Role Of Nogo-B In The LPS-induced Macrophage Immune Response And Its Mechanism

Backgrounds:Acute lung injury（ALI）is serious clinical disorders of respiratory system which can be attributed to biochemical and physical factors such as bacterial infection,injury,poisonous gas,pollutants,autoantibody,stomach acid,embolism,free fatty acids,etc.Despite advances in the past decades in the knowledge and treatment of ALI,the mortality remains unacceptably high,ranging from 27% to 45% and survivors suffer significant decrements in their quality of life.Currently,the mechanism of ALI is still unclear.Because of the high mortality,low cure rate and poor quality of life in patients who have ALI,proving the pathophysiology and finding effective treatments is particularly necessary.Alveolar Macrophages（AM）as the main innate immune cells of lung play an important role in the development of ALI.As we all know,macrophage,an important member of immune system,has significant functions in innate immune response and adaptive immune response.In addition,macrophages also play important roles in the process of injury repair,tissue remodeling and fibrosis.Nogo-B is one of the reticulon super-family proteins（RTNs）.Recently,the study of Nogo-B has been focused on its general role in regulating macrophage responses,including increasing chemokine-mediated monocyte/macrophages migration and infiltration,turning down the extent of ischemic injury and promoting wound healing.Marinet al.demonstrated a dramatic delay in the recruitment of macrophages in the Nogo-B gene disrupted mice in vivo,which was consistent with the findings by Kathrin et al.that Nogo-B deficiencywas associated with a reducedactivity of promoting macrophage homing and functional recovery.Thus,it was suggested that endogenous Nogo-B may be implicated in the control of macrophage activities and trafficking.However,whether endogenous Nogo-B is associated with the regulation of macrophage in immune response aroused by LPS is unclear.In the previous study we observed that the expression of Nogo-B in bronchial alveolar lavage fluid（BALF）,lung tissue,pulmonary vascular tissue and alveolar epithelial tissue of mice that had ALI was significantly reduced compared with normal mice,especially in BALF,and the main cellular components of BALF were AM.According to previous reports and our findings,we hypothesized that the expression of Nogo-B can regulate the immune response of macrophages by affecting the function of secretion,migration,antigen presentation and phagocytosis.In order to confirm the hypothesis and elucidate the molecular mechanism,we selected mouse macrophage cell line RAW264.7 as the research object and then explore the role of Nogo-B in manipulating macrophage activities and uncover the possible mechanism.Methods:1.RAW264.7 cells were cultured in DMEM containing 10% fetal bovine serum in a standard humidified incubator at 37℃ with 5% CO₂.After LPS stimulation（different concentrations）,Nogo-B expression in macrophages over different periods were detected to confirm the optimum concentration and effect time by Western blot.Confirm the optimum concentration and effect time.2.Up and down-regulation of Nogo-B in RAW264.7 by adenovirus mediated gene transfection and siRNA interference,respectively.After LPS（optimum concentration,1μg/ml）stimulation,inflammatory factors expression in macrophages were detectedby Elisa,cell migration abilities were examined by transwell assay,the major histocompatibility complex（MHC）Ⅰ/Ⅱ which reflects the antigen-presenting capacity of macrophages were testinged by flow cytometry and MSR1 expression by western blot at the optimal time.3.Investigate possible mechanisms of Nogo-B towards the function of macropahges.TLR4 expression on macrophages of different Nogo-B expression levels were assessed by flow cytometry.The phosphorylation of ERK,JNK and P38 of MAPK pathway in macrophages of different Nogo-B expression level were detected by Western Blot.Results:1.LPS stimulation of different concentrations to RAW264.7 will result in differences in Nogo-B expressionNogo-B expression in RAW264.7 declined in LPS concentration dependent manner.We found that as LPS concentration reached 1μg/ml,the Nogo-B expression declined to the bottom.And we regard 1μg/ml as the optimal concentration and used for time-course experiments.Western blot results showed that,in the time-course behavior,Nogo-B level was reduced gradually after LPS administration and fell to the lowest level at 24 hand remain till 48 h.2.Intervening of Nogo-B expression,the biological functions of RAW264.7 were changing LPS stimulation（1）After LPS stimulation,the level of cytokines（i.e.TNF-α,IL-1β,MCP-1 and TGF-β）excreted by Nogo-B over-expression macrophages significantly increased.While after silence of Nogo-B,the cytokines expression was suppressed.（2）At 24 hours after LPS stimulation,migration of RAW264.7cells with high Nogo-B expression were 1.8 times as that of the control;while in Nogo-B silencing group,migration of RAW264.7cells were 1.5 times less.（3）After LPS stimulation,MHC Ⅱ expression in the high Nogo-B expression group increased in a time dependent manner,while decreased in the Nogo-B silencing group compared with control.There was no significant difference about MHC Ⅰ expression level among groups.（4）At 24 hours after LPS stimulation,MSR1 expression increased in the high Nogo-B expression group and declined significantly in the Nogo-B silencing group.3.Nogo-B Affected Cell-Surface Expression of TLR4 and Regulated MAPK Pathway ActivationAfter LPS stimulation,we found the mean fluorescence intensity of TLR4 on the surface of RAW264.7 was significantly increased when Nogo-B over expressed,and markedly lower when Nogo-b was deficiency.The same result can be found in phosphorylation of ERK,JNK and P38.Conclusion:1.The expression of Nogo-B in macrophages can be affected by LPS stimulation and the most optimal concentration for LPS to stimulate RAW264.7 is 1μg/ml,in which the expression of Nogo-B reduces markedly.2.The expression of Nogo-B is positively correlated with cytokine production,migration,antigen-presenting and Scavenging Capacity of Macrophages.3.Nogo-B may affect macrophage immune response by regulating cell-surface expression of TLR4 and MAPK pathway activation.