Abstract
In an experimental asthma model, the activation of TLR4 by bacterial LPS occasionally exacerbates allergic inflammation through the production of Th2 cytokines, and mast cells have been suggested to play a central role in this response. However, the detailed mechanism underlying how LPS/TLR4 stimulates the production of Th2 cytokines, especially IL-13, remains unclear in mast cells. In the current study, we observed that the expression levels of leukotriene B4 receptor-2 (BLT2) and the synthesis of its ligands were highly upregulated in LPS-stimulated bone marrow–derived mast cells and that BLT2 blockade with small interfering RNA or a pharmacological inhibitor completely abolished IL-13 production, suggesting a mediatory role of the BLT2 ligand–BLT2 axis in LPS/TLR4 signaling to IL-13 synthesis in mast cells. Moreover, we demonstrated that MyD88 lies upstream of the BLT2 ligand–BLT2 axis and that this MyD88-BLT2 cascade leads to the generation of reactive oxygen species through NADPH oxidase 1 and the subsequent activation of NF-κB, thereby mediating IL-13 synthesis. Interestingly, we observed that costimulation of LPS/TLR4 and IgE/FcεRI caused greatly enhanced IL-13 synthesis in mast cells, and blockading BLT2 abolished these effects. Similarly, in vivo, the IL-13 level was markedly enhanced by LPS administration in an OVA-induced asthma model, and injecting a BLT2 antagonist beforehand clearly attenuated this increase. Together, our findings suggest that a BLT2-linked cascade plays a pivotal role in LPS/TLR4 signaling for IL-13 synthesis in mast cells, thereby potentially exacerbating allergic response. Our findings may provide insight into the mechanisms underlying how bacterial infection worsens allergic inflammation under certain conditions.
Introduction
Recently, much attention has been focused on the role of TLRs in the pathogenesis of allergic asthma. For example, TLR4 activation by LPS inhalation, particularly at low doses, exacerbates allergic airway inflammation by activating mast cells and promoting Th2 responses (1–4). Mast cells have been suggested to play a pivotal role in this response, as LPS-exacerbated airway inflammation and Th2 cytokine production were not observed in mast cell–deficient mice (5). The adoptive transfer of bone marrow–derived mast cells (BMMCs) from wild type (WT) but not from TLR4-deficient mice restored the increased allergic airway inflammation and Th2 cytokine production in mast cell–deficient mice, suggesting a central role of TLR4 in mast cells in the bacterial infection–induced exacerbation of the allergic response (1, 2, 5). Th2 cytokines produced by TLR4 activation in mast cells are important regulators of the allergic inflammatory response (2, 5). For example, TLR4 activation by LPS in mast cells was shown to be dependent on MyD88-NF-κB cascade for Th2 cytokine production (6, 7). Recently, the association of eicosanoid, especially leukotriene B4 receptor-2 (BLT2), with allergen-induced Th2 cytokine production in mast cells has been also established (8). However, the role of BLT2 in the LPS/TLR4 signaling cascade responsible for the production of Th2 cytokines in mast cells, especially IL-13, remains unclear.
BLT2 has been demonstrated as critical for the synthesis of Th2 cytokines, such as IL-13, during IgE/FcεRI–mediated mast cell activation in the allergic response (8). In addition, the levels of BLT2 ligands, leukotriene B4 (LTB4) and 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], and their synthesizing enzymes, 5-lipoxygenase (LO) and 12-LO, were highly elevated in allergic asthma, thereby mediating the allergic response (9–12). Importantly, IL-13 synthesis in genetically mast cell–deficient mice was significantly lower than that in WT mice in an OVA-induced asthma model (13). However, IL-13 production was restored to the levels observed in WT mice after the adoptive transfer of normal BMMCs into mast cell–deficient mice but was not restored in BLT2-null (BLT2−/−) BMMC-reconstituted mast cell–deficient mice in the OVA asthma model, suggesting a mediatory role of BLT2 in mast cells for the synthesis of IL-13 in the asthmatic allergic response (8, 13).
In the current study, we investigated whether BLT2 is also involved in LPS/TLR4 signaling for IL-13 synthesis in mast cells, and found that IL-13 production in LPS-stimulated BMMCs is highly dependent on BLT2. Blockading BLT2 clearly attenuated LPS-induced IL-13 synthesis in BMMCs. We also found that MyD88 lies upstream of BLT2 and that this MyD88-BLT2 cascade mediates the consequent activation of Nox1-ROS-NF-κB signaling in LPS-stimulated BMMCs, thereby mediating IL-13 production. In addition, we observed that costimulation of LPS/TLR4 and IgE/FcεRI caused greatly enhanced IL-13 synthesis in BMMCs, and that these effects were abolished by blockading BLT2. Finally, we observed that IL-13 production was markedly increased by LPS administration in an OVA-induced asthma model, whereas this increase was not observed when a BLT2 antagonist was injected beforehand. Taken together, to our knowledge our results demonstrate, for the first time, that the TLR4-MyD88-BLT2-Nox1-ROS-NF-κB cascade contributes to the production of IL-13 by LPS/TLR4 stimulation in mast cells. Thus, considering the previously reported role of BLT2 in mediating the IgE/FcεRI allergic signal for Th2 cytokine synthesis (8), BLT2 likely serves as a potential connection between innate and allergic signals in mast cells. Our findings may provide insight into the mechanisms underlying how the innate immune response to bacterial LPS worsens the allergic response under certain conditions.
Materials and Methods
Reagents
LPS (Escherichia coli serotype O55:B5), monoclonal anti-DNP-specific IgE (clone SPE-7), DNP-conjugated BSA (DNP-BSA), DMSO, and diphenyleneiodonium (DPI) were obtained from Sigma (St. Louis, MO). DCFDA was obtained from Molecular Probes (Eugene, OR), and AACOCF3, baicalein, MK886, and Bay11-7082 were obtained from Calbiochem (La Jolla, CA). The BLT1 antagonist U75302 (13, 14) and the BLT2 antagonist LY255283 (13, 14) were obtained from Cayman Chemical (Ann Arbor, MI). RPMI 1640, antibiotic and antimycotic compounds, and nonessential amino acids were obtained from Invitrogen (Grand Island, NY) (8).
BMMC culture and stimulation
BMMCs were isolated from the femurs of mice (8–12 wk old) as previously described (8, 13). The isolated BMMCs were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, nonessential amino acids, 50 μM 2-ME, sodium pyruvate, and HEPES buffer containing 20 ng/ml stem cell factor and 10 ng/ml IL-3 (R&D Systems, Minneapolis, MN) for 4–8 wk at 37°C and 5% CO2 (8). The purity of the cells was >97%, as determined at 4 wk by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ) using a FITC-conjugated rat anti-mouse CD117 (c-Kit) mAb (eBioscience, San Diego, CA). The BMMCs were stimulated with LPS (0.1 μg/ml) in cytokine-free complete medium for the indicated time periods. For costimulation with LPS/TLR4 and IgE/FcεRI, the cells were sensitized overnight with an optimal concentration (1 μg/ml) of monoclonal anti-DNP IgE in complete medium and stimulated with LPS (0.1 μg/ml), DNP-BSA (50 ng/ml), or LPS (0.1 μg/ml) + DNP-BSA (50 ng/ml) in cytokine-free complete medium for the indicated time periods.
Semiquantitative RT-PCR analysis
Total cellular RNA was extracted from BMMCs or homogenized lung tissue using easy-BLUE (Intron, Sungnam, Korea), and 2 μg of the extracted RNA was reverse transcribed using Moloney Murine Leukemia Virus Reverse Transcriptase (Beams Bio, Kyunggi, Korea). MyD88, BLT1, BLT2, NADPH oxidase 1 (Nox1), NADPH oxidase 4 (Nox4), IL-13, and GAPDH were then amplified using a PCR Pre-Mix Kit (Intron). To semiquantitatively analyze the transcripts, we first identified the optimal PCR conditions for the linear amplification of GAPDH. The primers for MyD88, BLT1, BLT2, Nox1, IL-13, and GAPDH were previously described (8, 15). The primers (forward and reverse) for Nox4 were 5′-GGGTCCACAGCAGAAAACTC-3′ and 5′-GAAGCCCATTTGAGGAGTCA-3′. All data were normalized to the abundance of GAPDH.
Measurement of LTB4, 12(S)-HETE, and IL-13
BMMCs were stimulated with LPS (0.1 μg/ml) in cytokine-free complete medium for the indicated times, after which the conditioned media were collected and immediately frozen. The levels of LTB4 and 12(S)-HETE were quantified using ELISA kits (Amersham Biosciences, Buckinghamshire, U.K.) according to the manufacturer’s instructions. IL-13 levels in collected cell-free medium or in the supernatants of bronchoalveolar lavage fluid (BALF) were quantified using ELISA kits (R&D Systems) according to the manufacturer’s instructions. BALF was collected as previously described (13) with slight modifications. In brief, BALF was obtained from mouse lungs using 0.7 ml of PBS after tracheal cannulation. The collected BALF was centrifuged at 1000 × g for 5 min. The supernatant was then collected and stored at −80°C until the IL-13 ELISA.
RNA interference of MyD88, BLT1, BLT2, and Nox1
To knockdown MyD88, BLT1, BLT2, and Nox1, BMMCs were transfected with MyD88 (Bioneer, Daejeon, Korea), BLT1 (Bioneer), BLT2 (Bioneer), or Nox1 small interfering RNA (siRNA) (Invitrogen) using an MP-100 MicroPorator (Digital Bio, Seoul, Korea) according to the manufacturer’s instructions. In brief, 2 × 106 cells in 100 μl of resuspension buffer (Invitrogen, Carlsbad, CA) containing control siRNA (scrambled) (50 nM), MyD88 siRNA (50 nM), BLT1 siRNA (50 nM), BLT2 siRNA (50 nM), or Nox1 siRNA (50 nM) were electroporated by applying two pulses of 1400 V for 30 ms (13). The cells were cultured in complete medium without antibiotics for 24 h, and the mRNA levels of MyD88, BLT1, BLT2, or Nox1 were then analyzed by RT-PCR to evaluate the level of interference.
Overexpression of MyD88
BMMCs (2 × 106) were transiently transfected with 1 μg of expression vectors for MyD88 (pCMV-Flag-MyD88, kindly provided by T. Renno) (15) or with the corresponding empty vector (pCMV-Flag) using the MP-100 MicroPorator (Digital Bio) in accordance with the manufacturer’s instructions.
Immunoblot analysis
Lysate protein samples were heated at 95°C for 5 min and separated by SDS-PAGE. The separated proteins were then electrophoretically transferred to a polyvinylidene difluoride membrane for 1 h at 100 V. The membrane was exposed for 1 h to TBS containing 0.05% Tween-20 and 5% dried nonfat milk, followed by incubation for 1 h with primary Abs at a dilution of 1:2000 (or 1:4000 for Abs against β-actin or 1:1000 for Abs against 12-LO) in TBS containing 0.05% Tween-20. The membrane was then incubated for 1 h at room temperature with HRP-conjugated secondary Abs prior to the detection of immune complexes using an ECL kit (Amersham Biosciences, Little Chalfont, U.K.). Abs against 5-LO were obtained from BD Biosciences, Abs against 12-LO were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and Abs against p-cytosolic phospholipase A2 (p-cPLA2), p-IκBα, and β-actin (loading control) were obtained from Cell Signaling Technology (Danvers, MA). Size estimates for proteins were obtained using m.w. standards obtained from Thermo Scientific (Rockford, IL).
Measurement of reactive oxygen species
Intracellular reactive oxygen species (ROS) levels were determined by measuring DCF fluorescence with a flow cytometer (FACSCalibur; BD Biosciences) as previously described (8, 14). In brief, BMMCs (2 × 106) were exposed to LPS (0.1 μg/ml) for 30 min, incubated for 15 min with the ROS-sensitive fluorophore DCFDA (10 μM), washed with PBS, and then immediately analyzed.
Immunization and challenge of mice
Female BALB/c mice (7 wk old; 18–20 g) were obtained from Orient Bio (Seoungnam, Korea) were distributed into the following groups: Saline/Saline (S/S; the saline-treated group), Saline/LPS (S/L; the LPS-treated group), OVA/OVA (O/O; the OVA-treated group), and OVA/OVA + LPS (O/OL; the OVA + LPS-treated group). Immunization and challenge were performed as previously described (16), with slight modifications. The immunization and challenge protocol used in this study is shown in Supplemental Fig. 1. In brief, BALB/c mice were immunized by an i.p. injection of OVA (5 μg) with 1 mg of adjuvant aluminum hydroxide (Pierce, Rockford, IL) on days 0 and 7. Then, the mice were challenged consecutively three times intranasally with low-dose LPS (0.1 μg), OVA (5 μg), or OVA (5 μg) + LPS (0.1 μg) on days 14, 15, and 16. LY255283 (10 mg/kg) or the vehicle control (DMSO) was administered i.p. 1 h before every challenge. The mice were sacrificed on day 17 to assess airway inflammation. All animals were housed under 12:12 h light/dark conditions at a density of four to five mice per static polycarbonate microisolator cage on disposable bedding. Wire-lidded food hoppers within the cages were filled to capacity with rodent chow, and the mice had access to bottle-supplied water. The study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of Korea University. The protocol was approved by the Committee on the Ethics of Animal Experiments of Korea University.
BALF cells and lung tissue histological analysis
Inflammatory cells in the BALF were collected by centrifugation (1000 × g for 5 min) and washed once in PBS. Smears of BALF cells were prepared with a cytospin (Hanil Science, Kyunggi, Korea). The smears were then stained with H&E. Cells in each of four different random locations were counted using a microscope. Excised lungs were fixed (10% formaldehyde) and embedded in paraffin. Sections 6 μm thick were mounted on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA) and stained with H&E. A quantitative histological analysis performed by five independent, blinded investigators was employed to measure the degree of inflammation. The degree of lung inflammation was evaluated on a subjective scale from 0 to 3, as described elsewhere (13). Grade 0 indicated that no inflammation was detectable; grade 1 indicated the occurrence of occasional cuffing with inflammatory cells; grade 2 indicated that most bronchi or vessels were surrounded by a thin layer (one to five cells thick) of inflammatory cells; and grade 3 indicated that most bronchi or vessels were surrounded by a thick layer (more than five cells thick) of inflammatory cells. Total lung inflammation was defined as the average of the peribronchial and perivascular inflammation scores. All images were acquired using a BX51 microscope (Olympus) equipped with a DP71 digital camera (Olympus).
Statistical analysis
Data are presented as the mean ± SD and were analyzed by Student t test and one-way ANOVA for comparisons between two or more groups, respectively. A p value <0.05 was considered statistically significant.
Results
LPS stimulates IL-13 production via BLT2 upregulation in BMMCs
In accordance with previous reports (4, 6), we observed significantly elevated IL-13 production in LPS-stimulated BMMCs (Fig. 1A–C). In addition, BLT2 expression was markedly increased by LPS in these cells (Fig. 1B). We then examined whether upregulated BLT2 plays a role in IL-13 production in LPS-stimulated BMMCs. For these experiments, we transiently transfected BMMCs with BLT2-specific siRNA, and found that blockading BLT2 with siRNA resulted in a considerable suppression of LPS-induced IL-13 production (∼60% decrease in transcript level; ∼40% decrease in protein level) (Fig. 1D, 1E). Under the same experimental conditions, BLT1 siRNA had no effect on LPS-induced IL-13 production (Supplemental Fig. 2), suggesting a specific mediatory role of BLT2 in this signal. In addition, we generated BLT2−/− mice and isolated BLT2−/− BMMCs from these mice, as previously described (13). LPS-induced IL-13 production was again considerably suppressed in the BLT2−/− BMMCs compared with cells isolated from WT mice (∼50% decrease) (Fig. 1F). Furthermore, the BLT2 antagonist LY255283 greatly suppressed LPS-induced IL-13 production (∼50% decrease), whereas the BLT1 antagonist U75302 had no effect (Fig. 1G). Together, these data suggest that BLT2, but not BLT1, is implicated in IL-13 production in LPS-stimulated BMMCs.
LPS-stimulated IL-13 production is dependent on BLT2 in BMMCs. (A–C) BMMCs were stimulated with LPS (0.1 μg/ml) for the indicated time periods. (A) IL-13 secretion was determined using specific ELISA kits after 12 h. (B and C) BMMCs were stimulated with LPS (0.1 μg/ml), after which total cellular RNA was isolated and transcript levels were assessed by semiquantitative RT-PCR (B). Cell-free supernatants were collected and subjected to ELISA analysis for IL-13 (C). (D and E) BMMCs were transfected with 50 nM control (siCont) or BLT2 siRNA (siBLT2). After 24 h, cells were stimulated with LPS (0.1 μg/ml) for 3 h (D) or 12 h (E). Thereafter, total cellular RNA was isolated, and transcript levels of IL-13 and BLT2 were assessed by semiquantitative RT-PCR (D). Additionally, cell-free supernatants were collected and subjected to ELISA analysis for IL-13 (E). (F) WT (BLT2+/+) and BLT2 null (BLT2−/−) BMMCs were stimulated with LPS (0.1 μg/ml) for 12 h. Cell-free supernatants were collected, and IL-13 levels were determined by ELISA. (G) BMMCs were first incubated with LY255283 (10 μM) or U75302 (1 μM) for 30 min and then stimulated with LPS (0.1 μg/ml) for 12 h to induce IL-13 secretion. All quantitative data are shown as the mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001. ns, not significant.
LPS-stimulated IL-13 production is dependent on BLT2 in BMMCs. (A–C) BMMCs were stimulated with LPS (0.1 μg/ml) for the indicated time periods. (A) IL-13 secretion was determined using specific ELISA kits after 12 h. (B and C) BMMCs were stimulated with LPS (0.1 μg/ml), after which total cellular RNA was isolated and transcript levels were assessed by semiquantitative RT-PCR (B). Cell-free supernatants were collected and subjected to ELISA analysis for IL-13 (C). (D and E) BMMCs were transfected with 50 nM control (siCont) or BLT2 siRNA (siBLT2). After 24 h, cells were stimulated with LPS (0.1 μg/ml) for 3 h (D) or 12 h (E). Thereafter, total cellular RNA was isolated, and transcript levels of IL-13 and BLT2 were assessed by semiquantitative RT-PCR (D). Additionally, cell-free supernatants were collected and subjected to ELISA analysis for IL-13 (E). (F) WT (BLT2+/+) and BLT2 null (BLT2−/−) BMMCs were stimulated with LPS (0.1 μg/ml) for 12 h. Cell-free supernatants were collected, and IL-13 levels were determined by ELISA. (G) BMMCs were first incubated with LY255283 (10 μM) or U75302 (1 μM) for 30 min and then stimulated with LPS (0.1 μg/ml) for 12 h to induce IL-13 secretion. All quantitative data are shown as the mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001. ns, not significant.
BLT2 ligand synthesis is necessary for LPS-induced IL-13 production in BMMCs
Next, we examined the roles of BLT2 ligands [LTB4 and 12(S)-HETE] in IL-13 production in LPS-activated BMMCs. Pretreating cells with the 5-LO inhibitor MK886 or the 12-LO inhibitor baicalein clearly suppressed IL-13 production (∼40% decrease in transcript level and a ∼40% decrease in protein level with MK886; ∼70% decrease in transcript level and ∼60% decrease in protein level with baicalein) (Fig. 2A, 2B). The cPLA2 inhibitor AACOCF3 also suppressed LPS-induced IL-13 synthesis (∼50% decrease in transcript level; ∼30% decrease in protein level) (Fig. 2A, 2B). These results suggest that cPLA2, 5-LO, and 12-LO, which are necessary for the synthesis of BLT2 ligands, are involved in IL-13 production in LPS-stimulated BMMCs. We next tested the effect of blockading 5-LO or 12-LO with specific siRNAs. As shown in Fig. 2C–F, 5-LO or 12-LO knockdown suppressed the LPS-stimulated production of IL-13 (∼40% decrease in transcript level and ∼50% decrease in protein level with si5-LO; ∼60% decrease in transcript level and ∼40% decrease in protein level with si12-LO), suggesting that the BLT2 ligand–BLT2 cascade is necessary for LPS-induced IL-13 synthesis in BMMCs. In agreement with this, we observed that the levels of BLT2 ligands [LTB4 and 12(S)-HETE] were also elevated by LPS stimulation (Fig. 2G, 2H). In addition, the levels of 5-LO and 12-LO (Fig. 2I), as well as the activation of cPLA2, as revealed by its phosphorylation (Fig. 2I), were markedly increased in response to LPS stimulation in BMMCs. Taken together, these results suggest that BLT2 ligands [LTB4 and 12(S)-HETE], which are produced via the consecutive actions of cPLA2 and either 5-LO or 12-LO, are necessary for IL-13 production in LPS-stimulated BMMCs.
Synthesis of BLT2 ligands [LTB4 and 12(S)-HETE] is necessary for IL-13 production in LPS-stimulated BMMCs. (A) BMMCs were first incubated with AACOCF3 (20 μM), MK886 (5 μM), or baicalein (20 μM) for 30 min then stimulated with LPS (0.1 μg/ml) for 3 h, after which total RNA was isolated and subjected to semiquantitative RT-PCR. (B) Culture supernatants of BMMCs treated as shown in (A) were stimulated with LPS (0.1 μg/ml) for 12 h to induce IL-13 secretion. (C and D) BMMCs were transfected with 50 nM siCont or 5-LO siRNA (si5-LO). After 24 h, the cell lysates were prepared, and the level of 5-LO protein was assessed by an immunoblot assay. The data shown are representative of three independent experiments with similar results (right panel of C). Cells transfected with siCont or si5-LO were stimulated with LPS (0.1 μg/ml) for 3 h, after which total RNA was isolated and subjected to semiquantitative RT-PCR (left panel of C). Additionally, cell-free supernatants were collected after 12 h and subjected to ELISA analysis for IL-13 (D). (E and F) BMMCs were transfected with 50 nM siCont or si12-LO. After 24 h, the immunoblot assay was performed to detect the 12-LO protein level. The data shown are representative of three independent experiments with similar results (right panel of E). Cells transfected with siCont or si12-LO were stimulated with LPS (0.1 μg/ml) for 3 h, after which total RNA was isolated and subjected to semiquantitative RT-PCR (left panel of E). Additionally, cell-free supernatants were collected after 12 h and subjected to ELISA analysis for IL-13 (F). (G and H) BMMCs were stimulated with LPS (0.1 μg/ml) for 12 h, after which secreted LTB4 (G) and 12(S)-HETE (H) were assayed into the culture medium using specific ELISA kits. All quantitative data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (I) BMMCs were stimulated with LPS (0.1 μg/ml) for the indicated time periods, after which cell lysates were prepared and subjected to an immunoblot analysis with Abs to p-cPLA2, 5-LO, or 12-LO. The data shown are representative of three independent experiments with similar results.
Synthesis of BLT2 ligands [LTB4 and 12(S)-HETE] is necessary for IL-13 production in LPS-stimulated BMMCs. (A) BMMCs were first incubated with AACOCF3 (20 μM), MK886 (5 μM), or baicalein (20 μM) for 30 min then stimulated with LPS (0.1 μg/ml) for 3 h, after which total RNA was isolated and subjected to semiquantitative RT-PCR. (B) Culture supernatants of BMMCs treated as shown in (A) were stimulated with LPS (0.1 μg/ml) for 12 h to induce IL-13 secretion. (C and D) BMMCs were transfected with 50 nM siCont or 5-LO siRNA (si5-LO). After 24 h, the cell lysates were prepared, and the level of 5-LO protein was assessed by an immunoblot assay. The data shown are representative of three independent experiments with similar results (right panel of C). Cells transfected with siCont or si5-LO were stimulated with LPS (0.1 μg/ml) for 3 h, after which total RNA was isolated and subjected to semiquantitative RT-PCR (left panel of C). Additionally, cell-free supernatants were collected after 12 h and subjected to ELISA analysis for IL-13 (D). (E and F) BMMCs were transfected with 50 nM siCont or si12-LO. After 24 h, the immunoblot assay was performed to detect the 12-LO protein level. The data shown are representative of three independent experiments with similar results (right panel of E). Cells transfected with siCont or si12-LO were stimulated with LPS (0.1 μg/ml) for 3 h, after which total RNA was isolated and subjected to semiquantitative RT-PCR (left panel of E). Additionally, cell-free supernatants were collected after 12 h and subjected to ELISA analysis for IL-13 (F). (G and H) BMMCs were stimulated with LPS (0.1 μg/ml) for 12 h, after which secreted LTB4 (G) and 12(S)-HETE (H) were assayed into the culture medium using specific ELISA kits. All quantitative data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (I) BMMCs were stimulated with LPS (0.1 μg/ml) for the indicated time periods, after which cell lysates were prepared and subjected to an immunoblot analysis with Abs to p-cPLA2, 5-LO, or 12-LO. The data shown are representative of three independent experiments with similar results.
MyD88 lies upstream of BLT2 in LPS-induced signaling for IL-13 production
A previous study demonstrated that MyD88 mediates LPS-induced IL-13 synthesis in RBL-2H3 cells (6). In addition, we recently reported that MyD88 lies upstream of BLT2 in several cell types (15, 17). Therefore, we examined the involvement of MyD88 as a molecule upstream of BLT2 in the LPS-induced signaling for IL-13 production in BMMCs. First, we examined the effect of MyD88 knockdown with siRNA on the LPS-induced IL-13 synthesis. As shown in Fig. 3A and 3B, IL-13 production was greatly attenuated by MyD88 siRNA in LPS-stimulated BMMCs (∼50% decrease in transcript level; ∼70% decrease in protein level). In addition, we observed that the elevated levels of BLT2 and its ligands after LPS stimulation were greatly reduced by MyD88 siRNA (Fig. 3A, 3C, 3D). Consistent with these results, the increased levels of 5-LO and 12-LO, as well as the level of cPLA2 phosphorylation, were markedly attenuated by MyD88 siRNA in LPS-stimulated BMMCs (Fig. 3E). To further examine the role of MyD88 in the production of IL-13, we transiently transfected BMMCs with MyD88 expression plasmids. The overexpression of MyD88 alone significantly increased IL-13 levels in BMMCs, and this elevated IL-13 production was significantly reduced by the BLT2 antagonist LY255283 (∼40% decrease in transcript level; ∼50% decrease in protein level) (Fig. 3F, 3G). Together, these results suggest that MyD88 lies upstream of BLT2 and contributes to LPS-induced IL-13 production in mast cells.
MyD88 lies upstream of BLT2 in LPS signaling for IL-13 production in BMMCs. (A) Cells were transfected with 50 nM siMyD88 or siCont, incubated for 24 h, and then incubated with LPS (0.1 μg/ml) for 3 h. Total RNA was isolated and subjected to a semiquantitative RT-PCR analysis of the IL-13, BLT2, and MyD88 mRNA levels. (B) Cells were transfected and incubated as shown in (A) and stimulated with LPS (0.1 μg/ml) for 12 h, followed by assays of IL-13 release. (C and D) Cells were transfected and incubated as shown in (A) and stimulated with LPS (0.1 μg/ml) for 12 h, after which the LTB4 (C) and 12(S)-HETE (D) levels released into the culture medium were assayed. (E) Cells transfected with siCont or siMyD88 were stimulated with LPS (0.1 μg/ml) for 3 h, after which cell lysates were prepared and subjected to an immunoblot analysis with Abs to p-cPLA2, 5-LO, or 12-LO. The results shown are representative of three independent experiments with similar results. (F and G) Cells were transfected with an expression plasmid for MyD88 and then incubated for 24 h. (F) Cells transfected with the MyD88 expression plasmid were incubated with LY255283 (10 μM) for 30 min, after which total RNA was isolated and subjected to semiquantitative RT-PCR. (G) Cells were transfected and incubated as shown in (F) and incubated with LY255283 (10 μM) for 24 h, followed by assays of IL-13 release. All quantitative data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01.
MyD88 lies upstream of BLT2 in LPS signaling for IL-13 production in BMMCs. (A) Cells were transfected with 50 nM siMyD88 or siCont, incubated for 24 h, and then incubated with LPS (0.1 μg/ml) for 3 h. Total RNA was isolated and subjected to a semiquantitative RT-PCR analysis of the IL-13, BLT2, and MyD88 mRNA levels. (B) Cells were transfected and incubated as shown in (A) and stimulated with LPS (0.1 μg/ml) for 12 h, followed by assays of IL-13 release. (C and D) Cells were transfected and incubated as shown in (A) and stimulated with LPS (0.1 μg/ml) for 12 h, after which the LTB4 (C) and 12(S)-HETE (D) levels released into the culture medium were assayed. (E) Cells transfected with siCont or siMyD88 were stimulated with LPS (0.1 μg/ml) for 3 h, after which cell lysates were prepared and subjected to an immunoblot analysis with Abs to p-cPLA2, 5-LO, or 12-LO. The results shown are representative of three independent experiments with similar results. (F and G) Cells were transfected with an expression plasmid for MyD88 and then incubated for 24 h. (F) Cells transfected with the MyD88 expression plasmid were incubated with LY255283 (10 μM) for 30 min, after which total RNA was isolated and subjected to semiquantitative RT-PCR. (G) Cells were transfected and incubated as shown in (F) and incubated with LY255283 (10 μM) for 24 h, followed by assays of IL-13 release. All quantitative data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01.
Nox1 lies downstream of BLT2 in LPS-induced signaling for IL-13 production
We previously reported that Nox-derived ROS generation lies downstream of BLT2 and mediates its signal (8, 15). Thus, we hypothesized that the mechanism underlying BLT2-mediated IL-13 production in LPS-stimulated BMMCs may involve a Nox-ROS cascade. To test this idea, we measured the levels of ROS in response to LPS. We found that LPS stimulates elevated levels of ROS generation (Fig. 4A). Next, we examined the effect of DPI, an inhibitor of Nox-like flavoproteins, e.g., Nox (13), on LPS-induced IL-13 production. As shown in Fig. 4B and 4C, DPI significantly inhibited LPS-induced IL-13 production (∼70% decrease in transcript level; ∼40% decrease in protein level), suggesting that Nox likely plays a role in the LPS signaling pathway for IL-13 synthesis. To examine whether Nox is indeed associated with LPS-induced ROS generation, we examined the expression levels of Nox isotypes following LPS stimulation. Incubation with LPS induced the upregulation of Nox1 and Nox4 mRNA (Fig. 4D) but had no influence on Nox2 mRNA levels (data not shown). Interestingly, when MyD88 expression was knocked down by siRNA, the LPS-induced Nox1 upregulation was markedly attenuated (Fig. 4E), but the Nox4 mRNA levels remained unchanged (Fig. 4E). Moreover, MyD88 siRNA clearly attenuated the LPS-induced ROS generation in BMMCs (Fig. 4F). Additionally, BLT2 knockdown with siRNA clearly suppressed the LPS-induced Nox1 upregulation (Fig. 4G) and ROS generation (Fig. 4H); thus, taken together, these results suggest that Nox1 has a potential mediatory role downstream of BLT2 in the LPS signaling pathway. Therefore, we next examined the role of Nox1 in LPS-induced IL-13 production and ROS generation. To do this, we transiently transfected with Nox1 siRNA and analyzed the levels of IL-13 and ROS in response to LPS stimulation. When Nox1 was knocked down by siRNA, LPS-induced IL-13 production was significantly attenuated (∼40% decrease in transcript level; ∼30% decrease in protein level) (Fig. 4I, 4J). Additionally, LPS-induced ROS generation was attenuated by Nox1 siRNA in BMMCs (Fig. 4K), suggesting that the Nox1-derived ROS are downstream mediators of BLT2 in the LPS-induced signaling for IL-13 production.
LPS-stimulated IL-13 synthesis is mediated by a BLT2-Nox1-ROS cascade in BMMCs. (A) Cells were incubated with LPS (0.1 μg/ml) for 30 min; DCFDA (10 μM) was added to the cells and incubated for 15 min before the ROS measurements. Intracellular ROS levels were measured by a FACS analysis of DCF fluorescence. (B) Cells were pretreated for 30 min with DPI (1 μM) and then stimulated for 3 h with LPS (0.1 μg/ml), followed by an analysis of total RNA. (C) Cells were exposed as shown in (B) and incubated with LPS (0.1 μg/ml) for 12 h. IL-13 release into the culture medium was then assayed. (D) Cells were stimulated with LPS (0.1 μg/ml) for 3 h. Then, total cellular RNA was isolated, and the Nox1 and Nox4 transcript levels were assessed by semiquantitative RT-PCR. The data shown are representative of three independent experiments with similar results. (E) Cells were transfected with 50 nM siMyD88 or siCont, incubated for 24 h, and then incubated with LPS (0.1 μg/ml) for 3 h. Total RNA was then analyzed to detect the transcript levels of Nox1, Nox4, and MyD88. (F) Cells were transfected and incubated as shown in (E) and stimulated with LPS for 30 min; DCFDA (10 μM) was added to the cells and incubated for 15 min before the ROS measurements. Intracellular ROS levels were measured by a FACS analysis of DCF fluorescence. (G) Cells were transfected with 50 nM siBLT2 or siCont, incubated for 24 h, and then incubated with LPS (0.1 μg/ml) for 3 h. Total RNA was then analyzed to detect the Nox1 and BLT2 transcript levels. (H) Cells were transfected and incubated as shown in (G) and stimulated with LPS for 30 min; DCFDA (10 μM) was added to the cells and cultured for 15 min before the ROS measurements. Intracellular ROS levels were measured by a FACS analysis of DCF fluorescence. (I–K) Cells were transfected with 50 nM Nox1 siRNA (siNox1) or siCont, incubated for 24 h, and then incubated with LPS (0.1 μg/ml) for 3 h (I), 12 h (J), or 30 min (K). (I) The cells were then harvested for the detection of IL-13 and Nox1 transcript levels. (J) The IL-13 level in the culture medium was determined using ELISA. (K) DCFDA (10 μM) was added to the cells and incubated for 15 min before the ROS measurements. Intracellular ROS levels were measured by a FACS analysis of DCF fluorescence. All quantitative data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01.
LPS-stimulated IL-13 synthesis is mediated by a BLT2-Nox1-ROS cascade in BMMCs. (A) Cells were incubated with LPS (0.1 μg/ml) for 30 min; DCFDA (10 μM) was added to the cells and incubated for 15 min before the ROS measurements. Intracellular ROS levels were measured by a FACS analysis of DCF fluorescence. (B) Cells were pretreated for 30 min with DPI (1 μM) and then stimulated for 3 h with LPS (0.1 μg/ml), followed by an analysis of total RNA. (C) Cells were exposed as shown in (B) and incubated with LPS (0.1 μg/ml) for 12 h. IL-13 release into the culture medium was then assayed. (D) Cells were stimulated with LPS (0.1 μg/ml) for 3 h. Then, total cellular RNA was isolated, and the Nox1 and Nox4 transcript levels were assessed by semiquantitative RT-PCR. The data shown are representative of three independent experiments with similar results. (E) Cells were transfected with 50 nM siMyD88 or siCont, incubated for 24 h, and then incubated with LPS (0.1 μg/ml) for 3 h. Total RNA was then analyzed to detect the transcript levels of Nox1, Nox4, and MyD88. (F) Cells were transfected and incubated as shown in (E) and stimulated with LPS for 30 min; DCFDA (10 μM) was added to the cells and incubated for 15 min before the ROS measurements. Intracellular ROS levels were measured by a FACS analysis of DCF fluorescence. (G) Cells were transfected with 50 nM siBLT2 or siCont, incubated for 24 h, and then incubated with LPS (0.1 μg/ml) for 3 h. Total RNA was then analyzed to detect the Nox1 and BLT2 transcript levels. (H) Cells were transfected and incubated as shown in (G) and stimulated with LPS for 30 min; DCFDA (10 μM) was added to the cells and cultured for 15 min before the ROS measurements. Intracellular ROS levels were measured by a FACS analysis of DCF fluorescence. (I–K) Cells were transfected with 50 nM Nox1 siRNA (siNox1) or siCont, incubated for 24 h, and then incubated with LPS (0.1 μg/ml) for 3 h (I), 12 h (J), or 30 min (K). (I) The cells were then harvested for the detection of IL-13 and Nox1 transcript levels. (J) The IL-13 level in the culture medium was determined using ELISA. (K) DCFDA (10 μM) was added to the cells and incubated for 15 min before the ROS measurements. Intracellular ROS levels were measured by a FACS analysis of DCF fluorescence. All quantitative data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01.
NF-κB activation lies downstream of the MyD88-BLT2-Nox1-ROS cascade in LPS-induced signaling for IL-13 production
ROS have been reported to regulate the activity of NF-κB (18), a principal transcription factor involved in IL-13 expression (19). In addition, we recently reported that BLT2 regulates NF-κB activation in mast cells (13, 14). Therefore, we examined the involvement of NF-κB as a molecule downstream of the MyD88-BLT2-Nox1-ROS cascade in the LPS-stimulated signaling for IL-13 synthesis in BMMCs. First, we examined the effects of the NF-κB inhibitor Bay11-7082 on LPS-induced IL-13 synthesis. As shown in Fig. 5A and 5B, IL-13 production was greatly inhibited by pretreatment with Bay11-7082 in LPS-stimulated BMMCs (∼70% decrease in transcript level; ∼50% decrease in protein level). We also found that MyD88 siRNA resulted in a marked attenuation of LPS-induced NF-κB activation, as indicated by reduced IκBα phosphorylation levels (Fig. 5C). To analyze the role of BLT2 in LPS-induced NF-κB activation, we knocked down BLT2 using siRNA. As shown in Fig. 5D, BLT2 siRNA clearly attenuated LPS-induced NF-κB activation. Similarly, pretreatment with LY255283 also resulted in a marked attenuation of LPS-induced NF-κB activation in BMMCs (Fig. 5E). Additionally, Nox1 knockdown markedly inhibited NF-κB activation in LPS-stimulated BMMCs (Fig. 5F). Taken together, these results indicate that a MyD88-BLT2-Nox1-NF-κB–dependent signaling pathway contributes to IL-13 production in BMMCs.
NF-κB activation is necessary for IL-13 synthesis in BMMCs. (A and B) Cells were first incubated with Bay11-7082 (20 μM) for 30 min and then stimulated for 3 h (A) or 12 h (B) with LPS (0.1 μg/ml). Total RNA was then isolated for IL-13 transcript level analysis (A), or the level of IL-13 released into the culture medium was assayed (B). Data are shown as the mean ± SD of three independent experiments. **p < 0.01. (C) BMMCs were transfected with siMyD88 or siCont and incubated for 24 h, after which semiquantitative RT-PCR was performed to detect the MyD88 transcript levels (right panel). Cells transfected with siCont or siMyD88 were stimulated with LPS (0.1 μg/ml) for 1 h, after which the cell lysates were subjected to an immunoblot analysis with Abs to the indicated proteins. The data shown are representative of three independent experiments with similar results (left panel). (D) BMMCs were transfected with siBLT2 or siCont and incubated for 24 h, after which semiquantitative RT-PCR was performed to detect the BLT2 transcript levels (right panel). Cells transfected with siCont or siBLT2 were stimulated with LPS (0.1 μg/ml) for 1 h, after which the cell lysates were subjected to an immunoblot analysis, as shown in (C). The results shown are representative of three independent experiments with similar results (left panel). (E) BMMCs were first incubated with LY255283 (10 μM) for 30 min and then stimulated with LPS (0.1 μg/ml) for 1 h, after which the cell lysates were subjected to an immunoblot analysis, as shown in (C). The data shown are representative of three independent experiments with similar results. (F) BMMCs were transfected with siNox1 or siCont and incubated for 24 h, after which semiquantitative RT-PCR was performed to detect the Nox1 transcript levels (right panel). Cells transfected with siCont or siNox1 were stimulated with LPS (0.1 μg/ml) for 1 h, after which the cell lysates were subjected to an immunoblot analysis, as shown in (C). The data shown are representative of three independent experiments with similar results (left panel).
NF-κB activation is necessary for IL-13 synthesis in BMMCs. (A and B) Cells were first incubated with Bay11-7082 (20 μM) for 30 min and then stimulated for 3 h (A) or 12 h (B) with LPS (0.1 μg/ml). Total RNA was then isolated for IL-13 transcript level analysis (A), or the level of IL-13 released into the culture medium was assayed (B). Data are shown as the mean ± SD of three independent experiments. **p < 0.01. (C) BMMCs were transfected with siMyD88 or siCont and incubated for 24 h, after which semiquantitative RT-PCR was performed to detect the MyD88 transcript levels (right panel). Cells transfected with siCont or siMyD88 were stimulated with LPS (0.1 μg/ml) for 1 h, after which the cell lysates were subjected to an immunoblot analysis with Abs to the indicated proteins. The data shown are representative of three independent experiments with similar results (left panel). (D) BMMCs were transfected with siBLT2 or siCont and incubated for 24 h, after which semiquantitative RT-PCR was performed to detect the BLT2 transcript levels (right panel). Cells transfected with siCont or siBLT2 were stimulated with LPS (0.1 μg/ml) for 1 h, after which the cell lysates were subjected to an immunoblot analysis, as shown in (C). The results shown are representative of three independent experiments with similar results (left panel). (E) BMMCs were first incubated with LY255283 (10 μM) for 30 min and then stimulated with LPS (0.1 μg/ml) for 1 h, after which the cell lysates were subjected to an immunoblot analysis, as shown in (C). The data shown are representative of three independent experiments with similar results. (F) BMMCs were transfected with siNox1 or siCont and incubated for 24 h, after which semiquantitative RT-PCR was performed to detect the Nox1 transcript levels (right panel). Cells transfected with siCont or siNox1 were stimulated with LPS (0.1 μg/ml) for 1 h, after which the cell lysates were subjected to an immunoblot analysis, as shown in (C). The data shown are representative of three independent experiments with similar results (left panel).
LPS-induced IL-13 production in the allergic response is significantly attenuated by BLT2 blockade in vivo
Previously, we reported that BLT2 mediates IgE/FcεRI-induced signaling for the synthesis of Th2 cytokines, such as IL-13, in BMMCs (8). Thus, we speculated that BLT2 may play a connective role between the LPS/TLR4 and IgE/FcεRI signaling pathways, leading to IL-13 production in BMMCs. Interestingly, we observed that costimulation with LPS/TLR4 and IgE/FcεRI caused greatly enhanced IL-13 synthesis in BMMCs (Fig. 6A–C), and these effects were markedly attenuated by blockading BLT2 with siRNA or an antagonist (∼30% decrease in transcript level and ∼50% decrease in protein level with siBLT2; ∼50% decrease in protein level with LY255283) (Fig. 6A–C). Next, we tested whether BLT2 plays a role in the LPS-induced signaling for IL-13 production in vivo, and we clearly observed that BLT2 inhibition with the BLT2 antagonist LY255283 significantly reduced the IL-13 levels in the S/L- (∼30% decrease in transcript level; ∼70% decrease in protein level), O/O- (∼50% decrease in transcript level; ∼40% decrease in protein level), and O/OL-treated mice (∼30% decrease in transcript level; ∼60% decrease in protein level) (Fig. 6D, 6E). The numbers of eosinophils in the BALF were significantly reduced by LY255283 in the S/L-, O/O-, and O/OL-treated mice compared with the S/S control mice (Fig. 6F). Additionally, airway inflammation was also markedly reduced by LY255283 administration, as determined by histological and quantitative analysis of inflammation scores (Fig. 6G, 6H).
BLT2 is a connection that links LPS/TLR4 and IgE/FcεRI signaling for IL-13 production. (A and B) BMMCs were transfected with 50 nM siBLT2 or siCont and incubated for 24 h, after which semiquantitative RT-PCR was performed to detect the BLT2 transcript levels (right panel of A). Cells transfected with siCont or siBLT2 were sensitized with anti-DNP IgE and were stimulated with LPS (0.1 μg/ml), DNP-BSA (50 ng/ml), or LPS (0.1 μg/ml) + DNP-BSA (50 ng/ml). (A) After 3 h, total cellular RNA was isolated, and the IL-13 transcript levels were assessed by semiquantitative RT-PCR (left panel of A). Additionally, after 12 h, cell-free supernatants were collected and subjected to ELISA analysis for IL-13 (B). (C) BMMCs were first incubated with LY255283 (10 μM) for 30 min and then sensitized and stimulated as shown in (A) for 12 h to induce IL-13 secretion. All quantitative data are shown as the mean ± SD of three independent experiments. #p < 0.001 versus no treatment, *p < 0.05, **p < 0.01. (D–H) S/S mice, S/L mice, O/O mice, and O/OL mice were sacrificed on day 17 to assess airway inflammation. (D) The lungs of the mice were homogenized, RNA was isolated, and the IL-13 and BLT2 transcript levels were assessed by semiquantitative RT-PCR. (E) BALF IL-13 levels were analyzed using a specific ELISA kit. Data are shown as the mean ± SD (n = 4–5 per group). &p < 0.05, ‡p < 0.01, #p < 0.001 versus the corresponding S/S control group, *p < 0.05, **p < 0.01 versus the group indicated. (F) Infiltration of eosinophils into the BALF. Eosinophils in the BALF were obtained using a cytospin and stained with H&E (n = 4–5 per group). ND, not detected. ‡p < 0.01 versus the corresponding S/S control group, *p < 0.05, **p < 0.01 versus the group indicated. (G) Airway inflammation in the lung tissues was histologically analyzed. Lungs were excised, fixed, and stained with H&E (n = 4–5 per group). Scale bars, 50 μm. (H) Peribronchial and perivascular lung inflammation was measured, and total lung inflammation was defined as the average of the peribronchial and perivascular inflammation scores (n = 4–5 per group). #p < 0.001 versus the corresponding S/S control group, *p < 0.05, **p < 0.01, ***p < 0.001 versus the group indicated. (I) Scheme of intracellular signaling responsible for LPS/TLR4-induced IL-13 synthesis dependent on BLT2 in mast cells.
BLT2 is a connection that links LPS/TLR4 and IgE/FcεRI signaling for IL-13 production. (A and B) BMMCs were transfected with 50 nM siBLT2 or siCont and incubated for 24 h, after which semiquantitative RT-PCR was performed to detect the BLT2 transcript levels (right panel of A). Cells transfected with siCont or siBLT2 were sensitized with anti-DNP IgE and were stimulated with LPS (0.1 μg/ml), DNP-BSA (50 ng/ml), or LPS (0.1 μg/ml) + DNP-BSA (50 ng/ml). (A) After 3 h, total cellular RNA was isolated, and the IL-13 transcript levels were assessed by semiquantitative RT-PCR (left panel of A). Additionally, after 12 h, cell-free supernatants were collected and subjected to ELISA analysis for IL-13 (B). (C) BMMCs were first incubated with LY255283 (10 μM) for 30 min and then sensitized and stimulated as shown in (A) for 12 h to induce IL-13 secretion. All quantitative data are shown as the mean ± SD of three independent experiments. #p < 0.001 versus no treatment, *p < 0.05, **p < 0.01. (D–H) S/S mice, S/L mice, O/O mice, and O/OL mice were sacrificed on day 17 to assess airway inflammation. (D) The lungs of the mice were homogenized, RNA was isolated, and the IL-13 and BLT2 transcript levels were assessed by semiquantitative RT-PCR. (E) BALF IL-13 levels were analyzed using a specific ELISA kit. Data are shown as the mean ± SD (n = 4–5 per group). &p < 0.05, ‡p < 0.01, #p < 0.001 versus the corresponding S/S control group, *p < 0.05, **p < 0.01 versus the group indicated. (F) Infiltration of eosinophils into the BALF. Eosinophils in the BALF were obtained using a cytospin and stained with H&E (n = 4–5 per group). ND, not detected. ‡p < 0.01 versus the corresponding S/S control group, *p < 0.05, **p < 0.01 versus the group indicated. (G) Airway inflammation in the lung tissues was histologically analyzed. Lungs were excised, fixed, and stained with H&E (n = 4–5 per group). Scale bars, 50 μm. (H) Peribronchial and perivascular lung inflammation was measured, and total lung inflammation was defined as the average of the peribronchial and perivascular inflammation scores (n = 4–5 per group). #p < 0.001 versus the corresponding S/S control group, *p < 0.05, **p < 0.01, ***p < 0.001 versus the group indicated. (I) Scheme of intracellular signaling responsible for LPS/TLR4-induced IL-13 synthesis dependent on BLT2 in mast cells.
Discussion
In the current study, we found that LPS-stimulated IL-13 synthesis is mediated by a BLT2-dependent pathway in BMMCs. In addition, we identified that MyD88, a pivotal regulator of the TLR4 signaling pathway, acts upstream of BLT2 and that the Nox1-ROS-NF-κB cascade acts downstream of BLT2, contributing to LPS-induced IL-13 synthesis in mast cells. Our findings may provide insight into the mechanisms underlying how innate immunity contributes to the allergic response.
Mast cells have been shown to play a pivotal role in both the innate immune and adaptive allergic immune responses. As a part of the innate immune system, mast cells have been found to express various TLRs (3). In particular, TLR4 is recognized by LPS, a key component of the outer membrane of Gram-negative bacteria (2, 4). LPS is ubiquitously present in the environment; it has been detected in polluted air and even in house dust (20, 21). Mast cells are most common at sites that are exposed to the external environment, such as the skin, gut, and airways (3). In these locations, they encounter and respond to pathogens, such as LPS, thereby initiating the innate immune defense response (3). Recently, LPS/TLR4-driven innate immunity in mast cells has been suggested to contribute to the allergic response under certain conditions. For example, a low dose of inhaled LPS induced Th2 responses to allergens in a mouse model of allergic sensitization, but not in mast cell–deficient mice or in TLR4-knockout mice, and BMMC transfer experiments using TLR4-knockout mast cells indicated that the effect of LPS on mast cells was mediated by TLR4 (2). Similarly, mast cells stimulated with LPS could produce Th2 cytokines such as IL-13 (22). In addition, low doses of LPS have been previously shown to augment eosinophilic inflammation in mice treated with OVA (23). These results are consistent with the clinical observation that microbial infections later in life occasionally exacerbate established asthma (24, 25). However, different studies have suggested that exposure to high doses of LPS could induce a Th1 response, which counter-balances the proallergic Th2 response (23, 26). Thus, depending on the exposure dose, timing, or route of administration, among other factors, there could be opposing roles of LPS/TLR4 in terms of the allergic response (26, 27). In any event, in our experimental model we observed that a low LPS dose could stimulate the production of IL-13 in BMMCs. Additionally, the low LPS dose significantly augmented eosinophilic inflammation as well as the production of IL-13 in mice challenged intranasally with OVA (Fig. 6). IL-4 and IL-13 are major Th2 cytokines and are considered to have similar activity (28). Interestingly, we could not detect IL-4 production after LPS stimulation in mast cells (data not shown), suggesting that the mechanisms for IL-4 and IL-13 production may be distinct. Indeed, it has been reported that IL-13 is not always coexpressed with other Th2 cytokines, such as IL-4 in mast cells and Th2 cells (2, 4).
In the current study, our results clearly suggest that IL-13 synthesis is largely mediated in a BLT2-dependent manner in LPS-stimulated mast cells (Fig. 1D–G). We also demonstrated that the synthesis of the BLT2 ligands LTB4 and 12(S)-HETE is critical for IL-13 production in LPS-stimulated mast cells (Fig. 2), suggesting that these BLT2 ligands may act in an autocrine or paracrine manner through BLT2. Consistent with the proposed action of the BLT2 ligand–BLT2 axis in LPS-induced IL-13 production, our previous study demonstrated that BLT2 overexpression alone significantly increased the levels of IL-13 production, and the addition of BLT2 ligands [LTB4 and 12(S)-HETE] further enhanced IL-13 production (8). Moreover, we demonstrated that MyD88 lies upstream of the BLT2 ligand–BLT2 axis (Fig. 3), thereby mediating the synthesis of IL-13. We also found that the Nox1-ROS cascade plays a role in BLT2-mediated IL-13 production in BMMCs (Fig. 4G, 4H), and that NF-κB is downstream of the MyD88-BLT2-Nox1 cascade for signaling IL-13 production (Fig. 5C–F). Consistent with our findings, a recent study demonstrated that ROS generation induced by LPS could lead to the activation of the redox-sensitive transcription factor NF-κB (18), one of the principal transcription factors involved in IL-13 expression (19). We also previously reported that BLT2 regulates NF-κB activation in mast cells (13, 14). Together, these results suggest that Nox1 upregulation and subsequent ROS generation lie downstream of BLT2 and contribute to the increased IL-13 synthesis in LPS-stimulated mast cells.
Recently, BLT2 has been demonstrated to be critical for the synthesis of Th2 cytokines, such as IL-13, during IgE/FcεRI-mediated mast cell activation in the allergic response (8). Therefore, we suspect that BLT2 mediates both LPS/TLR4 and IgE/FcεRI signaling for IL-13 production in mast cells. Consistent with this idea, costimulation with LPS/TLR4 and IgE/FcεRI caused greatly enhanced IL-13 synthesis in mast cells, and these effects were completely abolished by BLT2 blockade (Fig. 6A–C). Similarly, in vivo, the level of IL-13 was markedly increased by LPS administration in OVA-induced allergic asthma, and injecting a BLT2 antagonist beforehand clearly attenuated this increase. Taken together, our findings suggest that BLT2 likely acts as a potential connection between the innate and adaptive immune systems, leading to a Th2 response, at least in mast cells, as proposed in the hypothetical model (Fig. 6I). This model proposes that MyD88 lies downstream of TLR4, thus mediating LPS/TLR4 signaling for IL-13 production, as shown in Fig. 3. However, IgE/FcεRI signaling for IL-13 production was not affected by MyD88 knockdown in BMMCs (Supplemental Fig. 3).
In summary, our study shows that BLT2 plays a critical role in LPS-induced IL-13 production in mast cells via a TLR4-MyD88-BLT2-Nox1-ROS-NF-κB–linked cascade. Our findings not only suggest that BLT2 mediates LPS-triggered signaling for IL-13 production in mast cells, but may also provide insight into the mechanisms underlying how an infection can worsen allergic inflammation.
Footnotes
This work was supported by Bio and Medical Technology Development Program Grants 2012 M3A9C5048709 and 2012M3A9C1053532 and Mid-Career Researcher Program Grant 2017R1A2B4002203 through the National Research Foundation funded by the Ministry of Science, Information and Communication Technologies, and Future Planning, Republic of Korea. This work was also supported by Basic Science Research Grant 2015R1D1A1A01057757 through the National Research Foundation funded by the Ministry of Education and the BK21 Plus Program (College of Life Sciences and Biotechnology, Korea University), as well as by a Korea University Grant.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- BALF
bronchoalveolar lavage fluid
- BLT2
leukotriene B4 receptor-2
- BMMC
bone marrow–derived mast cell
- DNP-BSA
DNP-conjugated BSA
- DPI
diphenyleneiodonium
- LO
lipoxygenase
- LTB4
leukotriene B4
- Nox1
NADPH oxidase 1
- O/O
OVA/OVA
- O/OL
OVA/OVA+LPS
- p-cPLA2
p-cytosolic phospholipase A2
- ROS
reactive oxygen species
- 12(S)-HETE
12(S)-hydroxyeicosatetraenoic acid
- siRNA
small interfering RNA
- S/L
Saline/LPS
- S/S
Saline/Saline
- WT
wild type.
References
Disclosures
The authors have no financial conflicts of interest.