Macmoondongtang modulates Th1-/Th2-related cytokines and alleviates asthma in a murine model

Soon-Young Lee; Bossng Kang; So-Hyeon Bok; Seung Sik Cho; Dae-Hun Park

doi:10.1371/journal.pone.0224517

. 2019 Dec 2;14(12):e0224517. doi: 10.1371/journal.pone.0224517

Macmoondongtang modulates Th1-/Th2-related cytokines and alleviates asthma in a murine model

Soon-Young Lee ^1,^#, Bossng Kang ^2,^#, So-Hyeon Bok ¹, Seung Sik Cho ^3,^*, Dae-Hun Park ^1,^*

Editor: Michal A Olszewski⁴

PMCID: PMC6886797 PMID: 31790411

Abstract

Objective

Macmoondongtang has been used as a traditional medicine to treat pulmonary disease in Korea. However, the mechanism underlying its therapeutic effect has yet to be reported. In the present study, the role of macmoondongtang as a respiratory medicine, especially as an anti-asthmatic agent, has been attributed to the down-regulation of interleukin (IL)-4 and tumor necrosis factor (TNF)-α.

Materials & methods

BALB/c mice were divided into five groups: control, asthma-induced control, dexamethasone treatment, treatment with 150 mg/kg macmoondongtang, and treatment with 1500 mg/kg macmoondongtang. To evaluate the anti-asthmatic effect of macmoondongtang, we investigated its suppressive or inhibitory effects against typical asthmatic changes such as differential cell count in bronchioalveolar fluid (BALF), serum IgE levels, lung morphology, expression of Th1/Th2 cell transcription factors such as T-bet and GATA-3, and Th1-/Th2-/Th17-related cytokines such as interferon (IFN)-γ, IL-12p40, IL-4, -5, -13, TNF-α, and IL-6. The active ingredients in macmoondongtang were further analyzed.

Results

Macmoondongtang treatment down-regulated serum IgE level, a very important marker of hyper-responsiveness. It reversed typical morphological changes such as mucous hypersecretion, lung epithelial cell hyperplasia, and inflammatory cell infiltration near bronchioalveolar space and veins. Macmoondongtang significantly decreased neutrophil count in BALF, as well as reduced T-bet, IFN-γ, and TNF-α expression in the lung. It also showed a dose-dependent control of inflammatory cells in BALF, controlled the expression of IL-12, IL-4, and IL-5 genes in the lung, and the protein expression of IL12p40, GATA-3, IL-4, IL-5, and IL-13. The component analysis revealed glycyrrhizin and liquiritin as the active ingredients.

Conclusions

Macmoondongtang treatment alleviates asthma symptoms and modulate the Th1-/Th2- related cytokines. Glycyrrhizin and liquiritin could be the major the active therapeutic components.

Introduction

In 2013, the World Health Organization reported that at least 200 million individuals worldwide were diagnosed with asthma. The allergens were classified into two categories: indoor agents (such as pet dander, dust mites, tobacco smoke, and so forth) and outdoor agents (e.g., pollen, environmental pollutants, cold temperature, and so forth) [1]. Asthma is a type I allergy, and anaphylaxis. It is hard to completely eradicate and its symptoms are very diverse, ranging from cough to apnea. Patients with severe allergy may die from pulmonary obstruction and lack of gas exchange due to hypersecretion of mucus, overgrowth of pulmonary subepithelial cells, enlarged basement membrane, and accumulation of inflammatory cells near bronchioles and vessels [2,3].

Among several theories of asthma occurrence, the imbalance between Th1 and Th2 cells plays a significant role in increasing the risk [2]. Cytokines such as interferon gamma (IFN-γ) that induce the release of Th1 cells are associated with the severity of asthma [4]. Interleukin 12 (IL-12) is an important cytokine that mediates Th1 cell differentiation and decreases Th2 cell proliferation [5–7]. Th2 cells release specific cytokines such as IL-4, IL-5, IL-13, and tumor necrosis factor-alpha (TNF-α) while IL-6 and IL-1β are Th17-related cytokines [8]. IL-4 is induced by GATA-3, a transcription factor with several bioactive functions, including stimulation of Th2-related cytokine expression [9], regulation of IgE levels [10], and so on. The level of IL-6 in asthma patients is substantially higher than in normal persons [11]. As one of the important factors that regulate asthma severity, IL-6 induces IL-4 up-regulation and stimulates Th17 cell differentiation [12]. IL-13 is linked to morphological changes in respiratory organs and B cell activation [13–16]. TNF-α is synthesized by macrophages [17]. It recruits neutrophils and eosinophils [18], stimulates T cell activation [19], and finally establishes airway hyperresponsiveness [20].

Inhaled corticosteroids are usually used to inhibit clinical asthma symptoms such as cough, respiratory depression, etc [21]. Asthma occurrence is higher in early childhood and elderly population than in the young and grown-up groups [1]. Inhaled corticosteroids are associated with serious adverse effects such as growth inhibition [22], cataracts, glaucoma, hypertension, hyperlipidemia, peptic ulcers, myopathy, and immunological suppression [23]. Therefore, efforts to discover new anti-asthmatic agents are ongoing and trials to investigate the mode of action on suppression or inhibition against asthma occurrence among the traditional medicines-related pulmonary diseases have increased.

Several hundred years ago in Korea, a medical encyclopedia known as Donguibogam was composed [24]. Donguibogam introduced macmoondongtang, a traditional prescription for the treatment of respiratory illness. Macmoondongtang is composed of plants such as Glycyrrhizae radix, Oryza sativa, Zizyphus jujube Inermis, Ophiopogon japonicas, Pinellia ternate, and Panax ginseng. Although macmoondongtang has been used to treat pulmonary disease recently, its mode of action has yet to be reported. Therefore, the objective of this study was to analyze the possible anti-asthmatic effect of macmoondongtang and the underlying mechanism of action.

Materials and methods

Preparation of macmoondongtang

Macmoondongtang (Batch No. 14010, hot water extract-powder form) was manufactured by Hankuk Inspharm, Ltd. (Jeonnam, Korea) based on the prescription indicated in Donguibogam [24] and used in the present study. Macmoondongtang is generally available as an over-the-counter (OTC) drug. It contains G. radix, O. sativa, Z. jujube Inermis, O. japonicas, P. ternate, and P. ginseng. The dosage used in this study was calculated based the drug composition. The human equivalent dose (HED) for mice is 12.3 and the safety factor is 10×[25]. In this study, we applied 1/100 and 1/10 dosages for mouse based on therapeutic human dosage.

Identification of anti-asthmatic compounds in macmoondongtang

To analyze the levels of anti-asthmatic compounds in macmoondongtang, appropriate amounts of glycyrrhizin and liquiritin were accurately weighed and dissolved in methanol in 100 mL volumetric flasks to obtain stock solutions of 100 μg/mL. Solutions were subsequently serially diluted two-fold to 3.125 μg/mL. A sample measuring 0.5 g was dissolved in 10 mL methanol and sonicated to expedite the dissolution of particles. Subsequently, 1 mL was transferred to a volumetric flask and diluted with 9 mL of mobile phase A. The final concentration of macmoondongtang was 50 mg/mL. HPLC analysis was performed using an Alliance 2695 HPLC system (Waters, Milford, MA, USA) equipped with a photodiode array detector. A reverse phase column (C18, 5 μm, 150 mm × 5 mm) was used with a mobile phase consisting of a mixture of solvent A (acetonitrile) and B (0.2% acetic acid). The mobile phase conditions for glycyrrhizin were as follows: 0–35 min, gradient elution from 15/85 to 35/65 v/v; 35–50 min, gradient elution from 35/65 to 100/0 v/v; and 50–55 min, from 100/0 to 15/85 v/v. The mobile phase conditions for liquiritin were as follows: 0–8 min, gradient elution from 10/90 to 20/80 v/v; 8–20 min, gradient elution from 20/80 to 25/75 v/v; 20–21 min, from 25/75 to 100/ v/v; and 21–30 min, from 100/0 to 10/90 v/v. The flow rate was 1.0 mL/min, and the injection volume was 10 μL.

Animal experiments

The animal study investigating the anti-asthmatic effect of macmoondongtang was followed by a confirmative study. For each study, 35 female BALB/c mice were purchased from Samtako Korea (Osan, Korea) and divided into five groups according to the treatment: vehicle control (tap water), ovalbumin (OVA)-treated group, OVA-treated group exposed to 1 mg/kg dexamethasone, OVA-treated group exposed to 150 and 1500 mg/kg macmoondongtang. The following doses were injected intraperitoneally into all animals except the control group: 20 μg OVA (Sigma-Aldrich, St. Louis, MO, USA) and 1 mg aluminum hydroxide hydrate (Sigma-Aldrich) in 500 μL saline. A week after injection of the second booster dose, all animals except the control group were exposed to OVA including a first inhalation dose for 5 days after OVA challenge. The animals were treated orally with tap water, dexamethasone, and 2 doses of macmoondongtang.

Ethics statement

This animal study was conducted following approval by the Institutional Animal Care and Use Committee of Dongshin University (Animal Study Approval No. 2014-08-02).

Bronchoalveolar fluid (BALF) and serum analysis

Bronchioalveolar fluid (BALF) was collected from all mice under anesthesia on a day after the final treatment. BALF was collected using 0.4 mL cold phosphate-buffered saline (PBS). The white blood cell (WBC) count and differential blood count were performed with the collected fluid using Hemavet Multispecies Hematology System (Drew Scientific Inc, Waterbury, CT, USA). Inflammatory cells in BALF were stained with Diff-Quick method [26].

Serum IgE levels were evaluated using ELISA.

Histopathological analysis

In order to observe the morphological changes, lungs were fixed in formaldehyde, dehydrated in ethanol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. In order to compare the levels of released glycoprotein, periodic acid Schiff (PAS) stain was used. Subsequently, the images were acquired with Axioscope A1 (Carl Zeiss, Gottingen, Germany).

Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunoassay (ELISA)

In order to evaluate changes in the cDNA levels of IFN-γ, IL-12p40, IL-4, IL-5, IL-13, TNF-α, and IL-6, which are related to asthma induction, RT-PCR analysis was conducted as reported in our previous study protocol (Lee et al., 2017). Total RNA was extracted from the lung using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Total RNA (100 ng) was used as a template for the reaction. Primers were synthesized for RT-PCR as shown in Table 1. The RT-PCR cycles consisted of denaturation at 95°C for 5 s and annealing/extension at 65°C for 30 s for 40 cycles using with QTOWER2.2. (Analytik Jena AG, Thuringia, Germany). The serum levels of IgE were measured using a specific mouse IgE enzyme-linked immunosorbent assay kit (BD bioscience, 555248, San Jose, CA, USA) according to the manufacturer's protocols.

Table 1. Primer sequences for RT-PCR.

Genes	Primer sequences
IFN-γ	Forward	`5′-GGCCATCAGCAACAACATAAG-3′`
IFN-γ	Reverse	`5′-GTTGACCTCAAACTTGGCAATAC-3′`
IL-12p	Forward	`5′-GGACCAAAGGGACTATGAGAAG-3′`
IL-12p	Reverse	`5′-CTTCCAACGCCAGTTCAATG-3′`
IL-4	Forward	`5′-ACAGGAGAAGGGACGCCAT-3′`
IL-4	Reverse	`5′-GAAGCCCTACAGACGAGCTCA-3′`
IL-5	Forward	`5′-TGCATCAGGGTCTCAAGTATTC-3′`
IL-5	Reverse	`5′-GGATGCTAAGGTTGGGTATGT-3′`
IL-13	Forward	`5′-CAGCCCTCAGCCATGAAATA-3′`
IL-13	Reverse	`5′-CTTGAGTGTGTAACAGGCCATTCT-3′`
IL-6	Forward	`5′-GATAAGCTGGAGTCACAGAAGG-3′`
IL-6	Reverse	`5′-TTGCCGAGTAGATCTCAAAGTG-3′`
TNF-α	Forward	`5′-CTGAGTTCTGCAAAGGGAGAG-3′`
TNF-α	Reverse	`5′-CCTCAGGGAAGAATCTGGAAAG-3′`
GAPDH	Forward	`5′-GTGGAGTCATACTGAACATGTAG-3′`
GAPDH	Reverse	`5′-AATGGTGAAGGTCGGTGTG-3′`

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Immunofluorescent analysis

In order to localize the expression of Th1 cell transcription factor, T-bet, and Th2 cell transcription factor, GATA-3 immunofluorescent analysis was conducted. Only four groups were evaluated: control, OVA, dexamethasone plus OVA treatment, and 1500 mg/kg macmoondongtang plus OVA treatment. Prior to the antibody binding step, the same materials were used for immunohistochemical analysis; however, rabbit anti-mouse T-bet (Biorbyt, orb7075, Cambridge, UK) or goat anti-mouse GATA-3 (OriGene, TA305795, Rockville, MD, USA) were used as primary antibodies for 1 h at room temperature. The slides were incubated for 2 h with FITC-conjugated anti-rabbit IgG (Jackson Immunoresearch, 315-095-003, West Grove, PA, USA) or Alexa Fluor^® 555-conjugated anti-goat IgG (ThermoFisher Scientific, A-21127, Waltham, MA, USA). The cells were counterstained with DAPI (ThermoFisher Scientific, 62249, Waltham, MA, USA). The images were obtained using a K1-Fluo confocal microscope (Nanoscope System, Daejeon, Korea).

Immunohistochemical analysis

In order to analyze the changes in the levels of asthma-related protein, immunohistochemistry was performed using antibodies such as IFN-γ (Santa Cruz, sc-74104), IL-12p40 (Santa Cruz, sc-57258), IL-4 (Santa Cruz, sc-73318), IL-5 (Santa Cruz, sc-7887), IL-13 (Santa Cruz, sc-1776), IL-6 (Santa Cruz, sc-1265), and TNF-α (BioVision, 3053R-100, Milpitas, CA, USA) and the dilution rate ranged from 1:100 to 1:200. The slides were incubated with a biotinylated pan-specific secondary antibody for 10 min and reacted with the streptavidin-peroxidase complex for 5 min (Vector Laboratories Universal Quick Kit, Burlingame, Canada). Signals were detected using 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution. Cells were counterstained with Mayer’s hematoxylin. Subsequently, the cells were imaged using Axioscope A1 (Carl Zeiss).

Statistical analysis

Results are expressed as the mean ± standard deviation (SD). Group differences were evaluated via one-way analysis of variance followed by Dunnett’s multiple comparison tests. Significance was considered at p < 0.01 or p < 0.05.

Results

Macmoondongtang contains two anti-asthmatic markers: Glycyrrhizin and liquiritin

HPLC analyses were performed to identify the anti-asthmatic compounds and immune modulators in macmoondongtang. Typical HPLC chromatograms and their retention times are shown in Fig 1. Glycyrrhizin (0.38 ± 0.002%), and liquiritin (0.02 ± 0.0002%) were identified using HPLC analysis.

Fig 1 — (A) standard- liquiritin and paeoniflorin; (B) *macmoondongtang*, 50 mg/mL, (C) standard-glycyrrhizin; (D) macmoondongtang, 25 mg/mL.

Macmoondongtang suppresses ovalbumin-induced hyperproliferation of inflammatory cells and immune cells

OVA treatment significantly boosts the levels of inflammatory and immune cells such as white blood cells, eosinophils, and neutrophils in the lung [27]. Macmoondongtang induced a dose-dependent suppression of inflammatory cells, which were increased by OVA treatment (Fig 2A and S1 File). We used dexamethasone as a positive control during the treatment with macmoondongtang as it is generally used to manage acute asthma exacerbation [28]. As shown in Fig 2B–2D, the WBC, eosinophil, and neutrophil counts in the dexamethasone treatment group were significantly decreased compared with the OVA-induced asthma group. However, macmoondongtang treatment suppressed their proliferation dose-dependently. Especially, the number of neutrophils in the group treated with1500 mg/kg macmoondongtang was significantly decreased (Fig 2D) similar to the group treated with dexamethasone.

Fig 2 — (A) *Macmoondongtang* effectively decreased OVA-induced proliferation of inflammatory cells. (B) *Macmoondongtang* dose-dependently down-regulated the population of WBCs. (C) *Macmoondongtang* effectively suppressed the number of eosinophils. (D) *Macmoondongtang* significantly and dose-dependently decreased neutrophil proliferation. a, vehicle control; b, asthma induction; c, dexamethasone; d, 150 mg/kg/day *macmoondongtang*; e, 1500 mg/kg/day *macmoondongtang*. Each bar represents the mean ± SD (n = 8). *p < 0.05 vs. control; **p < 0.001 vs. control; ^$p < 0.05 vs. asthma induction; ^$$p < 0.01 vs. asthma induction; ^#p < 0.05 vs. dexamethasone. Scale Bar = 100 μm. Magnification, ×400.

Macmoondongtang induces dose-dependent suppression of IgE

IgE levels usually surge in allergy and increase significantly in asthma [29]. Serum IgE levels in OVA-induced asthma group (60.17 ± 8.135 ng/mL) were significantly increased compared to the levels (13.98 ± 3.877 ng/mL) in the control (Fig 3 and S1 File). Although the serum IgE level in dexamethasone-treated group was higher than in the control, the increased level of IgE induced by OVA treatment was down-regulated to 34.00 ± 8.351 ng/mL (57% compared with OVA treatment) after dexamethasone treatment. Macmoondongtang controlled the IgE level in a dose-dependent manner: 54.91 ± 10.141 ng/mL in the group treated with 150 mg/kg and 44.35 ± 9.618 ng/mL in the group exposed to 1500 mg/kg. There was no significant difference in serum IgE levels between groups treated with dexamethasone and 1500 mg/kg of macmoondongtang, which suggested that 1500 mg/kg macmoondongtang was adequate to treat asthma patients.

Fig 3 — Serum IgE level in the group treated with 1500 mg/kg *macmoondongtang* was compared with that of dexamethasone-treated group. Each bar represents the mean ± SD (n = 16). **p < 0.001 vs. control; ^$p < 0.05 vs. asthma induction; ^$ $p < 0.01 vs. asthma induction; ^#p < 0.05 vs. dexamethasone.

Macmoondongtang dose-dependently inhibits typical morphological changes in OVA-induced asthma model

To evaluate morphological changes in the lung, H&E staining was performed (Fig 4A). The OVA-treated group showed typical morphological changes such as inflammatory cell infiltration (purple color) near bronchioles and vessels, pulmonary hyperplasia (thickening inner bronchiole), and mucous hypersecretion (purple color) in the bronchiole (Fig 4Bb) compared with the control (Fig 4Aa). Dexamethasone controlled OVA-induced morphological changes in the lung by decreasing inflammatory cell infiltration near bronchioles and vessels and inhibiting basal membrane thickening of inner bronchioles and mucous secretion (Fig 4Ac). It Fewer differences in lung morphology were detected between groups treated with OVA (Fig 4Ab) and 150 mg/kg macmoondongtang (Fig 4Ad). However, OVA-induced asthmatic changes in the lung were reversed by treatment with 1500 mg/kg macmoondongtang (Fig 4Ae), although the recovery was less than that induced by dexamethasone.

In the case of asthma patients, breathing difficulty is usually observed due to factors such as pulmonary base membrane thickening due to epithelial cell hyperplasia and mucous hypersecretion [16]. Mucous hypersecretion in the bronchiole can directly inhibit breathing, and therefore, is a very important parameter for the evaluation of asthma severity. To determine the effect of macmoondongtang on mucous secretion, PAS staining was conducted (Fig 4B). In the control, no mucus was detected in the bronchiole (Fig 4Ba). However, in the OVA-treated group (Fig 4Bb) and the group treated with 150 mg/kg macmoondongtang (Fig 4Bd), the bronchiole was almost obstructed by mucus (purple color). There was little difference in mucus levels between the groups treated with dexamethasone (Fig 4Bc) and 1500 mg/kg macmoondongtang (Fig 4Be). These results indicate that macmoondongtang dose-dependently suppresses mucous secretion.

Macmoondongtang effectively induces inactivation of both Th1 and Th2 cell transcription factors

Asthma is caused by the imbalance of Th1 and Th2 cells [2]. T-bet is a Th1 cell transcription factor [30, 31] while GATA-3 is a Th2 cell transcription factor [9]. GATA-3 immunofluorescent assay was conducted to evaluate the changes in the activation levels of Th1 cell transcription factor, T-bet, and Th2 cell transcription factor (Fig 5). The activated transcription factors are translocated from the cytoplasm to nuclei. The distribution of T-bet (green color, Fig 5B) was similar to that of GATA-3 (red color, Fig 5C). OVA treatment activated both Th1 and Th2 transcription factors in the nuclei (Fig 5Db). However, in the other groups, both T-bet and GATA-3 remained localized to the cytoplasm (Fig 5Da, 5Dc & 5Dd). These results indicate that macmoondongtang inhibited the activation of both Th1 and Th2 cell transcription factors.

Fig 5 — (A) Nuclei are shown in blue color by DAPI staining. (B) Images displaying results of T-bet staining by FITC. (C) GATA-3 staining by AlexaFluor® 555. (D) Merged images of nuclei stained with DAPI, T-bet stained with FITC, and GATA-3 stained with Alexa Fluor® 555. Treatment with dexamethasone and *macmoondongtang* completely abrogated the activation of both Th1 cell transcription factor T-bet and Th2 cell transcription factor GATA-3. a, vehicle control; b, asthma induction; c, dexamethasone; d,1500 mg/kg/day macmoondongtang. Scale Bar = 100 μm. Magnification, ×1000.

Macmoondongtang significantly suppresses both gene and protein expression of IFN-γ and IL-12

To analyze the levels of Th1-related cytokines such as IFN-γ and IL-12, we conducted RT-PCR (Fig 6A and S1 File) and IHC staining (Fig 6B and 6C). As shown in Fig 6A, macmoondongtang effectively and dose-dependently inhibited IFN-γ gene expression, which was dramatically up-regulated by OVA treatment. IFN-γ gene expression in the group treated with 1500 mg/kg macmoondongtang was similar to that of the group treated with dexamethasone. Similar to changes in the expression of IFN-γ gene, the protein expression induced by OVA treatment was also suppressed by macmoondongtang (Fig 6B). Changes in gene and protein expression of IL12p40 were similar to those of IFN-γ (Fig 6A and 6C).

Macmoondongtang controls the expression of IL-4 and IL-5, but not IL-13, in a dose-dependent manner

OVA treatment significantly suppressed the expression of IL-5 and IL-13 genes, but not that of IL-4 gene, although it varied depending on treatment materials such as sterilized tap water (control), dexamethasone, and macmoondongtang (Fig 7A and S1 File). However, macmoondongtang significantly down-regulated the expression of IL-5 gene alone. The gene expression of Th2-related cytokines differed from their protein levels in the respiratory system (Fig 7B–7D). Although the IL-5 gene expression was statistically significant after macmoondongtang treatment, the protein expression of all Th2-related cytokines in the present study such as IL-4, IL-5, and IL-13 was dose-dependently suppressed by macmoondongtang treatment. The levels of IL-4 (Fig 7Be), IL-5 (Fig 7Ce) and IL-13 proteins (Fig 7De) in the group treated with 1500 mg/kg macmoondongtang were significantly decreased compared with those of the dexamethasone-treated group (Fig 7Cc & 7Dc).

Fig 7 — *Macmoondongtang* significantly and effectively inhibited the gene expression of *IL-5* (A) and IL-5 protein level (C). The treatment dose-dependently controlled the IL-4 protein expression (B). Similar changes at the gene (A) and protein levels of IL-13 (D) were observed as those for IL-5. a, vehicle control; b, asthma induction; c, dexamethasone; d,150 mg/kg/day *macmoondongtang*; e, 1500 mg/kg/day *macmoondongtang*. Each bar represents the mean ± SD (n = 8). *p < 0.05 vs. control; **p < 0.001 vs. control; ^$p < 0.05 vs. asthma induction; ^#p < 0.05 vs. dexamethasone. Scale Bar = 50 μm. Magnification, ×200.

Macmoondongtang modulates the expression of TNF-α, but not IL-6

OVA treatment increased the levels of only TNF-α gene (Fig 8A and S1 File). Dexamethasone significantly decreased the expression of TNF-α gene while macmoondongtang dose-dependently suppressed its level. Especially, treatment with 1500 mg/kg macmoondongtang suppressed the expression of TNF-α gene similar to that of dexamethasone-treated group. Macmoondongtang down-regulated TNF-α expression in a dose-dependent manner (Fig 8B). However, it did not suppress the level of IL-6, which was increased by OVA treatment (Fig 8Cc, 8Cd & 8Ce). The TNF-α level was decreased by treatment with 1500 mg/kg macmoondongtang (Fig 8Be), similar to that of dexamethasone treatment (Fig 8Bc).

Discussion

Environmental allergens inducing asthma are classified into two types: indoor allergens (for e.g., pet dander, dust mite, tobacco smoke, etc.) and outdoor allergens (for e.g., pollen, environmental pollutants, cold temperature, etc.) [32]. At first, antigen-presenting cells (APCs) recognize repeated invasion by allergens, leading to asthma. Studies have been performed to verify the relation between type 1 allergy and IgE, an important and representative biomarker [29, 33]. Accordingly, IgE increase is a very important factor that triggers asthma, suggesting the need to decrease IgE expression induced by OVA exposure. Immune response is critical to maintain the homeostasis of Th1 and Th2 cells in living organisms. The hypothesis of Th1/Th2 balance for immune system was introduced in the 1980s [34]. Asthma is one of the diseases triggered by Th1/Th2 imbalance and according to the theory [35], the balance between Th1 cells and Th2 cells is generally maintained. However, an up-regulation of Th2 cells including related cytokines may trigger asthma. IFN-γ and IL-12 are Th1 cytokines, whereas IL-4, IL-5, and IL-13 are Th2 cytokines, and IL-6 and TNF-α are Th17-related cytokines[10].

T-bet is a factor associated with Th1 cell transcription. T-bet and IFN-γ show a positive feed-back [30, 36]. IL-12p40 is one of the key factors regulating Th1-related cytokines in that it specifically stimulates IFN-γ production in asthma [5]. As shown in Fig 6, changes in IFN-γ gene and protein levels were very similar to those of IL-12p40 while macmoondongtang effectively inhibited their increases.

In asthma patients, the Th2-related cytokines such as IL-4, IL-5, and IL-13 are usually increased [37–39]. GATA-3 is a Th2 cell transcription factor. IL-4 increases its activation. Consequentially, the regulation of GATA-3 and IL-4 produces Th2-related cytokines [9]. Macmoondongtang not only significantly inactivated both Th1 and Th2 transcription factors (Fig 5), but also suppressed gene and protein levels of IFN-γ and IL-4 directly linked to each transcription factor (Figs 6A, 6B, 7A and 7B).

Recognition of APCs by Th2 cells not only releases cytokines such as IL-4, IL-5, and IL-13 that stimulate IgE surge via B cell activation, but also increases the eosinophil population induced by IL-5 to initiate airway remodeling [33, 40]. In the lung, increased IL-13 expression is extensively associated with airway hyper-responsiveness such as inflammation, mucous hypersecretion, epithelial fibrosis, and airway obstruction [16]. Macmoondongtang controlled the levels of all Th2-related cytokines investigated such as IL-4, IL-5, and IL-13. It specifically suppressed the expression of IL-4 and IL-5 (Fig 7A–7C). These results explain the anti-asthmatic effect of macmoondongtang mediated via suppression of serum IgE level (Fig 3) to reverse the typical morphological changes associated with asthma such as epithelial hyperplasia, mucous hypersecretion, and airway obstruction (Fig 4).

Th17-related cytokines such as TNF-α, IL-6, and IL-1β are important in the control of asthma onset [41]. TNF-α is one of the chemo-attractants for neutrophils and eosinophils [18]. It induces death of eosinophils via oxidative stress in pulmonary epithelial cells [42]. Although eosinophil numbers are increases in many asthma patients, other patients show a surge in neutrophil levels [43]. In the present study, macmoondongtang effectively suppressed the proliferation of eosinophils and neutrophils in BALF and inhibited eosinophil infiltration near bronchioles and alveoli, and vessels via TNF-α modulation (Fig 2). The expression of IL-6 in asthma patients is significantly increased [44]. The IL-6 induced by allergens stimulates the release of IL-4 and IL-13 from Th2 cells [12]. IL-6 may be one of the key factors maintaining the balance of helper T cells to control Treg cells or Th17 cells [44].

Several reports suggest the anti-asthmatic effects of herbal extracts [45–47] or compounds derived from natural products [48]. However, the bioactivity of may be associated with specific compounds derived from herbs or mushrooms. The anti-asthmatic effect was attributed to bioactive ingredient(s) in the natural product. Macmoondongtang used in this study was obtained as an extract from several herbs such as Glycyrrhizae radix, Oryza sativa, Zizyphus jujube Inermis, Ophiopogon japonicas, Pinellia ternate, and Panax ginseng. A review of literature suggests that macmoondongtang contains anti-asthmatic markers such as glycyrrhizin and anti-inflammatory markers such as liquiritin. To determine the bioactive compounds in macmoondongtang, HPLC analysis was conducted. Results showed that glycyrrhizin (0.38%) and liquiritin (0.02%) were present in the macmoondongtang extract. Glycyrrhizin is one of the major compounds in Glycyrrhiza uralensis and Glycyrrhiza glabra. Ram et al. (2006) reported that glycyrrhizin exhibits an immunomodulatory effect in a mouse model of asthma [49]. Oral administration of glycyrrhizin (2.5 to 20 mg/kg) to OVA-induced mice resulted in a suppression of IL-4, IL-5, IFN-γ, and IgE levels. In the present study, we identified liquiritin content in macmoondongtang extract. Liquiritin modulates inflammation via inhibition of pro-inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase (COX)-2, TNF-α, IL-1β, and IL-6 [50, 51]. Thus, liquiritin occurring in macmoondongtang may alleviate the inflammatory symptoms caused by asthma. Although G. auralensis contains glycyrrhizin and G. glabra has liquiritin, each herb exhibits a typical anti-asthmatic effect.

Ram et al. (2006) have reported that an oral dose starting with 5 mg/kg of liquiritin was effective in mice [49]. In the present study, we found that macmoondongtang resulted in an anti-asthmatic effect at a dose of 1500 mg/kg in our animal model. The dose of 1500 mg/kg corresponds to 5.7 mg/kg of glycyrrhizin. Thus, the curative dose in the present study was consistent with that of the previous report, suggesting that glycyrrhizin contained in macmoondongtang is a possible therapeutic candidate for the management of asthma. Furthermore, we evaluated a daily oral dose of 7.3 g macmoondongtang for treatment of asthma. The therapeutically effective oral dose of macmoondongtang in mouse was 1500 mg/kg/day. The conversion factor between human and mouse is 12.3 [21]. Therefore, if the effective dose of macmoondongtang in mice is 1500 mg/kg/day, the human equivalent dose is 7300 mg/60kg/day. The recommended daily intake of macmoondongtang for human is 9 g. Thus, the optimal dose for asthma treatment is less than its daily intake and then we could confirm that oral intake of macmoondongtang at a dose of 7.3 g might prevent the occurrence of asthma.

From the results we concluded that Macmoondongtang treatment alleviates asthma symptoms and modulate the Th1-/Th2- related cytokines. Glycyrrhizin and liquiritin could be the major the active therapeutic components.

Supporting information

S1 File. This file is raw data for making graphs in Fig 2, Fig 3, Fig 6, Fig 7 and Fig 8.

(XLSX)

Click here for additional data file.^{(55.3KB, xlsx)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP: Ministry of Science, ICT & Future Planning) (Grant Nos. NRF-2015R1D1A1A01059523 to D-HP and NRF-2017R1C1B5015187 to SSC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0224517.r001

Decision Letter 0

Michal A Olszewski

24 Jul 2019

PONE-D-19-17023

Macmoondongtang modulates asthma-related changes via modulation of TNF-a and T-bet & IFN-r in an asthma murine model

PLOS ONE

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Reviewer #1: Comment #1

The manuscript entitled with “Macmoondongtang modulates asthma-related changes via modulation of TNF-a and T-bet & IFN-r in an asthma murine model” brought some interesting findings. However, it is not convincible for its description and conclusion with several reasons as follows:

1) Macmoondongtang has been used as a traditional prescription for pulmonary disease in Korea. However, there is no report about the mechanism involved in its effect. In the present study, the authors described how Macmoondongtang as a repiratory medicine has effect on Ashma in a murine model. Since Macmoondongtang is a traditional prescription for pulmonary disease, surprisingly, there is no any information on how Macmoondongtang has been clinically used in Ashma treatment at all. So, it is really weird for me to choose Ashma instead of the other disease models for study on Macmoondongtang.

2) According to the results, it looks like that Macmoondongtang play role in modulation of expression of Th1, Th2 and Th17-related cytokines. Why did the manuscript choose a tittle as modulation of TNF-a and T-bet & IFN-r? It doesn't make any sense to me.

3) The results showed that Macmoondongtang contained glycyrrhizin and liquiritin. Theoretically glycyrrhizin and liquiritin could have some role in modulation of the cytokine expression. But how did you exclude effects of some unknown components in Macmoondongtang.

Comment #2

Has the statistical analysis been performed appropriately and rigorously? No Although the manuscript showed thirty-fine mice were divided into five groups, it was not clear of how many mice had being used in each figure. So, it is very confused about how they calculated the data without showing “n” in each figure.

Comment #3

Is the manuscript presented in an intelligible fashion and written in standard English? No

There are a lot of places to be rewrote in a standard English in this manuscript, for example, ”It is important to decrease IgE expression increased by ovalbumin treatment”, “T-bet is a factor for Th1 cell transcription…., to the end of this paraph”, and so on.

Some minor comments:

1) For material and methods, there were a lot of description to be improved, for example, which samples were used for RNA extraction? how the samples were prepared for IFA? and so on.

2) What’s magnitude for those microscopic figures? And the quality of those microscopic figures should be also improved.

Reviewer #2: In this study, Lee et al. reported that macmoondongtang, a traditional prescription for treating respiratory disease, could modulate host immune responses during OVA-induced asthma in a mice model. They reported that macmoondongtang treatment could reduce serum IgE level, mucus hypersecretion, inflammatory cell infiltration in the lungs. They also reported that macmoondongtang treatment leads to reduced expression of Th1/Th2 transcriptional factors as well as related cytokines such as IFN-g, IL-12, IL-4 and IL-5, IL-13 on mRNA level or protein level. The data is interesting; however, the manuscript has severe limitations that the authors need to deal with before publications.

1. The written Englished should be revised significantly. There are many grammar mistakes in the manuscript.

2. The conclusions are overstated and need revision. For example,

a. it is not convincing that glycyrrhizin and liquiritin were the effective ingredients of macmoondongtang.

b. There is no data supporting the title: “Macmoondongtang modulates asthma-related changes via modulation of TNF-a and Tbet & IFN-g in an asthma murine model”. They showed correlation, nut not causality.

3. The reviewer also concerned about the quality of some data. For example, fig 5, it is hard to tell the localization of the transcriptional factors (Tbet and GATA3) at the current magnification. Fig 6, 7, 8, it would be nice to quantify the IHC signals.

4. Methods are missing: RNA isolation, cDNA synthesis, qPCR methods, Elisa for IgE,

5. Fig 1, please also include the control HPLC data for glycyrrhizin and liquiritin.

6. For statistical analysis, how many times have these experiments been repeated? It says mean ± SEM in the figure legends but mean ± SD in the Materias and Methods, which one is correct?

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2019 Dec 2;14(12):e0224517. doi: 10.1371/journal.pone.0224517.r002

Author response to Decision Letter 0

19 Aug 2019

Reviewer #1

Comment #1

ans) Thank you for your kind advise.

Macmoondongtang has been used as oriental medicine in Korea and China and is still commercialized. Macmoondongtang was generally used to cure bronchitis, asthma, pneumonia, cough, and sputum production. The mechanism of anti-asthmatic activity of Macmoondongtang has not been reported. Therefore, we have demonstrated the anti-asthmatic effect of Macmoondongtangin animal experiments used commercial products (see Materials and methods). The effective dose of Macmoondongtang in animal studies was determined to be 1500 mg/kg. We calculated the dose of human based on the results of animal experiments. Human dose was included in the daily intake range of commercial products (see, discussion section). The important facts in this manuscript are that anti-asthmatic effect has been demonstrated within the stable dose range of Macmoondongtang, and the action mechanism has also been clearly identified.

Ans) Thank you so much for your comments and I agreed with your comments. I think that the manuscript title should be changed to “Macmoondongtang controls the asthmatic changes via modulation of Th1-/Th2-/Th17-related factors in an asthma murine model”.

ans) Thank you for your kind advise. The effective dose of glycyrrhizin was reported to be 2.5-20 mg/kg in the mouse model (Ram et al) and liquiritin has been reported to be efficacious at a minimum dose of 5 mg/kg in mouse model (Ram et al). We reported that in the discussion section that Macmoondongtang contained 0.38% glycyrrhizin. Glycyrrhizin in Macmoondongtang was calculated based on the content and correlated with animal experimental results. The dose of 1500 mg/kg Macmoondongtang is equivalent to 5.7 mg/kg glycyrrhizin. These results are correlated with previous reports (efficacy range 2.5 - 20 mg/kg). It can be interpreted that glycyrrhizin present in Macmoondongtang shows the main anti-asthmatic effect. Based on the content of liquiritin, the effective dose (5 mg/kg or more) is insufficient. Therefore, liquiritin was thought to be a cofactor for anti-asthmatic effect.

Comment #2

Ans) Thank you so much for your advices. This study was conducted twice time using with 70 heads, for one study 35 mice had been used and using with same method the other study was done. I added this method in the Materials and Methods section and used numbers of animals for each graph was described in the Figure Legends.

Comment #3

Is the manuscript presented in an intelligible fashion and written in standard English? No

Ans) Thank you so much for your advice. Although this manuscript had been edited by professional editing company (I attached the certificate file of editing) revised manuscript would be done by editing company again.

Some minor comments:

1)For material and methods, there were a lot of description to be improved, for example, which samples were used for RNA extraction? how the samples were prepared for IFA? and so on.

Ans) Thank you so much for your comments and I add the description about materials and methods in the Section.

2) What’s magnitude for those microscopic figures? And the quality of those microscopic figures should be also improved.

Ans) Thank you so much for your advice and I added the magnification in each photo. For improving the photos’ quality, I adjusted them again.

Reviewer #2

In this study, Lee et al. reported that macmoondongtang, a traditional prescription for treating respiratory disease, could modulate host immune responses during OVA-induced asthma in a mice model. They reported that macmoondongtang treatment could reduce serum IgE level, mucus hypersecretion, inflammatory cell infiltration in the lungs. They also reported that macmoondongtang treatment leads to reduced expression of Th1/Th2 transcriptional factors as well as related cytokines such as IFN-g, IL-12, IL-4 and IL-5, IL-13 on mRNA level or protein level. The data is interesting; however, the manuscript has severe limitations that the authors need to deal with before publications.

1. The written Englished should be revised significantly. There are many grammar mistakes in the manuscript.

2. The conclusions are overstated and need revision. For example,

a. it is not convincing that glycyrrhizin and liquiritin were the effective ingredients of macmoondongtang.

ans) Thanks for your kind advise. Macmoondongtang has been used as oriental medicine in Korea and China and is still commercialized. Macmoondongtang was generally used to cure bronchitis, asthma, pneumonia, cough, and sputum production. The mechanism of anti-asthmatic activity of Macmoondongtang has not been reported. Therefore, we have demonstrated the anti-asthmatic effect of Macmoondongtangin animal experiments used commercial products (see Materials and methods). The effective dose of Macmoondongtang in animal studies was determined to be 1500 mg/kg. We calculated the dose of human based on the results of animal experiments. Human dose was included in the daily intake range of commercial products (see, discussion section). The important facts in this manuscript are that anti-asthmatic effect has been demonstrated within the stable dose range of Macmoondongtang, and the action mechanism has also been clearly identified.

liquiritin and glycyrrhizin were designated as the main marker of Macmoondongtang and the test method for content analysis

(see. https://oasis.kiom.re.kr/oasis/pres/prdetailView2.jsp?idx=32&selectname=null&srch_menu_nix=null#view04) was defined in KFDA (Korea Food & Drug Administration). In this study, we confirmed that liquiritin and glycyrrhizin are markers for quality control and one of the effective markers.

b. There is no data supporting the title: “Macmoondongtang modulates asthma-related changes via modulation of TNF-a andTbet& IFN-g in an asthma murine model”. They showed correlation, nut not causality.

Ans) Thank you so much for your comments. We focused the localization of specific protein in the lung tissue such as in nucleus or in cytoplasm and I evaluated the localization of them based on the criteria that in dissimilar to general color when the different colors of light exist are summed-up they change the white color. Therefore, in the asthma-induction group we could evaluated that both of T-bet (green color) and GATA-3 (red color) existed in the nucleus (blue color) as in the nucleus the white spots were found compared to the other groups.

4. Methods are missing: RNA isolation, cDNA synthesis, qPCR methods, Elisa for IgE,

Ans) Thank you so much for your comments and I add the description about materials and methods in the Section.

5. Fig 1, please also include the control HPLC data for glycyrrhizin and liquiritin

Ans) Thank you for your kind advise. we modified the figure 1 according to reviewers’ recommendation.

6. For statistical analysis, how many times have these experiments been repeated? It says mean ± SEM in the figure legends but mean ± SD in the Materias and Methods, which one is correct?

Ans) Thank you so much for your comment and there were typos. I amended the expression as “mean ± SD”.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0224517.r003

Decision Letter 1

Michal A Olszewski

6 Sep 2019

PONE-D-19-17023R1

Macmoondongtang alleviates asthma via modulation of Th1-/Th2-/Th17-related cytokines in a murine model of asthma

PLOS ONE

Dear Professor Park,

While the manuscript has improved, both reviewers remain concerned about the over-interpretation of findings/speculative conclusions not fully supported by the data. In order to comply with PLOS publication policy the conclusions need to fully support by the data presented. Although, your data identified a link between the therapeutic effects of Macmoondongtang and certain group of cytokine responses, but they are insufficient to proof that it is the mechanism of drug action. Thus, the conclusions and title of the manuscript need to be adjusted as suggested by the Reviewer 2.

We would appreciate receiving your revised manuscript by Oct 21 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Michal A Olszewski, DVM, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Major comments:

After revise, the manuscript has been improved by the authors. But two major things still remain to be improved, as follows:

1, the writing must be improved. 1) introduction, special paragraph 2 and 3, is not so clear to this study; 2) discussion, which has been improved is not enough. Furthermore, some description is misleading, such as the synergistic role between glycyrrhizin and liquiritin unless there are some experimental finding available.

2, the microscropic figures are in a low quality. My previous comment has mentioned this problem. The authors' response was that it had been adjusted. But there are not clear, I don't know why.

Minor comments:

1, there are still some grammar mistakes to be checked.

2, ref #8, is not completed.

Reviewer #2: In this revised manuscript, the authors report that macmoondongtang could regulate host immune response in a murine model of asthma. Specifically, mice treated with macmoondongtang show decreased neutrophil count in BALF, reduced T-bet, IFN-g, and TNF-a, IL-12, IL-4, and IL-5 genes expression in the lung. While most of my previous concerns have been addressed, the reviewer still has some minor comments about several conclusions in the manuscript. The authors provide no data supports that modulation of Th1/Th2 cytokines is the mechanism of how Macmoondongtang alleviates asthma.

1. I would suggest change the title to “Macmoondongtang modulates Th1-/Th2-related cytokines and alleviates asthma in a murine model”.

2. I would suggest modify the conclusions to: “Macmoondongtang treatment alleviates asthma symptoms and modulate the Th1-/Th2- related cytokines. Glycyrrhizin and liquiritin could be the major the active therapeutic components”.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. 2019 Dec 2;14(12):e0224517. doi: 10.1371/journal.pone.0224517.r004

Author response to Decision Letter 1

17 Sep 2019

Reviewer #1: Major comments:

After revise, the manuscript has been improved by the authors. But two major things still remain to be improved, as follows:

Ans) Thank you so much for your comments and I amended several parts depended on your comments. As I agreed with your suggestions and omitted the sentence about the synergistic effects of glycyrrhizin and liquiritin.

2, the microscropic figures are in a low quality. My previous comment has mentioned this problem. The authors' response was that it had been adjusted. But there are not clear, I don't know why.

Ans) Thank you so much for your comments and all of figures were prepared as tiff files. Although all of them are made which met on the publication criteria during uploading the manuscript they might be unclearly changed. Please understand my situation.

Minor comments:

1, there are still some grammar mistakes to be checked.

Ans) The manuscript was edited by editing company and I attached the certificate of edit.

2, ref #8, is not completed.

Ans) Thank you so much for your comment and I amended the reference #8.

1. I would suggest change the title to “Macmoondongtang modulates Th1-/Th2-related cytokines and alleviates asthma in a murine model”.

Ans) Thank you so much for your suggestion and I amended the manuscript’s title.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0224517.r005

Decision Letter 2

Michal A Olszewski

8 Oct 2019

PONE-D-19-17023R2

Macmoondongtang modulates Th1-/Th2-related cytokines and alleviates asthma in a murine model

PLOS ONE

Dear Professor Park,

Thank you for submitting your manuscript to PLOS ONE. The manuscript has greatly improved and virtually all the issues have been resolved with one exception of "overlooked" conclusion adjustment as noted by the Reviewer 2. Therefore, we invite you to complete the final round of revisions and submit the revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Nov 22 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Michal A Olszewski, DVM, PhD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: My comment 2 is not addressed in the revised manuscript. Please change the conlusions on page 3.

I would suggest to modify the conclusions to: “Macmoondongtang treatment alleviates asthma symptoms and modulate the Th1-/Th2- related cytokines. Glycyrrhizin and liquiritin could be the major the active therapeutic components”.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. 2019 Dec 2;14(12):e0224517. doi: 10.1371/journal.pone.0224517.r006

Author response to Decision Letter 2

10 Oct 2019

Reviewer #2:

1. My comment 2 is not addressed in the revised manuscript. Please change the conlusions on page 3. I would suggest to modify the conclusions to: “Macmoondongtang treatment alleviates asthma symptoms and modulate the Th1-/Th2- related cytokines. Glycyrrhizin and liquiritin could be the major the active therapeutic components”.

Ans) Thank you so much for your suggestion and I amended the conclusion part in the abstract.

Attachment

Submitted filename: Response to Reviewers comment.docx

Click here for additional data file.^{(15.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0224517.r007

Decision Letter 3

Michal A Olszewski

16 Oct 2019

Macmoondongtang modulates Th1-/Th2-related cytokines and alleviates asthma in a murine model

PONE-D-19-17023R3

Dear Dr. Park,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Michal A Olszewski, DVM, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0224517.r008

Acceptance letter

Michal A Olszewski

14 Nov 2019

PONE-D-19-17023R3

Macmoondongtang modulates Th1-/Th2-related cytokines and alleviates asthma in a murine model

Dear Dr. Park:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Michal A Olszewski

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. This file is raw data for making graphs in Fig 2, Fig 3, Fig 6, Fig 7 and Fig 8.

(XLSX)

Click here for additional data file.^{(55.3KB, xlsx)}

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Attachment

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Macmoondongtang modulates Th1-/Th2-related cytokines and alleviates asthma in a murine model

Soon-Young Lee

Bossng Kang

So-Hyeon Bok

Seung Sik Cho

Dae-Hun Park

Roles

Abstract

Objective

Materials & methods

Results

Conclusions

Introduction

Materials and methods

Preparation of macmoondongtang

Identification of anti-asthmatic compounds in macmoondongtang

Animal experiments

Ethics statement

Bronchoalveolar fluid (BALF) and serum analysis

Histopathological analysis

Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunoassay (ELISA)

Table 1. Primer sequences for RT-PCR.

Immunofluorescent analysis

Immunohistochemical analysis

Statistical analysis

Results

Macmoondongtang contains two anti-asthmatic markers: Glycyrrhizin and liquiritin

Fig 1. HPLC results of macmoondongtang.

Macmoondongtang suppresses ovalbumin-induced hyperproliferation of inflammatory cells and immune cells

Fig 2. Macmoondongtang treatment suppressed infiltration of both inflammatory cells and immune cells such as white blood cells (WBCs), eosinophils, and neutrophils.

Macmoondongtang induces dose-dependent suppression of IgE

Fig 3. Macmoondongtang treatment effectively suppressed serum IgE level in a dose-dependent manner.

Macmoondongtang dose-dependently inhibits typical morphological changes in OVA-induced asthma model

Fig 4. Macmoondongtang dose-dependently inhibited typical morphological changes in the lungs of mice with OVA-induced asthma.

Macmoondongtang effectively induces inactivation of both Th1 and Th2 cell transcription factors

Fig 5. Macmoondongtang effectively induced the inactivation of both Th1 and Th2 cell transcription factors.

Macmoondongtang significantly suppresses both gene and protein expression of IFN-γ and IL-12

Fig 6. Treatment with macmoondongtang significantly down-regulated gene and protein expression of IFN-γand IL-12p40.

Macmoondongtang controls the expression of IL-4 and IL-5, but not IL-13, in a dose-dependent manner

Fig 7. Macmoondongtang dose-dependently suppressed Th2-related cytokines such as IL-4, IL-5, and IL-13.

Macmoondongtang modulates the expression of TNF-α, but not IL-6

Fig 8. Macmoondongtang treatment significantly inhibited TNF-α gene and protein expression, but not that of IL-6.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Michal A Olszewski

Roles

Author response to Decision Letter 0

Decision Letter 1

Michal A Olszewski

Roles

Author response to Decision Letter 1

Decision Letter 2

Michal A Olszewski

Roles

Author response to Decision Letter 2

Decision Letter 3

Michal A Olszewski

Roles

Acceptance letter

Michal A Olszewski

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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