Mechanism Of Lipopolysaccharide-Induced Lung Fibroblast Phenotypic Transformation And Aberrant Proliferation

The formation and development of pulmonary fibrosis are associated with acute lunginjury (ALI) and acute respiratory distress syndrome (ARDS), which is characterized byaberrant proliferation and activation of lung fibroblasts. Lipopolysaccharide (LPS), acomponent of bacteria membranes, plays an important role in the development ofpulmonary fibrosis. LPS could directly induce lung fibroblast activation and proliferationthrough Toll-like receptor4(TLR4), a specific receptor of LPS and downstreamintracellular signal transduction pathways. However, there is considerable controversyregarding the effect of LPS on fibroblast proliferation, which suggested that fibroblastsmight have heterogeneous subpopulations that play distinct roles in fibrotic responses.According to the expression status of thymocyte differentiation antigen1(Thy-1), acell surface glycoprotein on the cell surface, lung fibroblasts can be divided into twosubpopulations: Thy-1-positive [Thy-1(+)] and Thy-1-negative [Thy-1(-)] cells. Thy-1presents on normal lung fibroblasts, but is absent in lung tissue from the patients ofidiopathic pulmonary fibrosis (IPF). Thy-1(-) fibroblasts have an increased propensity tofibrogenic responses after various fibrogenic mediator stimulation. It has been reported thatepigenetic control, such as histone deacetylation, is involved in the regulation of Thy-1gene expression. On the other hand, several studies found that LPS could manipulate theepigenetic regulation of gene expression by histone acetylation. However, it is not clearwhether the epigenetic regulation of Thy-1expression is required for LPS-induced lungfibroblast proliferation.In the present study, based on the cellular model of LPS-induced lung fibroblastproliferation, we identify the inducible effect of LPS on lung fibroblast phenotypictransformation and aberrant proliferation at first. Then, we investigated the epigeneticmechanism related to the association of LPS-induced TLR4and Thy-1-related lungfibroblast phenotype transformation and aberrant proliferation through the depletion ofTLR4gene with RNA interference (RNAi). Objective: To identify the inducible effect of lipopolysaccharide (LPS) on lungfibroblast phenotypic transformation and aberrant proliferation during pulmonary fibrosisin the early stages of acute lung injury (ALI).Methods: Primary cultured mouse lung fibroblasts were seeded into96-well plateswith the density of1×104/ml. After being cultured for48h, the cells were randomlydivided into4groups (n=3each): PBS control group (group C) and LPS0.01μg/ml group(group LPS0.01), LPS0.1μg/ml group (group LPS0.1), and LPS1.0μg/ml group (groupLPS1.0). PBS was added to the96-well plates in group C. LPS with the finalconcentrations of0.01,0.1and1μg/ml were added to the96-well plates in groupsLPS0.01, LPS0.1and LPS1.0, respectively. After being incubated for0,6,24,48and72h(T0-4), the proliferation of the cells was measured by CCK-8assay and Thy-1mRNAexpression was detected by real-time PCR.Results: Compared with group C, the proliferation of the cells was significantlyincreased, while Thy-1mRNA expression was down-regulated at T3,4in groups LPS0.01,LPS0.1and LPS1.0(P<0.05).Conclusion: LPS results in abnormal proliferation of mouse lung fibroblasts throughdown-regulating Thy-1mRNA expression, indicating that endoxemia can inducepulmonary fibrosis. Objective: To design, construct and identify lentivirus vector of TLR4siRNA(Lentivirus-TLR4-siRNA) for the following study.Methods: Designed and prepared three plasmids containing the sequence of TLR4siRNA and then enclosed them with lentivirus. After being transfected into primarycultured mice lung fibroblast, the most effective lentivirus vector of TLR4siRNA wasselected by Real-time PCR. Results: Three plasmids containing the sequence of TLR4siRNA was constructedsuccessfully after identification with Real-time PCR and sequencing. Then it was enclosedwith lentivirus and therefore formulated the lentivirus vector of TLR4siRNA (Lentivirus-TLR4-siRNA). The most effective one (Target2＃), with titer of8×107TU/ml andinhibition efficiency of about80%, was selected by Real-time PCR.Conclusion: The lentivirus vector of TLR4siRNA (Lentivirus-TLR4-siRNA) couldinhibit expression of TLR4in primary cultured mice lung fibroblast effectively and,thereby, be applied in the following study. Objective: To identify the Mechanism of LPS-induced Thy-1(+) lung fibroblastphenotypic transformation and aberrant proliferation.Methods: Based on the cellular model of LPS-induced lung fibroblast proliferation,we observed dynamic changes of cellular proliferation and Thy-1expression at the geneticand cellular level in cultured mouse lung fibroblasts from0to72hours after beingchallenged with LPS. Then we studied the epigenetic mechanisms of LPS-regulated Thy-1(+) lung fibroblast phenotypic transformation and proliferation. We focused on the effectof LPS on histone deacetylation in the promoter region of Thy-1gene and expression ofThy-1gene thorough TLR4activaction. We also attempt to inhibit LPS-induced Thy-1gene silencing through the depletion of TLR4gene with RNA interference (RNAi).Results: BrdU assay showed that at0,6and24hours after LPS challenge, theamount of DNA synthesis was similar to that in the control group at the same time point,but it significantly increased from48to72hours after LPS challenge (P <0.05).The results of Real-time PCR and Western blot confirmed that control lung fibroblastswere predominantly Thy-1-positive. Thy-1expression in lung fibroblasts decreased from24hours and disappeared between48-72hours after LPS challenge, suggesting lungfibroblasts experienced phenotypic transformation from Thy-1(+) cells to Thy-1(-) one.Real-time PCR and Western-blot showed that the expression level of Thy-1mRNA andprotein in lung fibroblasts significantly decreased72hours after LPS challenge. However,the LPS-induced Thy-1expression inhibition was absent upon TLR4gene knockdown. Western-blot showed that Ace-H3and Ace-H4decreased significantly in the LPS-challenged lung fibroblasts when compared with the non-LPS-challenged cells, indicatingthe decrease of histone acetylation. However, all the above changes could be attenuated bydepletion of TLR4gene with TLR4-siRNA-lentivirus transfection.Conclusion: Our studies showed that epigenetic regulation of Thy-1gene expressionwere involved in LPS-induced lung fibroblast aberrant proliferation through histonemodification in the Thy-1gene promoter region and sequential lung fibroblast phenotypetransformation via LPS’s specific receptor, TLR4, which contributes to the pathogenesis ofLPS-induced lung fibroblast proliferation and pulmonary fibrosis.