Quercetin negatively regulates IL-1β production in Pseudomonas aeruginosa-infected human macrophages through the inhibition of MAPK/NLRP3 inflammasome pathways

Abstract

Pseudomonas aeruginosa remains a leading cause of nosocomial and serious life-threatening infections, and contributes to increased mortality in immunocompromised individuals. P. aeruginosa infection triggers host immune response and often provokes potent inflammatory mediators, which do not necessarily eradicate the causative pathogen. On the other hand, it causes severe airway damage and eventually decreased lung function. Such unfavorable outcomes of inflammatory injury have necessitated the development of novel effective agents that can combat with P. aeruginosa-mediated inflammation. Herein, we investigated the potential of quercetin in regulating P. aeruginosa-induced inflammation, with particular emphasized on the interleukin-1β (IL-1β). Our results showed that quercetin exerted the potent inhibitory activity against the production of IL-1β in macrophages infected by live P. aeruginosa PAO1, without exhibiting cytotoxicity. According to our settings, such the potent inhibitory activity of quercetin was clearly demonstrated through its ability to efficiently inhibit IL-1β during P. aeruginosa infection, pre- or even post-infection. In addition, quercetin strongly suppressed MAPK signaling pathway by inhibiting phosphorylation of the p38 MAPK and JNK2, and molecular docking study supported well with this observation. Moreover, quercetin reduced the NLRP3 expression and inhibited the P. aeruginosa-mediated cleavage of caspase-1 as well as mature IL-1β. These results thus indicated that quercetin inhibition of P. aeruginosa-induced IL-1β production is mediated by suppressing the initial priming step and by inhibiting the NLRP3 inflammasome activation. Taken together, our findings demonstrated the promising regulatory activity of quercetin against IL-1β production in P. aeruginosa-infected macrophages, and indicated that quercetin has the potential to be effective as a novel therapeutic agent for treatment of P. aeruginosa-induced inflammation.

Fig 1. Effect of quercetin on P. aeruginosa growth.

P. aeruginosa strain PAO1 was treated with quercetin at concentrations ranged from 20–100 μM. After incubation at 37°C for 24 h, bacterial growth was measured at OD600. Values are expressed as mean ± SEM of three independent experiments.

Fig 2. Effect of quercetin on cell viability of THP-1 macrophages.

THP-1 macrophages were treated with quercetin at the concentrations ranged from 20–100 μM or vehicle for 48 h and cell viability was assessed by an MTT assay. Values are expressed as mean ± SEM of three independent experiments.

Fig 3. Inhibitory effects of quercetin on IL-1β production in P. aeruginosa-infected THP-1 macrophages.

THP-1 macrophages were treated with quercetin (20–100 μM) at the same time as P. aeruginosa PAO1 (MOI = 10) for 4 h (A), or 2 h prior to P. aeruginosa exposure (B), or 2 h after P. aeruginosa exposure (C), and the levels of IL-1β were measured by sandwich ELISA. Values are represented as mean ± SEM of at least three independent experiments. ### p < 0.001 compared with the unstimulated THP-1 macrophages. ** p < 0.01 and *** p < 0.001 compared with the P. aeruginosa PAO1-infected THP-1 macrophages.

Fig 4. Effects of quercetin on the expression of MAPK pathway-related proteins in THP-1 macrophages infected by P. aeruginosa.

THP-1 macrophages were treated P. aeruginosa PAO1 at an MOI of 10 in the presence or absence of quercetin (40–100 μM) for 30 min and protein expression was evaluated by Western blotting (A). Bar diagrams showing densitometric analysis of the relative expression of p-p38/β-actin (B) and p-JNK (54 kDa)/β-actin (C), quantified using ImageJ software. Values are represented as mean ± SEM of three independent experiments. # p < 0.05 and ### p < 0.001 compared with the unstimulated THP-1 macrophages. * p < 0.05 and *** p < 0.001 compared with the P. aeruginosa PAO1-infected THP-1 macrophages.

Fig 5. Molecular docking and pose generation.

Chemical structure of quercetin is shown (A). A docking study was performed as described in Materials and Methods. Quercetin was docked with p38 MAPK structure (PDB ID: 5XYY) (B), and the JNK2 structure (PDB ID: 3E7O) (C). The proteins are shown in gray cartoon representation, interacting residues are shown in stick representation, the docked quercetin is represented as black ball and sticks, and hydrogen bonds are indicated as broken lines.

Fig 6. Effects of quercetin on the expression of NLRP3 proteins and IL-1β synthesis in P. aeruginosa-infected THP-1 macrophages.

THP-1 macrophages were treated P. aeruginosa PAO1 (MOI = 10) in the presence or absence of quercetin (40–100 μM) for 4 hr and protein expression was determined by Western blotting (A). Bar diagrams showing densitometric analysis of the relative expression of caspase-1/β-actin (B), IL-1β/β-actin (C) and NLRP3/β-actin (D), quantified using ImageJ software. Values are represented as mean ± SEM of three independent experiments. ### p < 0.001 compared with the unstimulated THP-1 macrophages. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the P. aeruginosa PAO1-infected THP-1 macrophages.