Punicalagin as a novel selective aryl hydrocarbon receptor (AhR) modulator upregulates AhR expression through the PDK1/p90RSK/AP-1 pathway to promote the anti-inflammatory response and bactericidal activity of macrophages

Abstract

Aryl hydrocarbon receptor (AhR) plays an important role in inflammation and immunity as a new therapeutic target for infectious disease and sepsis. Punicalagin (PUN) is a Chinese herbal monomer extract of pomegranate peel that has beneficial anti-inflammatory, antioxidant and anti-infective effects. However, whether PUN is a ligand of AhR, its effect on AhR expression, and its signaling pathway remain poorly understood. In this study, we found that PUN was a unique polyphenolic compound that upregulated AhR expression at the transcriptional level, and regulated the AhR nongenomic pathway. AhR expression in lipopolysaccharide-induced macrophages was upregulated by PUN in vitro and in vivo in a time- and dose-dependent manner. Using specific inhibitors and siRNA, induction of AhR by PUN depended on sequential phosphorylation of 90-kDa ribosomal S6 kinase (p90RSK), which was activated by the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphoinositide-dependent protein kinase (PDK)1 pathways. PUN promoted p90RSK-mediated activator protein-1 (AP-1) activation. AhR knockout or inhibitors reversed suppression of interleukin (IL)-6 and IL-1β expression by PUN. PUN decreased Listeria load and increased macrophage survival via AhR upregulation. In conclusion, we identified PUN as a novel selective AhR modulator involved in AhR expression via the MEK/ERK and PDK1 pathways targeting p90RSK/AP-1 in inflammatory macrophages, which inhibited macrophage inflammation and promoted bactericidal activity.

Introduction

Infectious diseases, such as sepsis, are an important global health problem. As a syndrome caused by underlying infection, sepsis causes life-threatening organ dysfunction by dysregulation of the host response to infection [1]. The critical pathological mechanisms of sepsis are related to excessive inflammation caused by activation of the immune system and diminished ability to eliminate pathogenic bacteria. Within this context, macrophages perform a pivotal role and act as key target cells for therapeutic intervention [2]. The aryl hydrocarbon receptor (AhR) is a basic helix–loop–helix transcription factor. Accumulating evidence shows that AhR has a variety of physiological effects, and is related to the occurrence and development of various diseases [3]. The pivotal roles of AhR in human physiology and pathophysiology imply that it is a promising therapeutic target [4]. Recently, AhR has been shown to have a dual impact characterized by anti-inflammatory regulation and facilitation of bactericidal function in macrophages, which is a potential new interventional target in sepsis and infectious diseases.

Previous reports have demonstrated the pivotal role of AhR in inflammation and immunity. AhR activation contributes to the establishment of endotoxin tolerance, and septic mice characterized by AhR activation are resistant to lethal attack by Gram-positive and Gram-negative bacteria [5]. AhR senses distinct bacterial virulence factors and controls antibacterial responses, and acts as a major sensor of infection dynamics, coordinating host defense according to infection status [6, 7]. Recent studies have also shown that AhR is a proviral host factor and a candidate therapeutic target for SARS-CoV-2 [8].

AhR signaling pathways include genomic and nongenomic pathways [9]. Inactive AhR exists in complex form with Hsp90, p23, and Src in the cytoplasm, and activated AhR regulates downstream molecules through genomic and nongenomic signaling pathways [10]. Ligands bind to AhR and activate the genomic pathway by promoting AhR translocation to the nucleus. The formation of active heterodimers between AhR and AhR nuclear translocator (ARNT) in the nucleus promotes downstream target gene expression, including cytochrome P450 (CYP)1A1 [10]. In addition to the classical genomic pathway of AhR activation, several alternative nongenomic pathways are completely dependent on the presence of AhR, but do not require nuclear translocation of AhR and binding to dioxin response elements, and their regulatory targets are on the cytoplasm or cell membrane [11]. The genomic pathway tends to mediate the toxicological effects of AhR, while nongenomic pathways are mainly involved in inflammation and immunoregulation [11]. Therefore, it is a more accurate intervention strategy to search for regulators that selectively regulate AhR cytoplasmic expression and nongenomic pathways.

Many AhR ligands (synthetic and natural) have been found. Since its discovery, AhR has been characterized as a xenosensor, specifically activated by classical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), thereby causing harmful effects to host organisms [12]. Therefore, scientists have been hunting for nonclassical AhR ligands that are less harmful. Natural AhR ligands such as resveratrol, quercetin, and curcumin from polyphenolic compounds possess low toxicity while still maintaining efficacy via the AhR pathway [13]. Punicalagin (PUN) is a Chinese herbal monomer and the main polyphenol component in pomegranate peel extract, which exhibit diverse activities, including antimicrobial, antioxidant, anti-inflammatory, and antitumor activities [14,15,16]. Several studies have shown the roles of PUN in the modulation of inflammation-related disorders [17]. Despite growing interest in the biological and pharmacological activities of PUN, its pharmacological mechanisms and their effects on AhR signaling remain poorly understood.

The 90-kDa ribosomal S6 kinase (p90RSK) family is composed of a group of highly homologous serine/threonine kinases that regulate a wide range of cellular processes, such as cell growth, survival, proliferation, and motility. The p90RSK kinases are directly phosphorylated by extracellular signal-regulated kinase (ERK) and phosphoinositide-dependent protein kinase (PDK)1, resulting in their activation. p90RSK activation induces the downstream gene phosphorylation and transcription of c-fos, which is a part of activator protein-1 (AP-1) transcription factor family [18]. The AhR signaling pathway interconnects with various other pathways, such as ERK and AP-1 [19, 20]. The AhR ligands, including PM10 [21] and indoxyl sulfate [22], upregulate AP-1 activity in an AhR-dependent manner. Furthermore, latent membrane protein 2A inhibits the expression and pathway activation of AhR by inducing phosphorylation of ERK [23].

In this study, we screened PUN as a selective AhR modulator from polyphenolic compounds that upregulated AhR expression, which was different from resveratrol, curcumin, and quercetin. We also found that PUN promoted AhR expression at the transcriptional level, mainly regulating the AhR non-genomic pathway. Activation of p90RSK was a checkpoint for AhR upregulation by PUN and lipopolysaccharide (LPS), which upregulated AhR via the mitogen-activated protein kinase kinase (MEK)/ERK and PDK1 pathways targeting c-fos. Finally, AhR upregulation by PUN inhibited inflammation and promoted bactericidal activity in macrophages.

Materials and methods

Materials and reagents

LPS (Escherichia coli 0111: B4) (L4130) and thioglycollate medium (T9032) were procured from Sigma-Aldrich (St. Louis, USA). Punicalagin (A0226), resveratrol (A0051), quercetin (A0083) and curcumin (A0086) were provided by Chengdu MUST bio-technology co, LTD (Chengdu, China). Inhibitor PD98059 (S1177) and GSK2334470 (S7087) were purchased from Selleck (Houston, Texas, USA). Inhibitor SCH772984 (HY-50846), LJH685 (HY-19712) and T-5224 (HY-12270) were purchased from Med chem express (New Jersey, USA). Inhibitor CH-223191 (C8124), Benzopyrene (B1760) and β-Naphthoflavone (N3633) were purchased from Sigma-Aldrich. THP-1 were obtained from Procell Life Science& Technology Co. Ltd. (CL-0233, Wuhan, China). Inhibitor U0126 (S1901), nuclear and cytoplasmic protein extraction kit (P0028), BCA protein assay kit (ST2222), Cell lysis buffer for Western and IP (P0013) and Protease/Phosphatase Inhibitor Cocktail (P1051) were provided by Beyotime (Shanghai, China).

Animals

Adult C57BL/6 mice (average body weight of 20 ~ 22 g, aged 6 ~ 8 weeks) were acquired from the of Daping Hospital’s (Chongqing, China) Experimental Animal Center. The AhR-KO mice were derived from AhR heterozygote mice that were obtained from Jackson's laboratory. All animal experiments were conducted in the SPF animal room of Daping Hospital's Experimental Animal Center (SYXK20170002), whose ethics approval number is AMUWEC20211429.

Extract mice peritoneal macrophages and cells culture

Mice were injected with 4% thioglycolate broth three days ahead, the peritoneal macrophages obtained by washing the abdominal cavity with PBS and adhering to a Petri dish for 2 h. After the suspension cells were washed away by PBS, adherent macrophages were collected. Macrophage and THP-1 cells were cultured in RPMI-1640 with 10% FBS, penicillin, and streptomycin. Cells were maintained at 37 °C in a 5% CO₂ humidified environment.

Western blotting

Cells lysed in buffer (20mM Tris, 150mM NaCl, 1% Triton X-100) for 15 min on ice, debris removed by centrifugation. Protein quantified by BCA assay. Equal protein per sample analyzed on 10% Tris-Glycine extended stain-free gels. Total protein stains used as loading controls for quantitative protein analysis. SDS gels transferred to PVDF membranes, blocked with 5% skim milk for 1 h, and incubated with primary antibodies overnight at 4 °C. Next day, membranes washed and probed with HRP-conjugated secondary antibody for 1 h. Immunoreactive bands were observed, identified, and quantitatively evaluated using enhanced chemiluminescence detection system. Detailed quantification was performed by Image J software for densitometric analysis. The antibodies information were listed in Table S1.

Quantitative reverse transcription PCR analysis

Total RNA was extracted from macrophages using the RNA rapid extraction kit (BioTeke, Beijing, China). The cDNA was synthesized from 1 μg RNA, and qRT-PCR was performed with SYBR Premix (TaKaRa). All the primer sequences used for each targeted gene were provided in Supporting Information Table S2.

Flow cytometry

Cells were stained with FITC-F4/80 and BV605-CD11b antibodies, and incubated 15 min in the dark. Fixed and permeabilized with Fixation/Permeabilization Solution Kit (BD, Biosciences) for 2 h. Stained with APC-AhR or APC-isotype antibodies for 30 min. Washed and centrifuged, cells resuspended in PBS and analyzed by flow cytometry. The antibodies information were listed in Table S3.

Immunofluorescence and confocal analysis

Cells on Confocal Petri Dish were fixed for 30 min with 4% Paraformaldehyde, permeabilized for 10 min with 0.5% Triton X-100, and blocked for 60 min with QuickBlock™ Blocking Buffer. Cells were incubated with AhR (GTX22770, GeneTex) or Phospho-ERK (4370, CST) antibodies overnight at 4 °C, then incubated with Alexa Fluor 647-conjugated or 480-conjugated secondary antibody for 1 h. DAPI Staining Solution was added for 10 min. Imaging was performed using a laser-scanning confocal microscope. After staining the nucleus with DAPI, the images were then collected by laser scanning confocal microscope for analysis (Olympus).

RNA interference

Cells were transfected with 100 nM of scrambled or p90RSK-specific siRNAs (Ribobio) using riboFECTTM CP Reagent in six-well plates. After 48 h, siRNA and oligofectamine mixtures were discarded. The mRNA and protein expression of p90RSK were detected by qPCR or Western blotting to confirm the efficiency of gene interference. The siRNA sequences information were listed in Table S4.

EMSA

The DNA–nuclear protein binding activity was evaluated by electrophoresis on a native gel. The expression of the complex on the gel was then measured using the Odyssey Scanning system. The DNA-binding sequences of AhR and AP-1 on the promoter were synthesized into infrared ray 700 labeled probes, and their information is listed in Table S5.

Preparation of Listeria cells

Listeria monocytogenes (ATCC19115) was grown in LB medium for 16 h at 37 °C on an orbital shaker. After washing with 0.9% NaCl and centrifugation, bacterial sediments were resuspended in sterile glycerine to form a 1 × 10⁸ CFU/mL bacterial solution.

In vitro bactericidal assay

Macrophages from wild-type and AhR-KO mice were collected and seeded to the 12-well plates with 1 × 10⁶ cells per well, and were pretreated with PUN for 12 h. Discard the culture medium and wash three times with sterile PBS. Peritoneal macrophages were infected with Listeria for 1 h (MOI = 1). The medium was discarded and washed, then the extracellular bacteria were killed with the antibiotic culture medium. The cells were dissolved in 200µL 0.5% TritonX-100 for 5 min, the supernatant was centrifuged, and the precipitate was re-suspended in 100µL sterile PBS. After 10 times dilution, 30µL bacterial solution was applied to LB medium and incubate at 37 ℃ for 18 h. The quantitative enumeration of bacterial colonies was performed using the colony-forming unit (CFU) method.

Analysis of cytotoxicity

Lactate dehydrogenase cytotoxicity test kit purchased from Beyotime (C0016). Prepare macrophages into 1 × 10⁵ cell/mL dilution solution. Seeded to the 96-well plates with 100 μL cell dilution solution per wells. Cells were pretreated with PUN for 12 h and subsequently challenged with Listeria for additional 24 h (MOI = 1). The LDH assay was performed in accordance with the product manual.

Transcriptome sequencing

Macrophages RNA was extracted using the TRIzol extraction method. After the concentration and purity of RNA were qualified, sequencing was performed on the second-generation sequencing platform. According to the read count of each gene, the relative quantitative analysis of gene expression and enrichment analysis of differential genes were carry out. When comparing between groups, the genes with a P value less than 0.05 and a fold change greater than 2 were identified as significantly differentially expressed genes. The analysis and illustration of results were performed using Majorbio Cloud Software.

Molecular docking and affinity prediction

AhR consisted of PASA and PASB two key domains, the latter of which was a target for conventional drugs. Adopting AlphaFold2 forecasted crystal structure. The PASB crystal structure was downloaded in the https:/ww.alphafold.ebi.ac.uk/entry/P30561. The two-dimensional structure of each compound was obtained from pubchem, and Gaussian 09 was used to optimize the molecule, B3LYP density functional was used to optimize the molecule, 6-31 g (d) was selected as the base group, and aqueous solution was selected as the solution. AutoDock Vina was used to dock each compound with the crystal structure separately. The molecular size of each compound was calculated using VEGA ZZ3.2.2.

Statistical analyses

Data were presented as mean ± standard error (SEM). The number of independent experiments is indicated by N. Statistical analyses were performed using appropriate tests for multiple comparisons (one-way ANOVA) or for pairwise comparisons (t-test). All statistical analyses were conducted using Prism version 8.02 (GraphPad Software). of P values < 0.05 were considered statistically significant.

Results

PUN screened from polyphenolic compounds was a selective AhR modulator

Previous studies have reported that the plant polyphenol flavonoids resveratrol (RES), quercetin (QUE) and curcumin (CUR) act as ligands of AhR and induce expression of CYP1A1 [24]. We conducted a comparative examination of the effects of PUN and the above compounds on AhR expression in LPS-induced mouse macrophages. As reported previously [9, 25], AhR expression was increased by LPS in peritoneal macrophages. We found that PUN alone or combined with LPS upregulated expression of AhR, and their combination was more potent (Fig. 1A, B). However, treatment with resveratrol, quercetin, and curcumin, alone or in combination with LPS, did not cause any significant change in AhR expression (Fig. 1A, B). To evaluate the binding of PUN to AhR, we used molecular docking techniques to evaluate the affinity of polyphenol compounds to AhR. Compared with TCDD, resveratrol, quercetin, and curcumin had a significantly lower affinity for AhR, decreasing by a factor of 4–10 times. The affinity of PUN for AhR was reduced by ~ 40 times compared with TCDD (Fig. 1C and S1A). We discovered that TCDD penetrated the hydrophobic pocket of AhR. In contrast, PUN was too large to enter the hydrophobic pocket, thus, it did not activate AhR (Fig. 1D). Benzo (a) pyrene (Bap) and β-naphthoflavone (βNAF) are agonists of AhR, which can promote the nuclear translocation of AhR. However, PUN did not induce AhR nuclear translocation but rather promoted its cytoplasmic expression (Fig. 1E). The downstream factors of AhR, including CYP-related genes, were not affected by PUN, regardless of CYP1B1 (Fig. 1F, G, and S1B, C). Additionally, PUN did not affect the DNA binding activity between nucleoproteins and dioxin response elements on the AhR promoter (Fig. 1H). Taken together, PUN screened from polyphenolic compounds was a selective AhR modulator that upregulated AhR expression, but did not induce nuclear translocation and downstream gene transcription of AhR.

Fig. 1

Punicalagin screened from polyphenolic compounds was selective AhR modulator. A Western blot analysis of AhR in peritoneal macrophages were induced with LPS (10 μg/mL) for 12 h after pre-treated with Punicalagin (50 μM), Resveratrol (25 μM), Quercetin (10 μM) and Curcumin (10 μM) for 1 h. B The relative abundance of protein AhR was calculated by loading control normalization (n = 3 bands per group). C Predicted affinities of TCDD, Punicalagin, Resveratrol, Quercetin, and Curcumin compounds to AhR proteins. D Evaluated the affinity of TCCD/PUN to AhR with molecular docking techniques. E AhR expression in the cytoplasm and nucleus was analyzed by Western blot. Peritoneal macrophages were pretreated with PUN for 1 h and then induced with LPS for 8 h. Alternatively, macrophages were induced with LPS for 8 h and then treated with Benzo (a) pyrene (Bap) (10 μM) and β-Naphthoflavone (βNAF) (1 μM) for 0.5 h. F-G Expression of AhR downstream related genes in peritoneal macrophages. F Transcriptome analysis; (G) qRT-PCR analysis (n = 3 groups per group). H Macrophages were stimulated with or without LPS for 12 h and PUN pretreated 1 h, nuclear-extract proteins were then prepared and incubated with AhR-binding site probe. Binding activity was measured using EMSA. The experiment was repeated three times, the data are shown as mean ± SEM. Combined results are compared using an one-way ANOVA test. N.S P > 0.05, ∗ P < 0.05, ∗ ∗ P < 0.01

PUN upregulated AhR expression in LPS-induced macrophages in vivo and in vitro

Several studies have demonstrated that AhR inhibited expression of interleukin (IL)-6 and IL-1β [26], and stimulated expression of IL-10 in inflammatory macrophages [9]. Therefore, we focused on the effect of PUN on AhR in inflammatory macrophages. In vivo, intraperitoneal injection of PUN increased the percentage of AhR-positive peritoneal macrophages in LPS-induced mice (Fig. 2A). In vitro, we confirmed that PUN alone or combined with LPS upregulated AhR protein expression (Fig. 2B) and mRNA expression (Fig. 2C). Following PUN pretreatment, AhR level were significantly increased at both 8 h and 12 h (Fig. 2D). AhR expression was initially triggered at a PUN concentration of 12.5 μM, with a maximum level observed at 50 μM (Fig. 2E). Therefore, AhR expression in macrophages treated with LPS was significantly augmented by PUN in a time- and dose-dependent manner (Fig. 2D, E). Compared with LPS-stimulated macrophages, the mean fluorescence intensity (MFI) of AhR was significantly enhanced in PUN-stimulated macrophages (Fig. 2F). Furthermore, the majority of the increased AhR was localized to the cytoplasm (Fig. 2F). In human THP-1 cells, we also demonstrated that PUN has the effect of up-regulating AhR expression (Fig. 2G). In summary, PUN exhibited the ability to augment AhR expression in inflammatory macrophages.

Fig. 2

Punicalagin upregulated AhR expression in LPS-induced macrophages in vivo and in vitro. A Flow cytometry analysis of AhR⁺ peritoneal macrophages in mice (n = 12). The mice were pretreated with 12.5 mg/kg PUN or equal volume PBS for 12 h and then stimulated with 10 mg/kg LPS for 8 h. The results are compared using t test. B Western blot analysis of AhR in the cell lysates. The cells were pretreated with PUN (50 μM) for 1 h, and then induced with LPS (10 µg/mL) for 12 h. Gray value analysis of relative abundance of AhR normalized to loading control. C The AhR mRNA levels were detected with qRT-PCR. The cells were pretreated with PUN (50 μM) for 1 h, and then induced with LPS (10 µg/mL) for 4 h or 8 h. D Western blot analysis of AhR in the cell lysates. Peritoneal macrophages were pretreated with PUN (50 μM) for 1 h, and then induced with LPS (10 µg/mL) for for different times (8 h, 12 h). E Western blot analysis of AhR in the cell lysates. Peritoneal macrophages were stimulated either with different doses of PUN (12.5 μM, 25 μM, 50 μM) for 1 h, and then induced with LPS (10 µg/mL) for 12 h. F AhR protein expression was measured with immunofluorescence. Cells were pretreted with 50 μM PUN for 1 h and induced by 1mg/mL LPS for 8 h, then immunostained with AhR specific antibody (Purple), cell nucleus stained with DAPI (Blue). Bar = 20 µm. The fluorescence intensity of AhR was measured (n = 6 sheets per group). G The THP-1 cells were pretreated with PUN (50 μM) for 1 h, and then induced with 10 µg/mL LPS for 12 h. Western blotting analysis with AhR antibodies. Gray value analysis of relative abundance of AhR normalized to loading control. The experiment was repeated three times. The data are shown as mean ± SEM. Combined results are compared using one-way ANOVA test or t test, ∗ P < 0.05

AP-1 was involved in AhR induction by PUN

Activated nuclear factor (NF)-κB is a key mediator of AhR expression [9, 27]. We found that the increase in AhR expression induced by PUN was not associated with NF-κB (Fig. S2A), prompting us to investigate additional transcription factors involved in AhR regulation. The AhR gene promoter contains an AP-1 binding site [28], and inhibition of AP-1 activity reduces AhR expression [29], suggesting the correlation between AP-1 and AhR expression. AP-1 is a transcription factor, usually composed of a heterodimer of the proteins c-fos and c-jun. Compared with untreated cells, serine phosphorylation of c-fos was increased in PUN-treated cells following LPS stimulation, while PUN had a tendency to increase the serine phosphorylation of c-jun (Fig. 3A). Within 0.5–2 h after LPS stimulation, phosphorylation of c-fos in macrophages pretreated with PUN increased significantly (Fig. 3B). Our analysis of the JASPAR database [30] indicated that the sequence spanning -697 to -687 bp on the AhR promoter would have the highest c-fos binding score (Fig. 3C). Next, we confirmed by electrophoresis mobility shift assay that PUN significantly enhanced the ability of nuclear protein to bind to AhR promoter in LPS-stimulated macrophages (Fig. 3D). We explored whether the suppression of c-fos affected AhR expression, using an AP-1 inhibitor (T-5224), which significantly inhibited phosphorylation of c-fos (Fig. 3E and S2B). Compared with control cells, AhR mRNA (Fig. 3F) and protein (Fig. 3G) expression levels were significantly lower in LPS- and PUN-stimulated cells exposed to T-5224. Above all, AP-1 was thought to play a crucial role in the triggering of AhR induction by PUN in macrophages exposed to LPS.

Fig. 3

AP-1 was involved in AhR induction by PUN. A The cells were pretreated with PUN (50 μM) for 1 h, and induced with LPS (10 µg/mL) for 30 min. cell lysates subjected to western blotting analysis with total c-fos, phospho-c-fos (Ser32), total c-jun, phospho-c-jun (Ser63). Gray value analysis of relative abundance of phospho-c-fos (Ser32) and phospho-c-jun (Ser63). B The cells were pretreated with PUN (50 μM) for 1 h, and induced with LPS (10 µg/mL) for 30 min, 1 h, 2 h. Cell lysates subjected to western blotting analysis with phospho-c-fos (Ser32). C ~ G PUN promoted AhR expression through transcription factor of c-fos. C The AhR promoter contains a binding site for the transcription factor c-fos. D Macrophages were stimulated with PUN pretreated 1 h and LPS for 0.5 h, nuclear-extract proteins were then prepared and incubated with AP-1-binding site probe. Binding activity was measured using EMSA. E Macrophages were pretreated with transcription factor AP-1 inhibitor T-5224 (10 μM) for 2 h, and then pretreated with PUN (50 μM) for 1 h, and induced with LPS (10 µg/mL) for 12 h. Cell lysates subjected to western blotting analysis with total c-fos, phospho-c-fos (Ser32). F The mRNA expression of AhR was analyzed by qRT-PCR after the application of transcription factor AP-1 inhibitor T-5224. G The protein expression of AhR was analyzed by western blotting after the application of T-5224. Gray value analysis of relative abundance of AhR normalized to loading control. The experiment was repeated three times. The data are shown as mean ± SEM. Combined results are compared using one-way ANOVA test. N.S P > 0.05, ∗ P < 0.05

MEK/ERK pathway, but not ERK phosphorylation and nuclear translocation, mediated PUN induction of AhR expression in LPS-induced macrophages

To explore the molecular mechanism of upregulation of AhR by PUN, we performed transcriptome sequencing on macrophages treated with LPS, PUN, or both. As shown in the Venn diagram, among the 1291 differentially expressed genes affected by LPS stimulation, 197 were detected in response to PUN treatment (Fig. 4A). Enrichment analysis of the KEGG signaling pathway suggested possible correlation with phosphoinositide 3-kinase (PI3K)/AKT, NF-κB, and mitogen-activated protein kinase (MAPK) signaling pathways (Fig. 4B). However, we revealed that pretreatment of macrophages with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) did not suppress PUN-induced AhR expression (Fig. S2A). After pretreatment with MEK (U0126 and PD98059) and ERK (SCH772984) inhibitors, the expression of AhR coactivated by LPS and PUN was significantly decreased (Figs. 4C–E and S2C, D). MEK (U0126) and ERK (SCH772984) inhibitors also downregulated phosphorylation of c-fos activated by LPS and PUN (Fig. S3C–F). In comparison to LPS, expression of phosphorylated MEK and total MEK was significantly augmented by PUN (Fig. 4F). Remarkably, PUN decreased the extent of ERK phosphorylation in LPS-stimulated macrophages (Fig. 4G, H) and reduced the translocation of phosphorylated ERK into the nucleus (Fig. 4I). Similarly, PUN also reduced phosphorylation of mitogen- and stress-activated kinase 1, a molecule downstream of ERK (Fig. S2E). These results supported the hypothesis that the MEK/ERK pathway, but not ERK phosphorylation and nuclear translocation, mediated PUN induction of AhR expression in LPS-induced macrophages. This suggests that additional transcription factors mediate the effect of PUN on upregulation of AhR expression.

Fig. 4

MEK-ERK pathway, but not ERK phosphorylation and nuclear translocation, mediated PUN induction of AhR expression in LPS-induced macrophages. A Venn diagram was used to analyze the differential gene sets between the two groups, LPS vs Control and LPS vs LPS + PUN. B KEGG enrichment analysis signaling pathway enriched by differential expression genes. C ~ E Peritoneal macrophages were pretreated with MEK inhibitors U0126 (20 μM) or PD98059 (10 μM), ERK inhibitors SCH772984 (1 μM) for 1 h and then pretreated with PUN (50 μM) for 1 h, and then treated with LPS (10 µg/mL) for 12 h, cells lysates subjected to western blotting analysis with AhR antibodies. Gray value analysis of relative abundance of AhR normalized to loading control. F ~ I Peritoneal macrophages were pretreated with PUN (50 μM) for 1 h and then treated with LPS (10 µg/mL) for 30 min. Cells lysates subjected to western blotting analysis the protein levels of MEK / p-MEK (F), ERK / p-ERK (G), and ERK / p-ERK in the nucleus and cytoplasm respectively(I). Gray value analysis of relative abundance of p-ERK in nucleus normalized to loading control. H Cells were immunostained with p-ERK antibody (Green) and co-stained with DAPI (Blue) to detect nucleus. Bar = 20 µm. The experiment was repeated three times. The data are shown as mean ± SEM. Combined results are compared using one-way ANOVA test or t test. ∗ P < 0.05, ∗ ∗ P < 0.01

p90RSK activation was critical for AhR expression in macrophages increased by PUN

Previous studies have reported that ERK activated p90RSK, and phosphorylated p90RSK translocated into the nucleus to stimulate activation of the downstream transcription factor c-fos [18, 31, 32]. p90RSK contains four essential phosphorylation sites (Ser221, Thr359, Ser380, and Thr573) (Fig. 5A) [18]. In this study, PUN promoted LPS-induced phosphorylation of Ser221, Thr359, Ser380, and Thr573 of p90RSK in macrophages (Fig. 5B). PUN significantly increased the translocation of phosphorylated p90RSK (Ser221) into the nucleus, while exerting no influence on the nuclear translocation of phosphorylated p90RSK (Thr573) (Fig. 5C). Additionally, we explored the potential impact of p90RSK inhibitor (LJH685) on the regulation of AhR by detecting the phosphorylation sites (Ser221, Thr359, Ser380, and Thr573) of p90RSK activated by LPS + PUN (Fig. 5D). Consistent with our hypothesis, we observed a decrease in the expression of AhR and phosphorylation of c-fos in PUN-stimulated cells following treatment with a p90RSK inhibitor (Figs. 5E and S3G, H). Subsequently, we utilized siRNA knockdown of p90RSK to identify the individual with the highest knockdown efficiency for subsequent experimentation (Fig. 5F). AhR expression induced by LPS and PUN was decreased in macrophages treated with p90RSK siRNA compared with macrophages treated with scrambled siRNA (Fig. 5G). In addition, MEK (U0126) and ERK (SCH772984) inhibitors also downregulated phosphorylation of p90RSK activated by LPS and PUN (Fig. S3A, B), suggesting that p90RSK was regulated by upstream MEK/ERK in promoting AhR expression. These results supported the hypothesis that p90RSK activation was critical for AhR expression in macrophages increased by LPS and PUN.

Fig. 5

p90RSK activation was critical for AhR expression in macrophages increased by PUN. A Structure diagram of the RSK, containing the S221, T359, S380 and T573 phosphorylation sites. B ~ C Peritoneal macrophages cells were treated with LPS (10 µg/mL) for 30 min after pretreated with PUN (50 μM) for 1 h. Cells lysates used for western blotting analysis. B The protein level of phospho-p90RSK (Thr573), phospho-p90RSK (Ser380), phospho-p90RSK (Thr359), phospho-p90RSK (Ser221) and RSK. C The protein level of p-p90RSK (Thr753) and p-p90RSK (Ser221) in cytoplasm and nucleus. D ~ E Peritoneal macrophages were pretreated with p90RSK inhibitors LJH685 (10 μM) for 1 h and then pretreated with PUN (50 μM) for 1 h, and then treated with LPS (10 µg/mL) for 0.5 h or 12 h, cells lysates subjected to western blotting analysis. D The protein level of phospho-p90RSK (Thr573), phospho-p90RSK (Ser380), phospho-p90RSK (Thr359), phospho-p90RSK (Ser221). E The protein level of AhR. F Expression of RSK protein after RSK silencing in macrophages by Western blotting analysis. G Western blotting detected the AhR protein level after RSK silencing and pretreated with (50 μM) PUN for 1 h and then induced by 10 µg/mL LPS for 12 h. All the bands gray value analysis of relative abundance of protein normalized to loading control. The experiment was repeated three times. The data are shown as mean ± SEM. Combined results are compared using one-way ANOVA test. ∗ P < 0.05

PUN promoted p90RSK mediated AhR expression through the PDK1 pathway in LPS-induced macrophages

Complete activation of p90RSK necessitated the cooperative effect of the MEK/ERK and PDK1 signaling pathways [18, 33]. The former has been demonstrated to mediate PUN-induced AhR expression. Consequently, we examined the effect of PUN on the PDK1 signaling pathway. PUN promoted PDK1 phosphorylation and total PKD1 protein expression (Fig. 6A). We further investigated the impact of PDK1 inhibition on AhR expression using PDK1 inhibitor (GSK2334470). This significantly counteracted the phosphorylation of PDK1 triggered by LPS + PUN (Fig. 6B). When PUN-activated cells were treated with GSK2334470, AhR expression was significantly reduced compared with control cells (Fig. 6C, D). GSK2334470 also decreased the phosphorylation of p90RSK, c-fos, and c-jun activated by LPS + PUN (Fig. 6E–G). These results identified that PDK1 was involved in the induction of AhR by PUN in LPS-induced macrophages.

Fig. 6

PUN promoted p90RSK mediated AhR expression through PDK1 pathway in LPS-induced macrophages. A, B Peritoneal macrophages cells were pretreated with PUN (50 μM) for 1 h, and then treated with LPS (10 µg/mL) for 30 min. Cells lysates subjected to western blotting analysis with total PDK1, phospho-PDK1. B ~ G Before cells were treated with LPS and PUN at the same dose and time, cells were pretreated with PDK1 inhibitor GSK2334470 (5 nM) for 1 h. Cells lysates subjected to western blotting analysis. B PDK1 and phospho-PDK1. C AhR. D Gray value analysis of relative abundance of AhR normalized to loading control. E phospho-p90RSK (Thr573), phospho-p90RSK (Ser380), phospho-p90RSK (Thr359) and phospho-p90RSK (Ser221). F c-fos and phospho-c-fos(s32). G c-jun and phospho-c-jun(s63). The experiment was repeated three times. The data are shown as mean ± SEM. Combined results are compared using one-way ANOVA test. ∗ P < 0.05

AhR upregulated by PUN inhibited macrophage inflammation and promoted bactericidal activity

Recently, it has been found that AhR negatively regulates the inflammatory response of macrophages, and contributes to the bactericidal capacity of macrophages [6, 34, 35]. In LPS-induced macrophages, PUN inhibited mRNA expression levels of inflammatory cytokines IL-6, IL-1β, and IL-27, but after pretreatment with AhR inhibitor CH223191, the fold change by PUN on reducing these inflammatory factors was partially reversed (Fig. 7A–C). The above results were also confirmed in AhR knockout macrophages (Fig. 7D). More significantly, in AhR knockout macrophages, the effect of PUN in reducing IL-6 and IL-1β was almost completely eliminated (Fig. 7E, F). In Listeria-infected macrophages, AhR promoted bacterial clearance by enhancing reactive oxygen species (ROS) production and inhibited apoptosis by inducing apoptosis inhibitor of macrophage expression [34]. Therefore, we investigated whether PUN enhanced macrophage clearance of Listeria monocytogenes and inhibited macrophage death in an AhR-dependent manner. PUN promoted Listeria clearance in wild-type macrophages, but this effect was reversed in AhR knockout macrophages (Fig. 7G, H). Similarly, the effect of PUN on reducing the cell death rate of Listeria-induced macrophages was partially reversed in AhR knockout macrophages (Fig. 7I). These results suggested that AhR upregulated by PUN inhibited macrophage inflammation and promoted bactericidal activity.

Fig. 7

AhR upregulated by PUN inhibits macrophage inflammation and promotes bactericidal activity. A ~ C Peritoneal macrophages cells were pretreated with AhR inhibitor CH-223191 and then treated with LPS (10 µg/mL) for 4 h after pretreated with PUN (50 μM) for 1 h. qRT-PCR analysis IL-6, IL-β and IL-27 mRNA expression level. D ~ F WT and AhR-KO peritoneal macrophages treated with LPS (10 µg/mL) for 4 h after pretreated with PUN (50 μM) for 1 h. qRT-PCR analysis AhR, IL-6 and IL-β mRNA expression level. G ~ I WT and AhR-KO peritoneal macrophages treated with Listeria monocytogenes for 1 h after pretreated with PUN (50 μM) for 1 h. G Results of intracellular bacterial plating. H Statistical results of bacterial loads. I Amount of LDH released from macrophages infected with Listeria. The experiment was repeated three times. The data are shown as mean ± SEM. Combined results are compared using one-way ANOVA test or t test. N.S P> 0.05, P < 0.05, ∗P < 0.01

Discussion

At present, AhR ligands can be broken down into endogenous and exogenous forms [4]. Endogenous ligands include those produced by the human body or gut microbiota, while exogenous ligands come from dietary compounds, environmental contaminants, drugs, and synthetic compounds. AhR activation by varying ligand classes results in context-specific outcomes. Natural plant compounds are preferred to synthetic pharmaceuticals due to their lower toxicity, higher bioavailability, and low cost. Polyphenols, abundant in plants, exert potent antioxidant and anti-inflammatory effects for disease prevention and health maintenance [36]. Polyphenolic compounds are AhR ligands belonging to a class of dietary compounds, including the currently known resveratrol, quercetin, and curcumin [24]. Resveratrol, a class of phytochemicals found particularly in certain plants when exposed to stress or injury, has been shown to be an antagonist of AhR [37]. Quercetin, a natural flavonoid, acts as an AhR agonist [37] and regulates CYP1A1 gene expression [24, 38]. Curcumin is derived from the plant Curcuma longa, and is a yellow spice and coloring agent that activates AhR [37]. In this study, we found that PUN, a polyphenol compound derived from pomegranate peel extract, was not an agonist or inhibitor of AhR like other polyphenol compounds, but rather a selective modulator. Previous studies have classified AhR ligands as agonists or antagonists based primarily on their ability to promote or inhibit AhR nuclear translocation. Recently, a third class of AhR ligands has been discovered [39, 40]. These compounds are known as selective AhR modulators (SAhRMs), displaying anti-inflammatory activity that is mainly mediated by nongenomic signaling pathways [12, 13]. The selected synthetic compound SGA360, as an SAhRM, significantly suppresses ear swelling and multiple inflammatory gene expression in mice [41], and inhibits the acute inflammatory response of macrophages, thus alleviating arthritis and reducing the mortality of endotoxic shock in mice [42]. Our results demonstrated that PUN enhanced AhR expression but did not promote AhR nuclear transport or gene transcription in macrophages. In addition, PUN inhibited macrophage inflammation and promoted bactericidal activity in an AhR-dependent manner. Therefore, given the regulation of AhR and its signaling pathway by PUN, we concluded that PUN could be defined as an SAhRM like SGA360.

Although ligand binding is crucial for the activation of AhR, the amount of AhR expression determines ligand activation and subsequent nongenomic effects. AhR knockout mice have increased sensitivity to septic shock and higher levels of inflammatory cytokines in AhR-deficient macrophages after LPS stimulation [43]. AhR is compensatively elevated in LPS-stimulated macrophages through the NF-κB pathway, resulting in inhibition of macrophage inflammatory response [9]. Expression of AhR in microglia is upregulated during ischemia, triggering neuroinflammation that subsequently causes vasogenic edema [44]. The metabolites of 1-methoxypyrene [45] and L-kynurenine [46] dose-dependently activate the AhR pathway, thus mediating different biological effects. However, the effects of polyphenolic compounds on AhR expression remain poorly understood. This study found that treatment with resveratrol, quercetin, and curcumin alone or in combination with LPS did not show any significant changes in AhR expression. We found for the first time that PUN, which is a polyphenol compound, upregulated the expression of AhR either alone or in combination with LPS, and combination treatment was more effective. Therefore, the discovery of PUN as a selective modulator that upregulated AhR expression has implications for the treatment of diseases targeted by AhR.

To investigate the molecular mechanism of upregulation of AhR by PUN, the results of transcriptome sequencing suggested that it is related to NF-κB, MAPK, and PI3K/AKT signaling pathways. Our team previously reported that LPS increased AhR expression in macrophages through the NF-κB signaling pathway [9], but Xu et al. demonstrated that PUN was capable of inhibition of NF-κB activation [16]. We also found that the NF-κB inhibitor PDTC did not reverse the upregulatory effect of PUN on AhR. These data indicate that the NF-κB signaling pathway is not involved in PUN-mediated AhR expression. Regarding the MAPK pathway, our results were consistent with previous studies [16], in which PUN inhibited the phosphorylation of EKR1/2 in LPS-induced macrophages. Surprisingly, MEK (U0126 and PD98059) and ERK (SCH772984) inhibitors reversed upregulation of AhR by PUN, suggesting that additional transcription factors might be involved in PUN-induced upregulation of AhR. Upon screening, we found that PUN promoted LPS-induced phosphorylation of Ser221, Thr359, Ser380, and Thr573 of p90RSK in macrophages. The p90RSK inhibitor LJH685, which was designed to bind to the N-terminal ATP domain of p90RSK kinase, has been shown to inhibit PUN-induced upregulation of AhR expression in macrophages but not LPS-induced AhR expression. The above effect was confirmed in macrophages treated with p90RSK siRNA. Activation of this key molecule, p90RSK, is essential for PUN-induced AhR expression in macrophages. As far as we know, this is the first study to demonstrate the involvement of p90RSK in the regulation of AhR expression. The p90RSK protein kinases, which include p90RSK1–4, belong to the highly conserved AGC kinase family. These kinases regulate various cellular processes and are activated by the Ras/ERK signaling cascade [18, 47]. Interestingly, in Fig. 5D and E, we found that LPS-induced levels of p-p90RSK(Thr573) and p-p90RSK(Ser380) increased more after treatment with the p90RSK inhibitor LJH685, as well as the expression level of AhR. p90RSK contains C-terminal and N-terminal kinase domains [48], and all isoforms have the four crucial phosphorylation sites at Ser221, Ser363, Ser380, and Thr573 [18]. The activation process of p90RSK is achieved through a sequential phosphorylation cascade at Thr573 and Ser221, respectively, by ERK and PDK1 [49]. After complete activation of p90RSK, Ser737 located on the KIM motifs exhibits self phosphorylation, resulted in ERK separation from Thr573 site of p90RSK. Therefore, a negative feedback loop is formed to prevent the continued activation of p90RSK. LJH685 is an N-terminal inhibitor of p90RSK kinase. It is possible that LJH685 prevented the negative feedback loop of p90RSK, which resulted in the sustained Thr573 and Ser380 activation of p90RSK and the subsequent elevation of AhR. Purified p90RSK protein can be fully activated by phosphorylated ERK and phosphorylated PDK1 in vitro [50]. Our research demonstrated that PDK1 (GSK2334470), MEK (U0126), and ERK (SCH772984) inhibitors effectively suppressed PUN-mediated AhR expression, by blocking phosphorylation of p90RSK induced by LPS and PUN. In alignment with earlier studies [47, 51], we confirmed that PDK1 and MEK/ERK synergistically fully activated full-length p90RSK, thereby contributing to the enhancement effect of AhR.

Previous studies focused on the regulation of the AP-1 signaling pathway by AhR, influencing the expression of various members of the AP-1 family in keratinocytes [20]. On the one hand, AhR ligand indolyl sulfate activated AhR and then upregulated E-selectin expression in vascular endothelium by promoting AP-1 transcriptional activity [22]. On the other hand, TCDD-activated AhR inhibited AP-1 protein expression in B cells [52]. However, there is evidence that AP-1 might also be associated with AhR expression. AP-1 upregulates TH17/ILC3 genes, including AhR, in lymphoma cells, so AP-1 inhibition leads to AhR reduction [29]. As indicated in the JASPAR database [30], we proposed that the binding checkpoint of the AP-1 family member c-fos on the AhR promoter was a crucial regulatory mechanism. Our hypothesis was verified through experimental evidence, which indicated that AP-1 played a key role in regulating AhR expression by PUN in LPS-induced macrophages. Finally, we proposed for the first time a new synergistic model to upregulate AhR expression. The partial activation of p90RSK by the LPS-activated MEK/ERK signaling pathway was the basis for its full activation. PUN promoted the full activation of p90RSK through the PDK1 pathway, and then synergistically enhanced c-fos-mediated AhR expression with LPS (Fig. 8).

Fig. 8

A new synergistic model of AhR up-regulated by PUN against inflammation and infection in inflammatory macrophages. The activation of p90RSK served as a critical checkpoint for AhR upregulation by PUN and LPS, which induced AhR upregulation by activating MEK/ERK and PDK1 pathways that ultimately targeted c-fos. The partial activation of p90RSK by LPS-activated MEK-ERK signaling pathway was the basis for its full activation. PUN promoted the full activation of p90RSK through PDK1 pathway, and then synergistically enhanced c-fos mediated AhR expression with LPS. Furthermore, AhR up-regulated by PUN could inhibit macrophage inflammation and promote its bactericidal activity

AhR serves as a critical regulator of both inflammatory responses and microbial defense [6, 8, 53]. AhR is intricately connected to the functional regulation of macrophages. In particular, activation of AhR influences the M1/M2 polarization of macrophages [54]. In inflammatory macrophages, AhR decreases the expression of IL-6, tumor necrosis factor-α, IL-12p40, and IL-1β [25, 43, 55], and promotes IL-10 expression in a ligand-dependent or nondependent manner [9, 56]. Our previous study also showed that the increased level of AhR in post-traumatic macrophages substantially decreased the production of inflammatory cytokines [57]. The present study showed that the AhR was a vital regulator in controlling macrophage response and its expression within macrophages was critical for maintaining homeostasis and during inflammatory responses. Nevertheless, although there are reports of anti-inflammatory drugs that target AhR, additional research is needed to discover more potent natural regulators.

Previous research showed that PUN treatment effectively combated inflammation in RAW264.7 cells exposed to LPS by reducing Toll-like-receptor-4-related MAPK and NF-κB activation and interfering with the FoxO3a/autophagy signaling pathway [16, 58]. PUN is also beneficial in minimizing pathological inflammation by suppressing M1 phenotype polarization and pyroptosis [59]. We demonstrated that PUN inhibited the production of proinflammatory cytokines IL-6, IL-1β, and IL-27 in a partially AhR-dependent manner. In AhR-inhibitor-pretreated or AhR knockout macrophages, the inhibitory effect of PUN on inflammatory cytokines was partially reversed. Our results demonstrated the anti-inflammatory mechanism of PUN, suggesting that PUN also regulates expression of AhR, which might further inhibit the inflammatory response of macrophages through a nongenomic mechanism of AhR. In macrophages, AhR upregulation by PUN played a crucial role in modulating the secretory function of these cells.

Recent evidence suggests that AhR and its pathway play a crucial role in combating common bacterial infections. TCDD improves survival and reduces lung bacterial load in mice infected with Streptococcus pneumoniae by activating AhR [60, 61]. In the Salmonella Typhimurium infection model in mice, sublethal doses of LPS enhanced resistance to infection by activating AhR [5]. MOTS-c [62], benzothiazole [63], coal tar [64], and other substances exerted an AhR-dependent killing or inhibitory effect on Staphylococcus aureus. AhR ligands tryptophan and Lactobacillus plantarum increased intestinal AhR level and ROS generation, and improved alveolar macrophage phagocytosis of drug-resistant Pseudomonas aeruginosa [65]. Intestinal microbiota-produced indole-3-propionic acid activated AhR and promoted macrophage phagocytosis, reducing bacterial burden in mice [66]. The above results highlight the critical function of AhR in combating extracellular bacteria, while it is also key in combatting intracellular bacteria. For instance, AhR also participated in macrophage infection by Mycobacterium tuberculosis, triggering innate immune response against the bacteria, and upregulating IL-23 and IL-22 production [67]. L. monocytogenes is a Gram-positive, facultative intracellular bacterium that causes severe illness and is commonly studied as a model intracellular pathogen. It is typically engulfed by macrophages, which are essential for L. monocytogenes killing [34]. Kimura et al. showed that AhR protected against macrophage cell death and promoted ROS production for L. monocytogenes confinement and clearance. Selective AhR-ligand activation may be ideal for treatment of listeriosis. Our group found that AhR expression in macrophages correlated with L. monocytogenes clearance, and that PUN enhanced macrophage clearance of L. monocytogenes and inhibited macrophage death in an AhR-dependent manner. Our findings confirmed previous studies and underscored the unique role of SAhRMs in regulating biological functions such as AhR bactericidal activity. The aforementioned research suggested that AhR served as a significant factor in the treatment of pathogenic infections and the regulation of inflammatory reactions.

The current study had several limitations that need to be considered. The impact of PUN on AhR was exclusively observed in macrophages. Future research will explore the potential of PUN in regulating AhR activity in other cells, including T cells and dendritic cells. Our investigation concentrated on understanding the transcriptional mechanisms used by PUN to augment AhR expression, but the question of whether PUN also influences AhR protein degradation remains to be answered. Moreover, the upstream mechanism of upregulation of AhR by PUN, particularly which receptor mediates this effect, remains to be studied.

Conclusions

We found for the first time that PUN, screened from polyphenols, upregulated AhR expression and mainly affected its non-genomic pathway, providing a new natural regulator for regulating AhR expression in cells. In addition, c-fos mediated the transcriptional regulation of AhR in macrophages. It was confirmed that activation of p90RSK served as a critical checkpoint for AhR upregulation by PUN and LPS, which induced AhR upregulation by activating the MEK/ERK and PDK1 pathways that ultimately targeted c-fos. Furthermore, AhR upregulated by PUN inhibited macrophage inflammation and promoted bactericidal activity (Fig. 8). PUN as a selective regulator of AhR has potential application in the treatment of various diseases targeting AhR.