Investigation of Protein Lysine Acetylation in Antiviral Innate Immunity.

Protein lysine acetylation involved in the antiviral innate immunity contributes to the regulation of antiviral inflammation responses, including type 1 interferon production and interferon-stimulated gene expression. Thus, investigation of acetylated antiviral proteins is vital for the complete understanding of inflammatory responses to viral infections. Immunoprecipitation (IP) assay with anti-targeted-protein antibody or with acetyl-lysine affinity beads followed by immunoblot provides a classical way to determine the potential modified protein in the antiviral innate pathways, whereas mass spectrometry can be utilized to identify the accurate acetylation lysine residues or explore the acetyl-proteomics. We demonstrate here comprehensive methods of protein lysine acetylation determination in virus-infected macrophages and embryonic fibroblast cells or proteins-overexpressed HEK 293 T cells in the context of antiviral innate immunity.

Dysregulation of lysine acetylation in the pathogenesis of digestive tract cancers and its clinical applications.

Recent advances in high-resolution mass spectrometry-based proteomics have improved our understanding of lysine acetylation in proteins, including histones and non-histone proteins. Lysine acetylation, a reversible post-translational modification, is catalyzed by lysine acetyltransferases (KATs) and lysine deacetylases (KDACs). Proteins comprising evolutionarily conserved bromodomains (BRDs) recognize these acetylated lysine residues and consequently activate transcription. Lysine acetylation regulates almost all cellular processes, including transcription, cell cycle progression, and metabolic functions. Studies have reported the aberrant expression, translocation, and mutation of genes encoding lysine acetylation regulators in various cancers, including digestive tract cancers. These dysregulated lysine acetylation regulators contribute to the pathogenesis of digestive system cancers by modulating the expression and activity of cancer-related genes or pathways. Several inhibitors targeting KATs, KDACs, and BRDs are currently in preclinical trials and have demonstrated anti-cancer effects. Digestive tract cancers, including encompass esophageal, gastric, colorectal, liver, and pancreatic cancers, represent a group of heterogeneous malignancies. However, these cancers are typically diagnosed at an advanced stage owing to the lack of early symptoms and are consequently associated with poor 5-year survival rates. Thus, there is an urgent need to identify novel biomarkers for early detection, as well as to accurately predict the clinical outcomes and identify effective therapeutic targets for these malignancies. Although the role of lysine acetylation in digestive tract cancers remains unclear, further analysis could improve our understanding of its role in the pathogenesis of digestive tract cancers. This review aims to summarize the implications and pathogenic mechanisms of lysine acetylation dysregulation in digestive tract cancers, as well as its potential clinical applications.

Histone Deacetylase 3 Is Involved in Maintaining Queen Hallmarks of a Termite.

The role of epigenetics in regulating caste polyphenism in social insects has been debated. Here, we tested the importance of histone de/acetylation processes for the maintenance of queen hallmarks like a high fecundity and a long lifespan. To this end, we performed RNA interference experiments against histone deacetylase 3 (HDAC3) in the termite Cryptotermes secundus. Fat body transcriptomes and chemical communication profiles revealed that silencing of HDAC3 leads to signals indicative of queen hallmarks. This includes fostering of queen signalling, defence against ageing and a reduction of life-shortening IIS (insulin/insulin-like growth factor signalling) and endocrine JH (juvenile hormone) signalling via Kr-h1 (Krüppel-homologue 1). These observed patterns were similar to those of a protein-enriched diet, which might imply that histone acetylation conveys nutritional effects. Strikingly, in contrast to solitary insects, reduced endocrine JH signalling had no negative effect on fecundity-related vitellogenesis in the fat bodies. This suggests an uncoupling of longevity pathways from fecundity in fat bodies, which can help explain queens' extraordinary lifespans combined with high fecundity.

Investigating the role of HSP90 in cancer cell phenotypic plasticity.

"What are the mechanisms driving tumor evolution under the selective pressure of chemotherapeutics?" The emerging importance of epigenetic gene regulation in cancer progression necessitates not only our understanding of which genes are potential targets but also what mechanisms are employed in targeting those genes. Understanding the mechanisms that promote the evolution of the normal genome and epigenome is central to understanding how cancer cells adapt to chemotherapy. Our previous investigations have shown that heat shock protein 90 (HSP90) has a critical role in epigenetic gene regulation through histone acetylation and phenotypic plasticity. We recently extended these results in an A549 lung cancer model to test the role of HSP90 in the plasticity of cells regarding multi-drug resistance and epithelial-to-mesenchymal transition phenotypes. HSP90 is over-expressed in multiple cancers with poor prognosis. We propose that inhibition of HSP90 results in lower phenotypic plasticity of cancer cells making them more susceptible to chemotherapeutic intervention. Here we review the context of our results in the broader field of evolution of these phenotypes.

Improving regulatory T cell-based therapy: insights into post-translational modification regulation.

Regulatory T (Treg) cells are pivotal for maintaining immune homeostasis and play essential roles in various diseases, such as autoimmune diseases, graft-versus-host disease (GVHD), tumors, and infectious diseases. Treg cells exert suppressive function via distinct mechanisms including inhibitory cytokines, granzyme or perforin-mediated cytolysis, metabolic disruption, and suppression of dendritic cells. Forkhead Box P3 (FOXP3), the characteristic transcription factor, is essential for Treg cell function and plasticity. Cumulative evidence has demonstrated that FOXP3 activity and Treg cell function are modulated by a variety of post-translational modifications (PTMs), including ubiquitination, acetylation, phosphorylation, methylation, glycosylation, poly(ADP-ribosyl)ation, and uncharacterized modifications. This review describes Treg cell suppressive mechanisms and summarizes the current evidence on PTM regulation of FOXP3 and Treg cell function. Understanding the regulatory role of PTMs in Treg cell plasticity and function will be helpful in designing therapeutic strategies for autoimmune diseases, GVHD, tumors, and infectious diseases.

ZNF521 promotes acute myeloid leukemogenesis by suppressing the expression and acetylation of SMC3.

Zinc finger protein 521 (ZNF521) participates in the self-renewal of hematopoietic stem cells, and its abnormal expression has been implicated to promote leukemia. However, the specific role of ZNF521 in leukemia has not been fully understood. In this study, we aimed to further elucidate its role. Using acute leukemia cell line THP-1, we demonstrated that knocking down ZNF521 inhibited leukemia cell proliferation, promoted apoptosis, and induced cell arrest in G2/M phase. Interestingly, we also observed the upregulation of SMC3 expression and acetylation, as well as the downregulation of histone deacetylases 8 (HDAC8), CDK2, and CDK6. The proliferation inhibition was reversed by knocking down SMC3, suggesting the key role of SMC3 reduction in ZNF521 elevated proliferation. Conversely, ZNF521 overexpression in HL-60 cells resulted in enhanced proliferation and inhibited apoptosis. Furthermore, we discovered that ZNF521 can interact with HDAC8, which deacetylates SMC3, and the interaction promotes proliferation and suppresses apoptosis. Notably, when HDAC8 was knocked down or its activity was inhibited by a HDAC8 inhibitor, the previous observed trend was reversed. Consequently, ZNF521 plays a critical role in acute myeloid leukemogenesis by reducing the expression and acetylation of SMC3. Overall, this study sheds light on the potential for targeted treatment in highly ZNF521 expressed acute myeloid leukemia, providing a valuable clue for precise and effective therapeutic approaches.

Acetylation of Oleanolic Acid Dimers as a Method of Synthesis of Powerful Cytotoxic Agents.

Oleanolic acid, a naturally occurring triterpenoid compound, has garnered significant attention in the scientific community due to its diverse pharmacological properties. Continuing our previous work on the synthesis of oleanolic acid dimers (OADs), a simple, economical, and safe acetylation reaction was performed. The newly obtained derivatives (AcOADs, 3a-3n) were purified using two methods. The structures of all acetylated dimers (3a-3n) were determined based on spectral methods (IR, NMR). For all AcOADs (3a-3n), the relationship between the structure and the expected directions of pharmacological activity was determined using a computational method (QSAR computational analysis). All dimers were also tested for their cytotoxic activity on the SKBR-3, SKOV-3, PC-3, and U-87 cancer cell lines. HDF cell line was applied to evaluate the Selectivity Index of the tested compounds. All cytotoxic tests were performed with the application of the MTT assay. Finally, all dimers of oleanolic acid were subjected to DPPH and CUPRAC tests to evaluate their antioxidant activity. The obtained results indicate a very high level of cytotoxic activity (IC50 for most AcOADs below 5.00 µM) and a fairly high level of antioxidant activity (Trolox equivalent in some cases above 0.04 mg/mL).

LINC01232 promotes ARNTL2 transcriptional activation and inhibits ferroptosis of CRC cells through p300/H3K27ac.

Aim: This study investigated the role of lncRNA LINC01232 in ferroptosis of colorectal cancer (CRC).Materials & methods: Real time quantitative polymerase chain reaction or western blot experiments were performed to examine relevant mRNAs and proteins expression. The kit assays evaluated malondialdehyde, iron, Fe2+ and glutathione levels. ROS levels were verified by flow cytometry. Chromatin immunoprecipitation and RNA immunoprecipitation analysis monitored the correlation among LINC01232, H3K27ac, p300 and ARNTL2.Results: LINC01232 or ARNTL2 knockdown facilitated erastin-induced ferroptosis. The interaction between LINC01232 and p300 resulted in the enhancement of H3K27ac levels at ARNTL2 promoter to promote ARNTL2 transcriptional activity. ARNTL2 overexpression reversed the promoting effect of LINC01232 knockdown on ferroptosis.Conclusion: LINC01232 inhibited the ferroptosis in CRC by epigenetically upregulating the transcriptional activity of ARNTL2.

Colorectal cancer (CRC) is a malignant disease of the digestive tract that occurs worldwide, which has high morbidity and mortality but has not effective targeted therapy. Ferroptosis has emerged as a new target for treating CRC since its proposed in 2012. Long noncoding RNAs are noncoding RNAs with a length greater than 200 nucleotides and their role in ferroptosis of cancer cells has attracted more and more attention in recent years. Herein, our study explored the effect of long noncoding RNA LINC01232 on CRC progression. This research exhibited the relationship between LINC01232 and the ferroptosis at the occurrence and development of CRC, which is expected to provide a potential therapeutic target for the treatment of CRC.

miRNA-105-5p regulates the histone deacetylase HDAC2 through FOXG1 to affect the malignant biological behavior of triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is a specific subtype of breast cancer (BC). Some potential molecular targets have been identified, and miR-105-5p was found to be abnormally expressed in TNBC tissues. OBJECTIVE: The objective of this study was to probe the effect of miR-105-5p on TNBC via FOXG1/HDAC2-mediated acetylation. METHODS: An animal model of TNBC was established by injecting BC cells into the axillary area of nude mice. The levels of miR-105-5p, FOXG1, HDAC2, Bcl-2, Bax, and Ki67 were detected via RT‒qPCR, Western blotting and immunohistochemistry. Flow cytometry, CCK-8, Transwell and colony formation assays were used to measure apoptosis, proliferation and migration, respectively. Total histone acetylation levels were measured by ELISA. The binding of FOXG1 to HDAC2 was detected by co-immunoprecipitation. The binding relationship between miR-105-5p and FOXG1 was verified using a dual-luciferase reporter gene assay. RESULTS: In this study, miR-105-5p and HDAC2 were highly expressed in the MDA-MB-231 and BT-549 BC cell lines, whereas FOXG1 was expressed at low levels. The inhibition of miR-105-5p inhibited the proliferation and migration of MDA-MB-231 and BT-549 cells and promoted their apoptosis. Bioinformatics analysis revealed that miR-105-5p and FOXG1 had a negative targeting regulatory relationship. FOXG1 overexpression had a similar effect on cancer cells as the inhibition of miR-105-5p. Moreover, experiments revealed that FOXG1 and HDAC2 could bind to each other and that HDAC2 overexpression or treatment with the histone acetyltransferase inhibitor Garcinol weakened the effect of FOXG1 overexpression. In addition, FOXG1 knockdown inhibited the effect of the miR-105-5p inhibitor, while Garcinol treatment further enhanced the effect of FOXG1 knockdown, inhibited histone acetylation, promoted the proliferation and migration of cancer cells, and inhibited apoptosis. Moreover, the in vivo results confirmed the in vitro results. CONCLUSION: miR-105-5p promotes HDAC2 expression by reducing FOXG1, inhibits histone acetylation, and aggravates the malignant biological behavior of TNBC cells.

KAT tales: Functions of Gcn5 and PCAF lysine acetyltransferases in SAGA and ATAC.

The Allis group identified Gcn5 as the first transcription-related lysine acetyltransferase in 1996, providing a molecular "missing link" between chromatin organization and gene regulation. This review will focus on functions subsequently identified for Gcn5 and the closely related PCAF protein, in the context of two major complexes, SAGA and ATAC, and how the study of these enzymes informs long standing questions regarding the importance of lysine acetylation.

Histone acetyltransferases as promising therapeutic targets in glioblastoma resistance.

Glioblastoma (GBM) is a fatal adult brain tumor with an extremely poor prognosis. GBM poses significant challenges for targeted therapies due to its intra- and inter-tumoral heterogeneity, a highly immunosuppressive microenvironment, diffuse infiltration into normal brain parenchyma, protection by the blood-brain barrier and acquisition of therapeutic resistance. Recent studies have implicated epigenetic modifiers as key players driving tumorigenesis, resistance, and progression of GBM. While the vast majority of GBM research on epigenetic modifiers thus far has focused predominantly on elucidating the functional roles and targeting of DNA methyltransferases and histone deacetylases, emerging evidence indicates that histone acetyltransferases (HATs) also play a key role in mediating plasticity and therapeutic resistance in GBM. Here, we will provide an overview of HATs, their dual roles and functions in cancer as both tumor suppressors and oncogenes and focus specifically on their implications in GBM resistance. We also discuss the technical challenges in developing selective HAT inhibitors and highlight their promise as potential anti-cancer therapeutics for treating intractable cancers such as GBM.

Structural Analysis of Mammalian Sialic Acid Esterase.

Sialic acid esterase (SIAE) catalyzes the removal of O-acetyl groups from sialic acids found on cell surface glycoproteins to regulate cellular processes such as B cell receptor signalling and apoptosis. Loss-of-function mutations in SIAE are associated with several common autoimmune diseases including Crohn's, ulcerative colitis, and arthritis. To gain a better understanding of the function and regulation of this protein, we determined crystal structures of SIAE from three mammalian homologs, including an acetate bound structure. The structures reveal that the catalytic domain adopts the fold of the SGNH hydrolase superfamily. The active site is composed of a catalytic dyad, as opposed to the previously reported catalytic triad. Attempts to determine a substrate-bound structure yielded only the hydrolyzed product acetate in the active site. Rigid docking of complete substrates followed by molecular dynamics simulations revealed that the active site does not form specific interactions with substrates, rather it appears to be broadly specific to accept sialoglycans with diverse modifications. Based on the acetate bound structure, a catalytic mechanism is proposed. Structural mapping of disease mutations reveals that most are located on the surface of the enzyme and would only cause minor disruptions to the protein fold, suggesting that these mutations likely affect binding to other factors. These results improve our understanding of SIAE biology and may aid in the development of therapies for autoimmune diseases and cancer.

RPS6KA1 is a histone acetylation-related oncoprotein in acute myeloid leukemia which is targeted by afzelin.

BACKGROUND: Histone acetylation plays a critical role in the progression of acute myeloid leukemia (AML). This study aimed to explore the prognostic significance and biological implications of histone acetylation-related genes in AML and to identify potential oncoproteins and therapeutic compounds. METHODS: Genes associated with AML and histone acetylation were identified using the TCGA-LAML and IMEx Interactome databases. A histone acetylation-related risk model was developed using the least absolute shrinkage and selection operator method. The prognostic value of the model was evaluated through Kaplan-Meier survival analysis, time-dependent receiver operating characteristic curve, univariate and multivariate Cox regression, and nomogram calibration. Key genes were identified using random forest, support vector machine, and multivariate Cox analysis. Molecular docking was employed to assess the binding affinity between ribosomal protein S6 kinase A1 (RPS6KA1) and potential compounds. Furthermore, the effects of RPS6KA1 and afzelin on the malignant behaviors and downstream pathways of AML cells were validated through in vitro experiments. RESULTS: A risk model composed of 6 genes, including HDAC6, CREB3, KLF13, GOLGA2, RPS6KA1 and ZMIZ2, was established, demonstrating strong prognostic predictive capability. Among these, RPS6KA1 emerged as a key risk factor linked to histone acetylation status in AML. Elevated RPS6KA1 expression was observed in AML samples and was associated with poor prognosis. RPS6KA1 knockdown suppressed AML cell proliferation, migration, and invasion, induced G0/G1 phase arrest, and promoted apoptosis. Additionally, RPS6KA1 was identified as a potential target for afzelin, which exhibited anti-AML activity by inactivating RPS6KA1. CONCLUSION: Histone acetylation status is closely associated with AML patient prognosis. RPS6KA1 acts as an oncoprotein in AML, facilitating disease progression. Afzelin may represent a novel therapeutic agent for AML by targeting RPS6KA1, which requires validation by clinical trials.

Epigenetic regulation of the human GDAP1 gene.

Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene are linked to Charcot-Marie-Tooth (CMT) disease, a hereditary neurodegenerative condition. The protein encoded by this gene is involved in mitochondrial fission and calcium homeostasis. Recently, GDAP1 has also been implicated in the survival of patients with certain cancers. Despite its significant role in specific cellular processes and associated diseases, the mechanisms regulating GDAP1 expression are largely unknown. Here, we show for the first time that methylation of the CpG island in the proximal promoter of the GDAP1 gene inhibits its activity. Treating cells with low GDAP1 expression using methyltransferase and HDAC inhibitors induced the expression of this gene and its encoded protein. This induction was associated with promoter demethylation and increased association of acetylated histones with the GDAP1 promoter. Thus, we identified a mechanism that could be used to manipulate GDAP1 expression.

PDHX acetylation facilitates tumor progression by disrupting PDC assembly and activating lactylation mediated gene expression.

Deactivation of the mitochondrial pyruvate dehydrogenase complex (PDC) is important for the metabolic switching of cancer cell from oxidative phosphorylation to aerobic glycolysis. Studies examining PDC activity regulation have mainly focused on the phosphorylation of pyruvate dehydrogenase (PDH, E1), leaving other post-translational modifications (PTMs) largely unexplored. Here, we demonstrate that the acetylation of Lys 488 of pyruvate dehydrogenase complex component X (PDHX) commonly occurs in hepatocellular carcinoma (HCC), disrupting PDC assembly and contributing to lactate-driven epigenetic control of gene expression. PDHX, an E3-binding protein (E3BP) in the PDC, is acetylated by the p300 at Lys 488, impeding the interaction between PDHX and dihydrolipoyl transacetylase (DLAT, E2), thereby disrupting PDC assembly to inhibit its activation. PDC disruption results in the conversion of most glucose to lactate, contributing to the aerobic glycolysis and H3K56 lactylation-mediated gene expression, facilitating tumor progression. These findings highlight a previously unrecognized role of PDHX acetylation in regulating PDC assembly and activity, linking PDHX Lys 488 acetylation and histone lactylation during HCC progression and providing a potential biomarker and therapeutic target for further development.

Butyrate and propionate are microbial danger signals that activate the NLRP3 inflammasome in human macrophages upon TLR stimulation.

Short-chain fatty acids (SCFAs) are immunomodulatory compounds produced by the microbiome through dietary fiber fermentation. Although generally considered beneficial for gut health, patients suffering from inflammatory bowel disease (IBD) display poor tolerance to fiber-rich diets, suggesting that SCFAs may have contrary effects under inflammatory conditions. To investigate this, we examined the effect of SCFAs on human macrophages in the presence of Toll-like receptor (TLR) agonists. In contrast to anti-inflammatory effects under steady-state conditions, we found that butyrate and propionate activated the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome in the presence of TLR agonists. Mechanistically, these SCFAs prevented transcription of FLICE-like inhibitory protein (cFLIP) and interleukin-10 (IL-10) through histone deacetylase (HDAC) inhibition, triggering caspase-8-dependent NLRP3 inflammasome activation. SCFA-driven NLRP3 activation was potassium efflux independent and did not result in cell death but rather triggered hyperactivation and IL-1ß release. Our findings demonstrate that butyrate and propionate are bacterially derived danger signals that regulate NLRP3 inflammasome activation through epigenetic modulation of the inflammatory response.

Construction of a pumpless gravity-driven vascularized Skin-on-a-Chip for the study of hepatocytotoxicity in percutaneous exposure to exogenous chemicals.

The utilization of existing Skin-on-a-Chip (SoC) is constrained by the complex structures, the multiplicity of auxiliary devices, and the inability to evaluate exogenous chemicals that are hepatotoxic after percutaneous metabolism. In this study, a gravity-driven SoC without any auxiliary devices was constructed for the hepatocytotoxicity study of exogenous chemicals. The SoC possesses 3 layers of culture chambers, from top to bottom, for human skin equivalent (HSE), Human Umbilical Vein Endothelial Cells (HUVEC) and hepatocytes (HepG2), and the maintenance and expression capacity of the corresponding cells on the SoC were verified by specificity parameters. The reactivity of the SoC to exogenous chemicals was verified by 2-aminofluorene (2-AF). The SoC can realistically simulate the in vivo exposure process of exogenous chemicals that are percutaneously exposed and metabolized into the bloodstream and then to the liver to produce toxicity, and it can achieve the same effects on transcriptome as those of animal tests at lower exposure levels while examining multiple toxicological targets of the skin, vascular endothelial cells, and hepatocytes. Both in terms of species similarity, the principles of reduction, replacement and refinement (3R), or the level of exposure suggest that the present SoC has a degree of replacement for animal models in assessing exogenous chemicals, especially those that are hepatotoxic after percutaneous metabolism.

Identification of a novel histone acetylation-related long non-coding RNA model combined with qRT-PCR experiments for prognosis and therapy in gastric cancer.

Gastric cancer (GC) is considered a global health crisis due to the scarcity of early diagnostic methods. Numerous studies have substantiated the involvement of histone acetylation imbalance in the progression of diverse tumor types. The potential roles of long non-coding RNA (lncRNA) in improving prognostic, predictive as well as therapeutic approaches in cancers have made it a major hotspot in recent years. Nevertheless, existent studies have never concerned the prognostic and clinical value of histone acetylation-related lncRNAs (HARlncs) in GC. Based on the aforementioned rationale, we developed a prognostic model incorporating four HARlncs-AC114730.1, AL445250.1, LINC01778, and AL163953.1-which demonstrated potential as an independent predictor of prognosis. Subsequently, GC patients were stratified into high-risk and low-risk groups. The low-risk group exhibited significantly higher overall survival (OS) compared to the high-risk group. Based on the analyses of the tumor microenvironment (TME) and immune responses, significant differences were observed between the two risk groups in terms of immune cell infiltration, immune checkpoint (ICP) expression, and other TME alterations. Furthermore, the sensitivity of GC patients to some chemotherapeutic drugs and the discrepant biological behaviors of three tumor clusters were studied in this model. In summary, we developed an effective HARlncs model with the objective of offering novel prognostic prediction methods and identifying potential therapeutic targets for GC patients.

Upregulation of Mitochondrial Sirt3 and Alleviation of the Inflammatory Phenotype in Macrophages by Estrogen.

BACKGROUND: Aging and comorbidities like type 2 diabetes and obesity contribute to the development of chronic systemic inflammation, which impacts the development of heart failure and vascular disease. Increasing evidence suggests a role of pro-inflammatory M1 macrophages in chronic inflammation. A shift of metabolism from mitochondrial oxidation to glycolysis is essential for the activation of the pro-inflammatory M1 phenotype. Thus, reprogramming the macrophage metabolism may alleviate the pro-inflammatory phenotype and protect against cardiovascular diseases. In the present study, we hypothesized that the activation of estrogen receptors leads to the elevation of the mitochondrial deacetylase Sirt3, which supports mitochondrial function and mitigates the pro-inflammatory phenotype in macrophages. MATERIALS AND METHODS: Experiments were performed using the mouse macrophage cell line RAW264.7, as well as primary male or female murine bone marrow macrophages (BMMs). Macrophages were treated for 24 h with estradiol (E2) or vehicle (dextrin). The effect of E2 on Sirt3 expression was investigated in pro-inflammatory M1, anti-inflammatory/immunoregulatory M2, and naïve M0 macrophages. Mitochondrial respiration was measured by Seahorse assay, and protein expression and acetylation were determined by western blotting. RESULTS: E2 treatment upregulated mitochondrial Sirt3, reduced mitochondrial protein acetylation, and increased basal mitochondrial respiration in naïve RAW264.7 macrophages. Similar effects on Sirt3 expression and mitochondrial protein acetylation were observed in primary female but not in male murine BMMs. Although E2 upregulated Sirt3 in naïve M0, pro-inflammatory M1, and anti-inflammatory/immunoregulatory M2 macrophages, it reduced superoxide dismutase 2 acetylation and suppressed mitochondrial reactive oxygen species formation only in pro-inflammatory M1 macrophages. E2 alleviated the pro-inflammatory phenotype in M1 RAW264.7 cells. CONCLUSIONS: The study suggests that E2 treatment upregulates Sirt3 expression in macrophages. In primary BMMs, female-specific Sirt3 upregulation was observed. The Sirt3 upregulation was accompanied by mitochondrial protein deacetylation and the alleviation of the oxidative and pro-inflammatory phenotype in M1 macrophages. Thus, the E2-Sirt3 axis might be used in a therapeutic strategy to fight chronic systemic inflammation and prevent the development of inflammation-linked diseases.