Blimp-1 Orchestrates Macrophage Polarization and Metabolic Homeostasis via Purine Biosynthesis in Sepsis

Blimp-1 Orchestrates Macrophage Polarization and Metabolic Homeostasis via Purine Biosynthesis in Sepsis | 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 Blimp-1 Orchestrates Macrophage Polarization and Metabolic Homeostasis via Purine Biosynthesis in Sepsis Liuluan Zhu, Rui Li, Qiushi Qin, Wenjuan Peng, Lan Li, Yujia Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4903330/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Feb, 2025 Read the published version in Cell Death & Disease → Version 1 posted 9 You are reading this latest preprint version Abstract Sepsis is a life-threatening condition characterized by a dysregulated immune response to infection, leading to systemic inflammation and organ dysfunction. Macrophage polarization plays a critical role in pathogenesis of sepsis, and the influence of B lymphocyte-induced maturation protein-1 (Blimp-1) on this polarization is an underexplored yet pivotal aspect. This study aimed to elucidate the role of Blimp-1 in macrophage polarization and metabolism during sepsis. Using a murine cecal ligation and puncture model, we observed elevated Blimp-1 expression in M2 macrophages. Knockdown of Blimp-1 in this model resulted in decreased survival rates, exacerbated tissue damage, and impaired M2 polarization, underscoring its protective role in sepsis. In vitro studies with bone marrow-derived macrophages, RAW264.7, and THP-1 cells further demonstrated Blimp-1 promotes M2 polarization and modulates key metabolic pathways. Metabolomics and dual-luciferase assays revealed Blimp-1 significantly influences purine biosynthesis and the downstream Ornithine cycle, which are essential for M2 macrophage polarization. Our findings unveil a novel mechanism by which Blimp-1 modulates macrophage polarization through metabolic regulation, presenting potential therapeutic targets for sepsis. This study highlights the significance of Blimp-1 in orchestrating macrophage responses and metabolic adaptations in sepsis, offering valuable insights into its role as a critical regulator of immune and metabolic homeostasis. Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages Biological sciences/Immunology/Infectious diseases sepsis macrophage polarization Blimp-1 cellular metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Sepsis is a life-threatening condition characterized by organ dysfunction resulting from a dysregulated host response to infection 1 and responsible for about 11 million deaths annually, accounting for nearly 20% of global mortality 2 . The pathogenesis of sepsis is multifaceted, involving intricate interactions between infectious microorganisms and the host, alongside various pathways including infection, inflammation, hypoxia, and metabolic reprogramming 3 . A critical mechanism underlying sepsis is the imbalance of inflammatory response, which significantly influences its progression 4 . Therefore, early intervention in inflammatory response disorders may be effective in reducing sepsis-related mortality. Macrophages play a pivotal role in mediating inflammatory responses and primarily contain two functional subsets: pro-inflammatory (M1) and anti-inflammatory (M2) macrophages. M1 macrophages are characterized by producing pro-inflammatory cytokines, or mediators such as TNF-α, IL-1α/β, IL-6, IL-12, and iNOS 5 . M2 macrophages express arginase-1 (ARG1) and secrete anti-inflammatory cytokines IL-10 and TGF-β. In sepsis, excessive activation of M1 macrophages can exacerbate inflammation and organ damage, while M2 macrophages promote the resolution of inflammation, tissue repair, and organ recovery. Given the critical role of the M1/M2 polarization in sepsis 6 , understanding the regulatory mechanisms governing macrophage polarization is essential for elucidating sepsis pathology. B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcription suppressor of IFN-β encoded by the Prdm1 gene 7 , 8 . Overexpression of Blimp-1 in pro-monocytic cells induces partial macrophage differentiation, characterized by the expression of surface markers such as CD11b and CD11c 9 . Blimp-1 was identified as a marker for a specific macrophage population located near the microbe-exposed surface in the colon 10 . Recent studies have shown CCL8 is a direct target of Blimp-1 in mononuclear macrophages, with elevated CCL8 levels observed in Prdm1 fl / fl mice 11 . Our previous study claimed Blimp-1 represses the production of several inflammatory cytokines, including IL-1β, IL-6, and IL-18, by directly binding to the genomic region and restricting the nuclear translocation and transcriptional activity of NF-κB 12 . However, the potential regulatory role of Blimp-1 in macrophage polarization and its significance in sepsis represent a critical gap in current understanding. Emerging evidence indicates energy metabolism plays a crucial role in regulating macrophage polarization and function 13 . Generally, pro-inflammatory M1 macrophages primarily rely on glycolysis to generate energy for innate immune responses, while M2 macrophages depend more on mitochondrial oxidative phosphorylation and the tricarboxylic acid cycle to satisfy their energy requirements 14 . During M2 polarization, transcription factors such as PPARγ and PGC-1β are critical for mitochondrial respiration and fatty acid oxidative metabolism 15 , 16 . Additionally, transketolase (TKT) is important for glycometabolism 17 , while glutamate-pyruvate transaminase (GPT) and glycine amidinotransferase (GATM) are involved in amino acid metabolism 18 . Notably, Blimp-1 may participate in cellular metabolism, thereby modulating inflammatory marker expression in quiescent macrophages 19 . For instance, Blimp-1 regulates IL-10 expression in group 2 innate lymphoid cells (ILC2s), which exhibit a metabolic dependence on glycolysis for IL-10 production 20 . These findings suggest that Blimp-1 may regulate macrophage polarization through cellular metabolic pathways, thus influencing inflammatory responses in sepsis. In this study, we aim to elucidate the effects of Blimp-1 on macrophage polarization in sepsis from the perspective of cellular metabolism. We inhibited Blimp-1 expression and measured its effects on macrophage polarization phenotypes and functions both in vitro and in vivo . Subsequently, we utilized the metabolomics technology to explore the metabolic mechanisms by which Blimp-1 regulates macrophage polarization. We conducted dual-luciferase reporter assay to validate the metabolic pathways regulated by Blimp-1 in macrophages. In summary, our findings clarify a previously unknown mechanism by which Blimp-1 plays a key role in orchestrating macrophage polarization during sepsis. Results Blimp-1 expression is elevated in sepsis-associated macrophages We had sucessfully established a cecal ligation and puncture (CLP) sepsis model in C57BL/6J mice. Compared to sham-operated controls, CLP mice exhibited significant histopathological lesions in liver and lungs, along with increased serum levels of hepatic injury biomarkers and inflammatory cytokines (Fig. 1 A- 1 C). Importantly, the peritoneal lavage fluid of CLP mice contained a higher percentage of macrophages, with Blimp-1 expression specifically elevated in the M2 subset (Fig. 1 D- 1 F). The mRNA level of Blimp-1 was also significantly increased in these cells (Fig. 1 G). This selective upregulation of Blimp-1 in M2 macrophages, but not in CD4 + T cells, CD8 + T cells, or B cells (Fig. 1 H, Fig. S1 A & 1B). Blimp-1 knockdown exacerbates sepsis by impairing M2 polarization To elucidate the functional significance of Blimp-1 upregulation, we knocked down its expression using shRNA-Blimp-1 and assessed the impact on CLP mice (as depicted in Fig. 2 A). Three weeks post-injection, the survival rate of CLP mice was significantly lower in the Blimp-1 knockdown group (Fig. 2 B). Histopathological analysis revealed exacerbated liver and lung tissue damage (Fig. 2 C). These findings underscore the protective role of Blimp-1 in modulating the inflammatory response and tissue repair during sepsis. Following shRNA-Blimp-1 intervention, the peritoneal lavage fluid of CLP mice exhibited an elevated total macrophage count, yet a diminished proportion of M2 macrophages, as evidenced by reduced presence of CD206 + and CD206 + Blimp-1 + cells (Fig. 2 D- 2 F). In contrast, Blimp-1 expression in other immune cells, such as CD4 + T cells, CD8 + T cells, and B cells, remained unaffected in both peritoneal lavage fluid (Fig. 2 G) and spleen grinding fluid (Fig. 2 H). These observations suggest that Blimp-1's protective effect in sepsis may depend on its regulatory role in M2 macrophage polarization. Blimp-1 promotes monocyte-macrophage differentiation and M2 polarization We next assessed the temporal expression of Blimp-1 during macrophage differentiation and polarization from bone marrow cells of naïve mice (as depicted in Fig. 3 A). Blimp-1 expression was significantly higher in monocytes compared to B cells and granulocytes (Fig. 3 B), and its mRNA levels progressively increased alongside bone marrow-derived macrophage (BMDM) differentiation and maturation (Fig. 3 C). Upon polarization, M1 macrophages exhibited high secretion of IL-1β, IL-6, TNF-α, and IL-12 (Fig. S2 A); whereas M2 macrophages secreted high levels of TGF-β, CCL17, and CCL22 (Fig. S2 B). Notably, Blimp-1 mRNA levels were specifically elevated in M2 macrophages compared to M0 and M1 counterparts (Fig. 3 D), indicating a potential role for Blimp-1 in the differentiation and M2 polarization of macrophages. To further dissect Blimp-1's influence on macrophage polarization, THP-1 monocytic cells were induced to differentiate into macrophages and subsequently transfected with a Blimp-1 expressing plasmid. The expression level of Blimp-1 was identified by real-time fluorescent quantitative PCR (Fig. 3 E). Overexpression of Blimp-1 causes morphological changes consistent with M2 macrophages, including the appearance of extended pseudopods (Fig. 3 F), and a significant increase in CD206 expression, as well as increased mRNA levels of M2 markers such as MRC1, MSR1, ARG1 and IL10 (Fig. 3 G & 3 H). Similar results were obtained with RAW264.7 macrophages overexpressing Blimp-1, where an increase in mRNA levels of Msr1, Mrc1, Arg1, Ppargc1b, Il10, Ido1, Pdl1 , and Pdl2 was observed (Fig. 3 I- 3 K). These findings collectively highlight the critical role of Blimp-1 in driving macrophage differentiation and the acquisition of the M2 phenotype. Blimp-1 is essential for energy metabolism in M2 macrophages Further investigation of Blimp-1's regulatory role in macrophage polarization was conducted by knocking down its expression in RAW264.7 cell (as depicted in Fig. 4 A). Compared to shRNA-NC control, shRNA-Blimp-1 effectively reduced Blimp-1 expression, particularly in M2 macrophages (Fig. 4 B). This knockdown resulted in a significant decrease in the proportion of M2 (CD206 + ) macrophages and a dramatic reduction in the mRNA levels of M2 markers Mrc1, Msr1 , and Arg1 (Fig. 4 C & 4 D). In contrast, the proportion of M1 (CD86 + ) macrophages and the mRNA levels of M1 markers Il12b, Nos2 , and Tnfa remained unchanged (Fig. 4 C & 4 E), underscoring the specific role of Blimp-1 in M2 macrophage polarization and function. We also examined the expression of genes pivotal to mitochondrial respiration, fatty acid oxidative metabolism, and glycometabolism. The robust expression levels of Pparg, Ppargc1b, Tkt, Gatm , and Gpt2 in M2 macrophages, which were notably diminished following Blimp-1 knockdown (Fig. 5 A). Conversely, overexpression of Blimp-1 in RAW264.7 macrophages resulted in elevated mRNA levels for these genes (Fig. 5 B). Employing the Seahorse XF analyzer, we conducted metabolic assays substantiated the impact of Blimp-1 on mitochondrial function. The knockdown of Blimp-1 corresponded with a reduction in mitochondrial oxygen consumption rates (OCRs) in M2 macrophages (Fig. 5 C). This reduction is indicative of a decrease in the maximal respiration, mitochondrial ATP production, and spare respiratory capacity (Fig. 5 D). Furthermore, the extracellular acidification rate (ECAR), a measure of glycolytic activity, was diminished after shRNA-Blimp-1 intervention (Fig. 5 E), reflecting lower basal and maximum glycolysis levels (Fig. 5 F). Collectively, these findings underscore the capacity of Blimp-1 to facilitate M2 macrophage polarization through the modulation of key metabolic enzyme expressions. Blimp-1 modulates the metabolome of M2 macrophages We furtherly conducted a non-targeted metabolomics analysis on RAW264.7 cells with Blimp-1 knockdown and identified a total of 121 metabolites (Fig. 6 A). An Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) clearly distinguished the metabolite profiles between the shRNA-Blimp-1 and shRNA-NC groups (Fig. 6 B). Differential metabolite analysis revealed significant differences in amino acids and nucleotides between the two groups (Fig. 6 C). The differential metabolites were displayed in Fig. 6 D & Table S1 , with those exceeding a importance in projection (VIP) scores score of 1 considered significant. Notably, the levels of Ornithine and multi-amino acids increased following Blimp-1 inhibition (Fig. 6 D, below panel). Subsequently, we conducted a quantitative metabolomics analysis targeting nucleotide metabolism and amino acid metabolism and identified 9 differential nucleotide metabolites and 22 differential amino acid metabolites between groups (Table S2 ). OPLS-DA clearly distinguished the nucleotide and amino acid metabolite of shRNA-Blimp-1 and shRNA-NC groups (Fig. 6 E & 6 F). Metabolic pathway enrichment analysis using the Small Molecule Blimp-1 knockdown significantly impacted GDP exchange, UDP exchange and ATP diphosphate hydrolase in M2 macrophages (Fig. 6 G), as well as ammonia recycling, glutamine metabolism, urea cycle and glycine and serine metabolism (Fig. 6 H). These findings suggest that Blimp-1 influences the metabolic pathways of nucleotides and amino acids in M2 macrophages. Blimp-1 enhances purine biosynthesis and the downstream Ornithine cycle in M2 macrophages Building on the differential metabolite analysis, we concentrated on purine biosynthesis, nucleotide metabolism, and the Ornithine cycle. Blimp-1 knockdown led to a down-regulation of most metabolites involved in purine biosynthesis (Fig. 7 A). The expression levels of genes encoding key enzymes in this pathway, such as Ppat , Pnp , Gda , and Xdh , were significantly reduced with Blimp-1 inhibition (Fig. 7 B). Following Blimp-1 knockdown, the content of metabolites in the Ornithine cycle and the mRNA levels of key enzymes were significantly diminished (Fig. 7 C & 7 D). Additionally, the concentrations of nucleotide metabolites GDP, GTP, ATP, and ADP were markedly reduced (Fig .7E). Blimp-1 directly enhances the transcriptional activity of promoters for PPAT , GDA , XDH , APRT , ASS1 , ASL , and SMA (Fig. 7 F). Collectively, these results demonstrated that Blimp-1 regulates purine biosynthesis and the downstream Ornithine cycle by promoting the transcription of key enzyme genes in M2 macrophages (Fig. 7 G). Discussion A central component of sepsis pathophysiology is the polarization of macrophages. Macrophages undergo distinct polarization states (M1 and M2) that significantly influence sepsis outcomes. Nrf2 has been shown to play a protective role in sepsis-induced pulmonary injury and inflammation by modulating autophagy and NF-κB/PPARγ-mediated macrophage polarization 21 . Similarly, Krüppel-like transcription factors reduce macrophage glycolysis and inflammatory cytokine secretion by suppressing the M1 macrophage polarized phenotype 22 . Our recent work revealed that Blimp-1 inhibits the secretion of inflammatory cytokines via multiple toll-like receptors 12 , which elucidate a novel role for Blimp-1 in macrophage polarization during sepsis. Macrophage metabolic remodeling is essential for adapting to different polarized states and their functional changes 13 , 14 , 15 , 17 . The polarization of M2 macrophages involves alterations in several metabolic pathways, including glucose, amino acid, fatty acid, and nucleotide metabolism. Specifically, in fatty acid metabolism, PGC-1β collaborates with PPARγ to inhibit the secretion of inflammatory factors in M1 macrophages and to promote the M2 polarization 23 . The mechanism may involve PPARγ and PGC-1β directly regulating ARG1 expression, with their activation playing a crucial role in mitochondrial respiration and fatty acid oxidation in M2 macrophages 15 , 16 . In glucose metabolism, TKT, a key enzyme in the pentose phosphate pathway downstream of D-fructose-1.6 diphosphate, is upregulated in M1 macrophages along with glycolysis-related enzymes; however, this does not affect PPAT-related purine synthesis or cell proliferation 24 . In amino acid metabolism, GATM and GPT2, which are involved in the glutamate pathway, facilitate glutaminolysis, leading to α-ketoglutarate accumulation and M2 polarization 25 , 26 . In our study, Blimp-1 suppression led to a significant reduction in the mRNA expression of Pparg, Ppargc1b, Tkt, Gatm , and Gpt2 in M2 macrophages, while Blimp-1 overexpression resulted in a marked increase in these genes. These findings suggest that Blimp-1 regulates M2 macrophage polarization by modulating key metabolic enzyme expressions. Enhanced glycolysis during M2 macrophage polarization provides a metabolic basis for improved mitochondrial oxidative phosphorylation. M2 macrophages sustain their metabolism primarily through the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation, resulting a more robust oxidative metabolic profile compared to M1 macrophages 27 . Our results demonstrated that Blimp-1 knockdown led to substantial inhibition of both basal respiration and maximum respiratory capacity in mitochondrial oxidative phosphorylation, as well as glycolytic function. These results confirm that Blimp-1 regulates both mitochondrial oxidative phosphorylation and glycolysis in macrophages. Our results claimed that Blimp-1 enhances the transcription of PPAT, ASS1, and ASL, affecting purine biosynthesis and the Ornithine cycle in M2 macrophages. Previous studies have linked the Ornithine cycle with M2 polarization 14 . Notably, the expression of ARG1, a marker of M2 macrophages, is associated with the Ornithine cycle, while citrulline consumption is indicative of M1 polarization 28 . Although research on purine biosynthesis regulation in macrophage polarization is limited, both purine metabolism and the Ornithine cycle depend on glutamine, a star amino acid that regulates macrophage function 26 , 29 . Glutamine contributes α-ketoglutaric acid to the tricarboxylic acid cycle and facilitates acetylation modification of histones through acetyl-CoA, activating α-ketoglutaric acid-dependent epigenetic regulation and thereby influencing M2 macrophage polarization 30 . Actually, glutamine is the hub of purine biosynthesis and Ornithine cycle, connecting the metabolism of two kinds of biological macromolecules. Our study not only confirmed the regulatory mechanism of glutamine metabolism by Blimp-1, but also more comprehensively mapped the characteristics of glutamine-centered purine biosynthesis and Ornithine cycle metabolism in M2 macrophages. By elucidating the transcriptional regulation mechanism of Blimp-1 on various metabolic enzymes, this study revealed the metabolic regulation of M2 polarization and the pathological mechanism of sepsis progression. While our study provides valuable insights into the role of Blimp-1 in sepsis pathogenesis, several limitations warrant consideration. First, the study predominantly utilized animal models, necessitating validation in human samples. Second, a mouse model of Blimp-1 conditional knockout in macrophages is needed to elucidate the exact mechanism of Blimp-1 for metabolic reprogramming, including detailed dynamics and interactions with other regulatory pathways. Third, due to the lack of suitable ChIP antibodies, the direct binding site and strength of Blimp-1 to target gene promoters cannot be evaluated in vivo . Additionally, longitudinal studies are needed to assess the long-term effects of Blimp-1 modulation on immune function and host recovery in sepsis. In conclusion, our study unveils a novel regulatory role for Blimp-1 in macrophage polarization during sepsis, mediated through modulation of purine biosynthesis and the Ornithine cycle. These findings expand our understanding of the complex interplay between metabolism and immune responses in sepsis pathology. Materials and Methods Culture of BMDMs Bone marrow cells were isolated from C57BL/6 male mice aged 6–8 weeks, re-suspended with DMEM complete culture medium, and uniformly planted on 10-cm petri dishes. Cells were cultured in DMEM complete culture medium containing 10 ng/mL M-CSF for 7 days to obtain adherent growth of M0 macrophages. Then, 100 ng/mL LPS and 40 ng/mL INF-γ were used to induce 2-day polarization into M1 macrophages, or 40 ng/mL IL-4 and 20 ng/mL IL-13 were used to induce 2-day polarization into M2 macrophages. Cell culture and treatment RAW264.7 and 293T cells were cultured with DMEM medium (Gibco, USA) supplemented with 10% FBS and 1% penicillin/streptomycin solution (Gibco, USA). THP-1 cells were cultured and induced to differentiate into M0 macrophages by 50 ng/mL PMA (P8139, Sigma-Aldrich) for 48 h. Differentiated THP-1 or RAW264.7 cells were transiently transfected with Blimp-1 expressing plasmid using Lipofectamine 3000 Transfection Reagent (L3000150, Thermo Fisher Scientific, USA) for 48 h. The Blimp-1 shRNA and a non-specific control (NC) shRNA were integrated into the lentiviral vector pWSLV-Sh08-GFP-Puro (Noweton Bioscience, China), and the lentiviral particles infected RAW264.7 cells or BMDMs as previously described 12 . Mice, sepsis model and in vivo intervention Male C57BL/6 mice (8 weeks old) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Science (Beijing, China). The sepsis model was established by CLP procedure as previously described 31 . Blimp-1 shRNA and control shRNA were integrated into the vector pAAV-U6-shRNA-CMV bGlobin-eGFP-3Flag (Shanghai Genechem Company, China). Mice were intraperitoneally injected with AAV particles carrying shRNA-Blimp-1 or shRNA-NC at a titer of 5.00E + 11 v.g/mL, and fed for three weeks before CLP operation. The shRNA-Blimp-1 and shRNA-NC oligonucleotides (5’-3’) refer to our previous report 12 . Splenic cell isolation Spleen was excised and minced in PBS. Minced spleen suspension was filtered through a 70-µm filter, spun at 1200 rpm for 5 min. The cell precipitation was collected and centrifuged at 1200 rpm for 5 min. The cell precipitates were collected and split in RBC lysate (BioLegend, USA) for 3 min and then rotated at 1200 rpm with PBS for 5 min. The supernatant was discarded and washed again with PBS, and an appropriate number of cells were resuspended in the FACS buffer for flow cytometry staining. Peritoneal lavage fluid extraction The abdominal skin of mice was cut open, the peritoneum was exposed, the abdominal cavity was irrigated with 5 mL 0.9% NaCl. The normal saline was sucked out after full shaking for 90 sec, the supernatant was rotated at 1200 rpm for 5 min and the supernatant was discarded. The cells were resuspended with FACS buffer and stained for flow cytometry. Flow cytometry Cells were washed and collected by centrifuging at 1200 rpm for 5 min, and resuspended with FACS buffer. The following mAbs were used: Blimp-1-PE (BD Biosciences, USA), CD86-PE-Cy7 (Tonbo Bioscience San Diego, USA), CD45-APC-Cy7 (BioLegend, USA), F4/80-FITC/APC (eBioscience, USA), CD11b-BV510 (BD Biosciences), CD3- eFluor450 (BD Biosciences), CD19-PE-Cy7 (BD Biosciences), CD8-FITC (eBioscience), CD4-Percp (eBioscience), CD206-APC/BV421 (BioLegend), Gr-1-FITC (BD Biosciences), CD48-Percp (Elabscience, USA). Data acquisition and analysis were performed using a CantoII flow cytometer (BD Biosciences) and FlowJo Software (version 10.1; Tree Star, USA). Multiple cytokine assay Cytokines in the BMDM culture supernatant and mouse serum were detected using LEGENDplexTM MU Macrophage/Microglia Panel (13-plex) w/VbP Reagent (#740846, BioLegend). The data analysis and processing were carried out on the BioLegend website. Serum alanine aminotransferase and aspartate aminotransferase measurement Mouse serum samples were collected and the activities of serum aspartate transaminase (AST) and alanine transaminase (ALT) were determined using an automatic analyzer (Model 7600 Series, Hitachi, Japan). Hematoxylin-eosin (H&E) staining After the mice were sacrificed, liver and lung samples were separated and fixed with 4% paraformaldehyde for 24 h, subsequently embedded in paraffin, sliced (4–5 µm). Histologic sections of tissues were stained with H&E staining analysis. RNA isolation, reverse transcription, and realtime PCR Total RNA was extracted with Total RNA Kit (Omega Biotek, China) and reverse transcribed using the PrimeScript™ RT reagent Kit (TaKaRa, Japan). Amplification was performed using the Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific, USA). The relative expression level of each transcript was normalized to murine GAPDH. The primers were listed in Table S3. Plasmid construction, transfection, and dual-luciferase assay The promoter luciferase reporters for PRPS1, PPAT, PNP, GDA, XDH, APRT, ASS1, ASL and SMS were constructed using pGL3-basic-Luc-wt plasmid. Blimp-1 cDNA was cloned into pcDNA3.1 + plasmid as previous report 32 . The promoter luciferase reporter, Blimp-1 expressing plasmid, and pRL-TK internal reference were transfected into 293T cells with PEI 40K Transfection Reagent (Servicebio, China) for 4–6 h. After 24 h, cell lysate was collected and detected using the Dual-Luciferase® Reporter Assay System (Promega). Seahorse XF glycolytic stress and mitochondrial stress test Mitochondrial respiratory capacity and glycolytic function of M2 macrophages were measures using Seahorse XF Glycolysis Stress Test Kit (#103020-100, Agilent, USA) and Seahorse XF Cell Mito Stress Test Kit (#103015-100, Agilent) on a Seahorse XFe24 Analyzer (Agilent). Non-targeted and targeted metabolomics analysis 1×10 7 RAW264.7 cells were centrifuged at 4℃ at 200–1000 rcf for 10 min. The cell pellets were lysed ultrasonically and centrifuged at 18000 g for 20 min. Ten microliter supernatants were mixed with 70 µl borate buffer and 20 µl 6-minoquinolyl-N-hydroxysuccinimidyl carbamate derivatization reagent. The mixture was shaken at 1200 rpm for 10 min, then added 900 µl ultrapure water and mixed well. Using liquid chromatography mass spectrometry (LC-MS) and Metabo-Profile's self-built database, non-targeted metabolomics analysis of metabolites in samples was performed qualitatively and quantitatively. The target metabolomics of amino acids and nucleotides in the samples were subjected to ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) assay for quantification. Statistics analysis All statistical analyses were performed using GraphPad Prism 9.0 software. The Kolmogorov-Smirnov test was used to inspect the normality and homogeneity of variance of all data. For two-group comparison, P values were derived from the Student t-test to determine differences between groups with normally distributed data and Mann-Whitney nonparametric test with other data. Declarations Conflict of Interest The authors declare no conflict of interest. ETHICS All animal procedures were approved by the Institutional Animal Care and Use Committee of Capital Medical University. Author Contributions R.L. and Q.Q. performed most experiments and analyzed the data. Q.Q. and W.P. wrote the draft of the manuscript. L.Z. designed experiments, supervised the study and revised the manuscript. L.L. assisted in the cellular experiments. Y.L. and Y.Z. assisted in the mouse model preparation. Acknowledgement This work was supported by the National Natural Science Foundation of China (grant numbers, 82372189, 82072295 and 82172128) and the Beijing High-Level Public Health Technical Talent Training Program (Discipline Backbone Talent 02–32). We would warmly thank Prof. Aibin He from Peking University for technical advice. References Srzić I, Nesek Adam V, Tunjić Pejak D. SEPSIS DEFINITION: WHAT'S NEW IN THE TREATMENT GUIDELINES. Acta clinica Croatica 2022, 61 (Suppl 1) : 67-72. Rudd KE, Johnson SC, Agesa KM, Shackelford KA, Tsoi D, Kievlan DR , et al. Global, regional, and national sepsis incidence and mortality, 1990-2017: analysis for the Global Burden of Disease Study. Lancet (London, England) 2020, 395 (10219) : 200-211. Wasyluk W, Zwolak A. Metabolic Alterations in Sepsis. Journal of clinical medicine 2021, 10 (11). Huang M, Cai S, Su J. The Pathogenesis of Sepsis and Potential Therapeutic Targets. International journal of molecular sciences 2019, 20 (21). 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Frontiers in immunology 2021, 12: 664871. Mornata F, Pepe G, Sfogliarini C, Brunialti E, Rovati G, Locati M , et al. Reciprocal interference between the NRF2 and LPS signaling pathways on the immune-metabolic phenotype of peritoneal macrophages. Pharmacology research & perspectives 2020, 8 (4) : e00638. Zhou W, Hu G, He J, Wang T, Zuo Y, Cao Y , et al. SENP1-Sirt3 signaling promotes α-ketoglutarate production during M2 macrophage polarization. Cell reports 2022, 39 (2) : 110660. Zhu Y, Zhang S, Sun J, Wang T, Liu Q, Wu G , et al. Cigarette smoke promotes oral leukoplakia via regulating glutamine metabolism and M2 polarization of macrophage. International journal of oral science 2021, 13 (1) : 25. Huang SC, Smith AM, Everts B, Colonna M, Pearce EL, Schilling JD , et al. Metabolic Reprogramming Mediated by the mTORC2-IRF4 Signaling Axis Is Essential for Macrophage Alternative Activation. Immunity 2016, 45 (4) : 817-830. Mao Y, Shi D, Li G, Jiang P. Citrulline depletion by ASS1 is required for proinflammatory macrophage activation and immune responses. Molecular cell 2022, 82 (3) : 527-541.e527. Hu X, Ma Z, Xu B, Li S, Yao Z, Liang B , et al. Glutamine metabolic microenvironment drives M2 macrophage polarization to mediate trastuzumab resistance in HER2-positive gastric cancer. Cancer communications (London, England) 2023, 43 (8) : 909-937. Zhu Y, Chen X, Lu Y, Xia L, Fan S, Huang Q , et al. Glutamine mitigates murine burn sepsis by supporting macrophage M2 polarization through repressing the SIRT5-mediated desuccinylation of pyruvate dehydrogenase. Burns & trauma 2022, 10: tkac041. Wang B, Zhu L, Jia B, Zhao C, Zhang J, Li F , et al. Sepsis induces non-classic innate immune memory in granulocytes. Cell reports 2023, 42 (9) : 113044. Zhu L, Kong Y, Zhang J, Claxton DF, Ehmann WC, Rybka WB , et al. Blimp-1 impairs T cell function via upregulation of TIGIT and PD-1 in patients with acute myeloid leukemia. Journal of hematology & oncology 2017, 10 (1) : 124. Additional Declarations (Not answered) Supplementary Files FigS1.tif FigS2.tif SupplementalData.docx Cite Share Download PDF Status: Published Journal Publication published 06 Feb, 2025 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 20 Sep, 2024 Review # 1 received at journal 14 Sep, 2024 Review # 2 received at journal 07 Sep, 2024 Reviewer # 2 agreed at journal 29 Aug, 2024 Reviewer # 1 agreed at journal 29 Aug, 2024 Reviewers invited by journal 27 Aug, 2024 Submission checks completed at journal 19 Aug, 2024 Editor assigned by journal 17 Aug, 2024 First submitted to journal 17 Aug, 2024 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. 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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-4903330","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":346038563,"identity":"1879f6bb-6609-426a-8a34-41020c5f802b","order_by":0,"name":"Liuluan Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYDACCQbGAwkMFkAW8wGoUAJBLQxALUCSgQ2mlBgtYJKBx4A4LfKzewwOPGyTSOyf3fPxw8+cwwz87DkGDD934NbCOOeMwYFEoJYZd85uluzddphBsueNAWPvGdxamCVyIFoabuRuY2YEajG4kWPAzNiGWwsbTMv8GznPwFrsCWnhgWnZcCOHDWKLBAEtEhJpBQcSzkkYb7yRZgz0SzqPxJlnBQd78WiRn5G88eGPMhvZeTeSH374uc1ajr89eeODn3i0YLoURBwgQcMoGAWjYBSMAiwAAE5pUq1qDHpkAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9078-2576","institution":"Beijing Ditan Hospital, Capital Medical University","correspondingAuthor":true,"prefix":"","firstName":"Liuluan","middleName":"","lastName":"Zhu","suffix":""},{"id":346038564,"identity":"932d9d54-1d26-429b-92eb-16e7f660bd42","order_by":1,"name":"Rui Li","email":"","orcid":"","institution":"Beijing Ditan Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Li","suffix":""},{"id":346038565,"identity":"1699c205-64f9-4f3a-af6a-0ae498c1a83a","order_by":2,"name":"Qiushi Qin","email":"","orcid":"","institution":"Beijing Ditan Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qiushi","middleName":"","lastName":"Qin","suffix":""},{"id":346038566,"identity":"0f81d062-9682-4887-82ec-9a7a84fcac89","order_by":3,"name":"Wenjuan Peng","email":"","orcid":"","institution":"Beijing Ditan Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenjuan","middleName":"","lastName":"Peng","suffix":""},{"id":346038567,"identity":"051936dc-d761-4aca-8a3a-d405187db044","order_by":4,"name":"Lan Li","email":"","orcid":"","institution":"Beijing Ditan Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lan","middleName":"","lastName":"Li","suffix":""},{"id":346038568,"identity":"4e71abee-16f2-404a-920d-f083dcfe60cf","order_by":5,"name":"Yujia Liu","email":"","orcid":"","institution":"Beijing Ditan Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yujia","middleName":"","lastName":"Liu","suffix":""},{"id":346038569,"identity":"cfac56da-56d3-42cd-8651-c416a6d8b9b3","order_by":6,"name":"Yue Zhang","email":"","orcid":"","institution":"Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-08-13 01:42:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4903330/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4903330/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-07405-6","type":"published","date":"2025-02-06T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":65325724,"identity":"619f3504-c182-4941-8ad2-4c1c059ca102","added_by":"auto","created_at":"2024-09-26 06:04:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3732647,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlimp-1 expression was significantly increased in macrophages in CLP mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e H\u0026amp;E staining of liver and lungs in CLP and sham mice. The liver tissue displayed a disordered lobular structure, swollen and deformed hepatocytes, and dispersed binuclear hepatocytes (red arrow). The alveolar space was widened with inflammatory cells infiltration and substantial leakage of red blood cells (red arrow). Scale bar = 50 μm. \u003cstrong\u003e(B)\u003c/strong\u003e Serum levels of ALT and AST. \u003cstrong\u003e(C)\u003c/strong\u003e Serum levels of inflammatory cytokines. \u003cstrong\u003e(D)\u003c/strong\u003e Percentage of macrophages in peritoneal lavage fluid, assessed by flow cytometry. \u003cstrong\u003e(E)\u003c/strong\u003e Geometric mean of M1 (CD86\u003csup\u003e+\u003c/sup\u003e) and M2 (CD206\u003csup\u003e+\u003c/sup\u003e) macrophages in peritoneal lavage fluid, assessed by flow cytometry. \u0026nbsp;\u003cstrong\u003e(F)\u003c/strong\u003e Geometric mean of Blimp-1 in macrophages with different polarization phenotypes in peritoneal lavage fluid, analyzed by flow cytometry. \u003cstrong\u003e(G)\u003c/strong\u003e The mRNA levels of \u003cem\u003eBlimp-1\u003c/em\u003e in peritoneal lavage fluid cells, measured by real-time PCR. \u003cstrong\u003e(H)\u003c/strong\u003e Geometric mean of Blimp-1 in CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, B cells, and macrophages in the peritoneal lavage fluid of mice. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4903330/v1/06ca637ac5acbd6bb46dd80a.png"},{"id":65326729,"identity":"81769a16-fac4-432e-99ba-fd8aa6ef9cb7","added_by":"auto","created_at":"2024-09-26 06:12:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3907277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eShRNA-Blimp-1 aggravated multiple organ damages in CLP mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Diagram of CLP mouse model construction and experiment timeline. \u003cstrong\u003e(B)\u003c/strong\u003e The survival rates of CLP mice intraperitoneally injected with adeno-associated virus (AAV) carrying shRNA-NC or shRNA-Blimp-1, respectively (n = 10 per group). \u003cstrong\u003e(C)\u003c/strong\u003e H\u0026amp;E staining of liver and lungs in mice. Scale bar = 50 μm. Red arrows indicate hepatic lobular structure damage, inflammatory cell infiltration and aggregation, and hepatocyte swelling and deformation in the liver. Blue arrows indicate destroyed alveolar cavity with inflammatory cell infiltration and congestion in the lungs. \u003cstrong\u003e(D)\u003c/strong\u003e Percentage of total macrophages and M2 macrophages in peritoneal lavage fluid. \u003cstrong\u003e(E)\u003c/strong\u003e Geometric mean of CD206 in macrophages from peritoneal lavage fluid. \u003cstrong\u003e(F)\u003c/strong\u003e Percent of CD206\u003csup\u003e+\u003c/sup\u003eBlimp-1\u003csup\u003e+\u003c/sup\u003e macrophages in peritoneal lavage fluid. \u003cstrong\u003e(G)\u003c/strong\u003e Geometric mean of Blimp-1 in CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, and B cells from peritoneal lavage fluid. \u003cstrong\u003e(H)\u003c/strong\u003e Geometric mean of Blimp-1 in CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, and B cells in the spleen. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4903330/v1/00620beb7572045d3a1b56b4.png"},{"id":65325720,"identity":"cbc8d40e-dc3f-436e-8537-051348eb3fb9","added_by":"auto","created_at":"2024-09-26 06:04:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3237825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of Blimp-1 promoted macrophage differentiation and M2 polarization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Diagram of experimental process of bone marrow-derived macrophage (BMDM) differentiation and polarization. \u003cstrong\u003e(B)\u003c/strong\u003eGeometric mean of Blimp-1 inB cells, granulocytes, and monocytes from bone marrow of naïve mice. \u003cstrong\u003e(C)\u003c/strong\u003eThe mRNA levels of \u003cem\u003eBlimp-1\u003c/em\u003e during BMDM differentiation. \u003cstrong\u003e(D)\u003c/strong\u003e The mRNA levels of \u003cem\u003eBlimp-1\u003c/em\u003e in different polarization types of BMDMs. \u003cstrong\u003e(E)\u003c/strong\u003e Morphology of THP-1 cells, differentiated THP-1 cells (dTHP-1), and differentiated THP-1 cells with Blimp-1 overexpression (dTHP-1 + bLIMP-1). Scale bar = 20 μm. \u003cstrong\u003e(F)\u003c/strong\u003eThe mRNA levels of \u003cem\u003eBlimp-1\u003c/em\u003e in dTHP-1 cells transfected with \u003cem\u003eBlimp-1\u003c/em\u003e plasmid or vehicle. \u003cstrong\u003e(G)\u003c/strong\u003e Geometric mean of CD206 in dTHP-1 cells with Blimp-1 overexpression. \u003cstrong\u003e(H)\u003c/strong\u003e The mRNA levels of \u003cem\u003eMRC1, MSR1, ARG1\u003c/em\u003e and\u003cem\u003e IL10\u003c/em\u003ein dTHP-1 cells with Blimp-1 overexpression. \u003cstrong\u003e(I)\u003c/strong\u003e The mRNA levels of \u003cem\u003eBlimp-1\u003c/em\u003e in RAW264.7 cells transfected with \u003cem\u003eBlimp-1\u003c/em\u003e plasmid or vehicle. \u003cstrong\u003e(J)\u003c/strong\u003eMorphology of RAW264.7 cells. Scale bar = 10 μm. \u003cstrong\u003e(K)\u003c/strong\u003e The mRNA levels of M2 marker genes in RAW264.7 cells. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4903330/v1/802e890ec231a7cc9608ce87.png"},{"id":65326731,"identity":"dc896d10-65f6-439d-9f69-450b8f2073f5","added_by":"auto","created_at":"2024-09-26 06:12:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1568286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eShRNA-Blimp-1 attenuated M2 macrophage polarization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Diagram of inducing polarization of RAW264.7 cells. \u003cstrong\u003e(B)\u003c/strong\u003e The mRNA levels of \u003cem\u003eBlimp-1\u003c/em\u003e in macrophages infected with lentivirus carrying shRNA-NC or shRNA-Blimp-1. \u003cstrong\u003e(C)\u003c/strong\u003e Percentages of M1 and M2 macrophages. \u003cstrong\u003e(D)\u003c/strong\u003e The mRNA levels of \u003cem\u003eMrc1, Msr1 \u003c/em\u003eand\u003cem\u003e Arg1 \u003c/em\u003ein M0 and M2 macrophages. \u003cstrong\u003e(E)\u003c/strong\u003eThe mRNA levels of \u003cem\u003eIl12b, Nos2 \u003c/em\u003eand\u003cem\u003e Tnfa \u003c/em\u003ein M0 and M1 macrophages. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4903330/v1/0d9dd88f67ecab235be1df6d.png"},{"id":65325725,"identity":"cf9852b8-2ff6-4749-8f34-eec247c9eca5","added_by":"auto","created_at":"2024-09-26 06:04:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":556040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlimp-1 regulated energy metabolism and metabolic marker expressions in M2 macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The mRNA levels of \u003cem\u003ePparg, Ppargc1b, Tkt, Gatm \u003c/em\u003eand\u003cem\u003e Gpt2 \u003c/em\u003ein M2 macrophages infected with lentivirus carrying shRNA-NC or shRNA-Blimp-1. \u003cstrong\u003e(B)\u003c/strong\u003e The mRNA levels of \u003cem\u003ePparg, Ppargc1b, Tkt, Gatm \u003c/em\u003eand\u003cem\u003e Gpt2 \u003c/em\u003ein RAW264.7 macrophages transfected with or without \u003cem\u003eBlimp-1\u003c/em\u003eexpressing plasmid. \u003cstrong\u003e(C)\u003c/strong\u003e Oxygen consumption rate (OCR) curve of the Seahorse Mito Stress Test in M2 macrophages infected with lentivirus carrying shRNA-NC or shRNA-Blimp-1. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of OCR parameters in M2 macrophages. \u003cstrong\u003e(E) \u003c/strong\u003eExtracellular acidification rate (ECAR) curve of the Seahorse Glycolytic Test in M2 macrophages infected with lentivirus carrying shRNA-NC or shRNA-Blimp-1. \u003cstrong\u003e(F)\u003c/strong\u003e Quantification of ECAR parameters in M2 macrophages. FCCP: Trifluoromethoxy carbonyl cyanide phenylhydrazone; Rot: rotenone; AA: antimycin A; 2-DG: 2-Deoxy-d-Glucose. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4903330/v1/a9ab4a456618cc8b73b354a5.png"},{"id":65325729,"identity":"e60aee9b-3a8d-421a-bddf-9ec9eb1ba88d","added_by":"auto","created_at":"2024-09-26 06:04:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2568593,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eShRNA-Blimp-1 altered metabolome in M2 macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAW264.7 cells were induced by IL-4 and IL-13 to polarize towards M2 macrophages, followed by infection with lentivirus carrying shRNA-NC or shRNA-Blimp-1. \u003cstrong\u003e(A)\u003c/strong\u003e Classification of the 121 metabolites identified in non-targeted metabolome analysis. \u003cstrong\u003e(B)\u003c/strong\u003e OPLS-DA Model Discrimination for non-targeted metabolome. \u003cstrong\u003e(C)\u003c/strong\u003eHeatmap of metabolites for quantitative identification in the non-targeted metabolome. The color scale indicates the level of metabolite accumulation, while the class indicates the metabolites. \u003cstrong\u003e(D)\u003c/strong\u003e Variable importance in projection (VIP) scores comparing shRNA-Blimp-1 and shRNA-NC groups in non-targeted metabolome analysis. VIP ≥ 1.0, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003cstrong\u003e (E)\u003c/strong\u003e OPLS-DA Model Discrimination for nucleotide-targeted metabolome.\u003cstrong\u003e (F)\u003c/strong\u003e OPLS-DA Model Discrimination for amino acid-targeted metabolome.\u003cstrong\u003e(G)\u003c/strong\u003e SMPDB metabolic pathway enrichment analysis of the differential metabolites in the nucleotide-targeted metabolome (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003e(H)\u003c/strong\u003e KEGG metabolic pathway enrichment analysis of the differential metabolites in the amino acid-targeted metabolome (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4903330/v1/a57bb0dd6e658f69bda9e522.png"},{"id":65326730,"identity":"2e0ae66c-a87f-40cc-8111-85eeab6ef107","added_by":"auto","created_at":"2024-09-26 06:12:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1909162,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlimp-1 regulated purine biosynthesis and the Ornithine cycle in M2 macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAW264.7 cells were induced by IL-4 and IL-13 to polarize towards M2 macrophages, followed by infection with lentivirus carrying shRNA-NC or shRNA-Blimp-1. \u003cstrong\u003e(A)\u003c/strong\u003e Differential abundance of metabolites in the purine biosynthesis pathway. \u003cstrong\u003e(B)\u003c/strong\u003e The mRNA levels of key metabolic enzymes in purine biosynthesis pathway. \u003cstrong\u003e(C)\u003c/strong\u003e Differential abundance of metabolites in the Ornithine cycle. \u003cstrong\u003e(D)\u003c/strong\u003e The mRNA levels of key metabolic enzymes in the Ornithine cycle. \u003cstrong\u003e(E)\u003c/strong\u003e Differential abundance of nucleotides GTP, GDP, ATP, and ADP. \u003cstrong\u003e(F)\u003c/strong\u003e Dual-luciferase reporter assay of metabolic enzyme gene promoters in 293T cells. The cells were transfected with \u003cem\u003eBlimp-1\u003c/em\u003e expressing plasmid or the vehicle, along with reporters of \u003cem\u003ePRPS1\u003c/em\u003e, \u003cem\u003ePPAT\u003c/em\u003e, \u003cem\u003ePNP\u003c/em\u003e, \u003cem\u003eGDA\u003c/em\u003e, \u003cem\u003eXDH\u003c/em\u003e, \u003cem\u003eAPRT\u003c/em\u003e, \u003cem\u003eASS1\u003c/em\u003e, \u003cem\u003eASL\u003c/em\u003e, and \u003cem\u003eAMS\u003c/em\u003e promoters, respectively. \u003cstrong\u003e(G)\u003c/strong\u003e Diagram of the differential metabolic pathway in M2 macrophages. Green indicates enzymes with upregulated expression by Blimp-1; yellow indicates metabolites with increased abundance by Blimp-1. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4903330/v1/49a7a2d8c5a7d93f07e63cb0.png"},{"id":65326734,"identity":"fab383d6-49f1-4450-bf61-5daec0ae2a7f","added_by":"auto","created_at":"2024-09-26 06:12:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1193097,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA proposed schematic diagram of the regulatory mechanism of Blimp-1 on macrophage metabolism–polarization–function and its protective effect in sepsis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen the body is suffered from polymicrobial infection, the\u003cstrong\u003e \u003c/strong\u003eBlimp-1 expression in macrophages will be enhanced and resulted in promoting M2 polarization, as well as invigorating purine biosynthesis and the downstream Ornithine cycle through Glutamine pathway, which furtherly suppressed secretion of the inflammatory cytokine, and finally protecting the organs (liver and lung) from damage in sepsis.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-4903330/v1/b22ddcefeec6380e9fba59e7.png"},{"id":75695238,"identity":"98259f2f-854d-4215-add7-e09fe3c76f2a","added_by":"auto","created_at":"2025-02-07 08:08:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29579443,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4903330/v1/fa8249fa-1996-4254-9535-1252be5457aa.pdf"},{"id":65326909,"identity":"791ef232-d46d-481d-b2ac-d95d6f1f9d80","added_by":"auto","created_at":"2024-09-26 06:20:15","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2461656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4903330/v1/27b1c012d8b06673a9a3c891.tif"},{"id":65326733,"identity":"d9cc777f-a956-4156-92d5-2e8e3b0392e2","added_by":"auto","created_at":"2024-09-26 06:12:15","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1767080,"visible":true,"origin":"","legend":"","description":"","filename":"FigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4903330/v1/d32da04f9612dc15b5ffa44a.tif"},{"id":65325721,"identity":"8700caf3-0f32-46d5-bde4-8b710d874475","added_by":"auto","created_at":"2024-09-26 06:04:14","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":24652,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalData.docx","url":"https://assets-eu.researchsquare.com/files/rs-4903330/v1/5c50f9a0cb3b14469b1c951a.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Blimp-1 Orchestrates Macrophage Polarization and Metabolic Homeostasis via Purine Biosynthesis in Sepsis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSepsis is a life-threatening condition characterized by organ dysfunction resulting from a dysregulated host response to infection \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and responsible for about 11\u0026nbsp;million deaths annually, accounting for nearly 20% of global mortality \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The pathogenesis of sepsis is multifaceted, involving intricate interactions between infectious microorganisms and the host, alongside various pathways including infection, inflammation, hypoxia, and metabolic reprogramming \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. A critical mechanism underlying sepsis is the imbalance of inflammatory response, which significantly influences its progression \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Therefore, early intervention in inflammatory response disorders may be effective in reducing sepsis-related mortality.\u003c/p\u003e \u003cp\u003eMacrophages play a pivotal role in mediating inflammatory responses and primarily contain two functional subsets: pro-inflammatory (M1) and anti-inflammatory (M2) macrophages. M1 macrophages are characterized by producing pro-inflammatory cytokines, or mediators such as TNF-α, IL-1α/β, IL-6, IL-12, and iNOS \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. M2 macrophages express arginase-1 (ARG1) and secrete anti-inflammatory cytokines IL-10 and TGF-β. In sepsis, excessive activation of M1 macrophages can exacerbate inflammation and organ damage, while M2 macrophages promote the resolution of inflammation, tissue repair, and organ recovery. Given the critical role of the M1/M2 polarization in sepsis \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, understanding the regulatory mechanisms governing macrophage polarization is essential for elucidating sepsis pathology.\u003c/p\u003e \u003cp\u003eB lymphocyte-induced maturation protein-1 (Blimp-1) is a transcription suppressor of IFN-β encoded by the \u003cem\u003ePrdm1\u003c/em\u003e gene \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Overexpression of Blimp-1 in pro-monocytic cells induces partial macrophage differentiation, characterized by the expression of surface markers such as CD11b and CD11c \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Blimp-1 was identified as a marker for a specific macrophage population located near the microbe-exposed surface in the colon \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Recent studies have shown CCL8 is a direct target of Blimp-1 in mononuclear macrophages, with elevated CCL8 levels observed in \u003cem\u003ePrdm1\u003c/em\u003e\u003csup\u003efl\u003c/sup\u003e/\u003csup\u003efl\u003c/sup\u003e mice \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Our previous study claimed Blimp-1 represses the production of several inflammatory cytokines, including IL-1β, IL-6, and IL-18, by directly binding to the genomic region and restricting the nuclear translocation and transcriptional activity of NF-κB \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, the potential regulatory role of Blimp-1 in macrophage polarization and its significance in sepsis represent a critical gap in current understanding.\u003c/p\u003e \u003cp\u003eEmerging evidence indicates energy metabolism plays a crucial role in regulating macrophage polarization and function \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Generally, pro-inflammatory M1 macrophages primarily rely on glycolysis to generate energy for innate immune responses, while M2 macrophages depend more on mitochondrial oxidative phosphorylation and the tricarboxylic acid cycle to satisfy their energy requirements \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. During M2 polarization, transcription factors such as PPARγ and PGC-1β are critical for mitochondrial respiration and fatty acid oxidative metabolism \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Additionally, transketolase (TKT) is important for glycometabolism \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, while glutamate-pyruvate transaminase (GPT) and glycine amidinotransferase (GATM) are involved in amino acid metabolism \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Notably, Blimp-1 may participate in cellular metabolism, thereby modulating inflammatory marker expression in quiescent macrophages \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. For instance, Blimp-1 regulates IL-10 expression in group 2 innate lymphoid cells (ILC2s), which exhibit a metabolic dependence on glycolysis for IL-10 production \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These findings suggest that Blimp-1 may regulate macrophage polarization through cellular metabolic pathways, thus influencing inflammatory responses in sepsis.\u003c/p\u003e \u003cp\u003eIn this study, we aim to elucidate the effects of Blimp-1 on macrophage polarization in sepsis from the perspective of cellular metabolism. We inhibited Blimp-1 expression and measured its effects on macrophage polarization phenotypes and functions both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Subsequently, we utilized the metabolomics technology to explore the metabolic mechanisms by which Blimp-1 regulates macrophage polarization. We conducted dual-luciferase reporter assay to validate the metabolic pathways regulated by Blimp-1 in macrophages. In summary, our findings clarify a previously unknown mechanism by which Blimp-1 plays a key role in orchestrating macrophage polarization during sepsis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBlimp-1 expression is elevated in sepsis-associated macrophages\u003c/h2\u003e \u003cp\u003eWe had sucessfully established a cecal ligation and puncture (CLP) sepsis model in C57BL/6J mice. Compared to sham-operated controls, CLP mice exhibited significant histopathological lesions in liver and lungs, along with increased serum levels of hepatic injury biomarkers and inflammatory cytokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Importantly, the peritoneal lavage fluid of CLP mice contained a higher percentage of macrophages, with Blimp-1 expression specifically elevated in the M2 subset (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The mRNA level of \u003cem\u003eBlimp-1\u003c/em\u003e was also significantly increased in these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). This selective upregulation of Blimp-1 in M2 macrophages, but not in CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, or B cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA \u0026amp; 1B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBlimp-1 knockdown exacerbates sepsis by impairing M2 polarization\u003c/h2\u003e \u003cp\u003eTo elucidate the functional significance of Blimp-1 upregulation, we knocked down its expression using shRNA-Blimp-1 and assessed the impact on CLP mice (as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Three weeks post-injection, the survival rate of CLP mice was significantly lower in the Blimp-1 knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Histopathological analysis revealed exacerbated liver and lung tissue damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These findings underscore the protective role of Blimp-1 in modulating the inflammatory response and tissue repair during sepsis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing shRNA-Blimp-1 intervention, the peritoneal lavage fluid of CLP mice exhibited an elevated total macrophage count, yet a diminished proportion of M2 macrophages, as evidenced by reduced presence of CD206\u003csup\u003e+\u003c/sup\u003e and CD206\u003csup\u003e+\u003c/sup\u003eBlimp-1\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). In contrast, Blimp-1 expression in other immune cells, such as CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, and B cells, remained unaffected in both peritoneal lavage fluid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) and spleen grinding fluid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). These observations suggest that Blimp-1's protective effect in sepsis may depend on its regulatory role in M2 macrophage polarization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBlimp-1 promotes monocyte-macrophage differentiation and M2 polarization\u003c/h2\u003e \u003cp\u003eWe next assessed the temporal expression of Blimp-1 during macrophage differentiation and polarization from bone marrow cells of na\u0026iuml;ve mice (as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Blimp-1 expression was significantly higher in monocytes compared to B cells and granulocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and its mRNA levels progressively increased alongside bone marrow-derived macrophage (BMDM) differentiation and maturation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Upon polarization, M1 macrophages exhibited high secretion of IL-1β, IL-6, TNF-α, and IL-12 (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA); whereas M2 macrophages secreted high levels of TGF-β, CCL17, and CCL22 (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Notably, \u003cem\u003eBlimp-1\u003c/em\u003e mRNA levels were specifically elevated in M2 macrophages compared to M0 and M1 counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), indicating a potential role for Blimp-1 in the differentiation and M2 polarization of macrophages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further dissect Blimp-1's influence on macrophage polarization, THP-1 monocytic cells were induced to differentiate into macrophages and subsequently transfected with a \u003cem\u003eBlimp-1\u003c/em\u003e expressing plasmid. The expression level of Blimp-1 was identified by real-time fluorescent quantitative PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Overexpression of Blimp-1 causes morphological changes consistent with M2 macrophages, including the appearance of extended pseudopods (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), and a significant increase in CD206 expression, as well as increased mRNA levels of M2 markers such as \u003cem\u003eMRC1, MSR1, ARG1\u003c/em\u003e and \u003cem\u003eIL10\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Similar results were obtained with RAW264.7 macrophages overexpressing Blimp-1, where an increase in mRNA levels of \u003cem\u003eMsr1, Mrc1, Arg1, Ppargc1b, Il10, Ido1, Pdl1\u003c/em\u003e, and \u003cem\u003ePdl2\u003c/em\u003e was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). These findings collectively highlight the critical role of Blimp-1 in driving macrophage differentiation and the acquisition of the M2 phenotype.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBlimp-1 is essential for energy metabolism in M2 macrophages\u003c/h2\u003e \u003cp\u003eFurther investigation of Blimp-1's regulatory role in macrophage polarization was conducted by knocking down its expression in RAW264.7 cell (as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Compared to shRNA-NC control, shRNA-Blimp-1 effectively reduced Blimp-1 expression, particularly in M2 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This knockdown resulted in a significant decrease in the proportion of M2 (CD206\u003csup\u003e+\u003c/sup\u003e) macrophages and a dramatic reduction in the mRNA levels of M2 markers \u003cem\u003eMrc1, Msr1\u003c/em\u003e, and \u003cem\u003eArg1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC \u0026amp; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In contrast, the proportion of M1 (CD86\u003csup\u003e+\u003c/sup\u003e) macrophages and the mRNA levels of M1 markers \u003cem\u003eIl12b, Nos2\u003c/em\u003e, and \u003cem\u003eTnfa\u003c/em\u003e remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC \u0026amp; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), underscoring the specific role of Blimp-1 in M2 macrophage polarization and function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also examined the expression of genes pivotal to mitochondrial respiration, fatty acid oxidative metabolism, and glycometabolism. The robust expression levels of \u003cem\u003ePparg, Ppargc1b, Tkt, Gatm\u003c/em\u003e, and \u003cem\u003eGpt2\u003c/em\u003e in M2 macrophages, which were notably diminished following Blimp-1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Conversely, overexpression of Blimp-1 in RAW264.7 macrophages resulted in elevated mRNA levels for these genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Employing the Seahorse XF analyzer, we conducted metabolic assays substantiated the impact of Blimp-1 on mitochondrial function. The knockdown of Blimp-1 corresponded with a reduction in mitochondrial oxygen consumption rates (OCRs) in M2 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This reduction is indicative of a decrease in the maximal respiration, mitochondrial ATP production, and spare respiratory capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Furthermore, the extracellular acidification rate (ECAR), a measure of glycolytic activity, was diminished after shRNA-Blimp-1 intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), reflecting lower basal and maximum glycolysis levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Collectively, these findings underscore the capacity of Blimp-1 to facilitate M2 macrophage polarization through the modulation of key metabolic enzyme expressions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBlimp-1 modulates the metabolome of M2 macrophages\u003c/h2\u003e \u003cp\u003eWe furtherly conducted a non-targeted metabolomics analysis on RAW264.7 cells with Blimp-1 knockdown and identified a total of 121 metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). An Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) clearly distinguished the metabolite profiles between the shRNA-Blimp-1 and shRNA-NC groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Differential metabolite analysis revealed significant differences in amino acids and nucleotides between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The differential metabolites were displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD \u0026amp; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, with those exceeding a importance in projection (VIP) scores score of 1 considered significant. Notably, the levels of Ornithine and multi-amino acids increased following Blimp-1 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, below panel).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we conducted a quantitative metabolomics analysis targeting nucleotide metabolism and amino acid metabolism and identified 9 differential nucleotide metabolites and 22 differential amino acid metabolites between groups (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). OPLS-DA clearly distinguished the nucleotide and amino acid metabolite of shRNA-Blimp-1 and shRNA-NC groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE \u0026amp; \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Metabolic pathway enrichment analysis using the Small Molecule Blimp-1 knockdown significantly impacted GDP exchange, UDP exchange and ATP diphosphate hydrolase in M2 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG), as well as ammonia recycling, glutamine metabolism, urea cycle and glycine and serine metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). These findings suggest that Blimp-1 influences the metabolic pathways of nucleotides and amino acids in M2 macrophages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBlimp-1 enhances purine biosynthesis and the downstream Ornithine cycle in M2 macrophages\u003c/h2\u003e \u003cp\u003eBuilding on the differential metabolite analysis, we concentrated on purine biosynthesis, nucleotide metabolism, and the Ornithine cycle. Blimp-1 knockdown led to a down-regulation of most metabolites involved in purine biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The expression levels of genes encoding key enzymes in this pathway, such as \u003cem\u003ePpat\u003c/em\u003e, \u003cem\u003ePnp\u003c/em\u003e, \u003cem\u003eGda\u003c/em\u003e, and \u003cem\u003eXdh\u003c/em\u003e, were significantly reduced with Blimp-1 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Following Blimp-1 knockdown, the content of metabolites in the Ornithine cycle and the mRNA levels of key enzymes were significantly diminished (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC \u0026amp; \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Additionally, the concentrations of nucleotide metabolites GDP, GTP, ATP, and ADP were markedly reduced (Fig .7E). Blimp-1 directly enhances the transcriptional activity of promoters for \u003cem\u003ePPAT\u003c/em\u003e, \u003cem\u003eGDA\u003c/em\u003e, \u003cem\u003eXDH\u003c/em\u003e, \u003cem\u003eAPRT\u003c/em\u003e, \u003cem\u003eASS1\u003c/em\u003e, \u003cem\u003eASL\u003c/em\u003e, and \u003cem\u003eSMA\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Collectively, these results demonstrated that Blimp-1 regulates purine biosynthesis and the downstream Ornithine cycle by promoting the transcription of key enzyme genes in M2 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eA central component of sepsis pathophysiology is the polarization of macrophages. Macrophages undergo distinct polarization states (M1 and M2) that significantly influence sepsis outcomes. Nrf2 has been shown to play a protective role in sepsis-induced pulmonary injury and inflammation by modulating autophagy and NF-κB/PPARγ-mediated macrophage polarization \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Similarly, Kr\u0026uuml;ppel-like transcription factors reduce macrophage glycolysis and inflammatory cytokine secretion by suppressing the M1 macrophage polarized phenotype \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Our recent work revealed that Blimp-1 inhibits the secretion of inflammatory cytokines \u003cem\u003evia\u003c/em\u003e multiple toll-like receptors \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, which elucidate a novel role for Blimp-1 in macrophage polarization during sepsis.\u003c/p\u003e \u003cp\u003eMacrophage metabolic remodeling is essential for adapting to different polarized states and their functional changes \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The polarization of M2 macrophages involves alterations in several metabolic pathways, including glucose, amino acid, fatty acid, and nucleotide metabolism. Specifically, in fatty acid metabolism, PGC-1β collaborates with PPARγ to inhibit the secretion of inflammatory factors in M1 macrophages and to promote the M2 polarization \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The mechanism may involve PPARγ and PGC-1β directly regulating ARG1 expression, with their activation playing a crucial role in mitochondrial respiration and fatty acid oxidation in M2 macrophages \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn glucose metabolism, TKT, a key enzyme in the pentose phosphate pathway downstream of D-fructose-1.6 diphosphate, is upregulated in M1 macrophages along with glycolysis-related enzymes; however, this does not affect PPAT-related purine synthesis or cell proliferation \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In amino acid metabolism, GATM and GPT2, which are involved in the glutamate pathway, facilitate glutaminolysis, leading to α-ketoglutarate accumulation and M2 polarization \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In our study, Blimp-1 suppression led to a significant reduction in the mRNA expression of \u003cem\u003ePparg, Ppargc1b, Tkt, Gatm\u003c/em\u003e, and \u003cem\u003eGpt2\u003c/em\u003e in M2 macrophages, while Blimp-1 overexpression resulted in a marked increase in these genes. These findings suggest that Blimp-1 regulates M2 macrophage polarization by modulating key metabolic enzyme expressions.\u003c/p\u003e \u003cp\u003eEnhanced glycolysis during M2 macrophage polarization provides a metabolic basis for improved mitochondrial oxidative phosphorylation. M2 macrophages sustain their metabolism primarily through the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation, resulting a more robust oxidative metabolic profile compared to M1 macrophages \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Our results demonstrated that Blimp-1 knockdown led to substantial inhibition of both basal respiration and maximum respiratory capacity in mitochondrial oxidative phosphorylation, as well as glycolytic function. These results confirm that Blimp-1 regulates both mitochondrial oxidative phosphorylation and glycolysis in macrophages.\u003c/p\u003e \u003cp\u003eOur results claimed that Blimp-1 enhances the transcription of PPAT, ASS1, and ASL, affecting purine biosynthesis and the Ornithine cycle in M2 macrophages. Previous studies have linked the Ornithine cycle with M2 polarization \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Notably, the expression of ARG1, a marker of M2 macrophages, is associated with the Ornithine cycle, while citrulline consumption is indicative of M1 polarization \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Although research on purine biosynthesis regulation in macrophage polarization is limited, both purine metabolism and the Ornithine cycle depend on glutamine, a star amino acid that regulates macrophage function \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Glutamine contributes α-ketoglutaric acid to the tricarboxylic acid cycle and facilitates acetylation modification of histones through acetyl-CoA, activating α-ketoglutaric acid-dependent epigenetic regulation and thereby influencing M2 macrophage polarization \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Actually, glutamine is the hub of purine biosynthesis and Ornithine cycle, connecting the metabolism of two kinds of biological macromolecules. Our study not only confirmed the regulatory mechanism of glutamine metabolism by Blimp-1, but also more comprehensively mapped the characteristics of glutamine-centered purine biosynthesis and Ornithine cycle metabolism in M2 macrophages. By elucidating the transcriptional regulation mechanism of Blimp-1 on various metabolic enzymes, this study revealed the metabolic regulation of M2 polarization and the pathological mechanism of sepsis progression.\u003c/p\u003e \u003cp\u003eWhile our study provides valuable insights into the role of Blimp-1 in sepsis pathogenesis, several limitations warrant consideration. First, the study predominantly utilized animal models, necessitating validation in human samples. Second, a mouse model of Blimp-1 conditional knockout in macrophages is needed to elucidate the exact mechanism of Blimp-1 for metabolic reprogramming, including detailed dynamics and interactions with other regulatory pathways. Third, due to the lack of suitable ChIP antibodies, the direct binding site and strength of Blimp-1 to target gene promoters cannot be evaluated \u003cem\u003ein vivo\u003c/em\u003e. Additionally, longitudinal studies are needed to assess the long-term effects of Blimp-1 modulation on immune function and host recovery in sepsis.\u003c/p\u003e \u003cp\u003eIn conclusion, our study unveils a novel regulatory role for Blimp-1 in macrophage polarization during sepsis, mediated through modulation of purine biosynthesis and the Ornithine cycle. These findings expand our understanding of the complex interplay between metabolism and immune responses in sepsis pathology.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCulture of BMDMs\u003c/h2\u003e \u003cp\u003eBone marrow cells were isolated from C57BL/6 male mice aged 6\u0026ndash;8 weeks, re-suspended with DMEM complete culture medium, and uniformly planted on 10-cm petri dishes. Cells were cultured in DMEM complete culture medium containing 10 ng/mL M-CSF for 7 days to obtain adherent growth of M0 macrophages. Then, 100 ng/mL LPS and 40 ng/mL INF-γ were used to induce 2-day polarization into M1 macrophages, or 40 ng/mL IL-4 and 20 ng/mL IL-13 were used to induce 2-day polarization into M2 macrophages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatment\u003c/h2\u003e \u003cp\u003eRAW264.7 and 293T cells were cultured with DMEM medium (Gibco, USA) supplemented with 10% FBS and 1% penicillin/streptomycin solution (Gibco, USA). THP-1 cells were cultured and induced to differentiate into M0 macrophages by 50 ng/mL PMA (P8139, Sigma-Aldrich) for 48 h. Differentiated THP-1 or RAW264.7 cells were transiently transfected with \u003cem\u003eBlimp-1\u003c/em\u003e expressing plasmid using Lipofectamine 3000 Transfection Reagent (L3000150, Thermo Fisher Scientific, USA) for 48 h. The \u003cem\u003eBlimp-1\u003c/em\u003e shRNA and a non-specific control (NC) shRNA were integrated into the lentiviral vector pWSLV-Sh08-GFP-Puro (Noweton Bioscience, China), and the lentiviral particles infected RAW264.7 cells or BMDMs as previously described \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMice, sepsis model and\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003eintervention\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMale C57BL/6 mice (8 weeks old) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Science (Beijing, China). The sepsis model was established by CLP procedure as previously described \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Blimp-1 shRNA and control shRNA were integrated into the vector pAAV-U6-shRNA-CMV bGlobin-eGFP-3Flag (Shanghai Genechem Company, China). Mice were intraperitoneally injected with AAV particles carrying shRNA-Blimp-1 or shRNA-NC at a titer of 5.00E\u0026thinsp;+\u0026thinsp;11 v.g/mL, and fed for three weeks before CLP operation. The shRNA-Blimp-1 and shRNA-NC oligonucleotides (5\u0026rsquo;-3\u0026rsquo;) refer to our previous report \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSplenic cell isolation\u003c/h2\u003e \u003cp\u003eSpleen was excised and minced in PBS. Minced spleen suspension was filtered through a 70-\u0026micro;m filter, spun at 1200 rpm for 5 min. The cell precipitation was collected and centrifuged at 1200 rpm for 5 min. The cell precipitates were collected and split in RBC lysate (BioLegend, USA) for 3 min and then rotated at 1200 rpm with PBS for 5 min. The supernatant was discarded and washed again with PBS, and an appropriate number of cells were resuspended in the FACS buffer for flow cytometry staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePeritoneal lavage fluid extraction\u003c/h2\u003e \u003cp\u003eThe abdominal skin of mice was cut open, the peritoneum was exposed, the abdominal cavity was irrigated with 5 mL 0.9% NaCl. The normal saline was sucked out after full shaking for 90 sec, the supernatant was rotated at 1200 rpm for 5 min and the supernatant was discarded. The cells were resuspended with FACS buffer and stained for flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eCells were washed and collected by centrifuging at 1200 rpm for 5 min, and resuspended with FACS buffer. The following mAbs were used: Blimp-1-PE (BD Biosciences, USA), CD86-PE-Cy7 (Tonbo Bioscience San Diego, USA), CD45-APC-Cy7 (BioLegend, USA), F4/80-FITC/APC (eBioscience, USA), CD11b-BV510 (BD Biosciences), CD3- eFluor450 (BD Biosciences), CD19-PE-Cy7 (BD Biosciences), CD8-FITC (eBioscience), CD4-Percp (eBioscience), CD206-APC/BV421 (BioLegend), Gr-1-FITC (BD Biosciences), CD48-Percp (Elabscience, USA). Data acquisition and analysis were performed using a CantoII flow cytometer (BD Biosciences) and FlowJo Software (version 10.1; Tree Star, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMultiple cytokine assay\u003c/h2\u003e \u003cp\u003eCytokines in the BMDM culture supernatant and mouse serum were detected using LEGENDplexTM MU Macrophage/Microglia Panel (13-plex) w/VbP Reagent (#740846, BioLegend). The data analysis and processing were carried out on the BioLegend website.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSerum alanine aminotransferase and aspartate aminotransferase measurement\u003c/h2\u003e \u003cp\u003eMouse serum samples were collected and the activities of serum aspartate transaminase (AST) and alanine transaminase (ALT) were determined using an automatic analyzer (Model 7600 Series, Hitachi, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin-eosin (H\u0026amp;E) staining\u003c/h2\u003e \u003cp\u003eAfter the mice were sacrificed, liver and lung samples were separated and fixed with 4% paraformaldehyde for 24 h, subsequently embedded in paraffin, sliced (4\u0026ndash;5 \u0026micro;m). Histologic sections of tissues were stained with H\u0026amp;E staining analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation, reverse transcription, and realtime PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted with Total RNA Kit (Omega Biotek, China) and reverse transcribed using the PrimeScript\u0026trade; RT reagent Kit (TaKaRa, Japan). Amplification was performed using the Power SYBR\u0026reg; Green PCR Master Mix (Thermo Fisher Scientific, USA). The relative expression level of each transcript was normalized to murine GAPDH. The primers were listed in Table S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction, transfection, and dual-luciferase assay\u003c/h2\u003e \u003cp\u003eThe promoter luciferase reporters for PRPS1, PPAT, PNP, GDA, XDH, APRT, ASS1, ASL and SMS were constructed using pGL3-basic-Luc-wt plasmid. \u003cem\u003eBlimp-1\u003c/em\u003e cDNA was cloned into pcDNA3.1\u0026thinsp;+\u0026thinsp;plasmid as previous report \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The promoter luciferase reporter, \u003cem\u003eBlimp-1\u003c/em\u003e expressing plasmid, and pRL-TK internal reference were transfected into 293T cells with PEI 40K Transfection Reagent (Servicebio, China) for 4\u0026ndash;6 h. After 24 h, cell lysate was collected and detected using the Dual-Luciferase\u0026reg; Reporter Assay System (Promega).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eSeahorse XF glycolytic stress and mitochondrial stress test\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eMitochondrial respiratory capacity and glycolytic function of M2 macrophages were measures using Seahorse XF Glycolysis Stress Test Kit (#103020-100, Agilent, USA) and Seahorse XF Cell Mito Stress Test Kit (#103015-100, Agilent) on a Seahorse XFe24 Analyzer (Agilent).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eNon-targeted and targeted metabolomics analysis\u003c/h2\u003e \u003cp\u003e1\u0026times;10\u003csup\u003e7\u003c/sup\u003e RAW264.7 cells were centrifuged at 4℃ at 200\u0026ndash;1000 rcf for 10 min. The cell pellets were lysed ultrasonically and centrifuged at 18000 g for 20 min. Ten microliter supernatants were mixed with 70 \u0026micro;l borate buffer and 20 \u0026micro;l 6-minoquinolyl-N-hydroxysuccinimidyl carbamate derivatization reagent. The mixture was shaken at 1200 rpm for 10 min, then added 900 \u0026micro;l ultrapure water and mixed well. Using liquid chromatography mass spectrometry (LC-MS) and Metabo-Profile's self-built database, non-targeted metabolomics analysis of metabolites in samples was performed qualitatively and quantitatively. The target metabolomics of amino acids and nucleotides in the samples were subjected to ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) assay for quantification.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eStatistics analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism 9.0 software. The Kolmogorov-Smirnov test was used to inspect the normality and homogeneity of variance of all data. For two-group comparison, \u003cem\u003eP\u003c/em\u003e values were derived from the Student t-test to determine differences between groups with normally distributed data and Mann-Whitney nonparametric test with other data.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eETHICS\u003c/h2\u003e \u003cp\u003e All animal procedures were approved by the Institutional Animal Care and Use Committee of Capital Medical University.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eR.L. and Q.Q. performed most experiments and analyzed the data. Q.Q. and W.P. wrote the draft of the manuscript. L.Z. designed experiments, supervised the study and revised the manuscript. L.L. assisted in the cellular experiments. Y.L. and Y.Z. assisted in the mouse model preparation.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grant numbers, 82372189, 82072295 and 82172128) and the Beijing High-Level Public Health Technical Talent Training Program (Discipline Backbone Talent 02\u0026ndash;32). We would warmly thank Prof. Aibin He from Peking University for technical advice.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSrzić I, Nesek Adam V, Tunjić Pejak D. 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Macrophage polarization plays a critical role in pathogenesis of sepsis, and the influence of B lymphocyte-induced maturation protein-1 (Blimp-1) on this polarization is an underexplored yet pivotal aspect. This study aimed to elucidate the role of Blimp-1 in macrophage polarization and metabolism during sepsis. Using a murine cecal ligation and puncture model, we observed elevated Blimp-1 expression in M2 macrophages. Knockdown of Blimp-1 in this model resulted in decreased survival rates, exacerbated tissue damage, and impaired M2 polarization, underscoring its protective role in sepsis. \u003cem\u003eIn vitro\u003c/em\u003e studies with bone marrow-derived macrophages, RAW264.7, and THP-1 cells further demonstrated Blimp-1 promotes M2 polarization and modulates key metabolic pathways. Metabolomics and dual-luciferase assays revealed Blimp-1 significantly influences purine biosynthesis and the downstream Ornithine cycle, which are essential for M2 macrophage polarization. Our findings unveil a novel mechanism by which Blimp-1 modulates macrophage polarization through metabolic regulation, presenting potential therapeutic targets for sepsis. This study highlights the significance of Blimp-1 in orchestrating macrophage responses and metabolic adaptations in sepsis, offering valuable insights into its role as a critical regulator of immune and metabolic homeostasis.\u003c/p\u003e","manuscriptTitle":"Blimp-1 Orchestrates Macrophage Polarization and Metabolic Homeostasis via Purine Biosynthesis in Sepsis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-26 06:04:09","doi":"10.21203/rs.3.rs-4903330/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-09-20T14:18:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-14T07:47:12+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-07T05:56:39+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-30T02:04:43+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-29T18:51:03+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-08-28T03:14:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-19T11:08:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-17T12:43:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2024-08-17T12:43:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e679776b-57df-41ab-b6bb-379a1c49b1f6","owner":[],"postedDate":"September 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":36688200,"name":"Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages"},{"id":36688201,"name":"Biological sciences/Immunology/Infectious diseases"}],"tags":[],"updatedAt":"2025-02-07T08:08:07+00:00","versionOfRecord":{"articleIdentity":"rs-4903330","link":"https://doi.org/10.1038/s41419-025-07405-6","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-02-06 05:00:00","publishedOnDateReadable":"February 6th, 2025"},"versionCreatedAt":"2024-09-26 06:04:09","video":"","vorDoi":"10.1038/s41419-025-07405-6","vorDoiUrl":"https://doi.org/10.1038/s41419-025-07405-6","workflowStages":[]},"version":"v1","identity":"rs-4903330","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4903330","identity":"rs-4903330","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}