Tumor Necrosis Factor Related Apoptosis Inducing Ligand(TRAIL) Signaling Is Crucial For The Development Of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a large group of interstitial lung diseases,the etiology of which remains unknown. It’s characterized by epithelial injury,pulmonary inflammation, formation of distinctive fibroblasts/myofibroblasts andexcessive extracellular matrix accumulation. The most common clinical features ofIPF include progressing exertional dyspnea (difficulty breathing on exercise),peripheral pattern of reticulonodular opacification or honeycombing in X ray orHRCT, restrictive ventilatory dysfunction, reduced diffusing capacity for carbonmonoxide (DLCO) and hyoxemia. The prognosis of patients with IPF is poor, with amedian survival of only3–5years.Unfortunately, the mechamisms how this diseasedevelops are not fully understand and there is no effective therapeutic approach forthis deadly disorder. Therefore, numerous overseas and domestic studies haveexplored the pathogenesis of and therapeutic approaches for IPF. In recent years,some researchers found that tumor necrosis factor-related apoptosis inducingfactor(TRAIL) has an important regulatory role in acute and chronic airwaysinflammation. Our preliminary published studies have already shown that TRAILdependent MID1-mediated regulation of PP2A contributes to the development inrhinovirus-induced airways inflammation. However, how TRAIL signaling involvesin the development of idiopathic pulmonary fibrosis remains obscure.ObjectiveTo identify how TRAIL involves in the development of pulmonary fibrosis invivo and in vitro and elucidate the mechanisms how TRAIL regulates downstreamsignaling pathways; To study the role of PP2A activation(AALs) onanti-inflammatory and anti-fibrosis in pulmonary fibrosis, providing a new thoughtand direction for clinical diagnosis and treatment of IPF. Methods⑴To establish pulmonary fibrosis mice model using TRAIL knockout andwildtype mice intratracheally bleomycin administered; Lung function measurementswere performed using Buxco-Forced Pulmonary Maneuvers;To evaluate the fibroticdegree through observing alveolitis of different stages, inflammation, fibrotic changesin the histological slides, detecting the mRNA expressions of collagen α2(I) usingqPCR, evaluating apoptosis in the lungs using TUNEL staining and measuring theprotein levels of TRAIL using ELISA. All above support the hypothesis that TRAILcould increase the development of pulmonary fibrosis in mice.⑵After establishing pulmonary fibrosis mice model, measure the mRNAexpressions of MID1, TGF-β,MCP1and MMP9using qPCR and measure thephosphorylation activities of PP2A using ELISA, which identifies the mechanismsthat how TRAIL regulates downstream signaling pathway.⑶AALs(PP2A activation) was instilled intranasally to intervene bleomycininduced pulmonary fibrosis after establishing fibrotic model, two ways of drug givenwere adopted, one-time AALs pretreatment and successive administration. Lung functionmeasurements were performed using Buxco-Forced Pulmonary Maneuvers;Toevaluate the fibrotic degree through observing alveolitis of different stages,inflammation, fibrotic changes in the histological slides, detecting the mRNAexpressions of collagen α2(I) using qPCR, evaluating apoptosis in the lungs usingTUNEL staining and measuring the phosphorylation activities of PP2A using ELISA.All above will explain how PP2A activation affect the fibrotic model.⑷Primary mice fibroblast culture and passage, to study the role of rTRAIL andAALs on proliferation of fibroblasts using MTT assay and to measure the mRNAexpression of MID-1,TGF-β and collagen α2(I) using quantitative PCR.⑸To collect the human data, serum and lung biopsies and to measure the proteinlevels of TRAIL in the serum and MID1,PP2A activities in the lungs by ELISA. Toanalyze the correlation between TRAIL, MID1and lung function using Pearson’scorrelation coefficient.Results⑴TUNEL-positive cells in bleomycin-treated lungs were significantly increasedat1day. The numbers of apoptotic cells decreased at4days after bleomycinchallenge. In the lung parenchyma, signs of epithelial cell damage, local hemorrhage, edema and inflammatory cell infiltration were evident by8days after bleomycintreatment. By21days after bleomycin challenge,evidence of both inflammation andfibrosis was present in most parenchymal tissue fields, and the deposition of collagenpeaked as determined by trichrome staining for collagen.⑵WT mice instilled with bleomycin(WT BLM) had a significant increase incollagen compared with the saline control (P<0.01) according to the scores of imageanalysis and mRNA expression of collagen α2(I) and had a significant decrease inpeak compliance and vital capacity compared with control group(P<0.01).TRAIL-/-(TKO BLM) mice had less fibrosis and better lung function(P<0.05).⑶The mRNA expressions of MID1and related factors(TGF-β、MCP1、MMP9)were increased in WT BLM group compared to control group（P<0.05）and they weredecreased in TKO BLM group.While PP2A in lung homogenates was deactivated inWT BLM compared to control group（P<0.05）;PP2A was activated in TKO BLMgroup,the difference was statistically significant.⑷The fibrotic degrees in WT BLM AAL(-1)(AALs treatment24h beforebleomycin challenge) were similar with mice treated with bleomycin only(WT BLM)(P<0.001),indicating that continuous drug intervention could improve mice lungfunction;the mRNA expressions of TGF-β、MCP1、MMP9were decreased, indicatingthat AALs(PP2A activation) could decrease the release of inflammatory factor andintervene the development of fibrosis.⑸rTRAIL at lower doses was found to stimulate collagenα2(I)mRNAexpression and the peak collagen secretion was observed at doses1ng/ml.Thissuggests that rTRAIL at concentrations below the apoptosis-inducing threshold couldstimulate fibroblast proliferation. Fibroblasts were incubated with1ng/ml rTRAIL andfound that they increased collagen α2(I) and TGFβ mRNA expression. The expressionof collagenα2(I) mRNA were decreased after1μM AALs chanllege.⑹Serum levels of TRAIL were increased in patients with IPF compared tocontrols(P<0.001）and Serum levels of TRAIL of IPF patients correlate with DLcovalues(r2=0.7528,P<0.0001,n=14) and RV(r2=0.7268,P=0.0001,n=14).The proteinlevels of MID-1were increased in the lung biopsies of IPF patients(P<0.05),positively correlated with serum TRAIL(r2=0.8717,P=0.0007,n=8) and negatively correlated with DLco values(r2=0.8903,P=0.0004,n=8) and RV(r2=0.7561,P=0.005,n=8). PP2A activities of IPF patients were decreased compared to controls（P<0.001）.Conclusion⑴TRAIL signalling through MID1mediated PP2A increases the expression ofTGF-β, MCP1and MMP9, which plays a fundamental role in the development ofairway remodeling and pulmonary fibrosis. Knockout gene TRAIL or PP2Aactivation(AALs) daily treatment could reduce deposition of collagen in the lung andimproved the lung function which may present novel therapeutic targets for thetreatment of human idiopathic pulmonary fibrosis.⑵Low dose of rTRAIL could increase the proliferation of mice primarypulmonary fibroblasts and PP2A activation intervene the expression of collagen insome extent.⑶TRAIL signaling involves in the development of IPF, the protein levels ofTRAIL in the serum of IPF patients could be a new auxiliary diagnostic method forIPF and one of measuring standards to evaluate fibrotic degrees.