All materials, kits, and antibodies utilized in this research are listed in Table 1 .

C57BL/6 HMGN2 knockout (HMGN2-/-) mice were generated by Beijing Biocytogen Co., Ltd. This knockout model eliminates HMGN2 expression at the genome-wide level. C57BL/6 Wildtype (WT) mice were obtained from Chengdu Dossy Experimental Animals Co., Ltd. All mice were maintained under standard conditions at the animal center of University of Electronic Science and Technology of China. All necessary measures were taken to avoid or minimize discomfort, pain, or distress to the animals. Animal procedures were approved by the Institutional Animal Care and Use Committee of University of Electronic Science and Technology of China (NO.1061420210305013).

Mus musculus macrophage RAW264.7 cells were purchased from ATCC and stored in our lab, cells were cultured with DMEM medium containing 10% fetal bovine serum, 100 μg/ml streptomycin and 100 U/ml penicillin at 37°C in a standard cell incubator with 5% CO₂. Bacteria strains Escherichia coli 19138, Klebsiella Pneumoniae K33, Uropathogenic Escherichia coli J96 are preserved in our lab. Bacteria were cultured in Luria-Bertani (LB) broth for overnight to reach the logarithmic phase. The concentration of bacteria suspensions was measured by the absorbance at 600nm.

Genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system was used to construct the HMGN2 gene deletion cell line. Briefly, Lenti-Crispr-V2-gRNA-hmgn2 plasmid were transfected into RAW264.7 cells with transfection reagent (poly plus) according to the product manual. The small guide RNA (sgRNA) sequence (5’-AGG CTG GCT TAC CTT CGC AGG GG-3’) was designed by https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design website. After 48 hours, puromycin was used to select genome-edited cells. Five days later, limiting dilution analysis was used to select the HMGN2 knockout monoclonal cell. Western blot analysis and DNA sequencing were used to identify HMGN2 knockout cells. For HMGN2/CD14 double knockout cell line construction, HMGN2 knockout cells were used following the protocol above, the sgRNA sequence 5’-CTT TCC TCG TCT AGC TCG CA-3’ was used for mouse CD14 deletion.

Wildtype and HMGN2 knockout RAW264.7 cells were seeded into 96-well plates for 5000 cells per well and cultured in standard condition (DMEM medium, 37°C, 5% CO₂) without any treatment for 24 hours, or treated with LPS (1μg/mL) for 24 hours. 10μL cell counting kit (CCK)-8 solution was added to each well and incubated at 37°C for two hours. The absorbance value at 450nm was measured by using a microplate analyzer and the corresponding cell number was calculated.

Total RNA was extracted using the Total RNA Isolation Kit according to the protocol provided by manufacturer. 0.5μg RNA were used for cDNA synthesis, cDNA was then applied for real time quantitative PCR amplification following the manufacturer’s instructions. Primers used in this study were listed in Table 2 .

Cells were stimulated with LPS for 24 h, and cells were washed with clean PBS for three times, then cells were lysed with RIPA buffer containing Protease inhibitors and phosphatase inhibitors for 30min at 4°C. Cell debris were removed by centrifuge. The concentration of protein was measured with BCA reagent according to the protocol. The obtained protein was then separated by SDS-PAGE and then transferred to the PVDF membrane and then incubated with specific antibodies overnight, followed by incubate with the peroxidase-conjugated goat anti rabbit secondary antibody. The immunoreactive bands were then analyzed.

RAW264.7 cells seeded into the slides. After being stimulated with LPS for the indicated time respectively, cells were washed three times with precooled PBS for 3 times, then fixed with 4% paraformaldehyde for 15 minutes, followed by permeation with Triton for 20 minutes, then sealed with goat serum for 30 minutes, and incubated overnight with HMGN2 (or F4/80) primary antibody. At the second day, cells were washed and then stained with fluorescent secondary antibodies for one hour. Then the nuclei were stained with DAPI solution and observed under a fluorescence microscope.

Cells were collected and suspended in staining buffer. After three times wash, cells were counted and the F4/80, CD11b antibodies were added at optimal concentrations and incubated on ice for 20min in dark. Then cells were washed for three times and the flow cytometric analysis were performed as instructions. For HMGN2 staining, the HMGN2 primary antibody was added and incubated with cells for 30min, then cells were washed for three times and then incubated with FITC- conjugated secondary antibody for 30min. Cells were then analyzed.

Shortly, the supernatant after LPS stimulation was collected, centrifuged at 12000 rpm for 10 minutes. For serum collection, blood was collected and left to rest for two hours, then centrifuged at 3500 rpm for 10 minutes. The concentration of CD14 were measured by CD14 ELISA kit according to the manufacturer instruction. The concentration of NO was measured by Total Nitric Oxide Assay Kit.

One day in advance, WT and HMGN2-/- RAW264.7 cells were inoculated into 24-well plate, and the Fluorescein-conjugated pHrodo™ Red E. coli BioParticles (Invitrogen) were added into each well at a ratio of 1:10 (cell:particles) and mixed well. The cells were placed in incubators at 37°C for three hours. The phagocytosis ability of the cells was detected by flow cytometry and fluorescence microscopy.

RAW264.7 cells were inoculated at 2×10⁵/well into a 24-well plate and then stimulated with LPS for 24 hours. bacteria (Escherichia coli 19138, Klebsiella Pneumoniae K33, Uropathogenic Escherichia coli J96) were added and co-incubated with RAW264.7 cells for different time and different MOI (multiplicity of infection, number ratio of bacteria to cells). Then Triton-100 was added and cells were lysed for 30 minutes, cells and fluid were next collected, diluted and plated on the LB plates. Bacterial colonies were counted.

Shortly, CD14 promoter was inserted in PGL3-basic plasmid which expressed Renilla luciferase. WT and HMGN2-/- RAW264.7 cells were inoculated into 96-well plate at 2×10⁴, cells were co-transfected with PGL3-mCD14 promoter plasmid and internal reference PRL-SV40 plasmid. 48 hours later, cells were stimulated with LPS for 24h, then the fluorescence values of Renilla luciferase and firefly luciferase were detected and calculated according to the protocol.

ChIP assay was applied and performed as reported previously (9). Shortly, initially, conduct formaldehyde cross-linking on WT and HMGN2 knockout cells to fix the protein-DNA complexes. Subsequently, lyse the cells and fragment the chromatin DNA into 150-900bp small pieces through sonication. Next, introduce H3K4me3, H3K27ac, H3K9ac, H3K27me3, or IgG antibodies for immunoprecipitation to capture the target protein-DNA complexes. Incorporate Protein A/G beads to bind the antibody-target protein-DNA complexes and collect them via precipitation. Wash the precipitated complexes to eliminate non-specific binding and acquire the enriched target protein-DNA complexes. Then, reverse the cross-linking and purify the enriched DNA fragments. Take 2% of the Input DNA, the DNA after immunoprecipitation (IP) with the target protein antibody (H3K4me3, H3K27ac, H3K9ac, H3K27me3), and the DNA after IP with the IgG antibody as templates respectively. Add the ChIP primers corresponding to the target genes to be detected ( Table 2 ) respectively, conduct quantitative PCR reactions, and obtain their respective Ct values. Calculate the ChIP enrichment efficiency according to the following formula: Signal relative to Input = 2% × 2(Ct [2%Input Sample]– Ct [IP Sample]).

For in vivo infection, 2×10⁵ bacteria (Escherichia coli 19138) were injected intraperitoneally (i.p.), 24 hours later, abdominal cells are collected by collecting abdominal flushing fluid, the lung, spleen, liver were anatomically separated out and grinded into single cell suspension, and then plated into LB-plate, the relative survival of bacteria was calculated using the plate colony count method (n=5).

All data in this study were processed by means of GraphPad Prism 7 and are presented as the mean ± standard deviation (SD). All data were analyzed through two-tailed unpaired Student’s t-tests for comparisons between two groups. For multiple comparisons, one- or two-way Analysis of Variances (ANOVA) followed by Bonferroni post hoc tests were employed. When P < 0.05, the difference between groups was regarded as statistically significant. For all cellular results, a minimum of three independent and repeated experiments were carried out. In the animal experiments, at least four mice were utilized.

The expression of HMGN2 varies in different immune microenvironments, it can be either promoted or inhibited to regulate various physiological or pathological reactions (1, 9, 22). To investigate the impact of bacterial infection on HMGN2 expression in macrophages, we stimulated the mouse RAW264.7 macrophage cell line with Escherichia coli (E. coli) endotoxin lipopolysaccharide (LPS). Subsequently, we detected HMGN2 expression through western blot analysis ( Figures 1A, B ), RT-qPCR ( Figure 1C ), and immunofluorescence assays ( Figure 1D ). The results indicated that the expression of HMGN2 was significantly decreased by more than half in LPS-stimulated macrophages after 48 or 72 hours. However, we also discovered that short-term LPS stimulation (1ng/ml and 10 ng/ml for 24 hours) could promote the mRNA expression of HMGN2, suggesting a complex regulatory mechanism of HMGN2 in macrophages during LPS stimulation.

We further established a murine model of lung infection to verify the alteration in HMGN2 expression within bacteria-infected macrophages in vivo. Specifically, we surgically administered an intratracheal injection of E. coli strain 19138 into the lung tissues of C57BL/6 mice. Twenty-four hours post-injection, the expression of HMGN2 in macrophages of mouse lung tissue was detected via flow cytometry ( Figure 1E ) and immunofluorescence ( Figure 1F ).

The findings revealed that the expression of HMGN2 in macrophages of E. coli-infected lung tissue was 35% lower compared to that in uninfected mice (treated with phosphate - buffered saline, PBS). These results suggest that HMGN2 may play a crucial role in macrophages infected by bacteria.

In subsequent experiments, the cells were treated with 1μg/mL of LPS for 24 hours. This treatment corresponds to the maximum concentration and the longest treatment duration that do not induce alterations in the expression of HMGN2. As such, it effectively circumvents any interference stemming from changes in the basal level of HMGN2.

To investigate the role of HMGN2 in macrophages, we utilized clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology to establish a HMGN2 knockout mouse RAW264.7 cell line. In the HMGN2 knockout cells, a deletion of two bases on the fourth exon of the HMGN2 gene (indicated in red font) was induced ( Figure 2A ). The western blot results demonstrated the absence of HMGN2 in the knockout cells ( Figure 2B ). Additionally, the CCK-8 assay results indicated that there was no significant change in the cell number when compared with wild-type RAW264.7 cells (WT) after 24 hours of culture under physiological (UT) or LPS stimulation conditions ( Figure 2C ).

Macrophages were incubated with live E. coli strain 19138. The impact of HMGN2 knockout on the bactericidal capacity of macrophages was detected via the in-vitro bacterial killing assay ( Figure 2D ). Following incubation with macrophages, the number of live bacteria remaining in the HMGN2-KO group was significantly lower than that in the WT group ( Figures 2E–G ), indicating an enhanced bactericidal capacity of HMGN2-KO RAW264.7 macrophages. In addition to E. coli, the influence of HMGN2 knockout on the bactericidal capacity of macrophages was also investigated using other common pathogens, including uropathogenic Escherichia coli (UPEC) and Klebsiella pneumoniae (K. pneumoniae). The results were consistent with those obtained for the E. coli strain. HMGN2-KO macrophages demonstrated stronger bactericidal capabilities during UPEC ( Figures 2H, I ) and K. pneumoniae infection ( Figures 2J, K ), with fewer live bacteria remaining. These findings suggest that HMGN2 knockout can enhance the bacterial-killing capacity of RAW264.7 macrophages.

Macrophages represent a crucial class of phagocytic cells. To explore whether HMGN2 knockout affects the bacterial-killing capacity of macrophages by influencing their phagocytic function, we detected the phagocytic ability of HMGN2-knockout cells. We found that the phagocytic ability of HMGN2-KO cells was significantly stronger than that of WT cells ( Figure 3A ) by using fluorescent E. coli biological particles and flow cytometry. A similar phenotype was also observed in the result of immunofluorescence result ( Figure 3B ). To identify the downstream target genes of HMGN2 that govern the phagocytic function of macrophages, we then analyzed the gene expression associated with phagocytosis and discovered that most phagocytic molecules were upregulated in HMGN2-knockout macrophages upon LPS stimulation. These molecules included Cd14, Rac family small GTPase 1(Rac1), cell division cycle 42 (Cdc42), Ras homolog family member A (RhoA), WASP sctin nucleation promoting factor (Wasp), Supervillin, Semaphorin 3E (Sema3e), TIAM Rac1 associated GEF 1 (Tiam1), and PlexinA1. Among them, CD14 was the gene with the most significant upregulation, showing more than a two-fold increase in HMGN2-knockout macrophages ( Figure 3C ).

CD14 is a crucial component of phagocytes (23–25). Subsequently, we further employed RT - qPCR and western blot techniques to investigate the impact of HMGN2 knockout on the expression of CD14 in unstimulated (UT) and LPS-stimulated RAW264.7 macrophages. The RT-qPCR results indicated that the mRNA level of CD14 in LPS-stimulated cells was significantly elevated by approximately 3.6 times compared with the UT group. Moreover, the CD14 expression in LPS - stimulated HMGN2-knockout cells was increased by twofold compared with LPS-stimulated WT cells ( Figure 3D ). The western blot results were consistent with those of RT - qPCR. Specifically, the protein level of CD14 in LPS - stimulated HMGN2-KO cells was increased by approximately 1.5 times compared with the LPS-stimulated WT group ( Figures 3E, F ).

As a pattern recognition receptor, CD14 exists in two distinct forms. One is soluble CD14 (sCD14), which is released from cells and dissolved in the extracellular fluid. The other is membranous CD14 (mCD14), which is located in the cell membrane. Although the working patterns of sCD14 and mCD14 differ slightly, both can regulate similar immune functions of macrophages. These functions include mediating bacterial phagocytosis and binding to LPS to activate downstream signaling pathways. Both sCD14 and mCD14 play crucial roles in the immune functions of macrophages during bacterial infection (24–26).

To investigate which type of CD14 is influenced by HMGN2, the cell culture supernatant was collected for the detection of the sCD14 level via the ELISA assay. The results indicated that the sCD14 level in the supernatant of LPS-stimulated HMGN2-KO cells were 1.2-fold higher than that in the supernatant of LPS-stimulated WT cells ( Figure 3G ). Meanwhile, we isolated the membrane protein of macrophages. It was found that the expression level of mCD14 was significantly elevated by 2.1-fold in LPS-stimulated HMGN2 knockout macrophages ( Figures 3H, I ). These results indicate that HMGN2 knockout can enhance the expression of both soluble and membrane CD14.

To verify whether CD14 is implicated in the HMGN2 - mediated bactericidal processes, we utilized CRISPR-Cas9 technology to establish the HMGN2-CD14 double-knockout RAW264.7 cell line (HMGN2^-/-/CD14 ^-/-) based on HMGN2-KO cells ( Figure 3J ). The impact of HMGN2^-/-/CD14 ^-/- on the bactericidal function of macrophages was detected via an in-vitro bacterial-killing assay. As shown in Figure 3K , the number of live bacteria remaining in the HMGN2^-/-/CD14 ^-/- cells was higher than that in the HMGN2 knockout cells ( Figure 3K ). This indicates that CD14 is involved in the regulation of the bactericidal function of RAW264.7 cells mediated by HMGN2.

As a chromatin structural protein, HMGN2 has been reported to regulate gene expression by influencing the histone modification of transcriptional regulatory elements, such as promoters and enhancers (1). In order to study the mechanism of HMGN2 on CD14 expression, we initially explored the regulatory effect of HMGN2 knockout on the CD14 promoter by dual luciferase reporter system ( Figure 4A ). The pGL3 plasmid inserted with the CD14 promoter sequence was transfected into WT and HMGN2-knockout macrophages. Significantly higher luciferase activity was detected in the HMGN2 knockout macrophages. Specifically, under the stimulation of LPS, the luciferase activity in HMGN2-knockout macrophages were nearly twice that of WT cells. This result demonstrates that the transcription efficiency of the CD14 promoter in RAW264.7 cells was enhanced after HMGN2 knockout.

Histone epigenetic modification represents one of the crucial mechanisms by which HMGN2 regulates the efficiency of gene transcription. This includes modifications such as H3K4me3, H3K27ac, H3K9ac, and H3K27me3. Elevated levels of H3K4me3, H3K27ac, and H3K9ac enhance transcription efficiency, while an increased level of H3K27me3 inhibits transcription efficiency ( Figure 4B ) (27). Initially, we analyzed the protein levels of these four histone modifications via Western blot. It was found that the protein levels of total H3K4me3, H3K9ac, H3K27Ac, and H3K27me3 in HMGN2 - knockout macrophages exhibited no significant alterations compared to those in WT cells ( Figure 4C ). Subsequently, chromatin immunoprecipitation (ChIP) was employed to detect the impact of HMGN2 knockout on histone modification in the CD14 promoter region. We discovered that, following LPS stimulation, the enrichment levels of H3K4me3, H3K27ac, and H3K9ac at multiple sites in HMGN2 - knockout macrophages were increased compared to those in WT cells ( Figures 4D, G ). Notably, H3K4me3 demonstrated the most significant elevation, with increased enrichment around positions -5, -50, -100, -450, -900, and -1000 upstream of the transcription start site ( Figure 4D ). H3K9ac also showed increased enrichment at positions -50 and -900 upstream of the transcription start site ( Figure 4E ), and H3K27ac exhibited increased enrichment at position -900 upstream of the transcription start site ( Figure 4F ). These findings indicate that, under the stimulation of LPS, HMGN2 knockout enhances CD14 transcriptional efficiency by promoting histone modifications, including H3K4me3, H3K27ac, and H3K9ac, in the CD14 promoter region.

The regulation of gene transcription level through histone modification is accomplished by altering the interaction between the promoter and transcription factors. It has been reported that the cAMP-response element binding protein (CREB), which serves as an important transcription regulator for promoting CD14 gene transcription, can enhance the CD14 transcription level by interacting with the CD14 promoter region (28). To further confirm whether the knockout of HMGN2 can enhance the transcription regulation efficiency of the CD14 gene by facilitating the enrichment of the transcription factor CREB in the promoter region near the -900 and -1000 sites, a ChIP assay was utilized to detect the enrichment level of CREB in the CD14 promoter region ( Figure 4H ). The results indicated that in LPS-stimulated macrophages, HMGN2-KO cells demonstrated an 83.4-fold higher enrichment level of CREB transcription factors on the CD14 promoter. Specifically, this enrichment level was five times that of the WT group ( Figure 4I ).

In addition to phagocytosis, CD14, serving as a crucial co-receptor of LPS, is also capable of activating intracellular signals via downstream signaling pathways such as the MAPK and NF-κB. Consequently, it regulates the transcription of immune - related genes (24, 25, 29). Hence, the higher CD14 expression induced by HMGN2 knockout may also facilitate the high expression of antimicrobial substances, thereby enhancing the bactericidal effect of macrophages.

Nitric oxide (NO) is a crucial molecule in the bactericidal function of macrophages. It can be released from activated macrophages to the extracellular environment and directly interact with pathogenic microorganisms to exert bactericidal effects (30). We found that no production was significantly enhanced in HMGN2-knockout macrophages. RT-qPCR showed that the iNOS expression in HMGN2-knockout macrophages was about 18.5 times higher than that in the WT ( Figure 5A ). Western blot yielded similar results. After 24-hour LPS stimulation, the iNOS level in HMGN2-knockout macrophages was nearly three times that in the WT ( Figures 5B, C ). Additionally, the NO level in the HMGN2-knockout macrophage culture medium was nearly 1.8 times that of the WT ( Figure 5D ). These results suggest that HMGN2 knockout enhances iNOS expression and promotes NO production.

Next, we used HMGN2/CD14 double-knockout RAW264.7 macrophages to detect whether HMGN2 promotes the expression of iNOS through CD14. The RT-qPCR results ( Figure 5E ) and western blot results ( Figures 5F, G ) both showed that, after knocking out CD14 on the basis of HMGN2 knockout, the originally elevated iNOS expression was significantly inhibited. This indicates that CD14 knockout can reverse the HMGN2 knockout-induced elevated iNOS expression. In other words, CD14 is involved in the regulation of HMGN2-mediated iNOS expression.

To verify whether NO is the effector molecule in the enhanced bactericidal ability of macrophages resulting from HMGN2 knockout, cells were treated with the iNOS inhibitor L-NAME. Subsequently, an in-vitro bacterial killing assay was employed to detect the impact of L-NAME on the bactericidal function of macrophages. As shown in Figure 5H , in the control group (CTR, without inhibitor), HMGN2 knockout enhanced the bactericidal function of macrophages. However, after treatment with L-NAME, this enhancing effect was neutralized. This indicates that NO may be a crucial effector molecule in the bactericidal ability of macrophages promoted by HMGN2 knockout.

Under inflammatory conditions, CD14-mediated iNOS expression is regulated via MAPK and NF-κB pathways (24, 25, 31). To confirm the involvement of the MAPK pathway in HMGN2-mediated NO production, macrophages were treated with extracellular-regulated protein kinases (ERK) inhibitor (PD98059), c-Jun N-terminal kinase (JNK) inhibitor (SP600125), p38 mitogen - activated protein kinase (P38) inhibitor (SB203580), and an MAPK downstream transcription factor activator protein-1 (AP-1) inhibitor (T-5224). Western blot was used to detect iNOS expression, and the results showed that inhibitor treatment reversed the increased iNOS expression induced by HMGN2 knockout ( Figures 5I–L ). We also employed inhibitors of the NF-κB signaling pathway and did not observe similar reverse outcomes (not shown). These findings indicate that the MAPK signaling pathway is crucial for HMGN2-mediated regulation of macrophage NO production.

We established a murine bacterial infection model to investigate the impact of HMGN2 knockout on the ability of mice to eliminate pathogenic microorganisms. E. coli was intraperitoneally injected into WT and HMGN2-knockout mice. Twenty - four hours after infection, the peritoneal lavage fluid was collected, and the lung, liver, and spleen tissues of the mice were dissected. The number of live bacteria in each tissue was detected using the in-vivo bacterial killing assay ( Figure 6A ). The results demonstrated that, compared with WT mice, the number of live bacteria remaining in the peritoneal cavity of HMGN2 knockout mice was decreased by 85% ( Figures 6B, C ). There was scarcely any live E. coli remaining in the lung tissue of HMGN2 knockout mice ( Figure 6D ). The number of live bacteria remaining in the liver of HMGN2 knockout mice was decreased by 90% ( Figure 6E ), and the number of live bacteria remaining in the spleen of HMGN2 knockout mice was decreased by 80% ( Figure 6F ). These findings indicated that HMGN2 knockout significantly enhanced the clearance of infected bacteria in mice.

To verify whether the knockout of HMGN2 promotes the production of CD14 and NO in vivo, the serum was collected 24 hours after bacterial treatment. The serum levels of CD14 and NO were determined using the ELISA method and the Griess Reagent method, respectively. It was found that the levels of CD14 ( Figure 6G ) and NO ( Figure 6H ) in HMGN2-knockout mice were significantly higher than those in WT mice. This indicates that the knockout of HMGN2 also promotes the production of CD14 and NO in vivo.