Ragweed pollen extract intensifies lipopolysaccharide-induced priming of NLRP3 inflammasome in human macrophages

Abstract

Ragweed pollen extract (RWE) possesses intrinsic NADPH oxidase activity that induces oxidative stress by initiating the production of intracellular reactive oxygen species (ROS). The ROS are important contributors to the manifestation of allergic inflammation; furthermore, concomitant exposure to an allergen and an endotoxin trigger a stronger inflammatory response. One of the main pro-inflammatory cytokines produced in inflammatory responses is interleukin-1β (IL-1β), and its production is associated with caspase-1-containing inflammasome complexes. Intracellular ROS have been implicated in NLRP3 inflammasome-mediated IL-1β production, therefore, we aimed to study whether RWE influences the function of NLRP3 inflammasome. Here we describe that, in the presence of NADPH, RWE significantly elevates lipopolysaccharide-induced IL-1β production of THP-1 cells as well as human primary macrophages and dendritic cells. We also demonstrate that increased IL-1β production is mediated through NLRP3 inflammasome in THP-1 macrophages. We provide evidence that RWE elevates cytosolic ROS level in these cells, and ROS inhibitors abolish IL-1β production. Furthermore, we show that RWE enhances lipopolysaccharide-induced gene transcription/expression of pro-IL-1β and key components of the inflammasome via a ROS-dependent mechanism.

Figure 1

Ragweed pollen extract (RWE) enhances lipopolysaccharide (LPS) -induced interleukin-1β (IL-1β) production by macrophages and dendritic cells in the presence of NADPH. (a) THP-1 macrophages were stimulated for 24 hr with an increasing amount (10, 30 and 100 μg/ml) of RWE in the absence or presence of LPS and 100 μm NADPH, as indicated. The supernatants were collected and their IL-1β content was measured using an ELISA method in triplicates. The experiment was repeated five times and results of a representative set are provided. (b) Human monocyte-derived macrophages and dendritic cells were treated with 500 ng/ml LPS in the absence or presence of 100 ug/ml RWE and 100 μm NADPH. Twenty-four hours after treatment supernatants were collected and their IL-1β content was determined using an ELISA method in triplicates. Experiments were repeated four times and results were combined. Mean ± SD values are provided. *P < 0·05, **P < 0·005.

Figure 2

Ragweed pollen extract (RWE) leads to intracellular reactive oxygen species (ROS) production and ROS inhibitors abolish interleukin-1β (IL-1β) production. (a) THP-1 cells were loaded with H₂DCFDA, treated with various combinations of 100 μg/ml RWE, 100 μm NADPH and 1000 ng/ml lipopolysaccharide (LPS), and changes in the intracellular ROS level were measured using flow cytometry for the indicated time interval; 1 mm H₂O₂ was used as a positive control. Mean intensity of fluorescence was calculated from the positive area defined by the stained cells. (b) THP-1 cells were pre-treated with 30 mm NAC, 300 μm MitoTempo or 10 μm DPI for 1 hr then treated with 100 ng/ml LPS in the presence or absence of 10 μg/ml RWE and 100 μm NADPH. Twenty-four hours after treatment the secreted IL-1β was measured from the collected supernatants in triplicates by an ELISA method. Results were obtained in three independent experiments, and a representative result set is shown. *P < 0·1, **P < 0·01, ***P < 0·001.

Figure 3

Caspase-1 inhibition and NLRP3 silencing abolish ragweed pollen extract (RWE) -enhanced lipopolysaccharide (LPS) -induced interleukin-1β (IL-1β) production. (a) THP-1 macrophages were treated with 100 ng/ml LPS and 10 μg/ml RWE in the absence or presence of the caspase-1-specific inhibitor Z-YVAD-FMK (20 μm), for 24 hr. IL-1β content of the supernatants was determined using an ELISA method. (b–d) THP-1 cells were electroporated with scrambled (SCR) small interfering (si) RNA, as negative control or siRNA specific for NLRP3. Two days after electroporation, cells were stimulated with LPS (100 ng/ml), RWE (10 μg/ml) and NADPH (100 μm) for 24 hr. After harvesting the cells, RNA was purified and NLRP3 transcription was measured by quantitative real-time PCR (b), while NLRP3 protein expression of NLRP3 was detected by Western blot technique (Alexis Biochemicals) (c). Gene expression is shown as the ratio of the studied transcripts relative to human cyclophilin expression (± SD) measured in triplicates. Equal amount of protein sample loading was verified by detecting β-actin protein expression (rabbit polyclonal antibody, Sigma-Aldrich). (d) The concentration of secreted IL-1β was measured from the supernatant of the cells in triplicates using an ELISA method. Results were obtained in three independent experiments, and a representative result set is shown. * P < 0·05, ** P < 0·005.

Figure 4

Ragweed pollen extract (RWE) increases NLRP3, caspase-1 and interleukin-1β (IL-1β) gene transcription or expression of lipopolysaccharide (LPS) -treated cells. THP-1 macrophages were treated with LPS (100 ng/ml), RWE and NADPH in the absence or presence of RWE (10 μg/ml) and NADPH (100 μm). Twenty-four hours following treatment cells were collected and gene expression of (a) NLRP3, (c) caspase-1 and (g) pro-IL-1 β was determined using quantitative RT-PCR. Protein expression was determined by Western blot technique using antibodies recognizing (b) NLRP3, (d) procaspase-1, (f) caspase-1 and (h) IL-1β. Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (anti-mouse and anti-rabbit, Amersham). Gene expression is shown as the ratio of NLRP3, caspase-1 or pro-IL-1β transcripts relative to human cyclophilin expression (± SD) measured in triplicates. For Western blot an equal amount of protein sample loading was verified by detecting β-actin protein expression. Protein levels were estimated by densitometric analysis of the bands using KODAK 1D Image Analysis Software. A representative immunoblot is also shown for NLRP3, caspase-1 and IL-1β. Densitometry values provided are the average of Western blot runs of three independent experiments. (e) From the lysate of THP-1 macrophages the activity of caspase-1 enzyme was determined using fluorometric peptide substrate in triplicates. Mean ± SD values of three independent experiments are shown. *P < 0·1, **P < 0·01

Figure 5

Ragweed pollen extract (RWE)-induced gene expression is NADPH-dependent and is suppressed by a reactive oxygen species (ROS)-inhibitor. THP-1 macrophages were treated for 24 hr with the compounds as indicated, DPI was used in 10 μm. After harvesting the cells, the gene expression of (a) pro-interleukin-1β (pro-IL-1β), (b) caspase-1 and (c) NLRP3 was determined by quantitative real-time PCR. Gene expression is shown as the ratio of the studied transcripts relative to human cyclophilin expression (± SD) measured in triplicates. Results were obtained from four independent experiments, and a representative result set is shown.*P < 0·05, **P < 0·005, ***P < 0·0005.

Figure 6

Ragweed pollen extract (RWE) triggers stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), p38 mitogen-activated protein kinase (MAPK) and AP-1 signalling. (a, c) THP-1 macrophages were treated with 30 μg/ml RWE in the absence or presence of 100 μm NADPH, or (b, d) with 100 ng/ml lipopolysaccharide (LPS) in the absence or presence of RWE plus NADPH. Five hours after treatment, cells were harvested, protein lysates were prepared and the phosphorylation of (a, b) p38 MAPK, SAPK/JNK, (c, d) c-Jun (phosphorylated at serine 73 or 63) and c-Fos; furthermore total c-Jun and c-Fos were analysed by Western blot method. To verify the loading of equal amounts of protein sample, the β-actin protein expression was detected (c, d) or total p38 and SAPK/JNK were used as loading controls (a, b). Results obtained from three independent experiments, and one representative Western blot are shown.