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

doi:10.21203/rs.3.rs-4903330/v1

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

https://doi.org/10.21203/rs.3.rs-4903330/v1

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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

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.

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. 1A-1C). 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. 1D-1F). The mRNA level of Blimp-1 was also significantly increased in these cells (Fig. 1G). This selective upregulation of Blimp-1 in M2 macrophages, but not in CD4⁺ T cells, CD8⁺ T cells, or B cells (Fig. 1H, Fig. S1A & 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. 2A). Three weeks post-injection, the survival rate of CLP mice was significantly lower in the Blimp-1 knockdown group (Fig. 2B). Histopathological analysis revealed exacerbated liver and lung tissue damage (Fig. 2C). 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. 2D-2F). 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. 2G) and spleen grinding fluid (Fig. 2H). 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. 3A). Blimp-1 expression was significantly higher in monocytes compared to B cells and granulocytes (Fig. 3B), and its mRNA levels progressively increased alongside bone marrow-derived macrophage (BMDM) differentiation and maturation (Fig. 3C). Upon polarization, M1 macrophages exhibited high secretion of IL-1β, IL-6, TNF-α, and IL-12 (Fig. S2A); whereas M2 macrophages secreted high levels of TGF-β, CCL17, and CCL22 (Fig. S2B). Notably, Blimp-1 mRNA levels were specifically elevated in M2 macrophages compared to M0 and M1 counterparts (Fig. 3D), 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. 3E). Overexpression of Blimp-1 causes morphological changes consistent with M2 macrophages, including the appearance of extended pseudopods (Fig. 3F), and a significant increase in CD206 expression, as well as increased mRNA levels of M2 markers such as MRC1, MSR1, ARG1 and IL10 (Fig. 3G & 3H). 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. 3I-3K). 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. 4A). Compared to shRNA-NC control, shRNA-Blimp-1 effectively reduced Blimp-1 expression, particularly in M2 macrophages (Fig. 4B). 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. 4C & 4D). In contrast, the proportion of M1 (CD86⁺) macrophages and the mRNA levels of M1 markers Il12b, Nos2, and Tnfa remained unchanged (Fig. 4C & 4E), 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. 5A). Conversely, overexpression of Blimp-1 in RAW264.7 macrophages resulted in elevated mRNA levels for these genes (Fig. 5B). 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. 5C). This reduction is indicative of a decrease in the maximal respiration, mitochondrial ATP production, and spare respiratory capacity (Fig. 5D). Furthermore, the extracellular acidification rate (ECAR), a measure of glycolytic activity, was diminished after shRNA-Blimp-1 intervention (Fig. 5E), reflecting lower basal and maximum glycolysis levels (Fig. 5F). 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. 6A). An Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) clearly distinguished the metabolite profiles between the shRNA-Blimp-1 and shRNA-NC groups (Fig. 6B). Differential metabolite analysis revealed significant differences in amino acids and nucleotides between the two groups (Fig. 6C). The differential metabolites were displayed in Fig. 6D & 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. 6D, 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. 6E & 6F). 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. 6G), as well as ammonia recycling, glutamine metabolism, urea cycle and glycine and serine metabolism (Fig. 6H). 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. 7A). 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. 7B). Following Blimp-1 knockdown, the content of metabolites in the Ornithine cycle and the mRNA levels of key enzymes were significantly diminished (Fig. 7C & 7D). 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. 7F). 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. 7G).

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.

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

1×10⁷ 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.

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.

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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
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Reviewer #1 agreed at journal
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You are reading this latest preprint version

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

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Version 1

Abstract

Figures

Introduction

Results

Blimp-1 expression is elevated in sepsis-associated macrophages

Blimp-1 knockdown exacerbates sepsis by impairing M2 polarization

Blimp-1 promotes monocyte-macrophage differentiation and M2 polarization

Blimp-1 is essential for energy metabolism in M2 macrophages

Blimp-1 modulates the metabolome of M2 macrophages

Blimp-1 enhances purine biosynthesis and the downstream Ornithine cycle in M2 macrophages

Discussion

Materials and Methods

Culture of BMDMs

Cell culture and treatment

Splenic cell isolation

Peritoneal lavage fluid extraction

Flow cytometry

Multiple cytokine assay

Serum alanine aminotransferase and aspartate aminotransferase measurement

Hematoxylin-eosin (H&E) staining

RNA isolation, reverse transcription, and realtime PCR

Plasmid construction, transfection, and dual-luciferase assay

Seahorse XF glycolytic stress and mitochondrial stress test

Non-targeted and targeted metabolomics analysis

Statistics analysis

Declarations

Conflict of Interest

ETHICS

Author Contributions

Acknowledgement

References

Additional Declarations

Supplementary Files

Status:

Version 1