Irf7 aggravates prostatitis by promoting Hif-1α-mediated glycolysis to facilitate M1 polarization

doi:10.21203/rs.3.rs-5284649/v1

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Irf7 aggravates prostatitis by promoting Hif-1α-mediated glycolysis to facilitate M1 polarization

https://doi.org/10.21203/rs.3.rs-5284649/v1

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Background: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common disorder associated with voiding symptoms and pain in the pelvic or perineal area. Macrophages, particularly the pro-inflammatory M1 subtype, are crucial in the initiation of CP/CPPS. Interferon regulatory factor 7 (Irf7) has been implicated in promoting M1 polarization, which contributes to the onset and progression of autoimmunity. However, the role of Irf7 in the etiology and progression of CP/CPPS remains unclear.

Method: We established the experimental autoimmune prostatitis (EAP) mouse model by subcutaneous injection of prostate antigen combined with complete Freund's adjuvant. We analyzed prostate, spleen, and blood samples to evaluate prostate inflammation, Irf7 expression levels, glycolysis, and M1 polarization. Our findings suggest that Irf7 exacerbates the development of EAP by enhancing Hif-1α transcription, thereby increasing glycolysis and M1 polarization. Further investigations included sh-Irf7 intervention, Dimethyloxalylglycine (a Hif-1α agonist), and in vitro M1 polarization experiments. We also employed ChIP assays, dual-luciferase reporter assays, and q-PCR to explore if Irf7 could directly interact with the Hif-1α promoter in macrophages.

Results: In the EAP mouse and cell models, elevated Irf7 expression was observed in inflamed tissues and cells. Reducing Irf7 expression decreased M1 cell glycolysis by inhibiting the nuclear translocation of Hif-1α, thus mitigating M1 cell polarization. Additionally, Irf7 was identified as a transcription factor in the cytoplasm that regulates Hif-1α transcription by interacting with its promoter in macrophages, confirmed through ChIP and dual-luciferase assays. Co-culturing macrophage cells with 3T3 fibroblasts with reduced Irf7 levels resulted in decreased fibrosis, and a significant reduction in prostate tissue fibrosis was noted in mice with Irf7 knockdown.

Conclusion: Our findings indicate that Irf7 can contribute to the development and progression of CP/CPPS by promoting glycolysis, which can enhance both M1 polarization as well as interstitial fibrosis in the prostate. This process was found to be mediated by the upregulation of Hif-1α transcription, presenting new potential therapeutic targets for managing CP/CPPS.

Chronic prostatitis/chronic pelvic pain syndrome

Macrophages

Irf7

Hif-1α

Glycolysis

Prostatitis is a prevalent urogenital disorder primarily affecting middle-aged and young men, with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) constituting over 90% of cases^[1]. A recent study revealed that the prevalence of CP/CPPS symptoms, including pelvic or perineal pain and sexual dysfunction, is 8.2%^[2]. The conventional treatments for CP/CPPS, such as anti-inflammatory drugs and alpha-adrenergic blockers have been less effective due to the complex and not fully understood pathogenesis of CP/CPPS^[3]. Therefore, a thorough exploration of the fundamental process of CP/CPPS is urgently needed.

Monocyte-derived macrophages are crucial immune cells involved in host defense, tissue homeostasis, the occurrence and development of inflammation, and tissue repair in response to tissue damage^[4]. Triggered by internal changes like infections or medical treatments, macrophages can shift into various functional states. Classically activated macrophages (M1) primarily enhance inflammation, while alternatively activated macrophages (M2) help mitigate inflammation and facilitate tissue repair^[5]. Consequently, precise control over macrophage activation is essential for disease management and preserving tissue equilibrium. Recent studies have shown that the glucose metabolism pathway is intricately linked to the polarization and function of macrophages^[4]. In M1 macrophages, increased glycolysis not only quickly generates energy but also supports the pentose phosphate pathway. This pathway produces NADPH, which is necessary for creating reactive oxygen species (ROS), thereby amplifying oxidative stress^{[4, 6, 7]}.

Hypoxia-Inducible Factor 1-alpha (Hif-1α) is a critical transcription factor that is activated under low oxygen conditions to support cellular adaptation to hypoxia and control the expression of numerous genes involved in angiogenesis, metabolic regulation, and apoptosis^{[8, 9]}. Numerous studies have highlighted the pivotal role of Hif-1α in the regulation of glycolysis with regulating the transcription of glycolysis-related genes to increase the utilization of glucose in hypoxic conditions^[10]. This process is referred to as anaerobic glycolysis, which generates ATP, allowing cells to survive under hypoxic conditions^{[4, 11]}. Hif-1α influences cellular metabolism by regulating genes like Glut1, Hk2, and Pkm2, thus enabling cells to adapt to hypoxic conditions^[10]. Consequently, understanding the role of Hif-1α in the regulation of glycolysis is vital in medical research.

Interferon regulatory factor 7 (Irf7), as an important member of Irf family and significantly impacts immune responses by enhancing the activation and proliferation of immune cells during inflammation^[12–14]. Previous studies have shown that Irf7 can exacerbate the severity of inflammation and fibrosis in various inflammatory conditions, such as systemic sclerosis^[12], liver inflammation^[15], acute kidney injury^[14], atherosclerosis^[13] and et al. However, the role of Irf7 in prostatitis and the mechanisms behind its effects have yet to be elucidated.

This study investigated the regulatory role of Irf7 in CP/CPPS. By comparing RNA-sequencing data from normal and experimental autoimmune prostatitis (EAP) mice groups, we noted differential expression of Irf7. Immunofluorescence performed on prostate tissues from these groups showed predominant Irf7 expression in macrophages. To assess its impact on CP/CPPS, we knocked down Irf7 in macrophages and EAP mouse models, observing that Irf7 could enhance M1 macrophage polarization. Further mechanistic studies indicated that Irf7 influences lactate metabolism by upregulating Hif-1α transcription, which subsequently promoted M1 polarization and interstitial fibrosis in the prostate, contributing to the onset and progression of CP/CPPS. Therefore, we propose that Irf7 can effectively trigger CP/CPPS development by enhancing glycolysis, which in turn can stimulate M1 polarization and prostate fibrosis through the activation of Hif-1α transcription.

2. 16 ELISA assay

We performed the ELISA assay for mouse serum according to the instructions provided in the kit.

2.1 Reagents

2.1.1 Preparation of Animals

The study utilized 6-week-old NOD immunodeficient mice, with prostate antigens extracted from adult male Sprague-Dawley rats for the experimental model. The rats were sourced from Nanjing JiCui YaoKang, and all animals were kept at the Animal Experimental Center of Anhui Medical University. They had access to water and food ad libitum and were maintained on a 12-hour light-dark cycle. The animal experimental protocols received approval from the Animal Care and Use Committee of Anhui Medical University and adhered to Chinese guidelines for laboratory animal welfare and ethics, along with the NIH Guide for the Care and Use of Laboratory Animals.

2.1.2 Establishment of the EAP Mice Models

A prostatitis model was established in NOD mice using a secondary immunization method. The mice were acclimatized for five days before the experiment, which lasted 28 days. On days 0 and 28, the mice received subcutaneous injections of prostate antigens from SD rats at six sites, including the armpits, groin, and lower back. The antigens were processed by pulverizing the prostates and combining them with complete Freund's adjuvant.

For the IRF7 gene knockdown, adenovirus-associated vectors were administered via the tail vein at a volume of 100 µl per mouse, with a control group receiving Sh-NC-AAV at the same dosage. Prostate modeling was initiated 14 days after the knockdown. Drug administration commenced two days before the second modeling phase, using Dimethyloxalylglycine (DMOG) (an HIF-1α activator from Glpbio), administered intraperitoneally at 25 mg/kg daily.

The mice were euthanized on day 42 of the modeling phase, and pelvic stimulation responses were evaluated one day earlier. A positive pain response was identified by any of the following behaviors: (a) severe abdominal contractions, (b) immediate scratching or licking of the stimulated area, or (c) jumping.

2.2 Cell Treatment

Immortalized bone marrow-derived macrophages (iBMDM) cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were kept at 37°C in a humidified incubator with an atmosphere of 95% oxygen and 5% carbon dioxide. M1 phenotype polarization of macrophages was induced using 100 ng/ml LPS (sourced from MCE) for 24 hours. IRF7 knockdown was performed using a lentiviral vector, with successfully modified cells being selected using puromycin.

2.3 Immunofluorescence

The procedure began with de-paraffinization of the embedded tissue samples, followed by antigen retrieval and a blocking step. Primary antibodies, such as INOS, IRF7, HIF-1α, F480, and α-SMA, were applied and incubated overnight at 4°C. This was followed by a two-hour incubation with secondary antibodies at room temperature. 4',6-diamidino-2-phenylindole (DAPI) staining was performed for 10 minutes in a dark environment at room temperature to highlight nuclei. The slides were then sealed with an anti-fluorescence quenching agent and analyzed using a fluorescence microscope.

2.4 Hematoxylin-Eosin (HE) Staining

For H&E staining, tissue sections were first deparaffinized until clear, followed by staining with hematoxylin to accentuate cell nuclei and eosin to emphasize cytoplasm. After staining, the samples were dehydrated, cleared, and then mounted on slides. These prepared slides were examined under a light microscope to assess tissue structure and identify any changes in cell morphology.

2.5 Sirius Red staining

First, tissue sections on glass slides were dewaxed and rehydrated. These sections were then immersed in a Sirius Red staining solution for one hour before being rinsed with distilled water. They underwent differentiation in a 0.5% acetic acid solution, followed by dehydration using ethanol, and clearance in xylene. A mounting medium was then applied to the slides, and coverslips were positioned on top. The final step involved examining the collagen fibers, which had been stained with Sirius Red, under a light microscope to assess their structure.

2.6 Masson Staining

Tissue sections were subjected to deparaffinization and dehydration, followed by staining with Weigert's iron hematoxylin, Biebrich scarlet-acid fuchsin, phosphotungstic/phosphomolybdic acid, and aniline blue solutions. Differentiation was achieved using acetic acid, followed by additional dehydration and mounting with an appropriate medium. The stained sections were then examined under a light microscope to assess the distribution and structure of collagen, muscle fibers, and other tissues.

Flow 2.7 Cytometry Analysis

The prepared cell samples were collected into flow tubes and washed three times with PBS. Extracellular antibodies CD11b, MHC II, and F4/80 were then added, and the samples were incubated at room temperature for one hour. After the incubation, the samples were washed three more times with PBS and subsequently analyzed using flow cytometer.

2.8 Western Blotting

The protein extraction process involved collecting cell or tissue samples and disrupting the membranes with lysis buffer, followed by centrifugation to obtain the supernatant containing the proteins. The protein concentration was measured, and samples were stored. For nuclear protein extraction, nuclear lysis buffer was used to disrupt membranes and separate cytoplasmic proteins, followed by centrifugation to collect the nuclear protein supernatant. In the Western blotting process, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a membrane, and blocked. The membrane was then incubated with primary and secondary antibodies. Detection was achieved through chemiluminescence or staining to visualize the target proteins.

2.9 RNA Extraction

The RNA extraction procedure involved collecting samples, disrupting the membranes, and adding stabilizing agents. Extraction was then performed using either phenol-chloroform or column methods. RNA was precipitated with ethanol or isopropanol, followed by washing to remove impurities. Finally, the RNA was resuspended in RNase-free water for subsequent experiments.

2.10 RT-qPCR

Utilizing TAKARA's qPCR kit for quantitative PCR experiments involved initially preparing essential materials, including the Master Mix, primers, probes, and cDNA template. The PCR mix was then meticulously configured and dispensed into the PCR plate. It was subsequently subjected to the PCR program within a real-time fluorescence quantitative PCR instrument.

2.11 Seahorse XF Assays

The basic steps of the Seahorse experiment involved seeding cells onto Seahorse assay plates for attachment, followed by calibrating the Seahorse instrument with calibration buffer. The cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured. Cells were then treated with compounds to analyze their effects on cellular bioenergetics. Finally, OCR and ECAR data were collected and analyzed to understand the metabolic characteristics of the cells.

2.12 Lactate and Nitric Oxide (NO) Detection

The experiment employed BioVision assay kits, following the manufacturer's protocols. The cells were cultured in a 96-well plate, and lactate or nitric oxide (NO) detection reagents were added to both standards and test samples. After the reactions, absorbance was measured with an enzyme-linked immunosorbent assay (ELISA) reader to determine lactate or NO concentrations from established standard curves. Sample concentrations were also evaluated using a Bicinchoninic Acid (BCA) assay kit.

2.13 ROS and 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) Detection

The test samples were thoroughly mixed with an ROS probe or 2-NBDG and then incubated at 37°C for 30 minutes in a water bath. Afterwards, the samples were transferred to flow cytometry tubes, washed three times with PBS, and analyzed using a flow cytometer to assess the intensity of ROS generation and intracellular glucose uptake.

2.14 Chromatin Immunoprecipitation (ChIP) Assay

ChIP assay was used to study protein-DNA interactions and involved several key steps. Initially, cells were treated to crosslink proteins to DNA, preserving their interactions. Following this, the cells were lysed to release the chromatin. Specific antibodies were then employed to immunoprecipitate the target protein along with its associated DNA fragments. The resulting complexes were washed to eliminate nonspecific binding materials. After purification, the crosslinks between DNA and proteins were reversed, allowing for the separation of DNA from proteins. Finally, the purified DNA was extracted for further analysis, such as PCR or sequencing. ChIP provided valuable insights into the roles of protein-DNA interactions in gene regulation.

2.15 Dual-luciferase Reporter Assay

The dual-luciferase reporter assay uses firefly and Renilla luciferase as reporter genes to study gene expression. The experimental construct was co-transfected into cells, which were then lysed, and luciferase activity was measured using specific substrates. Firefly luciferase reported on the activity of the experimental promoter, while Renilla luciferase served as a control. By normalizing firefly luciferase activity to Renilla luciferase, researchers were able to accurately quantify promoter activity.

3.1 Irf7 expression was significantly increased in M1 polarized macrophages and EAP

We downloaded three datasets of M1-polarized macrophages from the GEO database (GSE208667, GSE211683, and GSE200210), and concurrently, we performed RNA-seq sequencing on prostate tissues from both normal group mice and EAP group mice. The overlap of these four datasets is depicted in Fig. 1A. We also conducted GSEA enrichment analysis to explore the relationship between immune infiltration and chronic prostatitis, as shown in Fig. 1B, aiming to identify crucial pathogenic genes involved in prostatitis. Notably, genes from the Irf family, which are associated with immune-related diseases, displayed elevated expressions of Irf1 and Irf7 in both macrophages and prostate tissues. While previous studies have discussed the role of Irf1 in macrophages, the effects and mechanisms of Irf7 on macrophages remain unexplored. This spurred our interest in the potential mechanisms through which the Irf7 gene could influence M1 polarization of macrophages and subsequently promote chronic prostatitis. To explore the effect of Irf7 on the polarization of macrophages, comprehensive experimental validations were undertaken. Initial immuno-fluorescent staining indicated co-expression of Irf7 and iNOS following M1 polarization (Fig. 1C). Secondly, PCR and Western blot analysis showed that Irf7 was upregulated at both the RNA and protein levels (Fig. 1D-1F). Additionally, animal studies revealed a significant increase in Irf7 expression levels in EAP mice compared to normal mice, as shown in Figs. 1G-1J. These findings collectively underscore the critical role of Irf7 in M1 polarized macrophages and in the pathophysiology of EAP in mice.

3.2 Irf7 deficiency inhibited M1 polarization

In order to analyze the effect of Irf7 on M1 polarization, we decreased the expression of Irf7 in macrophages with LPS stimulation, and observed the changes in inflammatory cytokines. Figures 2A and 2B demonstrate the successful reduction of Irf7 expression following LPS stimulation. This downregulation led to a decrease in inflammatory markers (IL-6, IL-1β, and TNF-α) and M1 macrophage polarization markers (iNOS and Arg1), as shown in Fig. 2C. Western blot analysis further supported the role of Irf7 in inhibiting M1 polarization by displaying changes in iNOS and Arg1 expression levels (Fig. 2D). Flow cytometry confirmed that reducing Irf7 expression decreased the proportion of M1 cells, as depicted in Figs. 2E and 2F. Additionally, we measured the intracellular concentrations of ROS and NO, key mediators released during macrophage activation. The results shown that Irf7 affect the inflammatory capacity and activation status of macrophages (Fig. 2G-2H). Clearly, Irf7 is pivotal in promoting inflammation, driving macrophages towards a pro-inflammatory M1 phenotype, and intensifying the severity of inflammation.

3.3 Irf7 can transcriptionally regulate Hif-1α to reprogram macrophage glycolysis

During macrophage polarization, an increase in glycolysis facilitates rapid energy acquisition, enhancing pro-inflammatory responses. We hypothesized that glycolysis would decrease with the downregulation of Irf7. Measurements of 2-NBDG uptake and lactate production confirmed that that knocking down the Irf7 gene inhibited glycolysis in macrophages (Fig. 3A, 3B). Furthermore, hippocampal experiments demonstrated that inhibiting Irf7 lowers the extracellular acidification rate, thus reducing glycolytic flux and capacity, regardless of LPS stimulation (Fig. 3D). Extensive PCR analysis revealed that Irf7 influences the expression of key glycolytic genes (Fig. 3E), suggesting that potential role of Irf7 in glycolysis extends beyond individual genes to a regulatory network that impacts the entire glycolytic pathway. Given the role of Hif-1α as a crucial transcription factor in regulating glycolysis during M1 macrophage polarization^[16], we explored its interaction with Irf7. PCR results indicate that Irf7 can affect the expression of Hif-1α (Fig. 3F). Further investigations using ChIP and dual-luciferase reporter assays validated the interaction and regulatory influence of Irf7 on three binding sites associated with Hif-1α(Fig. 3G-3H). Western blot results confirmed increased nuclear translocation of Hif-1α, enhancing its role in regulating glycolytic gene expression (Fig. 3I). Additionally, immunofluorescence studies indicated a reduction in the co-expression of intracellular Hif-1α and iNOS levels (Fig. 3J). In summary, these findings demonstrated that Irf7 can effectively promote M1 polarization by augmenting glycolysis, with Hif-1α serving as a critical downstream target in this regulatory pathway.

3.4 Hif-1α agonist enhanced Irf7-mediated M1 polarization and glycolysis in macrophages

To further explore the impact of Hif-1α on macrophage polarization and glycolysis induced by Irf7, we treated cells with knocked down Irf7 with DMOG, a well-known Hif-1α activator. As shown in Fig. 4A-4C, activation of Hif-1α enhanced the expression of inflammatory factors and the inflammatory response in macrophages with reduced Irf7 expression. This was corroborated by the protein analysis showing elevated levels of iNOS and Arg1(Fig. 4D-4E). Flow cytometric analysis of cells with reduced Irf7 also showed an increased proportion of M1 macrophages following Hif-1α activation (Fig. 4F). Additionally, we investigated the influence of DMOG on glycolytic metabolism mediated by Irf7, revealing a noticeable rise in lactate production and glucose uptake capacity (Fig. 4G-4H). Seahorse XF assays demonstrated that DMOG increased the extracellular acidification rate in macrophages with suppressed Irf7 (Fig. 4I). By employing a glycolysis inhibitor, 2-deoxy-D-glucose (2-DG), to obstruct glucose availability, we observed a reduction in the expression of pro-inflammatory factors in macrophages(Fig. 4J). These experimental results suggest that Irf7 can regulate the glycolysis process through Hif-1α and thereby changes the M1 polarization state.

3.5 Irf7 deficiency reduced macrophage infiltration

A prostatitis model was established in EAP mice, and the Irf7 gene was knocked down through tail vein injection of an adenoviral vector. Western blot analysis confirmed a significant reduction in Irf7 expression in the EAP mice, as shown in Fig. 5A. To assess the effects of Irf7 knockdown on inflammation, histological examinations were performed using HE staining, alongside inflammation scoring and pain response evaluations (Figs. 5B-5D). The findings consistently demonstrated that reducing Irf7 expression led to a marked decrease in the severity of inflammation in the EAP model. Additionally, PCR analysis and ELISA of serum inflammatory cytokine levels were employed to measure serum levels of inflammatory cytokines and to evaluate inflammatory factor expression in tissue samples. These analyses further confirmed that lowering Irf7 expression alleviated inflammation in EAP mice, as illustrated in Figs. 5E and 5F. Lastly, immunofluorescence staining for the macrophage marker F4/80 revealed that Irf7 knockdown significantly reduced macrophage infiltration in the prostate (Fig. 5G). These results collectively support the hypothesis that Irf7 plays a critical role in regulating prostatitis by modulating macrophage activity and inflammatory responses.

3.6 Silencing Irf7 alleviated EAP-induced prostatic fibrosis

Chronic inflammation in the prostate often triggers excessive proliferation of fibrous connective tissue, ultimately leading to prostatic fibrosis. Based on this, we hypothesized that knocking down Irf7 could not only reduce inflammation but also alleviate prostate fibrosis in EAP mice. To test this, we performed Masson and Sirius Red staining, which revealed that Irf7 knockdown significantly reduced the degree of fibrosis in the prostate tissue of EAP mice (Fig. 6A). We next examined the expression levels of key fibrosis-related markers, including α-SMA, collagen, and TIMP1, in the prostate of EAP mice before and after Irf7 knockdown using PCR and Western blot analysis. Our results indicated that compared to EAP mice without Irf7 knockdown, the expression of these markers was significantly reduced at both the RNA and protein levels in the Irf7-knockdown group (Figs. 6B-6C). These findings suggest that Irf7 plays a role in promoting prostate fibrosis, likely through the modulation of Hif-1α. To further investigate the mechanism by which Irf7 influences fibrosis, we conducted co-culture experiments with macrophages and fibroblasts (Fig. 6D). PCR and Western blot analyses of these co-cultured cells yielded consistent results, demonstrating that both Irf7 and Hif-1α regulate macrophage activation and fibroblast transformation, key processes in fibrosis development (Figs. 6E-6F). Immunofluorescence staining (Fig. 6G) further confirmed that the expression of Irf7 in macrophages had a substantial effect on the expression of α-SMA in co-cultured fibroblasts, indicating that macrophage-derived Irf7 influences fibroblast activation. In summary, these experimental findings suggest that Irf7 can regulate macrophage polarization through Hif-1α, which in turn can modulate the extent of prostatic fibrosis. By targeting Irf7, it may be possible to not only reduce inflammation but also prevent or mitigate the fibrotic changes associated with chronic prostatitis.

3.7 Irf7 deficiency alleviated M1 infiltration and Hif-1α activation in prostates of EAP mice

Immunofluorescence analysis of prostate tissue in mice revealed a marked reduction in the infiltration of M1 macrophages in the Irf7 knockdown group (Fig. 7A). This observation was further corroborated by PCR and Western blot analyses, which produced consistent results that aligned with the immunofluorescence findings (Figs. 7B-7C). These data suggest that Irf7 may exacerbate macrophage infiltration in the prostate tissues of EAP mice. Moreover, flow cytometry analysis demonstrated that Irf7 actively promotes macrophage polarization towards the M1 phenotype (Fig. 7D). In the stromal tissues of EAP mice, Hif-1α expression in macrophages increased following Irf7 suppression (Fig. 7E), providing in vivo evidence of potential involvement of Hif-1α in the regulation of M1 macrophage polarization by Irf7. Furthermore, evaluation of total and nuclear Hif-1α protein levels across experimental groups revealed that Irf7 significantly influences downstream Hif-1α, leading to enhanced nuclear localization. This, in turn, was found to play a critical role in the pathophysiology of prostatitis (Fig. 7F). Collectively, these findings suggest that Irf7 can enhances M1 macrophage polarization through its interaction with Hif-1α, thereby contributing to the development and progression of EAP (Fig. 7G). This pathway underscores the central role of Irf7 in regulating macrophage activity and its impact on chronic inflammation in prostatitis.

CP/CPPS is a common urogenital disease among middle-aged and young men. Despite the availability of various therapeutic approaches, their clinical efficacy has remained limited due to the intricate and poorly understood nature of the disease. As a result, it is crucial to conduct in-depth investigations to uncover the underlying mechanisms and identify key risk factors contributing to CP/CPPS, with the ultimate goal of improving treatment outcomes. The current study has demonstrated that Irf7 induced the occurrence and development of CP/CPPS, with the stimulation of M1 polarization and interstitial fibrosis of prostates. Mechanistic insights from this study showed that Irf7 could enhance glycolysis in macrophages by upregulating the transcription of Hif-1α, which in turn promoted M1 polarization. These discoveries highlight Irf7 as a potential therapeutic target for modulating macrophage activity and mitigating the pathological features of CP/CPPS, offering new avenues for more effective clinical interventions.

Irf7 is a key transcription factor that plays diverse roles in various inflammatory diseases. It has been shown to contribute to the progression of inflammation through mechanisms such as promoting tissue fibrosis, facilitating the differentiation of inflammatory cells, or inhibiting autophagy, as seen in conditions like systemic sclerosis and lung inflammation^{[17, 18]}. Recent studies has validated that Irf7 can regulate the transcription of glycolysis-related genes to modulate macrophage polarization^{[14, 19–21]}. While the critical involvement of Irf7 in different forms of inflammation has been well-documented^{[22, 23]}, its role in CP/CPPS remains unexplored. Macrophages have been widely recognized as key players in the onset and progression of CP/CPPS, with M1 macrophages in particular driving the inflammatory response and worsening disease severity^{[24, 25]}. Therefore, we examined the impact of Irf7 on the occurrence and development of CP/CPPS and found that Irf7 was closely associated with both the occurrence and development of CP/CPPS by stimulating M1 polarization, as evidenced by more severe inflammation of the prostate and increased pain sensitivity compared to mice with the normal control. These findings suggest that Irf7 plays a pivotal role in the pathogenesis of CP/CPPS by promoting M1 macrophage activity, further exacerbating inflammation and disease symptoms.

Extensive research has demonstrated that cells can enhance glucose utilization by increasing glycolysis, a metabolic process that plays a key role in supporting inflammation by promoting the differentiation of inflammatory cells and contributing to oxidative inactivation^{[26, 27]}. Furthermore, previous studies have suggested that Irf7 can regulate glycolysis by regulating the transcription of various glycolytic genes^[28–31]. At the same time, other findings have confirmed that glycolysis is crucial in driving M1 macrophage polarization and sustaining the immune functions of M1 macrophages. Therefore, we investigated the oxidative levels, glycolysis-related genes, and metabolic profiles of macrophages with and without Irf7 knockdown. Our results revealed that Irf7 significantly influences M1 polarization by enhancing the transcription of multiple classical glycolytic genes. These findings suggest that Irf7 not only regulates the glycolytic pathway but also plays an essential role in macrophage polarization by driving metabolic reprogramming, thus linking metabolism with inflammation and immune activation.

Hif-1α, a key transcription factor activated in response to hypoxia, plays a pivotal role in regulating glycolysis. Previous studies have shown that Hif-1α influences macrophage polarization by enhancing glycolysis through the upregulation of glycolytic enzymes such as Hk2, phosphoglycerate kinase 1, and lactate dehydrogenase A (LDHA), thereby promoting inflammatory responses^{[32, 33]}. In the current study, we discovered that Irf7 promotes M1 macrophage polarization by enhancing glycolysis through the upregulation of Hif-1α transcription, which, in turn, increases the expression of several glycolysis-related genes. This was further confirmed using an Hif-1α inhibitor, demonstrating the role of Irf7 in sensitizing glycolysis via Hif-1α. Furthermore, based on the presence of binding sequences on promoters or enhancers of various genes, Irf7 is known to regulate gene transcription by binding to specific response elements within the promoters of its target genes. In our study, we found that Irf7 acts as a transcription factor by directly binding to the promoter region of Hif-1α in macrophages, a mechanism that has not been previously reported. This innovative finding highlights the potential role of Irf7 in modulating the transcriptional activity of Hif-1α, providing further insights into the regulatory pathways that drive macrophage polarization and inflammation.

Several studies have established that fibrosis plays a critical role in the progression of CP/CPPS, with research indicating that reducing stromal fibrosis can alleviate the condition by inhibiting processes like ferroptosis^[34] or osteopontin^[35]. Previous studies have demonstrated that Irf7 can regulate fibrotic protein expression at various levels^[36–38]and inhibition of Irf7 can effectively reduce tissues fibrosis to alleviate systemic sclerosis and chronic hepatitis^{[12, 39]}. In the present study, we discovered that Irf7 has a direct impact on prostate fibrosis. Knockdown experiments, conducted both in vivo and in vitro, revealed a significant decrease in fibrotic tissue when Irf7 expression was suppressed. Our findings show that Irf7 promotes the transcription of Hif-1α, which enhances glycolysis, ultimately driving M1 macrophage polarization and exacerbating prostatitis-associated fibrosis. This mechanism significantly contributes to the severity of CP/CPPS. Notably, this is the first time that the possible role of Irf7 in facilitating prostate fibrosis through the Hif-1α-glycolysis axis has been reported. These insights provide a new understanding of the molecular pathways driving CP/CPPS and highlight Irf7 as a potential therapeutic target for mitigating both inflammation and fibrosis in this condition.

In summary, our study demonstrated that Irf7 can enhance the transcription of Hif-1α to facilitate glycolysis, which subsequently facilitated glycolysis and promoted M1 macrophage polarization, ultimately aggravating CP/CPPS. This newly identified Irf7/Hif-1α/glycolysis pathway appears to play a crucial role in the pathogenesis of CP/CPPS, suggesting that targeting this axis could offer novel therapeutic strategies for treating the condition.

Ethics approval and consent to participate in previous study

All animal experiments were approved by the Animal Care and Use Committee of Anhui Medical University (Approval No. LLSC20211051).

Consent for publication

All authors have approved submission of the manuscript to this journal.

Funding

This study was supported by the National Natural Science Foundation of China (82300872, 82270818 and 82170787) and Natural Science Foundation of Anhui Province (23080850B63).

Author's Contributions

Jing Chen: Writing – review & editing, Funding acquisition, Conceptualization.

Tong Meng: Writing – original draft, Data curation.

Yi Zhang:Validation. Huihui Wang: Data curation. Weikang Wu: Data curation. Wei Peng: Data curation. JiaBing Yue: Data curation. Cong Huang: Supervision. Wanqing Liu:Data curation Chaozhao Liang: Validation, Funding acquisition Cheng Yang: Validation, Funding acquisition

Competing interests

The authors declare that there are no conflict of interests.

Availability of Data and Material

All data associated with this study are available from the corresponding author with reasonable request.

Acknowledgments

We thank the participants enrolled in this study for time and contributions.

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Editorial decision: Major Revision
13 Nov, 2024
Reviewers agreed at journal
21 Oct, 2024
Reviewers invited by journal
21 Oct, 2024
Editor assigned by journal
20 Oct, 2024
First submitted to journal
17 Oct, 2024

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Irf7 aggravates prostatitis by promoting Hif-1α-mediated glycolysis to facilitate M1 polarization

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Abstract

Figures

1. Introduction

2. 16 ELISA assay

2. Materials and methods

2.1 Reagents

2.1.1 Preparation of Animals

2.1.2 Establishment of the EAP Mice Models

2.2 Cell Treatment

2.3 Immunofluorescence

2.4 Hematoxylin-Eosin (HE) Staining

2.5 Sirius Red staining

2.6 Masson Staining

2.8 Western Blotting

2.9 RNA Extraction

2.10 RT-qPCR

2.11 Seahorse XF Assays

2.12 Lactate and Nitric Oxide (NO) Detection

2.13 ROS and 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) Detection

2.14 Chromatin Immunoprecipitation (ChIP) Assay

2.15 Dual-luciferase Reporter Assay

3. Results

3.1 Irf7 expression was significantly increased in M1 polarized macrophages and EAP

3.2 Irf7 deficiency inhibited M1 polarization

3.3 Irf7 can transcriptionally regulate Hif-1α to reprogram macrophage glycolysis

3.4 Hif-1α agonist enhanced Irf7-mediated M1 polarization and glycolysis in macrophages

3.5 Irf7 deficiency reduced macrophage infiltration

3.6 Silencing Irf7 alleviated EAP-induced prostatic fibrosis

3.7 Irf7 deficiency alleviated M1 infiltration and Hif-1α activation in prostates of EAP mice

4. Discussion

5. Conclusion

Declarations

References

Status:

Version 1