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NF-κB-mediated enhancement of H3K27me3 through EZH2: a mechanism to suppress pyocyanin-induced autophagy in macrophages

European Journal of Medical Research volume 30, Article number: 346 (2025) Cite this article

Abstract

Background

Histone modification is a key mechanism of epigenetic regulation. Our previous study demonstrated that histone H3 acetylation at lysine 27 (H3K27ac) promotes pyocyanin (PYO)-induced autophagy in macrophages. However, the regulatory role of H3K27 trimethylation (H3K27me3) in this process remains unclear.

Methods

THP-1 macrophages were treated with PYO, and autophagy was assessed by evaluating LC3B II expression and autophagosome formation. The expression of EZH2 and JMJD3 was analyzed to identify the key enzyme responsible for regulating H3K27me3. Nuclear-cytoplasmic fractionation and co-immunoprecipitation were performed to determine the distribution of NF-κB and its interaction with H3K27me3. To explore the role of H3K27me3 in PYO-induced autophagy, cells were co-treated with PYO and EZH2 inhibitors (EI1 or CPI-169), and the transcription of ULK1, BECN1, and MAP1LC3B was analyzed using ChIP-qPCR. Similarly, to assess the role of NF-κB, cells were co-treated with PYO and the NF-κB nuclear translocation inhibitor curcumin, followed by ChIP–qPCR analysis. Finally, the reciprocal transcriptional regulation between NF-κB and H3K27me3 was further investigated.

Results

PYO increases LC3B II expression and autophagosome formation in THP-1 macrophages. It also elevates H3K27me3 levels by upregulating EZH2 expression, while JMJD3 remains unchanged. Co-treatment with EZH2 inhibitors reduces H3K27me3 levels, leading to increased LC3B II expression and enhanced autophagosome formation. ChIP–qPCR analysis shows that H3K27me3 enrichment at the ULK1 and MAP1LC3B promoters correlates with reduced transcription, whereas BECN1 remains unaffected. PYO promotes nuclear translocation of NF-κB and enhances its interaction with H3K27me3. ChIP–qPCR further reveals that NF-κB represses the transcription of ULK1 and MAP1LC3B and upregulates EZH2 transcription, which contributes to increased H3K27me3 levels and further suppression of autophagy-related gene expression.

Conclusions

The NF-κB/EZH2/H3K27me3 axis plays a pivotal role in suppressing PYO-induced autophagy in macrophages by repressing the transcription of ULK1 and MAP1LC3B.

Graphical Abstract

Introduction

Pseudomonas aeruginosa (PA), a Gram-negative, rod-shaped γ-proteobacterium, is a common pathogen responsible for hospital-acquired infections. It can cause acute and chronic infections, such as cystic fibrosis, ventilator-associated pneumonia, urinary tract and osteoarticular infections [1, 2]. The World Health Organization (WHO) has categorized PA as a 'critical' pathogen due to its carbapenem-resistant nature, highlighting the urgent need for new drug development [3]. Pyocyanin (PYO), a key virulence factor secreted by PA, disrupts multiple cellular processes in host cells, including electron transport, cellular respiration, energy metabolism, gene expression, and innate immune responses [4].

Studies have shown that PYO induces autophagy in various cell types, including bronchial and bladder epithelial cells and astrocytes [5,6,7,8]. Histone H3 trimethylation at lysine 27 (H3K27me3), known to function as a transcriptional repressor, can either activate and inhibit autophagy, and its levels are regulated by the methyltransferase enhancer of zeste homolog 2 (EZH2) and the demethylase jumonji domain-containing protein 3 (JMJD3) [9,10,11,12]. Our previous research demonstrated that PYO enhances the transcription of the unc-51 like autophagy activating kinase 1 (ULK1) gene via acetylation of HMGN2 and H3K27, promoting autophagy in RAW264.7 macrophages [13]. In addition, under PYO stimulation, an increase in LC3B II expression in THP-1 macrophages was observed as well [13]. However, whether H3K27me3 levels change during PYO-induced autophagy in THP-1 macrophages and whether they play a regulatory role in this autophagy process has not yet been reported.

NF-κB, a pro-inflammatory transcription factor, regulates H3K27me3 levels through its influence on methylation-modifying enzymes [14, 15]. It has been shown that NF-κB and its associated signaling molecules can activate or suppress autophagy, depending on the stimulus and biological context [16]. However, the specific mechanism by which NF-κB regulate H3K27me3 and its effect on PYO-induced autophagy in THP-1 macrophages remains to be elucidated.

In this study, we stimulated THP-1 macrophages with PYO to investigate the specific molecular pathways through which NF-κB influences H3K27me3 levels and how this affects the transcription of autophagy-related genes. Understanding these mechanisms is crucial, given the significant clinical impact of PA-associated hospital-acquired infections.

Methods

Antibodies and reagents

Rabbit polyclonal antibodies against H3K27me3, EZH2, NF-κB, JMJD3, Histone H3, and HRP-labelled anti-rabbit IgG were obtained from Cell Signaling Technology, USA, LC3B and β-actin were procured from Abways, China, and Alexa 488 (goat anti-rabbit, Green) was purchased from Servicebio, China. 4,6-Diamidino-2-phenylindole (DAPI), PYO (Cat# P0046), Phorbol 12-myristate 13-acetate (PMA), Triton X-100, glutaraldehyde, and osmium tetroxide were obtained from Sigma-Aldrich, USA. Chloroquine (CQ; Cat# HY-17589A), El1 (Cat# HY-15573), CPI-169 (Cat# HY-15956A), and Curcumin (Cat# HY-N0005) were acquired from MedChemExpress, USA. RPMI 1640 medium was obtained from Gibco, USA; fetal bovine serum (FBS) from PAN-biotech, Germany; and Penicillin–Streptomycin from Solarbio, China. The All-in-One cDNA Synthesis SuperMix was purchased from TransScript, China; the 2xSYBR Green RT-qPCR Master Mix from Thermo Scientific, USA; and the Nuclear and Cytoplasmic Protein Extraction Kit from Beyotime, China. The UNIQ-10 Column Total RNA Purification Kit was obtained from Sangon Biotech, China; the SimpleChIP® Enzymatic Chromatin IP Kit from Cell Signaling Technology, USA; and Protein A/G PLUS-Agarose beads from Santa Cruz Biotechnology, USA. Additional reagents included RIPA lysis buffer, phosphatase inhibitor, PMSF, and BSA (all from Beyotime, China), a protease inhibitor cocktail (Bimake, China), a BCA Protein Assay Kit (KeyGen Biotech, China), DMSO (Sigma-Aldrich, USA), the Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, USA), and EMbed 812 resin (Electron Microscopy Sciences, USA). Unless otherwise specified, all other reagents were of analytical grade.

Cell lines and cell culture

THP-1 cells (Chinese academy of Sciences, Cat# SCSP-567), a human acute monocytic leukemia cell line, were selected for analysis in this study. The cells were cultured in RPMI 1640 medium (Gibco, Cat# 11875101) containing 12% FBS (PAN-biotech, Cat# P30-3302; inactivated at 56 °C for 30 min) and antibiotics (100 U/mL Penicillin and 100 µg/mL Streptomycin; Solarbio, Cat# P1400), and incubated at 37 °C in a 5% CO2 and humidity-saturated environment. Suspension THP-1 monocytes were seeded in well plates and differentiated into adherent THP-1 macrophages by treating with 100 ng/mL PMA (Sigma-Aldrich, Cat# 79346) for 48 h, followed by a 24-h incubation in RPMI-1640 medium without PMA.

For PYO treatment, THP-1 macrophages were treated with 50 μM PYO for 6 h. In subsequent experiment, cells were treated with 10 μM CQ for 12 h, 1 nM CPI-169 for 24 h, 1 μM EI1 for 24 h, or 10 μM Curcumin for 12 h, depending on the experimental design. DMSO was used as a vehicle control at a final concentration of 0.1% for all treatments.

RNA extraction and RT–qPCR analysis

Total RNA from THP-1 macrophages was extracted using the UNIQ-10 Column Total RNA Purification Kit (Sangon Biotech, Cat# B511361), following the manufacturer’s instructions. RNA purity and concentration were assessed with a Nano Spectrophotometer (Implen, Germany). cDNA was synthesized using All-in-One cDNA Synthesis SuperMix (TransScript, Cat# ABIN5519497), and amplification was carried out in a RT–qPCR instrument (Bio-Rad CFX96, USA) following the Maxima SYBR Green qPCR Master Mix protocol (Thermo Scientific, Cat# K0252). ACTB was set as an internal reference, and the relative quantification of target gene expression was determined by the 2−ΔΔCt method, with normalization to ACTB. The relative gene expression levels in each experimental group were normalized to that of the control group. The specific primer sequences used for RT–qPCR are listed in Supplementary Table 1.

Protein extraction and co-immunoprecipitation (Co-IP)

THP-1 macrophages were lysed using RIPA buffer (Beyotime, Cat# P0013B) containing protease inhibitors (Bimake, Cat# B14012), phosphatase inhibitors (Beyotime, Cat# P1081), and PMSF (Beyotime, Cat# ST506). The lysates were centrifuged at 12000 × rpm for 15 min at 4 °C, and the supernatant was collected for protein extraction. Primary antibodies against H3K27me3 (1:50; Cell Signaling Technology, Cat# 9733, RRID: AB_2626029) and NF-κB (1:100; Cell Signaling Technology, Cat# 8242; RRID: AB_10859369) were added to the protein solution and incubated at 4 °C overnight, followed by incubation with Protein A/G PLUS-Agarose beads (Santa Cruze, Cat# sc-2003) for 2–4 h. The protein–antibody–bead complexes were gently washed three times with RIPA buffer, then denatured by boiling for the subsequent Western blot analysis.

Western blot

For Western blot analysis, THP-1 macrophages were lysed using RIPA buffer for protein extraction. Protein concentrations were determined using a BCA Protein Assay Kit (KeyGen Biotech, Cat# KGP903). Samples were denatured by boiling for 5 min, and equal amounts of protein (20 µg/well) were loaded onto SDS–PAGE gels. After electrophoresis, proteins were transferred onto PVDF membranes and blocked with 5% BSA (Beyotime, Cat# ST023) for 2 h at room temperature. Membranes were then incubated overnight at 4 °C with primary antibodies against H3K27me3 (1:1000), EZH2 (1:1000; Cell Signaling Technology, Cat# 5246; RRID: AB_10694683), NF-κB (1:1000), JMJD3 (1:1000; Cell Signaling Technology, Cat# 3457; RRID: AB_1549620), H3 (1:2000; Cell Signaling Technology, Cat# 4499; RRID: AB_10544537), LC3B (1:2000; Abways, Cat# CY5992), and β-actin (1:10000; Abways, Cat# AB0035). After washing with TBST, membranes were incubated with HRP-labelled Anti-rabbit IgG (1:3000; Cell Signaling Technology, Cat# 7074; RRID: AB_2099233) at room temperature for 2 h. The immunoreactive bands were visualized by enhanced chemiluminescence (Merck Millipore, Cat# WBKLS0500) and imaged using a ChemiDoc™ MP Imaging System (Bio-Rad, USA).

Immunofluorescence

THP-1 macrophages were seeded onto coverslips. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and then permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Cat# T8787) for 15 min. Following three washes with PBS, cells were blocked with 5% BSA for 1–2 h. The coverslips were incubated overnight at 4 °C with primary antibody against LC3B (1:200), and then with Alexa Fluor® 488-conjugated Goat anti-rabbit IgG (1:500; Servicebio, Cat# GB25303; RRID: AB_2910224) for 1 h at room temperature in the dark. Nuclei were stained with DAPI (Sigma-Aldrich, Cat# D9542) for 5 min. Finally, the samples were examined under a fluorescence microscope (Olympus FV-10000, Japan).

Transmission electron microscopy

THP-1 macrophages were fixed in 0.1M PBS with 2.5% glutaraldehyde (Sigma-Aldrich, Cat# G5882) and 2% paraformaldehyde at room temperature for 2 h, followed by post-fixation with 1% osmium tetroxide (Sigma-Aldrich, Cat# 201030) for 2 h. Then, dehydrated in a graded series of ethanol, the cells were embedded in EMbed 812 (Electron Microscopy Sciences, Cat# 149000) and sectioned into ultra-thin slices of about 70 nm. The sections were subsequently double-stained with 3% uranyl acetate and lead citrate, and the autophagy structures were observed under transmission electron microscopy (EM900, Carl Zeiss, Oberkochen, Germany).

Nuclear and cytoplasmic protein extraction

PMSF was added to both the cytoplasmic (Beyotime, Cat# P0028) and nuclear protein extraction reagents (Beyotime, Cat# P0028) to achieve a final concentration of 1mM. Cells were harvested using a scraper and centrifuged at 500 g for 3 min. To each 20 μL cell pellet, 200 μL of cytoplasmic protein extraction reagent was added. The mixture was vortexed for 15 s, chilled for 10 min, vortexed again at maximum speed for 10 s, and then centrifuged at 14,000 g at 4 °C for 10 min to separate cytoplasmic proteins from the supernatant, which was immediately transferred to pre-cooled tubes. The remaining pellet, containing the nuclei, was resuspended in 50–100 μL of nuclear protein extraction reagent, vortexed and chilled as before, and centrifuged to collect nuclear proteins in the final supernatant for further analysis or storage.

Chromatin immunoprecipitation (ChIP)

ChIP–qPCR was performed with SimpleChIP® Enzymatic Chromatin IP Kit (Cell Signaling Technology, Cat# 9003) according to the manufacturer’s protocol. THP-1 macrophages were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by chromatin digestion using Micrococcal nuclease (0.5 μL per tube) and fragmentation into 150–900 bp segments via sonication (Pico, Belgium) for 12 cycles (5 s on/off). Target primary antibodies against H3K27me3 (1:50) and NF-κB (1:100) and Normal IgG were added to the corresponding ChIP samples. Purified DNA was amplified using Maxima SYBR Green qPCR Master Mix. Data were represented as the percentage of input DNA. The primer sequences used for amplifying the gene promoter regions in ChIP–qPCR are detailed in Supplementary Table 2.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8 software (GraphPad, USA). Group comparisons were conducted using the t test or one-way analysis of variance (ANOVA) with Bonferroni post hoc tests. Differences were considered statistically significant when the P value was less than 0.05.

Results

PYO induces autophagy in THP-1 macrophages

We previously reported that PYO induces autophagy in RAW264.7 cells and upregulates the expression of the autophagy marker LC3B II in THP-1 macrophages [13]. Given that THP-1 is a human-derived macrophage cell line, it is of significant interest to investigate whether PYO can induce autophagy in THP-1 macrophages and whether histone modifications are involved in this process. In this study, we first examined whether PYO could induce autophagy in THP-1 macrophages as well. We observed an increase in LC3B II expression in the PYO group, accompanied by LC3B-positive puncta formation (Fig. 1A–C). Transmission electron microscopy further revealed an increased number of both double- and multi-membranous autophagosomes in the PYO group (Fig. 1D). Given that LC3B II levels are affected by lysosomal degradation at the end of the autophagic flux, and typically increase when lysosomal degradation is inhibited [17], we, therefore, pretreated cells with CQ, an inhibitor of the autophagic flux, to assess this effect. Increased LC3B II expression was observed in the PYO + CQ group compared to PYO alone (Fig. 1E, F), indicating that PYO enhances the autophagic flux in THP-1 macrophages. These results suggest that PYO induces autophagy in THP-1 macrophages.

Fig. 1
figure 1

PYO induces autophagy in THP-1 macrophages. A Western blot analysis of LC3B II expression in THP-1 macrophages treated with 50 μM PYO for 6 h, with DMSO as the vehicle control. B Densitometric analysis of relative LC3B II/LC3B I levels normalized to the DMSO group. C Confocal microscopy images displaying the intracellular LC3B-positive puncta (green) and DAPI-stained nuclei (blue) under DMSO or PYO treatment, at 630 × magnification; scale bar = 10 μm. D Transmission electron microscopic images of autophagosome formation following DMSO or PYO treatment. The right panel (at 6000 ×) is an enlargement of the black boxed area in the left panel (at 1500 ×), with arrows denoting double- or multi-membranous autophagosomes; scale bar = 5 μm. E Western blot of LC3B II in THP-1 macrophages treated with PYO and/or CQ (10 μM for 12 h). F Densitometric analysis corresponding to panel (E), showing relative LC3B II/LC3B I levels normalized to the DMSO group. Data are presented as mean ± SD, **P < 0.01, n = 3

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PYO upregulates H3K27me3 levels via EZH2

Next, we applied Western blot analysis to investigate the impact of PYO on the expression levels of H3K27me3, EZH2, and JMJD3 in THP-1 macrophages. Compared with the DMSO control group, PYO treatment increased H3K27me3 and EZH2 expression, without affecting JMJD3 (Fig. 2A–F). In contrast, EZH2 inhibition with CPI-169 and EI1 reduced both EZH2 and H3K27me3 levels compared to the PYO group (Fig. 2G–J; Supplementary Fig. 1A, B). These findings indicate that PYO promotes the upregulation of H3K27me3 in THP-1 macrophages predominantly by increasing EZH2 expression.

Fig. 2
figure 2

PYO upregulates H3K27me3 levels through EZH2. Western blot analysis of A H3K27me3, C EZH2, and E JMJD3 expression in THP-1 macrophages treated with 50 μM PYO for 6 h, with DMSO as control. Densitometric analysis showing H3K27me3 (B), EZH2 (D), and JMJD3 levels (F), normalized to the DMSO group. Western blot analysis of H3K27me3 in THP-1 macrophages treated with G PYO and/or CPI-169 (1 nM for 24 h), and with (I) PYO and/or EI1 (1 μM for 24 h). Densitometric analysis corresponding to panels (G) and (I) are shown in (H) and (J), respectively, displaying the relative H3K27me3 levels normalized to the DMSO group. Data are presented as mean ± SD, **P < 0.01, ***P < 0.001, n = 3

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H3K27me3 inhibits PYO-induced autophagy in THP-1 macrophages

Moreover, LC3B II expression (Fig. 3A–D) and LC3B-positive puncta formation (Fig. 3E) were further increased in the PYO + CPI-169 and PYO + EI1 groups compared to the PYO group, suggests that H3K27me3 inhibits autophagy induced by PYO in THP-1 macrophages.

Fig. 3
figure 3

H3K27me3 Inhibits autophagy in THP-1 macrophages. Western blot analysis of LC3B II expression in THP-1 macrophages treated with A PYO and/or CPI-169 (1 nM for 24 h), and C PYO and/or EI1 (1 μM for 24 h). B, D Densitometric analysis showing relative LC3B II/LC3B I levels normalized to the DMSO group. E Confocal microscopy images showing intracellular LC3B-positive puncta (green) and DAPI-stained nuclei (blue) in THP-1 macrophages treated with DMSO, PYO, PYO + EI1, PYO + CPI-169, EI1, or CPI-169, at 630 × magnification; scale bar = 10 μm. Data are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, n = 3

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H3K27me3 represses the transcription of autophagy-related genes

To further explore H3K27me3’s role in autophagy, we investigated its regulatory effect on the transcription of key autophagy-related genes. RT–qPCR analysis identified a notable upregulation of ULK1, BECN1, and MAP1LC3B in the PYO group, with ULK1 and MAP1LC3B showing even more pronounced increases in the PYO + CPI-169 and PYO + EI1 groups, whereas BECN1 levels remained unchanged (Fig. 4A–F). In addition, ChIP–qPCR analysis on the ULK1 and MAP1LC3B promoter regions revealed a marked decrease in the binding of H3K27me3 to the DNA in the PYO group, with further reduction observed in the PYO + CPI-169 and PYO + EI1 groups (Fig. 4G, H). These findings indicate that H3K27me3 plays a repressive role in the transcription of ULK1 and MAP1LC3B. Under PYO stimulation, there is a decreased recruitment of H3K27me3 to the promoter regions of ULK1 and MAP1LC3B in THP-1 macrophages, thus alleviating its transcriptional repression effects on these genes.

Fig. 4
figure 4

H3K27me3 represses autophagy-related gene transcription. RT–qPCR analysis of transcriptional levels in THP-1 macrophages: A ULK1, B BECN1, and C MAP1LC3B treated with PYO and/or EI1 (1 μM for 24 h); D ULK1, E BECN1, and F MAP1LC3B treated with PYO and/or CPI-169 (1 nM for 24 h). ChIP–qPCR analysis of the enrichment of H3K27me3 at the promoter regions of G ULK1 and H MAP1LC3B genes. Data are presented as mean ± SD, with DMSO as the control. n.s. not significant, *P < 0.05, **P < 0.01, ***P < 0.001, n = 3

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PYO induces nuclear translocation of NF-κB and enhances its interaction with H3K27me3

We continued our investigation with Western blot analysis to examine the impact of PYO on NF-κB expression in THP-1 macrophages. While total NF-κB levels remained largely unchanged compared to the DMSO group, PYO treatment notably upregulated the levels of p-NF-κB (p56) (Fig. 5A, C). Furthermore, nuclear–cytoplasmic fractionation analysis revealed a marked increase in nuclear NF-κB following PYO treatment (Fig. 5B, D). To further explore the molecular interactions, we conducted Co-IP assays, which revealed that the interaction between NF-κB and H3K27me3 was enhanced in the PYO group, as demonstrated by IP results from both H3K27me3 (Fig. 5E, F) and NF-κB (Fig. 5G, H). Collectively, these findings suggest that PYO promote NF-κB activation and nuclear translocation, and enhances its interaction with H3K27me3, without affecting total NF-κB levels.

Fig. 5
figure 5

PYO induces nuclear translocation of NF-κB and enhances its interaction with H3K27me3. A Western blot analysis of total NF-κB and phosphorylated (p-NF-κB) expression levels in THP-1 macrophages following DMSO or PYO treatment. B Western blot analysis of cytoplasmic and nuclear NF-κB levels, using β-actin and H3 as cytoplasmic and nuclear markers, respectively. Densitometric analysis showing the relative levels of total NF-κB and p-NF-κB (C), as well as cytoplasmic and nuclear NF-κB (D), normalized to the DMSO group. The interaction between NF-κB and H3K27me3 proteins was examined using Co-IP assay, with E NF-κB precipitated using an anti-H3K27me3 antibody and G H3K27me3 precipitated using an anti-NF-κB antibody. Densitometric analysis for F IP H3K27me3 and H IP NF-κB expression levels normalized to the DMSO group. Data are presented as mean ± SD, n.s. not significant, *P < 0.05, **P < 0.01, ***P < 0.001, n = 3

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NF-κB inhibits PYO-induced autophagy in THP-1 macrophages

To elucidate the role of NF-κB in autophagy regulation, we pretreated THP-1 macrophages with Curcumin, known to inhibit NF-κB nuclear translocation. Western blot analysis of nuclear–cytoplasmic fractionation showed that NF-κB nuclear translocation was significantly reduced in the PYO + Curcumin group compared with the PYO group alone (Fig. 6A, C). In addition, Western blot analysis indicated an increase in LC3B II expression in the PYO + Curcumin group (Fig. 6B, D). Furthermore, we performed ChIP–qPCR analysis on the promoter regions of ULK1 and MAP1LC3B to assess whether NF-κB regulates the transcription of these genes. This analysis demonstrated a significant decrease in NF-κB binding to the DNA at the aforementioned promoter regions, more significantly in the PYO + Curcumin group than in the PYO group alone (Fig. 6E, F). Collectively, these findings indicate that NF-κB inhibits autophagy induced by PYO in THP-1 macrophages.

Fig. 6
figure 6

NF-κB inhibits autophagy in THP-1 macrophages. A Western blot analysis of cytoplasmic and nuclear NF-κB expression levels in THP-1 macrophages treated with PYO and/or Curcumin (10 μM for 12 h), using β-actin and H3 as respective cytoplasmic and nuclear markers. B Western blot analysis of LC3B II expression in THP-1 macrophages treated with PYO and/or Curcumin. Densitometric analysis showing cytoplasmic and nuclear NF-κB (C), and LC3B II/LC3B I levels (D), normalized to the DMSO group. ChIP–qPCR analysis of the enrichment of NF-κB at the promoter regions of E ULK1 and F MAP1LC3B genes. Data are presented as mean ± SD, n.s. not significant, *P < 0.05, **P < 0.01, n = 3

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NF-κB promotes EZH2 gene transcription to upregulate H3K27me3 levels

In this final experiment, we explored the potential reciprocal regulation between H3K27me3 and NF-κB. We began by pretreating THP-1 macrophages with the EZH2 inhibitor EI1. RT–qPCR analysis revealed no significant differences in NFKB1 transcription levels across all the groups (Fig. 7A), and ChIP–qPCR analysis also showed consistent H3K27me3 binding at the NFKB1 gene promoter regions among groups (Fig. 7B). Therefore, these findings suggest that H3K27me3 levels do no regulate NFKB1 transcription.

Fig. 7
figure 7

NF-κB promotes EZH2 gene transcription to upregulate H3K27me3 expression. A RT–qPCR analysis of NFKB1 transcription levels and B ChIP–qPCR analysis of H3K27me3 enrichment at the NFKB1 gene promoter region in THP-1 macrophages treated with PYO and/or EI1 (1 μM for 24 h). C RT–qPCR analysis of EZH2 transcription levels and D ChIP–qPCR analysis of NF-κB enrichment at the EZH2 gene promoter region in THP-1 macrophages treated with PYO and/or Curcumin (10 μM for 12 h). E Western blot analysis of H3K27me3 and EZH2 expression in THP-1 macrophages treated with PYO and/or Curcumin. F Densitometric analysis of H3K27me3 and EZH2 levels normalized to the DMSO group. Data are presented as mean ± SD, n.s. not significant, *P < 0.05, **P < 0.01, ***P < 0.001, n = 3

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Subsequently, we pretreated THP-1 macrophages with Curcumin, an inhibitor that prevents the nuclear translocation of NF-κB. RT–qPCR analysis revealed a significant increase in EZH2 transcription in the PYO group compared to the DMSO control, an effect that was diminished by Curcumin in the PYO + Curcumin group (Fig. 7C). This suggests that the increased nuclear translocation of NF-κB appears to enhance EZH2 transcription. Further ChIP–qPCR analysis demonstrated increased NF-κB binding at the EZH2 gene promoter in the PYO group, an effect that Curcumin attenuated in the PYO + Curcumin group (Fig. 7D), thus indicating NF-κB’s direct involvement in regulating EZH2 transcription. Moreover, western blot analysis showed reduced EZH2 and H3K27me3 expression in the PYO + Curcumin group compared to the PYO group (Fig. 7E, F). Collectively, these results highlight the pivotal role of NF-κB in the upregulation of H3K27me3 levels through the transcriptional activation of the EZH2 gene.

Discussion

Autophagy plays a double-edged sword role in the interactions between bacterial pathogens and host cells. On one hand, some bacterial pathogens can escape phagosomes and enter autophagosomes, aiding their survival and replication [18]. On the other hand, autophagy helps host cells capture pathogens that escape into the cytoplasm and degrade them in autolysosomes [18, 19]. In this study, we explored the regulatory roles of H3K27me3 and NF-κB in PYO-induced autophagy in THP-1 macrophages. We found that PYO induces autophagy in these cells and promotes the nuclear translocation of NF-κB, which enhances EZH2 gene transcription and increases H3K27me3 levels. The elevated H3K27me3, through its enhanced interaction with NF-κB, suppresses the transcription of the autophagy-related genes ULK1 and MAP1LC3B, thereby restraining the autophagic process. In contrast, in PYO-induced RAW264.7 macrophages, PYO enhances the transcription of the ULK1 and promotes autophagy by increasing the levels of HMGN2 acetylation and H3K27 acetylation (H3K27ac) [13]. Since H3K27me3 and H3K27ac are classical histone marks associated with transcriptional repression and activation, respectively, these two studies together elucidate the mechanism of PYO-induced autophagy in macrophages from the complementary perspectives of histone methylation and acetylation.

Wei et al. reported that under nutrient-rich conditions, EZH2 catalyzes an increase in H3K27me3 and suppresses the transcription of negative regulators of the mTOR pathway, including TSC2, DEPTOR, RHOA, and GPI [20]. This downregulation of TSC2 by EZH2 leads to mTOR activation and inhibition of autophagy, while EZH2 inhibition promotes autophagy [20]. A study demonstrated that Mycobacterium tuberculosis, through EZH2 and histone deacetylase HDAC3, increases H3K27me3 and decreases H3K27ac, inhibiting autophagy in RAW 264.7 cells and promoting bacterial survival [21]. In line with these findings, this study showed that increased H3K27me3 levels inhibit PYO-induced autophagy in THP-1 macrophages. However, Yang et al. found that elevated homocysteine enhances H3K27me3 levels through EZH2, suppressing CFTR (cystic fibrosis transmembrane conductance regulator) gene transcription, promoting autophagy, and exacerbating liver injury in mice [11]. We, therefore, speculate that the increase in H3K27me3 levels, catalyzed by EZH2, may have dual effects on autophagy, either promoting or inhibiting it. This variability likely arises from specific molecular mechanisms that vary across cell types, tissues, and pathological conditions, as well as genetic and environmental factors.

The role of NF-κB in macrophage autophagy is complex. In RAW264.7 macrophages, Nrf2 promotes autophagy by inhibiting NF-κB nuclear translocation [22]. Wang et al. found that palmitic acid blocks autophagic flux and upregulates NF-κB signaling, promoting macrophages inflammation and contributing to non-alcoholic steatohepatitis [23]. Similarly, our results show that NF-κB inhibits PYO-induced autophagy in THP-1 macrophages by suppressing the transcription of ULK1 and MAP1LC3B. Conversely, Zhong et al. demonstrated that NF-κB mitigates NLRP3 inflammasomes overactivation in macrophages by promoting mitochondrial autophagy [24]. We consider that autophagy, as a protective mechanism, can be activated during inflammation to eliminate inflammatory products, damaged organelles, and proteins, preventing cell death caused by excessive inflammation. The discrepancies in the NF-κB-autophagy interplay likely stem from variations in stimuli, cell types, and environmental contexts.

Numerous studies have demonstrated the reciprocal regulation between NF-κB and H3K27me3. Wu et al. found that NF-κB interacts with EZH2-mediated hypermethylation of H3K27me3, contributing to wheezing in children with pulmonary inflammation [25]. In addition, EZH2-mediated H3K27me3/SOCS3/TRAF6/NF-κB signaling is linked to neuroinflammation in rats after subarachnoid hemorrhage [26]. Furthermore, DZNep, an EZH2 inhibitor, activates NF-κB signaling in bone marrow-derived macrophages via the EZH2–H3K27me3–Foxc1 axis, enhancing osteoclast formation [27]. In this study PYO stimulation in THP-1 macrophages enhanced the interaction between NF-κB and H3K27me3. NF-κB also promoted H3K27me3 levels by upregulating EZH2 gene transcription, but H3K27me3 did no influence NFKB1 gene transcription.

In conclusion, our findings demonstrate that PYO induces autophagy in THP-1 macrophages while activating intrinsic inhibitory pathways. Specifically, PYO promotes NF-κB nuclear translocation and enhances its interaction with H3K27me3. NF-κB upregulates EZH2 gene expression, increasing H3K27me3 levels and suppressing the transcription of ULK1 and MAP1LC3B genes, thereby inhibiting autophagy These insights into the host–pathogen interactions may inform therapeutic strategies to combat PA-associated hospital-acquired infections by targeting cell-intrinsic autophagy regulatory pathways in macrophages.

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Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 82201353), the Science and Technology Bureau of Nanchong City, Sichuan Province, China (Grant No. 22SXZRKX0008), and the School-Level Scientific Research Development Plan Project of North Sichuan Medical College (Grant Nos. CBY23-QNA12, CBY21-QD20).

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  1. Ji Wang, Min Bian and Simin Liang contributed as the first co-authors.

Authors and Affiliations

  1. Department of Anesthesiology, Affiliated Hospital of North Sichuan Medical College, Nanchong, China

    Ji Wang, Min Bian & Xiaowei Yi

  2. Department of Anesthesiology, Beijing Anzhen Nanchong Hospital, Capital Medical University & Nanchong Central Hospital/the Second Clinical Medical Institution, North Sichuan Medical College, Nanchong, 637000, China

    Simin Liang & Yu Du

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Contributions

Conceptualization: Wang J, Bian M, Liang SM, Yi XW, Du Y Data curation: Wang J, Bian M, Liang SM,Yi XW, Du Y. Formal analysis: Bian M, Liang SM, Yi XW. Funding acquisition: Wang J, Yi XW, Du Y. Investigation: Wang J, Bian M, Liang SM, Yi XW, Du Y. Methodology: Wang J, Bian M, Liang SM, Yi XW, Du Y. Validation: Du Y. Visualization: Du Y. Writing—original draft: Wang J. Writing—review & editing: Wang J, Bian M, Liang SM, Yi XW, Du Y.

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Supplementary Information

40001_2025_2614_MOESM1_ESM.docx

Supplementary material 1: Table 1 Primer sequences for RT-qPCR. Table 2 Primer sequences of gene promoters. Fig. 1 Inhibitory efficiency of EZH2 inhibitors in THP-1 macrophages. (A) Western blot analysis of EZH2 expression in THP-1 macrophages treated with CPI-169 (1 nM) or El1 (1 μM) for 24 h, using DMSO as the vehicle control. (B) Densitometric analysis showing relative EZH2 expression normalized to the DMSO group. Data are presented as mean ± SD, ***P < 0.001, n=3.

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Wang, J., Bian, M., Liang, S. et al. NF-κB-mediated enhancement of H3K27me3 through EZH2: a mechanism to suppress pyocyanin-induced autophagy in macrophages. Eur J Med Res 30, 346 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40001-025-02614-3

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European Journal of Medical Research

ISSN: 2047-783X

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