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RSC Advances Apr 2024Ferroptosis is a newly discovered iron-dependent form of regulated cell death associated with high levels of hydroxyl radical (˙OH) production. Meanwhile, lysosome...
Ferroptosis is a newly discovered iron-dependent form of regulated cell death associated with high levels of hydroxyl radical (˙OH) production. Meanwhile, lysosome dysfunction has been shown to be one of the causes of ferroptosis. Although a variety of ˙OH-responsive fluorescent probes have been developed for detecting intracellular ˙OH in living cells, there are still only few lysosome-targeted probes to monitor the variation in lysosomal ˙OH levels during ferroptosis. Herein, we report a novel ˙OH-specific fluorescent probe HCy-Lyso, which is composed of the hydrocyanine and morpholine moiety. Upon treatment with ˙OH, its hydrocyanine unit was converted to the corresponding cyanine group, thus leading to a large π-conjugation extension of HCy-Lyso, accompanied by a significant fluorescence off-on response. Moreover, after reacting with ˙OH in an acidic environment, the protonation product of HCy-Lyso exhibits a higher fluorescence enhancement, which is suitable for detecting lysosomal ˙OH variation. HCy-Lyso has been utilized for imaging endogenous ˙OH in living cells under phorbol myristate acetate (PMA) stimuli and monitoring the changes in lysosomal ˙OH levels during ferroptosis. Thus, our study proposes a new strategy to design lysosome-targeted and ˙OH-responsive fluorescent probes to investigate the relationship between lysosomes and ferroptosis.
PubMed: 38650686
DOI: 10.1039/d4ra00562g -
BMC Microbiology Apr 2024Mycobacteria bloodstream infections are common in immunocompromised people and usually have disastrous consequences. As the primary phagocytes in the bloodstream,...
BACKGROUND
Mycobacteria bloodstream infections are common in immunocompromised people and usually have disastrous consequences. As the primary phagocytes in the bloodstream, monocytes and neutrophils play critical roles in the fight against bloodstream mycobacteria infections. In contrast to macrophages, the responses of monocytes infected with the mycobacteria have been less investigated.
RESULTS
In this study, we first established a protocol for infection of non-adherent monocyte-like THP-1 cells (i.e. without the differentiation induced by phorbol 12-myristate 13-acetate (PMA) by bacillus Calmette-Guérin (BCG). Via the protocol, we were then capable of exploring the global transcriptomic profiles of non-adherent THP-1 cells infected with BCG, and found that NF-κB, MAPK and PI3K-Akt signaling pathways were enhanced, as well as some inflammatory chemokine/cytokine genes (e.g. CCL4, CXCL10, TNF and IL-1β) were up-regulated. Surprisingly, the Akt-HIF-mTOR signaling pathway was also activated, which induces trained immunity. In this in vitro infection model, increased cytokine responses to lipopolysaccharides (LPS) restimulation, higher cell viability, and decreased Candida albicans loads were observed.
CONCLUSIONS
We have first characterized the transcriptomic profiles of BCG-infected non-adherent THP-1 cells, and first developed a trained immunity in vitro model of the cells.
Topics: Humans; Monocytes; BCG Vaccine; Trained Immunity; Proto-Oncogene Proteins c-akt; THP-1 Cells; Phosphatidylinositol 3-Kinases; Cytokines; Mycobacterium bovis
PubMed: 38643095
DOI: 10.1186/s12866-024-03191-x -
Veterinary Parasitology Jun 2024Neutrophils, a crucial element of the host defense system, develop extracellular traps against helminth parasites. Neutrophils accumulate around the larvae of Toxocara...
Neutrophils, a crucial element of the host defense system, develop extracellular traps against helminth parasites. Neutrophils accumulate around the larvae of Toxocara canis (T. canis) in the tissues of the organism. This study aimed to determine the reaction in canine neutrophils after incubation with infective stage T. canis larvae (L3) in vitro. Most L3 were still active and moved between the extracellular traps (NETs) after 60-min incubation. NETs were not disintegrated by L3 movement. The L3 was only immobilized by NETs, entrapped larvae were still motile between the traps at the 24 h incubation. NETs were observed not only to accumulate around the mouth, excretory pole or anus but also the entire body of live L3. The extracellular DNA amount released from the canine neutrophils after being induced with phorbol 12-myristate 13-acetate was not affected by T. canis excretory/secretory products obtained from 250 L3. To the Authors'knowledge, the extracellular trap structures was firstly observed in canine neutrophils against T. canis L3 in vitro. NETs decorated with myeloperoxidase, neutrophil elastase and histone (H3) were observed under fluorescence microscope. There were not significant differences in the amount of extracellular DNA (P > 0.05), but the morphological structure of NETs was different in the live and head-inactivated T. canis larvae.
Topics: Animals; Extracellular Traps; Dogs; Toxocara canis; Neutrophils; Larva; Dog Diseases; Toxocariasis
PubMed: 38640875
DOI: 10.1016/j.vetpar.2024.110186 -
Frontiers in Immunology 2024B cell transcriptomic signatures hold promise for the early prediction of vaccine-induced humoral immunity and vaccine protective efficacy. We performed a longitudinal...
B cell transcriptomic signatures hold promise for the early prediction of vaccine-induced humoral immunity and vaccine protective efficacy. We performed a longitudinal study in 232 healthy adult participants before/after a 3 dose of MMR (MMR3) vaccine. We assessed baseline and early transcriptional patterns in purified B cells and their association with measles-specific humoral immunity after MMR vaccination using two analytical methods ("per gene" linear models and joint analysis). Our study identified distinct early transcriptional signatures/genes following MMR3 that were associated with measles-specific neutralizing antibody titer and/or binding antibody titer. The most significant genes included: the interleukin 20 receptor subunit beta/ gene (a subunit receptor for IL-24, a cytokine involved in the germinal center B cell maturation/response); the phorbol-12-myristate-13-acetate-induced protein 1/, the brain expressed X-linked 2/ gene and the B cell Fas apoptotic inhibitory molecule/, involved in the selection of high-affinity B cell clones and apoptosis/regulation of apoptosis; as well as (encoding the B lymphocyte-derived IL-16 ligand of CD4), involved in the crosstalk between B cells, dendritic cells and helper T cells. Significantly enriched pathways included B cell signaling, apoptosis/regulation of apoptosis, metabolic pathways, cell cycle-related pathways, and pathways associated with viral infections, among others. In conclusion, our study identified genes/pathways linked to antigen-induced B cell proliferation, differentiation, apoptosis, and clonal selection, that are associated with, and impact measles virus-specific humoral immunity after MMR vaccination.
Topics: Adult; Humans; Measles-Mumps-Rubella Vaccine; Immunity, Humoral; Longitudinal Studies; Antibodies, Viral; Measles; Gene Expression Profiling; Nerve Tissue Proteins
PubMed: 38633249
DOI: 10.3389/fimmu.2024.1358477 -
Journal of Interferon & Cytokine... Apr 2024This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from...
Activated and Naïve Allogenic Human Placental Mesenchymal Stromal Cells Exert an Immunomodulatory Effect on Hidradenitis Suppurativa Patient Peripheral Blood Mononuclear Cells.
This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from patients with hidradenitis suppurativa (HS). Blood samples were collected from 3 healthy and 3 patients with HS. Isolated PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin solution. The PBMCs of patients with HS were co-cultured with naïve MSCs (n-MSCs), activated with tumor necrosis factor (TNF)-α (10 ng/mL) and interferon (IFN)-γ (10 ng/mL) MSCs (a-MSCs), or adalimumab (30 μg/mL). The division index (proliferation inhibition) of PBMCs was analyzed by flow cytometry using the Proliferation Modeling tool after 5 days of coculture. The relative inflammatory gene expression dynamics and cytokine secretion were quantified in triplicate using real-time polymerase chain reaction (PCR) and Luminex assays. PBMCs from the HS control group showed statistically significant increases in interleukin (IL)-6 and IFN-γ cytokine concentrations and gene expression when compared with healthy subjects. Statistically significant reduction of the division index was found in the a-MSCs group ( = 0.04). Also, the Luminex assay revealed significantly reduced proinflammatory cytokine concentrations of IL-9 ( = 0.022) and IL-17A ( = 0.022) in the a-MSCs group with the same trend of numerical lowering in n-MSCs group when compared to HS control. The results of real-time PCR revealed a numerical increase in the expression of the , and genes in both the a-MSCs and n-MSCs groups compared with the HS control. In conclusion, our findings suggest that MSCs can effectively curb PBMCs proliferation and suppress the production of inflammatory cytokines. Moreover, the preactivation of MSCs with IFN-γ and TNF-α before use can enhance their therapeutic effectiveness. Nevertheless, a larger sample size is imperative to validate these results.
PubMed: 38607317
DOI: 10.1089/jir.2024.0035 -
Zhongguo Xue Xi Chong Bing Fang Zhi Za... Feb 2024To investigate the expression of neutrophil extracellular traps (NETs) and phagocytic function in the peripheral blood of patients with hepatic alveolar echinococcosis...
OBJECTIVE
To investigate the expression of neutrophil extracellular traps (NETs) and phagocytic function in the peripheral blood of patients with hepatic alveolar echinococcosis (HAE), and to examine their correlations with clinical inflamma tory indicators and liver functions.
METHODS
A total of 50 patients with HAE admitted to Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qinghai University from August 2022 to June 2023 were enrolled, while 50 age- and gender-matched healthy individuals from the Centre for Healthy Examinations of the hospital during the same period served as controls. The levels of NETs markers neutrophil myeloperoxidase (MPO) and neutrophil elastase (NE) were measured using enzyme-linked immunosorbent assay (ELISA). Peripheral blood neutrophils were isolated using density gradient centrifugation, stimulated using phorbol 12-myristate 13 acetate (PMA), and the levels of MPO and citrullination histone H3 (CitH3) released by neutrophils were quantified using flow cytometry. The phagocytic functions of neutrophils were examined using flow cytometry. In addition, the correlations of MPO and NE levels with clinical inflammatory indicators and liver biochemical indicators were examined using Spearman correlation analysis among HAE patients.
RESULTS
The peripheral blood plasma MPO[(417.15 ± 76.08) ng/mL vs. (255.70 ± 80.84) ng/mL; = 10.28, < 0.05], NE[(23.16 ± 6.75) ng/mL vs. (11.92 ± 3.17) ng/mL; = 10.65, < 0.05]and CitH3 levels[(33.93 ± 18.93) ng/mL vs. (19.52 ± 13.89) ng/mL; = 4.34, < 0.05]were all significantly higher among HAE patients than among healthy controls, and a lower phagocytosis rate of neutrophils was detected among HAE patients than among healthy controls[(70.85 ± 7.32)% vs. (94.04 ± 3.90)%; = 20.18, < 0.05], and the ability to produce NETs by neutrophils was higher among HAE patients than among healthy controls following PMA stimulation. Pearson correlation analysis showed that the phagocytosis rate of neutrophils correlated negatively with platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), interleukin-6 (IL-6) level and C-reactive protein (CRP) level ( = -0.515 to -0.392, all values < 0.05), and the MPO and NE levels positively correlated with inflammatory markers NLR, PLR, CRP and IL-6 ( = 0.333 to 0.445, all values < 0.05) and clinical liver biochemical indicators aspartic transaminase, alanine aminotransferase, direct bilirubin and total bilirubin among HAE patients ( = 0.290 to 0.628, all values < 0.001).
CONCLUSIONS
Excessive formation of NETs is found among HAE patients, which affects the phagocytic ability of neutrophils and results in elevated levels of inflammatory indicators. NETs markers may be promising novel biomarkers for early diagnosis, monitoring, and severity assessment of liver disease.
Topics: Humans; Extracellular Traps; Interleukin-6; Echinococcosis, Hepatic; Neutrophils; Tetradecanoylphorbol Acetate; Bilirubin
PubMed: 38604682
DOI: 10.16250/j.32.1374.2023172 -
Biomolecules & Therapeutics May 2024In this study, we investigated the efficacy of kaempferol (a flavonoid found in plants and plant-derived foods such as kale, beans, tea, spinach and broccoli) on...
In this study, we investigated the efficacy of kaempferol (a flavonoid found in plants and plant-derived foods such as kale, beans, tea, spinach and broccoli) on vascular contractibility and aimed to clarify the detailed mechanism underlying the relaxation. Isometric contractions of divested muscles were stored and linked with western blot analysis which was carried out to estimate the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to estimate the effect of kaempferol on the RhoA/ROCK/CPI-17 pathway. Kaempferol conspicuously impeded phorbol ester-, fluoride- and a thromboxane mimetic-derived contractions regardless of endothelial nitric oxide synthesis, indicating its direct effect on smooth muscles. It also conspicuously impeded the fluoride-derived elevation in phospho-MYPT1 rather than phospho-CPI-17 levels and phorbol 12,13-dibutyrate-derived increase in phospho-CPI-17 and phospho-ERK1/2 levels, suggesting the depression of PKC and MEK activities and subsequent phosphorylation of CPI-17 and ERK1/2. Taken together, these outcomes suggest that kaempferol-derived relaxation incorporates myosin phosphatase retrieval and calcium desensitization, which appear to be modulated by CPI-17 dephosphorylation mainly through PKC inactivation.
PubMed: 38589300
DOI: 10.4062/biomolther.2023.186 -
Inflammation Research : Official... Jun 2024Mast cells (MCs), as the fastest immune responders, play a critical role in the progression of neuroinflammation-related diseases, especially in depression. Quercetin...
OBJECTIVE AND DESIGN
Mast cells (MCs), as the fastest immune responders, play a critical role in the progression of neuroinflammation-related diseases, especially in depression. Quercetin (Que) and kaempferol (Kae), as two major diet-derived flavonoids, inhibit MC activation and exhibit significant antidepressant effect due to their anti-inflammatory capacity. The study aimed to explore the mechanisms of inhibitory effect of Que and Kae on MC activation, and whether Que and Kae suppress hippocampal mast cell activation in LPS-induced depressive mice.
SUBJECTS AND TREATMENT
In vitro assays, human mast cells (HMC-1) were pretreated with Que or Kae for 1 h, then stimulated by phorbol 12-myristate 13-acetate (PMA) and 2,5-di-t-butyl-1,4-benzohydroquinone (tBHQ) for 3 h or 12 h. In vivo assays, Que or Kae was administered by oral gavage once daily for 14 days and then lipopolysaccharide (LPS) intraperitoneally injection to induce depressive behaviors.
METHODS
The secretion and expression of TNF-α were determined by ELISA and Western blotting. The nuclear factor of activated T cells (NFAT) transcriptional activity was measured in HMC-1 stably expressing NFAT luciferase reporter gene. Nuclear translocation of NFATc2 was detected by nuclear protein extraction and also was fluorescently detected in HMC-1 stably expressing eGFP-NFATc2. We used Ca imaging to evaluate changes of store-operated calcium entry (SOCE) in HMC-1 stably expressing fluorescent Ca indicator jGCamP7s. Molecular docking was used to assess interaction between the Que or Kae and calcium release-activated calcium modulator (ORAI). The hippocampal mast cell accumulation and activation were detected by toluidine blue staining and immunohistochemistry with β-tryptase.
RESULTS
In vitro assays of HMC-1 activated by PtBHQ (PMA and tBHQ), Que and Kae significantly decreased expression and secretion of TNF-α. Moreover, NFAT transcriptional activity and nuclear translocation of NFATc2 were remarkably inhibited by Que and Kae. In addition, the Ca influx mediated by SOCE was suppressed by Que, Kae and the YM58483 (ORAI inhibitor), respectively. Importantly, the combination of YM58483 with Que or Kae had no additive effect on the inhibition of SOCE. The molecular docking also showed that Que and Kae both exhibit high binding affinities with ORAI at the same binding site as YM58483. In vivo assays, Que and Kae significantly reversed LPS-induced depression-like behaviors in mice, and inhibited hippocampal mast cell activation in LPS-induced depressive mice.
CONCLUSIONS
Our results indicated that suppression of SOCE/NFATc2 pathway-mediated by ORAI channels may be the mechanism of inhibitory effect of Que and Kae on MC activation, and also suggested Que and Kae may exert the antidepressant effect through suppressing hippocampal mast cell activation.
Topics: Animals; Mast Cells; NFATC Transcription Factors; Lipopolysaccharides; Kaempferols; Hippocampus; Humans; Male; Quercetin; Depression; Cell Line; Signal Transduction; Mice; Calcium; Calcium Channels; Mice, Inbred C57BL; Antidepressive Agents
PubMed: 38587532
DOI: 10.1007/s00011-024-01876-7 -
Virology Journal Apr 2024Although macrophages are now recognized as an essential part of the HIV latent reservoir, whether and how viral latency is established and reactivated in these cell...
BACKGROUND
Although macrophages are now recognized as an essential part of the HIV latent reservoir, whether and how viral latency is established and reactivated in these cell types is poorly understood. To understand the fundamental mechanisms of viral latency in macrophages, there is an urgent need to develop latency models amenable to genetic manipulations and screening for appropriate latency-reversing agents (LRAs). Given that differentiated THP-1 cells resemble monocyte-derived macrophages in HIV replication mechanisms, we set out to establish a macrophage cell model for HIV latency using THP-1 cells.
METHODS
We created single-cell clones of THP-1 cells infected with a single copy of the dual-labeled HIV in which a codon switched eGFP (csGFP) is under the control of the HIV-1 5' LTR promoter, and a monomeric Kusabira orange 2 (mKO2) under the control of cellular elongation factor one alpha promoter (EF1α). Latently infected cells are csGFP, mKO2 while cells with actively replicating HIV (or reactivated virus) are csGFP,mKO2. After sorting for latently infected cells, each of the THP-1 clones with unique integration sites for HIV was differentiated into macrophage-like cells with phorbol 12-myristate 13-acetate (PMA) and treated with established LRAs to stimulate HIV reactivation. Monocyte-derived macrophages (MDMs) harboring single copies of HIV were used to confirm our findings.
RESULTS
We obtained clones of THP-1 cells with latently infected HIV with unique integration sites. When the differentiated THP-1 or primary MDMs cells were treated with various LRAs, the bromodomain inhibitors JQ1 and I-BET151 were the most potent compounds. Knockdown of BRD4, the target of JQ1, resulted in increased reactivation, thus confirming the pharmacological effect. The DYRK1A inhibitor Harmine and lipopolysaccharide (LPS) also showed significant reactivation across all three MDM donors. Remarkably, LRAs like PMA/ionomycin, bryostatin-1, and histone deacetylase inhibitors known to potently reactivate latent HIV in CD4 + T cells showed little activity in macrophages.
CONCLUSIONS
Our results indicate that this model could be used to screen for appropriate LRAs for macrophages and show that HIV latency and reactivation mechanisms in macrophages may be distinct from those of CD4 + T cells.
Topics: Humans; Virus Latency; Virus Activation; Transcription Factors; HIV Infections; Nuclear Proteins; HIV-1; Macrophages; CD4-Positive T-Lymphocytes; Bromodomain Containing Proteins; Cell Cycle Proteins
PubMed: 38581045
DOI: 10.1186/s12985-024-02343-9 -
NanoImpact Apr 2024The increasing application of quantum dots (QDs) increases interactions with organisms. The inflammatory imbalance is a significant manifestation of immunotoxicity....
The increasing application of quantum dots (QDs) increases interactions with organisms. The inflammatory imbalance is a significant manifestation of immunotoxicity. Macrophages maintain inflammatory homeostasis. Using macrophages differentiated by phorbol 12-myristate 13-acetate-induced THP-1 cells as models, the study found that low-dose (5 μM) cadmium telluride QDs (CdTe-QDs) hindered monocyte-macrophage differentiation. CD11b is a surface marker of macrophage, and the addition of CdTe-QDs during induction resulted in a decrease in CD11b expression. Moreover, exposure of differentiated THP-1 macrophage (dTHP-1) to 5 μM CdTe-QDs led to the initiation of M1 polarization. This was indicated by the increased surface marker CD86 expression, along with elevated level of NF-κB and IL-1β proteins. The potential mechanisms are being explored. The transcription factor EB (TFEB) plays a significant role in immune regulation and serves as a crucial regulator of the autophagic lysosomal pathway. After exposed to CdTe-QDs, TFEB activation-mediated autophagy and M1 polarization were observed to occur simultaneously in dTHP-1. The mTOR signaling pathway contributed to TFEB activation induced by CdTe-QDs. However, mTOR-independent activation of TFEB failed to promote M1 polarization. These results suggest that mTOR-TFEB is an advantageous target to enhance the biocompatibility of CdTe-QDs.
Topics: Tellurium; Quantum Dots; Cadmium Compounds; Humans; Macrophages; TOR Serine-Threonine Kinases; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; THP-1 Cells; Autophagy; Cell Differentiation; Signal Transduction
PubMed: 38579989
DOI: 10.1016/j.impact.2024.100505