The Antimicrobial Peptide MPX Promotes the Maturation of Dendritic Cells by Enhancing Glycolysis

The Antimicrobial Peptide MPX Promotes the Maturation of Dendritic Cells by Enhancing Glycolysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Antimicrobial Peptide MPX Promotes the Maturation of Dendritic Cells by Enhancing Glycolysis Xiaowei Du, Bo Wen, Ruibiao Wang, Chengshui Liao, Weiyu Luo, Wei Zhang, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5867452/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Antimicrobial peptide MPX is derived from wasp venom, known for its antibacterial activity against a wide range of bacteria. Researches have indicated that antimicrobial peptide could induce dendritic cell maturation through interaction with surface receptors, thereby activating antigen presentation and initiating immune responses, which indirectly contributes to antimicrobial effects. However, the specific mechanism by which the antimicrobial peptide MPX activates dendritic cells remains unclear. The aim of this study was to investigate how MPX activates dendritic cells and thus enhances antigen presentation.In the present study, it was found that dendritic cells exhibited a time-dependent internalization of the antimicrobial peptide MPX. MPX internalized by dendritic cells increased the expression of maturation markers such as CD80, CD86, and promoted the secretion of the pro-inflammatory factors IL-1β and IL-18, whereas it had a relatively mild effect on TNF-α.Furthermore, MPX also altered metabolic pathways of dendritic cells, favoring glycolysis and thereby activating them to subsequently promote CTL cell proliferation. To explore the molecular mechanisms underlying MPX’s regulation of dendritic cell activation, RNA-seq analysis was performed. The results showed that following MPX treatment dendritic cells exhibited had 90 differentially expressed genes (DEGs) compared to the control group, with enrichment observed in metabolism and biosynthesis processes. These findings not only provide insights into the potential applications of MPX in infection control but also emphasize its significant role in dendritic cell-mediated immune responses. In summary, this study has demonstrated that the MPX can activate dendritic cells, promote T-cell proliferation, and present new avenues for utilizing antimicrobial peptides in immune modulation within the host. Biological sciences/Immunology Biological sciences/Immunology/Innate immunity antimicrobial peptides dendritic cell maturation MPX immune regulation glycolysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Antimicrobial peptides (AMPs) are short cationic peptides capable of regulating the host's immune response, serving as a crucial component in the innate immune system[ 1 ]. They function within the immune system by regulating signaling pathways and inflammatory responses, while also modulating both innate and adaptive cell-mediated immune responses[ 2 – 4 ]. For example, immune cells such as macrophages, mast cells, and T cells exhibit direct chemotactic responses to AMPs and their derivatives[ 5 – 7 ]. Interestingly, under varying experimental conditions, antimicrobial peptides have been observed to induce or inhibit the activation of dendritic cells. A study found that antimicrobial peptides mBD2 and mBD14 could promote the maturation of mouse-derived dendritic cells through TLR4, which serves as the receptor for bacterial lipopolysaccharide (LPS)[8; 9]. Coincidentally, another study found that the cathelicidin LL-37 can induce the maturation of human-derived dendritic cells in vitro [ 10 ]. Mastoparan-X (MPX), a member of the mastoparan antimicrobial peptide family isolated from the venom of Vespidae insects, is a 14-amino acid peptide known for its broad antibacterial spectrum, stable physical and chemical properties, and potent bactericidal effects against Gram-positive as well as Gram-negative bacteria. MPX has been suggested to effectively reduce the host's inflammatory response[ 11 – 15 ]. Dendritic cells are the most powerful antigen-presenting cells (APCs) in the organism, and current studies have confirmed that AMPs not only have antimicrobial effects, but also can participate in the immune process of the organism by promoting dendritic cell maturation, it has been documented in the literature that antimicrobial peptides can promote the activation of dendritic cells[ 16 ], but the specific mechanism by which MPX promotes the maturation of dendritic cells is not clear.. Antigen presentation is widely acknowledged as a pivotal process for eliciting effective immune responses against pathogens and tumors. Dendritic cells are recognized within the body as the most efficient APCs[ 17 – 19 ]. Depending on the expression levels of co-stimulatory molecules such as CD40 and CD80, dendritic cells can be classified into immature dendritic cells (imDCs) and mature DCs (mDCs)[ 20 ]. ImDCs primarily inhabit peripheral tissues, relying on mechanisms like receptor-mediated endocytosis and receptor-independent phagocytosis, which enable robust capture of antigens, including those from pathogens and newly expressed antigens induced by malignant transformation. Upon encountering antigens or other activating stimuli, dendritic cells undergo maturation and migrate to lymph nodes[21; 22]. This maturation process involves significant production of pro-inflammatory cytokines and increased expression of co-stimulatory molecules. Mature dendritic cells effectively present processed antigenic fragments to T cells, thereby initiating immune responses. Research has demonstrated that antimicrobial peptides have the ability to modulate dendritic cells functions and induce specific antigen immune responses, thereby linking innate and adaptive immune responses. For example, human lactoferrin (hLF-11) can induce dendritic cells to express HLA class II antigens and dectin-1, promoting differentiated dendritic cells to produce inflammatory cytokines such as IL-6 and IL-10, while significantly increasing their phagocytic activity against Candida. Additionally, lactoferrin itself can activate the TLR4 pathway, thereby stimulating macrophages and dendritic cells[ 23 ]. In other study, Human beta-defensin 3 (hBD3) can induce the phenotypic maturation of LC-DCs, resulting in the upregulation of CCR7and mediating chemotaxis to CCL19 and CCL21, thereby endowing hBD3 with excellent immunostimulatory properties[ 24 ]. Mouse beta-defensin 2 (mBD2) promotes the maturation of dendritic cells by activating the TLR4 receptor for bacterial lipopolysaccharide and stimulates dendritic cells to express high levels of co-stimulatory molecules (e.g., CD40, CD80, CD86), major histocompatibility complex class II, and the chemokine receptor CCR7[ 25 ]. LL-37 can interact with Toll-like receptors on dendritic cells, thereby modulating immune responses through enhanced endocytic activity, increased co-stimulatory molecule expression, enhanced pro-inflammatory cytokine secretion, and the regulation of T cell differentiation[ 26 ]. Considering the above, MPX holds immense potential in aiding host infection control and immune response regulation, with broad prospects for application. However, the specific mechanisms by which MPX influences dendritic cell maturation remain unclear. This study aims to explore the impact of antimicrobial peptide MPX on dendritic cell maturation and its regulation of inflammatory responses. The studys contribute novel insights to the field of immunology and provide vigorous support for the future development of dendritic cell-based immune therapies against infections. 2 Materials and Methods 2.1 Reagents Pyruvate detection kit, Pyruvate Kinase Assay Kit, glucose test kit, and lactate test kit were purchased from Nanjing Jiancheng Bioengineering Institute (China). The ATP test kit was purchased from Solarbio (China). The dendritic cells were retained in the laboratory. 2.2 Peptide Synthesis and Sequence, cells. MPX (H-INWKGIAAMAKKLL-NH2; Mw: 1556.01) was synthesized by Shanghai Jielu Biochemical Co., Ltd. (China) using solid-phase N-9-fluorenylmethoxycarbonyl (Fmoc) strategy and high-performance liquid chromatography (HPLC) purification, with a purity of up to 98%. The cell line used in the experiment is the bone marrow-derived dendritic cell line DC2.4. 2.3 Laser Confocal Microscopy Mouse dendritic cells were cultured in DMEM (containing 10% serum and 1% double antibodies) and incubated in a 37°C, 5% CO 2 incubator to ensure cell attachment to the culture dish. Then, dendritic cells were seeded at a density of 1×10 5 cells/well in laser confocal culture dishes and allowed to adhere overnight. FITC-labled MPX drug at a concentration of 10 µg/mL was added to the laser confocal culture dishes, and dendritic cells were incubated for different time periods (1h, 2h, 3h). A blank control group was also set up. After incubation, the culture dishes were washed three times with PBS, each time for 5 min, to ensure the removal of residual culture medium. Subsequently, 250 µL of 4% paraformaldehyde was added to fix the cells in each well at room temperature for 15 min, followed by thorough washing with PBS. Finally, 300 µL of dye containing DAPI (concentration 3 µg/mL) was added to each well for nuclear staining for 5 min. After five washes with PBS, images were observed and captured using a laser confocal microscope, and Zen software was used to process the images. 2.4 Cell Viability Assay Cell Counting Kit-8 (CCK-8) method was performed to assess cell viability. Initially, cells in logarithmic growth phase were seeded in a 96-well plate and randomly divided into different groups: blank control group (0 µg/mL) and various concentrations of MPX treatment groups (5/10/20/40/80 µg/mL). After cell attachment in the culture dish, the respective concentration of MPX was added, and cells were further cultured for 24 h. Subsequently, 10 µL of CCK-8 solution was added to each well, and the culture dish was incubated for 2 h. Then, the absorbance was measured at 450 nm using an enzyme meter. Each experiment was repeated three times to ensure reliable results. The calculation of relative cell activity is as follows: (Experimental group OD value / Blank group OD value) × 100%. 2.5 Western Blot (WB) Total cellular proteins were extracted using Upload Buffer Protein Upload Buffer (Beyotime, China). Subsequently, the proteins were subjected to SDS-PAGE gel electrophoresis under reducing conditions, using a 10% SDS-PAGE gel. The proteins were then transferred to a PVDF membrane. The PVDF membrane was blocked in 5% skim milk for 2 h and then incubated overnight at 4°C with antibodies CD80 (1:1000, CST, USA), CD86 (1:1000, CST), MHC I (1:1000, Abcam, USA). β-Actin (1:1000, CST) was used as a loading control. Afterward, the membrane was treated with horseradish peroxidase-conjugated IgG and visualized using an enhanced chemiluminescence (ECL) reagent (Solarbio, China). The protein bands were analyzed using ImageJ software 1.8.0. 2.6 RT-qPCR Dendritic cells were cultured in six-well plates at a density of 1×10 6 cells/cm 2 for 24h, then treated with MPX and LPS for 12h and the cells were collected. Total RNA was extracted from dendritic cells using the Total RNA Extraction Kit (Solarbio). The RNA concentration and quality were assessed. The CD80, CD86, MHC II, IL-18, IL-1β, TNF-α RNA samples were reverse-transcribed into cDNA using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kits (Thermo Fisher,USA). Subsequently, used the Yisheng QuantiNova SYBR Green PCR kit to detect the expression of genes such as CD80 , CD86 , MHC II , IL-β , TNF-α, IL-18 . Primer sequences were designed using Primer6 software. A fluorescent quantitative PCR instrument from Applied Biosystems was used, and all measurements were performed in triplicate in three independent experiments. The SYBR Green PCR conditions were as follows: initial denaturation at 95°C for 2 min, followed by 40 cycles, each comprising 5 s at 95°C, 30 s at 60°C, and 15 s at 95°C. GAPDH was used as a reference gene, and the 2 −ΔΔCT method was used to calculate the relative gene expression level, normalized to the GAPDH mRNA level[ 27 ]. The primer sequences in S1: 2.7 Allogeneic Mixed Lymphocyte Reaction Dendritic cells were resuspended in complete culture medium and co-cultured with CTL cells at a 1:5 ratio in a 96-well plate. Two control groups were set up, one with CTL cells added alone, and the other with complete culture medium added alone. All culture groups were incubated at 37°C for 3 d, and then CCK-8 reagent (20 µL/sample) was added, followed by further incubation for 4h. A microplate reader was used to measure the absorbance of each group at 450 nm. Additionally, the stimulation index (SI) of each group was calculated using the formula SI = (experimental sample A value - complete culture medium A value) / (CTL A value - complete culture medium A value).Results for each group were obtained from experiments with three replicates to ensure reliability. 2.8 Glycolysis Dendritic cells were first digested with trypsin to prepare a single-cell suspension. Then, these single cells were evenly seeded in six-well culture dishes, and cultured until the cell fusion density reached over 90%. Subsequently, complete culture medium was prepared with a solution containing 10 µg/mL MPX and 1 µg/mL LPS. This solution was used to culture dendritic cells for 12h. A control group with pure culture medium without MPX and LPS was also set up. After incubation, the cell supernatant was collected, and the respective assay kits (Nanjing Jiancheng ) were used to determine the levels of lactate, Pyruvate, pyruvate kinase, glucose, and ATP in the supernatant. In addition, RT-qPCR was used to detect the expression levels of GLUT1 and HK2 genes related to glycolysis. The primer sequences in S1: 2.9 Differential gene expression analysis Dendritic cells were divided into an untreated group and an antimicrobial peptide MPX-treated group, with each group containing three replicate samples from the same experiment. In the experiment, RNA was first extracted according to standard steps, and its quality and concentration were detected by spectrophotometer. Qualified RNA samples were sent to Azenta for library construction and expression profiling. High-throughput RNA sequencing was performed using the Illumina NovaSeq 6000 platform, and gene expression levels were calculated using HTSeq. Differentially expressed genes (DEGs) were identified by DESeq2 software, and the screening criteria were adjusted p-value less than 0.05 and absolute value of log2 fold change greater than 1. Subsequently, functional enrichment analyses of the GO and KEGG pathways were performed[ 28 – 30 ], and the protein interaction networks of DEGs were analyzed using the STRING database and Cytoscape software. The list of top upregulated and downregulated molecules identified by transcriptomic analysis is presented in S2. Sequencing results have been uploaded to NCBI, the SRA database ID SRR31850248-SRR31850253. Finally, eight randomly selected DEGs ( Apoo-ps , Myh4 , Myl1 , Car3 , Ndrg1 , Car9 , Adm and Nos2 ) were validated by RT-qPCR to ensure the reliability of the transcriptome sequencing results. The primer sequences in S1: 2.10 Data Analysis All experiments were performed with 3–6 replicate samples. All data are presented as mean ± SD. Statistical data and charts were obtained using GraphPad Prism 8.3.0 software. One-way analysis of variance (ANOVA) was used to determine statistical significance. Statistical significance was considered as follows: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. 3 Results 3.1 Evaluation of Phagocytosis of Antimicrobial Peptide FITC-labled MPX by Dendritic Cells Qualitative observations using laser confocal microscopy revealed that FITC-labeled antimicrobial peptide MPX enters dendritic cells (DCs) via passive transport mechanisms. As illustrated in Fig. 1, intracellular fluorescence intensity demonstrated a time-dependent increase over the 0–3 hour period, indicating efficient uptake of MPX by DCs. This dynamic process provides compelling evidence for the immune responsiveness of dendritic cells to antimicrobial peptides and underscores the critical role of temporal factors in mediating biological interactions. Fig. 1 : Fluorescence Intensity Changes of FITC-Labeled MPX Internalized by Dendritic Cells at Different Time Points Detected by Laser Confocal Microscopy 3.2 Regulation of Dendritic Cell Activation-Related Protein Expression by MPX First, the effect of MPX on the viability of dendritic cells was assessed using the CCK-8 assay. As shown in Fig. 2 A, the cell viability remained above 85% after MPX treatment, indicating a minimal effect on cell viability, thus allowing further experiments. Next, changes in the expression of activation markers in dendritic cells after MPX treatment were observed. The effects of MPX on the expression of markers in dendritic cells were evaluated from two perspectives: different concentrations of MPX treatment (Fig. 2 B- 2 D) and different durations (Fig. 2 E- 2 G). Mature dendritic cells (mDC) were identified using Western blot analysis of CD80, CD86, and MHC I. The results showed that the expression of CD80 and CD86 was increased in the MPX-treated group at different concentrations and in the positive control LPS (1 µg/mL)-treated group. And the expression level decreased at MPX concentration of 20 µg/mL, suggesting that high concentration of MPX may inhibit the expression of markers. Therefore, 10 µg/mL of MPX was selected for subsequent experiments.Under the condition of a 10 µg/mL concentration, the expression levels of MHC I and CD80 both showed an increasing trend with different treatment durations and reached a peak at 12h of treatment. Therefore, for subsequent experiments, dendritic cells were treated with the antimicrobial peptide MPX at a concentration of 10 µg/mL for 12 hours. 3.3 The Regulation of Surface Markers and Inflammatory Cytokines Expression in Dendritic Cell Activation by Antimicrobial Peptide MPX Focused on studying the impact of MPX (10 µg/mL) treatment on dendritic cells activation and cytokine expression, using LPS treatment as a positive control. First, examined the expression of CD80 , CD86 , and MHC II , molecules typically considered markers of dendritic cell activation. Consistent with the previous Western blot results, after MPX treatment, the expression of CD80 , CD86 , and MHC II all significantly increased (Fig. 3 A- 3 C), indicating a marked activation of dendritic cells under MPX treatment. This suggests that MPX may play a critical role in antigen presentation by dendritic cells and T cell activation. Furthermore, monitored changes in the expression of pro-inflammatory cytokines. Following MPX treatment, the expression levels of IL-1β and IL-18 significantly increased (Fig. 3 D- 3 F). The elevation of these cytokines suggests that MPX treatment may induce an inflammatory response, which may have an important role in immune responses. However, unlike the former two, the effect of MPX on tumor necrosis factor TNF-α expression (Fig. 3 E) was relatively mild, indicating that MPX may have different regulatory effects on different cytokines. In summary, observations reveal that after MPX treatment, dendritic cells exhibit an activated state, as confirmed by the upregulation of CD80 , CD86 , and MHC II . Furthermore, the elevation of pro-inflammatory cytokines IL-1β and IL-18 suggests that MPX treatment may induce an inflammatory response. However, the impact of MPX on TNF-α expression is relatively minor. These findings provide a strong basis for further research on the effects of MPX on the immune system, particularly in the context of dendritic cell-mediated antigen presentation and immune regulation. 3.4 Allogeneic Mixed Lymphocyte Reaction To evaluate the capacity of dendritic cells to stimulate the proliferation of allogeneic T cells, conducted an allogeneic mixed lymphocyte reaction assay using CTL cells. As shown in Fig. 3 G, the dendritic cell groups treated with LPS and MPX (LPS group and MPX group) both exhibited higher stimulation indices, revealed that dendritic cells supplemented with the antimicrobial peptide MPX could significantly induce T cell proliferation, indicating that MPX can effectively promote the activation of dendritic cells. 3.5 Glycolysis Experiment During dendritic cell activation, metabolic reprogramming occurs, shifting cellular metabolism from oxidative phosphorylation (OXPHOS) to glycolysis—a critical hallmark of DC activation. To investigate this, we analyzed changes in glycolytic substrates, products, and enzymes in cell culture media, which indirectly indicated that MPX significantly enhances DC glycolytic activity. Specifically, MPX-treated DCs showed elevated lactate levels reflecting accelerated production (Fig. 4A), reduced pyruvate levels suggesting metabolic redirection of carbon flux (Fig. 4B), and an increased lactate-to-pyruvate ratio indicating adaptive redox adjustments (Fig. 4C). Concurrently, pyruvate kinase upregulation underscored MPX's role in pyruvate metabolism (Fig. 4D), while decreased extracellular glucose levels confirmed enhanced uptake to fuel glycolysis (Fig. 4E). Corresponding ATP increases directly linked glycolytic enhancement to elevated biosynthesis (Fig. 4F), supported by GLUT1/HK2 upregulation confirming augmented glucose uptake and phosphorylation (Fig. 4G-4H). Fig. 4 : Changes in key glycolytic substrates, products, enzymes in the culture medium, and related gene expression within dendritic cells after treatment with antimicrobial peptide MPX. A: lactic acid content. B: pyruvate content. C: ratio of lactic acid to pyruvate. D: pyruvate kinase content. E: glucose content. F: ATP content. G: Glut1 gene expression. H: HK2 gene expression. 3.6 Differential Gene Expression Analysis with GO and KEGG Enrichment Analysis To investigate the changes in gene expression profiles in dendritic cells upon treatment with antimicrobial peptide MPX, then utilized the FeatureCounts software to calculate the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) expression values based on read counts. The DESeq2 software was employed to compute the fold change (FC) of each gene at different time points post-infection compared to the control group. A threshold of FDR (False Discovery Rate) ≤ 0.05 and |Log2FC| ≥ 1 was applied to identify differentially expressed genes (DEGs) in response to MPX treatment relative to the control group (Fig. 5 A- 5 B).In comparison to general descriptions of gene or transcript properties provided by functional annotations, enrichment analysis serves to elucidate the functions and KEGG pathways of genes at the lowest levels. Furthermore, Fisher's test is employed to determine the significant enrichment of gene functions and KEGG pathways for the DEGs. Enrichment analysis offers the most detailed information regarding gene functions and KEGG pathways. GO terms and KEGG pathways are separately subjected to enrichment analysis using GOatools and KOBAS tools. GO terms and KEGG pathways with a corrected p-value of ≤ 0.05 are considered enriched. Enriched GO terms are categorized into three groups: Biological Processes (BP), Cellular Components (CC), and Molecular Functions (MF) (Fig. 5 C- 5 D). The results show that MPX mainly affects the metabolic pathway after the effect of DCs, consistent with the previous test proving that MPX can change its glycolysis levels, and did provide the basis of later experiments. 3.7 Protein-Protein Interaction (PPI) Analysis of DEGs and RT-qPCR Validation The PPI network analysis was performed for DEGs in the experimental group treated with MPX and the control group, based on FDR (False Discovery Rate) ≤ 0.05 and |Log2FC| ≥ 2. To gain a deeper understanding of the biological relevance of the Differentially Expressed Genes (DEGs), PPI network analysis of the DEGs was conducted using the STRING database (Fig. 6 A). The Cytoscape software was utilized to visualize the protein-protein interaction network of the DEGs (Fig. 6 B). The PPI network analysis identified a total of 123 interaction relationships among 90 genes within the DEGs from the control group and the MPX-treated group.To validate the reproducibility and repeatability of the DEGs identified from the transcriptome sequencing, eight genes were randomly selected for RT-qPCR validation. These genes included Apoo-ps , Myh4 , Myl1 , Car3 , Ndrg1 , Car9 , Adm , and Nos2. The results demonstrated (Fig. 6 C- 6 D) that the differential expression of these genes was significant and consistent with the direction of gene expression changes identified through RNA-seq. This suggests that the DEGs obtained through transcriptome sequencing are reliable. 3.8 Validation of Nos2 Downregulation and Glycolysis-Related Metabolite Changes in Dendritic Cells Treated with MPX Based on transcriptome sequencing results, the downregulated gene Nos2 was selected for validation. Since ferrous citrate (FC) has been reported as an agonist of Nos2 that promotes its gene expression[ 31 ], measured glycolysis-related products (glucose, lactate, pyruvate) in culture media across experimental groups. Notably, FC treatment led to decreased supernatant lactate levels, increased pyruvate and glucose concentrations, and reduced lactate-to-pyruvate ratios—phenotypes opposite to MPX effects—indicating suppressed dendritic cell glycolysis (Fig. 7 A-D). These results confirm that Nos2 serves as a critical gene in dendritic cell activation, validating the accuracy of transcriptome sequencing findings. 4 Discussion Dendritic cells play an integral role when pathogens invade, and their main task is to promote antigen presentation and activate T cells, thus ensuring the continuity of the antigen-specific immune response. Studies have shown that certain drugs, such as oligofructose, the Chinese herb astragalus polysaccharide, and the antimicrobial peptide LL-37, can promote dendritic cell differentiation, maturation, and function[ 32 ]. Unlike conventional drugs, the antimicrobial peptide MPX has the ability to disrupt the integrity of bacterial membranes, increase membrane permeability, and alter membrane potential, which leads to leakage of intracellular material and ultimately produces a bactericidal effect. More notably, MPX neither induces bacterial resistance nor causes damage to host cells and organs. Both MPX and immune cells play important roles in defense against infections. The aim of this experiment was to verify whether MPX can effectively induce the maturation and function of dendritic cells through in vitro experiments. The results showed that MPX significantly increased the expression levels of maturation markers (e.g., CD 80, CD 86, and MHC I) on dendritic cells cultured in vitro . In addition, MPX treatment significantly increased the production of interleukin factors such as IL-1β and IL-18 . However, the increase in TNF-α expression induced by MPX treatment was not as pronounced as that of LPS, suggesting that the inflammatory response triggered by MPX may not be as strong as that of LPS. Mature dendritic cells not only express higher levels of co-stimulatory molecules and MHC-I during antigen presentation, but also play a key role in the antigen-specific T-cell immune response, promoting the activation of cytotoxic and helper T cells. These findings provide a new scientific basis for resistance to pathogen invasion, as well as strong support for the development of dendritic cell-based anti-tumor immunotherapy. When dendritic cells perceive changes in homeostasis caused by pathogens or tissue-derived inflammatory signals, they switch from a resting state to an active state. Activated dendritic cells require increased biological energy and biosynthesis for enhanced protein and membrane synthesis to facilitate dendritic cells maturation. In this process, dendritic cells typically shift their metabolism from catabolism, characterized by fatty acid oxidation (FAO) and mitochondrial respiration, to anabolic metabolism, concurrently increasing glycolytic activity while reducing oxidative phosphorylation (OXPHOS)[ 33 ]. Through glycolysis experiments, observed a decrease in pyruvic acid content and an increase in pyruvic acid kinase, lactate, and other indicators, indicating enhanced glycolytic activity in dendritic cells treated with MPX[ 34 ]. These findings suggest that MPX can significantly induce dendritic cells maturation. Furthermore, previous studies have shown that Mycobacterium tuberculosis negatively regulates the activation status of dendritic cells through the HIF-1-NOS2 axis, consistent with the significantly downregulated expression of the NOS2 gene in our transcriptome sequencing results[ 35 ]. This suggests that MPX may promote maturation by modulating dendritic cells metabolism. However, the underlying mechanisms of this process require further in-depth research. RNA-Seq is a high-throughput method used for transcriptome profiling analysis that provides precise measurements of transcript and isoform expression levels compared to other methods[ 36 ]. The mechanism by which antimicrobial peptide MPX triggers dendritic cell maturation is quite complex, and RNA-Seq provides an effective tool for the systematic study of the interactions between dendritic cells and antimicrobial peptides and the impact of antimicrobial peptides on the host immune system. In this study, we sequenced the transcriptome of dendritic cells after 12h of MPX incubation using the NovaSeq platform and identified differentially expressed genes (DEGs). RT-qPCR analysis of randomly selected genes showed a strong correlation between the expression changes of these genes and those identified by RNA-Seq, supporting the reliability of the RNA-Seq analysis data. DEG analysis revealed that among the down-regulated differential genes in dendritic cells after treatment with the antimicrobial peptide MPX, NOS2 was associated with metabolic changes in dendritic cells. This suggests that MPX may alter the metabolic processes of dendritic cells, thereby promoting their self-activation. These results provide a plausible explanation for the regulation of dendritic cell activation by MPX. 5 Conclusion In this study, the beneficial effects of MPX on dendritic cell maturation were successfully demonstrated for the first time in vitro . After MPX treatment, dendritic cells could effectively promote T cell proliferation and recruit immune cells to a certain extent. Subsequently, transcriptome analysis of MPX-treated dendritic cells revealed dynamic changes in the expression of different genes, which contributed to our understanding of the mechanism of interaction between MPX and dendritic cells. Thus, this study provides valuable insights for the future development of dendritic cell-based anti-infection immunotherapies. However, further studies in vitro and in vivo are needed to gain a more comprehensive understanding of the mechanism of action of MPX and its potential value in clinical applications. Declarations Funding This work was supported by National Natural Science Foundation of China (32172862); National Key R&D Program of China (2021YFD1301200); Outstanding Youth Foundation of He’nan Scientific Committee (222300420043); Science and Technology Innovative Research Team in Higher Educational Institutions of Henan Province (24IRTSTHN035); the joint fund of science and technology research and development plan in Henan province (225200810044). Competing Interests The authors declare that there are no conflicts of interest. Author Contributions Conceptualization, C. L.; Data curation, X. D.; Formal analysis, W. Z., C. Z. and Y. W.; Investigation, L. Z.; Methodology, R. W. and B. W.; Project administration, K. D. and L. W.; Resources, X. L. and J. S.; Supervision, H. S.; Visualization, J. H. and Y. B.; Writing – original draft, W. L. and X. D.; Writing – review & editing, Y.W, X. W. and L. H.. All authors reviewed the manuscript. Data Availability The datasets generated and/or analysed during the current study are available in the NCBI SRA repository, the SRA database ID SRR31850248-SRR31850253.The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Consent to participate Informed consent was obtained from all individual participants included in the study. Consent to publish Not applicable Ethical Approval and consent to participate Not applicable Acknowledgement Not applicable References Duarte-Mata, D. I. & Salinas-Carmona, M. C. Antimicrobial peptides immune modulation role in intracellular bacterial infection. Front. Immunol. 14 , 1119574. https://doi.org/10.3389/fimmu.2023.1119574 (2023). Xu, D. & Lu, W. Defensins: a double-edged sword in host immunity. Front. Immunol. 11 , 764. https://doi.org/10.3389/fimmu.2020.00764 (2020). Weinberg, A., Jin, G., Sieg, S. & McCormick, T. S. The yin and yang of human beta-defensins in health and disease. 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Mycobacterium tuberculosis-infected alveolar epithelial cells modulate dendritic cell function through the hif-1alpha-nos2 axis. J. Leukoc. Biol. 108 , 1225–1238. https://doi.org/10.1002/JLB.3MA0520-113R (2020). Wang, Z., Gerstein, M. & Snyder, M. Rna-seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10 , 57–63. https://doi.org/10.1038/nrg2484 (2009). Additional Declarations No competing interests reported. Supplementary Files S1.docx S2.xlsx originalpicture.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5867452","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":445079803,"identity":"84597273-88e4-4c88-9cfd-37a96c25055b","order_by":0,"name":"Xiaowei Du","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Xiaowei","middleName":"","lastName":"Du","suffix":""},{"id":445079804,"identity":"af3383fa-d9ac-4b92-9515-a9ae497aaa8a","order_by":1,"name":"Bo Wen","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Wen","suffix":""},{"id":445079805,"identity":"fcd0883a-843d-4680-9f33-bee7681117c7","order_by":2,"name":"Ruibiao Wang","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Ruibiao","middleName":"","lastName":"Wang","suffix":""},{"id":445079808,"identity":"6333fca5-6a4e-44be-9a9f-48bbb5f8db77","order_by":3,"name":"Chengshui Liao","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Chengshui","middleName":"","lastName":"Liao","suffix":""},{"id":445079812,"identity":"798299e6-10e9-4087-9b00-c238f22d3194","order_by":4,"name":"Weiyu Luo","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Weiyu","middleName":"","lastName":"Luo","suffix":""},{"id":445079813,"identity":"1d950bed-4aaf-41de-b71f-0c6a3fda1170","order_by":5,"name":"Wei Zhang","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhang","suffix":""},{"id":445079814,"identity":"968b9807-4785-44d1-ba34-20bdac1c6778","order_by":6,"name":"Chunling Zhu","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, 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Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Longfei","middleName":"","lastName":"Zhang","suffix":""},{"id":445079818,"identity":"a9d0f22c-1812-4169-926e-d4f54cbe94f4","order_by":10,"name":"Xuehan Liu","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Xuehan","middleName":"","lastName":"Liu","suffix":""},{"id":445079819,"identity":"725b9e24-44e0-4a3c-aa39-eb77a8589dd9","order_by":11,"name":"Jiyuan Shen","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Jiyuan","middleName":"","lastName":"Shen","suffix":""},{"id":445079820,"identity":"ac53c97c-2adf-4322-8525-91b2f6bbc8b1","order_by":12,"name":"Yundi Wu","email":"","orcid":"","institution":"School of Biomedical Engineering, State Key Laboratory of Marine Resource Utilization in the South China Sea, Hainan University, Haikou","correspondingAuthor":false,"prefix":"","firstName":"Yundi","middleName":"","lastName":"Wu","suffix":""},{"id":445079821,"identity":"2211d2ac-2ed9-437f-aad6-e4cfcf78c21d","order_by":13,"name":"Xilong Wu","email":"","orcid":"","institution":"School of Biomedical Engineering, State Key Laboratory of Marine Resource Utilization in the South China Sea, Hainan University, Haikou","correspondingAuthor":false,"prefix":"","firstName":"Xilong","middleName":"","lastName":"Wu","suffix":""},{"id":445079822,"identity":"3ed7dcdc-d767-48c3-aa85-20dcb68a0aa2","order_by":14,"name":"Hao Li","email":"","orcid":"","institution":"Institute of Farmland Irrigation, Chinese Academy of Agricultural Sciences, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Li","suffix":""},{"id":445079823,"identity":"8be8f542-5399-4e1b-a6f6-9378c72d589a","order_by":15,"name":"Jianhe Hu","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Jianhe","middleName":"","lastName":"Hu","suffix":""},{"id":445079824,"identity":"f9697024-d540-4c83-94cf-416a58a8bb77","order_by":16,"name":"Yueyu Bai","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Yueyu","middleName":"","lastName":"Bai","suffix":""},{"id":445079825,"identity":"cf771aef-c507-45a8-baea-bd627e898449","order_by":17,"name":"Ke Ding","email":"","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Ding","suffix":""},{"id":445079826,"identity":"2b79bbf3-6428-44d4-afa2-9d9ff48f3276","order_by":18,"name":"Lei Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIie3RMQrCMBTG8RcexCXaNW4e4UGhU8GDuGTK5u5QsFCwY2dv4RFaHjgVvELBC/QADjaDc9/o8P5ThvyGfAHQtH9sA9AD+WuHyJOMYCKX0txbG0lMAMZoHi938CJBjENvbow5OyCoypOA2JCILXjbT/CM53qNFOwoEVfwLpCpWUKyORGfN468kLhlsTESoZQc2VIaOXheRg6St+zb5j0vXxmyjnmaq3KdpPDzOwXJdU3TNG29LxO8Pf5BcUQpAAAAAElFTkSuQmCC","orcid":"","institution":"College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang","correspondingAuthor":true,"prefix":"","firstName":"Lei","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-01-20 16:12:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5867452/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5867452/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81239614,"identity":"0c17a984-f628-4ae4-85a3-8a79a839eef8","added_by":"auto","created_at":"2025-04-23 21:18:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":381858,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence Intensity Changes of FITC-Labeled MPX Internalized by Dendritic Cells at Different Time Points Detected by Laser Confocal Microscopy\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5867452/v1/7e945d0b2079896f9cdd575b.png"},{"id":81239617,"identity":"792ba256-2eb9-4611-b24c-c1ad738dd12b","added_by":"auto","created_at":"2025-04-23 21:18:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":265158,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of different concentrations of MPX on the viability of dendritic cells, as well as the expression changes of surface markers (CD80, CD86, MHC I) on dendritic cells induced by MPX at different time points or concentrations detected by Western Blot. A: The effects of different concentrations of MPX on the activity of dendritic cells. B-D: Western blot detection of the expression of dendritic cell surface markers (CD80, CD86, MHC I) and gray value analysis results after different concentrations of MPX treatment. E-G: Western blot detection of the expression of dendritic cell surface markers (CD80, CD86, MHC I) and gray value analysis results after different times of MPX treatment\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5867452/v1/5318f85d4a3a4da5846129f4.png"},{"id":81239616,"identity":"61bd4396-8ed0-48e9-ad65-34ee09dc1383","added_by":"auto","created_at":"2025-04-23 21:18:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":202225,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of antimicrobial peptide MPX on the expression of dendritic cell surface markers and inflammatory cytokines, as well as its impact on CTL proliferation in co-culture, detected by RT-qPCR technology. A-C: The detection of the expression of dendritic cell surface markers and inflammatory cytokines following MPX and LPS treatment by RT-qPCR technology. D-F: Detection of the expression of inflammatory cytokines in dendritic cells following treatment with antimicrobial peptide MPX and LPS by RT-qPCR technology. G: Stimulation Indices of Dendritic Cells on Allogeneic T Cell Proliferation.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5867452/v1/f91c709f34d854a6af119579.png"},{"id":81239620,"identity":"14a43cff-0135-4720-bc3b-13eb2da6d6d2","added_by":"auto","created_at":"2025-04-23 21:18:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":239079,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in key glycolytic substrates, products, enzymes in the culture medium, and related gene expression within dendritic cells after treatment with antimicrobial peptide MPX. A: lactic acid content. B: pyruvate content. C: ratio of lactic acid to pyruvate. D: pyruvate kinase content. E: glucose content. F: ATP content. G: Glut1 gene expression. H: HK2 gene expression.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5867452/v1/32c4a9fd10b9a8c4331f0685.png"},{"id":81240061,"identity":"8749de61-d5e5-46c2-8043-bb0953de0fde","added_by":"auto","created_at":"2025-04-23 21:26:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":348724,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential Gene Expression and Functional Enrichment Analysis of Dendritic Cells Treated with Antimicrobial Peptide MPX. A: Volcano plot of global DEGs in different groups. B: heat map of global DEGs in different groups. C: GO enrichment analysis of DEGs in different groups. D: KEGG Pathway enrichment analysis of DEGs in different groups.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5867452/v1/2c57270b010513a1b6b180bc.png"},{"id":81239626,"identity":"04674c63-31c4-4d2d-9693-dda61ff3453b","added_by":"auto","created_at":"2025-04-23 21:18:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":446850,"visible":true,"origin":"","legend":"\u003cp\u003ePPI Network Analysis and Validation of DEGs in Dendritic Cells Treated with MPX. A-B: PPI network analysis of DEGs between the Control and MPX groups, constructed using the STRING database and visualized with Cytoscape software. C-D: Expression level of eight randomly selected gene validated by RT-qPCR.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5867452/v1/180e0bf072342d05d6bb53c8.png"},{"id":81240070,"identity":"854e9880-f252-4a2a-afab-1b9c743c30f7","added_by":"auto","created_at":"2025-04-23 21:26:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":159488,"visible":true,"origin":"","legend":"\u003cp\u003eThe Impact of \u003cem\u003eNos2\u003c/em\u003e Agonist FC on Glycolytic Products in Dendritic Cells.A: lactic acid content. B: pyruvate content. C: ratio of lactic acid to pyruvate. D: Glucose content.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5867452/v1/2fa34ee9177960eab650ae10.png"},{"id":82508767,"identity":"e801fca9-6f2b-426a-b7fc-4f7bd9b339b5","added_by":"auto","created_at":"2025-05-12 10:09:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3051889,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5867452/v1/eb8ecfde-2cfa-4788-9123-5e4b08655008.pdf"},{"id":81240058,"identity":"0cb5801b-4265-48ea-a888-d75f2d751d7a","added_by":"auto","created_at":"2025-04-23 21:26:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14115,"visible":true,"origin":"","legend":"","description":"","filename":"S1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5867452/v1/f533b76890df206a4f663023.docx"},{"id":81240060,"identity":"05d713d9-2d10-4b07-901d-7f5c854e94ca","added_by":"auto","created_at":"2025-04-23 21:26:02","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":28203,"visible":true,"origin":"","legend":"","description":"","filename":"S2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5867452/v1/cc7378ca412ef951234fd97c.xlsx"},{"id":81239624,"identity":"e98eb74b-85d7-4959-b510-59c68612a931","added_by":"auto","created_at":"2025-04-23 21:18:02","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":533341,"visible":true,"origin":"","legend":"","description":"","filename":"originalpicture.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5867452/v1/f4a12c7f503394bdbaba681b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Antimicrobial Peptide MPX Promotes the Maturation of Dendritic Cells by Enhancing Glycolysis","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAntimicrobial peptides (AMPs) are short cationic peptides capable of regulating the host's immune response, serving as a crucial component in the innate immune system[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They function within the immune system by regulating signaling pathways and inflammatory responses, while also modulating both innate and adaptive cell-mediated immune responses[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. For example, immune cells such as macrophages, mast cells, and T cells exhibit direct chemotactic responses to AMPs and their derivatives[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Interestingly, under varying experimental conditions, antimicrobial peptides have been observed to induce or inhibit the activation of dendritic cells. A study found that antimicrobial peptides mBD2 and mBD14 could promote the maturation of mouse-derived dendritic cells through TLR4, which serves as the receptor for bacterial lipopolysaccharide (LPS)[8; 9]. Coincidentally, another study found that the cathelicidin LL-37 can induce the maturation of human-derived dendritic cells \u003cem\u003ein vitro\u003c/em\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Mastoparan-X (MPX), a member of the mastoparan antimicrobial peptide family isolated from the venom of Vespidae insects, is a 14-amino acid peptide known for its broad antibacterial spectrum, stable physical and chemical properties, and potent bactericidal effects against Gram-positive as well as Gram-negative bacteria. MPX has been suggested to effectively reduce the host's inflammatory response[\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDendritic cells are the most powerful antigen-presenting cells (APCs) in the organism, and current studies have confirmed that AMPs not only have antimicrobial effects, but also can participate in the immune process of the organism by promoting dendritic cell maturation, it has been documented in the literature that antimicrobial peptides can promote the activation of dendritic cells[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], but the specific mechanism by which MPX promotes the maturation of dendritic cells is not clear.. Antigen presentation is widely acknowledged as a pivotal process for eliciting effective immune responses against pathogens and tumors. Dendritic cells are recognized within the body as the most efficient APCs[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Depending on the expression levels of co-stimulatory molecules such as CD40 and CD80, dendritic cells can be classified into immature dendritic cells (imDCs) and mature DCs (mDCs)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. ImDCs primarily inhabit peripheral tissues, relying on mechanisms like receptor-mediated endocytosis and receptor-independent phagocytosis, which enable robust capture of antigens, including those from pathogens and newly expressed antigens induced by malignant transformation. Upon encountering antigens or other activating stimuli, dendritic cells undergo maturation and migrate to lymph nodes[21; 22]. This maturation process involves significant production of pro-inflammatory cytokines and increased expression of co-stimulatory molecules. Mature dendritic cells effectively present processed antigenic fragments to T cells, thereby initiating immune responses.\u003c/p\u003e \u003cp\u003eResearch has demonstrated that antimicrobial peptides have the ability to modulate dendritic cells functions and induce specific antigen immune responses, thereby linking innate and adaptive immune responses. For example, human lactoferrin (hLF-11) can induce dendritic cells to express HLA class II antigens and dectin-1, promoting differentiated dendritic cells to produce inflammatory cytokines such as IL-6 and IL-10, while significantly increasing their phagocytic activity against \u003cem\u003eCandida.\u003c/em\u003e Additionally, lactoferrin itself can activate the TLR4 pathway, thereby stimulating macrophages and dendritic cells[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In other study, Human beta-defensin 3 (hBD3) can induce the phenotypic maturation of LC-DCs, resulting in the upregulation of CCR7and mediating chemotaxis to CCL19 and CCL21, thereby endowing hBD3 with excellent immunostimulatory properties[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Mouse beta-defensin 2 (mBD2) promotes the maturation of dendritic cells by activating the TLR4 receptor for bacterial lipopolysaccharide and stimulates dendritic cells to express high levels of co-stimulatory molecules (e.g., CD40, CD80, CD86), major histocompatibility complex class II, and the chemokine receptor CCR7[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. LL-37 can interact with Toll-like receptors on dendritic cells, thereby modulating immune responses through enhanced endocytic activity, increased co-stimulatory molecule expression, enhanced pro-inflammatory cytokine secretion, and the regulation of T cell differentiation[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsidering the above, MPX holds immense potential in aiding host infection control and immune response regulation, with broad prospects for application. However, the specific mechanisms by which MPX influences dendritic cell maturation remain unclear. This study aims to explore the impact of antimicrobial peptide MPX on dendritic cell maturation and its regulation of inflammatory responses. The studys contribute novel insights to the field of immunology and provide vigorous support for the future development of dendritic cell-based immune therapies against infections.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents\u003c/h2\u003e \u003cp\u003ePyruvate detection kit, Pyruvate Kinase Assay Kit, glucose test kit, and lactate test kit were purchased from Nanjing Jiancheng Bioengineering Institute (China). The ATP test kit was purchased from Solarbio (China). The dendritic cells were retained in the laboratory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Peptide Synthesis and Sequence, cells.\u003c/h2\u003e \u003cp\u003eMPX (H-INWKGIAAMAKKLL-NH2; Mw: 1556.01) was synthesized by Shanghai Jielu Biochemical Co., Ltd. (China) using solid-phase N-9-fluorenylmethoxycarbonyl (Fmoc) strategy and high-performance liquid chromatography (HPLC) purification, with a purity of up to 98%. The cell line used in the experiment is the bone marrow-derived dendritic cell line DC2.4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Laser Confocal Microscopy\u003c/h2\u003e \u003cp\u003eMouse dendritic cells were cultured in DMEM (containing 10% serum and 1% double antibodies) and incubated in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator to ensure cell attachment to the culture dish. Then, dendritic cells were seeded at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well in laser confocal culture dishes and allowed to adhere overnight. FITC-labled MPX drug at a concentration of 10 \u0026micro;g/mL was added to the laser confocal culture dishes, and dendritic cells were incubated for different time periods (1h, 2h, 3h). A blank control group was also set up. After incubation, the culture dishes were washed three times with PBS, each time for 5 min, to ensure the removal of residual culture medium. Subsequently, 250 \u0026micro;L of 4% paraformaldehyde was added to fix the cells in each well at room temperature for 15 min, followed by thorough washing with PBS. Finally, 300 \u0026micro;L of dye containing DAPI (concentration 3 \u0026micro;g/mL) was added to each well for nuclear staining for 5 min. After five washes with PBS, images were observed and captured using a laser confocal microscope, and Zen software was used to process the images.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell Viability Assay\u003c/h2\u003e \u003cp\u003eCell Counting Kit-8 (CCK-8) method was performed to assess cell viability. Initially, cells in logarithmic growth phase were seeded in a 96-well plate and randomly divided into different groups: blank control group (0 \u0026micro;g/mL) and various concentrations of MPX treatment groups (5/10/20/40/80 \u0026micro;g/mL). After cell attachment in the culture dish, the respective concentration of MPX was added, and cells were further cultured for 24 h. Subsequently, 10 \u0026micro;L of CCK-8 solution was added to each well, and the culture dish was incubated for 2 h. Then, the absorbance was measured at 450 nm using an enzyme meter. Each experiment was repeated three times to ensure reliable results. The calculation of relative cell activity is as follows: (Experimental group OD value / Blank group OD value) \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Western Blot (WB)\u003c/h2\u003e \u003cp\u003eTotal cellular proteins were extracted using Upload Buffer Protein Upload Buffer (Beyotime, China). Subsequently, the proteins were subjected to SDS-PAGE gel electrophoresis under reducing conditions, using a 10% SDS-PAGE gel. The proteins were then transferred to a PVDF membrane. The PVDF membrane was blocked in 5% skim milk for 2 h and then incubated overnight at 4\u0026deg;C with antibodies CD80 (1:1000, CST, USA), CD86 (1:1000, CST), MHC I (1:1000, Abcam, USA). β-Actin (1:1000, CST) was used as a loading control. Afterward, the membrane was treated with horseradish peroxidase-conjugated IgG and visualized using an enhanced chemiluminescence (ECL) reagent (Solarbio, China). The protein bands were analyzed using ImageJ software 1.8.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 RT-qPCR\u003c/h2\u003e \u003cp\u003eDendritic cells were cultured in six-well plates at a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e for 24h, then treated with MPX and LPS for 12h and the cells were collected. Total RNA was extracted from dendritic cells using the Total RNA Extraction Kit (Solarbio). The RNA concentration and quality were assessed. The CD80, CD86, MHC II, IL-18, IL-1β, TNF-α RNA samples were reverse-transcribed into cDNA using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kits (Thermo Fisher,USA). Subsequently, used the Yisheng QuantiNova SYBR Green PCR kit to detect the expression of genes such as \u003cem\u003eCD80\u003c/em\u003e, \u003cem\u003eCD86\u003c/em\u003e, \u003cem\u003eMHC II\u003c/em\u003e, \u003cem\u003eIL-β\u003c/em\u003e, \u003cem\u003eTNF-α, IL-18\u003c/em\u003e. Primer sequences were designed using Primer6 software. A fluorescent quantitative PCR instrument from Applied Biosystems was used, and all measurements were performed in triplicate in three independent experiments. The SYBR Green PCR conditions were as follows: initial denaturation at 95\u0026deg;C for 2 min, followed by 40 cycles, each comprising 5 s at 95\u0026deg;C, 30 s at 60\u0026deg;C, and 15 s at 95\u0026deg;C. GAPDH was used as a reference gene, and the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method was used to calculate the relative gene expression level, normalized to the GAPDH mRNA level[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The primer sequences in S1:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Allogeneic Mixed Lymphocyte Reaction\u003c/h2\u003e \u003cp\u003eDendritic cells were resuspended in complete culture medium and co-cultured with CTL cells at a 1:5 ratio in a 96-well plate. Two control groups were set up, one with CTL cells added alone, and the other with complete culture medium added alone. All culture groups were incubated at 37\u0026deg;C for 3 d, and then CCK-8 reagent (20 \u0026micro;L/sample) was added, followed by further incubation for 4h. A microplate reader was used to measure the absorbance of each group at 450 nm. Additionally, the stimulation index (SI) of each group was calculated using the formula SI = (experimental sample A value - complete culture medium A value) / (CTL A value - complete culture medium A value).Results for each group were obtained from experiments with three replicates to ensure reliability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Glycolysis\u003c/h2\u003e \u003cp\u003eDendritic cells were first digested with trypsin to prepare a single-cell suspension. Then, these single cells were evenly seeded in six-well culture dishes, and cultured until the cell fusion density reached over 90%. Subsequently, complete culture medium was prepared with a solution containing 10 \u0026micro;g/mL MPX and 1 \u0026micro;g/mL LPS. This solution was used to culture dendritic cells for 12h. A control group with pure culture medium without MPX and LPS was also set up. After incubation, the cell supernatant was collected, and the respective assay kits (Nanjing Jiancheng ) were used to determine the levels of lactate, Pyruvate, pyruvate kinase, glucose, and ATP in the supernatant. In addition, RT-qPCR was used to detect the expression levels of \u003cem\u003eGLUT1\u003c/em\u003e and \u003cem\u003eHK2\u003c/em\u003e genes related to glycolysis. The primer sequences in S1:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Differential gene expression analysis\u003c/h2\u003e \u003cp\u003eDendritic cells were divided into an untreated group and an antimicrobial peptide MPX-treated group, with each group containing three replicate samples from the same experiment. In the experiment, RNA was first extracted according to standard steps, and its quality and concentration were detected by spectrophotometer. Qualified RNA samples were sent to Azenta for library construction and expression profiling. High-throughput RNA sequencing was performed using the Illumina NovaSeq 6000 platform, and gene expression levels were calculated using HTSeq.\u0026nbsp;Differentially expressed genes (DEGs) were identified by DESeq2 software, and the screening criteria were adjusted p-value less than 0.05 and absolute value of log2 fold change greater than 1. Subsequently, functional enrichment analyses of the GO and KEGG pathways were performed[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and the protein interaction networks of DEGs were analyzed using the STRING database and Cytoscape software. The list of top upregulated and downregulated molecules identified by transcriptomic analysis is presented in S2. Sequencing results have been uploaded to NCBI, the SRA database ID SRR31850248-SRR31850253. Finally, eight randomly selected DEGs (\u003cem\u003eApoo-ps\u003c/em\u003e, \u003cem\u003eMyh4\u003c/em\u003e, \u003cem\u003eMyl1\u003c/em\u003e, \u003cem\u003eCar3\u003c/em\u003e, \u003cem\u003eNdrg1\u003c/em\u003e, \u003cem\u003eCar9\u003c/em\u003e, \u003cem\u003eAdm\u003c/em\u003e and \u003cem\u003eNos2\u003c/em\u003e) were validated by RT-qPCR to ensure the reliability of the transcriptome sequencing results. The primer sequences in S1:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Data Analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed with 3\u0026ndash;6 replicate samples. All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical data and charts were obtained using GraphPad Prism 8.3.0 software. One-way analysis of variance (ANOVA) was used to determine statistical significance. Statistical significance was considered as follows: *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Evaluation of Phagocytosis of Antimicrobial Peptide FITC-labled MPX by Dendritic Cells\u003c/h2\u003e \u003cp\u003eQualitative observations using laser confocal microscopy revealed that FITC-labeled antimicrobial peptide MPX enters dendritic cells (DCs) via passive transport mechanisms. As illustrated in Fig.\u0026nbsp;1, intracellular fluorescence intensity demonstrated a time-dependent increase over the 0\u0026ndash;3 hour period, indicating efficient uptake of MPX by DCs. This dynamic process provides compelling evidence for the immune responsiveness of dendritic cells to antimicrobial peptides and underscores the critical role of temporal factors in mediating biological interactions.\u003cb\u003eFig.\u0026nbsp;1\u003c/b\u003e: Fluorescence Intensity Changes of FITC-Labeled MPX Internalized by Dendritic Cells at Different Time Points Detected by Laser Confocal Microscopy\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Regulation of Dendritic Cell Activation-Related Protein Expression by MPX\u003c/h2\u003e \u003cp\u003eFirst, the effect of MPX on the viability of dendritic cells was assessed using the CCK-8 assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the cell viability remained above 85% after MPX treatment, indicating a minimal effect on cell viability, thus allowing further experiments. Next, changes in the expression of activation markers in dendritic cells after MPX treatment were observed. The effects of MPX on the expression of markers in dendritic cells were evaluated from two perspectives: different concentrations of MPX treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and different durations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Mature dendritic cells (mDC) were identified using Western blot analysis of CD80, CD86, and MHC I. The results showed that the expression of CD80 and CD86 was increased in the MPX-treated group at different concentrations and in the positive control LPS (1 \u0026micro;g/mL)-treated group. And the expression level decreased at MPX concentration of 20 \u0026micro;g/mL, suggesting that high concentration of MPX may inhibit the expression of markers. Therefore, 10 \u0026micro;g/mL of MPX was selected for subsequent experiments.Under the condition of a 10 \u0026micro;g/mL concentration, the expression levels of MHC I and CD80 both showed an increasing trend with different treatment durations and reached a peak at 12h of treatment. Therefore, for subsequent experiments, dendritic cells were treated with the antimicrobial peptide MPX at a concentration of 10 \u0026micro;g/mL for 12 hours.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 The Regulation of Surface Markers and Inflammatory Cytokines Expression in Dendritic Cell Activation by Antimicrobial Peptide MPX\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFocused on studying the impact of MPX (10 \u0026micro;g/mL) treatment on dendritic cells activation and cytokine expression, using LPS treatment as a positive control. First, examined the expression of \u003cem\u003eCD80\u003c/em\u003e, \u003cem\u003eCD86\u003c/em\u003e, and \u003cem\u003eMHC II\u003c/em\u003e, molecules typically considered markers of dendritic cell activation. Consistent with the previous Western blot results, after MPX treatment, the expression of \u003cem\u003eCD80\u003c/em\u003e, \u003cem\u003eCD86\u003c/em\u003e, and \u003cem\u003eMHC II\u003c/em\u003e all significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), indicating a marked activation of dendritic cells under MPX treatment. This suggests that MPX may play a critical role in antigen presentation by dendritic cells and T cell activation. Furthermore, monitored changes in the expression of pro-inflammatory cytokines. Following MPX treatment, the expression levels of \u003cem\u003eIL-1β\u003c/em\u003e and \u003cem\u003eIL-18\u003c/em\u003e significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The elevation of these cytokines suggests that MPX treatment may induce an inflammatory response, which may have an important role in immune responses. However, unlike the former two, the effect of MPX on tumor necrosis factor \u003cem\u003eTNF-α\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) was relatively mild, indicating that MPX may have different regulatory effects on different cytokines.\u003c/p\u003e \u003cp\u003eIn summary, observations reveal that after MPX treatment, dendritic cells exhibit an activated state, as confirmed by the upregulation of \u003cem\u003eCD80\u003c/em\u003e, \u003cem\u003eCD86\u003c/em\u003e, and \u003cem\u003eMHC II\u003c/em\u003e. Furthermore, the elevation of pro-inflammatory cytokines \u003cem\u003eIL-1β\u003c/em\u003e and \u003cem\u003eIL-18\u003c/em\u003e suggests that MPX treatment may induce an inflammatory response. However, the impact of MPX on \u003cem\u003eTNF-α\u003c/em\u003e expression is relatively minor. These findings provide a strong basis for further research on the effects of MPX on the immune system, particularly in the context of dendritic cell-mediated antigen presentation and immune regulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Allogeneic Mixed Lymphocyte Reaction\u003c/h2\u003e \u003cp\u003eTo evaluate the capacity of dendritic cells to stimulate the proliferation of allogeneic T cells, conducted an allogeneic mixed lymphocyte reaction assay using CTL cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, the dendritic cell groups treated with LPS and MPX (LPS group and MPX group) both exhibited higher stimulation indices, revealed that dendritic cells supplemented with the antimicrobial peptide MPX could significantly induce T cell proliferation, indicating that MPX can effectively promote the activation of dendritic cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Glycolysis Experiment\u003c/h2\u003e \u003cp\u003eDuring dendritic cell activation, metabolic reprogramming occurs, shifting cellular metabolism from oxidative phosphorylation (OXPHOS) to glycolysis\u0026mdash;a critical hallmark of DC activation. To investigate this, we analyzed changes in glycolytic substrates, products, and enzymes in cell culture media, which indirectly indicated that MPX significantly enhances DC glycolytic activity. Specifically, MPX-treated DCs showed elevated lactate levels reflecting accelerated production (Fig.\u0026nbsp;4A), reduced pyruvate levels suggesting metabolic redirection of carbon flux (Fig.\u0026nbsp;4B), and an increased lactate-to-pyruvate ratio indicating adaptive redox adjustments (Fig.\u0026nbsp;4C). Concurrently, pyruvate kinase upregulation underscored MPX's role in pyruvate metabolism (Fig.\u0026nbsp;4D), while decreased extracellular glucose levels confirmed enhanced uptake to fuel glycolysis (Fig.\u0026nbsp;4E). Corresponding ATP increases directly linked glycolytic enhancement to elevated biosynthesis (Fig.\u0026nbsp;4F), supported by GLUT1/HK2 upregulation confirming augmented glucose uptake and phosphorylation (Fig.\u0026nbsp;4G-4H).\u003cb\u003eFig.\u0026nbsp;4\u003c/b\u003e: Changes in key glycolytic substrates, products, enzymes in the culture medium, and related gene expression within dendritic cells after treatment with antimicrobial peptide MPX. A: lactic acid content. B: pyruvate content. C: ratio of lactic acid to pyruvate. D: pyruvate kinase content. E: glucose content. F: ATP content. G: Glut1 gene expression. H: HK2 gene expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Differential Gene Expression Analysis with GO and KEGG Enrichment Analysis\u003c/h2\u003e \u003cp\u003eTo investigate the changes in gene expression profiles in dendritic cells upon treatment with antimicrobial peptide MPX, then utilized the FeatureCounts software to calculate the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) expression values based on read counts. The DESeq2 software was employed to compute the fold change (FC) of each gene at different time points post-infection compared to the control group. A threshold of FDR (False Discovery Rate)\u0026thinsp;\u0026le;\u0026thinsp;0.05 and |Log2FC| \u0026ge; 1 was applied to identify differentially expressed genes (DEGs) in response to MPX treatment relative to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).In comparison to general descriptions of gene or transcript properties provided by functional annotations, enrichment analysis serves to elucidate the functions and KEGG pathways of genes at the lowest levels. Furthermore, Fisher's test is employed to determine the significant enrichment of gene functions and KEGG pathways for the DEGs. Enrichment analysis offers the most detailed information regarding gene functions and KEGG pathways. GO terms and KEGG pathways are separately subjected to enrichment analysis using GOatools and KOBAS tools. GO terms and KEGG pathways with a corrected p-value of \u0026le;\u0026thinsp;0.05 are considered enriched. Enriched GO terms are categorized into three groups: Biological Processes (BP), Cellular Components (CC), and Molecular Functions (MF) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The results show that MPX mainly affects the metabolic pathway after the effect of DCs, consistent with the previous test proving that MPX can change its glycolysis levels, and did provide the basis of later experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Protein-Protein Interaction (PPI) Analysis of DEGs and RT-qPCR Validation\u003c/h2\u003e \u003cp\u003eThe PPI network analysis was performed for DEGs in the experimental group treated with MPX and the control group, based on FDR (False Discovery Rate)\u0026thinsp;\u0026le;\u0026thinsp;0.05 and |Log2FC| \u0026ge; 2. To gain a deeper understanding of the biological relevance of the Differentially Expressed Genes (DEGs), PPI network analysis of the DEGs was conducted using the STRING database (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The Cytoscape software was utilized to visualize the protein-protein interaction network of the DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The PPI network analysis identified a total of 123 interaction relationships among 90 genes within the DEGs from the control group and the MPX-treated group.To validate the reproducibility and repeatability of the DEGs identified from the transcriptome sequencing, eight genes were randomly selected for RT-qPCR validation. These genes included \u003cem\u003eApoo-ps\u003c/em\u003e, \u003cem\u003eMyh4\u003c/em\u003e, \u003cem\u003eMyl1\u003c/em\u003e, \u003cem\u003eCar3\u003c/em\u003e, \u003cem\u003eNdrg1\u003c/em\u003e, \u003cem\u003eCar9\u003c/em\u003e, \u003cem\u003eAdm\u003c/em\u003e, and \u003cem\u003eNos2.\u003c/em\u003e The results demonstrated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) that the differential expression of these genes was significant and consistent with the direction of gene expression changes identified through RNA-seq.\u0026nbsp;This suggests that the DEGs obtained through transcriptome sequencing are reliable.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Validation of \u003cem\u003eNos2\u003c/em\u003e Downregulation and Glycolysis-Related Metabolite Changes in Dendritic Cells Treated with MPX\u003c/h2\u003e \u003cp\u003eBased on transcriptome sequencing results, the downregulated gene \u003cem\u003eNos2\u003c/em\u003e was selected for validation. Since ferrous citrate (FC) has been reported as an agonist of \u003cem\u003eNos2\u003c/em\u003e that promotes its gene expression[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], measured glycolysis-related products (glucose, lactate, pyruvate) in culture media across experimental groups. Notably, FC treatment led to decreased supernatant lactate levels, increased pyruvate and glucose concentrations, and reduced lactate-to-pyruvate ratios\u0026mdash;phenotypes opposite to MPX effects\u0026mdash;indicating suppressed dendritic cell glycolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). These results confirm that \u003cem\u003eNos2\u003c/em\u003e serves as a critical gene in dendritic cell activation, validating the accuracy of transcriptome sequencing findings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eDendritic cells play an integral role when pathogens invade, and their main task is to promote antigen presentation and activate T cells, thus ensuring the continuity of the antigen-specific immune response. Studies have shown that certain drugs, such as oligofructose, the Chinese herb astragalus polysaccharide, and the antimicrobial peptide LL-37, can promote dendritic cell differentiation, maturation, and function[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Unlike conventional drugs, the antimicrobial peptide MPX has the ability to disrupt the integrity of bacterial membranes, increase membrane permeability, and alter membrane potential, which leads to leakage of intracellular material and ultimately produces a bactericidal effect. More notably, MPX neither induces bacterial resistance nor causes damage to host cells and organs. Both MPX and immune cells play important roles in defense against infections. The aim of this experiment was to verify whether MPX can effectively induce the maturation and function of dendritic cells through \u003cem\u003ein vitro\u003c/em\u003e experiments.\u003c/p\u003e \u003cp\u003eThe results showed that MPX significantly increased the expression levels of maturation markers (e.g., CD 80, CD 86, and MHC I) on dendritic cells cultured \u003cem\u003ein vitro\u003c/em\u003e. In addition, MPX treatment significantly increased the production of interleukin factors such as \u003cem\u003eIL-1β\u003c/em\u003e and \u003cem\u003eIL-18\u003c/em\u003e. However, the increase in \u003cem\u003eTNF-α\u003c/em\u003e expression induced by MPX treatment was not as pronounced as that of LPS, suggesting that the inflammatory response triggered by MPX may not be as strong as that of LPS. Mature dendritic cells not only express higher levels of co-stimulatory molecules and MHC-I during antigen presentation, but also play a key role in the antigen-specific T-cell immune response, promoting the activation of cytotoxic and helper T cells. These findings provide a new scientific basis for resistance to pathogen invasion, as well as strong support for the development of dendritic cell-based anti-tumor immunotherapy.\u003c/p\u003e \u003cp\u003eWhen dendritic cells perceive changes in homeostasis caused by pathogens or tissue-derived inflammatory signals, they switch from a resting state to an active state. Activated dendritic cells require increased biological energy and biosynthesis for enhanced protein and membrane synthesis to facilitate dendritic cells maturation. In this process, dendritic cells typically shift their metabolism from catabolism, characterized by fatty acid oxidation (FAO) and mitochondrial respiration, to anabolic metabolism, concurrently increasing glycolytic activity while reducing oxidative phosphorylation (OXPHOS)[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Through glycolysis experiments, observed a decrease in pyruvic acid content and an increase in pyruvic acid kinase, lactate, and other indicators, indicating enhanced glycolytic activity in dendritic cells treated with MPX[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These findings suggest that MPX can significantly induce dendritic cells maturation. Furthermore, previous studies have shown that Mycobacterium tuberculosis negatively regulates the activation status of dendritic cells through the HIF-1-NOS2 axis, consistent with the significantly downregulated expression of the \u003cem\u003eNOS2\u003c/em\u003e gene in our transcriptome sequencing results[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This suggests that MPX may promote maturation by modulating dendritic cells metabolism. However, the underlying mechanisms of this process require further in-depth research.\u003c/p\u003e \u003cp\u003eRNA-Seq is a high-throughput method used for transcriptome profiling analysis that provides precise measurements of transcript and isoform expression levels compared to other methods[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The mechanism by which antimicrobial peptide MPX triggers dendritic cell maturation is quite complex, and RNA-Seq provides an effective tool for the systematic study of the interactions between dendritic cells and antimicrobial peptides and the impact of antimicrobial peptides on the host immune system. In this study, we sequenced the transcriptome of dendritic cells after 12h of MPX incubation using the NovaSeq platform and identified differentially expressed genes (DEGs). RT-qPCR analysis of randomly selected genes showed a strong correlation between the expression changes of these genes and those identified by RNA-Seq, supporting the reliability of the RNA-Seq analysis data. DEG analysis revealed that among the down-regulated differential genes in dendritic cells after treatment with the antimicrobial peptide MPX, \u003cem\u003eNOS2\u003c/em\u003e was associated with metabolic changes in dendritic cells. This suggests that MPX may alter the metabolic processes of dendritic cells, thereby promoting their self-activation. These results provide a plausible explanation for the regulation of dendritic cell activation by MPX.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn this study, the beneficial effects of MPX on dendritic cell maturation were successfully demonstrated for the first time \u003cem\u003ein vitro\u003c/em\u003e. After MPX treatment, dendritic cells could effectively promote T cell proliferation and recruit immune cells to a certain extent. Subsequently, transcriptome analysis of MPX-treated dendritic cells revealed dynamic changes in the expression of different genes, which contributed to our understanding of the mechanism of interaction between MPX and dendritic cells. Thus, this study provides valuable insights for the future development of dendritic cell-based anti-infection immunotherapies. However, further studies \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e are needed to gain a more comprehensive understanding of the mechanism of action of MPX and its potential value in clinical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (32172862); National Key R\u0026amp;D Program of China (2021YFD1301200); Outstanding Youth Foundation of He’nan Scientific Committee (222300420043); Science and Technology Innovative Research Team in Higher Educational Institutions of Henan Province (24IRTSTHN035); the joint fund of science and technology research and development plan in Henan province (225200810044).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, C. L.; Data curation, X. D.; Formal analysis, W. Z., C. Z. and Y. W.; Investigation, L. Z.; Methodology, R. W. and B. W.; Project administration, K. D. and L. W.; Resources, X. L. and J. S.; Supervision, H. S.; Visualization, J. H. and Y. B.; Writing – original draft, W. L. and X. D.; Writing – review \u0026amp; editing, Y.W, X. W. and L. H.. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available in the NCBI SRA repository, the SRA database ID SRR31850248-SRR31850253.The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDuarte-Mata, D. 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Genet.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 57\u0026ndash;63. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrg2484\u003c/span\u003e\u003cspan address=\"10.1038/nrg2484\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"antimicrobial peptides, dendritic cell maturation, MPX, immune regulation, glycolysis","lastPublishedDoi":"10.21203/rs.3.rs-5867452/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5867452/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial peptide MPX is derived from wasp venom, known for its antibacterial activity against a wide range of bacteria. Researches have indicated that antimicrobial peptide could induce dendritic cell maturation through interaction with surface receptors, thereby activating antigen presentation and initiating immune responses, which indirectly contributes to antimicrobial effects. However, the specific mechanism by which the antimicrobial peptide MPX activates dendritic cells remains unclear. The aim of this study was to investigate how MPX activates dendritic cells and thus enhances antigen presentation.In the present study, it was found that dendritic cells exhibited a time-dependent internalization of the antimicrobial peptide MPX. MPX internalized by dendritic cells increased the expression of maturation markers such as CD80, CD86, and promoted the secretion of the pro-inflammatory factors IL-1β and IL-18, whereas it had a relatively mild effect on TNF-α.Furthermore, MPX also altered metabolic pathways of dendritic cells, favoring glycolysis and thereby activating them to subsequently promote CTL cell proliferation. To explore the molecular mechanisms underlying MPX\u0026rsquo;s regulation of dendritic cell activation, RNA-seq analysis was performed. The results showed that following MPX treatment dendritic cells exhibited had 90 differentially expressed genes (DEGs) compared to the control group, with enrichment observed in metabolism and biosynthesis processes. These findings not only provide insights into the potential applications of MPX in infection control but also emphasize its significant role in dendritic cell-mediated immune responses. In summary, this study has demonstrated that the MPX can activate dendritic cells, promote T-cell proliferation, and present new avenues for utilizing antimicrobial peptides in immune modulation within the host.\u003c/p\u003e","manuscriptTitle":"The Antimicrobial Peptide MPX Promotes the Maturation of Dendritic Cells by Enhancing Glycolysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-23 21:17:57","doi":"10.21203/rs.3.rs-5867452/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e734ceb9-80e4-4108-b663-1894dd973aeb","owner":[],"postedDate":"April 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47389247,"name":"Biological sciences/Immunology"},{"id":47389248,"name":"Biological sciences/Immunology/Innate immunity"}],"tags":[],"updatedAt":"2025-05-12T10:08:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-23 21:17:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5867452","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5867452","identity":"rs-5867452","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}