Children and young adults have suffered from an extraordinary increase in allergic diseases and asthma worldwide (Pawankar, 2014). Chronic inflammatory responses in the airways are the main driving forces in asthma pathogenesis (Holgate, 2008). Thus, asthma is considered as a Th2-dominant allergic disorder by mast cells, eosinophils, cytokines, and IgE (Deo et al., 2010). Further, continuous, sustained environmental stimuli led to hyper-responsive airways (AHR), remodeling of airway walls, and hyperproduction of mucus through aberrant injury repair mechanisms. (Holgate, 2011). Currently, inhaled corticosteroids and oral leukotriene D₄ antagonists are very helpful to manage asthma symptoms in addition to long-acting β₂ agonists (O’Byrne et al., 2009). Investigation on aberrant injury-repair mechanism might provide us alternative therapeutic chances. A variety of environmental factors may induce oxidative stress, resulting in exacerbation of asthma conditions. Observed evidence suggests that asthma pathogenesis is influenced by oxidative stress and redox-regulated mechanisms (Hoshino et al., 2008). Furthermore, exogenously administered thioredoxin 1, a redox active protein, inhibited AHR and eosinophilia in ovalbumin (OVA)-sensitized and -challenged asthmatic mice (Ichiki et al., 2005). In human thioredoxin 1 transgenic mice, eosinophilic pulmonary inflammation and airway remodeling caused by chronic exposure of OVA was prevented (Imaoka et al., 2007). As an interesting aside, thioredoxin 1 targets apoptosis signal-regulating kinase 1 (ASK1), which is required for tumor necrosis factor (TNF)-α-induced apoptosis (Saitoh et al., 1998). It is normally bound and inhibited by the thiol-containing antioxidant thioredoxin 1, and oxidative stress modulates ASK1 binding through this site (Psenakova et al., 2020). Therefore, ASK1 is suggested as a signaling molecule in responses to oxidative stress (Psenakova et al., 2020). Furthermore, ASK1 phosphorylates MKK3, MKK4, MKK6 and MKK7 to activate MAP kinase p38 and c-Jun N-terminal kinase pathways (Pelaia et al., 2005). Because oxidative stress is involved in asthma pathogenesis, ASK1 gene deficiency was investigated in animal models. In ASK1 gene deficient mice, AHR was decreased in comparison to wild-type mice (Takada et al., 2013), suggesting involvement of oxidative stress and ASK1 in asthma pathogenesis. ASK1 gene-deficient mice also had lower bronchoalveolar lavage fluid (BALF) eosinophilia and mucus hyperproduction than wild-type mice when exposed to house dust mites (HDMs) (Maruoka et al., 2017). As of yet, no in vivo studies have been conducted on ASK1 inhibitors and allergic asthma. The current study examined whether selonsertib (formerly GS-4997), an oral ASK1 inhibitor that is highly selective and potent, suppressed OVA-induced allergic asthma, as selonsertib has clinically evident hemodynamic effects for nonalcoholic steatohepatitis (Loomba et al., 2018).

Materials

We purchased selonsertib from MedChemExpress (Cat no. HY-18938). Alum and OVA were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Culture of RBL-2H3 cells

US-based ATCC provided RBL-2H3 mast cells (Masassas, VA, USA). Cells were cultured as previously described (Lee et al., 2022).

BALB/c mice

The Daehan Biolink Corporation was located in Seoul, Korea, and provided female BALB/c mice that were five weeks old. The study protocol was approved by the Institutional Animal Care Committee of Kyung Hee University (KHSASP-23-011).

Degranulation assessment

Degranulation was determined by measuring β-hexosaminidase activity in RBL-2H3 cells. Human dinitrophenyl-albumin and mouse monoclonal immunoglobulin E for dinitrophenyl moiety were used to induce degranulation (Lee and Im, 2021b).

Immunocytochemistry of phospho-MAPKs

A total of 1x 105 RBL-2H3 cells per well were cultured on a slide with 8 wells (SPL, 12/CS 30408) for immunocytochemistry. An incubation for three hours with the DNP-IgE was followed by an overnight incubation at 37°C with the DNP-IgE. We treated cells with vehicle or selonsertib for 30 min, and then treated with human dinitrophenyl-albumin for 15 min. Methanol was used to fix the slides for 10 min at room temperature. PBS with 0.5% Tween-20 (PBS-T) was used for washing. PBS with 10% normal goat serum was used for blocking by incubating the sections for 30 min at room temperature. Phospho-p38 MAPK (Thr180, Tyr182) antibody (Invitrogen, Calsbard, CA, USA, 44-684G), phospho-JNK1/JNK2 (Thr183, Tyr185) antibody (Invitrogen, 44-682G), or phospho-ERK1/ERK2 (Thr202, Tyr204) antibody (Invitrogen, 36-8800) was used to label the sections at room temperature for 1 h following the blocking step. Biotinyl 2’ antibody (1:200, 5 μL/mL) in 1% serum-containing PBS-T was used to label the slides for 1 h at room temperature. In order to stain the samples, Vector Laboratories provided the kit (PK-6101, Burlingame, CA, USA). Incubation for 10 min with substrate (SK-4105, Vector Laboratories) enabled visualization of the peroxidase reaction product. Counterstaining with hematoxylin was performed on the slides. As a comparison between the total area and the brown stained area, ImageJ software (NIH, Bethesda, MD, USA) was used to calculate the brown stained area.

Asthma induction

We divided female BALB/c mice into the following groups (n=5): one control group received PBS, one asthmatic group received OVA, one group received selonsertib (30 mg/kg) before sensitization, and one group received selonsertib before challenge. OVA sensitization (D0 and 14) and challenge (D28, D29, and D30) were performed as previously described (Lee and Im, 2021a). A 30 min interval was allowed after the administration of selonsertib by intraperitoneal injection (D0 and D14) or the administration of selonsertib by intraperitoneal injection (D28, D29, and D30). We collected BALF on D32 (Lee and Im, 2021a).

Analysis of BALF cells

A Cellspin^® centrifuge (Hanil Electric, Seoul, Korea) was used to adhere immune cells from BALF to a glass slide. Fixed cells in MeOH were stained with May-Grünwald and Giemsa solution.

AHR measurement

Upon successful completion of the final OVA challenge, we used whole-body plethysmography (EMKA Technologies, Paris, France) to measure the enhanced pause (Penh) by increasing concentrations of methacholine (0-50 mg/ml). Penh is expressed as a percentage increase (Lee and Im, 2022).

Lung histology

A hematoxylin and eosin (H&E) stain was used to evaluate infiltration of cells within lung tissue and the presence of mucous-producing cells with periodic acid-Schiff (PAS) (Lee and Im, 2022). Based on the subjective score of 0-3, an observer who was not aware of the treatment measured the level of inflammation in the lungs. We counted the number of PAS-positive cells as a measurement of the mucous production (Lee and Im, 2022).

Immunohistochemistry of phospho-ASK1

A solution of 0.5% Triton X-100 in PBS was applied to lung tissue sections prior to immunohistochemical staining for phospho-ASK1 (Thr845) at room temperature for 30 min. Washing, blocking, labelling, visualization, and calculation were as same as the above-mentioned immunocytochemistry except the antibody for phospho-ASK1 (Thr845).

Measurement of serum IgE and BALF IL-13

The levels of IgE in serum and IL-13 in BALF were determined as described previously (Son et al., 2022).

Statistics

We used GraphPad Prism software (La Jolla, CA, USA) for the statistical analyses. AONVA followed by Tukey’s multiple comparison test for multiple groups was applied. SEM was used as data. When p values were below 0.05, statistical significance was accepted.

Selonsertib suppressed degranulation of RBL-2H3 mast cells

IgE-bound mast cells produce vesicles during asthma attacks that degranulate upon contact with antigens (Sibilano et al., 2014). As a result, many early-step mediators are released during degranulation, including histamine, leukotrienes D4, chemokines, cytokines, and neutral proteases (Warner and Kroegel, 1994). In RBL-2H3 mast cells exposed to HSA antigen, β-hexosaminidase activity was measured as an indicator of degranulation. Treatment of selonsertib inhibited the HSA-induced increase of β-hexosaminidase activity in the medium in a concentration-dependent manner (Fig. 1). When selonsertib was administered at either 30 or 50 μM levels, degranulation of mast cells was significantly inhibited (Fig. 1), which is in agreement with a previous report that GS-444217, another ASK1 inhibitor, suppressed IgE-mediated mast cell degranulation in both fetal liver and bone marrow mast cells (Rouillard, 2022).

Figure 1. Selonsertib inhibits phosphorylation of p38 MAPK, and JNK, but not ERK in RBL-2H3 cells. RBL-2H3 cells were cultured in 8-well slides. After sensitization with DNP-IgE for 18 h, RBL-2H3 cells were challenged with DNP-HSA. Selonsertib treatment was performed at the indicated concentrations 30 min before antigen challenge. The phosphorylation of p38 MAPK (Thr180, Tyr182), phosphorylation of JNK1/2 (Thr183, Tyr185), and phosphorylation of ERK1/2 (Thr202, Tyr204) was detected following fixation of the cells using specific antibodies. (A, C, E) Immunocytochemistry of RBL-2H3 cells. (B, D, F) Histograms were made by using ImageJ. Experiments were conducted in three times and 5 images per sample were analyzed. Values represent means ± the SEM (n=3). ***p<0.001 vs. the HSA-untreated group, ^#p<0.05, ^##p<0.01, ^###p<0.001 vs. the HSA-treated group.

Selonsertib suppressed activation of p38 MAPK and JNK but not ERK in RBL-2H3 mast cells

To confirm the ASK1 activation in RBL-2H3 mast cells, an immunocytochemistry was performed in RBL-2H3 cells. Phosphorylation of p38 MAPK was strongly detected in HSA-treated group as dark brown, and the degree of phospho-p38 MAPK staining was significantly suppressed by selonsertib treatment (Fig. 2A, 2B). As a result, the same method was employed to check whether JNK1/2 and ERK1/2 are activated. HSA treatment activated both MAPKs as shown in Fig. 2C-2F. It was found that selonsertib treatment inhibited JNK1/2 but not ERK1/2, which supports the previous finding that ASK1 signaling is involved (Pelaia et al., 2005).

Figure 2. Selonsertib reduces antigen-induced degranulation in RBL-2H3 mast cells. Dinitrophenyl-human serum albumin (DNP-HSA) was employed as a challenge after sensitization with anti-dinitrophenyl immunoglobulin E (DNP-IgE) for 18 h. A 30 min treatment with selonsertib was performed at the indicated concentrations before an antigen challenge was performed. Positive controls for antigen-induced degranulation show samples with IgE and HSA, and samples without IgE and HSA are seen for basal degranulation. The results are presented as means ± the standard error (SE) of three independent experiments. ***p<0.001 vs. the HSA-untreated group. ^##p<0.01, ^###p<0.001 vs. the HSA-treated group.

Selonsertib suppressed the AHR during OVA-induced asthma

Further investigation of selonsertib’s suppressive effects on mast cell degranulation in vivo was carried out in a BALB/c mouse model of allergic asthma induced by OVA. Intraperitoneal delivery of selonsertib (30 mg/kg) was performed 30 min before either OVA sensitization or challenge (Fig. 3A). To determine if selonsertib affected AHR, the Penh value was measured. A significant increase in Penh values was observed in OVA-sensitized mice at dosages compared to control mice indicating a successful asthma induction. A significant reduction in Penh values was achieved with both selonsertib treatments at dosages of 12.5 and 50 mg/mL methacholine in OVA-induced mice (Fig. 3B).

Figure 3. Effects of selonsertib treatment on airway hyper-responsiveness to methacholine in an OVA-induced murine asthma model. (A) Schematic drawing of experimental time course. (B) Airway hyper-responsiveness was measured as Penh (enhanced pause) in mice treated with selonsertib (30 mg/kg) or PBS after challenge with increasing concentrations of methacholine. PBS: PBS-treated mice, OVA: OVA-challenged mice, OVA+selonsertib sensitization: OVA-challenged mice treated selonsertib before sensitization, OVA+selonsertib challenge: OVA-challenged mice treated selonsertib before challenge. The results are presented as means ± the standard error of the mean (SEM) (n=5). *p<0.01 vs. the PBS-treated group, ^#p<0.05, ^##p<0.01 vs. the OVA-treated group.

Selonsertib suppressed the increase in eosinophil counts in the BALF

A collection of immune cells was made from the BALF on day 49 and cells were analyzed. Compared to PBS group, OVA group BALF consisted of more immune cells, mostly eosinophils and lymphocytes (Fig. 4A, 4B). A significant reduction in eosinophil and total cell numbers was shown in the group treated with selonsertib before antigen exposure, but not in a group treated with selonsertib before exposure to antigens (Fig. 4B). In contrast, macrophage counts were not significantly altered after OVA or selonsertib treatment, when eosinophil and lymphocyte counts were increased by OVA and decreased by selonsertib, respectively (Fig. 4A, 4B).

Figure 4. Selonsertib inhibits OVA-induced immune cell accumulation in BALF. (A) Mice were sensitized with OVA twice by i.p. injection on D0 and D14 and were subsequently challenged on D28, D29, and D30 with nebulized OVA. Selonsertib was administrated intraperitoneally at a dose of 30 mg/kg 30 min before the OVA sensitization or before the OVA challenge. The cells in the BALF were stained using May-Grünwald stain and counted. (B) Total cell counts, eosinophils, macrophages, and lymphocytes in the BALF. The results are presented as means ± the SEM cell count values (n=5). ***p<0.001 vs. the PBS-treated group, ^#p<0.05, ^##p<0.01, ^###p<0.001 vs. the OVA-treated group.

Selonsertib inhibited asthma-induced cytokine expression in the BALF

Due to the important role Th2 cells play in allergic asthma, the qPCR testing of BALF cells detected proinflammatory cytokines of type 2, IL-4, IL-5, and IL-13. As Th1 and Th17 cells also play a role in the chronic asthma stage, BALF cells were also analyzed for the mRNA levels of the Th1 cytokine IFN-γ and IL-17A in combination. As shown in Fig. 4, the mRNA levels of five cytokines in BALF immune cells were increased in the BALF cells of OVA group compared to PBS group. Fig. 5 shows that both selonsertib treatments suppressed the increase. In the OVA plus selonsertib before OVA challenge group (IL-4, IL-13, and IL-17A) as well as in the OVA plus selonsertib before OVA sensitization group (IL-13 and IL-17A), significant results were observed (Fig. 5).

Figure 5. Selonsertib treatment inhibits the mRNA expression of cytokines in BALF cells. Analysis of the mRNA expression of the Th2 cytokines IL-4, IL-5, and IL-13, the Th1 cytokine IFN-γ, and the Th17 cytokine IL-17A in the BALF cells. (A) IL-4, (B) IL-5, (C) IL-13, (D) IFN-γ, and (E) IL-17A. The relative mRNA levels of the cytokines were quantified relative to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The values are represented as means ± the SEM (n=5). *p<0.05, **p<0.01, ***p<0.001 vs. the PBS-treated group, ^#p<0.05, ^##p<0.01, ^###p<0.001 vs. the OVA-treated group.

Selonsertib suppressed morphologic changes and inflammation in the lungs

The effects of selonsertib on asthma histological changes were examined with H&E and PAS staining. Airway inflammation was observed in lung tissues from mice exposed to OVA (Fig. 6A). H&E staining of lung sections (Fig. 6A) revealed small, dark blue dots that were eosinophils. Compared to the PBS control group, the OVA group showed many eosinophils located in the peribronchial regions (Fig. 6A). On the other hand, airway inflammation decreased in mice treated with selonsertib before having OVA sensitized or challenged (Fig. 6A). An assessment of pulmonary inflammation was conducted on a subjective scale from 0 to 3. OVA-treated group had an average inflammation score of about 2.5, which was significantly reduced after selonsertib treatment (Fig. 6B).

Figure 6. Selonsertib protects against airway inflammation and mucin production. (A) H&E-stained sections of lung tissues from the PBS, OVA, and selonsertib (30 mg/kg)-treated OVA groups. The small navy-blue dots around the bronchioles are eosinophils. Eosinophils were rarely observed in the PBS group, whereas they accumulated extensively around the bronchioles in the OVA group (green arrows). (B) Periodic acid-Schiff (PAS)/hematoxylin-stained sections of lung tissues from the PBS, OVA, and selonsertib (30 mg/kg)-treated OVA groups. In PAS staining, mucin is stained purple. In the OVA group, a darker and thicker purple color was observed surrounding the bronchioles compared with that in the PBS group (red arrows). However, the eosinophil accumulation was less pronounced in the OVA+selonsertib group than in the OVA group. (C) Immunohistochemical staining of the lung tissue. Phospho-ASK1 (Thr845) antibody was applied on the lung tissue and phospho-ASK1 positive area appears to be brown. In the OVA group, a lot of brown areas appeared, as opposed to the PBS group. Selonsertib (30mg/kg)-treated OVA groups showed fewer brown area. (D) The lung inflammation was semi-quantitatively evaluated; histological findings were scored as described in the Materials and Methods section. (E) Mucous production was evaluated by counting the number of PAS-positive cells (red arrows) per mm of bronchioles (n=5 per group). (F) Histogram was made by using ImageJ. The IHC stained area, which appears to be brown, compared to the total tissue area was calculated and expressed as percentage. Phospho-ASK1 (Thr845)-positive staining was evaluated from 5 samples images per mouse. Values represent means ± the SEM (n=5). ***p<0.001 vs. the PBS-treated group, ^#p<0.05, ^###p<0.001 vs. the OVA-treated group.

In studies that used PAS staining to visualize lung samples, mucin hypersecretion was observed to be increased. In the OVA group (Fig. 6C), PAS-positive goblet cells were detected in dark violet spots surrounding the bronchioles, indicating hyperplasia of these cells and an increase in mucin production. There was a significant decrease in PAS-positive goblet cells around the bronchial airways with selonsertib treatment (Fig. 6C). We analyzed mucus production semi-quantitatively by counting PAS-positive bronchioles (Fig. 6B). A very small number of cells stained with PAS were found in the PBS-treated group. According to Fig. 6D, PAS-positive cells were found in approximately 110/mm in the OVA-treated group, and their numbers were significantly reduced by selonsertib treatment prior to sensitization or challenge.

IHC staining of lung tissue was conducted to confirm ASK1 activation in the lungs. In the PBS-treated group, phospho-ASK1 (Thr845) was rarely detected as brown color, while OVA-treated group strongly detected it as dark brown color (Fig. 6E). Using ImageJ (Fig. 6F), the phospho-ASK1 staining was calculated by dividing the brown color area by the total area. Fig. 6E and F show that both selonsertib treatments reduced phospho-ASK1 staining surrounding bronchial airways and peribronchial areas. This implys that ASK1 is activated in the lungs of OVA-induced allergic asthma model and selonsertib effectively inhibits the activation of ASK1.

Selonsertib inhibited asthma-induced cytokine expression in the lungs

Fig. 7 shows elevated levels of mRNA for these six cytokines in the lungs of the OVA group, and selonsertib treatment significantly suppressed the OVA-induced increases (Fig. 7A-7F). We also measured the level of expression of pro-inflammatory genes in the lungs, such as cyclooxygenase 2, induced nitric oxide synthase (iNOS), nuclear factor k-light-chain-enhancer of activated B cells (NF-κB), and GATA3, which is essential for the development of Th2 cells. Fig. 7G-7J illustrates elevated mRNA levels of the genes in the lungs of the OVA group, and selonsertib treatment both before OVA challenge and sensitization significantly suppressed the OVA-induced increase.

Figure 7. Selonsertib treatment inhibits the mRNA expression of cytokines in the lungs. Analysis of mRNA expression of the Th2 cytokines IL-4, IL-5, and IL-13, the Th1 cytokine IFN-γ, the Th17 cytokine IL-17A, IL-33, COX-2, iNOS, NF-κB, and GATA-3 in the lung tissues. (A) IL-4, (B) IL-5, (C) IL-13, (D) IFN-γ, (E) IL-17A, (F) IL-33, (G) COX-2, (H) iNOS, (I) NF-κB, and (J) GATA-3. The relative mRNA levels of the cytokines were quantified relative to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The values are represented as means ± the SEM (n=5). **p<0.01, ***p<0.001 vs. the PBS-treated group, ^#p<0.05, ^##p<0.01, ^###p<0.001 vs. the OVA-treated group.

Selonsertib suppressed OVA-induced increase of serum IgE titers and BALF IL-13 levels

Because the serum IgE levels are increased in asthma patients, the IgE levels were measured in the serum. Compared to the PBS group, the serum IgE levels in the OVA group were significantly higher. In contrast, the OVA+selonsertib group showed significantly lower serum IgE concentrations than the OVA only group (Fig. 8A). As shown in Fig. 8A, suppression was greater in the group treated with OVA+selonsertib before antigen challenge than in the group treated with OVA+selonsertib before antigen sensitization. In allergic asthma, Th2 cytokines play an important role by stimulating the recruitment of eosinophils, metaplasia, and hypersecretion of mucus. BALF protein levels of the Th2 cytokine IL-13 were measured using an ELISA method. The OVA-induced group had elevated IL-13 levels compared with the PBS-treated control group, and both selonsertib treatments reduced the elevation significantly (Fig. 8B).

Figure 8. Effect of selonsertib on IgE levels in serum and IL-13 levels in BALF. ELISA was used to measure the protein levels of IgE in serum (A) and IL-13 in the BALF (B). The results are presented as means ± the standard error of the mean (SEM) (n=5). **p<0.01, ***p<0.001 vs. the PBS-treated group, ^##p<0.01, ^###p<0.001 vs. the OVA-treated group.

We found that selonsertib, an ASK1 inhibitor, inhibited mast cell degranulation in vitro and OVA-induced allergic asthma in vivo. In fetal liver-derived mast cells as well as bone marrow-derived mast cells, another ASK1 inhibitor (GS-444217) suppressed mast cell degranulation, supporting selonsertib’s inhibitory effect (Rouillard, 2022). If ASK1 inhibitors suppress mast cell degranulation, asthmatic phenotypes are likely to be suppressed. Two previous in vivo studies using mice lacking the ASK1 gene support the inhibitory effects of selonsertib in the current study (Takada et al., 2013; Maruoka et al., 2017). ASK1 gene deficient mice showed reduced immune cell infiltration into airways, lower IgE levels, and lower AHR than wild-type mice in the same OVA-induced model (Takada et al., 2013), which is consistent with the present study. Further, BALF from mice lacking the ASK1 gene was moderately lower than the BALF from controls, but not for IL-4 and IFN-γ (Takada et al., 2013). As opposed to the results observed in mice carrying ASK1 gene deficient mice, we observed the decrease of IL-4 and IFN-γ along with IL-5 and IL-13. There is no explanation for why IL-4 and IFN-γ levels were reduced in mice treated with selonsertib, but not in mice deficient in ASK1. Possibly, the difference arises from the difference between mouse species and animal protocols, namely, C57BL/6 mice and BALB/c mice, and six-time versus two-time sensitizations (D0 and D14). ASK1 gene deficiency may cause compensatory adaptations that lead to a difference in immunity in mice with the ASK1 gene deficiency. ASK1 gene deficient mice had lower levels of eosinophil numbers in the BALF following exposure to house dust mites (HDM) and inflammatory cells infiltrating the lung than mice with the wild-type ASK1 gene (Maruoka et al., 2017). Although ASK1 gene deficient mice produced less mucus than wild-type mice, there were no significant differences between their lung tissues with regard to AHR and IL-4, IL-5, IL-13, and IFN-γ levels (Maruoka et al., 2017), which is quite contrasting to the results of the present study. We differ from Maruoka et al. (2017) in several aspects; for example, we used OVA rather than HDM as antigens, and we used BALB/c mice instead of C57BL/6 mice, as well as a different asthma protocol (intraperitoneal sensitization and aerial challenge versus intranasal sensitization and challenge). Compensatory adaptation to ASK1 gene deficiency during development period may change the inflammatory responses in the ASK1 gene deficient mice. Because selonsertib was applied very shortly before OVA sensitization or challenge, the results of the present study may reflect the real immune responses caused by temporal suppression of ASK1 activity.

Asthma involves many different types of cells, including dendritic cells, T and B lymphocytes, eosinophils, mast cells, macrophages, goblet cells, and smooth muscle cells. There have been studies on the functionality of ASK1 in several cell types, including dendritic cells, macrophages, mast cells, and smooth muscle cells. ASK1-deficient bone marrow-derived dendritic cells were significantly less sensitive to LPS-induced activation of p38 MAPK and subsequent release of IL-6, IL-1, and TNF-α (Matsuzawa et al., 2005). ASK1 and p38 MAPK were also involved in pro-inflammatory cytokine expression in RAW 264.7 macrophages after LPS treatment (Sujitha et al., 2018). Mast cells secreted less IL-13, IL-6, and CCL1 proteins after ASK1 inhibitor treatment (Rouillard, 2022). Inhibition of ASK1 reduced smooth muscle cell proliferation and migration in the airway wall induced by mitogens (Eapen et al., 2018). It is therefore likely that ASK1 can promote asthma pathogenesis by inducing allergic responses in those cells. According to our findings with selonsertib, inhibition of ASK1 in those cells may account for the current results. Particularly, we observed similar suppression of allergic asthma by both administration of selonsertib before sensitization and before challenge. During sensitization and challenge, ASK1 inhibition affects both dendritic cells and mast cells, contributing to suppression.

Based on functional roles of ASK1 in asthma pathogenesis and the in vivo suppressive efficacy of selonsertib, the present study suggests that selonsertib could be used therapeutically for allergic asthma in addition to a recent report demonstrating therapeutic efficacy of bleomycin-induced pulmonary fibrosis (Valenca et al., 2022).

This research was supported by the Basic Science Research Program of the Korean National Research Foundation funded by the Korean Ministry of Science, ICT, and Future Planning (NRF-2023R1A2C2002380).

The authors declare that there is no conflict of interest.

SY Han and DS Im: Designed the experiments; SY Han: Performed the experiments and analyzed the data; DS Im: Wrote the manuscript.