Comprehensive Lipid Profiling Recapitulates Enhanced Lipolysis and Fatty Acid Metabolism in Intimal Foamy Macrophages From Murine Atherosclerotic Aorta

Jae Won Seo,Kyu Seong Park,Gwang Bin Lee,Sang-eun Park,Jae-Hoon Choi,Myeong Hee Moon 대한면역학회 2023 Immune Network Vol.23 No.4

Lipid accumulation in macrophages is a prominent phenomenon observed in atherosclerosis. Previously, intimal foamy macrophages (FM) showed decreased inflammatory gene expression compared to intimal non-foamy macrophages (NFM). Since reprogramming of lipid metabolism in macrophages affects immunological functions, lipid profiling of intimal macrophages appears to be important for understanding the phenotypic changes of macrophages in atherosclerotic lesions. While lipidomic analysis has been performed in atherosclerotic aortic tissues and cultured macrophages, direct lipid profiling has not been performed in primary aortic macrophages from atherosclerotic aortas. We utilized nanoflow ultrahigh-performance liquid chromatography-tandem mass spectrometry to provide comprehensive lipid profiles of intimal non-foamy and foamy macrophages and adventitial macrophages from Ldlr−/− mouse aortas. We also analyzed the gene expression of each macrophage type related to lipid metabolism. FM showed increased levels of fatty acids, cholesterol esters, phosphatidylcholine, lysophosphatidylcholine, phosphatidylinositol, and sphingomyelin. However, phosphatidylethanolamine, phosphatidic acid, and ceramide levels were decreased in FM compared to those in NFM. Interestingly, FM showed decreased triacylglycerol (TG) levels. Expressions of lipolysis-related genes including Pnpla2 and Lpl were markedly increased but expressions of Lpin2 and Dgat1 related to TG synthesis were decreased in FM. Analysis of transcriptome and lipidome data revealed differences in the regulation of each lipid metabolic pathway in aortic macrophages. These comprehensive lipidomic data could clarify the phenotypes of macrophages in the atherosclerotic aorta.

Mycobacterium tuberculosis virulence protein ESAT-6 influences M1/M2 polarization and macrophage apoptosis to regulate tuberculosis progression

Sun Feng,Li Jiangbo,Cao Ling,Yan Cunzi 한국유전학회 2024 Genes & Genomics Vol.46 No.1

Background Tuberculosis (TB) is an infectious disease caused by infection with Mycobacterium tuberculosis (Mtb), and it remains one of the major threats to human health worldwide. To our knowledge, the polarization of M1/M2 macrophages were critical innate immune cells which play important roles in regulating the immune response during TB progression. Objective We aimed to explore the potential mechanisms of M1/M2 macrophage polarization in TB development. Methods THP-1 macrophages were treated with early secreted antigenic target of 6 kDa (ESAT-6) protein for an increasing time. The polarization profiles, apoptosis levels of M1 and M2 macrophages were detected by RT-qPCR, immunofluorescence, Western blot and flow cytometry. Results ESAT-6 initially promoted the generation of pro-inflammatory M1-polarized macrophages in THP-1 cells within 24 h, which were suppressed by further ESAT-6 treatment at 30–42 h. Interestingly, ESAT-6 continuously promoted M2 polarization of THP-1 cells, thereby maintaining the anti-inflammatory response in a time-dependent manner. In addition, ESAT-6 promoted apoptotic cell death in M1-polarized macrophages, which had little effects on apoptosis of M2-phenotype of macrophages. Then, the potential underlying mechanisms were uncovered, and we verified that ESAT-6 increased the protein levels of TLR4, MyD88 and NF-κB to activate the TLR4/MyD88/NF-κB pathway within 24 h, and this signal pathway was significantly inactivated at 36 h post-treatment. Interestingly, the following experiments confirmed that ESAT-6 TLR4/MyD88/NF-κB pathway-dependently regulated M1/M2 polarization and apoptosis of macrophage in THP-1 cells. Conclusion Our study investigated the detailed effects and mechanisms of M1/M2 macrophages in regulating innate responses during TB development, which provided a new perspective on the development of treatment strategies for this disease. Background Tuberculosis (TB) is an infectious disease caused by infection with Mycobacterium tuberculosis (Mtb), and it remains one of the major threats to human health worldwide. To our knowledge, the polarization of M1/M2 macrophages were critical innate immune cells which play important roles in regulating the immune response during TB progression. Objective We aimed to explore the potential mechanisms of M1/M2 macrophage polarization in TB development. Methods THP-1 macrophages were treated with early secreted antigenic target of 6 kDa (ESAT-6) protein for an increasing time. The polarization profiles, apoptosis levels of M1 and M2 macrophages were detected by RT-qPCR, immunofluorescence, Western blot and flow cytometry. Results ESAT-6 initially promoted the generation of pro-inflammatory M1-polarized macrophages in THP-1 cells within 24 h, which were suppressed by further ESAT-6 treatment at 30–42 h. Interestingly, ESAT-6 continuously promoted M2 polarization of THP-1 cells, thereby maintaining the anti-inflammatory response in a time-dependent manner. In addition, ESAT-6 promoted apoptotic cell death in M1-polarized macrophages, which had little effects on apoptosis of M2-phenotype of macrophages. Then, the potential underlying mechanisms were uncovered, and we verified that ESAT-6 increased the protein levels of TLR4, MyD88 and NF-κB to activate the TLR4/MyD88/NF-κB pathway within 24 h, and this signal pathway was significantly inactivated at 36 h post-treatment. Interestingly, the following experiments confirmed that ESAT-6 TLR4/MyD88/NF-κB pathway-dependently regulated M1/M2 polarization and apoptosis of macrophage in THP-1 cells. Conclusion Our study investigated the detailed effects and mechanisms of M1/M2 macrophages in regulating innate responses during TB development, which provided a new perspective on the development of treatment strategies for this disease.

Sulfatase 1 and sulfatase 2 as novel regulators of macrophage antigen presentation and phagocytosis

( Hyun-je Kim ),( Hee-sun Kim ),( Young-hoon Hong ) 영남대학교 의과대학 2021 Yeungnam University Journal of Medicine Vol.38 No.4

Background: Sulfation of heparan sulfate proteoglycans (HSPGs) is critical for the binding and signaling of ligands that mediate inflammation. Extracellular 6-O-endosulfatases regulate posttranslational sulfation levels and patterns of HSPGs. In this study, extracellular 6-O-endosulfatases, sulfatase (Sulf)-1 and Sulf-2, were evaluated for their expression and function in inflammatory cells and tissues. Methods: Harvested human peripheral blood mononuclear cells were treated with phytohemag-glutinin and lipopolysaccharide, and murine peritoneal macrophages were stimulated with interleukin (IL)-1β for the evaluation of Sulf-1 and Sulf-2 expression. Sulf expression in inflammatory cells was examined in the human rheumatoid arthritis (RA) synovium by immunofluorescence staining. The antigen presentation and phagocytic activities of macrophages were compared according to the expression state of Sulfs. Sulfs-knockdown macrophages and Sulfs-overexpressing macrophages were generated using small interfering RNAs and pcDNA3.1 plasmids for Sulf-1 and Sulf-2, respectively. Results: Lymphocytes and monocytes showed weak Sulf expression, which remained unaffected by IL-1β. However, peritoneal macrophages showed increased expression of Sulfs upon stimulation with IL-1β. In human RA synovium, two-colored double immunofluorescent staining of Sulfs and CD68 revealed active upregulation of Sulfs in macrophages of inflamed tissues, but not in lymphocytes of lymphoid follicles. Macrophages are professional antigen-presenting cells. The antigen presentation and phagocytic activities of macrophages were dependent on the level of Sulf expression, suppressed in Sulfs-knockdown macrophages, and enhanced in Sulfs-overexpressing macrophages. Conclusion: The results demonstrate that upregulation of Sulfs in macrophages occurs in response to inflammation, and Sulfs actively regulate the antigen presentation and phagocytic activities of macrophages as novel immune regulators.

Separation of Macrophages Using a Dielectrophoresis-Based Microfluidic Device

Ying Zhang,Shangyu Wang,Jie Chen,Fang Yang,Guiying Li 한국바이오칩학회 2020 BioChip Journal Vol.14 No.2

Macrophages are an important component of the immune system and play a key role in tissue damage repair and immune defense. As a potential biomarker, macrophages contribute to the early diagnosis and therapy of cancer and other disease. The isolation of macrophages is a key step in relevant research and clinical applications. However, conventional macrophage separation and purification methods rely on benchtop equipment and are time-consuming, inefficient, complicated, and immobile. Herein, we present a method based on dielectrophoresis (DEP) to rapidly separate and purify macrophages from micro-volume samples using a microfluidic device for liquid biopsy and point-of-care testing (POCT). The device can be mechanically manipulated easily for straightforward separation. The DEP force on macrophages was measured under different electric field conditions, then the macrophages were successfully separated from the mixture of macrophages (RAW264.7) and breast cancer cells (MCF-7) using the microfluidic chip. The purity of macrophages in the collections reached higher than 99%. The effects of flow rate and voltage on the separation efficiency of macrophages by DEP were further studied to optimize the separation conditions. This work proposes a new method for rapid separation of macrophages based on microfluidic systems, providing a technical foundation for macrophage-based disease monitoring and real-time rapid diagnosis.

Distinct Features of Brain-Resident Macrophages: Microglia and Non-Parenchymal Brain Macrophages

Lee, Eunju,Eo, Jun-Cheol,Lee, Changjun,Yu, Je-Wook Korean Society for Molecular and Cellular Biology 2021 Molecules and cells Vol.44 No.5

Tissue-resident macrophages play an important role in maintaining tissue homeostasis and innate immune defense against invading microbial pathogens. Brain-resident macrophages can be classified into microglia in the brain parenchyma and non-parenchymal brain macrophages, also known as central nervous system-associated or border-associated macrophages, in the brain-circulation interface. Microglia and non-parenchymal brain macrophages, including meningeal, perivascular, and choroid plexus macrophages, are mostly produced during embryonic development, and maintained their population by self-renewal. Microglia have gained much attention for their dual roles in the maintenance of brain homeostasis and the induction of neuroinflammation. In particular, diverse phenotypes of microglia have been increasingly identified under pathological conditions. Single-cell phenotypic analysis revealed that microglia are highly heterogenous and plastic, thus it is difficult to define the status of microglia as M1/M2 or resting/activated state due to complex nature of microglia. Meanwhile, physiological function of non-parenchymal brain macrophages remain to be fully demonstrated. In this review, we have summarized the origin and signatures of brain-resident macrophages and discussed the unique features of microglia, particularly, their phenotypic polarization, diversity of subtypes, and inflammasome responses related to neurodegenerative diseases.

High-plasticity mineral trioxide aggregate and its effects on M1 and M2 macrophage viability and adherence, phagocyte activity, production of reactive oxygen species, and cytokines

Vasconcellos Betânia Canal,Tavares Layara Cristine Tomaz,da Silva Danilo Couto,Fonseca Francielen Oliveira,Benetti Francine,Sobrinho Antônio Paulino Ribeiro,Tavares Warley Luciano Fonseca 대한치과보존학회 2023 Restorative Dentistry & Endodontics Vol.48 No.1

Objectives This study evaluated the effects of high-plasticity mineral trioxide aggregate (MTA-HP) on the activity of M1 and M2 macrophages, compared to white MTA (Angelus). Materials and Methods Peritoneal inflammatory M1 (from C57BL/6 mice) and M2 (from BALB/c mice) macrophages were cultured in the presence of the tested materials. Cell viability (MTT and trypan blue assays), adhesion, phagocytosis, reactive oxygen species (ROS) production, and tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β production were evaluated. Parametric analysis of variance and the non-parametric Kruskal-Wallis test were used. Results were considered significant when p < 0.05. Results The MTT assay revealed a significant decrease in M1 metabolism with MTA-HP at 24 hours, and with MTA and MTA-HP later. The trypan blue assay showed significantly fewer live M1 at 48 hours and live M2 at 48 and 72 hours with MTA-HP, compared to MTA. M1 and M2 adherence and phagocytosis showed no significant differences compared to control for both materials. Zymosan A stimulated ROS production by macrophages. In the absence of interferon-γ, TNF-α production by M1 did not significantly differ between groups. For M2, both materials showed higher TNF-α production in the presence of the stimulus, but without significant between-group differences. Likewise, TGF-β production by M1 and M2 macrophages was not significantly different between the groups. Conclusions M1 and M2 macrophages presented different viability in response to MTA and MTA-HP at different time points. Introducing a plasticizer into the MTA vehicle did not interfere with the activity of M1 and M2 macrophages. Objectives This study evaluated the effects of high-plasticity mineral trioxide aggregate (MTA-HP) on the activity of M1 and M2 macrophages, compared to white MTA (Angelus). Materials and Methods Peritoneal inflammatory M1 (from C57BL/6 mice) and M2 (from BALB/c mice) macrophages were cultured in the presence of the tested materials. Cell viability (MTT and trypan blue assays), adhesion, phagocytosis, reactive oxygen species (ROS) production, and tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β production were evaluated. Parametric analysis of variance and the non-parametric Kruskal-Wallis test were used. Results were considered significant when p < 0.05. Results The MTT assay revealed a significant decrease in M1 metabolism with MTA-HP at 24 hours, and with MTA and MTA-HP later. The trypan blue assay showed significantly fewer live M1 at 48 hours and live M2 at 48 and 72 hours with MTA-HP, compared to MTA. M1 and M2 adherence and phagocytosis showed no significant differences compared to control for both materials. Zymosan A stimulated ROS production by macrophages. In the absence of interferon-γ, TNF-α production by M1 did not significantly differ between groups. For M2, both materials showed higher TNF-α production in the presence of the stimulus, but without significant between-group differences. Likewise, TGF-β production by M1 and M2 macrophages was not significantly different between the groups. Conclusions M1 and M2 macrophages presented different viability in response to MTA and MTA-HP at different time points. Introducing a plasticizer into the MTA vehicle did not interfere with the activity of M1 and M2 macrophages.

The Macrophage-Specific Transcription Factor Can Be Modified Posttranslationally by Ubiquitination in the Lipopolysaccharide-Treated Macrophages

Jung, Jae-Woo,Choi, Jae-Chol,Kim, Jae-Yeol,Park, In-Won,Choi, Byoung-Whui,Shin, Jong-Wook,Christman, John William The Korean Academy of Tuberculosis and Respiratory 2011 Tuberculosis and Respiratory Diseases Vol.70 No.2

Background: Macrophages are one of the most important inflammatory cells in innate immunity. PU.1 is a macrophage-specific transcription factor. Ubiquitins are the ultimate regulator of eukaryotic transcription. The ubiquitination process for PU.1 is unknown. This study investigated the lipopolysaccharide (LPS)-induced activation of PU.1 and its relation to ubiquitins in the macrophages. Methods: Raw264.7 cells, the primary cultured alveolar, pulmonary, and bone marrow derived macrophages were used. The Raw264.7 cells were treated with MG-132, $NH_4Cl$, lactacytin and LPS. Nitric oxide and prostaglandin D2 and E2 were measured. Immunoprecipitation and Western blots were used to check ubiquitination of PU.1. Results: The PU.1 ubiquitination increased after LPS ($1{\mu}g$/mL) treatment for 4 hours on Raw264.7 cells. The ubiquitination of PU.1 by LPS was increased by MG-132 or $NH_4Cl$ pretreatment. Two hours of LPS treatment on macrophages, PU.1 activation was not induced nor increased with the inhibition of proteasomes and/or lysosomes. The ubiquitination of PU.1 was increased in LPS-treated Raw264.7 cells at 12- and at 24 hours. LPS-treated cells increased nitric oxide production, which was diminished by MG-132 or $NH_4Cl$. LPS increased the production of $PGE_2$ in the alveolar and peritoneal macrophages of wild type mice; however, $PGE_2$ was blocked or diminished in Rac2 null mice. Pretreatment of lactacystin increased $PGE_2$, however it decreased the $PGD_2$ level in the macrophages derived from the bone marrow of B57/BL6 mice. Conclusion: LPS treatment in the macrophages ubiquitinates PU.1. Ubiquitination of PU.1 may be involved in synthesis of nitric oxide and prostaglandins.

CD11b Deficiency Exacerbates Methicillin-Resistant Staphylococcus aureus-Induced Sepsis by Upregulating Inflammatory Responses of Macrophages

심현섭,정대철,김혜인,박성원,THAPABIKASH,권형주,이근욱 대한면역학회 2021 Immune Network Vol.21 No.2

Macrophages are important for the first line of defense against microbial pathogens. Integrin CD11b, which is encoded by Itgam, is expressed on the surface of macrophages and has been implicated in adhesion, migration, and cell-mediated cytotoxicity. However, the functional impact of CD11b on the inflammatory responses of macrophages upon microbial infection remains unclear. Here, we show that CD11b deficiency resulted in increased susceptibility to sepsis induced by methicillin-resistant Staphylococcus aureus (MRSA) infection by enhancing the pro-inflammatory activities of macrophages. Upon infection with MRSA, the mortality of Itgam knockout mice was significantly higher than that of control mice, which is associated with increased production of TNF-α and IL-6. In response to MRSA, both bone marrow-derived macrophages and peritoneal macrophages lacking CD11b produced elevated amounts of pro-inflammatory cytokines and nitric oxide. Moreover, CD11b deficiency upregulated IL-4-induced expression of anti-inflammatory mediators such as IL-10 and arginase-1, and an immunomodulatory function of macrophages to restrain T cell activation. Biochemical and confocal microscopy data revealed that CD11b deficiency augmented the activation of NF-κB signaling and phosphorylation of Akt, which promotes the functional activation of macrophages with pro-inflammatory and immunoregulatory phenotypes, respectively. Overall, our experimental evidence suggests that CD11b is a critical modulator of macrophages in response to microbial infection.

The Macrophage-Specific Transcription Factor Can Be Modified Posttranslationally by Ubiquitination in the Lipopolysaccharide-Treated Macrophages

( Jae Woo Jung ),( Jae Chol Choi ),( Jae Yeol Kim ),( In Won Park ),( Byoung Whui Choi ),( Jong Wook Shin ),( John William Christman ) 대한결핵 및 호흡기학회 2011 Tuberculosis and Respiratory Diseases Vol.70 No.2

Background: Macrophages are one of the most important inflammatory cells in innate immunity. PU.1 is a macrophage-specific transcription factor. Ubiquitins are the ultimate regulator of eukaryotic transcription. The ubiquitination process for PU.1 is unknown. This study investigated the lipopolysaccharide (LPS)-induced activation of PU.1 and its relation to ubiquitins in the macrophages. Methods: Raw264.7 cells, the primary cultured alveolar, pulmonary, and bone marrow derived macrophages were used. The Raw264.7 cells were treated with MG-132, NH4Cl, lactacytin and LPS. Nitric oxide and prostaglandin D2 and E2 were measured. Immunoprecipitation and Western blots were used to check ubiquitination of PU.1. Results: The PU.1 ubiquitination increased after LPS (1ug/mL) treatment for 4 hours on Raw264.7 cells. The ubiquitination of PU.1 by LPS was increased by MG-132 or NH4Cl pretreatment. Two hours of LPS treatment on macrophages, PU.1 activation was not induced nor increased with the inhibition of proteasomes and/or lysosomes. The ubiquitination of PU.1 was increased in LPS-treated Raw264.7 cells at 12- and at 24 hours. LPS-treated cells increased nitric oxide production, which was diminished by MG-132 or NH4Cl. LPS increased the production of PGE2 in the alveolar and peritoneal macrophages of wild type mice; however, PGE2 was blocked or diminished in Rac2 null mice. Pretreatment of lactacystin increased PGE2, however it decreased the PGD2 level in the macrophages derived from the bone marrow of B57/BL6 mice. Conclusion: LPS treatment in the macrophages ubiquitinates PU.1. Ubiquitination of PU.1 may be involved in synthesis of nitric oxide and prostaglandins.

Repression of PPARγ Reduces the ABCG2-mediated Efflux Activity of M2 Macrophages

( Chae Eun Kim ),( Ha Young Park ),( Jae-gook Shin ),( Su-kil Seo ) 대한결핵 및 호흡기학회 2020 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.128 No.-

Background Even though subclasses of macrophage have distinct roles during progression of infectious diseases, it remains poorly understood whether there is a subset-specific difference in drug responses. Here, we report that ABCG2 was expressed specifically in M2-like macrophages and that it controlled their efflux activities. Methods Macrophage subsets (M1/M2-like macrophages) are generated from THP-1 cells and CD14+ monocytes by the nomenclature of in vitro. ABCG2 expression is analyzed by real-time PCR and flow cytometry. The intracellular concentration is measured using Hoechst 33342 efflux assay. The binding of PPARγ in the Abcg2 promoter region is measured using chromatin immunoprecipitation assay. Results Abcg2 expression is markedly induced during polarization of PMA-primed macrophages toward an M2 type. IL-4 and IL- 13 induced Pparg expression through STAT6 and PPARγ in turn acted on the Abcg2 promoter for its transcription activation. Once polarized to M2-like macrophages, these cells had sustained PPARγ transcription activation of Abcg2 gene. Accordingly, interruption of this machinery by T0070907, an inverse agonist of PPARγ, was shown to be effective in Abcg2 downregulation and its efflux activity in M2-like macrophages. Conclusions Our Results implicate that ABCG2 of M2 macrophages may function as an important pump that plays a potential role in drug efflux and that T0070907 may be used to increase the efficacy of M2 macrophage-targeting drugs such as antibiotics.