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Cell Communication and Signaling : CCS Oct 2023Neutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative...
LRRK2 is involved in the chemotaxis of neutrophils and differentiated HL-60 cells, and the inhibition of LRRK2 kinase activity increases fMLP-induced chemotactic activity.
BACKGROUND
Neutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative phosphorylation (OXPHOS) during chemotaxis. However, the mechanism by which the energy supply changes from glycolysis to OXPHOS remains unknown. Leucine-rich repeat kinase 2 (LRRK2) is partially present in the outer mitochondrial membrane fraction. Lrrk2-deficient cells show mitochondrial fragmentation and reduced OXPHOS activity. We have previously reported that mitofusin (MFN) 2 is involved in chemotaxis and OXPHOS activation upon chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation in differentiated HL-60 (dHL-60) cells. It has been previously reported that LRRK2 binds to MFN2 and partially colocalizes with MFN2 at the mitochondrial membranes. This study investigated the involvement of LRRK2 in chemotaxis and MFN2 activation in neutrophils and dHL-60 cells.
METHODS
Lrrk2 knockout neutrophils and Lrrk2 knockdown dHL-60 cells were used to examine the possible involvement of LRRK2 in chemotaxis. Lrrk2 knockdown dHL-60 cells were used a tetracycline-inducible small hairpin RNA (shRNA) system to minimize the effects of LRRK2 knockdown during cell culture. The relationship between LRRK2 and MFN2 was investigated by measuring the GTP-binding activity of MFN2 in Lrrk2 knockdown dHL-60 cells. The effects of LRRK2 kinase activity on chemotaxis were examined using the LRRK2 kinase inhibitor MLi-2.
RESULTS
fMLP-induced chemotactic activity was reduced in Lrrk2 knockout neutrophils in vitro and in vivo. Lrrk2 knockdown in dHL-60 cells expressing Lrrk2 shRNA also reduced fMLP-induced chemotactic activity. Lrrk2 knockdown dHL-60 cells showed reduced OXPHOS activity and suppressed mitochondrial morphological change, similar to Mfn2 knockdown dHL-60 cells. The amount of LRRK2 in the mitochondrial fraction and the GTP-binding activity of MFN2 increased upon fMLP stimulation, and the MFN2 GTP-binding activity was suppressed in Lrrk2 knockdown dHL-60 cells. Furthermore, the kinase activity of LRRK2 and Ser935 phosphorylation of LRRK2 were reduced upon fMLP stimulation, and LRRK2 kinase inhibition by MLi-2 increased the migration to fMLP.
CONCLUSIONS
LRRK2 is involved in neutrophil chemotaxis and the GTP-binding activity of MFN2 upon fMLP stimulation. On the other hand, the kinase activity of LRRK2 shows a negative regulatory effect on fMLP-induced chemotactic activity in dHL-60 cells. Video Abstract.
Topics: Humans; Chemotaxis; Neutrophils; HL-60 Cells; Oxidative Phosphorylation; RNA, Small Interfering; Guanosine Triphosphate; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
PubMed: 37904222
DOI: 10.1186/s12964-023-01305-y -
BMC Infectious Diseases Jun 2022Passive immunization against SARS-CoV-2 limits viral burden and death from COVID-19; however, it poses a theoretical risk of disease exacerbation through...
BACKGROUND
Passive immunization against SARS-CoV-2 limits viral burden and death from COVID-19; however, it poses a theoretical risk of disease exacerbation through antibody-dependent enhancement (ADE). ADE after anti-SARS-CoV2 antibody treatment has not been reported, and therefore the potential risk and promoting factors remain unknown.
CASE PRESENTATION
A 75-year-old female was admitted to the emergency room with recurrent, unexplained bruises and leukocytopenia, anemia, and thrombocytopenia. Evaluation of a bone marrow biopsy established the diagnosis of an acute promyelocytic leukemia (APL). SARS-CoV-2 RT-PCR testing of nasal and throat swabs on admission was negative. During the routine SARS-CoV-2 testing of inpatients, our patient tested positive for SARS-CoV-2 on day 14 after admission without typical COVID-19 symptoms. Due to disease- and therapy-related immunosuppression and advanced age conferring a high risk of progressing to severe COVID-19, casirivimab and imdevimab were administered as a preemptive approach. The patient developed immune activation and cytokine release syndrome (CRS) occurring within four hours of preemptive anti-SARS-CoV2 antibody (casirivimab/imdevimab) infusion. Immune activation and CRS were evidenced by a rapid increase in serum cytokines (IL-6, TNFα, IL-8, IL-10), acute respiratory insufficiency, and progressive acute respiratory distress syndrome.
DISCUSSION AND CONCLUSION
The temporal relationship between therapeutic antibody administration and the rapid laboratory, radiological, and clinical deterioration suggests that CRS was an antibody-related adverse event, potentially exacerbated by APL treatment-mediated differentiation of leukemic blasts and promyelocytes. This case highlights the need for careful assessment of life-threatening adverse events after passive SARS-CoV-2 immunization, especially in the clinical context of patients with complex immune and hematological landscapes.
Topics: Aged; Antibodies, Monoclonal, Humanized; COVID-19; COVID-19 Testing; Cytokine Release Syndrome; Female; Humans; Leukemia, Promyelocytic, Acute; Respiratory Distress Syndrome; SARS-CoV-2
PubMed: 35692034
DOI: 10.1186/s12879-022-07513-0 -
Pharmaceuticals (Basel, Switzerland) Jan 2023Acute promyelocytic leukemia (APL) is phenotypically characterized by the accumulation of dysplastic promyelocytes, resulting from a cytogenetic condition due to the... (Review)
Review
Pharmaceutical/Clinical Strategies in the Treatment of Acute Promyelocytic Leukemia: All-Trans Retinoic Acid Encapsulation by Spray-Drying Technology as an Innovative Approach-Comprehensive Overview.
Acute promyelocytic leukemia (APL) is phenotypically characterized by the accumulation of dysplastic promyelocytes, resulting from a cytogenetic condition due to the balanced chromosomal translocation t(15;17)(q22;q21). Current first-line treatment of APL includes all-trans retinoic acid (all-trans RA), with or without arsenic trioxide, combined with chemotherapy, and a chemotherapy-free approach wherein arsenic trioxide is used alone or in combination with all-trans RA. The usage of all-trans RA revolutionized the treatment of APL, with survival rates of 80 to 90% being achieved. The mechanism of action of all-trans RA is based on regulation of gene transcription, promoting the differentiation of leukemic promyelocytes. Encapsulation technology has been explored as an innovative strategy to overcome the major drawbacks related to the all-trans RA oral administration in the APL treatment. The most recently published works on this subject highlight the development and optimization of carrier-based delivery systems based in microparticle formulations obtained by spray-drying to be used in the treatment of APL. The ultimate goal is to obtain a controlled delivery system for RA oral administration capable of providing a slow release of this bioactive compound in the intestinal lumen.
PubMed: 37259328
DOI: 10.3390/ph16020180 -
Leukemia Jul 2021Complex karyotype acute myeloid leukemia (CK-AML) has a dismal outcome with current treatments, underscoring the need for new therapies. Here, we report synergistic...
Complex karyotype acute myeloid leukemia (CK-AML) has a dismal outcome with current treatments, underscoring the need for new therapies. Here, we report synergistic anti-leukemic activity of the BCL-2 inhibitor venetoclax (Ven) and the asparaginase formulation Pegylated Crisantaspase (PegC) in CK-AML in vitro and in vivo. Ven-PegC combination inhibited growth of multiple AML cell lines and patient-derived primary CK-AML cells in vitro. In vivo, Ven-PegC showed potent reduction of leukemia burden and improved survival, compared with each agent alone, in a primary patient-derived CK-AML xenograft. Superiority of Ven-PegC, compared to single drugs, and, importantly, the clinically utilized Ven-azacitidine combination, was also demonstrated in vivo in CK-AML. We hypothesized that PegC-mediated plasma glutamine depletion inhibits 4EBP1 phosphorylation, decreases the expression of proteins such as MCL-1, whose translation is cap dependent, synergizing with the BCL-2 inhibitor Ven. Ven-PegC treatment decreased cellular MCL-1 protein levels in vitro by enhancing eIF4E-4EBP1 interaction on the cap-binding complex via glutamine depletion. In vivo, Ven-PegC treatment completely depleted plasma glutamine and asparagine and inhibited mRNA translation and cellular protein synthesis. Since this novel mechanistically-rationalized regimen combines two drugs already in use in acute leukemia treatment, we plan a clinical trial of the Ven-PegC combination in relapsed/refractory CK-AML.
Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Female; HL-60 Cells; Humans; K562 Cells; Leukemia, Myeloid, Acute; Male; Mice; Mice, Inbred NOD; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; U937 Cells
PubMed: 33199836
DOI: 10.1038/s41375-020-01080-6 -
MBio Jun 2020Posttranscriptional regulation plays an essential role in the quick adaptation of pathogenic bacteria to host environments, and RNases play key roles in this process by...
Posttranscriptional regulation plays an essential role in the quick adaptation of pathogenic bacteria to host environments, and RNases play key roles in this process by modifying small RNAs and mRNAs. We find that the endonuclease YbeY is required for rRNA processing and the bacterial virulence in a murine acute pneumonia model. Transcriptomic analyses reveal that knocking out the gene results in downregulation of oxidative stress response genes, including the catalase genes and Consistently, the mutant is more susceptible to HO and neutrophil-mediated killing. Overexpression of restores the bacterial tolerance to HO and neutrophil killing as well as virulence. We further find that the downregulation of the oxidative stress response genes is due to defective expression of the stationary-phase sigma factor RpoS. We demonstrate an autoregulatory mechanism of RpoS and find that mutation increases the level of a small RNA, ReaL, which directly represses the translation of through the 5' UTR of its mRNA and subsequently reduces the expression of the oxidative stress response genes. assays demonstrate direct degradation of ReaL by YbeY. Deletion of or overexpression of in the mutant restores the bacterial tolerance to oxidative stress and the virulence. We also demonstrate that YbeZ binds to YbeY and is involved in the 16S rRNA processing and regulation of and as well as the bacterial virulence. Overall, our results reveal pleiotropic roles of YbeY and the YbeY-mediated regulation of through ReaL. The increasing bacterial antibiotic resistance imposes a severe threat to human health. For the development of effective treatment and prevention strategies, it is critical to understand the mechanisms employed by bacteria to grow in the human body. Posttranscriptional regulation plays an important role in bacterial adaptation to environmental changes. RNases and small RNAs are key players in this regulation. In this study, we demonstrate critical roles of the RNase YbeY in the virulence of the pathogenic bacterium We further identify the small RNA ReaL as the direct target of YbeY and elucidate the YbeY-regulated pathway on the expression of bacterial virulence factors. Our results shed light on the complex regulatory network of and indicate that inference with the YbeY-mediated regulatory pathway might be a valid strategy for the development of a novel treatment strategy.
Topics: Animals; Bacterial Proteins; Endoribonucleases; Female; Gene Expression Regulation, Bacterial; HL-60 Cells; Humans; Lung; Mice; Mice, Inbred BALB C; Pseudomonas aeruginosa; RNA Processing, Post-Transcriptional; RNA, Bacterial; Sigma Factor; Virulence
PubMed: 32605982
DOI: 10.1128/mBio.00659-20 -
Molecules (Basel, Switzerland) Apr 2021Synthetic and natural ionophores have been developed to catalyze ion transport and have been shown to exhibit a variety of biological effects. We synthesized 24 aza- and...
Synthetic and natural ionophores have been developed to catalyze ion transport and have been shown to exhibit a variety of biological effects. We synthesized 24 aza- and diaza-crown ethers containing adamantyl, adamantylalkyl, aminomethylbenzoyl, and ε-aminocaproyl substituents and analyzed their biological effects in vitro. Ten of the compounds (, -, and ) increased intracellular calcium ([Ca]) in human neutrophils, with the most potent being compound (,'-[2-(1-adamantyl)acetyl]-4,10-diaza-15-crown-5), suggesting that these compounds could alter normal neutrophil [Ca] flux. Indeed, a number of these compounds (i.e., , -, and ) inhibited [Ca] flux in human neutrophils activated by -formyl peptide (MLF). Some of these compounds also inhibited chemotactic peptide-induced [Ca] flux in HL60 cells transfected with N-formyl peptide receptor 1 or 2 (FPR1 or FPR2). In addition, several of the active compounds inhibited neutrophil reactive oxygen species production induced by phorbol 12-myristate 13-acetate (PMA) and neutrophil chemotaxis toward MLF, as both of these processes are highly dependent on regulated [Ca] flux. Quantum chemical calculations were performed on five structure-related diaza-crown ethers and their complexes with Ca, Na, and K to obtain a set of molecular electronic properties and to correlate these properties with biological activity. According to density-functional theory (DFT) modeling, Ca ions were more effectively bound by these compounds versus Na and K. The DFT-optimized structures of the ligand-Ca complexes and quantitative structure-activity relationship (QSAR) analysis showed that the carbonyl oxygen atoms of the ,'-diacylated diaza-crown ethers participated in cation binding and could play an important role in Ca transfer. Thus, our modeling experiments provide a molecular basis to explain at least part of the ionophore mechanism of biological action of aza-crown ethers.
Topics: Aza Compounds; Calcium; Chemotaxis; Crown Ethers; Density Functional Theory; HL-60 Cells; Humans; Ligands; Models, Molecular; Neutrophils; Reactive Oxygen Species; Receptors, Formyl Peptide; Regression Analysis; Static Electricity; Thermodynamics
PubMed: 33921479
DOI: 10.3390/molecules26082225 -
Scientific Reports Apr 2021(-)-Epigallocatechin-3-gallate (EGCG), the major active polyphenol extracted from green tea, has been shown to induce apoptosis and inhibit cell proliferation, cell...
(-)-Epigallocatechin-3-gallate (EGCG), the major active polyphenol extracted from green tea, has been shown to induce apoptosis and inhibit cell proliferation, cell invasion, angiogenesis and metastasis. Herein, we evaluated the in vivo effects of EGCG in acute myeloid leukaemia (AML) using an acute promyelocytic leukaemia (APL) experimental model (PML/RARα). Haematological analysis revealed that EGCG treatment reversed leucocytosis, anaemia and thrombocytopenia, and prolonged survival of PML/RARα mice. Notably, EGCG reduced leukaemia immature cells and promyelocytes in the bone marrow while increasing mature myeloid cells, possibly due to apoptosis increase and cell differentiation. The reduction of promyelocytes and neutrophils/monocytes increase detected in the peripheral blood, in addition to the increased percentage of bone marrow cells with aggregated promyelocytic leukaemia (PML) bodies staining and decreased expression of PML-RAR oncoprotein corroborates our results. In addition, EGCG increased expression of neutrophil differentiation markers such as CD11b, CD14, CD15 and CD66 in NB4 cells; and the combination of all-trans retinoic acid (ATRA) plus EGCG yield higher increase the expression of CD15 marker. These findings could be explained by a decrease of peptidyl-prolyl isomerase NIMA-interacting 1 (PIN1) expression and reactive oxygen species (ROS) increase. EGCG also decreased expression of substrate oncoproteins for PIN1 (including cyclin D1, NF-κB p65, c-MYC, and AKT) and 67 kDa laminin receptor (67LR) in the bone marrow cells. Moreover, EGCG showed inhibition of ROS production in NB4 cells in the presence of N-acetyl-L-cysteine (NAC), as well as a partial blockage of neutrophil differentiation and apoptosis, indicating that EGCG-activities involve/or are in response of oxidative stress. Furthermore, apoptosis of spleen cells was supported by increasing expression of BAD and BAX, parallel to BCL-2 and c-MYC decrease. The reduction of spleen weights of PML/RARα mice, as well as apoptosis induced by EGCG in NB4 cells in a dose-dependent manner confirms this assumption. Our results support further evaluation of EGCG in clinical trials for AML, since EGCG could represent a promising option for AML patient ineligible for current mainstay treatments.
Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Catechin; Cell Differentiation; Humans; Leukemia, Experimental; Leukemia, Promyelocytic, Acute; Mice, Transgenic; NIMA-Interacting Peptidylprolyl Isomerase; Reactive Oxygen Species; Retinoic Acid Receptor alpha; Spleen; bcl-2-Associated X Protein; bcl-Associated Death Protein
PubMed: 33907248
DOI: 10.1038/s41598-021-88478-z -
Frontiers in Immunology 2023Dendritic cells (DCs) are readily generated from the culture of mouse bone marrow (BM) treated with either granulocyte macrophage-colony stimulating factor (GM-CSF) or...
Dendritic cells (DCs) are readily generated from the culture of mouse bone marrow (BM) treated with either granulocyte macrophage-colony stimulating factor (GM-CSF) or FMS-like tyrosine kinase 3 ligand (FLT3L). CD11cMHCII or CD11cMHCII cells are routinely isolated from those BM cultures and generally used as -generated DCs for a variety of experiments and therapies. Here, we examined CD11c cells in the BM culture with GM-CSF or FLT3L by staining with a monoclonal antibody 2A1 that is known to recognize mature or activated DCs. Most of the cells within the CD11cMHCII DC gate were 2A1 in the BM culture with GM-CSF (GM-BM culture). In the BM culture with FLT3L (FL-BM culture), almost of all the CD11cMHCII cells were within the classical DC2 (cDC2) gate. The analysis of FL-BM culture revealed that a majority of cDC2-gated CD11cMHCII cells exhibited a 2A1CD83CD115CXCR1 phenotype, and the others consisted of 2A1CD83CD115CXCR1 and 2A1CD83CD115CXCR1 cells. According to the antigen uptake and presentation, morphologies, and gene expression profiles, 2A1CD83CD115CXCR1 cells were immature cDC2s and 2A1CD83CD115CXCR1 cells were mature cDC2s. Unexpectedly, however, 2A1CD83CD115CXCR1 cells, the most abundant cDC2-gated MHCII cell subset in FL-BM culture, were non-DCs. Adoptive cell transfer experiments in the FL-BM culture confirmed that the cDC2-gated MHCII non-DCs were precursors to cDC2s, i.e., MHCII pre-cDC2s. MHCII pre-cDC2s also expressed the higher level of DC-specific transcription factor Zbtb46 as similarly as immature cDC2s. Besides, MHCII pre-cDC2s were generated only from pre-cDCs and common DC progenitor (CDP) cells but not from monocytes and common monocyte progenitor (cMoP) cells, verifying that MHCII pre-cDC2s are close lineage to cDCs. All in all, our study identified and characterized a new cDC precursor, exhibiting a CD11cMHCIICD115CXCR1 phenotype, in FL-BM culture.
Topics: Mice; Animals; Histocompatibility Antigens Class II; Bone Marrow; Granulocyte-Macrophage Colony-Stimulating Factor; CX3C Chemokine Receptor 1; Bone Marrow Cells; Dendritic Cells; Cell Differentiation; Receptor Protein-Tyrosine Kinases
PubMed: 38094300
DOI: 10.3389/fimmu.2023.1179981 -
PLoS Computational Biology Aug 2021Navigation of fast migrating cells such as amoeba Dictyostelium and immune cells are tightly associated with their morphologies that range from steady polarized forms... (Comparative Study)
Comparative Study
Navigation of fast migrating cells such as amoeba Dictyostelium and immune cells are tightly associated with their morphologies that range from steady polarized forms that support high directionality to those more complex and variable when making frequent turns. Model simulations are essential for quantitative understanding of these features and their origins, however systematic comparisons with real data are underdeveloped. Here, by employing deep-learning-based feature extraction combined with phase-field modeling framework, we show that a low dimensional feature space for 2D migrating cell morphologies obtained from the shape stereotype of keratocytes, Dictyostelium and neutrophils can be fully mapped by an interlinked signaling network of cell-polarization and protrusion dynamics. Our analysis links the data-driven shape analysis to the underlying causalities by identifying key parameters critical for migratory morphologies both normal and aberrant under genetic and pharmacological perturbations. The results underscore the importance of deciphering self-organizing states and their interplay when characterizing morphological phenotypes.
Topics: Animals; Cell Movement; Cell Polarity; Cell Shape; Cell Surface Extensions; Cells, Cultured; Cichlids; Computational Biology; Computer Simulation; Deep Learning; Dictyostelium; Fibroblasts; HL-60 Cells; Humans; Models, Biological
PubMed: 34383753
DOI: 10.1371/journal.pcbi.1009237 -
PLoS Biology Feb 2022Elevated circulating levels of growth differentiation factor 15 (GDF15) have been shown to reduce food intake and lower body weight through activation of hindbrain...
Elevated circulating levels of growth differentiation factor 15 (GDF15) have been shown to reduce food intake and lower body weight through activation of hindbrain receptor glial-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) in rodents and nonhuman primates, thus endogenous induction of this peptide holds promise for obesity treatment. Here, through in silico drug-screening methods, we found that small molecule Camptothecin (CPT), a previously identified drug with potential antitumor activity, is a GDF15 inducer. Oral CPT administration increases circulating GDF15 levels in diet-induced obese (DIO) mice and genetic ob/ob mice, with elevated Gdf15 expression predominantly in the liver through activation of integrated stress response. In line with GDF15's anorectic effect, CPT suppresses food intake, thereby reducing body weight, blood glucose, and hepatic fat content in obese mice. Conversely, CPT loses these beneficial effects when Gdf15 is inhibited by a neutralizing antibody or AAV8-mediated liver-specific knockdown. Similarly, CPT failed to reduce food intake and body weight in GDF15's specific receptor GFRAL-deficient mice despite high levels of GDF15. Together, these results indicate that CPT is a promising anti-obesity agent through activation of GDF15-GFRAL pathway.
Topics: Animals; Body Weight; Camptothecin; Cell Line; Cell Line, Tumor; Diet, High-Fat; Eating; Gene Expression Regulation; Glial Cell Line-Derived Neurotrophic Factor Receptors; Growth Differentiation Factor 15; HEK293 Cells; HL-60 Cells; Humans; MCF-7 Cells; Male; Mice, Inbred C57BL; Mice, Knockout; Mice, Obese; Obesity; PC-3 Cells; Mice
PubMed: 35202387
DOI: 10.1371/journal.pbio.3001517