Macrophage-activating lipopeptide-2 requires Mal and PI3K for efficient induction of heme oxygenase-1.

Xiaoxing You,Minjun Yu,Ranhui Li,Yanhua Zeng,Xiaohua Ma,Chuanhao Jiang,Jun He,Guangli Ou,Cuiming Zhu,Liangzhuan Liu,Liesong Chen,Yimou Wu,Chuen-Mao Yang

doi:10.1371/journal.pone.0103433

Abstract

AimsThis study is to investigate the mechanisms by which macrophage-activating lipopeptide-2 (MALP-2) induces heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme, in human monocytes.MethodsHuman monocytic THP-1 cells were cultured for transient transfection with plasmids and stimulation with MALP-2 for indicative time intervals. After incubation with MALP-2, cells were collected and disrupted, before being tested for promoter activity using luciferase assay. For analysis of proteins, immunoreactive bands were detected using an enhanced chemiluminescence Western blotting system, and the band intensity was measured by densitometryic analysis. For the detection of co-immunoprecipitation, SDS-PAGE was performed and the membranes were probed using respective antibodies. To investigate the cellular localization of NF-E2-related factor 2 (Nrf2), cells underwent immunofluorescence staining and confocal microscopy, and were analyzed using electrophoretic mobility shift assay.ResultsMALP-2-induced HO-1 expression and promoter activity were abrogated by transfection with dominant negative (DN) plasmids of TLR2 and TLR6, or their neutralizing antibodies. However, inhibition of MyD88 or transfection with the DN-MyD88 was insufficient to attenuate HO-1 expression. In contrast, mutation or silencing of MyD88 adapter-like (Mal) by DN-Mal or siRNA almost completely blocked HO-1 induction. Btk, c-Src and PI3K were also involved in MALP-2-induced HO-1 expression, as revealed by specific inhibitors LFM-A13, PP1 and LY294002, or by transfection with siRNA of c-Src. MALP-2-induced activation of PI3K was attenuated by transfection with DN mutant of Mal, and by pretreatment with LFM-A13 or PP1. Furthermore, MALP-2 stimulated the translocation of Nrf2 from the cytosol to the nucleus and Nrf2 binding to the ARE site in the HO-1 promoter, which could also be inhibited by pretreatment with a PI3K inhibitor, LY294002.ConclusionsThese results indicated that MALP-2 required TLR2/6, Btk, Mal and c-Src to activate PI3K, which in turn initiated the activation of Nrf2 for efficient HO-1 induction.

Highlights

Mycoplasma is a kind of the smallest cellular organisms that are capable of self-replicating and persist as obligate extracellular parasites [1,2]
We report that myeloid differentiation primary response 88 (MyD88) is not necessary for heme oxygenase (HO)-1 induction after macrophage-activating lipopeptide-2 (MALP-2) stimulation, but is strongly associated with MyD88 adapter-like (Mal) to mediate PI3K activation, which leads to the activation of nuclear factor (NF)-E2-related factor 2 (Nrf2) and induces heme oxygenase-1 (HO-1) expression
MALP-2induced HO-1 expression was inhibited by pretreatment with antiTLR2 or anti-TLR6 neutralizing antibodies, indicating the involvement of TLR2/6 in these responses (Fig. 1A, Fig. S1)

Summary

Introduction

Mycoplasma is a kind of the smallest cellular organisms that are capable of self-replicating and persist as obligate extracellular parasites [1,2]. Mycoplasma infects nearly 2 million people yearly [3], and is responsible for up to 40% of the community-acquired pneumonia diagnosed in children. During mycoplasma infection, invading pathogens interact with the local environment. Inflammatory cells are activated and secrete a spectrum of cytokines and chemokines [5,6]. These cytokines consist of a complicated synergetic or antistatic network and have been implicated in many disordered inflammatory diseases [7,8]

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Conclusion