Effect of Staphylea bumalda Leaf Extracts on Macrophage Activation through Toll-Like Receptor 2 and Toll-Like Receptor 4-dependent c-Jun N-terminal Kinase Activation

INTRODUCTION

Staphylea bumalda (S. bumalda, Staphyleacease) is a deciduous tree distributed in Korea, China, and Japan. The wood of S. bumalda is used in pegs, chopsticks, and firewood, and its young leaves are used for food. It was recently reported that the flower buds of S. bumalda exert neuroprotective activity against oxidative damage (Zhang et al., 2018). Although young S. bumalda leaves are used to treat respiratory diseases and inflammation, no scientific research has been conducted on the biological activity of S. bumalda.

Humans are exposed to various pathogenic microorganisms and viruses throughout their lives, but the host immune system protects the human body from these attacks (Hong et al., 2017; Hirayama et al., 2018).

Macrophages are representative immune cells that can phagocytose infectious agents and present antigens (Geng et al., 2018; Hirayama et al., 2018). These functions of macrophages are enhanced by pathogenic microorganisms or viruses and by immune-enhancing factors such as interferons (IFNs). Activated macrophages secrete various immunomodulators, including nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1); phagocytose infectious agents, such as pathogenic microorganisms and viruses; and exert cytotoxicity against cancer cells (Cheng et al., 2010; Liu et al., 2016; Gong et al., 2017). Furthermore, these immunomodulators secreted from activated macrophages increase the activity of helper T cells and natural killer cells (Lapaque et al., 2009; Guerriero, 2019) and promote B cell maturation and clone expansion, thus playing an important role in initial immune function (Craxton et al., 2003; Bordet et al., 2019).

Thus, macrophage activation is known to strengthen the body’s immune system by contributing to innate and adaptive immune responses (Hirayama et al., 2018). In addition, exploratory studies have been conducted to identify various natural products that induce macrophage activation (Cheng et al., 2010; Liu et al., 2016; Hong et al., 2017; Geng et al. 2018; Hirayama et al., 2018).

In this study, the effect of S. bumalda leaf extracts (SBL) on the production of immunomodulators in macrophages was evaluated and the related mechanisms of action were elucidated to investigate the immune-enhancing activity of SBL, thus contributing to the search for natural products that induce macrophage activation.

MATERIALS AND METHODS

12. Reverse transcription polymerase chain reaction (RT-PCR)

RT-PCR analysis was performed according to a previous method with some modifications (Geng et al., 2018).

Briefly, after all treatments were completed, total RNA was separated from cells using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and was quantitatively analyzed. Then, cDNA was synthesized from 1 ㎍ of total RNA using a Verso cDNA Kit (Thermo Scientific Inc., Pittsburgh, PA, USA). PCR was performed using a PCR Master Mix Kit (Promega, Madison, WI, USA) and the primer presented in Table 1.

Table 1.

Primer sequences used in this study.

The PCR results were visualized using agarose gel electrophoresis. The density of mRNA bands was calculated using UN-SCAN-IT gel version 5.1 (Silk Scientific Inc., Orem, UT, USA).

RESULTS

1. SBL promotes the production of immunomodulators in RAW264.7 cells

To evaluate the effect of SBL on the production of immunomodulators in macrophages, the levels of NO, iNOS, IL-1β, IL-6, TNF-α, and MCP-1 were investigated after treating cells with SBL for 24 h.

As shown in Fig. 1A, SBL increased NO production in a concentration-dependent manner. Since an excessive NO level results in macrophage death (Taylor et al., 2003), the cytotoxicity of SBL toward macrophages was evaluated. As shown in Fig. 1B, SBL showed no cytotoxicity.

Fig. 1.

Effect of SBL on the activation of RAW264.7 cells. RAW264.7 cells were treated with the indicated concentrations of SBL for 24 h. (A): NO level was measured using Griess assay. (B): Cell viability was measured using MTT assay. (C): mRNA level was measured using RT-PCR analysis. (D): Cytokine production was measured using ELISA kit. (E): phagocytic activity was measured using Neutral Red assay. *p < 0.05 compared to the cells without the treatment.

In addition, SBL increased the expression of major immunomodulators, such as iNOS, IL-1β, IL-6, TNF-α and MCP-1, in macrophages (Fig. 1C and 1D). It is known that these macrophage-secreted immunomodulators increase phagocytosis by macrophages, which is used as an indicator of macrophage activation (We et al., 2006).

Thus, we investigated the effect of SBL on macrophage phagocytosis through the neutral red assay. As shown in Fig. 1E, SBL was found to significantly increase macrophage phagocytosis. These results indicate that SBL activates macrophages (Fig. 1).

2. TLR2/4 are receptors related to immunomodulator production induced by SBL in RAW264.7 cells

Since TLR2/4 are known to be major receptors associated with macrophage activation (Li and Cherayil, 2003; Kakutani et al., 2012), the effect of TLR2/4 SBL-induced immunomodulator production was evaluated.

First, after suppressing TLR2 or TLR4 with C29 (TLR2 inhibitor) or TAK-242 (TLR4 inhibitor), cells were treated with SBL for 24 h. As shown in Fig. 2A-2D, TLR2 and TLR4 inhibition by C29 and TAK-242 dramatically attenuated NO production and the expression of iNOS, IL-1β, IL-6, and MCP-1 induced by SBL. In addition, we observed that SBL increased TLR2 and TLR4 expression (Fig. 2E and 2F).

Fig. 2.

Effect of TLR2 and TLR4 on SBL-mediated production of immunomodulators in RAW264.7 cells. (A, B, C, D) RAW264.7 cells were pretreated with C29 (TLR2 inhibitor) or TAK-242 (TLR4 inhibitor) for 2 h and then co-treated with SBL for 24 h. The level of NO or mRNA was measured using Griess assay and RT-PCR analysis, respectively. (E, F) RAW264.7 cells were treated with SBL for 24 h. mRNA level was measured using RT-PCR analysis. *p < 0.05 compared to the cells without the treatment.

The results shown in Fig. 2 indicate that SBL increases the production of immunomodulators by inducing TLR2/4 expression in the cells.

3. JNK is a major upstream kinase related to immunomodulator production induced by SBL in RAW264.7 cells

Since TLR2/4 are known to activate various signaling pathways, such as mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB), to induce the production of immunomodulators in macrophages (Shen et al., 2017; Zhang et al., 2017; Bai et al., 2019; Yang et al., 2019), we analyzed the signaling pathway related to SBL-mediated immunomodulator production in RAW264.7 cells.

RAW264.7 cells was pretreated with PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), or BAY 11-7082 (NF-κB inhibitor) to inhibit each signaling pathway, and then treated with SBL for 24 h. As shown in Fig. 3A, SBL induced NO production regardless of whether ERK1/2 and p38 were inhibited. However, JNK inhibition reduced the level of NO produced by SBL. In addition, the inhibition of NF-κB resulted in slight inhibition of SBL-mediated NO production (Fig. 3B). The results shown in Fig. 3A and 3B suggest that JNK is the major signaling pathway associated with SBL-mediated immunomodulator production in macrophages.

Fig. 3.

Effect of MAPKs and NF-κB signaling pathways on SBL-mediated production of immunomodulators in RAW264.7 cells. RAW264.7 cells were pretreated with PD98059 (10 μM, ERK1/2 inhibitor), SB203580 (10 μM, p38 inhibitor), SP600125 (10 μM, JNK inhibitor) or BAY 11-7082 (10 μM, NF-κB inhibitor) for 2 h and then co-treated with SBL. (A, B) NO level was measured using Griess assay. (C) mRNA level was measured using RT-PCR analysis. (D) RAW264.7 cells were pretreated with PD98059 (10 μM, ERK1/2 inhibitor), SB203580 (10 μM, p38 inhibitor), SP600125 (10 μM, JNK inhibitor) or BAY 11-7082 (10 μM, NF-κB inhibitor) for 2 h and then co-treated with SBL. mRNA level was measured using RT-PCR analysis. *p < 0.05 compared to the cells without the treatment. #p < 0.05 compared to the cells with SBL treatment alone.

Thus, we investigated the effect of JNK on the expression of other immunomodulators induced by SBL. As shown in Fig. 3C, JNK inhibition reduced the expression of iNOS, IL-1β, IL-6, and MCP-1. Although ERK1/2, p38, and NF-κB signaling had no effect on SBL-induced NO production, we investigated the effects of these pathways on the production of other immunomodulators, such as iNOS, IL-1β, IL-6, and MCP-1.

As shown in Fig. 4D, ERK1/2 inhibition by PD98059 blocked IL-6 production by SBL, while SB230580-induced p38 inhibition attenuated SBL-mediated production of iNOS, IL-1β, and IL-6. However, NF-κB inhibition by BAY 11-7082 did not affect immunomodulator production. Therefore, the results depicted in Fig. 3 demonstrate that JNK is a major upstream kinase related to SBL-mediated immunomodulator production.

Fig. 4.

Effect of TLR2 and TLR4 on SBL-mediated JNK activation in RAW264.7 cells. (A): RAW264.7 cells were treated with SBL for the indicated times. JNK phosphorylation was measured using Western blot analysis. (B and C): RAW264.7 cells were pretreated with C29 (TLR2 inhibitor) or TAK-242 (TLR4 inhibitor) for 2 h and then co-treated with SBL for 3 h. JNK phosphorylation was measured using Western blot analysis. (D): RAW264.7 cells were treated with SBL for the indicated times. ERK1/2 and p38 phosphorylation were measured using Western blot analysis. (E): RAW264.7 cells were pretreated with C29 (TLR2 inhibitor) or TAK-242 (TLR4 inhibitor) for 2 h and then co-treated with SBL for 3 h. ERK1/2 and p38 phosphorylation were measured using Western blot analysis. *p < 0.05 compared to the cells without the treatment. #p < 0.05 compared to the cells with SBL treatment alone.

5. The immunomodulator-inducing activity of SBL is similar to that of γ-PGA in RAW264.7 cells

As a well-known immune-enhancing agent, γ-PGA activates macrophages by increasing the production of immunomodulators (Jeon et al., 2015).

Thus, we compared the immunomodulator-inducing activity of SBL with that of γ-PGA to determine the degree of SBL’s immune-enhancing activity. As shown in Fig. 5A and 5B, the production of NO, iNOS, and IL-1β induced by SBL was found to be slightly higher than that induced by γ-PGA; conversely, SBL-mediated IL-6 and MCP-1 production was slightly lower than that mediated by γ-PGA. However, given the results overall, the immune-enhancing activity of SBL appears to be similar to that of γ-PGA.

Fig. 5.

Comparison of immune-enhancing activity of SBL and γ-PGA in RAW264.7 cells. RAW264.7 cells were treated with SBL and γ-PGA for 24 h. RAW264.7 cells were treated with SBL and γ-PGA for 24 h. (A): NO level was measured using Griess assay. (B): mRNA level was measured using RT-PCR analysis. *p < 0.05 compared to the cells without the treatment.

6. SBL inhibits LPS-induced overproduction of immunomodulators in RAW264.7 cells

If immunomodulator secretion by macrophages is excessive or abnormal, an inflammatory response occurs, with continual inflammation leading to chronic inflammation-related diseases.

Thus, we evaluated the effect of SBL on LPS-induced immunomodulator production in macrophages. As shown in Fig. 6A and 6B, SBL suppressed excessive production of NO, iNOS, and IL-1β. Since it is known that excessive immunomodulator production induced by LPS is also dependent on the activation of TLR2 or TLR4, we investigated the effect of SBL on LPS-induced expression of TLR2 and TLR4.

Fig. 6.

Anti-inflammatory activity of SBL in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pre-treated with SBL for 4 h and then co-treated with PBS or LPS for 20 h. (A): NO level was measured using Griess assay. (B and C): mRNA level was measured using RT-PCR analysis. *p < 0.05 compared to the cells without the treatment.

As shown in Fig. 6C, TLR2 and TLR4 expression was induced in cells treated with LPS alone, but SBL inhibited the expression of TLR2 and TLR4 induced by LPS. The results shown in Fig. 6 indicate that SBL suppresses excessive production of immunomodulators secreted by macrophages during excessive immune responses.

7. Analysis of total phenolic, flavonoid, and tannin contents

Since polyphenols, flavonoids, and tannins in natural products are known to enhance the body’s immune response to external pathogens (Kaneko et al., 2017; Ding et al., 2018; Reyes et al., 2018), we analyzed the contents of phenols, flavonoids, and tannins in SBL. As shown in Fig. 7A, the total phenolic and flavonoid contents were confirmed to be 40.1 ± 0.7 and 67.5 ± 0.7 ㎍/㎎ extract, respectively. Moreover, tannic acid was also analyzed in SBL, the contents of which were approximately 14.58 ± 1.27 ㎍/㎎ (RSD = 1.04) extracts.

Fig. 7.

Analysis of total phenolic compounds, total flavonoid compounds, and tannin contents of SBL. (A): Contents of total phenolic compounds and total flavonoid compounds. (B): HPLC Chromatogram of a tannic acid standard and SBL.

DISCUSSION

The innate immune response is the first defense system to protect the human body from harmful external pathogens, such as pathogenic microorganisms and viruses. Of the immune cells responsible for the innate immune response, macrophages are known to protect the human body through the process of recognizing, engulfing, and killing pathogenic microorganisms or viruses (Nau et al., 2002).

Activated macrophages secrete various immunomodulators, such as NO, iNOS, IL-1β, IL-6, TNF-α, and MCP-1, to combat pathogens. NO synthesized by iNOS has been reported to directly kill pathogenic microorganisms and cancer cells (Gong et al., 2017).

IL-1β is directly involved in the activation of T cells, NK cells, and B cells (Papayianni, 1996; Delgado et al., 2003). IL-6 is known to promote the proliferation and differentiation of T cells and B cells (Li et al., 2017). TNF-α promotes the production of various immunomodulators and enhances the function of macrophages and monocytes, thereby increasing immune system activity (Mosser, 2003). MCP-1 is directly involved in transferring monocytes to inflammatory sites and has been reported to be essential for the Th1 immune response (Huang et al., 2001).

Taken together, these reports suggest that the immunomodulators secreted by macrophages may enhance immune function. Here, we have shown that SBL increases the production of immunomodulators, such as NO, iNOS, IL-1β, IL-6, TNF-α, and MCP-1, and phagocytosis by macrophages (Fig. 1). These findings demonstrate that SBL can enhance immune functions through activating macrophages.

Macrophage activation begins by recognizing various stimuli, including pathogenic microorganisms and viruses. Macrophages have receptors, such as TLR2, TLR4 complement receptor 3, Mannose receptor, and Dectin-1, that recognize these stimuli on the cell membrane. In fact, many natural products have been reported to stimulate TLR2 and TLR4 in macrophages to induce immunomodulator production.

Physalis alkekengi L induces IL-6 and TNF-α through activation of TLR2 and TLR4 (Yang et al., 2019). It has been reported that TLR4 is involved the production of IL-6 and TNF-α by macrophages in Astragalus polysaccharides (Zhou et al., 2017). In addition, Cordyceps militaris polysaccharides have been reported to activate macrophages through TLR2 (Lee and Hong, 2011). Stimulation of TLR2 and TLR4 on macrophages activates intracellular downstream signaling pathways, such as the MAPK and NF-κB pathways, subsequently inducing immunomodulator production (Shen et al., 2017; Yang et al., 2019).

Taken together, these studies suggest that TLR2 and TLR4 may be important receptors for macrophage activation. Here, we demonstrated that SBL-induced immunomodulator production in macrophages was blocked by inhibiting TLR2 and TLR4 (Fig. 2) and by suppressing JNK (Fig. 3). Furthermore, SBL-induced JNK activation was attenuated when TLR2 and TLR4 were suppressed (Fig. 4). These results are evidence that SBL activates JNK through stimulating TLR2 and TLR4 in macrophages to induce immunomodulator production.

Macrophages have been reported to have biological functions in two aspects: Inflammatory response and immune enhancement (Kasimu et al., 2017). In other words, normal activation of macrophages contributes to enhancing immune response, but excessive production of immunomodulators through abnormal immune responses by macrophages leads to the development of diseases associated with chronic inflammation.

Thus, we investigated how SBL affects immunomodulator production when excessive immune response is induced by LPS. We observed that SBL inhibits the excessive production of immunomodulators in LPS-stimulated macrophages (Fig. 6). These results indicate that SBL may be able to suppress excessive immune response.

In fact, since LPS also induces the production of immunomodulators through activating TLR2 and TLR4, the result that SBL suppresses LPS-induced immunomodulator production was unexpected. However, we found that SBL inhibits LPS-induced TLR2 and TLR4 expression when compared to that of cells treated with LPS alone. Therefore, under normal conditions, SBL may act as an antagonist of TLR2/4, whereas in excessive immune response conditions caused by external stimuli such as LPS, SBL acts as an inhibitor of TLR2/4.

In addition, reports suggest that many natural products have both immune-enhancing activity and excessive immune response-suppressing activity. For example, mushrooms containing various polysaccharides and Tripterygium wilfordii have been reported to induce the production of various immunomodulators, including NO, in macrophages under normal conditions, but they inhibit immunomodulator production in the presence of external stimuli such as LPS (Lull et al., 2005; Shao et al., 2008).

In conclusion, the present results demonstrate that SBL has two immunoregulatory activities. First, SBL activates JNK through the stimulation of TLR2 and TLR4 and continuously increases the production of immunomodulators in macrophages, indicating that SBL can contribute to enhancing the immune function of the human body through activating macrophages. Second, SBL suppresses excessive immune response by blocking overproduction of immunomodulators during abnormal immune response. It is known that a weakened immune system can lead to infectious diseases and cancer. However, when the immune system is overactivated, chronic inflammatory diseases and autoimmune diseases can occur. Thus, the balance of the human immune system is important to maintain human health (Haddad et al., 2005).

Due to its immunostimulatory activity under normal conditions and immunosuppressive activity during excessive immune response, SBL may be used as a natural agent to maintain balance in the immune system.

Acknowledgments

This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1D1A3A03103685 and NRF-2018R1A6A1A03024862).

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