Study On Molecular Mechanisms Of Toll-like Receptor 2 In Phagocytosis And Autophagy And Allergic Airway Inflammation

Toll-like receptors (TLRs) are type I transmembrane proteins family which are responsible for sensing invading pathogens. To date,10 TLRs have been identified in humans. TLRs-regulated innate immune responses have been identified in the pathogenesis of multiple infections and inflammatory diseases. In the present study, we investigated the role of TLR2 in the following two aspects.1. Molecular mechanisms of TLR2-mediated phagocytosis and autophagy signaling pathways in Staphylococcus aureus-stimulated macrophages BackgroundTLR2 has been reported to participate in the recognition of S. aureus. However, whether macrophages can activate phagocytosis and autophagy in response to S. aureus infection in airway, the role of TLR2 in innate immune processes in S. aureus-stimulated macrophages and the underlying mechanisms as yet remain unclear.Objective1. Study activated level of phagocytic and autophagic signals in macrophages during S. aureus infection.2. Investigate the role of TLR2 and downstream potential signal cascade in phagocytosis and autophagy in S. aureus-stimulated macrophages.Methods1. Detect signal molecular expression and autophagy in S. aureus-stimulated macrophages at different timesMouse mononuclear macrophages RAW264.7 were stimulated with S. aureus for 15min, 30min,45min,60min,120min,240min. The expression of multiple signal molecules and autophagic proteins was analyzed by Western Blot. The level of cellular autophagy was analyzed by GFP-LC3 transient transfection using confocal laser scanning microscope (CLSM).2. Evaluate the effect of TLR2 siRNA on phagocytosis of S. aureus by macrophages and autophagy activationStimulation RAW264.7 cells with S. aureus for 1h with TLR2 siRNA, the amount of phagocytosis was examined by phagocytosis assay. The level of cellular autophagy was analyzed by GFP-LC3 transient transfection using CLSM. The expression of phagocytic and autophagic proteins was analyzed by Western Blot.3. Evaluate the effect of JNK inhibition on phagocytosis of S. aureus by macrophages and autophagy activationStimulation RAW264.7 cells with S. aureus for 1h with JNK specific inhibitor SP600125, the amount of phagocytosis was examined by phagocytosis assay. The level of cellular autophagy was analyzed by GFP-LC3 transient transfection using CLSM. The expression of phagocytic and autophagic proteins was analyzed by Western Blot.Results1. Activation of multiple signaling pathways and autophagy in S. aureus-stimulated macrophagesIncreased expression of P-JNK, P-ERK, P-p38 MAPKs, MyD88, P-Akt and GTP-Racl was found in RAW264.7 cells upon S. aureus infection. Among activated MAPKs, only phosphorylation of JNK was increased in a time-dependent manner. At the same time, Beclin-1 and LC3-Ⅱ proteins expression, and the number of GFP-LC3 punctate-structures per cell were upregulated in S. aureus-stimulated macrophages.2. TLR2 is critical for phagocytosis of S. aureus by macrophages and autophagy activationRAW264.7 cells treated with TLR2 siRNA showed a significant reduction in the phagocytosis of S. aureus, and decreases in the proteins expression of P-Akt, GTP-Racl and P-JNK while did not attenuate ERK or p38 phosphorylation upon S. aureus infection. TLR2 silence by siRNA in RAW264.7 cells notably decreased S. aureus-induced expression of Beclin-1 and LC3-Ⅱ proteins, and the number of GFP-LC3 punctate-structures per cell.3. TLR2-dependent JNK signaling pathway is involved in phagocytosis of S. aureus by macrophages and autophagy activationJNK inhibitor weakened the phagocytosis of S. aureus by RAW264.7 cells, and S. aureus-induced proteins expression of P-Akt and GTP-Racl. RAW264.7 cells treated with SP600125 showed reductions in the proteins expression of Beclin-1 and LC3-Ⅱ, the number of GFP-LC3 punctate-structures per cell upon S. aureus stimulation. Treatment of RAW264.7 cells with both TLR2 siRNA and JNK inhibitor SP600125 did not further decrease phagocytosis of S. aureus and autophagic level upon S. aureus stimulation in comparison with that in TLR2 siRNA treatment alone.Conclusions1. Activation of phagocytosis and autophagy in S. aureus-stimulated macrophages.2. TLR2 mediates phagocytosis and autophagy through JNK signaling pathway in S. aureus-stimulated macrophages, which propose a new feasible innate immune regulatory mechanism.2. Targeting inhibition of TLR2 knockout mice JNK signaling in allergic airway inflammation and autophagy activationBackgroundAccording to World Health Organization (WHO) estimates, over 300 million people suffer from bronchial asthma. The role of TLR2-induced innate immune response in the asthma pathogenesis remains controversial. In addition, the occurance of autophagy in asthma pathogenesis is little known. As one of the most characteristic clinical manifestations of asthma, the role of TLR2 in allergic airway inflammation, associated autophagy activation and potential signaling mechanisms need to be elucidated.Objective1. Investigate TLR2 expression in allergic airway inflammation model.2. Discuss the role of TLR2 and downstream potential signal cascade in airway allergic inflammation and autophagy activation.Methods1. Establish allergic airway inflammation model, detect TLR2 expression in the lung tissues of WT miceWT mice were sensitized and challenged with OVA. The expression level of TLR2 protein in the mice lung tissues was analyzed by Western Blot. The expression of TLR2 in the airway of mice was observed by immunofluorescence analysis using inverted fluorescence microscope.2. Evaluate the effect of TLR2 knockout in mice in allergic airway inflammation and autophagy activationTLR2-/- mice were sensitized and challenged with OVA. Histological analysis of mice airways were determined by HE staining for airway inflammation manifestations and by PAS staining for goblet cell hyperplasia and mucus production. The number of inflammatory cells in BALF was detected by Wright’s staining, and the levels of OVA sIgE in serum and TNF-α、IL-10 in BALF were investigated by ELISA. The expression of inflammatory signal molecules and autophagic proteins in the mice lung tissues was analyzed by Western Blot.3. Evaluate the effect of JNK inhibition in mice in allergic airway inflammation and autophagy activationMice were sensitized and challenged with OVA under JNK specific inhibitor SP600125 treatment. Histological analysis of mice airways were determined by HE staining for airway inflammation manifestations and by PAS staining for goblet cell hyperplasia and mucus production. The number of inflammatory cells in BALF was detected by Wright’s staining, and the levels of OVA sIgE in serum and TNF-α, IL-10 in BALF were investigated by ELISA. The expression of inflammatory signal molecules and autophagic proteins in the mice lung tissues was analyzed by Western Blot.Results1. Increased TLR2 expression in OVA-induced lung tissues of WT miceThe level of TLR2 expression which mainly located around the bronchus and alveolar wall was significantly increased in OVA-challenged mice lung tissues compared with control group.2. TLR2 knockout in mice alleviated OVA-induced airway inflammationUnder OVA challenge, in comparison with WT mice, the level of OVA sIgE in serum, airway inflammatory manifestations including peribronchial inflammatory infiltration, wall thickening, epithelial goblet cell hyperplasia and mucus production, the number of inflammatory cells and the level of TNF-a in BALF were all markedly weakened in TLR2-/-mice.3. TLR2 knockout in mice decreased the level of OVA-induced lung inflammatory and autophagic signal activationUnder OVA challenge, increased activated levels of inflammatory signal including MyD88, Akt, NF-κB and MAPK (JNK, ERK, p38) and autophagy were detected in WT mice. Compared with WT mice, the expression levels of inflammatory proteins including P-JNK, P-Akt and P-p65, autophagic proteins Beclin-1 and LC3-Ⅱ in OVA-induced lung tissues were dramatically decreased in TLR2-/- mice.4. JNK inhibition in mice reduced OVA-induced airway inflammationUnder OVA challenge, in comparison with WT mice, the level of OVA sIgE in serum, airway inflammatory manifestations including peribronchial inflammatory infiltration, wall thickening, epithelial goblet cell hyperplasia and mucus production, the number of inflammatory cells and the level of TNF-a in BALF were all weakened in WT-SP600125 mice. No obvious differences were found in OVA-induced lung inflammatory manifestations between TLR2-/- and TLR2-/- -SP600125 mice.5. JNK inhibition in mice down-regulated the level of OVA-induced lung inflammatory and autophagic signal activationCompared with WT mice, the levels of inflammatory signal activation including JNK, Akt and NF-κB, autophagic proteins Beclin-1 and LC3-Ⅱ expression in OVA-induced lung tissues were down-regulated in WT-SP600125 mice. No obvious differences were found in OVA-induced activation of lung inflammatory and autophagic signal between TLR2-/- and TLR2-/- -SP600125 mice.Conclusions1. Up-regulation of TLR2 expression in allergic airway inflammation model.2. TLR2 favors allergic airway inflammation through JNK signal with autophagy activation, which search out a new feasible signal target for preventive treatment of allergic airway inflammation.