P2Y12 antagonist attenuates eosinophilic inflammation and airway hyperresponsiveness in a mouse model of asthma

Leukotriene E4 (LTE4) that plays a key role in airway inflammation is expressed on platelets and eosinophils. We investigated whether blocking of the P2Y12 receptor can suppress eosinophilic inflammation in a mouse model of asthma because platelets and eosinophils share this receptor to be activated. BALB/c mice were sensitized by intraperitoneal injection of ovalbumin (OVA), followed by OVA nebulization. On each challenge day, clopidogrel, a P2Y12 antagonist was administered 30 min. before each challenge. Forty‐eight hours after the last OVA challenge, mice were assessed for airway hyperresponsiveness (AHR), cell composition and cytokine levels, including chemokine ligand 5 (CCL5), in bronchoalveolar lavage (BAL) fluid. EOL cells were treated with LTE4, with or without clopidogrel treatment, and intracellular and extracellular eosinophil cationic protein (ECP) expressions were measured to find the inhibiting function of P2Y12 antagonist on eosinophilic activation. The levels of P2Y12 expression were increased markedly in the lung homogenates of OVA‐sensitized and ‐challenged mice after platelet depletion. Administration of clopidogrel decreased AHR and the number of airway inflammatory cells, including eosinophils, in BAL fluid following OVA challenge. These results were associated with decreased levels of Th2 cytokines and CCL5. Histological examination showed that inflammatory cells as well as mucus‐containing goblet cells were reduced in clopidogrel‐administered mice compared to vehicle‐treated mice. Clopidogrel inhibited extracellular ECP secretion after LTE4 stimulation in EOL‐1 cells. Clopidogrel could prevent development of AHR and airway inflammation in a mouse model of asthma. P2Y12 can be a novel therapeutic target to the suppression of eosinophils in asthma.     

TLR-7 agonist attenuates airway reactivity and inflammation through Nrf2-mediated antioxidant protection in a murine model of allergic asthma

Toll-like receptors (TLRs) through innate immune system recognize pathogen associated molecular patterns and play an important role in host defense against bacteria, fungi and viruses. TLR-7 is responsible for sensing single stranded nucleic acids of viruses but its activation has been shown to be protective in mouse models of asthma. The NADPH oxidase (NOX) enzymes family mainly produces reactive oxygen species (ROS) in the lung and is involved in regulation of airway inflammation in response to TLRs activation. However, NOX-4 mediated signaling in response to TLR-7 activation in a mouse model of allergic asthma has not been explored previously. Therefore, this study investigated the role TLR-7 activation and downstream oxidant–antioxidant signaling in a murine model of asthma. Mice were sensitized with ovalbumin (OVA) intraperitoneally and treated with TLR-7 agonist, resiquimod (RSQ) intranasally before each OVA challenge from days 14 to 16. Mice were then assessed for airway reactivity, inflammation, and NOX-4 and nuclear factor E2-related factor 2 (Nrf2) related signaling [inducible nitric oxide synthase (iNOS), nitrotyrosine, lipid peroxides and copper/zinc superoxide dismutase (Cu/Zn SOD)]. Treatment with RSQ reduced allergen induced airway reactivity and inflammation. This was paralleled by a decrease in ROS which was due to induction of Nrf2 and Cu/Zn SOD in RSQ treated group. Inhibition of MyD88 reversed RSQ-mediated protective effects on airway reactivity/inflammation due to reduction in Nrf2 signaling. SOD inhibition produced effects similar to MyD88 inhibition. The current study suggests that TLR-7 agonist is beneficial and may be developed into a therapeutic option in allergic asthma.     

Ethyl gallate attenuates acute lung injury through Nrf2 signaling

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is the clinical syndrome of persistent lung inflammation caused by various direct and indirect stimuli. Despite advances in the understanding of disease pathogenesis, few therapeutic have emerged for ALI/ARDS. Thus, in the present study we evaluated the therapeutic potential of ethyl gallate (EG), a plant flavanoid in the context of ALI using in vivo (BALB/c) and in vitro models (human monocytes). Our in vivo data supports the view that EG alleviates inflammatory condition in ALI as significant reduction in BALF neutrophils, ROS, proinflammatory cytokines and albumin levels were observed with the single i.p of EG post LPS exposure. Also, histochemical analysis of mice lung tissue demonstrated that EG restored LPS stimulated cellular influx inside the lung airspaces. Unraveling the mechanism of action, our RT-PCR and western blot analysis suggest that enhanced expression of HO-1 underlies the protective effect of EG on ROS level in mice lung tissue. Induction of HO-1 in turn appears to be mediated by Nrf2 nuclear translocation and consequent activation and ablation of Nrf2 activity through siRNA notably abrogated the EG induced protective effect in LPS induced human monocytes. Furthermore, our results indicate that EG generated moderate amounts of H₂O₂ could induce Nrf2 translocation in the in vitro systems. However, given the insignificant amount of H₂O₂ recorded in the injected material in the in vivo system, additional mechanism for EG action could not be excluded. Nevertheless our results highlight the protective role of EG in ALI and provide the novel insight into its usefulness as a therapeutic tool for the treatment of ALI.     

4-Octyl itaconate regulates immune balance by activating Nrf2 and negatively regulating PD-L1 in a mouse model of sepsis

Introduction: Sepsis is a major global health challenge with high mortality rates and no effective treatment. Recent studies have suggested that sepsis may be associated with immune system dysfunction. Itaconate may exert anti-inflammatory effects via Nrf2 signaling. Although Nrf2 regulates oxidative/exogenous stress responses and inhibits inflammatory responses, the mechanism via which Nrf2 regulates immune checkpoints in sepsis remains unclear.Objectives: This study aimed to investigate the role of the Nrf2 signaling pathway in sepsis immunosuppression injury by exploring Nrf2 target genes in inflammatory macrophages in a mouse model of sepsis.Methods: We evaluated the effects of 4-octyl itaconate (OI) on pro-inflammatory and anti-inflammatory cytokines in a mouse model of sepsis and RAW264.7 cells. In addition, we investigated if OI could inhibit LPS-induced oxidative stress by activating Nrf2 signaling in vitro and in vivo.Results: OI reduced the release of pro-inflammatory cytokines and increased the release of anti-inflammatory cytokines, thereby inhibiting inflammation. OI increased glutathione synthase (GSS) expression by activating the Nrf2 signaling pathway to promote GSH synthesis, thus, inhibiting oxidative stress. OI inhibited the early release of inflammatory and oxidative stress-related factors to reduce tissue and organ injury in mice with sepsis, while Nrf2 interfered with PD-L1 induction and inhibited PD-L1 expression at an advanced stage to reduce the occurrence of sepsis immunosuppression.Conclusions: This study indicates that Nrf2 is a novel negative regulator of PD-L1 that functions at immune checkpoints and suggests an underlying mechanism for the anti-inflammatory process mediated by Nrf2.     

Rapamycin attenuates the paraquat-induced pulmonary fibrosis through activating Nrf2 pathway

Oxidative stress is a key regulator of idiopathic pulmonary fibrosis. Paraquat (PQ)-induced pulmonary fibrosis seriously endangers people's health. Rapamycin has been reported to alleviate PQ-induced pulmonary fibrosis, but its underlying mechanism is unclear. The nuclear factor E2-related factor 2 (Nrf2) plays an important regulatory role in the antioxidant therapy of PQ-induced pulmonary fibrosis. In this study, we tried to confirm that rapamycin attenuates PQ-induced pulmonary fibrosis by regulating Nrf2 pathway. In vivo, we proved that rapamycin could inhibit the degree of PQ-induced oxidant stress as well as enhanced the expression of Nrf2. In vitro, rapamycin decreased the upregulated effects of cell death and apoptosis, fibrosis-related factors expression and fibroblast-to-myofibroblast transformation by PQ treatment. In vivo, rapamycin treatment reduced fibrosis degree and the expression of fibrosis-related factors in lung tissues of rat treated PQ. Furthermore, we also found that Nrf2 knockdown reduced the inhibitory effect of rapamycin on PQ-induced pulmonary fibrosis, as well as decreased Nrf2 transfer from the cytoplasm into the nucleus. Our findings demonstrated that the protective effect of rapamycin is associated with the activation of the Nrf2 pathway in pulmonary fibrosis induced by PQ poisoning.     

Resolvin E1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma

Resolvin E1 (RvE1; 5S, 12R, 18R-trihydroxyeicosapentaenoic acid) is an anti-inflammatory lipid mediator derived from the omega-3 fatty acid eicosapentaenoic acid (EPA). It has been recently shown that RvE1 is involved in the resolution of inflammation. However, it is not known whether RvE1 is involved in the resolution of asthmatic inflammation. To investigate the anti-inflammatory effect of RvE1 in asthma, a murine model of asthma was studied. After RvE1 was administered to mice intraperitoneally, there were decreases in: airway eosinophil and lymphocyte recruitment, specific Th2 cytokine, IL-13, ovalbumin-specific IgE, and airway hyperresponsiveness (AHR) to inhaled methacholine. Moreover, RvE1-treated mice had significantly lower mucus scores compared to vehicle-treated mice based on the number of goblet cells stained with periodic acid-schiff (PAS). These findings provide evidence that RvE1 is a pivotal counterregulatory signal in allergic inflammation and offer novel multi-pronged therapeutic approaches for human asthma.     

Downregulation of leukotriene biosynthesis by thymoquinone attenuates airway inflammation in a mouse model of allergic asthma

Chronic airway inflammation is a key feature of bronchial asthma. Leukotrienes are potent inflammatory mediators that play a role in the pathophysiology of asthma, and their levels are elevated in the airways in response to allergen challenge. We examined the anti-inflammatory effect of thymoquinone (TQ), the active principle in the volatile oil of Nigella sativa seeds, on leukotriene (LT) biosynthesis in a mouse model of allergic asthma. Mice sensitized and challenged with ovalbumin (OVA) antigen had an increased amounts of leukotriene B4 and C4, Th2 cytokines, and eosinophils in bronchoalveolar lavage (BAL) fluid. In addition, there was also a marked increase in lung tissue eosinophilia and goblet cell numbers. Administration of TQ before OVA challenge inhibited 5-lipoxygenase, the main enzyme in leukotriene biosynthesis, expression by lung cells and significantly reduced the levels of LTB4 and LTC4. This was accompanied by a marked decrease in Th2 cytokines and BAL fluid and lung tissue eosinophilia, all of which are characteristics of airway inflammation. These results demonstrate the anti-inflammatory effect of TQ in experimental asthma.     

Herbal decoction Divya-Swasari-Kwath attenuates airway inflammation and remodeling through Nrf-2 mediated antioxidant lung defence in mouse model of allergic asthma

Background and purposeAsthma is a chronic respiratory disease orchestrated by immune and structural cells. Identification of novel therapeutic strategies are needed for asthma due to the limitations of existing therapies. We have validated the anti-inflammatory, anti-asthmatic and immunomodulatory therapeutic properties of herbal decoction, Divya-Swasari-Kwath (DSK) using mouse model of ovalbumin (OVA) induced allergic asthma.Methods and resultsHPLC analysis identified the presence of Rutin, Glycyrrchzin, Gallic acid, Cinnamic acid, Chlorogenic acid, Caffeic acid and Piperine as bioactive herbal metabolites in DSK. Therapeutic treatment with herbal decoction DSK significantly alleviated the pathological features of allergic asthma including inflammatory cell accumulation in Broncho-Alveolar Lavage (BAL) fluids, specifically lymphocytes and eosinophils, lung inflammation, oxidative stress, airway remodelling, and pro-inflammatory cytokine levels. H&E analysis of lung tissue sections identified attenuated inflammatory cell infiltration and thickening of bronchial epithelium by DSK. PAS staining and MT staining identified decrease in OVA-induced mucus hyper secretion and peri-bronchial collagen deposition respectively, upon DSK treatment. Treatment with DSK increased the mRNA expression of antioxidative defence gene Nrf-2 and its downstream target genes HO-1 and NQO-1. In the same line, biochemical analysis for the markers of oxidative/antioxidant system confirmed the restoration of activity of Catalase, GPx, SOD and EPO and the levels of GSH, GSSG, MDA and Nitrite in whole lungs. In line with PAS staining, DSK treatment decreased the OVA-induced expression of Muc5AC and Muc5B genes. DSK treatment reduced the steady state mRNA expression levels of IL-6, IL-1β, TNF-α, IL-4, -5, -33, IFN-γ in whole lung; and IL-6, TNF-α and IL-1β protein levels in BALF.ConclusionCollectively, our results suggest that herbal decoction DSK is effective in protecting against allergic airway inflammation and remodelling by regulating anti-oxidant mechanisms. We postulate that DSK could be the potential therapeutic option for allergic asthma management.     

Seven in Absentia Homolog 2 (Siah2) Protein Is a Regulator of NF-E2-related Factor 2 (Nrf2)

Under pathological conditions such as ischemia-reperfusion, Nrf2 acts as a key regulator of cellular oxidative response. Provided that Nrf2 is sensitive to hypoxia during ischemia, Nrf2 may affect reactive oxygen species metabolism during reoxygenation. In this study, hypoxia suppressed Nrf2 protein, and its hypoxic suppression was not recovered with knockdown of the Nrf2 repressor Keap1. Moreover, an Nrf2 mutant lacking the Keap1 binding domain was suppressed under hypoxia, suggesting that Keap1 does not contribute to hypoxic Nrf2 suppression. HIF-1α and Siah2 are both key regulators of hypoxic responses. Hypoxia induced the Siah2 protein. Although inhibition or knockdown of Siah2 prevented the suppression of Nrf2, knockdown of HIF-1α did not. Moreover, Siah2 interacted with Nrf2 through a binding motif, suggesting that Siah2 contributes to the suppression of Nrf2. Some cytosolic kinases also play important roles in Nrf2 regulation. In this study, PKC phosphorylates serine residues of Nrf2 during hypoxia. Knockdown of Siah2 rescued hypoxic decreases in an Nrf2 mutant that mimicked phosphorylation at serine 40 or lacked this phosphorylation site, suggesting that Siah2 contributes to the degradation of Nrf2 irrespective of its phosphorylation status. Moreover, knockdown of Siah2 attenuated ubiquitination of the Nrf2 mutant, suggesting that association of Siah2 with Nrf2 causes proteasome-mediated degradation of Nrf2.     

Azithromycin attenuates airway inflammation in a mouse model of viral bronchiolitis

Background

Viral bronchiolitis is the leading cause of hospitalization in young infants. It is associated with the development of childhood asthma and contributes to morbidity and mortality in the elderly. Currently no therapies effectively attenuate inflammation during the acute viral infection, or prevent the risk of post-viral asthma. We hypothesized that early treatment of a paramyxoviral bronchiolitis with azithromycin would attenuate acute and chronic airway inflammation.

Methods

Mice were inoculated with parainfluenza type 1, Sendai Virus (SeV), and treated daily with PBS or azithromycin for 7 days post-inoculation. On day 8 and 21 we assessed airway inflammation in lung tissue, and quantified immune cells and inflammatory mediators in bronchoalveolar lavage (BAL).

Results

Compared to treatment with PBS, azithromycin significantly attenuated post-viral weight loss. During the peak of acute inflammation (day 8), azithromycin decreased total leukocyte accumulation in the lung tissue and BAL, with the largest fold-reduction in BAL neutrophils. This decreased inflammation was independent of changes in viral load. Azithromycin significantly attenuated the concentration of BAL inflammatory mediators and enhanced resolution of chronic airway inflammation evident by decreased BAL inflammatory mediators on day 21.

Conclusions

In this mouse model of paramyxoviral bronchiolitis, azithromycin attenuated acute and chronic airway inflammation. These findings demonstrate anti-inflammatory effects of azithromycin that are not related to anti-viral activity. Our findings support the rationale for future prospective randomized clinical trials that will evaluate the effects of macrolides on acute viral bronchiolitis and their long-term consequences.     

Tryptase inhibition blocks airway inflammation in a mouse asthma model

Release of human lung mast cell tryptase may be important in the pathophysiology of asthma. We examined the effect of the reversible, nonelectrophilic tryptase inhibitor MOL 6131 on airway inflammation and hyper-reactivity in a murine model of asthma. MOL 6131 is a potent selective nonpeptide inhibitor of human lung mast cell tryptase based upon a beta-strand template (K(i) = 45 nM) that does not inhibit trypsin (K(i) = 1,061 nM), thrombin (K(i) = 23, 640 nM), or other serine proteases. BALB/c mice after i.p. OVA sensitization (day 0) were challenged intratracheally with OVA on days 8, 15, 18, and 21. MOL 6131, administered days 18-21, blocked the airway inflammatory response to OVA assessed 24 h after the last OVA challenge on day 22; intranasal delivery (10 mg/kg) had a greater anti-inflammatory effect than oral delivery (10 or 25 mg/kg) of MOL 6131. MOL 6131 reduced total cells and eosinophils in bronchoalveolar lavage fluid, airway tissue eosinophilia, goblet cell hyperplasia, mucus secretion, and peribronchial edema and also inhibited the release of IL-4 and IL-13 in bronchoalveolar lavage fluid. However, tryptase inhibition did not alter airway hyper-reactivity to methacholine in vivo. These results support tryptase as a therapeutic target in asthma and indicate that selective tryptase inhibitors can reduce allergic airway inflammation.     

Cholinergic anti-inflammatory pathway confers airway protection against oxidative damage and attenuates inflammation in an allergic asthma model

Asthma is characterized by the influx of inflammatory cells, especially of eosinophils as well as reactive oxygen species (ROS) production, driven by the release of the T helper 2 (Th2)-cell-associated cytokines. The cholinergic anti-inflammatory pathway (CAP) inhibit cytokines production and controls inflammation. Thus, we investigated the effects of pharmacological activation of CAP by neostigmine on oxidative stress and airway inflammation in an allergic asthma model. After the OVA challenge, mice were treated with neostigmine. We showed that CAP activation by neostigmine reduced the levels of pro-inflammatory cytokines (IL-4, IL-5, IL-13, IL-1β, and TNF-α), which resulted in a decrease of eosinophils influx. Furthermore, neostigmine also conferred airway protection against oxidative stress, attenuating ROS production through the increase of antioxidant defense, evidenced by the catalase (CAT) activity. We propose, for the first time, that pharmacological activation of the CAP can lead to new possibilities in the therapeutic management of allergic asthma.     

Apoptosis and airway inflammation in asthma

Asthma is a disease characterized by a chronic inflammation of the airways and by structural alterations of bron-chial tissues, often referred to as airway remodelling. The development of chronic airway inflammation in asthma depends upon the continuous recruitment of inflammatory cells from the bloodstream towards the bronchial mucosa and by their subsequent activation. It is however increasingly accepted that mechanisms involved in the regulation of the survival and apoptosis of inflammatory cells may play a central role in the persistent inflammatory process characterizing this disease. Increased cellular recruitment and activation, enhanced cell survival and cell:cell interactions are therefore the key steps in the development of chronic airway inflammation in asthma, and represent the major causes for tissue damge, repair and remodelling.     

Paeonol attenuates airway inflammation and hyperresponsiveness in a murine model of ovalbumin-induced asthma

Paeonol, the main active component isolated from Moutan Cortex, possesses extensive pharmacological activities such as anti-inflammatory, anti-allergic, and immunoregulatory effects. In the present study, we examined the effects of paeonol on airway inflammation and hyperresponsiveness in a mouse model of allergic asthma. BALB/c mice sensitized and challenged with ovalbumin were administered paeonol intragastrically at a dose of 100?mg/kg daily. Paeonol significantly suppressed ovalbumin-induced airway hyperresponsiveness to acetylcholine chloride. Paeonol administration significantly inhibited the total inflammatory cell and eosinophil count in bronchoalveolar lavage fluid. Treatment with paeonol significantly enhanced IFN-γ levels and decreased interleukin-4 and interleukin-13 levels in bronchoalveolar lavage fluid and total immunoglobulin E levels in serum. Histological examination of lung tissue demonstrated that paeonol significantly attenuated allergen-induced lung eosinophilic inflammation and mucus-producing goblet cells in the airway. These data suggest that paeonol exhibits anti-inflammatory activity in allergic mice and may possess new therapeutic potential for the treatment of allergic bronchial asthma.     

Fibrinogen is a co-antioxidant that supplements the vitamin E analog trolox in a model system

Objective: It appears that the atherosclerotic plaque is a prooxidant environment where some molecules that are normally antioxidants, including vitamins C and E, may act as prooxidants that contribute to atherosclerosis by oxidizing LDL. Some molecules can act as co-antioxidants to eliminate this prooxidant effect by recycling or other mechanisms of supplementation. Fibrinogen and other acute phase proteins found in the plaque are antioxidants. We hypothesized that fibrinogen can act as a co-antioxidant to supplement vitamin E thereby eliminating its oxidative effect under prooxidant conditions. We tested a model system for this hypothesis using the vitamin E analogue Trolox in a cell free system.

Methods: LDL was oxidized using 5 umol/l copper. Antioxidant conditions were achieved by adding the antioxidants immediately with LDL, while prooxidant conditions were created by adding antioxidants after a 40 min delay. Oxidation was monitored as the lag phase at 234 nm.

Results: Under antioxidant conditions, the protective effect of fibrinogen and Trolox combined together were about equal to the sum of the anitioxidant effects of each alone (additive), while under prooxidant conditions the combined protection was 54-200% greater (synergistic). These effects were different than those of vitamin C with Trolox in that under antioxidant conditions fibrinogen and Trolox were additive while vitamin C and Trolox showed strong synergistic effects, and in that unlike vitamin C and Trolox fibrinogen showed no prooxidant tendencies under prooxidant reaction conditions.

Conclusions: The data indicated that fibrinogen did act as a co-antioxidant to supplement Trolox and eliminate its prooxidant effect, most probably, by directly quenching the phenoxyl radical, because unlike vitamin C, fibrinogen did not appear to recycle vitamin E. But fibrinogen may act as a universal antioxidant, since unlike Trolox and vitamin C, it showed little tendency toward becoming a prooxidant.     

A broad-spectrum caspase inhibitor attenuates allergic airway inflammation in murine asthma model

Asthma is characterized by acute and chronic airway inflammation, and the severity of the airway hyperreactivity correlates with the degree of inflammation. Many of the features of lung inflammation observed in human asthma are reproduced in OVA-sensitized/challenged mice. T lymphocytes, particularly Th2 cells, are critically involved in the genesis of the allergic response to inhaled Ag. In addition to antiapoptotic effects, broad-spectrum caspase inhibitors inhibit T cell activation in vitro. We investigated the effect of the broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), on airway inflammation in OVA-sensitized/challenged mice. OVA-sensitized mice treated with z-VAD-fmk immediately before allergen challenge showed marked reduction in inflammatory cell infiltration in the airways and pulmonary blood vessels, mucus production, and Th2 cytokine production. We hypothesized that the caspase inhibitor prevented T cell activation, resulting in the reduction of cytokine production and eosinophil infiltration. Treatment with z-VAD-fmk in vivo prevented subsequent T cell activation ex vivo. We propose that caspase inhibitors may offer a novel therapeutic approach to T cell-dependent inflammatory airway diseases.     

Inhalation of sphingosine kinase inhibitor attenuates airway inflammation in asthmatic mouse model

Sphingosine 1-phosphate (S1P) produced by sphingosine kinase (SPHK) is implicated in acute immunoresponses, however, mechanisms of SPHK/S1P signaling in the pathogenesis of bronchial asthma are poorly understood. In this study, we hypothesized that SPHK inhibition could ameliorate lung inflammation in ovalbumin (OVA)-challenged mouse lungs. Six- to eight-week-old C57BL/6J mice were sensitized and exposed to OVA for 3 consecutive days. Twenty-four hours later, mice lungs and bronchoalveolar lavage (BAL) fluid were analyzed. For an inhibitory effect, either of the two different SPHK inhibitors, N,N-dimethylsphingosine (DMS) or SPHK inhibitor [SK-I; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole], was nebulized for 30 min before OVA inhalation. OVA inhalation caused S1P release into BAL fluid and high expression of SPHK1 around bronchial epithelial walls and inflammatory areas. DMS or SK-I inhalation resulted in a decrease in S1P amounts in BAL fluid to basal levels, accompanied by decreased eosinophil infiltration and peroxidase activity. The extent of inhibition caused by DMS inhalation was higher than that caused by SK-I. Like T helper 2 (Th2) cytokine release, OVA inhalation-induced increase in eotaxin expression was significantly suppressed by DMS pretreatment both at protein level in BAL fluid and at mRNA level in lung homogenates. Moreover, bronchial hyperresponsiveness to inhaled methacholine and goblet cell hyperplasia were improved by SPHK inhibitors. These data suggest that the inhibition of SPHK affected acute eosinophilic inflammation induced in antigen-challenged mouse model and that targeting SPHK may provide a novel therapeutic tool to treat bronchial asthma.