Edited by: Hans-Uwe Simon
Conversion of Th17-type into Th2-type inflammation by acetyl salicylic acid via the adenosine and uric acid pathway in the lung
Version of Record online: 22 MAR 2010
© 2010 John Wiley & Sons A/S
Volume 65, Issue 9, pages 1093–1103, September 2010
How to Cite
Moon, H.-G., Tae, Y.-M., Kim, Y.-S., Gyu Jeon, S., Oh, S.-Y., Song Gho, Y., Zhu, Z. and Kim, Y.-K. (2010), Conversion of Th17-type into Th2-type inflammation by acetyl salicylic acid via the adenosine and uric acid pathway in the lung. Allergy, 65: 1093–1103. doi: 10.1111/j.1398-9995.2010.02352.x
- Issue online: 4 AUG 2010
- Version of Record online: 22 MAR 2010
- Accepted for publication 26 January 2010
- acetyl salicylic acid;
To cite this article: Moon H-G, Tae Y-M, Kim Y-S, Gyu Jeon S, Oh S-Y, Song Gho Y, Zhu Z, Kim Y-K. Conversion of Th17-type into Th2-type inflammation by acetyl salicylic acid via the adenosine and uric acid pathway in the lung. Allergy 2010; 65: 1093–1103.
Background: Allergen-specific T-cell responses orchestrate airway inflammation, which is a characteristic of asthma. Recent evidence suggests that noneosinophilic asthma can be developed by mixed Th1 and Th17 cell responses when exposed to lipopolysaccharide (LPS)-containing allergens.
Objective: To evaluate the therapeutic or adverse effects of acetyl salicylic acid (ASA) on the expression of Th1-type and Th17-type inflammation induced by airway exposure to LPS-containing allergens.
Methods: Th1 + Th17 asthma and Th2 asthma mouse models were generated by intranasal sensitization with ovalbumin (OVA) and LPS and intraperitoneal sensitization with OVA and alum, respectively. Therapeutic or adverse effects were evaluated after allergen challenge using pharmacologic and transgenic approaches.
Results: Lung infiltration of eosinophils was enhanced in OVA/LPS-sensitized mice by ASA treatment, which was accompanied by the enhanced production of eotaxin. These changes were associated with the down-regulation of Th17 cell response, which was partly dependent on adenosine receptor A1 and A3 subtypes, but up-regulation of allergen-specific IL-13 production from T cells. Lung inflammation induced by LPS-containing allergen was markedly reduced in IL-13-deficient mice in the context of ASA treatment, but not without ASA. Meanwhile, adenosine levels in the lung were enhanced by ASA treatment. Moreover, lung infiltration of eosinophils induced by ASA treatment was reversed by co-treatment of a xanthine oxidase inhibitor (allopurinol).
Conclusion: These findings suggest that ASA changes Th17-type into Th2-type inflammation mainly via the adenosine and uric acid metabolic pathway in the lung.
Asthma is a complex syndrome with many clinical phenotypes in both adults and children. Its major characteristics include a variable degree of airflow obstruction, airway hyperresponsiveness, and eosinophilic or noneosinophilic inflammation (1). Mild and moderate asthma are related to eosinophilic lung inflammation and severe and persistent asthma may be associated with significant neutrophilic lung inflammation (1). In patients with difficult-to-control asthma, neutrophils rather than eosinophils are often predominant (2). Recently, we demonstrated that eosinophilic inflammation was causally associated with a Th2 cell response, while noneosinophilic (or neutrophilic) inflammation is associated with mixed Th1 and Th17 cell responses when the airway is exposed to lipopolysaccharide (LPS)-containing allergens (3, 4).
Acetyl salicylic acid (ASA), or aspirin, has been used for the treatment of inflammatory conditions for several decades. Acetyl salicylic acid is well-known to inhibit cyclooxygenase (COX) enzymes, which are related to the production of proinflammatory arachidonic acid (AA) metabolites (5). However, the action mechanisms of anti-inflammatory effects, other than the COX inhibition of ASA, have been proposed: ASA inhibits the activity of IkB kinase-β, thereby preventing activation by NF-kB involved in the pathogenesis of inflammation (6); ASA triggers anti-inflammatory 15-epi-lipoxin A4 (7) and induction of apoptosis of inflammatory cells via the MAP kinase pathway (8); ASA inhibits the accumulation of inflammatory cells in an adenosine-dependent manner (9).
Adenosine is a natural purine metabolite. Uric acid is the final oxidation (breakdown) product of purine metabolism and produced by xanthine oxidase from xanthine and hypoxanthine, which in turn are produced from purines, including adenosine (10). Uric acid is well-known to be associated with a variety of medical conditions (11). Moreover, recent data have suggested that uric acid is an endogenous innate trigger in the development of the Th2 cell response induced by an adjuvant of alum (aluminum hydroxide) (12). Uric acid induces innate immune responses through the NALP3-inflammasome complex (12), and although the relationship between ASA and uric acid was observed three decades ago (13), the role of uric acid in the anti-inflammatory effects of ASA is still unclear.
In the present study, we hypothesized that ASA modulates Th1-type or Th17-type inflammation in an adenosine-dependent manner, which may be related to uric acid production. To test this hypothesis, we evaluated the therapeutic effects of ASA and the role of the adenosine and uric acid metabolic pathway on the development of Th1-type or Th17-type inflammation in the lung.
Wild-type (WT) C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA), and IL-13-deficient (C57BL/6 background) mice were kindly donated by Jack A. Elias (Yale University, New Haven, CT). These mice were bred in an animal laboratory at Pohang University of Science and Technology (POSTECH, Pohang, Korea). Age- and sex-matched mice were used for animal experiments. Animal study protocols were approved by the Institutional Animal Care and Use Committee of POSTECH.
Chemicals and drugs
Lipopolysaccharide and ovalbumin (OVA) were purchased from Calbiochem (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Acetyl salicylic acid (aspirin–lysine) was obtained from Ilyang Pharmaceuticals (Seoul, Korea), and allopurinol was acquired from Sigma-Aldrich. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), 2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH 58261), 8-4-[[(4-Cyano) phenylcarbamoyl-methyl]oxy]phenyl]-1,3-di-(n-propyl) xanthine (MRS 1754), and 6-Ethyl-5-(ethylsulfanylcarbonyl)-2-phenyl-4-propylpyridine-3-carboxylic acid propyl ester (MRS 1523), which are antagonists of adenosine receptor (AR) A1, A2a, A2b, and A3 subtypes, respectively, were purchased from Sigma-Aldrich.
Protocols for a mixed Th1 + Th17 and a Th2 asthma mouse models
Lipopolysaccharide-depleted OVA, as an allergen, was prepared as previously described (14). To generate a mixed Th1 + Th17 asthma mouse model, we followed a previously described method (4). Briefly, 6-week-old mice were sensitized intranasally four times with 75 μg of OVA with LPS (10 μg) on days 0, 1, 2, and 7. To generate a conventional Th2 asthma mouse model, 6-week-old mice were sensitized intraperitoneally two times with OVA (75 μg) and alum (2 mg) on days 0 and 7. To generate allergic inflammation, intranasal OVA challenge was performed four times intranasally with 50 μg of OVA on days 14, 15, 21, and 22. Pharmacological intervention with drug candidates was performed during OVA challenge. Lung inflammation and immunologic and inflammatory parameters were evaluated after allergen (OVA) challenge.
Cellularity in bronchoalveolar lavage (BAL) fluid
Bronchoalveolar lavage cellularity was analyzed as described previously (3). Briefly, BAL cellularity was determined by counting 300 inflammatory cells after diluting the cell pellets with 50 μl phosphate-buffered saline (PBS). Inflammatory cells were classified as macrophages, lymphocytes, neutrophils, or eosinophils.
Histological examination of lung tissues
Lung sections were stained with hematoxylin and eosin (H&E) after pressure fixation with Streck solution (Streck Laboratories, La Vista, NE, USA). All sample slides were compared at the same magnification. Lung inflammation was assessed by the degree of peribronchiolar and perivascular inflammation as described previously (3).
Cytokine production from T cells after nonspecific and allergen-specific stimuli
Single-cell suspensions from spleens or lung tissues were prepared. Cells were incubated with nonspecific stimuli (anti-CD3 and anti-CD28 antibodies) for 6 h and with OVA (100 μg/ml) for 72 h. The levels of cytokines in the supernatants were measured.
Fluorescent-activated cell sorting (FACS) analysis and intracellular cytokine staining
To identify T cells recruited into the lung, FACS analysis was performed using antibodies for T-cell surface markers, including anti-CD3, anti-CD4, and anti-CD8. After preparing single-cell suspensions from the lung tissue, cells were counted as previously described (14). To identify individual cells of interest, 1 × 106 isolated cells were aliquoted into tubes and stained with fluorescein isothiocyanate (FITC)-conjugated cell surface markers (BD Biosciences Pharmingen, San Diego, CA, USA).
To determine intracellular cytokine levels, isolated T cells were incubated at 37°C for 3 h in RPMI medium containing 10% fetal bovine serum (FBS) and 2 μg/ml brefeldin A (Sigma-Aldrich). Cells were then washed in PBS containing 3% FBS and 0.1% NaN3, followed by fixation in PBS containing 4% formaldehyde for 20 min. After washing, the cells were permeabilized with 0.5% saponin (Sigma-Aldrich) in PBS for 10 min, centrifuged, re-suspended in 50 μl of the same solution, and stained with anti-IFN-γ, anti-IL-17, and anti-IL-4 antibodies for 30 min.
The cells were analyzed using a FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA), and the results were processed using CellQuest software (BD Biosciences). The number of cells in the lung was determined by multiplying total lung cells by the percentage of CD4+ or CD8+ T cells.
In vitro production of adenosine and adenosine monophosphate (AMP)
Raw 264.7 cells (5 × 105) were activated with LPS (100 ng/ml) for 2 h. The supernatant was washed away three times with fresh PBS, and different doses of ASA were treated with fresh RPMI media for 4 h. Then, the supernatants and cells were collected to measure the levels of AMP and adenosine by reversed-phase high-performance liquid chromatography (HPLC).
mRNA expression of candidate genes
The expression of adenosine deaminase (ADA) and adenosine kinase (AK) mRNA was quantified by real-time PCR and then normalized with β-actin.
Quantification of cytokine levels
Cytokines were measured by ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).
Quantification of AMP, adenosine, and uric acid levels
Adenosine monophosphate and adenosine assays were performed as previously described (15). Briefly, the frozen lungs were homogenized in 0.85 ml of ice-cold 0.4 N HClO4. The samples were placed on ice for 15 min and then centrifuged for 2 min at full speed in a centrifuge at 4°C. The supernatants were transferred to a clean tube, and 0.1 ml was removed for protein determination. The remaining volume (0.685 ml) was neutralized with 0.345 ml of 0.6 M KHCO3/0.72 M KOH. The samples were clarified by centrifugation for 2 min at full speed in a centrifuge at 4°C and then analyzed by HPLC. The amount of adenosine was calculated from the areas under the respective peaks using authentic standards and an internal peak to correct for variations in extraction efficiency and sample loading. Uric acid was assayed using the Quantichrom™ Uric Acid Assay kit (BioAssay Systems, Hayward, CA, USA).
Significant differences between the treatments were assessed using Student’s t-test, anova, or Wilcoxon’s rank-sums test. For multiple comparisons, anova was used initially, and when significant differences were found, individual t-tests or Wilcoxon’s rank-sums tests for pairs of groups were performed.
The therapeutic effect of ASA on the expression of lung inflammation and T-cell responses in the Th1 + Th17 asthma model
Our previous data showed that airway sensitization with LPS-containing allergens induces noneosinophilic lung inflammation, which results from mixed Th1 and Th17 cell responses (4). In the present study, we evaluated the effects of ASA treatment on the development of Th1 and Th17 inflammation in the Th1 + Th17 asthma model (Fig. 1A). To test this, ASA (18 mg/kg) was administered intraperitoneally during allergen challenge on days 14, 15, 21, and 22. In the present study, BAL cellularity showed that total inflammatory cell numbers were similar between OVA/LPS-sensitized mice in the ASA and sham treatments; however, lung infiltration of eosinophils was enhanced in ASA-treated, OVA/LPS-sensitized mice compared to sham-treated, OVA/LPS-sensitized mice (Fig. 1B). Lung histology also showed that peribronchiolar and perivascular infiltration of eosinophils was enhanced in the former group compared to the latter group (Fig. 1C). In terms of the production of mediators related with Th1, Th17, and Th2 cell responses, the present study showed that the levels of tumor necrosis factor-α (TNF-α) (an IL-17 downstream mediator) in BAL fluids were significantly lower in ASA-treated, OVA/LPS-sensitized mice than in sham-treated, OVA/LPS-sensitized mice, although the levels of IP-10 (an IFN-γ downstream mediator) were similar in the two groups; however, the levels of eotaxin (a Th2 cytokine downstream mediator) were enhanced in the former group compared to the latter group (Fig. 1D). These findings together suggest that ASA treatment increases the lung infiltration of eosinophils by enhancing the production of Th2 cytokine downstream mediators.
Next, we evaluated the production of Th1, Th17, and Th2 cytokines from lung T cells. To test this, the expression of IFN-γ, IL-17, and IL-4 in T cells isolated from lung tissues was evaluated 6 h after allergen challenges on day 21 with or without ASA (18 mg/kg) treatment, as shown in Fig. 1A. Intracellular cytokine staining showed that the numbers of IFN-γ+ CD4+ and IFN-γ+ CD8+ lung T cells were similar in OVA/LPS-sensitized mice treated with ASA and the sham treatment (Fig. 2A). The production of IFN-γ from lung T cells after stimulation with anti-CD3 and anti-CD28 antibodies was also similar in the two groups (Fig. 2B). In regard to the expression of Th17 cytokines, intracellular cytokine staining showed that the numbers of IL-17+ CD4+ and IL-17+ CD8+ lung T cells were significantly lower in ASA-treated, OVA/LPS-sensitized mice compared to sham-treated, OVA/LPS-sensitized mice (Fig. 2C). In addition, the production of IL-17 from lung T cells after anti-CD3 and anti-CD28 stimulation was significantly lower in the former group compared to the latter group (Fig. 2D). The expression of IL-4 in lung T cells was not enhanced by sensitization with OVA/LPS compared to OVA, irrespective of ASA treatment (data not shown). Taken together, these findings suggest that ASA treatment inhibits the expression of allergen-specific Th17 responses in the lung.
The therapeutic effects of ASA on the expression of Th2-type inflammation in the Th2 asthma model
We evaluated the effect of ASA treatment in the development of lung inflammation in the conventional Th2 asthma model (Fig. 3A). To test this, ASA (18 mg/kg) was applied intraperitoneally during allergen challenge on days 14, 15, 21, and 22. Bronchoalveolar lavage cellularity showed that lung infiltration of eosinophils was significantly higher in ASA-treated, OVA/alum-sensitized mice than in sham-treated, OVA/alum-sensitized mice (Fig. 3B). Lung histology also indicated that peribronchiolar and perivascular infiltration of eosinophils was enhanced in the former group compared to the latter group (Fig. 3C). These data suggest that ASA treatment aggravates eosinophilic inflammation induced by Th2 cell response.
The role of IL-13 in the development of lung inflammation in the Th2 asthma animal model
IL-13 is now considered to be crucial in the development of eosinophilic lung inflammation induced by Th2 cell response (16). In the present study, we evaluated the role of IL-13 in the development of lung inflammation in the Th2 asthma model (Fig. 3A) using IL-13-deficient mice. In the present study, BAL cellularity showed that lung infiltration of inflammatory cells, including eosinophils, was totally blocked in the absence of IL-13 (Fig. 3D). In addition, the production of eotaxin, enhanced by sensitization with OVA + alum, was completely abolished in the absence of IL-13 (Fig. 3E). These findings indicate that IL-13 is a key mediator in the development of lung inflammation induced by Th2 cell response.
The role of IL-13 in the development of lung inflammation in the Th1 + Th17 asthma model
To investigate the role of IL-13 in the development of eosinophilic infiltration enhanced by ASA treatment in the Th1 + Th17 asthma model, the allergen-specific production of IL-13 from T cells was evaluated 6 h after three OVA challenges on day 21 (Fig. 1A). This study showed that the production of IL-13 from spleen T cells 72 h after stimulation with OVA (100 μg/ml) was significantly higher in ASA-treated, OVA/LPS-sensitized mice compared to sham-treated, OVA/LPS-sensitized mice, although this production was not enhanced by sensitization with OVA/LPS compared to OVA alone (Fig. 4A).
Next, we evaluated the role of IL-13 in the development of eosinophilic infiltration enhanced by ASA treatment in the Th1 + Th17 asthma model. To test this, lung inflammation was evaluated 24 h after the final OVA challenge using IL-13-deficient mice, as shown in Fig. 1A. Bronchoalveolar lavage cellularity showed that lung infiltration by inflammatory cells, including eosinophils, was significantly lower in ASA-treated, OVA/LPS-sensitized IL-13-deficient mice than in ASA-treated, OVA/LPS-sensitized WT mice; however, in the case of the sham treatment, lung infiltration by inflammatory cells was similar in IL-13-deficient and WT mice sensitized with OVA/LPS (Fig. 4B). Lung histology showed that lung infiltration by inflammatory cells was markedly lower in ASA-treated, OVA/LPS-sensitized IL-13-deficient mice compared to ASA-treated, OVA/LPS-sensitized WT mice (Fig. 4C). In addition, the production of eotaxin enhanced by ASA treatment after OVA/LPS sensitization was completely inhibited in the absence of IL-13 (Fig. 4D). Taken together, these findings suggest that lung inflammation induced by airway sensitization with LPS-containing allergens is mainly dependent on IL-13 in the context of ASA treatment.
The effect of ASA on the production of adenosine and AMP
The anti-inflammatory effects of ASA have been suggested to be related to adenosine (9). First, we evaluated the levels of adenosine in lung tissues 2 h after the final allergen challenge with or without ASA treatment. This study showed that the levels of adenosine in lung tissues were enhanced in OVA/LPS-sensitized mice by treatment with ASA compared to the sham treatment (Fig. 5A). In regard to the mRNA expression of adenosine metabolism enzymes, the expression of ADA mRNA was enhanced by sensitization with OVA/LPS compared to OVA alone, and this enhanced expression was completely reversed by ASA treatment; however, the expression of AK mRNA was similar in these groups (Fig. 5B). These data suggest that ASA enhances adenosine production, possibly by inhibiting the expression of the adenosine-metabolizing enzyme ADA.
Next, we evaluated the effect of ASA on the in vitro production of adenosine and its upstream metabolite AMP from macrophages (Raw 264.7 cell line). To test this, different doses of ASA were treated with macrophages for 4 h after a 2-h stimulation with LPS (100 ng/ml). The levels of intracellular AMP were enhanced by ASA treatment in a dose-dependent manner, although the extracellular AMP levels were not altered by the ASA treatment (Fig. 5C). In terms of the production of adenosine, the extracellular adenosine levels were enhanced by the ASA treatment in a dose-dependent manner, although the intracellular adenosine levels were enhanced by only high doses of ASA (Fig. 5D). These findings together suggest that ASA enhances the levels of extracellular adenosine, possibly via up-regulation of intracellular AMP production.
The role of ARs on the development of Th17 cell response in the Th1 ± Th17 asthma model in context of ASA treatment
To investigate the role of ARs on the development of Th17 cell response, AR antagonists in combination were treated in the Th1 ± Th17 asthma model (Fig. 1A) in context of ASA treatment; for example, ARA2a ± ARA2b ± ARA3 antagonists in combination were used to evaluate ARA1-mediated effects. This study showed that the number of IL-17-producing cells in lung tissues 6 h after allergen challenge on day 21 was decreased by effects mediated by ARA1, ARA2-, and ARA3 subtypes, although enhanced by ARA2b-mediated effects (Fig. 5E). In addition, the production of IL-17 from lung T cells 6 h after anti-CD3 ± anti-CD28 stimuli was decreased by ARA1- and ARA3-mediated effects, although enhanced by ARA2b-mediated effects (Fig. 5F). These data together suggest that therapeutic effects of adenosine against Th17 cell response are mainly dependent on ARA1 and ARA3 subtype-mediated signals.
The role of uric acid in the development of ASA-enhanced IL-13-mediated inflammation in the Th1 + Th17 asthma model
Recent evidence indicates that alum, as an adjuvant, boosts the allergen-specific Th2 cell response by inducing uric acid (12), and ASA is known to induce the production of uric acid (17). This evidence led to the notion that conversion of Th17-type into Th2-type inflammation by ASA may be related to the production of uric acid by ASA treatment. First, we evaluated the levels of serum uric acid in the Th1 + Th17 asthma model with or without ASA treatment. This study showed that serum levels of uric acid were significantly higher after allergen challenge in OVA/LPS-sensitized mice by ASA treatment compared to the sham treatment, although these levels were similar between sham-treated, OVA/LPS-sensitized and OVA-sensitized mice (Fig. 6A).
Next, we evaluated the role of uric acid in the development of IL-13-mediated inflammation converted by ASA treatment in the Th1 + Th17 asthma model. To test this, OVA/LPS-sensitized mice were treated with ASA with or without a xanthine oxidase inhibitor (allopurinol, 1 mg/kg) during allergen challenge. Bronchoalveolar lavage cellularity and lung histology 24 h after allergen challenge showed that allopurinol treatment inhibited lung infiltration by inflammatory cells, including eosinophils, in ASA-treated, OVA/LPS-sensitized mice, although allopurinol treatment did not affect lung inflammation in sham-treated, OVA/LPS-sensitized mice (Fig. 6B,C). In addition, when evaluated 24 h after allergen challenge, the production of eotaxin enhanced by ASA treatment in the Th1 + Th17 asthma model was reversed by allopurinol co-treatment; however, the production of TNF-α inhibited by ASA in this model was unaltered by allopurinol (Fig. 6D). In addition, when evaluated 6 h after the allergen challenge, the expression levels of IFN-γ and IL-17 in lung T cells from ASA-treated, OVA/LPS-sensitized mice were not altered by treatment with allopurinol (Fig. 6E). In terms of the in vivo effects of allopurinol on the production of uric acid, the serum levels of uric acid 6 h after allergen challenge were significantly lower in ASA + allopurinol-treated, OVA/LPS-sensitized mice than in OVA/LPS-sensitized mice treated with ASA alone (Fig. 6F). These data together suggest that uric acid is a key mediator in the development of IL-13-mediated inflammation converted by ASA.
The present study indicates that ASA aggravates eosinophilic inflammation in the conventional Th2 asthma model. In addition, ASA converts Th17-type into Th2-type inflammation mainly via the IL-13-mediated pathway in the mixed Th1 + Th17 asthma model. Moreover, IL-13-mediated inflammation converted by ASA in the Th1 + Th17 asthma model appears to be related to the adenosine and uric acid pathway. To the best of our knowledge, this is the first study to report that ASA converts Th17 into Th2 inflammation, possibly via the adenosine–uric acid pathway.
Acetyl salicylic acid is one of the most widely used medications in the world, with an estimated 40 000 metric tons consumed each year (18). However, ASA use may also lead to ASA-exacerbated respiratory tract disease (AERD), which is a common and often underdiagnosed disease that affects up to 20% of the adult population with asthma (19). This condition is associated with patients having severe asthma who also have underlying nasal polyps and chronic hyperplastic eosinophilic sinusitis (19, 20). Several hypotheses regarding the pathogenesis of AERD have been proposed. The shunting hypothesis states that COX-1 inhibition shifts AA metabolism away from the production of protective prostanoids and toward cysteinyl leukotriene biosynthesis, resulting in bronchoconstriction and increased mucus production (21). The COX-2 hypothesis proposes that ASA causes a structural change in COX-2, which results in the generation of lipoxygenase pathway products (19, 21). In terms of genetic susceptibility, a great deal of evidence has demonstrated that genetic polymorphisms of the AA pathways are associated with AERD (22, 23). In addition, recent evidence showed that genetic polymorphisms of ARs were significantly associated with AERD (24). The present animal data showed that ASA aggravated eosinophilic inflammation in both Th1 ± Th17 and Th2 asthma models and that ASA also converted Th17-type into Th2-type inflammation mainly via adenosine-dependent manner. Moreover, inhalation of AMP, an upstream metabolite of adenosine in the purine pathway, can cause airway constriction and induce sputum eosinophilia (25). These findings together suggest that AERD can develop in patients with a genetic predisposition to Th2 cell response and/or eosinophilic inflammation.
Previous data indicate that ASA inhibits noneosinophilic or neutrophilic inflammation via an adenosine-dependent and COX-independent manner (9). However, the role of ASA in the development of Th17 cell response or Th17-type inflammation has been little studied. Recent data showed that prostaglandin E2 (PGE2), a COX metabolite, can directly promote the differentiation and proinflammatory functions of human and murine Th17 cells (26). Our present data show that ASA inhibits the expression of Th17 inflammation possibly via up-regulation of adenosine production. Moreover, recent data showed that colonic commensal bacteria promote colon tumorigenesis via activation of Th17 inflammation (27). A great deal of evidence has demonstrated that ASA inhibits tumor growth and metastases in animal models and reduces the risk of colorectal cancer in clinical trials (22, 28, 29). These data together suggest that ASA reduces the Th17 response, which can be related to a reduced risk of tumorigenesis because of Th17 inflammation.
Adenosine is a nucleoside, which is generated by ATP catabolism at sites of tissue injury including inflammation. Adenosine signals via G protein–coupled receptors which consist of four subtypes (ARA1, ARA2a, ARA2b, and ARA3) (30) and modulates immune and inflammatory process (31, 32). Elevated levels of adenosine are found in the BAL fluid from patients with asthma (33). Previous animal data indicate that high levels of adenosine in the lung cause IL-13-induced lung inflammation and remodeling mainly via ARA2b-mediated effects in ADA-deficient mice (31). In the present study, our data showed that ASA inhibited the expression of ADA mRNA and that the inhibitory effects of ASA against Th17 cell response were mainly dependent on ARA1 and ARA3 subtypes. These data together suggest that ASA inhibits Th17 cell response via ARA1- and ARA3-mediated signals, whereas enhances Th2 cell response partly via ARA2-mediated signal.
Uric acid has long been known to be a key causative agent of gout induction (34). Recent data showed that uric acid works as an extracellular stressor, which induces NALP3 inflammasome, which leads to Th2 inflammation in gout and a conventional allergic asthma mouse model (12, 35). Our present data showed that ASA augments the production of uric acid; moreover, Th2 inflammation converted by ASA in the Th1 + Th17 asthma model can be effectively blocked through the inhibition of uric acid production by a xanthine oxidase inhibitor (allopurinol). Taken together, these findings suggest that ASA converts Th17 into Th2 inflammation, possibly via the adenosine and uric acid metabolic pathway.
In summary, we can speculate the underlying mechanisms of ASA-induced conversion of Th17 into Th2 cell response, as shown in Fig. 7. When a subject inhales LPS-containing allergens, LPS-induced innate immune response polarizes naïve T cell into mixed Th1 and Th17 cells. Recruited Th17 cells produce cytokines, including IL-17 and TNF-α, which induce lung infiltration of inflammatory cells other than eosinophils. Injurious stress in recruited inflammatory cells induces ATP breakdown and then results in the increase in adenosine levels, which is further enhanced by ASA treatment via inhibition of adenosine-metabolizing enzyme (ADA). The ASA-enhanced adenosine then inhibits Th17 cell response, but enhances Th2 cell response. In addition, adenosine is metabolized into xanthine by the actions of metabolizing enzymes, and then xanthine metabolized into uric acid, which also enhances Th2 cell response, by xanthine oxidase. In conclusion, the present study suggests that ASA converts Th17-type into Th2-type inflammation via the up-regulation of the adenosine and uric acid metabolic pathway. In terms of clinical applications, adenosine and its receptor, IL-13-mediated signals, and uric acid metabolism pathway are good therapeutic targets against Th17-type inflammation in the context of ASA use.
We thank Jack A. Elias for the kind donation of IL-13-deficient mice, Jee-In Lim for providing secretarial assistance, and members of POSTECH animal facility for their helping with the animal experiments. This study was supported by grants from the Korea Ministry of Health & Welfare, Republic of Korea (A080711-0912-0000100) and the Korea Science and Education Foundation (RO1-2007-000-11026-0 and 20090081757).
Conflict of interest
The authors declare that they have no conflicts of interest.
- 35Alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome. J Immunol 2008;181:2755–2759., , , , , et al.