DW2008S and its major constituents from Justicia procumbens exert anti‐asthmatic effect via multitargeting activity

Abstract Our previous study revealed that the ethanolic extract of Justicia procumbens ameliorates ovalbumin‐induced airway inflammation and airway hyper‐responsiveness in a mouse model of asthma. However, the mechanism of action of the extract remains unknown. In this study, we prepared DW2008S, an optimized and standardized powder extracted from J. procumbens using anhydrous ethanol, and investigated its anti‐asthmatic effect and mechanism of action. Our results showed that DW2008S contains two major ingredients, justicidin A (JA) and justicidin B (JB), which selectively inhibit T helper 2 (Th2) cell responses in concanavalin A‐activated spleen cells and polarized Th2 cells. Blockade of T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine‐based inhibition motif domains (TIGIT) using a neutralizing antibody also selectively inhibited Th2 cell responses. Furthermore, DW2008S regulated TIGIT expression in the mice and cultured cells. Additionally, DW2008S and JA antagonized human adenosine receptor A3 (A3 AR), which mediates mast cell‐dependent inflammation and bronchoconstriction. DW2008S and JB inhibited human phosphodiesterase 4 (PDE4), which is known to cause bronchoconstriction; however, the required concentrations were higher than those needed to affect TIGIT . These findings suggest that DW2008S can potentially ameliorate Th2‐driven airway inflammation and bronchoconstriction through negative regulation of TIGIT and blockade of A3 AR and PDE4 activities.

through degranulation of mast cells, causing mucus production and bronchoconstriction. [3][4][5][6] Thus, many studies have developed antibodies for the treatment or prevention of allergic asthma by targeting Th2 immune pathophysiological factors. [7][8][9][10] However, the clinical efficacies of these drugs are limited to alleviation of the condition.
Moreover, reduction in the steroid dose is usually required because of poor improvement in lung function in patients with severe asthma. 11,12 The limited efficacies of these drugs may be due to their partial inhibition of Th2 responses. Thus, agents that target key molecules that negatively regulate Th2 cell differentiation or total Th2 responses must be developed. T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibition motif domains (TIGIT) is expressed on natural killer cells, memory T cells, activated T cells and regulatory T cells (Tregs). Furthermore, it is considered a novel immune checkpoint molecule. 13 TIGIT negatively regulates T cell responses through the effect of cluster of differentiation (CD) 112 or CD155 on dendritic cells or through intrinsic inhibitory effects such as inhibition of T cell proliferation or suppression of cytokine production in CD4 + T cells. 14 Recently, TIGIT + Tregs have been shown to selectively suppress Th1 and Th17 responses but not Th2 responses. 15 Moreover, TIGIT improves Th2 responses and allergic disease through the activity of CD155, whereas blockade of TIGIT with a neutralizing anti-TIGIT antibody exerts a therapeutic effect on allergic airway inflammation in a murine model of asthma. 16 However, there is still no chemical agent or herbal medicine that can selectively regulate Th2-driven allergic inflammation via TIGIT regulation.
Some molecules that regulate bronchoconstriction, such as adenosine receptors (ARs) and phosphodiesterases (PDEs), are useful for alleviating asthma and Th2-driven airway inflammation. Adenosine levels increase in the lungs of patients with allergic asthma, which contributes to bronchoconstriction and mast cell degranulation. 17 A 2A AR agonists, as well as A 1 AR, A 2B AR, and A 3 AR antagonists, show antiasthmatic effects by reducing inflammation, bronchoconstriction and mucus secretion in animal models. 18 A 2B AR and A 3 AR have been evaluated as important targets in the development of drugs for asthma treatment because of safety issues with other ARs. 19,20 PDE3 and PDE4 are involved in respiratory diseases. It is reported that PDE3, PDE4 and mixed PDE3/4 inhibitors significantly suppress allergen-induced contractions in passively sensitized airways in humans. 21 Thus, roflumilast, a selective PDE4 inhibitor that reduces exacerbation frequency and significantly improves pulmonary function, is used to treat patients with severe chronic obstructive pulmonary disease (COPD). 22 In a previous study, we showed that an anhydrous ethanolic extract of Justicia procumbens (DW2008) protected against allergic asthma by reducing the expression of Th2-type cytokines and alleviating methacholine-induced AHR in a mouse model of ovalbumin (OVA)-induced asthma. 23 However, the mechanisms underlying these effects remain unknown. In this study, we prepared DW2008S powder, which has better solubility and homogeneity characteristics than DW2008 has, and investigated its anti-asthmatic effect and mechanisms of action.

| High-performance liquid chromatography
(HPLC) analysis HPLC analysis of DW2008S was performed as previously described 23 using an Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA). Data were collected and processed using OpenLAB chromatography software. Ultraviolet (UV) detection was performed at 256 nm, and the injection volume was 10 lL. Peaks detected on the chromatogram for DW2008S were identified by comparing their retention times and UV spectra to those of pure compounds.

| Cell culture and Th cell differentiation
Spleen cells were collected from BALB/c mice as previously described. 23 Viable spleen cells were plated at a density of 5 9 10 6 cells/mL and cotreated with 5 lg/mL concanavalin A and test drugs for 48 hours. The levels of cytokines in the culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA) kits (Koma Biotech, Seoul, Korea).
| 2681 incubation of the plates for 72 hours. Next, the cells were transferred into new wells without anti-CD3/CD28 antibody, and the medium was replaced with one containing neutralizing antibodies (no cytokines). After 48 hours of incubation, fresh anti-CD3/CD28 antibody was added to the cells and incubation was continued for 48 hours. For Treg polarization, 5 ng/mL recombinant mouse transforming growth factor-b1 (R&D Systems) was added to the culture for 96 hours. Six-week-old female BALB/c mice were acclimated to the experimental conditions for 7 days and randomly assigned to treatment groups. The mice were sensitized with an intraperitoneal injection of saline containing OVA/aluminium hydroxide and then challenged with aerosol OVA using nebulizers ( Figure 1A). On the indicated days, the mice were administered DW2008S, montelukast and dexamethasone for 1 hour prior to a challenge with aerosol OVA. Serum was collected and stored at À70°C until analysis. Serum levels of total IgE and OVA-specific IgE were determined using ELISA kits (Koma Biotech). Bronchoalveolar lavage fluid (BALF) was collected by lavaging the trachea twice with a total of 1 mL of 19 Hank's balanced salt solution. Cells were pelleted by centrifuging BALF at 400 g for 5 minutes. The cells were resuspended in 0.5 mL of phosphate-buffered saline, after which total cell count was determined using a haemocytometer.
For differential cell counting, 0.1 mL of the cell suspension was cytospun onto a microscopic slide at 800 g for 3 minutes. The cells were then stained using Diff-Quik â staining solution kit (Sysmex, Kobe, Japan). Images were obtained from random areas, and differential cell counts were performed for >200 total cells in 1-6 images.
The levels of IL-4, IL-5 and IL-13 in BALF were determined using ELISA kits (R&D Systems). Airway resistance to inhaled methacholine was measured using a flexiVent â system (SCIREQ, Montreal, QC, Canada). At 48 hours after the last challenge, mice were anaesthetized and their tracheas were exposed and intubated with cannulas. Methacholine chloride (Sigma-Aldrich) was dissolved in normal saline (1.56-25 mg/mL) and administered to the mice in increasing doses (0, 1.56, 3.12, 6.25, 12.5 and 25 mg/mL). Airway resistance was recorded after each dose was administered.
Mediastinal lymph nodes (MLNs) were isolated from normal and asthmatic mice as previously described 24 and crushed for isolation of MLN cells.  Transcript levels of target genes were normalized to those of b-actin and expressed as fold changes relative to the indicated controls.

| Target screening
All target screening tests were performed by Eurofins Panlabs, Inc.
The composition of DW2008S was determined by HPLC. Two major constituents, JA and JB, were identified with retention times of 50.9 and 47.3 minutes, respectively ( Figure S1). To determine the efficacy of DW2008S against allergic airway inflammation, a mouse model of allergic asthma was established ( Figure 1A).
In the OVA asthma group treated with DW2008S, a dosedependent reduction in methacholine-induced AHR was observed.
At a dose of 20 mg/kg, DW2008S reduced AHR to a level equivalent to that obtained with a saturation dose (10 mg/kg) of montelukast, a positive control that antagonizes the leukotriene receptor. Furthermore, DW2008S at doses of 50 and 100 mg/kg significantly inhibited AHR. It was observed that the extent of inhibition was higher than that caused by montelukast and equivalent to that caused by dexamethasone, a representative glucocorticosteroid ( Figure 1B).
The DW2008S-treated group showed lower infiltration of inflammatory cells and eosinophils into the lungs than the OVA asthma group did ( Figure 1C). Additionally, DW2008S reduced serum total IgE and OVA-specific IgE levels in a dose-dependent manner ( Figure 1D).  Figure 1E). Figure 1F shows that DW2008S reduced mucus-positive areas and the thickness of airway smooth muscles in the lung tissues of the animals.
The results showed that JA and JB individually reduced the infiltration of total inflammatory cells and eosinophils into the lungs (P < .05 in each case, Figure 1G). Additionally, they inhibited increase in airway epithelium thickness and mucus production in the lung tissues (Figure 1H). The results indicate that at doses >50 mg/kg, DW2008S shows an anti-asthmatic effect that is superior to that of montelukast in a mouse model of asthma. In addition, our findings confirm that JA and JB are active constituents in DW2008S.
To elucidate the mechanism underlying the inhibitory effects of

| DW2008S negatively regulates TIGIT expression
Previous studies have revealed that TIGIT + Treg subsets do not affect Th2 cytokine levels in mouse models of OVA-induced allergic asthma. 15 In addition, TIGIT stimulates Th2 cell differentiation through its interaction with CD155 to promote the development of allergic disease. 16 Therefore, we investigated whether TIGIT is To investigate whether DW2008S is involved in TIGIT regulation, spleen cells were cotreated with concanavalin A and DW2008S, JA, JB, or a neutralizing TIGIT antibody for 48 hours. TIGIT mRNA level was increased by concanavalin A but reduced by DW2008S (Figure 3D). DW2008S and its two major constituents significantly reduced the number of TIGIT + GATA3 + Th2 cells in concanavalin Aactivated ( Figure 3E) and in vitro polarized Th2 cell populations (Figure 3F). In contrast, DW2008S and its two major constituents did not reduce the number of TIGIT À GATA3 + Th2 cells that had been  Figure 4B).
In addition, we investigated BALF TIGIT + IL-4 + Th2 cells. The results showed that CD4 + lymphocytes were undetectable in the BALF samples of normal mice; however, they increased in number in the BALF samples of OVA asthmatic mice ( Figure 4C). In the latter group, TIGIT + cells formed~50% of the total cell number, indicating that their proportion was much higher than that of MLN cells.

| DW2008S selectively inhibits A 3 AR and PDE4
To determine whether DW2008S and its constituents affect other targets in addition to TIGIT, we performed screening assays of 85 targets related to allergic, respiratory and inflammatory diseases. The results showed that DW2008S inhibited A 3 AR and PDE4 activities (Table 1).
Additionally, 20 lg/mL DW2008S antagonized A 3 AR activity by 96% but did not antagonize other AR isoforms in the FLIPR calcium flux assay (  Figure 5A). JA and JB were also tested in these assays to determine whether they are associated with A 3 AR antagonism and These results suggest that DW2008S plays a critical role in protecting against airway allergic inflammation and bronchoconstriction through negative regulation of TIGIT and blockade of A 3 AR and PDE4 activities ( Figure 6).

| DISCUSSION
Recent studies have provided evidence that TIGIT is an important factor in Th2-driven responses and Th1/Th2/Th17 cell balance. 16,25 However, no regulators of TIGIT, except for a few specific antibod- with the results of a previous study, which showed increased TIGIT expression in the MLNs of mice with Th2-driven allergic asthma, 16 we observed that TIGIT expression was elevated in the MLNs of mice with allergic asthma. However, TIGIT expression was restored to its normal level by DW2008S. The increase in TIGIT expression and its attenuation by DW2008S in mice with allergic asthma appear to be limited to FOXP3 À effector T cells, which seems to mostly result from fluctuations in number of TIGIT + IL-4 + Th2 cells.
TIGIT + Th2 cells may substantially contribute to the secretion of Th2 cytokines. DW2008S, JA, JB and an anti-TIGIT neutralizing antibody reduced Th2 cytokine expression and the number of TIGIT + GATA3 + Th2 cells but not TIGIT À GATA3 + Th2 cells in the concanavalin A-activated spleen cells. In the in vitro Th2 cell polarization test, the anti-TIGIT neutralizing antibody also reduced the level of IL-5 without changing the number of TIGIT À GATA3 + Th2 cells. The results are in agreement with those of previous studies, which shows that sorted TIGIT + Th2 cells secrete higher amounts of Th2 cytokines than TIGIT À Th2 cells do. 16 Tregs are well known to protect against the progression of asthma. 26,27 However, Tregs are also associated with autoimmune diseases such as rheumatoid arthritis, which is reminiscent of Th1/Th17driven inflammation. 28

CONFLI CTS OF INTERES TS
The authors declare that they have no conflicts of interests.