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Frontiers in Bioscience (Landmark... Mar 2023To investigate the effects of extract (GBE) on autophagy in human macrophages stimulated by cigarette smoke extract (CSE).
OBJECTIVE
To investigate the effects of extract (GBE) on autophagy in human macrophages stimulated by cigarette smoke extract (CSE).
METHODS
The human monocyte cell line U937 was cultured , and phorbol ester (PMA) was added to the cell culture medium to induce differentiation into human macrophages. CSE was prepared by traditional methods for experiments. The cells were divided into four groups: the blank group, the CSE model group, the GBE + CSE group, and the rapamycin + CSE group. Immunofluorescence was used to identify human macrophages, transmission electron microscopy was used to observe the ultrastructure of human macrophages in each group, ELISA was used to measure the amount of IL-6 and IL-10 in the supernatant from each group of cells, the mRNA levels of p62, ATG5, ATG7, and Rab7 were measured by real-time qPCR, and the protein expression levels of p62, ATG5, ATG7, and Rab7 were measured by Western blotting.
RESULTS
U937 cells were successfully differentiated into human macrophages after induction with PMA. The CSE model group had many more autophagosomes than the blank group. Compared with the CSE model group, the GBE + CSE group and the rapamycin + CSE group had significantly more autophagolysosomal. Compared with the other groups, the CSE model group had a higher level of IL-6 but a lower level of IL-10 in the supernatant ( 0.05). Compared with the blank group, the mRNA and protein expression levels of p62 in the CSE model group were significantly decreased, while the mRNA and protein expression levels of ATG5 and ATG7 were significantly increased in the CSE model group ( 0.05). No difference was found in the mRNA and protein expression levels of Rab7 between the blank group and the CSE model group. Compared with the CSE model group, the IL-6 level in the GBE + CSE group and the rapamycin + CSE group cell culture supernatant decreased significantly, p62 mRNA and protein expression significantly decreased, while ATG5, ATG7, and Rab7 mRNA and protein expression levels were significantly increased ( 0.05). Moreover, increased LC3-II/LC3-I ratio were also found in the GBE + CSE group and the rapamycin + CSE group compared with the CSE model group.
CONCLUSIONS
GBE could promote the fusion of autophagosomes and lysosomes in human macrophages, enhance the autophagy function of human macrophages, and reduce the damaging effect of CSE on the autophagy function of macrophages.
Topics: Humans; Interleukin-10; Cigarette Smoking; Interleukin-6; RNA, Messenger; Autophagy; Macrophages
PubMed: 37005757
DOI: 10.31083/j.fbl2803050 -
Frontiers in Immunology 2023Alpha-fetoprotein(AFP) is a cancer biomarker for the diagnosis of hepatocellular carcinoma(HCC); however, its role in macrophage polarization and phagocytosis remains...
Alpha-fetoprotein(AFP) is a cancer biomarker for the diagnosis of hepatocellular carcinoma(HCC); however, its role in macrophage polarization and phagocytosis remains unclear. In the present study, we explored the correlation between AFP regulation of macrophage function and the possible regulatory mechanisms. Human mononuclear leukemia cells (THP-1) and monocytes from healthy donors were used to analyze the effect of AFP on the macrophages' phenotype and phagocytosis. THP-1 cells and healthy human donor-derived monocytes were polarized into M0 macrophages induced by phorbol ester (PMA), and M0 macrophages were polarized into M1 macrophages induced by lipopolysaccharide(LPS) and interferon-γ(IFN-γ). Interleukin-4(IL-4) and interleukin-13(IL-13) were used to induce M0 macrophage polarization into M2 macrophages. Tumor-derived AFP(tAFP) stimulated M0 macrophage polarization into M2 macrophages and inhibited M1 macrophages to phagocytize HCC cells. The role of AFP in promoting macrophage polarization into M2 macrophages and inhibiting the M1 macrophages to phagocytize HCC cells may be involved in activating the PI3K/Akt signaling pathway. AFP could also enhanced the migration ability of macrophages and inhibited the apoptosis of HCC cells when co-cultured with M1-like macrophages. AFP is a pivotal cytokine that inhibits macrophages to phagocytize HCC cells.
Topics: Humans; alpha-Fetoproteins; Carcinoma, Hepatocellular; Phosphatidylinositol 3-Kinases; Liver Neoplasms; Macrophages; Interferon-gamma; Phenotype
PubMed: 36911723
DOI: 10.3389/fimmu.2023.1081572 -
International Journal of Molecular... Feb 2023Protein kinase C delta (PKC-δ) is an important signaling molecule in human cells that has both proapoptotic as well as antiapoptotic functions. These conflicting...
Protein kinase C delta (PKC-δ) is an important signaling molecule in human cells that has both proapoptotic as well as antiapoptotic functions. These conflicting activities can be modulated by two classes of ligands, phorbol esters and bryostatins. Phorbol esters are known tumor promoters, while bryostatins have anti-cancer properties. This is despite both ligands binding to the C1b domain of PKC-δ (δC1b) with a similar affinity. The molecular mechanism behind this discrepancy in cellular effects remains unknown. Here, we have used molecular dynamics simulations to investigate the structure and intermolecular interactions of these ligands bound to δC1b with heterogeneous membranes. We observed clear interactions between the δC1b-phorbol complex and membrane cholesterol, primarily through the backbone amide of L250 and through the K256 side-chain amine. In contrast, the δC1b-bryostatin complex did not exhibit interactions with cholesterol. Topological maps of the membrane insertion depth of the δC1b-ligand complexes suggest that insertion depth can modulate δC1b interactions with cholesterol. The lack of cholesterol interactions suggests that bryostatin-bound δC1b may not readily translocate to cholesterol-rich domains within the plasma membrane, which could significantly alter the substrate specificity of PKC-δ compared to δC1b-phorbol complexes.
Topics: Humans; Protein Kinase C-delta; Bryostatins; Isoenzymes; Phorbol Esters; Phorbols; Lactones
PubMed: 36902029
DOI: 10.3390/ijms24054598 -
Frontiers in Immunology 2023Neutrophil extracellular traps (NET) formation is one important host innate defense mechanism elicited by polymorphonuclear neutrophils (PMN). NETs are composed by...
Neutrophil extracellular traps (NET) formation is one important host innate defense mechanism elicited by polymorphonuclear neutrophils (PMN). NETs are composed by chromatin and proteins with microbicidal and signaling activity. So far, there is one report on triggered NETs in cattle, however, exact mechanisms, including signalling pathways and dynamics governing this reaction remain largely unknown. Recently, involvement of cell cycle proteins was demonstrated for phorbol myristate acetate (PMA)-triggered human PMN-derived NETs. Here, we studied the involvement of cell cycle proteins in -induced NETs in exposed bovine PMN. Through confocal and transmission electron microscopy we discovered that Ki-67 and lamin B1 signals are upregulated and relocated during -induced NETosis. Nuclear membrane disruption was also observed as a hallmark of NET formation in bovine PMN confronted with viable tachyzoites, mimicking some steps of mitosis. However, we did not observe centrosome duplication as previously described for human PMN-derived NET formation stimulated with PMA.
Topics: Humans; Cattle; Animals; Extracellular Traps; Cell Cycle Proteins; Toxoplasma; Neutrophils; Cell Cycle; Receptor Protein-Tyrosine Kinases; Tetradecanoylphorbol Acetate
PubMed: 36875070
DOI: 10.3389/fimmu.2023.1125667 -
Scientific Reports Feb 2023Macrophages (MQs) pro-inflammatory phenotype is triggered by gliadin peptides. Akkermansia muciniphila (A. muciniphila) showed to enhance the anti-inflammatory phenotype...
Macrophages (MQs) pro-inflammatory phenotype is triggered by gliadin peptides. Akkermansia muciniphila (A. muciniphila) showed to enhance the anti-inflammatory phenotype of MQs. This study aimed to investigate the anti-inflammatory effects of A. muciniphila, on gliadin stimulated THP-1 derived macrophages. THP-1 cell line monocytes were differentiated into MQs by phorbol 12-myristate 13-acetate (PMA). MQs were treated with A. muciniphila before and after stimulation with gliadin (pre- and post-treat). CD11b, as a marker of macrophage differentiation, and CD206 and CD80, as M1 and M2 markers, were evaluated by flow cytometry technique. The mRNA expression of TGF-β, IL-6, and IL-10 and protein levels of IL-10 and TNF-α were measured by RT-PCR and ELISA techniques, respectively. Results show an increased percentage of M1 phenotype and release of proinflammatory cytokines (like TNF-α and IL-6) by macrophages upon incubation with gliadin. Pre- and post-treatment of gliadin-stimulated macrophages with A. muciniphila induced M2 phenotype associated with decreased proinflammatory (IL-6, TNF-α) and increased anti-inflammatory (IL-10, TGF-β) cytokines expression relative to the group that was treated with gliadin alone. This study suggests the potential beneficial effect of A. muciniphila on gliadin-stimulated MQs and the importance of future studies focusing on their exact mechanism of action on these cells.
Topics: Interleukin-10; Gliadin; Tumor Necrosis Factor-alpha; Interleukin-6; Macrophages; Cytokines; Anti-Inflammatory Agents; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta
PubMed: 36828897
DOI: 10.1038/s41598-023-30266-y -
Biochemical and Biophysical Research... Apr 2023Skeletal muscle differentiation involves activation of quiescent satellite cells to proliferate, differentiate and fuse to form new myofibers; this requires coordination...
Skeletal muscle differentiation involves activation of quiescent satellite cells to proliferate, differentiate and fuse to form new myofibers; this requires coordination of myogenic transcription factors. Myogenic transcription is tightly regulated by various intracellular signaling pathways, which include members of the protein kinase D (PKD) family. PKD is a family of serine-threonine kinases that regulate gene expression, protein secretion, cell proliferation, differentiation and inflammation. PKD is a unique PKC family member that shares distant sequence homology to calcium-regulated kinases and plays an important role in muscle physiology. In this report, we show that class I histone deacetylase (HDAC) inhibition, and in particular HDAC8 inhibition, attenuated PKD phosphorylation in skeletal C2C12 myoblasts in response to phorbol ester, angiotensin II and dexamethasone signaling independent of changes in total PKD protein expression. As class I HDACs and PKD signaling are requisite for myocyte differentiation, these data suggest that HDAC8 functions as a potential feedback regulator of PKD phosphorylation to control myogenic gene expression.
Topics: Phosphorylation; Protein Kinase C; Signal Transduction; Myoblasts, Skeletal
PubMed: 36773343
DOI: 10.1016/j.bbrc.2023.02.010 -
Journal of Virology Feb 2023The ability of Epstein-Barr virus (EBV) to switch between latent and lytic infection is key to its long-term persistence, yet the molecular mechanisms behind this switch...
The ability of Epstein-Barr virus (EBV) to switch between latent and lytic infection is key to its long-term persistence, yet the molecular mechanisms behind this switch remain unclear. To investigate transcriptional events during the latent-to-lytic switch, we utilized Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to map cellular RNA polymerase (Pol) activity to single-nucleotide resolution on the host and EBV genome in three different models of EBV latency and reactivation. In latently infected Mutu-I Burkitt lymphoma (BL) cells, Pol activity was enriched at the Qp promoter, the EBER region, and the BHLF1/LF3 transcripts. Upon reactivation with phorbol ester and sodium butyrate, early-phase Pol activity occurred bidirectionally at CTCF sites within the LMP-2A, EBER-1, and RPMS1 loci. PRO-Seq analysis of Akata cells reactivated from latency with anti-IgG and a lymphoblastoid cell line (LCL) reactivated with small molecule C60 showed a similar pattern of early bidirectional transcription initiating around CTCF binding sites, although the specific CTCF sites and viral genes were different for each latency model. The functional importance of CTCF binding, transcription, and reactivation was confirmed using an EBV mutant lacking the LMP-2A CTCF binding site. This virus was unable to reactivate and had disrupted Pol activity at multiple CTCF binding sites relative to the wild-type (WT) virus. Overall, these data suggest that CTCF regulates the viral early transcripts during reactivation from latency. These activities likely help maintain the accessibility of the viral genome to initiate productive replication. The ability of EBV to switch between latent and lytic infection is key to its long-term persistence in memory B cells, and its ability to persist in proliferating cells is strongly linked to oncogenesis. During latency, most viral genes are epigenetically silenced, and the virus must overcome this repression to reactivate lytic replication. Reactivation occurs once the immediate early (IE) EBV lytic genes are expressed. However, the molecular mechanisms behind the switch from the latent transcriptional program to begin transcription of the IE genes remain unknown. In this study, we mapped RNA Pol positioning and activity during latency and reactivation. Unexpectedly, Pol activity accumulated at distinct regions characteristic of transcription initiation on the EBV genome previously shown to be associated with CTCF. We propose that CTCF binding at these regions retains Pol to maintain a stable latent chromosome conformation and a rapid response to various reactivation signals.
Topics: Humans; Binding Sites; Epstein-Barr Virus Infections; Gene Expression Regulation, Viral; Genome, Viral; Herpesvirus 4, Human; Virus Activation; Virus Latency; RNA-Dependent RNA Polymerase; Cell Line, Tumor; CCCTC-Binding Factor
PubMed: 36744959
DOI: 10.1128/jvi.01894-22 -
PKCβII activation requires nuclear trafficking for phosphorylation and Mdm2-mediated ubiquitination.Life Science Alliance Apr 2023PKCβII, a conventional PKC family member, plays critical roles in the regulation of a variety of cellular functions. Here, we employed loss-of-function approaches and...
PKCβII, a conventional PKC family member, plays critical roles in the regulation of a variety of cellular functions. Here, we employed loss-of-function approaches and mutants of PKCβII with altered phosphorylation and protein interaction behaviors to identify the cellular mechanisms underlying the activation of PKCβII. Our results show that 3-phosphoinositide-dependent protein kinase-1 (PDK1)-mediated constitutive phosphorylation of PKCβII at the activation loop (T500) is required for phorbol ester-induced nuclear entry and subsequent Mdm2-mediated ubiquitination of PKCβII, whereas ubiquitination of PKCβII is required for the PDK1-mediated inducible phosphorylation of PKCβII at T500 in the nucleus. After moving out of the nucleus, PKCβII interacts with actin, undergoes inducible mTORC2-mediated phosphorylation at the turn motif (T641), interacts with clathrin, and then translocates to the plasma membrane. This overall cascade of cellular events intertwined with the phosphorylation at critical residues and Mdm2-mediated ubiquitination in the nucleus and along with interactions with actin and clathrin plays roles that encompass the core processes of PKC activation.
Topics: Actins; Clathrin; Phosphorylation; Protein Kinase C beta; Ubiquitination; Proto-Oncogene Proteins c-mdm2
PubMed: 36717249
DOI: 10.26508/lsa.202201748 -
Polymers Jan 2023The aim of this research was to evaluate the effect of untreated and 5% aqueous NaOH solution-treated filler of the plant Jatropha Curcas L. on the mechanical properties...
The aim of this research was to evaluate the effect of untreated and 5% aqueous NaOH solution-treated filler of the plant Jatropha Curcas L. on the mechanical properties of adhesive bonds, especially in terms of their service life at different amplitudes of cyclic loading. As a result of the presence of phorbol ester, which is toxic, Jatropha oilseed cake cannot be used as livestock feed. The secondary aim was to find other possibilities for the utilization of natural waste materials. Another use is as a filler in polymer composites, that is, in composite adhesive layers. The cyclic loading of the adhesive bonds was carried out for 1000 cycles in two amplitudes, that is, 5-30% of the maximum force and 5-50% of the maximum force, which was obtained by the static tensile testing of the adhesive bonds with unmodified filler. The static tensile test showed an increase in the shear strength of the adhesive bonds with alkali-treated filler compared to the untreated filler by 3-41%. The cyclic test results did not show a statistically significant effect of the alkaline treatment of the filler surface on the service life of the adhesive bonds. Positive changes in the strain value between adhesive bonds with treated and untreated filler were demonstrated at cyclic stress amplitudes of 5-50%. SEM analysis showed the presence of interlayer defects in the layers of the tested materials, which are related to the oil-based filler used.
PubMed: 36679275
DOI: 10.3390/polym15020395 -
Frontiers in Immunology 2022The activation of NLRP3 inflammasome in macrophages has been proven to play a crucial role in the development of cardiovascular diseases. THP-1 monocytes can be...
BACKGROUND
The activation of NLRP3 inflammasome in macrophages has been proven to play a crucial role in the development of cardiovascular diseases. THP-1 monocytes can be differentiated to macrophages by incubation with phorbol-12-myristate 13-acetate (PMA), providing a suitable model for studies. However, PMA has been shown to have effects on the levels of IL-1β, the main mediator of NLRP3 inflammasome, while the effects on the other mediators of the inflammasome have not been reported before.
METHODS
THP-1 monocytes were incubated without (THP-1), with 5ng/ml PMA for 48h (PMA48h) or with 5ng/ml PMA for 48h plus 24h in fresh medium (PMArest). Morphological changes and the expression of macrophage surface markers (CD14, CD11b, CD36 and CD204) were evaluated by flow cytometry. Changes in intracellular levels of inflammasome components (NLRP3, ASC, pro-caspase-1, pro-IL1β) were analyzed by western blot and release of mature IL-1β in cell supernatant was analyzed by ELISA. ASC speck formation was determined by immunofluorescence.
RESULTS
After 48h incubation with PMA or subsequent rest in fresh medium, cells became adherent, and the differential expression of CD36, CD11b, CD14 and CD204 compared to THP-1 cells confirmed that PMArest resemble macrophages from a molecular point of view. Changes in the levels were detected in PMA48h group for all the NLRP3-related proteins, with increase of NLRP3 and pro-IL-1β and secretion of mature IL-1β. In PMArest, no pro-IL-1β and lower amounts of mature IL-1β were detected. No ASC speck was found in PMA treated groups, but the addition of a second stimulus to PMArest resulted in ASC speck formation, together with IL-1β production, confirming the responsiveness of the model.
CONCLUSION
Differentiation of THP-1 with 5ng/ml PMA followed by 24h resting period provides a model that morphologically and molecularly resembles macrophages. However, even at low concentrations, PMA induces production of IL-1β. The 24h rest period provides for down-regulation of pro-IL-1β in PMArest group, without affecting its ability to respond to a second stimulus through activation of inflammasome.
Topics: Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Myristates; Tetradecanoylphorbol Acetate; Macrophages; Acetates
PubMed: 36618426
DOI: 10.3389/fimmu.2022.958098