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International Journal of Molecular... Jun 2024Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an...
Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an outstanding contribution from the team of Prof. Greg Lemke. MerTK activity is implicated in a wide variety of functions involving the elimination of apoptotic cells and has recently been linked to cancers, auto-immune diseases, and atherosclerosis/stroke. In the retina, MerTK is required for the circadian phagocytosis of oxidized photoreceptor outer segments by the retinal-pigment epithelial cells, a function crucial for the long-term maintenance of vision. We previously showed that MerTK ligands carry the opposite role in vitro, with Gas6 inhibiting the internalization of photoreceptor outer segments while Protein S acts conversely. Using site-directed mutagenesis and ligand-stimulated phagocytosis assays on transfected cells, we presently demonstrate, for the first time, that Gas6 and Protein S recognize different amino acids on MerTK Ig-like domains. In addition, MerTK's function in retinal-pigment epithelial cells is rhythmic and might thus rely on the respective stoichiometry of both ligands at different times of the day. Accordingly, we show that ligand bioavailability varies during the circadian cycle using RT-qPCR and immunoblots on retinal and retinal-pigment epithelial samples from control and beta5 integrin knockout mice where retinal phagocytosis is arrhythmic. Taken together, our results suggest that Gas6 and Protein S might both contribute to refine the acute regulation of MerTK in time for the daily phagocytic peak.
Topics: c-Mer Tyrosine Kinase; Phagocytosis; Animals; Intercellular Signaling Peptides and Proteins; Protein S; Humans; Retina; Mice; Circadian Rhythm; Ligands; Retinal Pigment Epithelium; Receptor Protein-Tyrosine Kinases
PubMed: 38928335
DOI: 10.3390/ijms25126630 -
International Journal of Molecular... Jun 2024CD147 is upregulated in cancers, including aggressive T-ALL. Traditional treatments for T-ALL often entail severe side effects and the risk of relapse, highlighting the...
Engineered CD147-Deficient THP-1 Impairs Monocytic Myeloid-Derived Suppressor Cell Differentiation but Maintains Antibody-Dependent Cellular Phagocytosis Function for Jurkat T-ALL Cells with Humanized Anti-CD147 Antibody.
CD147 is upregulated in cancers, including aggressive T-ALL. Traditional treatments for T-ALL often entail severe side effects and the risk of relapse, highlighting the need for more efficacious therapies. ADCP contributes to the antitumor response by enhancing the ability of phagocytic cells to engulf cancer cells upon antibody binding. We aimed to engineer CD147 THP-1 cells and evaluated their differentiation properties compared to the wild type. A humanized anti-CD147 antibody, HuM6-1B9, was also constructed for investing the phagocytic function of CD147 THP-1 cells mediated by HuM6-1B9 in the phagocytosis of Jurkat T cells. The CD147 THP-1 was generated by CRISPR/Cas9 and maintained polarization profiles. HuM6-1B9 was produced in CHO-K1 cells and effectively bound to CD147 with high binding affinity (K: 2.05 ± 0.30 × 10 M). Additionally, HuM6-1B9 enhanced the phagocytosis of Jurkat T cells by CD147 THP-1-derived LPS-activated macrophages (M-LPS), without self-ADCP. The formation of THP-1-derived mMDSC was limited in CD147 THP-1 cells, highlighting the significant impact of CD147 deletion. Maintaining expression markers and phagocytic function in CD147 THP-1 macrophages supports future engineering and the application of induced pluripotent stem cell-derived macrophages. The combination of HuM6-1B9 and CD147 monocyte-derived macrophages holds promise as an alternative strategy for T-ALL.
Topics: Humans; Phagocytosis; Jurkat Cells; Basigin; Cell Differentiation; THP-1 Cells; Myeloid-Derived Suppressor Cells; Antibodies, Monoclonal, Humanized; Animals; CHO Cells; Cricetulus; Monocytes; Macrophages; CRISPR-Cas Systems
PubMed: 38928332
DOI: 10.3390/ijms25126626 -
International Journal of Molecular... Jun 2024Obesity and metabolic syndrome alter serum lipid profiles. They also increase vulnerability to viral infections and worsen the survival rate and symptoms after...
Obesity and metabolic syndrome alter serum lipid profiles. They also increase vulnerability to viral infections and worsen the survival rate and symptoms after infection. How serum lipids affect influenza virus proliferation is unclear. Here, we investigated the effects of lysophosphatidylcholines on influenza A virus (IAV) proliferation. IAV particles in the culture medium were titrated using extraction-free quantitative PCR, and viral RNA and protein levels were assessed using real-time PCR and Western blot, respectively. RNA sequencing data were analyzed using PCA and heatmap analysis, and pathway analysis was performed using the KEGG mapper and PathIN tools. Statistical analysis was conducted using SPSS21.0. LPC treatment of THP-1 cells significantly increased IAV proliferation and IAV RNA and protein levels, and saturated LPC was more active in IAV RNA expression than unsaturated LPC was. The functional analysis of genes affected by LPCs showed that the expression of genes involved in IAV signaling, such as suppressor of cytokine signaling 3 (SOCS3), phosphoinositide-3-kinase regulatory subunit 3 (PI3K) and AKT serine/threonine kinase 3 (AKT3), Toll-like receptor 7 (TKR7), and interferon gamma receptor 1 (IFNGR1), was changed by LPC. Altered influenza A pathways were linked with MAPK and PI3K/AKT signaling. Treatment with inhibitors of MAPK or PI3K attenuated viral gene expression changes induced by LPCs. The present study shows that LPCs stimulated virus reproduction by modifying the cellular environment to one in which viruses proliferated better. This was mediated by the MAPK, JNK, and PI3K/AKT pathways. Further animal studies are needed to confirm the link between LPCs from serum or the respiratory system and IAV proliferation.
Topics: Humans; Lysophosphatidylcholines; Virus Replication; MAP Kinase Signaling System; Influenza A virus; Macrophages; THP-1 Cells; Cell Differentiation; Influenza, Human; Signal Transduction; Animals
PubMed: 38928244
DOI: 10.3390/ijms25126538 -
International Journal of Molecular... Jun 2024Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to...
Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.
Topics: Animals; Sepsis; Neutrophils; Glycosylation; Immunoglobulin G; Mice; Disease Models, Animal; Cytokines; Immunoglobulin Fc Fragments; Mice, Inbred C57BL; Leukocyte Elastase; Male; Extracellular Traps; Glycoproteins
PubMed: 38928183
DOI: 10.3390/ijms25126478 -
International Journal of Molecular... Jun 2024Obesity is a risk factor for type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). Adipose tissue (AT) extracellular vesicles (EVs) could play a role in...
Extracellular Vesicles Secreted by Adipose Tissue during Obesity and Type 2 Diabetes Mellitus Influence Reverse Cholesterol Transport-Related Gene Expression in Human Macrophages.
Obesity is a risk factor for type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). Adipose tissue (AT) extracellular vesicles (EVs) could play a role in obesity and T2DM associated CVD progression via the influence of their specific cargo on gene expression in recipient cells. The aim of this work was to evaluate the effects of AT EVs of patients with obesity with/without T2DM on reverse cholesterol transport (RCT)-related gene expression in human monocyte-derived macrophages (MDMs) from healthy donors. AT EVs were obtained after ex vivo cultivation of visceral and subcutaneous AT (VAT and SAT, respectively). , , , , and mRNA levels in MDMs as well as in origine AT were determined by a real-time PCR. T2DM VAT and SAT EVs induced gene expression whereas and mRNA levels were simultaneously downregulated. mRNA levels also decreased in the presence of VAT EVs of obese patients without T2DM. In contrast and mRNA levels tended to increase with the addition of obese AT EVs. Thus, AT EVs can influence RCT gene expression in MDMs during obesity, and the effects are dependent on T2DM status.
Topics: Humans; Diabetes Mellitus, Type 2; Extracellular Vesicles; Obesity; Liver X Receptors; Macrophages; Cholesterol; ATP Binding Cassette Transporter, Subfamily G, Member 1; Adipose Tissue; PPAR gamma; Female; ATP Binding Cassette Transporter 1; Male; Middle Aged; Biological Transport; Gene Expression Regulation; Adult; RNA, Messenger
PubMed: 38928163
DOI: 10.3390/ijms25126457 -
International Journal of Molecular... Jun 2024We review the importance of monocytic differentiation and differentiation induction in non-APL (acute promyelocytic leukemia) variants of acute myeloid leukemia (AML), a... (Review)
Review
Monocytic Differentiation in Acute Myeloid Leukemia Cells: Diagnostic Criteria, Biological Heterogeneity, Mitochondrial Metabolism, Resistance to and Induction by Targeted Therapies.
We review the importance of monocytic differentiation and differentiation induction in non-APL (acute promyelocytic leukemia) variants of acute myeloid leukemia (AML), a malignancy characterized by proliferation of immature myeloid cells. Even though the cellular differentiation block is a fundamental characteristic, the AML cells can show limited signs of differentiation. According to the French-American-British (FAB-M4/M5 subset) and the World Health Organization (WHO) 2016 classifications, monocytic differentiation is characterized by morphological signs and the expression of specific molecular markers involved in cellular communication and adhesion. Furthermore, monocytic FAB-M4/M5 patients are heterogeneous with regards to cytogenetic and molecular genetic abnormalities, and monocytic differentiation does not have any major prognostic impact for these patients when receiving conventional intensive cytotoxic therapy. In contrast, FAB-M4/M5 patients have decreased susceptibility to the Bcl-2 inhibitor venetoclax, and this seems to be due to common molecular characteristics involving mitochondrial regulation of the cellular metabolism and survival, including decreased dependency on Bcl-2 compared to other AML patients. Thus, the susceptibility to Bcl-2 inhibition does not only depend on general resistance/susceptibility mechanisms known from conventional AML therapy but also specific mechanisms involving the molecular target itself or the molecular context of the target. AML cell differentiation status is also associated with susceptibility to other targeted therapies (e.g., CDK2/4/6 and bromodomain inhibition), and differentiation induction seems to be a part of the antileukemic effect for several targeted anti-AML therapies. Differentiation-associated molecular mechanisms may thus become important in the future implementation of targeted therapies in human AML.
Topics: Humans; Cell Differentiation; Leukemia, Myeloid, Acute; Mitochondria; Monocytes; Drug Resistance, Neoplasm; Molecular Targeted Therapy; Antineoplastic Agents
PubMed: 38928061
DOI: 10.3390/ijms25126356 -
International Journal of Molecular... Jun 2024Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of...
Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.
Topics: Lipopolysaccharides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Animals; Mice; Macrophages; Lipid A; Cytokines; Endopeptidase K; Electrophoresis, Polyacrylamide Gel
PubMed: 38928052
DOI: 10.3390/ijms25126345 -
Genes Jun 2024Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting the brain and spinal cord. Non-neuronal cells, including macrophages, may contribute to the...
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting the brain and spinal cord. Non-neuronal cells, including macrophages, may contribute to the disruption of motor neurons (MNs), neuromuscular junction dismantling and clinical signs of ALS. Understanding the modality and the effect of MNs-macrophage communication is pivotal. Here, we focus on extracellular vesicle (EVS)-mediated communication and, in particular, we analyze the response of macrophages. NSC-34 cells transfected with mutant SOD1 (G93A, A4V, G85R, G37R) and differentiated towards MN-like cells, and Raw 264.7 macrophages are the cellular models of the study. mSOD1 NSC-34 cells release a high number of vesicles, both large-lEVs (300 nm diameter) and small-sEVs (90 nm diameter), containing inflammation-modulating molecules, and are efficiently taken up by macrophages. RT-PCR analysis of inflammation mediators demonstrated that the conditioned medium of mSOD1 NSC-34 cells polarizes Raw 264.7 macrophages towards both pro-inflammatory and anti-inflammatory phenotypes. sEVs act on macrophages in a time-dependent manner: an anti-inflammatory response mediated by TGFβ firstly starts (12 h); successively, the response shifts towards a pro-inflammation IL-1β-mediated (48 h). The response of macrophages is strictly dependent on the SOD1 mutation type. The results suggest that EVs impact physiological and behavioral macrophage processes and are of potential relevance to MN degeneration.
Topics: Animals; Extracellular Vesicles; Mice; RAW 264.7 Cells; Superoxide Dismutase-1; Macrophages; Amyotrophic Lateral Sclerosis; Motor Neurons; Inflammation; Mutation; Transfection; Humans
PubMed: 38927671
DOI: 10.3390/genes15060735 -
Genes May 2024Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease mainly caused by mutations in the X-linked gene that abrogate reactive oxygen species (ROS)...
Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease mainly caused by mutations in the X-linked gene that abrogate reactive oxygen species (ROS) production in phagocytes and microbial defense. Gene repair using the CRISPR/Cas9 system in hematopoietic stem and progenitor cells (HSPCs) is a promising technology for therapy for CGD. To support the establishment of efficient and safe gene therapies for CGD, we generated a mouse model harboring a patient-derived mutation in the gene. Our CybbC517del mouse line shows the hallmarks of CGD and provides a source for Cybb-deficient HSPCs that can be used to evaluate gene-therapy approaches in vitro and in vivo. In a setup using Cas9 RNPs and an AAV repair vector in HSPCs, we show that the mutation can be repaired in 19% of treated cells and that treatment restores ROS production by macrophages. In conclusion, our CybbC517del mouse line provides a new platform for refining and evaluating novel gene therapies and studying X-CGD pathophysiology.
Topics: Granulomatous Disease, Chronic; Animals; Genetic Therapy; Mice; CRISPR-Cas Systems; Disease Models, Animal; NADPH Oxidase 2; Reactive Oxygen Species; Hematopoietic Stem Cells; Humans; Macrophages; Mutation
PubMed: 38927642
DOI: 10.3390/genes15060706 -
Biomedicines Jun 2024Hematoma clearance is critical for mitigating intracerebral hemorrhage (ICH)-induced brain injury. Multinucleated giant cells (MGCs), a type of phagocyte, and the...
Hematoma clearance is critical for mitigating intracerebral hemorrhage (ICH)-induced brain injury. Multinucleated giant cells (MGCs), a type of phagocyte, and the complement system may play a pivotal role in hematoma resolution, but whether the complement system regulates MGC formation after ICH remains unclear. The current study investigated the following: (1) the characteristics of MGC formation after ICH, (2) whether it was impacted by complement C3 deficiency in mice and (3) whether it also influenced hematoma degradation (hemosiderin formation). Young and aged male mice, young female mice and C3-deficient and -sufficient mice received a 30 μL injection of autologous whole blood into the right basal ganglia. Brain histology and immunohistochemistry were used to examine MGC formation on days 3 and 7. Hemosiderin deposition was examined by autofluorescence on day 28. Following ICH, MGCs were predominantly located in the peri-hematoma region exhibiting multiple nuclei and containing red blood cells or their metabolites. Aging was associated with a decrease in MGC formation after ICH, while sex showed no discernible effect. C3 deficiency reduced MGC formation and reduced hemosiderin formation. Peri-hematomal MGCs may play an important role in hematoma resolution. Understanding how aging and complement C3 impact MGCs may provide important insights into how to regulate hematoma resolution.
PubMed: 38927458
DOI: 10.3390/biomedicines12061251