Akt, mTOR and NF-κB pathway activation in Treponema pallidum stimulates M1 macrophages

https://doi.org/10.1016/j.intimp.2018.03.040Get rights and content

Highlights

  • T. pallidum induced inflammatory cytokine expression in a dose- and time-dependent manner.

  • T. pallidum promotes macrophage polarization toward the M1 phenotype.

  • Treponema pallidum stimulates M1 macrophage activation via the Akt-mTOR-NF-κB signaling pathway.

Abstract

The polarization of macrophages and the molecular mechanism involved during the early process of syphilis infection remain unknown. This study was conducted to explore the influence of Treponema pallidum (T. pallidum) treatment on macrophage polarization and the Akt-mTOR-NFκB signaling pathway mechanism involved in this process. M0 macrophages derived from the phorbol-12-myristate-13-acetate-induced human acute monocytic leukemia cell line THP-1 were cultured with T. pallidum. T. pallidum induced inflammatory cytokine (IL-1β and TNF-α) expression in a dose- and time-dependent manner. However IL-10 cytokine expression decreased at the mRNA and protein levels. Additionally, the expression of the M1 surface marker iNOS was up-regulated with incubation time, and the expression of the M2 surface marker CD206 was low (vs. PBS treated macrophages, P < 0.001) and did not fluctuate over 12 h. Further studies revealed that Akt-mTOR-NFκB pathway proteins, including p-Akt, p-mTOR, p-S6, p-p65, and p-IκBα, were significantly higher in the T. pallidum-treated macrophages than in the PBS-treated macrophages (P < 0.05). In addition, inflammatory cytokine expression was suppressed in T. pallidum-induced M1 macrophages pretreated with LY294002 (an Akt-specific inhibitor) or PDTC (an NF-κB inhibitor), while inflammatory cytokine levels increased in T. pallidum-induced M1 macrophages pretreated with rapamycin (an mTOR inhibitor). These findings revealed that T. pallidum promotes the macrophage transition to pro-inflammatory M1 macrophages in vitro. The present study also provides evidence that Akt, mTOR and NF-κB pathway activation in T. pallidum stimulates M1 macrophages. This study provides novel insights into the innate immune response to T. pallidum infection.

Introduction

The defensive strategies by which a host eliminates Treponema pallidum (T. pallidum), the etiologic agent of syphilis, are poorly understood. The host develops and resolves lesions, which are a direct manifestation of the inflammatory processes triggered by T. pallidum within compromised tissues [1]. Macrophages, a family of dynamic cells, are on the first line of defense against invading T. pallidum in compromised tissues, where they act as effectors of the immune response [1,2]. Macrophages internalize and kill T. pallidum [3] and coordinate their responses to clear spirochete from the infected tissues [4] and increase cytokine (such as tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β)) secretion in primary syphilis [5]. Macrophages exert various biological functions and undergo highly reversible and transient polarization processes in a broad spectrum of inflammatory conditions. For the sake of simplicity and efficiency, a model system has been established to identify macrophages on a continuum, with pro-inflammatory M1 macrophages representing one extreme and anti-inflammatory M2 macrophages representing the opposite extreme. M1 and M2 macrophages can serve distinct functions in regulating the inflammatory response [[6], [7], [8]].

Recent evidence has suggested that protein kinase B, also known as the Akt signaling pathway, plays crucial roles in macrophage activation and gene expression [9]. Additionally, Akt activation can initiate the downstream mammalian target of rapamycin (mTOR) pathway and regulate the macrophage polarization process [10]. Moreover, mTOR can activate IκB kinase and its downstream nuclear factor κB (NF-κB) to promote inflammatory factor secretion [11]. Evidence suggests that this activation occurs in a variety of inflammatory diseases, such as rheumatoid arthritis [12], atherosclerosis [13], and cancer [14]. However, the function of the Akt, mTOR and NF-κB signaling pathway in macrophages during the syphilis infection process is poorly understood. In this context, identifying macrophage polarization and the underlying molecular mechanisms involved in this process may offer novel potential treatment options for controlling the inflammatory response in syphilis patients.

In the present study, we performed a series of experiments using human monocytic cell line (THP-1)-derived macrophages to investigate macrophage polarization and inflammatory cytokine expression. Additionally, we explored the mechanism underlying the involvement of the Akt, mTOR and NF-κB signaling pathway in this process.

Section snippets

Differentiation of M0 macrophages

THP-1 cells (obtained from the American Type Culture Collection, Manassas, VA, USA) were incubated in Roswell Park Memorial Institute 1640 Medium (RPMI 1640 Medium; HyClone, Logan, Utah, USA) with 10% (v/v) heat-inactivated fetal bovine serum (Biological Industries Ltd., Kibbutz Beit Haemek, Israel). Phorbol 12-myristate 13-acetate (PMA; 50 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) was used to induce THP-1 cell differentiation into M0 macrophages in a humidified 5% CO2 atmosphere at 37 °C, and

T. pallidum induced inflammatory cytokine expression in a dose- and time-dependent manner

To investigate the effect of T. pallidum on macrophages, we incubated macrophages derived from THP-1 cells with T. pallidum at a MOI of 20:1, 50:1, 100:1, or 200:1 for 12 h. As shown in Fig. 1, T. pallidum markedly increased IL-1β and TNF-α mRNA and protein levels in macrophages in a dose-dependent manner. Macrophages expressed substantially higher IL-1β and TNF-α mRNA and protein levels (P < 0.001) and lower CD206 and IL-10 levels after T. pallidum treatment at different MOIs than after PBS

Discussion

Conceptually, syphilis is a spirochete-driven type of inflammation [16]. The cells in primary seropositive syphilis have the greatest ability to produce cytokines. Immunohistochemistry and molecular studies in humans have confirmed that primary lesions are enriched with natural killer cells, plasma cells, and macrophages capable of expressing mRNA for the cytokines IFN-γ, IL-2 and IL-12 [4,17]. Cytokines stimulate macrophages to destroy bacteria, suggesting that activated macrophages are

Conclusions

In summary, we show that T. pallidum promotes macrophage switching to pro-inflammatory M1 macrophages in vitro. In addition, the present study provides evidence demonstrating that Akt, mTOR and NF-κB pathway activation in T. pallidum stimulates M1 macrophages.

Acknowledgments

This work supported by the National Natural Science Foundation [grant numbers 81772260, 81771312, 81672094, 81471967, 81471231, 81401749, 81471970, 81201360, 81271335, 81101324, 81171625], the Key Projects for Province Science and Technology Program of Fujian [grant number 2018D0014], the National Science Foundation for Distinguished Young Scholars of Fujian [grant number 2014D001], the Major Special Projects for Serious Illness of Xiamen [grant number 3502Z20159016, 3502Z20179045] and the

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