Protective effects of a traditional herbal extract from Stellaria dichotoma var. lanceolata against Mycobacterium abscessus infections

Abstract

Stellaria dichotoma var. lanceolata (SdLv), a member of the Caryophyllaceae, is a traditional herbal medicine that has been used to treat fever, night sweats, and malaria in East Asia. Inflammation plays an essential role in both host defense and pathogenesis during infection by diverse intracellular pathogens. Herein, we showed that an herbal extract from SdLv effectively attenuated inflammatory responses from infection of Mycobacterium abscessus (Mab), but not Toxoplasma gondii (T. gondii). In primary murine macrophages, Mab infection resulted in the rapid activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK), as well as in the generation of proinflammatory cytokines, such as tumor necrosis factor α and interleukin-6, which were all significantly inhibited by pretreatment with SdLv. However, herbal extracts from Bupleurum chinense DC. (Buch) or Bupleurum falcatum L. (Bufa) did not affect M. abs-induced activation of proinflammatory responses. Importantly, we demonstrated that generation of intracellular reactive oxygen species, which are important signaling intermediaries in the activation of NF-κB and the MAPK signaling pathway, was rapidly increased in Mab-infected macrophages, and this was effectively suppressed by pretreatment with SdLv, but not Buch and Bufa. We further found that the treatment of Buch and Bufa, but not SdLv, led to the activation of NF-κB and the MAPK signaling pathway and the generation of intracellular reactive oxygen species. Moreover, oral administration of SdLv significantly reduced lethality in Mab-infected mice. Collectively, these results suggest the possible use of SdLv as an effective treatment for Mab infection.

About the Authors:

Su-Jin Bae

Contributed equally to this work with: Su-Jin Bae, Jae-Won Choi, Byung-Joon Park

Roles Formal analysis, Investigation, Methodology

Affiliations Department of Infection Biology, College of Medicine, Chungnam National University, Daejeon, Korea, Department of Medical Science, College of Medicine, Chungnam National University, Daejeon, Korea

Jae-Won Choi

Contributed equally to this work with: Su-Jin Bae, Jae-Won Choi, Byung-Joon Park

Roles Data curation, Investigation, Methodology, Validation, Visualization

Affiliations Department of Infection Biology, College of Medicine, Chungnam National University, Daejeon, Korea, Department of Medical Science, College of Medicine, Chungnam National University, Daejeon, Korea

Byung-Joon Park

Contributed equally to this work with: Su-Jin Bae, Jae-Won Choi, Byung-Joon Park

Roles Data curation, Investigation, Methodology, Validation

Affiliations Department of Infection Biology, College of Medicine, Chungnam National University, Daejeon, Korea, Department of Medical Science, College of Medicine, Chungnam National University, Daejeon, Korea

Jina Lee

Roles Methodology, Visualization

Affiliations Department of Infection Biology, College of Medicine, Chungnam National University, Daejeon, Korea, Department of Medical Science, College of Medicine, Chungnam National University, Daejeon, Korea

Eun-Kyeong Jo

Roles Conceptualization

Affiliations Department of Medical Science, College of Medicine, Chungnam National University, Daejeon, Korea, Department of Microbiology, College of Medicine, Chungnam National University, Daejeon, Korea

Young-Ha Lee

Roles Conceptualization

Affiliations Department of Infection Biology, College of Medicine, Chungnam National University, Daejeon, Korea, Department of Medical Science, College of Medicine, Chungnam National University, Daejeon, Korea

Sung-Bae Kim

Roles Formal analysis, Resources, Writing – original draft

* E-mail: [email protected] (JMY); [email protected] (SBK)

Affiliation: Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon, South Korea

Jae-Min Yuk

Roles Conceptualization, Funding acquisition, Investigation, Project administration, Validation, Writing – original draft

* E-mail: [email protected] (JMY); [email protected] (SBK)

Affiliations Department of Infection Biology, College of Medicine, Chungnam National University, Daejeon, Korea, Department of Medical Science, College of Medicine, Chungnam National University, Daejeon, Korea

ORCID logo http://orcid.org/0000-0002-0630-9194

Introduction

Natural products from plants have been used both clinically and as folk medicines for the treatment of various diseasesand have also been demonstrated to be an important resource of novel lead compounds [1, 2]. Stellaria dichotoma L. var. lanceolota Bunge (SdLv), generally called Yin Chai Hu, is commonly distributed in East Asia, including China and the Republic of Korea, as a staple herbal medicine used to treat fever and infantile malnutrition [3]. Moreover, recent studies reported that components isolated from SdLv contribute to various biological functions, including anti-inflammatory [4], anti-allergic [5], and antioxidant activities [6]. Bupleuri Radix includes plants in the genus, Bupleurum, which are commonly used to treat colds, fever, influenza, hepatitis, and malaria in East Asia [7, 8]. Among them, Bupleurum chinense DC. (Buch), also called Bei Chai Hu in Chinese, has been used to treat fever, influenza, and malaria [9, 10]. Numerous studies have also indicated that Buch-derived compounds, such as saikosaponins, flavonoids, and fatty acids, exhibit diverse physiological functions, including anti-inflammatory, antibiotic, antiviral, and immunomodulatory effects [11, 12]. Additionally, Bupleurum falcatum L. (Bufa), also called Sandao chaihu in Chinese, is a traditional Asian herbal medicine that improves stress-induced depression and anxiety-like behaviors in rats [13–15].

Ancient records of traditional Chinese medicine practices indicate that the dried roots of Buch and B. scorzonerifolium Willd. were prescribed as Radix Bupleuri [16, 17]. Moreover, Bufa and its related species are used in Korea as traditional medicines [18]. According to the Encyclopedia of Chinese Materia Medica, because of its similar morphology and name, SdLv was used as a substitute for Radix Bupleuri [16, 19]. However, because of noticeable differences with B. chinense in terms of medicinal effects, the latter was continuously used as a new herb and is currently known as Stellariae Radix in China. For these reasons, the species of Radix Bupleuri and its clinical applications are confusing [16, 17]. Thus, comparative studies to characterize the different effects of the three different species are needed.

Intracellular pathogens have evolved various strategies to invade, survive, and replicate inside a variety of cell types through the evasion of the host immune system [20, 21]. Mycobacterium abscessus (Mab) belongs to a group of rapidly-growing mycobacterium that cause a broad range of human infections, especially chronic lung disease in elderly patients with bronchiectasis and young patients with cystic fibrosis [22, 23]. Moreover, numerous cases of community- and hospital-acquired Mab infections have been widely reported throughout the world [24]. Importantly, Mab is known to be resistant to most antibiotics, including first-line anti-tuberculous drugs, resulting in a particularly high mortality rate because of treatment failures [22, 25]. Toxoplasma gondii (T. gondii) is an intracellular protozoan parasite that infects a broad range of warm-blooded animals, including avian and mammalian species [26]. Although T. gondi-infected healthy patients remain asymptomatic, T. gondii exposure in immunocompromised and congenitally infected patients may result in toxoplasmosis that can lead to high morbidity and mortality [27].

Pathogen-associated molecular pattern molecules derived from various microorganisms are recognized by pattern recognition receptor (PRR)-bearing cells, which then activate an inflammatory response that can contribute not only to host protective immunity but also to immunopathology [28, 29]. Mitogen-activating protein kinases (MAPKs) and nuclear factor (NF)-κB play important roles in promoting inflammatory responses following pathogen infection [30, 31]. Recent studies have reported that intracellular pathogens, including Mab and T. gondii, strongly induced various proinflammatory cytokines through MAPKs and NF-κB signaling pathways, thereby playing a key role in innate immunity [32–35]. Accumulating evidence has also suggested that reactive oxygen species (ROS) function as important intermediaries, regulating various cellular signaling pathways in biological and physiological processes [36]. Our previous studies suggested that cytosolic ROS were essential in activating inflammatory responses during mycobacterial and toxoplasma infections [32, 35, 37, 38]. These studies strongly suggested that MAPKs and NF-κB signaling pathways and intracellular ROS generation contributed to the outcomes of inflammatory responses.

In the present study, we examined the anti-inflammatory role and molecular mechanisms of traditional herbal medicines, including SdLv, Buch, and Bufa, in primary macrophages infected with intracellular Mab or T. gondii. Moreover, we further investigated the function of SdLv in the in vivo infection mouse model of Mab.

Materials and methods

Mice and cell preparation

Wild-type (WT) C57BL/6 mice were purchased from Koatech (Pyungtek, Korea) and maintained in a pathogen-free environment condition. Bone marrow-derived macrophages (BMDMs) were isolated from femurs of mice and differentiated for 5–7 days in medium containing macrophage colony-stimulating factor (25 μg/ml, R&D Systems Minneapolis, MN, USA), as described previously [35]. The culture medium consisted of Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco BRL, Grand Island, NY, USA), 1 mM sodium pyruvate, 50 U/mL penicillin, 50 μg/mL streptomycin, and 5 × 10−5 M β-mercaptoethanol. The human retinal pigment epithelial cell lines ARPE-19 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in DMEM supplemented with 10% FBS or with nutrient mixture F-12, 10% FBS and 1% Antibiotic-Antimycotic (Gibco BRL, Grand Island, NY, USA). ARPE-19 cells were passaged by 0.25% Trypsin-EDTA (Life Technologies, Carlsbad, CA, USA) every 2–3 days.

Preparation of the Buch, Bufa and SdLv

The roots of Bupleurum chinense DC., Bupleurum falcatum L. and Stellaria dichotoma var. lanceolata Bunge were purchased from Kwangmyeongdang Medicinal Herbs Co. (Ulsan, Korea). All samples were authenticated by evaluation of their microscopic and macroscopic characteristics by Dr. Goya Choi of the Korea Institute of Oriental Medicine (KIOM). The voucher specimens were deposited in the Korean Herbarium of Standard Herbal Resources at KIOM. (Index herbariorum code KIOM, Specimen No. 2-16-0424 (Buch), 2-16-0383 (Bufa), 2-16-0191(SdLv). Buch (779.31 g), Bufa (798.38 g) and SdLv (793.33 g) were extracted twice with 70% (v/v) ethanol using a 2 h reflux extraction at 80°C and then concentrated under reduced pressure. The 70% ethanol extracts were filtered through a standard sieve, evaporated to dryness, and freeze-dried. The yields were 12.21% Buch, 20.63% Bufa and 41.86% SdLv. The percentage yield was calculated by dividing the mass of product obtained (g) by the mass of sample (g). Prior to use, the lyophilized powders were dissolved in 0.25% sodium carboxymethyl cellulose (CMC; for in vivo analysis) or 0.01% DMSO (for in vitro analysis).

Preparation of Mab and T. gondii

Mab ATCC 19977 strain was obtained from ATCC (Manassas, VA, USA) and cultured as previously described [32]. Briefly, Mab was grown for 4 days, at 37°C on Middlebrook 7H9 broth (Difco, Sparks, MD, USA) supplemented with oleic acid-albumin-dextrose-catalase (OADC, Becton Dickinson, Sparks, MD, USA) and 0.05% Tween 80 (Sigma-Aldrich, St. Louise, MO, USA) and stored at −70°C until used. The number of viable bacteria on Middlebrook 7H10 agar (Difco, Sparks, MD, USA) was determine as colony-forming units (CFU).

Tachyzoites of T. gondii RH strain were maintained ARPE-19 cells for 2 or 3 days at 37°C, 5% CO2 and biweekly passaged in DMEM with 10% FBS, nutrient mixture F-12, antibiotics. To collect T. gondii tachyzoites, cell debris including ARPE-19 cells and parasites were washed in cold phosphate-buffered saline (PBS) and then resuspended in cold culture medium. The suspension was passed through a 27-gauge needle and a 5 μm pore size polycarbonate membrane (Millipore, Bedford, MA, USA) to remove host cells.

Reagents and antibody

Specific antibodies against phospho-ERK1/2 (9101), phospho-p38 (9211), phospho-SAPK/JNK (9251), IκBα (9242), and phospho-IKKα/β (2697) were purchased from Cell Signaling. Specific antibodies against β-tubulin (ab6046) were purchased from Abcam. All other reagents were purchased from Sigma-Aldrich, unless otherwise indicated.

Ethics statement

Animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Chungnam National University (CNU-00706) and at Chungnam National University Hospital (CNUH-017-A0012) and conformed to National Institutes of Health guidelines. Animal husbandry was provided by the staff of the IACUC under the guidance of supervisors who are certified Animal Technologists, and by the staff of the Animal Core Facility. Veterinary care was provided by IACUC faculty members and veterinary residents located on the Chungnam National University School of Medicine. The animals were fed standard rodent food and water ad libitum, and housed (maximum of 5 per cage) in sawdust-lined cages in an air-conditioned environment with 12-hour light/dark cycles. To minimize pain or illness of Mab-infected mice, the irreversible condition leading the inevitable death, such as loss of body condition, failure to drink, abnormal breathing, blood in the feces, was monitored daily and each mouse were euthanized with carbon dioxide [39].

Cell counting kit (CCK) 8 assay

The cytotoxicity effects of herbal extracts on BMDMs were determined using CCK 8 assay (Dojindo Molecular Technologies, CK04-11) according to manufacturer’s protocol. Briefly, BMDMs were seeded in 96-well plates and differentiated with M-CSF for 5 days as described in cell preparation. Cells were replaced with serum-free media and then incubated with various herbal extracts for 24 hours. Then, 10 μl of the CCK-8 solution was added and incubated for 1 hours at 37°C. Absorbance was measured at 450 nm using a microplate reader (SPECTRO star Nano, BMG Labtech, Ortenberg, Germany).

Experimental in vivo infection models

For in vivo experiments, we designed two model based on the infection rate of Mab. 1) Mice were orally administrated with SdLv (200 mg/kg) for 4 days, consecutively and then intravenously (i.v.) infected with Mab (5 × 108 CFU/mouse). On the 1 day after infection of Mab, mice were further administrated with SdLv for 3 days, consecutively. On the 4 day after infection of Mab, mice were euthanized, and bacterial burden and mRNA expression of Tnf were analyzed in the lungs and spleens, respectively. (2) Mice were orally administrated with SdLv (200 mg/kg) for 4 days, consecutively and then intravenously (i.v.) infected with Mab (5 × 109 CFU/mouse). On the 1 day after infection of Mab, mice were further administrated with SdLv for 7 days, consecutively. The body weight and survival of each mouse were monitored for 12 days. For a negative control, 0.25% CMC was orally administered instead of SdLv.

RNA extraction, real-time quantitative PCR, semi-quantitative RT-PCR, Western blot analysis, and enzyme-linked immunosorbent assays (ELISAs)

RNA extraction, real-time quantitative PCR, and semi-quantitative RT-PCR were performed as described previously [40]. The sequences of the primers used were as follows: mTNFα (forward: 5′-AGCACAGAAAGCATGATCCG-3′; reverse: 5′-CTGATGAGAGGGAGGCCATT-3), mIL-6 (forward: 5′-GGAAATTGGGGTAGGAAGGA-3′; reverse: 5′-CCGGAGAGGA-GACTTCACAG -3′), mIL-1β (forward: 5′-CTCCATGAGCTTTGTACAAGG-3′; reverse: 5′-TGCTGATGTACCAGTTGGGG-3′), mIL-12p40 (forward: 5′-GACCATCACTGTCAAAGAGTT-3′; reverse: 5′-AGGAAAGTCTTGTTTTTGAAA-3′), and mβ-actin (forward: 5′-TCATGAAGTG TGACGTTGACATCCGT-3′; reverse: 5′-CCTAGAAGCATTTGCGGTGCACGATG-3′).

For Western blot analysis, cell lysates were collected and lysed in PRO-PREP (iNtRON BIOTECHNOLOGY, Korea) containing additional set of phosphatase inhibitors. Protein concentration was determined using a BCA assay kit. Proteins (30 μg/each conditions) were immediately heated for 5 min at 100°C. Each sample was subjected to SDS-PAGE on gel containing 12% (w/v) acrylamide under reducing conditions. Separated proteins were transferred to PVDF membranes (Millipore Corp., Billerica, MA, USA), and then the membranes were blocked with 5% skim milk. Membranes were developed using chemiluminescence assay kit (ECL; Millipore Corp., Billerica, MA, USA) and subsequently analyzed using Chemiluminescence Imaging System (Davinch-K, Seoul, Korea). Data were analyzed using Alliance Mini HD6 (UVitec Cambridge, MA, USA).

In the sandwich ELISA, cell culture supernatants were analyzed using DuoSet antibody pairs (BD Pharmingen) for the detection of mouse tumor necrosis factor (TNF)-α and interleukin (IL)-6.

Measurement of ROS production

Level of intracellular ROS was determined by dihydroethidium (DHE, Calbiochem), 2′,7′-dichlorodihydroflurescein (DCFDA, Invitrogen), or CellROX Green reagent (Thermo Fisher Scientific, Waltham, MD, USA) [37]. Briefly, cells were incubated with 10 μM DHE for 30 min, 20 μM H2DCFDA for 30 min, 1 μM CellROX for 30 min at 37 xC and then analyzed by a confocal microscope (LSM 710; Zeiss), fluorescence microscope (SZX7; Olympus) or a FACS Calibur flow cytometer (Becton-Dickinson, San Josè, CA, USA).

Statistical analyses

Differences between averages were analyzed by a two-tails paired Student’s t-test with Bonferroni adjustment or log-rank test in survival and are presented as the means ± SD. Statistical comparisons were carried out using GraphPad Prism software (GraphPad Software, Inc. La Jolla, CA, USA). Differences were considered significant at p <0.05.

Results

SdLv has no cytotoxic effects on primary macrophages

To investigate the host protective roles of herbal extracts against intracellular pathogens, we first determined the cytotoxic effects of three candidates, including SdLv, Bufa, and Buch, in BMDMs. As shown in Fig 1, the stimulation of BMDMs with SdLv had no significant effect on either cell viability at various concentrations (5–200 μg/mL) over 24 hours (Fig 1C) or on solvent control-stimulated cells. However, cell viability was significantly decreased after treatment with 20 μg/mL Buch (Fig 1A) or 50 μg/mL Bufa (Fig 1B). Based on these results, we used concentrations of SdLv (5–200 μg/ml), Bufa (5–20 μg/ml), and Buch (5–10 μg/ml) that did not exert a cytotoxic effect.

[Figure omitted. See PDF.]

Fig 1. Cytotoxic effects of herbal extracts in BMDMs.

(A-C) BMDMs were treated with different concentrations of each herbal extract (5–200 μg/ml) for 24 h, and cell viability was evaluated using CCK-8 assay. Quantitative data for cell viability are representative of three independent experiments and are presented as means ± SD. *P < 0.05, ***P < 0.001 (two-tailed Student’s t-test), compared with SC-treated cells. SC, vehicle control (0.01% DMSO).

https://doi.org/10.1371/journal.pone.0207696.g001

SdLv attenuates Mab-induced generation of proinflammatory cytokines in primary macrophages

Based on the previous studies showing that alkaloid constituents from SdLv and exhibited anti-inflammatory [4] and anti-allergic effects [41], we examined the effects of herbal extracts, including SdLv, Bufa, and Buch, on the Mab-mediated activation of inflammatory responses. As shown in Fig 2A and 2B, we infected BMDMs with Mab for various time periods and then assessed the mRNA (Fig 2A) and protein (Fig 2B) expressions of inflammatory cytokines, such as TNF-α and IL-6. We also assessed the mRNA expression levels of the Tnf and Il6 genes at 18 hours after the infection of Mab in a MOI-dependent manner (Fig 2C).

[Figure omitted. See PDF.]

Fig 2. SdLv inhibits Mab-mediated the generation of TNF-α and IL-6 in BMDMs.

(A and B) BMDMs were infected with Mab (MOI = 5) for the indicated time periods (A) Cell lysates was collected and the mRNA expression of Tnf, Il6 and Il1b then measured using semiquantitative RT-PCR analysis. Actb (encoding β-actin) serves as a loading control throughout. (B) Culture supernatant was collected and the generation of TNF-α and IL-6 protein then were measured using ELISA assay. (C) BMDMs were infected with Mab (at MOI = 0.1, 1, 5 or 10) for 18 h. semiquantitative RT-PCR analysis of Tnf and Il6 mRNA. (D—F) BMDMs were stimulated with increasing concentration of SdLv (1 h, 5–200μg/ml), followed by Mab infection (MOI = 5) for 18 h. (D and E) Semi-quantitative RT-PCR (top) or Quantitative RT-PCR analysis (bottom) were assessed to evaluate the mRNA expression of Tnf (for D) and Il6 (for E). (F) Each culture supernatant was collected and the production of TNF-α and IL-6 were measured using ELISA assay. Data are representative of three independent experiments and are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test), compared with uninfected cells (B and C) or Mab-infected control cells (D—F). U, Untreated; SC, vehicle control (0.01% DMSO).

https://doi.org/10.1371/journal.pone.0207696.g002

To determine whether herbal extracts, including SdLv, Bufa, and Buch, inhibited the Mab-mediated generation of inflammatory cytokines, BMDMs were infected with Mab in the presence or absence of each herbal extract, and the expressions of TNF-α and IL-6 were evaluated by RT-PCR, qPCR, or an ELISA (Fig 2D–2F and Fig 3A–3D). Mab-induced mRNA expression of the Tnf (Fig 2D) and Il6 (Fig 2E) genes were significantly attenuated by pretreatment with SdLv in a concentration-dependent manner. Moreover, the secretions of TNF-α and IL-6 in culture supernatants after Mab infection were also decreased by pretreatment with SdLv in a concentration-dependent manner (Fig 2F). However, these inhibitory effects were not observed in the presence of Buch (Fig 3A and 3B) or Bufa (Fig 3C and 3D); rather, Mab-induced mRNA expressions of the Tnf and Il6 genes were significantly increased under some conditions when pretreated with Buch and Bufa. These results indicated that SdLv, but not Bufa and Buch, regulated Mab-mediated inflammatory responses.

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Fig 3. Mab-mediated the inflammatory response is not attenuated by pretreatment of Buch and Bufa.

(A and B) BMDMs were pretreated with Buch (2, 5, or 10μg/ml) for 1 h and then infected 18 h with Mab (MOI = 5). Total RNAs were extracted and subjected to quantitative RT-PCR analysis to measure the mRNA expression of Tnf (for A) and Il6 (for B). (C and D) BMDMs were pretreated with Bufa (2, 5, or 10μg/ml) for 1 h and then infected 18 h with Mab (MOI = 5). Total RNAs were extracted and subjected to quantitative RT-PCR analysis to measure the mRNA expression of Tnf (for C) and Il6 (for D). Data are representative of three independent experiments and are presented as means ± SD. *P < 0.05, **P < 0.01 (two-tailed Student’s t-test), compared with Mab-infected control cells. U, Untreated; SC, vehicle control (0.01% DMSO).

https://doi.org/10.1371/journal.pone.0207696.g003

T. gondii-mediated inflammatory responses are not altered by pretreatment of primary macrophages with SdLv

Because innate immune recognition of T. gondii, a well-known intracellular parasite, promotes host immunity and inflammatory responses similar to those of mycobacterial infection [42, 43], we determined the effects of SdLv in regulating the T. gondii-induced activation of proinflammatory responses in primary macrophages. We first evaluated the mRNA (S1A Fig) or protein (S1B Fig) expression levels of proinflammatory cytokines in BMDMs infected with T. gondii for the indicated time periods. We also further assessed the mRNA expression levels of the Tnf and Il6 genes at 48 hours after the infection of T. gondii in a MOI-dependent manner (S1C Fig).

Because SdLv has anti-inflammatory properties during Mab infection, we identified its roles in T. gondii infection. BMDMs were infected with T. gondii in the presence or absence of SdLv, and the expressions of TNF-α and IL-6 were evaluated by RT-PCR, qPCR, or ELISA (S1D–S1F Fig). As shown in S1D and S1E Fig, pretreatment of SdLv did not affect T. gondii-induced mRNA expression of the Tnf and Il6 genes compared with T. gondii-infected BMDMs pretreated with the solvent control. Moreover, the secretions of TNF-α and IL-6 in culture supernatants after T. gondii infection were also not altered by pretreatment with SdLv (S1F Fig). Together, these results indicated that SdLv, unlike infection with Mab, had no effects on the regulation of the inflammatory responses of T. gondii infection.

SdLv modulates the activation of c-Jun N-terminal kinase (JNK) and the p38 MAPK pathway in response to Mab but not to T. gondii

MAPKs are known to be essential kinases for the formation of transcription factor complex AP-1, which is involved in the activation of inflammatory responses upon diverse microbial infections [30, 44]. We therefore examined whether SdLv affected the MAPK signaling pathways activated by the infection of Mab or T. gondii. Primary macrophages infected with Mab (Fig 4A) or T. gondii (S2A Fig) showed rapid activation of all three MAPK subfamilies, including p38, extracellular signal-regulated kinases (ERK) 1/2, and JNK, within 30 minutes. Notably, Mab-mediated phosphorylation of p38 and JNK, but not ERK 1/2, were attenuated by pretreatment with SdLv in a concentration-dependent manner (Fig 4B). We next examined whether SdLv regulated the activation of three MAPK subfamilies in response to T. gondii infection in a similar manner as Mab infection. As shown in S2B Fig, T. gondii-mediated phosphorylation of p38, ERK 1/2, and JNK were not modulated by pretreatment with SdLv in BMDMs. We also found that Mab-mediated activations of these kinases were not attenuated by pretreatment with Bufa and Buch, but was rather enhanced in Mab-infected BMDMs pretreated with Buch (10 μg/ml) and Buch (5 and 10 ug/ml) (Fig 4C). Furthermore, we found that BMDMs treated with Buch or Buch showed the enhanced phosphorylation of ERK 1/2, p38, and JNK in a dose-dependent manner, whereas SdLv has no effects (Fig 4D). Taken together, these findings indicated that SdLv may selectively inhibit the Mab-mediated activation of inflammatory responses through regulation of the p38 and JNK signaling pathways.

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Fig 4. SdLv regulates Mab-induced the activation of JNK and p38 MAPK in BMDMs.

(A) BMDMs were infected with Mab (MOI = 5) for the indicated time periods. (B) BMDMs were infected with Mab (MOI = 5, 30 min) in the presence or absence of SdLv (5–200 μg/ml). (C) BMDMs were pretreated with Buch (2, 5, or 10 μg/ml), Bufa (2, 5, or 10 μg/ml), or SdLv (20, 50, or 100 μg/ml) for 1 h and subsequently infected with Mab (MOI = 5, 30 min). (D) BMDMs were treated with Buch (2, 5, 10 μg/ml), Bufa (2, 5, 10 μg/ml), or SdLv (20, 50, 100 μg/ml) for 1 hours. MAPKs activation was determined by western blotting analysis for phosphorylated ERK, p38, and JNK/SAPK antibody. Total protein was determined by monitoring β-tubulin as a loading control. Representative data from one of three independent experiments with similar results are shown. U, Untreated; SC, vehicle control (0.01% DMSO).

https://doi.org/10.1371/journal.pone.0207696.g004

SdLv attenuates the activation of the Mab-induced NF-κB signaling pathway in macrophages

NF-κB signaling is activated by the stimulation of diverse pathogens, including bacterial, viral, fungal, and parasitic agents, and is closely associated with the activation of inflammatory responses [44]. We showed that infection with Mab (Fig 5A) and T. gondii (S2C Fig) resulted in the rapid activation of the kinases, IKKα and IKKβ, leading to the degradation of the NF-κB inhibitor, IκB-α. To examine the inhibitory roles of SdLv in Mab-induced activation of NF-κB signaling, BMDMs were infected with Mab for 30 minutes in the presence of SdLv, and phosphorylation of IKKα/β and degradation of IκB-α were then evaluated using immunoblot assays. As shown in Fig 5B, pretreatment with SdLv in Mab-infected BMDMs effectively attenuated the activation of NF-κB signaling. However, these inhibitory effects were not only observed in Bufa- and Buch-pretreated cells, but were also activated in some conditions pretreated with Bufa and Buch (Fig 5C). Similar to those shown in MAPK signaling pathways (Fig 4D), the stimulation of Bufa or Buch, but not SdLv, resulted in the degradation of IκB-α and the phosphorylation of IKKαβ in primary murine macrophages (Fig 5D). We next examined the effects of SdLv in T. gondii-induced activation of NF-κB signaling expression. Similar to MAPKs signaling, T. gondii-mediated phosphorylation of IKKαβ and degradation of IκB-α were not attenuated by SdLv pretreatment of BMDMs (S2D Fig). Taken together, these results showed that SdLv effectively attenuated Mab-mediated activation of the NF-κB signaling pathway, which may be crucial for the regulation of inflammation.

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Fig 5. SdLv, but not Buch and Bufa, effectively attenuates the activation of NF-κB signaling in response to Mab.

(A) BMDMs were infected with Mab (MOI = 5) for the indicated time periods. (B) BMDMs were infected with Mab (MOI = 5, 30 min) in the presence or absence of SdLv (5–200 μg/ml). (C) BMDMs were pretreated with Buch (2, 5, or 10 μg/ml), Bufa (2, 5, or 10 μg/ml), or SdLv (20, 50, or 100 μg/ml) for 1 h and subsequently infected with Mab (MOI = 5, 30 min). (D) BMDMs were treated with Buch (2, 5, 10 μg/ml), Bufa (2, 5, 10 μg/ml), or SdLv (20, 50, 100 μg/ml) for 1 hours. Cell lysate were collected and protein expression of phosphorylated IKKαβ and total IκBα were determined by western blotting analysis. Total protein was determined by monitoring β-tubulin as a loading control. Representative data from one of three independent experiments with similar results are shown. U, Untreated; SC, vehicle control (0.01% DMSO).

https://doi.org/10.1371/journal.pone.0207696.g005

SdLv regulates Mab-induced generation of intracellular reactive oxygen species (ROS) in macrophages

Previous studies have reported that intracellular ROS are required for the activation of inflammatory responses as a signaling intermediate [35, 38, 45, 46]. Moreover, Lim et al., reported that enzyme extracts from Stellaria dichotoma had antioxidant properties [6]. We thus assessed the generation of intracellular ROS using oxidized DCFDA (for hydrogen peroxide), DHE (for superoxide anion), and CellROX (for detection of general oxidative stress) using flow cytometry, fluorescence microscopy, or laser-based confocal microscopy, respectively. Exposure of BMDMs to Mab resulted in rapid generation of intracellular superoxide within 10–30 minutes, with peak activation at 1 hour after infection (Fig 6A), with an increase of cellular oxidative stress (Fig 6B). Additionally, pretreatment with SdLv effectively attenuated the Mab-mediated activation of cellular oxidative stress (Fig 6C), intracellular superoxide production (Fig 6D), and intracellular hydrogen peroxide production (Fig 6E). However, the stimulation of Buch and Bufa mediated the significant increase of cellular oxidative stress in BMDMs. In addition, Mab-induced the activation of cellular oxidative stress was slightly increased in the presence of Buch or Bufa (S3 Fig). Together, these results indicated that Mab-induced activation of oxidative stress was attenuated by SdLv.

[Figure omitted. See PDF.]

Fig 6. Mab-induced intracellular ROS generation is attenuated by pretreatment of SdLv in BMDMs.

(A) BMDMs were infected with Mab (MOI = 5) for indicated time periods and were then stained with DHE (10 μM) for 30 min to measure intracellular superoxide using confocal microscopy. (scale bars = 50 μm) (B) BMDMs were infected with Mab (MOI = 5) for indicated time periods and were then stained with CellROX (1 μM) for 30 min to measure intracellular oxidative stress using flow cytometry. (C-E) BMDMs were infected with Mab (MOI = 5) for 30 min in a presence or absence of SdLv. H2O2 (1 mM) was used for positive control. (C) Cells were stained with CellROX (1 μM) for 30 min and intracellular oxidative stress were measured using flow cytometry. (D-E) Cells were stained with DHE (10 μM) for 30 min (scale bars = 50 μm for D) or H2DCFDA (20 μM) for 30 min (scale bars = 25 μm for E) to measure superoxide or hydrogen peroxide using fluorescence microscope, respectively. Data represent the means and SD of three independent experiments. *p < 0.05, ***p < 0.001 (two-tailed Student’s t-test), compared with uninfected cells (B) or Mab-infected cells (C). U, Untreated.

https://doi.org/10.1371/journal.pone.0207696.g006

SdLv inhibits proinflammatory responses and increases survival of an in vivo mouse infection model of Mab

To investigate the in vivo efficacy of SdLv, we used an Mab mouse intravenous infection model [47, 48]. As described in the Material and Methods section and Fig 7A and 7D, the mice that received SdLv (200 mg/kg) for 4 days were intravenously injected with Mab [5 × 108 CFU/mouse (Fig 7A–7C) or 5 × 109 CFU/mouse (Fig 7D–7F)] and then further treated with SdLv for the indicated time periods. As shown in Fig 7B, the bacterial burden in the spleen and lung tissues were not significantly altered in SdLv-administered mice. However, the Tnf mRNA expressions in the spleen and lung tissue of each mouse were significantly attenuated by the administration of SdLv (Fig 7C). Based on the anti-inflammatory roles of SdLv in vivo, we further determined the body weight (Fig 7E) and survival rate (Fig 7F) in each mouse infected by a high dose of Mab (5 × 109 CFU/mouse), as described in Fig 7D. As shown in Fig 7E and 7F, there was no change in the body weight between each experimental group (Fig 7E); however, the lethality associated with the infection of Mab was dramatically decreased in SdLv-administered mice compared with in solvent control-treated mice. Together, these in vivo findings indicated that SdLv improved the survival of mice through the regulation of Mab-induced inflammatory responses but not through bacterial clearance.

[Figure omitted. See PDF.]

Fig 7. SdLv contributes to the host protection through the regulation of proinflammatory responses in vivo infection models of Mab.

(A-C) Mice received each herbal extracts, such as Buch, Bufa, or SdLv (200 mg/kg; administrated orally) or a solvent control for 7 consecutive days and then infected with Mab (5 × 108 CFU/mouse, i.v.) at 3 days after first drug administration. The mice were sacrificed at 4 days after the infection of Mab. (A) In vivo experimental schedule to determine bacterial loads (for B) and Tnf mRNA expression (for C) in tissues. (B) In vivo mycobacterial burden of infected mice (n = 5 per group) in spleen (left) and liver (right) were determined by CFU assay. (C) Tnf mRNA expression in spleen and liver were evaluated using qPCR (n = 3 per group). (D-F) Mice received SdLv (200 mg/kg; administrated orally) for 11 consecutive days and then infected with Mab (5 × 109 CFU/mouse, i.v.) at 3 days after first drug administration. (D) In vivo experimental schedule. (E and F) The body weight (for D) or survival rate (for E) of each mouse (n = 5 per group) were monitored for 12 days. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, compared with control mice infected with Mab (two-tailed Student’s t-test (C) or log-rank test (F)). SC, vehicle control (0.25% CMC). CFU, colony-forming units.

https://doi.org/10.1371/journal.pone.0207696.g007

Discussion

To date, various efforts have been made to identify the function of major components and to characterize the underlying mechanisms to improve treatments for inflammatory disorders and infectious diseases. In East Asia, a variety of herbal extracts, including SdLv, Bufa, and Buch, have been used to treat fever-related illnesses, such as influenza and malaria [3, 8, 10]. Notably, diverse compositions including saikosaponin, fatty acids and essential oil in Bupleurum species and flavonoids, sterols, cyclic peptides, and β-carboline-type alkaloids in Stellaria dichotoma are important for their biological activities [6, 49]. Although recent studies have shown that these extracts possess diverse biological and physiological properties, the functions of these herbal extracts in mycobacterial or toxoplasma infection and their mechanisms of action have yet to be fully elucidated. In this study, we identified the effects of herbal medicines on the innate immune response to Mab infection in primary macrophages and in vivo in a mouse model.

Increasing evidence has shown that the interaction between innate immune cells and diverse pathogens rapidly trigger inflammation, thereby playing a crucial role in the activation of host protective immunity. Furthermore, an aberrant activation of inflammation is responsible for immunopathogenesis in various infectious or non-infectious diseases [44]. The Mab complex causes pulmonary disease in patients with cystic fibrosis, which is closely associated with the disruption of balance between inflammation and tissue remodeling during repeated bacterial infections [23, 50, 51]. Although TNF-α is an essential cytokine that controls mortality and bacterial growth during mycobacterial infection [52, 53], excessive production of TNF-α results in the impaired survival of mice despite sufficient bacterial clearance [54]. These studies suggested that the optimal regulation of inflammatory responses may be responsible for the improvement of mycobacteria-associated diseases. In this study, we found that SdLv effectively attenuated the Mab-induced mRNA and protein expression of proinflammatory cytokines in primary murine macrophages. Consistent with this finding, in vivo administration of SdLv significantly reduced the mRNA expression of TNF-α in the spleen and lung tissues of Mab-infected mice, although the bacterial burden in each tissue was not altered by SdLv treatment. Moreover, the lethality in Mab-infected mice was significantly improved by treatment with SdLv. These findings indicated that SdLv contributes to host protection against Mab infection by the regulation of inflammatory responses rather than by mycobacterial killing.

During mycobacterial infection, different PRRs recognize bacteria and/or bacterial-derived components and then activate intracellular signals leading to the generation of inflammatory cytokines and the initiation of adaptive immune responses [55]. Previous studies showed that MAPKs signaling pathways are crucial for the activation of inflammatory responses through toll like receptor (TLR) 2 in response to Mab [32, 33]. Additionally, during myeloid differentiation primary response gene 88 (MyD88) is also required for the production of TNF-α in BMDMs [33]. Consistent with these findings, M. abscessus subsp. massiliense (M. mas), which belongs to the Mab complex, induced the generation of TNF-α and IL-6 through MyD88- and JNK-dependent signaling pathways in murine macrophages [56]. In the present study, we found that SdLv specifically attenuated Mab-mediated activation of the JNK and p38 MAPK pathways but not of the ERK 1/2 pathways. However, SdLv did not inhibit T. gondii-induced production of inflammatory cytokines or activation of the three MAPK subfamilies. It would have been of interest if the inhibitory roles of SdLv had differed depending on the type of bacteria and protozoa. In acute toxoplasmosis, the hyperactivation of the inflammatory responses is mediated by extensive liver damage and lymphoid degeneration, which are closely related to lethality in mice [57]. The infection of the high virulence type 1 RH strain of T. gondii induced the activation of p38 MAPK and IL-12 production via a MyD88-independent manner in BMDMs [58]. Although we could not extend our findings to the role of MyD88-dependent inflammatory responses, our findings indicated the crucial role of SdLv in the regulation of inflammatory responses in response to Mab but not to T. gondii, which was mediated by the inhibition of the JNK and p38 MAPK signaling pathways in macrophages.

Stimulation of mycobacteria and their components induces the production of inflammatory cytokines through the NF-κB signaling pathway as well as the MAPK signaling pathway, which are crucial for the activation of innate and adaptive immunities [59]. Previous studies reported that Mab infection resulted in the translocation of NF-κB p65 into the nuclei [33, 48]. Moreover, the M. mas-induced NF-κB signaling pathway was required for the production of TNF-α and IL-6 in BMDMs [56]. Here, we found that SdLv inhibited the degradation of IκBα and the phosphorylation of IKKα/β in Mab-infected BMDMs.

Numerous studies have reported that intracellular ROS act as signaling molecules and play essential roles in regulating a broad range of biological responses [60]. Previous studies also reported that intracellular ROS generation was required for the production of proinflammatory cytokines via TRAF6-mediated activation of the ASK1-p38 pathway in response to TLR4 [61]. Moreover, ROS regulated TLR4-mediated IL-8 expression via a NF-κB signaling pathway in the human monocyte/macrophage cell line, THP-1 [62]. In a similar manner, intracellular ROS are also involved in the activation of the inflammatory response in M. bovis bacille Calmette-Guerin- or M. tuberculosis-infected macrophages [37, 38]. Moreover, Dectin-1-dependent activation of spleen tyrosine kinase is crucial for Mab-induced intracellular ROS generation and the production of inflammatory cytokines in BMDMs [32]. In response to M. mas, NADPH oxidase 2-induced ROS modulated the activation of the inflammatory response via the JNK-dependent signaling pathway [56]. Together, our data strongly support that SdLv plays an important role in the regulation of Mab-mediated intracellular ROS generation and it is thought to be effective against the infection of various atypical mycobacteria.

In the present study, we found that Mab infection induced the generation of inflammatory cytokines in primary murine macrophages, which was significantly attenuated by pretreatment with SdLv, but not Bufa or Buch. To elucidate the reasons for this disparity in the roles of these herbal extracts in Mab infection, we evaluated their cytotoxicity and effect on the activity of the intracellular signaling pathways that regulate the inflammatory response. The Bufa and Buch extracts had stronger cytotoxic effects at lower concentrations compared to the SdLv extract. Moreover, treatment for 1 h with Bufa or Buch, but not SdLv, led to activation of the NF-κB and MAPK signaling pathways and the generation of intracellular ROS. In future studies we plan to identify the active components that mediate cytotoxic and inflammatory effects in these compounds.

In summary, we identified novel functions of SdLv in regulating Mab-induced inflammatory responses by targeting the generation of ROS and the subsequent activation of MAPKs and NF-κB signaling pathways in macrophages. Additionally, SdLv had protective effects against Mab through the regulation of excessive inflammation in the spleen and lung tissues of mice. Our findings suggested the efficacy of SdLv not only against other mycobacterial infections but also for a variety of inflammatory diseases, including sepsis and inflammatory bowel disease, which are closely associated with uncontrolled oxidative stress. Because crude extracts isolated from SdLv contain diverse bioactive compounds, further studies should therefore identify the most effective compounds purified from herbal extracts, which can be used in the regulation of inflammatory responses and to evaluate possible clinical applications for the control of mycobacterial infections.

Supporting information

[Figure omitted. See PDF.]

S1 Fig. SdLv do not compromise T. gondii-mediated inflammatory responses in primary macrophages.

(A and B) BMDMs were infected with T. gondii (MOI = 1) for the indicated time periods (A) Cell lysates was collected and the mRNA expression of Tnf, Il6, Il1b and Il12p40 then measured using semiquantitative RT-PCR analysis. Actb (encoding β-actin) serves as a loading control throughout. (B) Culture supernatant was collected and the generation of TNF-α and IL-6 protein then were measured using ELISA assay. (C) BMDM were infected with T. gondii (at MOI = 0.1, 1, 5 or 10) for 18 h. semiquantitative RT-PCR analysis of Tnf and Il6 mRNA. (D—F) BMDMs were stimulated with increasing concentration of SdLv (1 h, 5–200μg/ml), followed by T. gondii (MOI = 1) for 18 h. (D and E) Semi-quantitative RT-PCR (top) or quantitative RT-PCR analysis (bottom) were assessed to evaluate the mRNA expression of Tnf (for D) and Il6 (for E). (F) Each culture supernatant was collected and the production of TNF-α and IL-6 were measured using ELISA assay. Data are representative of three independent experiments and are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test), compared with uninfected cells (B and C). U, Untreated; SC, vehicle control (0.01% DMSO).

https://doi.org/10.1371/journal.pone.0207696.s001

(TIF)

S2 Fig. T. gondii-mediated the activation of MAPK and NF-κB signaling is not inhibited by the pretreatment of SdLv in primary murine macrophages.

(A and C) BMDMs were infected with T. gondii (MOI = 1) for the indicated time periods. (B and D) BMDMs were infected with T. gondii (MOI = 1, 30 min) in the presence or absence of SdLv (A and B) Immunoblot analysis was performed to determine protein expression of phosphorylated ERK, p38, or JNK. β-tubulin served as a loading control. (C and D) Immunoblot analysis was performed to determine protein expression of total IκB-α and phosphorylated IKKα/β. Data are representative of three independent experiments. U, Untreated; SC, vehicle control (0.01% DMSO).

https://doi.org/10.1371/journal.pone.0207696.s002

(TIF)

S3 Fig. Mab-mediated the activation of intracellular oxidative stress is not inhibited by the pretreatment of Buch and Bufa in primary murine macrophages.

BMDMs were treated with Buch or Bufa for 1 hours and then infected with or without Mab (MOI = 5) for 30 min. H2O2 (1 mM) was used for positive control. Cells were stained with CellROX (1 μM) for 30 min and intracellular oxidative stress were measured using flow cytometry. Data are representative of three independent experiments. U, Untreated; SC, vehicle control (0.01% DMSO).

https://doi.org/10.1371/journal.pone.0207696.s003

(TIF)

Acknowledgments

We thank Yeeun Kim for providing the materials to determine ROS generation; Bo-In Kwon (Sangji University) for technical assistance; Hae-Sung Nam (Chungnam National University) for statistics analysis; Jaeyul Kwon (Chungnam National University) for scientific and language editing.

Citation: Bae S-J, Choi J-W, Park B-J, Lee J, Jo E-K, Lee Y-H, et al. (2018) Protective effects of a traditional herbal extract from Stellaria dichotoma var. lanceolata against Mycobacterium abscessus infections. PLoS ONE 13(11): e0207696. https://doi.org/10.1371/journal.pone.0207696

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