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1.
FASEB J ; 36(12): e22622, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36421039

ABSTRACT

Diabetes shortens the life expectancy by more than a decade, and the excess mortality in diabetes is correlated with the incidence of kidney disease. Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. Macrophage accumulation predicts the severity of kidney injury in human biopsies and experimental models of DKD. However, the mechanism underlying macrophage recruitment in diabetes glomeruli is unclear. Elevated plasma growth hormone (GH) levels in type I diabetes and acromegalic individuals impaired glomerular biology. In this study, we examined whether GH-stimulated podocytes contribute to macrophage accumulation. RNA-seq analysis revealed elevated TNF-α signaling in GH-treated human podocytes. Conditioned media from GH-treated podocytes (GH-CM) induced differentiation of monocytes to macrophages. On the other hand, neutralization of GH-CM with the TNF-α antibody diminished GH-CM's action on monocytes. The treatment of mice with GH resulted in increased macrophage recruitment, podocyte injury, and proteinuria. Furthermore, we noticed the activation of TNF-α signaling, macrophage accumulation, and fibrosis in DKD patients' kidney biopsies. Our findings suggest that podocytes could secrete TNF-α and contribute to macrophage migration, resulting in DKD-related renal inflammation. Inhibition of either GH action or TNF-α expression in podocytes could be a novel therapeutic approach for DKD treatment.


Subject(s)
Diabetic Nephropathies , Monocytes , Podocytes , Tumor Necrosis Factor-alpha , Animals , Humans , Mice , Monocytes/cytology , Podocytes/metabolism , Tumor Necrosis Factor-alpha/metabolism , Cell Differentiation
2.
Nature ; 609(7925): 166-173, 2022 09.
Article in English | MEDLINE | ID: mdl-35948634

ABSTRACT

During infection, inflammatory monocytes are thought to be key for bacterial eradication, but this is hard to reconcile with the large numbers of neutrophils that are recruited for each monocyte that migrates to the afflicted tissue, and the much more robust microbicidal functions of the neutrophils. However, unlike neutrophils, monocytes have the capacity to convert to situationally specific macrophages that may have critical functions beyond infection control1,2. Here, using a foreign body coated with Staphylococcus aureus and imaging over time from cutaneous infection to wound resolution, we show that monocytes and neutrophils are recruited in similar numbers with low-dose infection but not with high-dose infection, and form a localization pattern in which monocytes surround the infection site, whereas neutrophils infiltrate it. Monocytes did not contribute to bacterial clearance but converted to macrophages that persisted for weeks after infection, regulating hypodermal adipocyte expansion and production of the adipokine hormone leptin. In infected monocyte-deficient mice there was increased persistent hypodermis thickening and an elevated leptin level, which drove overgrowth of dysfunctional blood vasculature and delayed healing, with a thickened scar. Ghrelin, which opposes leptin function3, was produced locally by monocytes, and reduced vascular overgrowth and improved healing post-infection. In sum, we find that monocytes function as a cellular rheostat by regulating leptin levels and revascularization during wound repair.


Subject(s)
Leptin , Monocytes , Neovascularization, Physiologic , Staphylococcal Infections , Staphylococcus aureus , Wound Healing , Adipocytes/cytology , Adipocytes/metabolism , Animals , Cicatrix , Ghrelin/metabolism , Leptin/metabolism , Macrophages/cytology , Macrophages/metabolism , Mice , Monocytes/cytology , Monocytes/metabolism , Neutrophils/cytology , Neutrophils/immunology , Staphylococcal Infections/metabolism , Staphylococcal Infections/microbiology , Staphylococcal Infections/pathology , Staphylococcus aureus/physiology
3.
J Biol Chem ; 298(8): 102153, 2022 08.
Article in English | MEDLINE | ID: mdl-35718062

ABSTRACT

The generation of bispecific antibodies (bsAbs) targeting two different antigens opens a new level of specificity and, compared to mAbs, improved clinical efficacy in cancer therapy. Currently, the different strategies for development of bsAbs primarily focus on IgG isotypes. Nevertheless, in comparison to IgG isotypes, IgE has been shown to offer superior tumor control in preclinical models. Therefore, in order to combine the promising potential of IgE molecules with increased target selectivity of bsAbs, we developed dual tumor-associated antigen-targeting bispecific human IgE antibodies. As proof of principle, we used two different pairing approaches - knobs-into-holes and leucine zipper-mediated pairing. Our data show that both strategies were highly efficient in driving bispecific IgE formation, with no undesired pairings observed. Bispecific IgE antibodies also showed a dose-dependent binding to their target antigens, and cell bridging experiments demonstrated simultaneous binding of two different antigens. As antibodies mediate a major part of their effector functions through interaction with Fc receptors (FcRs) expressed on immune cells, we confirmed FcεR binding by inducing in vitro mast cell degranulation and demonstrating in vitro and in vivo monocyte-mediated cytotoxicity against target antigen-expressing Chinese hamster ovary cells. Moreover, we demonstrated that the IgE bsAb construct was significantly more efficient in mediating antibody-dependent cell toxicity than its IgG1 counterpart. In conclusion, we describe the successful development of first bispecific IgE antibodies with superior antibody-dependent cell toxicity-mediated cell killing in comparison to IgG bispecific antibodies. These findings highlight the relevance of IgE-based bispecific antibodies for clinical application.


Subject(s)
Antibodies, Bispecific , Antineoplastic Agents, Immunological , Immunoglobulin E , Monocytes , Animals , Antibodies, Bispecific/pharmacology , Antineoplastic Agents, Immunological/pharmacology , CHO Cells , Cricetinae , Cricetulus , Humans , Immunoglobulin E/pharmacology , Monocytes/cytology
4.
Nature ; 607(7919): 578-584, 2022 07.
Article in English | MEDLINE | ID: mdl-35636458

ABSTRACT

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.


Subject(s)
Brain , Fear , Leukocytes , Motor Neurons , Neural Pathways , Stress, Psychological , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Brain/cytology , Brain/physiology , COVID-19/immunology , Chemokines/immunology , Disease Susceptibility , Fear/physiology , Glucocorticoids/metabolism , Humans , Leukocytes/cytology , Leukocytes/immunology , Lymphocytes/cytology , Lymphocytes/immunology , Lymphoid Tissue/cytology , Lymphoid Tissue/immunology , Mice , Monocytes/cytology , Monocytes/immunology , Motor Neurons/cytology , Motor Neurons/physiology , Neutrophils/cytology , Neutrophils/immunology , Optogenetics , Orthomyxoviridae Infections/immunology , Paraventricular Hypothalamic Nucleus/physiology , SARS-CoV-2/immunology , Stress, Psychological/immunology , Stress, Psychological/physiopathology
5.
Int J Mol Sci ; 23(10)2022 May 20.
Article in English | MEDLINE | ID: mdl-35628558

ABSTRACT

Platelets (PLT) bind to a significant percentage of circulating monocytes and this immunomodulatory interaction is increased in several inflammatory and autoimmune conditions. The therapeutic blockage of IL-6 with Tocilizumab (TCZ) alters PLT and the phenotype and function of monocytes in rheumatoid arthritis (RA). However, the relationship between monocyte-PLT conjugates (CD14+PLT+) and clinical and immunological variables and the regulation of this interaction by IL-6 blockage are still unknown. Here, we compared the presence of monocyte-PLT conjugates (CD14+PLT+) and membrane CD162 expression using flow cytometry, and, by ELISA, the markers of PLT activation (sCD62P and sCD40L) in healthy donors (HD) and patients with long-standing RA before TCZ (baseline). We found higher percentages and absolute counts of CD14+PLT+, and higher plasmatic levels of sCD62P and sCD40L but lower CD162 expression on monocytes from RA patients than those from HD. Additionally, the levels of CD14+PLT+ inversely correlated with inflammatory parameters. Interestingly, 95% of patients with lower percentages of CD14+PLT+ and only 63% of patients with higher percentages of CD14+PLT+ achieved a EULAR-defined response at four weeks (p = 0.036). After TCZ, the percentage of CD14+PLT+ increased in 92% of RA patients who achieved 12 w-remission (p < 0.001). Our results suggest that the binding of PLTs has a modulatory effect, accentuated by the increased binding of PLTs to monocytes in response to the therapeutic blockage of IL-6.


Subject(s)
Antibodies, Monoclonal, Humanized , Arthritis, Rheumatoid , Blood Platelets , Monocytes , Antibodies, Monoclonal, Humanized/therapeutic use , Arthritis, Rheumatoid/drug therapy , Blood Platelets/cytology , Flow Cytometry , Humans , Interleukin-6/antagonists & inhibitors , Monocytes/cytology
6.
Front Immunol ; 13: 826047, 2022.
Article in English | MEDLINE | ID: mdl-35401562

ABSTRACT

Opening of the endothelial barrier and targeted infiltration of leukocytes into the affected tissue are hallmarks of the inflammatory response. The molecular mechanisms regulating these processes are still widely elusive. In this study, we elucidate a novel regulatory network, in which miR-125a acts as a central hub that regulates and synchronizes both endothelial barrier permeability and monocyte migration. We found that inflammatory stimulation of endothelial cells induces miR-125a expression, which consecutively inhibits a regulatory network consisting of the two adhesion molecules VE-Cadherin (CDH5) and Claudin-5 (CLDN5), two regulatory tyrosine phosphatases (PTPN1, PPP1CA) and the transcription factor ETS1 eventually leading to the opening of the endothelial barrier. Moreover, under the influence of miR-125a, endothelial expression of the chemokine CCL2, the most predominant ligand for the monocytic chemokine receptor CCR2, was strongly enhanced. In monocytes, on the other hand, we detected markedly repressed expression levels of miR-125a upon inflammatory stimulation. This induced a forced expression of its direct target gene CCR2, entailing a strongly enhanced monocyte chemotaxis. Collectively, cell-type-specific differential expression of miR-125a forms a synergistic functional network controlling monocyte trafficking across the endothelial barrier towards the site of inflammation. In addition to the known mechanism of miRNAs being shuttled between cells via extracellular vesicles, our study uncovers a novel dimension of miRNA function: One miRNA, although disparately regulated in the cells involved, directs a biologic process in a synergistic and mutually reinforcing manner. These findings provide important new insights into the regulation of the inflammatory cascade and may be of great use for future clinical applications.


Subject(s)
MicroRNAs , Monocytes , Endothelial Cells/cytology , Humans , Inflammation/metabolism , MicroRNAs/genetics , Monocytes/cytology , Permeability
7.
FASEB J ; 36(4): e22218, 2022 04.
Article in English | MEDLINE | ID: mdl-35218567

ABSTRACT

An immunoregulatory role of stem cells, often mediated by their secretome, has been claimed by several studies. Stem cell-derived extracellular vesicles (EVs) are crucial components of the secretome. EVs, a heterogeneous group of membranous vesicles released by many cell types into the extracellular space, are now considered as an additional mechanism for intercellular communication. In this study, we aimed at investigating whether human amniotic stem cell-derived extracellular vesicles (HASC-EVs) were able to interfere with inflammasome activation in the THP-1 cell line. Two subsets of HASC-EVs were collected by sequential centrifugation, namely HASC-P10 and HASC-P100. We demonstrated that HASC-EVs were neither internalized into nor undertake a direct interaction with THP-1 cells. We showed that HASC-P10 and P100 were able to intrinsically produce ATP, which was further converted to adenosine by 5'-nucleotidase (CD73) and ectonucleoside triphosphate diphosphohydrolase-1 (CD39). We found that THP-1 cells conditioned with both types of HASC-EVs failed to activate the NLRP3/caspase-1/inflammasome platform in response to LPS and ATP treatment by a mechanism involving A2a adenosine receptor activation. These results support a role for HASC-EVs as independent metabolic units capable of modifying the cellular functions, leading to anti-inflammatory effects in monocytic cells.


Subject(s)
Amniotic Fluid/cytology , Anti-Inflammatory Agents/pharmacology , Extracellular Vesicles/metabolism , Inflammasomes/antagonists & inhibitors , Inflammation/prevention & control , Monocytes/cytology , Stem Cells/cytology , Adenosine/metabolism , Amniotic Fluid/metabolism , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Monocytes/metabolism , Purinergic P1 Receptor Antagonists/pharmacology , Receptors, Purinergic P1/chemistry , Receptors, Purinergic P1/metabolism , Stem Cells/metabolism , THP-1 Cells
8.
Front Immunol ; 13: 842535, 2022.
Article in English | MEDLINE | ID: mdl-35185933

ABSTRACT

Myeloid-derived suppressor cells (MDSCs) are generated under biological stress such as cancer, inflammatory tissue damage, and viral infection. In recent years, with occurrence of global infectious diseases, new discovery on MDSCs functions has been significantly expanded during viral infection and COVID-19. For a successful viral infection, pathogens viruses develop immune evasion strategies to avoid immune recognition. Numerous viruses induce the differentiation and expansion of MDSCs in order to suppress host immune responses including natural killer cells, antigen presenting cells, and T-cells. Moreover, MDSCs play an important role in regulation of immunopathogenesis by balancing viral infection and tissue damage. In this review article, we describe the overview of immunomodulation and genetic regulation of MDSCs during viral infection in the animal model and human studies. In addition, we include up-to-date review of role of MDSCs in SARS-CoV-2 infection and COVID-19. Finally, we discuss potential therapeutics targeting MDSCs.


Subject(s)
Immunomodulation/immunology , Macrophages/immunology , Myeloid-Derived Suppressor Cells/immunology , Neutrophils/immunology , SARS-CoV-2/immunology , Animals , COVID-19/immunology , Disease Models, Animal , Humans , Immune Evasion/immunology , Macrophages/cytology , Monocytes/cytology , Monocytes/immunology , Myeloid-Derived Suppressor Cells/cytology
9.
Front Immunol ; 13: 827719, 2022.
Article in English | MEDLINE | ID: mdl-35145525

ABSTRACT

The lung tumor microenvironment plays a critical role in the tumorigenesis and metastasis of lung cancer, resulting from the crosstalk between cancer cells and microenvironmental cells. Therefore, comprehensive identification and characterization of cell populations in the complex lung structure is crucial for development of novel targeted anti-cancer therapies. Here, a hierarchical clustering approach with multispectral flow cytometry was established to delineate the cellular landscape of murine lungs under steady-state and cancer conditions. Fluorochromes were used multiple times to be able to measure 24 cell surface markers with only 13 detectors, yielding a broad picture for whole-lung phenotyping. Primary and metastatic murine lung tumor models were included to detect major cell populations in the lung, and to identify alterations to the distribution patterns in these models. In the primary tumor models, major altered populations included CD324+ epithelial cells, alveolar macrophages, dendritic cells, and blood and lymph endothelial cells. The number of fibroblasts, vascular smooth muscle cells, monocytes (Ly6C+ and Ly6C-) and neutrophils were elevated in metastatic models of lung cancer. Thus, the proposed clustering approach is a promising method to resolve cell populations from complex organs in detail even with basic flow cytometers.


Subject(s)
Flow Cytometry/methods , Fluorescent Dyes/chemistry , Lung Neoplasms/pathology , Staining and Labeling/methods , Animals , Antigens, Ly/genetics , Cell Line, Tumor , Dendritic Cells/cytology , Dendritic Cells/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Flow Cytometry/instrumentation , Genetic Heterogeneity , Humans , Macrophages, Alveolar/cytology , Macrophages, Alveolar/metabolism , Mice , Mice, Inbred C57BL , Monocytes/cytology , Monocytes/metabolism , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Neutrophils/cytology , Neutrophils/metabolism , Primary Cell Culture , Tumor Microenvironment
10.
BMC Res Notes ; 15(1): 31, 2022 Feb 10.
Article in English | MEDLINE | ID: mdl-35144666

ABSTRACT

OBJECTIVE: The endothelial cells overexpress the adhesion molecules in the leukocyte diapedesis pathway, developing vessel subendothelial molecular events. In this study, miR-194 and miR-27a were predicted and investigated on the expression of adhesion molecules in HUVEC cells. The SELE, SELP, and JAM-B adhesion molecules involved in the leukocyte tethering were predicted on the GO-enriched gene network. Following transfection of PEI-miRNA particles into HUVEC cells, the SELE, SELP, and JAM-B gene expression levels were evaluated by real-time qPCR. Furthermore, the monocyte-endothelial adhesion was performed using adhesion assay kit. RESULTS: In agreement with the prediction results, the cellular data showed that miR-27a and miR-194 decrease significantly the SELP and JAM-B expression levels in HUVECs (P < 0.05). Moreover, both the miRNAs suppressed the monocyte adhesion to endothelial cells. Since the miR-27a inhibited significantly the monocyte-endothelial adhesion (P = 0.0001) through the suppression of SELP and JAM-B thus it might relate to the leukocyte diapedesis pathway.


Subject(s)
Cell Adhesion , Human Umbilical Vein Endothelial Cells/cytology , MicroRNAs , Monocytes , Cell Adhesion Molecules/genetics , Cells, Cultured , Humans , MicroRNAs/genetics , Monocytes/cytology , P-Selectin
11.
Science ; 375(6584): eabi7377, 2022 03 04.
Article in English | MEDLINE | ID: mdl-35084939

ABSTRACT

Cerebrovascular diseases are a leading cause of death and neurologic disability. Further understanding of disease mechanisms and therapeutic strategies requires a deeper knowledge of cerebrovascular cells in humans. We profiled transcriptomes of 181,388 cells to define a cell atlas of the adult human cerebrovasculature, including endothelial cell molecular signatures with arteriovenous segmentation and expanded perivascular cell diversity. By leveraging this reference, we investigated cellular and molecular perturbations in brain arteriovenous malformations, which are a leading cause of stroke in young people, and identified pathologic endothelial transformations with abnormal vascular patterning and the ontology of vascularly derived inflammation. We illustrate the interplay between vascular and immune cells that contributes to brain hemorrhage and catalog opportunities for targeting angiogenic and inflammatory programs in vascular malformations.


Subject(s)
Blood Vessels/cytology , Brain/blood supply , Intracranial Arteriovenous Malformations/pathology , Transcriptome , Adult , Blood Vessels/pathology , Blood Vessels/physiology , Blood Vessels/physiopathology , Cells, Cultured , Cerebral Cortex/blood supply , Cerebral Hemorrhage/pathology , Cerebral Hemorrhage/physiopathology , Cerebrovascular Circulation , Endothelial Cells/cytology , Endothelial Cells/pathology , Endothelial Cells/physiology , Fibroblasts/cytology , Fibroblasts/physiology , Humans , Inflammation , Intracranial Arteriovenous Malformations/metabolism , Monocytes/cytology , Monocytes/physiology , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/physiology , Pericytes/cytology , Pericytes/physiology , RNA-Seq , Single-Cell Analysis
12.
Nat Commun ; 13(1): 192, 2022 01 11.
Article in English | MEDLINE | ID: mdl-35017482

ABSTRACT

A key challenge in analyzing single cell RNA-sequencing data is the large number of false zeros, where genes actually expressed in a given cell are incorrectly measured as unexpressed. We present a method based on low-rank matrix approximation which imputes these values while preserving biologically non-expressed genes (true biological zeros) at zero expression levels. We provide theoretical justification for this denoising approach and demonstrate its advantages relative to other methods on simulated and biological datasets.


Subject(s)
Algorithms , RNA/genetics , Sequence Analysis, RNA/statistics & numerical data , Animals , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Bronchi/cytology , Bronchi/metabolism , Datasets as Topic , Epithelial Cells/cytology , Epithelial Cells/metabolism , Humans , Killer Cells, Natural/cytology , Killer Cells, Natural/metabolism , Mice , Monocytes/cytology , Monocytes/metabolism , Primary Cell Culture , RNA/metabolism , RNA-Seq , Single-Cell Analysis , T-Lymphocytes/cytology , T-Lymphocytes/metabolism
13.
Int J Mol Sci ; 23(1)2021 Dec 21.
Article in English | MEDLINE | ID: mdl-35008429

ABSTRACT

Interleukin (IL)-25 is a cytokine released by airway epithelial cells responding to pathogens. Excessive production of reactive oxygen species (ROS) leads to airway inflammation and remodeling in asthma. Mitochondria are the major source of ROS. After stress, defective mitochondria often undergo selective degradation, known as mitophagy. In this study, we examined the effects of IL-25 on ROS production and mitophagy and investigated the underlying mechanisms. The human monocyte cell line was pretreated with IL-25 at different time points. ROS production was measured by flow cytometry. The involvement of mitochondrial activity in the effects of IL-25 on ROS production and subsequent mitophagy was evaluated by enzyme-linked immunosorbent assay, Western blotting, and confocal microscopy. IL-25 stimulation alone induced ROS production and was suppressed by N-acetylcysteine, vitamin C, antimycin A, and MitoTEMPO. The activity of mitochondrial complex I and complex II/III and the levels of p-AMPK and the mitophagy-related proteins were increased by IL-25 stimulation. The CCL-22 secretion was increased by IL-25 stimulation and suppressed by mitophagy inhibitor treatment and PINK1 knockdown. The Th2-like cytokine IL-25 can induce ROS production, increase mitochondrial respiratory chain complex activity, subsequently activate AMPK, and induce mitophagy to stimulate M2 macrophage polarization in monocytes.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Interleukin-17/metabolism , Macrophages/cytology , Mitochondria/metabolism , Monocytes/cytology , Reactive Oxygen Species/metabolism , Acetylcysteine/pharmacology , Antimycin A/pharmacology , Ascorbic Acid/pharmacology , Cell Polarity , Flow Cytometry , Humans , Interleukin-17/pharmacology , Macrophages/metabolism , Microscopy, Confocal , Mitophagy , Monocytes/metabolism , Organophosphorus Compounds/pharmacology , Phosphorylation/drug effects , Piperidines/pharmacology , THP-1 Cells
14.
ACS Appl Bio Mater ; 4(1): 441-450, 2021 01 18.
Article in English | MEDLINE | ID: mdl-35014295

ABSTRACT

Cancer stem cells (CSCs) are self-renewing and constitute the primary cause of cancer relapse post-cancer therapy. The CSC niche is composed of various nonmalignant stromal cells that support CSCs' survival during cancer chemoradiotherapy. Understanding the cross-talk between CSCs and stromal cells could pave the way for developing therapeutic strategies to eradicate CSCs. Traditionally, CSC research has been relying on animal models, which can give rise to complications and poor translation in clinical practice. An efficient model to co-culture CSCs and stromal cells is urgently needed. Hence, we leveraged our expertise in enriching CSCs from in vitro cell lines with a 3D alginate-based platform, as reported previously. We established a 3D co-culture system that allowed us to study the interactions between stromal cells and CSCs over an extended period. We showed that the self-renewal capacity and stemness of CSCs were significantly enhanced when co-cultured with 3D cultured human umbilical vein endothelial cells (HUVECs) or a human monocyte cell line (THP1). Strikingly, the expression of MDR1 in 3D co-cultured CSCs was upregulated, leading to enhanced chemotoxic drug tolerance. We suggest that our in vitro co-culture model can impact CSC research and clinical practice when the goal is to develop therapeutics that target and eradicate CSCs by targeting stromal cells.


Subject(s)
Alginates/chemistry , Coculture Techniques/methods , Neoplastic Stem Cells/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Culture Techniques, Three Dimensional , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic , Human Umbilical Vein Endothelial Cells , Humans , Hydrogels/chemistry , Mice , Monocytes/cytology , Monocytes/metabolism , Neoplastic Stem Cells/cytology , Paracrine Communication , Spheroids, Cellular/cytology , Spheroids, Cellular/metabolism , Up-Regulation
15.
FEBS Lett ; 596(4): 491-509, 2022 02.
Article in English | MEDLINE | ID: mdl-35007347

ABSTRACT

In autophagy, LC3-positive autophagophores fuse and encapsulate the autophagic cargo in a double-membrane structure. In contrast, lipidated LC3 (LC3-II) is directly formed at the phagosomal membrane in LC3-associated phagocytosis (LAP). In this study, we dissected the effects of autophagy inhibitors on LAP. SAR405, an inhibitor of VPS34, reduced levels of LC3-II and inhibited LAP. In contrast, the inhibitors of endosomal acidification bafilomycin A1 and chloroquine increased levels of LC3-II, due to reduced degradation in acidic lysosomes. However, while bafilomycin A1 inhibited LAP, chloroquine did not. Finally, EACC, which inhibits the fusion of autophagosomes with lysosomes, promoted LC3 degradation possibly by the proteasome. Targeting LAP with small molecule inhibitors is important given its emerging role in infectious and autoimmune diseases.


Subject(s)
Autophagosomes/drug effects , Autophagy/drug effects , Dendritic Cells/drug effects , Phagocytosis/drug effects , Proteasome Endopeptidase Complex/drug effects , Autophagosomes/metabolism , Autophagy/genetics , Cell Differentiation , Chloroquine/pharmacology , Class III Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Class III Phosphatidylinositol 3-Kinases/genetics , Class III Phosphatidylinositol 3-Kinases/metabolism , Dendritic Cells/cytology , Dendritic Cells/metabolism , Endosomes/drug effects , Endosomes/metabolism , Gene Expression Regulation , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Macrolides/pharmacology , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Monocytes/cytology , Monocytes/metabolism , Phagocytosis/genetics , Phagosomes/drug effects , Phagosomes/metabolism , Primary Cell Culture , Proteasome Endopeptidase Complex/metabolism , Pyridines/pharmacology , Pyrimidinones/pharmacology , Thiophenes/pharmacology , Zymosan/metabolism
16.
Biochem Biophys Res Commun ; 590: 89-96, 2022 01 29.
Article in English | MEDLINE | ID: mdl-34973535

ABSTRACT

Cholinergic anti-inflammatory pathway (CAP) describes a neuronal-inflammatory reflex centered on systemic cytokine regulation by α7 nicotinic acetylcholine receptor (α7nAChR) activation of spleen-residue macrophage. However, the CAP mechanism attenuating distal tissue inflammation, inducing a low level of systemic inflammation, is lesser known. In this study, we hypothesized that CAP regulates monocyte accessibility by influencing their adhesion to endothelial cells. Using RNA-seq analysis, we identified that α1,3-Fucosyltransferase 7 (FucT-VII), the enzyme required for processing selectin ligands, was significantly downregulated by α7nAChR agonist among other cell-cell adhesion genes. The α7nAChR agonist inhibited monocytic cell line U-937 binding to P-selectin and adhesion to endothelial cells. Furthermore, α7nAChR agonist selectivity was confirmed by α7nAChR knockdown assays, showing that FUT7 inhibition and adhesion attenuation by the agonist was abolished by siRNA targeting α7nAChR encoding gene. Consistently, FUT7 knockdown inhibited the adhesive properties of U-937 and prevented them to adhere to endothelial cells. Overexpression of FUT7 also abrogated the adhesion attenuation induced by GTS-21 indicating that FUT7 inhibition was sufficient for inhibiting adhesion by α7nAChR activation. Our work demonstrated that α7nAChR activation regulates monocyte adhesion to endothelial cells through FUT7 inhibition, providing a novel insight into the CAP mechanism.


Subject(s)
Fucosyltransferases/antagonists & inhibitors , Human Umbilical Vein Endothelial Cells/cytology , Monocytes/cytology , alpha7 Nicotinic Acetylcholine Receptor/metabolism , Benzylidene Compounds/pharmacology , Cell Adhesion/drug effects , Cell Adhesion/genetics , Down-Regulation/drug effects , Down-Regulation/genetics , Fucosyltransferases/metabolism , Gene Knockdown Techniques , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Monocytes/drug effects , Monocytes/metabolism , Pyridines/pharmacology , U937 Cells , alpha7 Nicotinic Acetylcholine Receptor/antagonists & inhibitors
17.
ACS Chem Biol ; 16(11): 2373-2386, 2021 11 19.
Article in English | MEDLINE | ID: mdl-34592097

ABSTRACT

The cyclotide T20K inhibits the proliferation of human immune cells and is currently in clinical trials for multiple sclerosis. Here, we provide novel functional data and mechanistic insights into structure-activity relationships of T20K. Analogs with partial or complete reduction of the cystine knot had loss of function in proliferation experiments. Similarly, an acyclic analog of T20K was inactive in lymphocyte bioassays. The lack of activity of non-native peptide analogs appears to be associated with the ability of cyclotides to interact with and penetrate cell membranes, since cellular uptake studies demonstrated fast fractional transfer only of the native peptide into the cytosol of human immune cells. Therefore, structural differences between cyclic and linear native folded peptides were investigated by NMR to elucidate structure-activity relationships. Acyclic T20K had a less rigid backbone and considerable structural changes in loops 1 and 6 compared to the native cyclic T20K, supporting the idea that the cyclic cystine knot motif is a unique bioactive scaffold. This study provides evidence that this structural motif in cyclotides governs bioactivity, interactions with and transport across biological membranes, and the structural integrity of these peptides. These observations could be useful to understand the structure-activity of other cystine knot proteins due to the structural conservation of the cystine knot motif across evolution and to provide guidance for the design of novel cyclic cysteine-stabilized molecules.


Subject(s)
Cyclotides/chemistry , Cyclotides/pharmacology , Cystine Knot Motifs , Immunosuppressive Agents/pharmacology , Cell Proliferation/drug effects , Cyclotides/metabolism , Humans , Immunosuppressive Agents/metabolism , Monocytes/cytology , Monocytes/drug effects , Protein Conformation
18.
J Clin Lab Anal ; 35(11): e24014, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34545629

ABSTRACT

BACKGROUND: Papillary thyroid carcinoma (PTC) is considered to be an inflammatory disease. This study aimed to investigate the association of monocyte to high-density lipoprotein cholesterol ratio (MHR) with PTC. METHODS: Clinical parameters from 300 patients with PTC and 552 patients with benign thyroid nodule were compared. Serum renal function and liver enzymes, fasting plasma glucose, lipid profile, and blood cell count were measured. RESULTS: Patients with PTC had a higher MONO (p < 0.001) and MHR (p < 0.001). There was a step-wise increase in the prevalence of PTC (p = 0.003) with the tertile of MHR. Logistic regression analysis revealed that MHR could be considered an independent risk factor (p < 0.001) in the case-control study and the cohort study. Pearson correlation analysis and simple linear regression analysis indicated that MHR was positively associated with neutrophil (NEU) and lymphocyte (LYM) count as well as neutrophil-to-lymphocyte ratio (NLR). Area under the curve (AUC) was 0.711. The optimal cutoff of MHR was 0.33 × 109 /mmol. CONCLUSION: This study identifies novel evidence that patients with PTC have a higher MHR. MHR is an independent risk factor for PTC. These findings support the application of MHR to predict, diagnose, and evaluate the occurrence of PTC.


Subject(s)
Cholesterol, HDL/blood , Monocytes/cytology , Thyroid Cancer, Papillary , Thyroid Neoplasms , Adult , Aged , Case-Control Studies , Female , Humans , Male , Middle Aged , Risk Factors , Thyroid Cancer, Papillary/blood , Thyroid Cancer, Papillary/epidemiology , Thyroid Cancer, Papillary/physiopathology , Thyroid Neoplasms/blood , Thyroid Neoplasms/epidemiology , Thyroid Neoplasms/physiopathology
19.
J Clin Lab Anal ; 35(11): e24007, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34545611

ABSTRACT

AIM: Hepatitis B virus-related decompensated cirrhosis (HBV-DeCi) has a high mortality rate, and it remains a challenge to predict its outcomes in clinical practice. We aimed to determine the association between monocyte-to-HDL-cholesterol ratio (MHR) and short-term prognosis in HBV-DeCi patients. METHODS: A total of 145 HBV-DeCi patients were enrolled. A multivariate analysis was performed to identify predictors of mortality. The findings were validated by a receiver operating characteristic analysis using the area under the curve (AUC). RESULTS: A total of 20 (13.8%) patients had died 30 days after admission. MHR was markedly increased in the non-survivors compared with the survivors. In the multivariate analysis, MHR was identified as an independent risk factor for mortality, with a significant predictive value (AUC = 0.825; sensitivity, 90.0%; specificity, 62.4%). CONCLUSIONS: Elevated MHR is associated with increased mortality rate in HBV-DeCi patients.


Subject(s)
Cholesterol, HDL/blood , Liver Cirrhosis , Liver Failure , Monocytes/cytology , Aged , Biomarkers/blood , Female , Humans , Liver Cirrhosis/blood , Liver Cirrhosis/complications , Liver Cirrhosis/diagnosis , Liver Cirrhosis/mortality , Liver Failure/blood , Liver Failure/diagnosis , Liver Failure/etiology , Liver Failure/mortality , Male , Middle Aged , Multivariate Analysis , Prognosis , ROC Curve
20.
J Nanobiotechnology ; 19(1): 281, 2021 Sep 20.
Article in English | MEDLINE | ID: mdl-34544425

ABSTRACT

BACKGROUND: Spinal cord injury (SCI) is damage to the central nervous system (CNS) that causes devastating complications from chronic pain to breathing problems. Unfortunately, few effective and safe treatments are known to relieve the damages of SCI. Nanomedicines are used for the treatment of SCI with relatively few side effects, but only depending on the delivery of additional drugs, which increase complexity to the treatment. Considering the urgent need for saving SCI patients, it is important to develop promising nanobiotechnology for relieving their pains. METHODS: The clinical survey was used to investigate SCI patients, thereafter the therapy plan was designed. The receiver-operating characteristics (ROC) curves of the prediction model were built to find symptoms after SCI. The treatment plan (i.e. immunosuppressive strategy) was designed by manufacturing therapies based on gold nanoclusters (AuNCs). The response of the immune cells (macrophages) was studied accordingly. The western blot, reactive oxygen species (ROS) activity assay, enzyme-linked immunosorbent assay (ELISA), quantitative real-time PCR (RT-qPCR), and immunochemical staining were used for evaluation of the in vivo and in vitro therapeutic effects. RESULTS: We found increased monocytes/macrophages (M/Ms) levels in 114 SCI subjects (44.7% with severe SCI complications) by the clinical survey. Additionally, the enhanced macrophage level was found to be closely related to the walking disorder after SCI. Since macrophages were central effector cells of the immune system, we assumed that the immune-suppressing strategies could be used for SCI therapy. Thereafter, AuNCs were stabilized by dihydrolipoic acid (DHLA) enantiomers (including DL-DHLA, R-DHLA; A racemic mixture (R and S) was denoted as DL; R and S refer to Rectus and Sinister), obtaining DL-DHLA-AuNCs and R-DHLA-AuNCs, respectively. In addition, zinc-modified DL-DHLA and R-DHLA stabilized AuNCs (i.e., DL-DHLA-AuNCs-Zn and R-DHLA-AuNCs-Zn) were investigated. Among these AuNCs, R-DHLA-AuNCs-Zn showed the most remarkable therapeutic effect for promoting the polarization of pro-inflammatory macrophages and reducing neuronal ROS-induced apoptosis and inflammation in vitro and in vivo; the lesion size was decreased and the survival rate of ventral neurons is higher. CONCLUSIONS: R-DHLA-AuNCs-Zn have comprehensive therapeutic capabilities, especially the immune-suppressing effects for the therapy of SCI, which is promising to relieve the pain or even recover SCI for the patients.


Subject(s)
Gold/chemistry , Metal Nanoparticles/therapeutic use , Spinal Cord Injuries/drug therapy , Zinc/chemistry , Animals , Cell Survival/drug effects , Humans , Lymphocytes/cytology , Lymphocytes/immunology , Macrophages/cytology , Macrophages/metabolism , Male , Metal Nanoparticles/chemistry , Metal Nanoparticles/toxicity , Mice , Monocytes/cytology , Monocytes/immunology , Neuroprotective Agents/chemistry , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Prognosis , RAW 264.7 Cells , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Spinal Cord Injuries/immunology , Spinal Cord Injuries/pathology , Stereoisomerism , Thioctic Acid/analogs & derivatives , Thioctic Acid/chemistry
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