| TAM receptors-Tyro3, Axl and Mer, constitute a unique subfamily of receptor tyrosine kinase(RTK), which is a single transmembrane protein. TAM receptors maintain the stability of the body’s immune environment through two basic biological functions(phagocytosis of apoptotic cells and inhibition of the innate immune response). In the present study, we investigated the role of Mer receptor tyrosine kinase(Mer TK) in the regulation of LTA-induced inflammatory response and phagocytosis of Staphylococcus aureus.一、Study on molecular mechanisms of Mer TK in the regulation of LTA-induced inflammatory responseBackground(LTA) and LPS represent two major PAMP molecules embedded in the cell wall of Gram-positive and Gram-negative bacteria, respectively. At present, it has been reported that Mer TK negatively regulates LPS-induced inflammatory response. However, in the LTA-induced inflammatory response, whether Mer TK has a similar role and underlying mechanisms remain to be elucidated.Objective1. Investigate the expression of proteins related to TLR2 pro-inflammatory signaling and Mer TK anti-inflammatory signaling in LTA-stimulated RAW264.7 macrophages.2. Explore the roles and potential mechanisms of Mer TK in the inflammatory response of LTA-stimulated macrophage.Methods1. Detect the expression of related proteins in LTA-stimulated macrophages by Western Blot Mouse mononuclear macrophages RAW264.7 were stimulated with 200 ng/ml LTA at the indicated time points(4h, 8h, 12 h, 24h). The expression of proteins related to TLR2 signaling and Mer TK signaling was analyzed by Western Blot.2. Effects of Gas6-neutralizing antibody on the LTA-induced Mer TK activation First, RAW264.7 cells were pretreated with 20 μg/ml of a specific Mer-blocking antibody for 1 h and then stimulated with 200 ng/ml LTA for 12 h or 400 ng/ml Gas6 for10 min. Second, RAW264.7 cells were pretreated with 5 ng/ml of the antibody neutralizing Gas6 for 1 h and then stimulated with 200 ng/ml LTA for 12 h. Finally, the expression of phospho-Mer TK was analyzed by Western Blot.3. Detect the expression of P-Mer TK and Mer TK by immunocytochemistry and Western Blot RAW264.7 cells were stimulated with 200 ng/ml LTA for 12 h with pretreatment with20 μg/ml of a specific Mer-blocking antibody or Ig G for 1 h. The expression of P-Mer TK and Mer TK was detected by immunocytochemistry and Western Blot.4. Effects of specific Mer-blocking antibody on the LTA-induced inflammatory response and the expression of related proteins in TLR2 and Mer TK signaling downstream First, RAW264.7 cells were stimulated with 200 ng/ml LTA for 48 h with pretreatment with 20 μg/ml of a specific Mer-blocking antibody or Ig G for 1 h. The levels of TNF-αand IL-6 in culture supernatants were measured by ELISA. Second, RAW264.7 cells were stimulated with 200 ng/ml LTA for 12 h with pretreatment with 20 μg/ml of a specific Mer-blocking antibody or Ig G for 1 h. The expression of related proteins in TLR2 and Mer TK signaling downstream was analyzed by Western Blot.5. Effects of PI3 K inhibitor LY294002 on the LTA-induced inflammatory response and the expression of related proteins in TLR2 signaling downstream First, RAW264.7 cells were stimulated with 200 ng/ml LTA for 48 h with or without pretreatment with 50μM LY294002 for 1 h. The levels of TNF-α and IL-6 in culture supernatants were measured by ELISA. Second, RAW264.7 cells were stimulated with200 ng/ml LTA for 12 h with or without 50 μM LY294002 pretreatment for 1 h. The expression of related proteins in TLR2 signaling downstream was analyzed by Western Blot.Results1. TLR2 and Mer TK signaling pathways are activated in LTA-stimulated RAW264.7 cells The downstream molecules of TLR2 signaling, such as My D88, IκB-α, p65 and ERK1/2, were activated in LTA-stimulated RAW264.7 cells. Meanwhile, The downstream molecules of Mer TK signaling, such as Akt and SOCS3, were also activated. Their activation with the exception of ERK1/2 is a time-dependent manner.2. LTA-induced activation of Mer TK is Gas6-dependent First, the specific Mer-blocking antibody was able to inhibit LTA- or Gas6-induced phosphorylation of Mer TK in RAW264.7 cells. Second, pretreatment with Gas6-neutralizing antibody significantly inhibited LTA-induced phosphorylation of Mer TK.3. Mer TK negatively regulates LTA- induced inflammatory response Pretreatment with the specific Mer-blocking antibody further enhanced LTA-induced TNF-α and IL-6 production in RAW264.7 cells. Moreover, pretreatment with the specific Mer-blocking antibody further enhanced LTA-induced phosphorylation of IκB-α and p65.4. The LTA-induced inflammatory response is negatively regulated by Mer TK signaling via the PI3K/Akt pathway and SOCS3 protein Akt and SOCS3 are activated in a Mer TK-dependent manner in LTA-stimulated RAW264.7 cells. Meanwhile, pretreatment with the specific Mer-blocking antibody inhibited LTA-induced activation of Akt and SOCS3. Moreover, the PI3K/Akt pathway and SOCS3 protein inhibited TLR2-mediated inflammatory response.Conclusions1. LTA induces the activation of TLR2 pro-inflammatory signaling and Mer TK anti-inflammatory signaling in RAW264.7 cells.2. Mer TK negatively regulates LTA-induced inflammatory response via PI3K/Akt pathway and SOCS3 protein.二、 Study on the role of Mer TK in macrophage phagocytosis of S. aureusBackground At present, Many receptors that are employed for the engulfment of apoptotic cells are also used for the recognition and phagocytosis of bacteria., including SR-A, CD14,integrin, complement receptor and Fc receptor expressed on the surface of macrophages.In addition, Mer TK expressed on the surface of macrophages is essential for the rapid phagocytosis of apoptotic cells. Therefore, we investigated whether Mer TK are also involved in the phagocytosis of bacteria.Objective1. Detect the expression of proteins related to the phagocytic signaling in S.aureus-stimulated RAW264.7 macrophages.2. Study on the role of Mer TK in the phagocytosis of S. aureus by macrophages.Methods1. Detect the expression of related proteins in S. aureus-stimulated macrophages by Western Blot RAW264.7 macrophages were stimulated with S. aureus at the indicated time points(20min, 40 min, 60 min, 120min). The expression of related proteins was analyzed by Western Blot.2. Explore the effects of Mer TK on the phagocytosis of S. aureus in the aspects of protein molecular level and morphology, respectively RAW264.7 cells were stimulated with S. aureus(MOI = 10)with pretreatment with 20μg/ml of a specific Mer-blocking antibody or Ig G for 1 h. First, the expression levels of P-FAK and GTP-Rac1 related to phagocytosis were detected by Western Blot. Second,the ability of RAW264.7 cells to devour S. aureus was evaluated by phagocytosis test.Results1. A variety of receptor molecules were activated in S. aureus-stimulated RAW264.7 cells Stimulating RAW264.7 macrophages with S. aureus triggered the activation of a variety of receptor molecules on the surface of macrophages, including TLR2 signaling pathway(My D88, JNK, ERK and p38, p65), SR-A and Mer TK. Moreover, RAW264.7cells with S. aureus led to the activation of these proteins in a time-dependent manner.2. FAK and Rac1 proteins related to phagocytosis were activated in S.aureus-stimulated RAW264.7 cells At the same time, stimulating RAW264.7 cells with S. aureus also induced the activation of FAK and Rac1 proteins related to phagocytosis. Moreover, RAW264.7cells with S. aureus led to the activation of these proteins in a time-dependent manner.3. The specific Mer-blocking antibody had little effect on the S. aureus-induced activation of FAK and Rac1RAW264.7 cells were stimulated with S. aureus(MOI = 10)with pretreatment with 20μg/ml of a specific Mer-blocking antibody or Ig G for 1 h. The expression levels of P-FAK and GTP-Rac1 related to phagocytosis were detected by Western Blot. We found that the specific Mer-blocking antibody had no significant effect on the expression levels of P-FAK and GTP-Rac1 in S. aureus-stimulated macrophages.4. Mer TK had no significant effect on the phagocytic ability of macrophages In order to further confirm the role of Mer TK in macrophage phagocytosis of S. aureus,the ability of RAW264.7 cells to devour S. aureus was evaluated by phagocytosis test.We found that the specific Mer-blocking antibody had no significant effect on the ability of RAW264.7 cells to devour the S. aureus.Conclusions1. Stimulating macrophages with S. aureus induces activation of the phagocytic signaling and phosphorylation of Mer TK.2. However, Mer TK is not important for phagocytosis of S. aureus by macrophages. |
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