar macrophage(AM) in miceObjective:To study the effect of pulmonary fibrosis on expressions of IRHOM2 and TNF-a in type II alveolar epithelial cell and macrophage in mice.Method:C57BL/6 mice were randomly divided into control group (Con) and pulmonary fibrosis group (Fb). Bleomycin(BLM,4mg/kg) was given to Fb mice on Day 1, while Con mice were treated with saline (2ml/kg). AT-II and MA were isolated to evaluate the levels of TNF-a and IRHOM2 by ELISA and Western Blot on Day 14.Results:Compared to the Con group, both TNF-a and IRHOM2 were upregualated in MA of Fb group. The expressions of free TNF-a and IRHOM2 decreased in AT-II of Fb group.Conclusion:Pulmonary fibrosis was induced by BLM with increased levels of TNF-a and IRHOM2 in MA, and decreased expressions of free TNF-a and IRHOM2 in AT-II.Part 2. The role of cytokine in the variation of IRHOM2 in pulmonary fibrosis mice and the role of IRHOM2 in epithelial-mesenchymal transition (EMT) in cell line of A549Objectives:To study what the reason for the change of IRHOM2 and the effect of IRHOM2 in EMT.Method:Cells were isolated from C57BL/6 mice, then randomly divided into control group (AT-Con), cytokine group(AT-Fb) which involved cytokine in culture medium, BLM group(AT-BLM) which involved BLM in culture medium, TNF-a group(AT-TNF) which involved TNF-a in culture medium. Cells were isolated to detect the expression of IRHOM2 by Western Blot.Human type 2 alveolar epithelial cell line A549 was divided into control group (A-Con), EMT group(A-EMT) which involved BLM in culture medium, IRHOM2 knockdown group(A-RNAi) which was pre-treated with lentivirus. Cells were isolated to detect the expressions of a-SMA and collagen-I by Western Blot.Results:Compared with AT-Con group, the expression of IRHOM2decreased in AT-Fb and AT-TNF group. The levels of a-SMA and collagen-I in A-RNAi group were expressed between A-Con and A-EMT groups.Conclusion:The inflammatory cytokine, such as TNF-a, may play a key role IRHOM2 in pulmonary fibrosis mice by reducing the expression of IRHOM2. The knockdown of IRHOM2 could down regulate the markers of EMT, which suggested that it relieves the degree of fibrosis.