The regulatory effect of acetylation of HMGN2 and H3K27 on pyocyanin‐induced autophagy in macrophages by affecting Ulk1 transcription

Abstract Pyocyanin (PYO) is a major virulence factor secreted by Pseudomonas aeruginosa, and autophagy is a crucial homeostatic mechanism for the interaction between the pathogens and the host. It remains unknown whether PYO leads to autophagy in macrophages by regulating histone acetylation. The high mobility group nucleosomal binding domain 2 (HMGN2) has been reported to regulate the PYO‐induced autophagy and oxidative stress in the epithelial cells; however, the underlying molecular mechanism has not been fully elucidated. In this study, PYO was found to induce autophagy in macrophages, and the mechanism might be correlated with the up‐regulation of HMGN2 acetylation (HMGN2ac) and the down‐regulation of H3K27 acetylation (H3K27ac) by modulation of the activities of acetyltransferases and deacetylases. Moreover, we further demonstrated that the up‐regulated HMGN2ac enhances its recruitment to the Ulk1 promoter, while the down‐regulation of H3K27ac reduces its recruitment to the Ulk1 promoter, thereby promoting or inhibiting the transcription of Ulk1. In conclusion, HMGN2ac and H3K27ac play regulatory roles in the PYO‐induced autophagy in macrophages.


| INTRODUC TI ON
Pseudomonas aeruginosa (PA), a Gram-negative pathogenic bacterium of the genus Pseudomonas, accounts for a wide variety of acute and chronic infections. Among the other systems in the body, the respiratory system is most susceptible to PA infection. 1 PA can also infect the urinary system, blood system and central nervous system. 2 Pyocyanin (PYO) is a major virulence factor secreted by PA. 3 PYO can impose oxidative stress on the host cells and cause inflammatory damage by up-regulating the pro-inflammatory factors such as TNFα and IL- 8. 4 Autophagy is a crucial homeostasis mechanism involved in many physiological and pathological processes. 5,6 PA has been well documented to induce autophagy in macrophages, mast cells and bronchial epithelial cells. [7][8][9] Current studies on autophagy pertaining to PYO infection have been restricted to the astrocytes and bronchial epithelium. 10,11 Therefore, whether PYO can lead to autophagy in macrophages remains to be clarified.
Manipulating the host cellular epigenetics is one of the notable strategies by which bacteria evade immune responses. And it has been verified that H4K16ac plays an important role in the process of autophagy. 12 Subsequent studies have indicated that H2AK5ac, H2BK15ac, H3K9ac, H3K56ac and H4K5ac also regulated autophagy. [13][14][15] However, whether PA or PYO regulates autophagy via epigenetic manipulation has not yet been elucidated.
The high mobility group nucleosomal binding proteins (HMGNs) are the only non-histone nuclear proteins capable of binding to nucleosomes, where HMGN2 is a critical member. 16 We have previously reported that HMGN2 has antibacterial and antiviral effect. 17,18 Moreover, our previous studies have also found that HMGN2 can mediate antimicrobial peptide expression, 19 induce cytoskeleton rearrangement 20 and bacterial internalization, 21 and modulate oxidative stress 22 and autophagy, 23 all of which contribute to innate immune responses.
This study aimed to investigate whether PYO induces autophagy in macrophages, to detect the changes in the acetylation of HMGN2 and H3K27 in RAW 264.7 cells both with and without Hmgn2 knockout under PYO treatment and to explore the interactions between HMGN2ac and H3K27ac. We further explored the autophagyrelated genes that are regulated by modification in the acetylation of HMGN2 and H3K27. Since PA/PYO nosocomial infection is a challenging problem in clinical medicine, this study would be of great clinical significance.
Alexa Fluor 488-labelled goat anti-rabbit IgG (GB25303) was purchased from Servicebio. The All-in-One cDNA Synthesis SuperMix was obtained from Bimake Corporation. The 2xSYBR Green RT- were purchased from Sigma. The foetal calf serum (FBS) was purchased from PAN-Biotech (Germany), and the penicillin and streptomycin were obtained from Solarbio (Beijing, China).

| Cell culture
The murine macrophage-like cell line RAW 264.7 and human monocytic cell line THP-1 were obtained from the Institute of Cell Biology, Chinese Academy of Sciences. The Hmgn2-knockout (KO) RAW 264.7 cells were prepared using the CRISPR/Cas9 technique in our laboratory. 24 The suspended THP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate for 48 h to differentiate into adherent macrophages. The cells were maintained in RPMI 1640 medium supplemented with 12% heat-inactivated FBS, 100 U/ ml penicillin and 100 mg/ml streptomycin and cultured in 5% CO 2 at 37°C.

| Isolation of murine peritoneal macrophages (PM)
Animal experiments were performed according to protocols approved by the Animal Care and Use Committee of Sichuan University, China. Female BALB/c mice (6-8 weeks old) were purchased from Dossy Corporation (Chengdu, China). The peritoneumderived macrophages were isolated from the peritoneal cavity of mice, as previously described. 25 Briefly, the mice were killed and decapitated 72 h after receiving a 3 ml intraperitoneal injection of 3% thioglycollate. A small incision along the midline of the abdomen was immediately made, and the intact peritoneal wall was exposed. Five millilitres of the pre-cooled serum-free medium was injected into the abdominal cavity with a syringe and needle. Using the same syringe and needle, the lavage fluid from the lower peritoneal cavity was aspirated. Next, the lavage fluid was transferred into a pre-cooled 50-ml conical polypropylene centrifuge tube. This procedure was performed three to five times, and the peritoneal exudate cells were centrifuged at 1500 × g for 15 min. The cells were resuspended, counted and inoculated into the cell culture plates. The non-adherent cells were washed away with phosphate buffered saline (PBS) after 12 h.

| RNA extraction and quantitative real-time PCR (RT-qPCR)
Total RNA from cells was extracted using the UNIQ-10 Trizol Total RNA Isolation Kit (Sangon Biotech), and its quality and quantity were measured with a spectrophotometer (NanoPhotometer, Implen).
The cDNA was synthesized using the All-in-One cDNA Synthesis SuperMix kit, and amplification reactions were performed using the SYBR Green RT-qPCR Master Mix on a fluorescence RT-qPCR system (CFX96, Bio-Rad). In this study, the Actb gene was chosen as the internal control, and the relative quantification of gene expression in each group was calculated using the 2 −△△CT method. The primer sequences used for RT-qPCR are listed in Table 1.

| Protein extraction and immunoprecipitation (IP) or Co-immunoprecipitation (Co-IP)
The IP was used to detect HMGN2ac, while the Co-IP was performed to observe the interaction between HMGN2 and H3K27ac. Briefly, the proteins were extracted from the cells by using the RIPA cell lysis buffer containing protease inhibitor (Biotool), phosphatase inhibitor (Biotool) and PMSF (Beyotime) and then centrifuged at 4°C and 12000× rpm for 15 min. The protein supernatants were incubated with primary antibodies overnight at 4°C and then co-incubated with protein A/G agarose (Beyotime) for 4 h. The protein-antibody-beads complex was washed three times with RIPA cell lysis buffer, boiled and subjected to Western blotting analysis.

| Protein-modified mass spectrometry
After washing with PBS, the total protein was extracted from the cells. The acetylation sites of protein peptides were detected following reductive alkylation, proteolysis and protein extraction, desalting and freeze-drying, liquid chromatography-mass spectrometry (LC-MS) assay, and data analysis (Trump, Inc., China).
Primers, as shown in Table 2, were designed to mutate the K2 and K4 sites of the HMGN2 amino acid sequence to arginine (2K-R) or to mutate the K53, K55 and K56 sites to arginine (3K-R). The WT HMGN2 plasmids, together with the aforementioned primers, were added to the reaction system and amplified according to the manufacturer's instructions (TransGen Biotech). The PCR products were transformed into competent cells, and the 2K-R HMGN2 and 3K-R HMGN2 plasmids were obtained by sequencing.
The plasmids with a final concentration of 1 μg/ml were then transfected into the KO RAW 264.7 cells using the jetPRIME kit (Polyplus-transfection, France) according to the manufacturer's recommendations, and the culture medium was changed 6 h later. The transfection efficiency was verified 24 h post-transfection by using a fluorescence microscope (Olympus FV-10000).

| Western blotting (WB) analysis
Proteins from cells were extracted using pre-cooled RIPA lysis buffer (Beyotime), and the concentrations were measured using a bicinchoninic acid protein assay kit (KeyGen Biotech). Equal amounts of total proteins (20 μg) were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. After blocking with 5% bovine serum albumin (BSA) for 2 h at room temperature, the membranes were incubated with the appropriate concentrations of primary antibodies overnight at 4°C. After several washes, the membranes were incubated with the corresponding secondary antibodies for 2 h at room temperature and then visualized using enhanced chemiluminescence detection kit (Merck Millipore) on the ChemiDoc™ MP image system (Bio-Rad).

| Immunofluorescence staining
After washing with PBS, the cells were fixed with 4% paraformaldehyde for 20 min, followed by permeabilization with 0.5% Triton X-100 for 15 min. Next, the cells were blocked with 5% BSA for 2 h at room temperature. Subsequently, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with fluorescently labelled secondary antibodies at room temperature for 1 h in the dark. Nuclei were stained with DAPI. Finally, staining was visualized using a fluorescent microscope (Olympus FV-10000) or a confocal microscope (Zeiss LSM710).

| Transmission electron microscopy
The transmission electron microscopy (TEM) was used to directly observe autophagy-specific structures. The cells were fixed in 2% glutaraldehyde in sodium cacodylate buffer overnight at 4°C and processed for TEM according to a previously described method. 26

| Chromatin immunoprecipitation (ChIP) assay
The chromatin immunoprecipitation analysis was carried out using

| Statistical analysis
Data in this study were expressed as mean ± standard deviation (SD) and analysed using the GraphPad Prism 7 software. Values of P less than 0.05 were considered to be statistically significant, while n.s indicated no statistical difference.

| PYO induces autophagy in macrophages
To investigate whether PYO induced autophagy in RAW 264.7 cells, we first examined the protein levels of LC3B II and p62 in the RAW 264.7 cells in response to the different doses and time of PYO treatment ( Figure 1A

| PYO up-regulates HMGN2ac in the macrophages
We then applied an IP assay to determine whether PYO induced the acetylation modification of HMGN2. After enrichment with TA B L E 2 Sequences of primers for mutation of HMGN2. VPKGKK. Next, we compared these peptide sequences to the mouse-derived HMGN2 amino acid sequences (UniProt number: P09602) and found that the lysine acetylation sites were K2, K4, K53, K55 and K56 ( Figure 2E), all of which are located in the nuclear localization signal (NLS). 28 In addition, we performed an identical IP assay on the PM and THP-1 cells, and the results showed that PYO up-regulated HMGN2ac in both types of macrophages ( Figure 2F-G). We planned to further explore the regulatory roles of these lysine acetylation sites on autophagy in subsequent experiments.

| Influence of HMGN2ac on PYO-induced autophagy in RAW 264.7 cells
Herein, we first performed WB assay to analyse the LC3B II protein level in KO RAW 264.7 cells and wild-type (WT) RAW 264.7 cells which were exposed to PYO. Although the protein level of LC3B In contrast, after treatment with PYO, the protein level of LC3B II was significantly lower in the 5K-R group than in the WT group ( Figure 3F-G), indicating that simultaneous acetylation of these five sites in the HMGN2 NLS region might be related to autophagy caused by PYO.

| PYO down-regulates H3K27ac in macrophages
Previous studies have demonstrated that PA induces acetylation modification of histone, 29 and the knockout of the Hmgn2 gene affected the expression of H3K27ac, 30 which participates in the regulation of transcription of autophagy-related genes. 31 We, therefore, conducted a WB assay to examine the H3K27ac protein level in the RAW 264.7 cells and THP-1 cells that were exposed to PYO.
Exposure of the RAW 264.7 cells and THP-1 cells to PYO caused a significant reduction in H3K27ac levels compared to the exposure to DMSO (Figure 4A-B)

| HMGN2ac does not interact with H3K27ac
To test the involvement of HMGN2ac in the regulation of H3K27ac (S555) in the RAW 264.7 cells exposed to PYO at different timepoints. The results showed that the expression of p-ULK1 peaked at 1 h and then gradually declined from 2 h to 6 h, but was still higher at 6 h than that in the DMSO group ( Figure 6A-B). Consistently, the p-ULK1 protein levels in the PM cells were significantly increased upon PYO treatment for 2 h and 6 h ( Figure 6C-D). Taken

| D ISCUSS I ON
Oxidative stress and inflammation have been reported as core events during PA infection. 32,35,36 Our previous study has shown that HMGN2 attenuates PYO-induced oxidative stress via the Nrf2 signalling cascade and inhibits the internalization of PA in the lung epithelial cells. 22 In addition, our previous data from the study on bladder epithelial cell line 5637 infected with Escherichia coli also indicated that HMGN2 induces autophagy by activating the AMPK/ ULK1 pathway, which facilitates the proliferation of Escherichia coli. 23 Macrophages are one of the most important innate immune cells.
Autophagy induced by pathogen infection is known to be involved in a series of biological events, including macrophage activation and polarization, apoptosis, mitochondrial metabolism, inflammasome assembly, the release of inflammatory cytokines, bacterial clearance and autophagic death. Therefore, in the present study, we established a PYO-induced autophagy model in macrophages to explore the role and mechanism of HMGN2ac and H3K27ac during autophagy.
In this study, we verified that PYO can induce autophagy in macrophages from several aspects, including detecting the autophagy markers with WB assay and observing the formation of autophagosomes by using the confocal microscopy and TEM, as well as the introduction of autophagy flux inhibitors, such as CQ (inhibits the fusion of autophagosomes and lysosomes) and 3-MA (inhibits the formation of autophagosomes). During autophagy, p62 delivers ubiquitinated proteins or bacteria to autophagosomes for degradation and is subsequently degraded in the autolysosome. 37  improve the survival of bacteria, which is supposed to be the biological meaning of the elevated p62 levels observed in this study. 39,40 PA has been shown to induce autophagy in the epithelial cells and macrophages. 8 It has been verified that the P300/CBP-associated factor (PCAF) and p300 mediate the acetylation modification of HMGN2. 42 Unfortunately, our data showed that there was no interaction between HMGN2ac and H3K27ac and that they may independently participate in the regulation of autophagy. However, the above conclusion is based only on indirect evidence, and the exact relationship between HMGN2ac and H3K27ac needs to be further studied.
ULK1/ATG1, among the first of over 30 autophagy-related proteins identified, 47 is the key promoter of autophagy. 34 The stress signal kinase AMPK and protein kinase mTORC1 were found to positively and negatively regulate Ulk1, respectively, participating in the modulation of cellular responses to a wide range of stimuli, such as nutrition and starvation. 48 Our previous experiments confirmed that HMGN2 regulates autophagy in the bladder epithelial cells through the AMPK/ULK1 pathway. 23 In this study, our data showed that PYO regulates the transcription of Ulk1 by down-regulating H3K27ac and up-regulating HMGN2ac, which indicates an important role of HMGN2ac and H3K27ac in the maintenance of homeostasis of Ulk1 transcription.
In conclusion, we found that PYO induced autophagy in macrophages by up-regulating HMGN2ac and down-regulating H3K27ac.
We further demonstrated that the up-regulated HMGN2ac promoted its recruitment at the Ulk1 promoter region, which ultimately enhanced the transcription of Ulk1, while the down-regulation of H3K27ac resulted in a decrease in its recruitment at the Ulk1 promoter region, thereby suppressing the transcription of Ulk1. Overall, HMGN2ac and H3K27ac can regulate PYO-induced macrophage autophagy.

ACK N OWLED G EM ENTS
This work was supported by grants from the Scientific Research

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.