| Background and Objective: Exposure to mycoplasma may lead to pathological injury through an inappropriate inflammatory response. Previous studies have indicated that the mycoplasma derived membrane lipoproteins or their derived analogues such as macrophage-activating lipopeptide-2(MALP-2), exert a monocyte/macrophage-stimulating substance and play a crucial role in immune response and the pathogenesis of mycoplasma. Heme oxygenase-1(HO-1) is the rate-limiting enzyme catalyze the degradation of heme into CO, Fe2+ and bilirubin. HO-1 and its metabolites exhibit many immune-modulating effects under the condition of inflammatory and stress response. Reports also have been demonstrated many pathogen-associated molecular patterns(PAMPs) derived from pathogenic microbes can induce monocytes/macrophages and dendritic cells expression of HO-1 to protect against the excessive immune response. In the previous studies, we have demonstrated that MALP-2could induce the THP-1 cells expression of HO-1 after Nrf2 activation to modulate cyclooxygenase-2(COX-2) synthesis. However, the upper signaling pathways remain to be elucidated. The aim of this study is designated to investigate the mechanism underlying MALP-2-induced HO-1 expression, and the effect of HO-1 on cytokines secretion in THP-1cells.Methods:(1) THP-1 cells were cultured in vitro and were pretreated with the functional neutralizing anti-Toll-like receptor 2(TLR2) or anti-TLR6 antibodies for 1h, or transfected with dominant negative plasmids of TLR2(DN-TLR2) or TLR6, and then stimulated with5μg/m L MALP-2 for 16 h, the expression of HO-1 was measured by Western blot; Cells were cotransfected with DN-TLR2 or DN-TLR6 and p GL3-HO-1-luc reporter gene, and then stimulated with MALP-2 for 8h.The luciferase activity derived from HO-1 activation was detected to demonstrate the involvement of TLR2 and TLR6 in HO-1 expression.(2)THP-1 cells were stimulated with MALP-2 for 0~30min and lysed. The lysates were subjected to Western blot to analyze the phosphorylation of c-Src; Cells were preincubated with the c-Src inhibitor PP1 for 1h, or transfected with c-Src specific si RNA, western blot were used for the requirement of c-Src in MALP-2-induced HO-1 expression.(3) THP-1cells were stimulated with MALP-2 for 0~20min, phosphorylation of the Bruton’s tyrosine kinase(Btk) was detected by western blot; And then cells were pretreated with different concentration of PP1, or transfected with c-Src si RNA, phosphorylation of Btk was measured by Western blot to analyze the effect of c-Src on Btk activation. In addition, the Btk inhibitor LFM-A13 was used to analyze the effect of Btk on HO-1expression and promoter activity;(4) THP-1 cells were transfected with si RNA or dominant negative plasmid for My D88 or Mal, the expression of HO-1 protein and activation of the promoter was detected by Western blot and luciferase assay, respectively, to investigate the role of My D88 and Mal in mediation of HO-1 expression;(5) Cells were stimulated with MALP-2 for 0~60min, activation of Akt was detected by Western blot; To observe the involvement of c-Src, Btk, Mal, My D88 in Akt activation,cells were preincubated with PP1 and LFM-A13, or transfected with si RNA for Mal and My D88 before MALP-2 stimulation, Western blot was used to detect the phosphorylation of Akt; THP-1 cells were preincubated with the PI3 K inhibitor LY294002 for 1h, HO-1 level was measured by Western blot to demonstrate of the requirement of PI3 K in HO-1 expression; To analyze the interaction between c-Src, Btk, Mal,My D88 and p85α, co-immunoprecipitation was used to detect the formation of the c-Src/Btk/Mal/My D88/p85α complex;(6) Cells were incubated with LY294002 or transfected with My D88 si RNA,immunofluorescence staining and electrophoretic mobility shift assay were used to observe the nuclear translocation and the DNA-binding activity, respectively;(7) To investigate the role of HO-1 in cytokines secretion, cells were transfected with si RNA of HO-1 and Nrf2, or pretreated with HO-1 inhibitor Zinc protoporphyrin(Zn PP). After stimulated by MALP-2, the expression of TNF-α and IL-6 m RNA were detected by real-time PCR, secretion of TNF-α, IL-6, IL-10, and the NF-κB activity were measured by ELISA; The HO-1 was induced by transfection of the pc DNA3.1-HO-1, or by treatment of the Cobalt protoporphyrin(Co PP) or Sulforaphane(SFN), secretion of TNF-α and IL-1β, and the activity of NF-κB were detected by ELISA.Results: Our results demonstrated that:(1) MALP-2-induced HO-1expression was inhibited by pretreatment with anti-TLR2 or anti-TLR6 neutralizing antibodies. This hypothesis was further supported by transfection of dominant negative plasmids of TLR2 or TLR6. Moreover,mutation of TLR2 or TLR6 also inhibited HO-1 promoter activity induced by MALP-2;(2) The c-Src was phosphorylated 5 min after MALP-2stimulation. In addition, pretreatment with a c-Src inhibitor PP1 significantly attenuated MALP-2-induced HO-1expression in aconcentration-dependent manner. Furthermore, transfection of THP-1cells with c-Src si RNA significantly down-regulated c-Src and subsequently led to a decrease of HO-1 protein expression in response to MALP-2.(3) Btk from stimulated cells exhibited increased phosphorylation within 10 min of stimulation; Pretreatment of THP-1cells with PP1, or transfection of the c-Src-specific si RNA blocked MALP-2-induced Btk phosphorylation; In addition, pretreatment of cells with LFM-A13 significantly inhibited the up-regulation of HO-1 protein in response to MALP-2;(4) Transfection of THP-1 cells with neither a dominant negative plasmid of My D88 nor My D88 si RNA could significantly block HO-1 expression, while silencing of Mal by si RNA almost completely blocked HO-1 induction; Although both of DN-My D88 and DN-Mal decreased the promoter activity of HO-1 to some degree, but the luciferase activity in cells transfected with DN-Mal was much lower than that in My D88 mutant cells;(5) MALP-2 induced phosphorylation of Akt at 5 min, and the peak was reached at 15 min, this could be suppressed by pretreatment with PP1 and LFM-A13; In addition,transfection with si RNA of Mal severely impaired Akt phosphorylation after stimulation by MALP-2. In contrast, Akt phosphorylation in My D88 si RNA-transfected cells was significantly decreased at 15 min after stimulation. Interestingly, as stimulation continues, Akt phosphorylation reoccurred after about 60 min and lasted until 120 min; Moreover,MALP-2-induced HO-1 expression was abrogated by pretreatment with LY294002; Furthermore, MALP-2- induced HO-1 expression in THP-1cells was mediated by formation of a c-Src/Btk/Mal/My D88/p85α;(6)Treatment with MALP-2 led to marked increase of the Nrf2 immunofluorescence signal in the nucleus and binding to the ARE promoter, when the cells were pretreated with LY294002, nucleartranslocationof Nrf2 and binding to the ARE promoter sequence was significantly attenuated. While cells were transfected with My D88 si RNA,nuclear translocation of Nrf2 and the Nrf2-ARE-binding complex was almost not affected.(7) Transfection of si RNA of HO-1 and Nrf2up-regulated MALP-2-induced TNF-α and IL-6 expression and secretion,and NF-κB activation, but the IL-10 was not affected; Similar results have been obtained by treatment of the HO-1 inhibitor, Zn PP; Cell expression of the HO-1 by transfected with the pc DNA3.1-HO-1 plasmid,or induction of HO-1 by Co PP or SFN decreased the secretion of TNF-αand IL-1β, and NF-κB activation after MALP-2 stimulation.Conclustions:(1) Mycoplasma MALP-2 induces THP-1 cells expression of HO-1 through the TLR2,6/c-Src/Btk/Mal/PI3K/Nrf2pathways;(2) MALP-2-induced expression of HO-1 protects against excessive inflammatory responses in human monocytes. |
