Phorbol 12-myristate 13-acetate Research Articles

PCN‐134(Fe)‐Gated Organic Photoelectrochemical Transistor with Unique Dual‐Directional Signaling

AbstractRecently, increasing attention has been paid to exploring the unique characteristics of newly emerged organic photoelectrochemical transistors (OPECT) for biological application and neuromorphic engineering due to their effectiveness for light‐driven ionic regulation of channels interfaced with rich biochemical reactions. The observation of interesting signaling reversion is reported—the realization of dual‐directional signaling with enhanced signal resolution—enabled by porous coordination networks (PCN)‐134(Fe) in the presence of varying H2O2 concentrations, which is totally different from exiting unidirectional signaling in OPECT. The underlying mechanism of such behavior is attributed to the change in the polarity of the gate photocurrent and is explained from the perspective of potential distribution. The practical application of this device is then studied by detecting the phorbol myristate acetate (PMA)‐induced secretion of H2O2 from three kinds of human breast cells. This study features the realization of dual‐directional signaling, which is expected to catalyze a new category of OPECT operation with unknown possibilities.

MiR-525-5p modulates cell proliferation, cell cycle, and apoptosis in Burkitt's lymphoma by targeting MyD88 and regulating the NF-κB signaling pathway.

MiR-525-5p functions as an oncomiRNA or tumor suppressor, and has been reported in various cancer types, including laryngeal squamous cell carcinoma, glioma, breast cancer, and cervical cancer. However, the biological functions and precise mechanisms of miR-525-5p remain unclarified in Burkitt's lymphoma (BL). This study aimed to explore the roles of miR-525-5p in BL, with the goal of ascertaining its regulatory effects on the nuclear factor-kappaB (NF-κB) signaling pathway by targeting Myeloid differentiation factor 88 (MyD88). The expression levels of miR-525-5p and MyD88 were measured by quantitative real-time PCR and immunohistochemical staining, respectively. The effects of miR-525-5p overexpression on BL cell proliferation, colony-forming, and migration were evaluated by cell counting kit-8, soft agar colony-forming, and transwell assays, while cell cycle and cell apoptosis were analyzed by flow cytometry. Possible interactions between miR-525-5p and MyD88 was examined via luciferase reporter assay. The expression of MyD88 and NF-κB signaling pathway-related proteins, including p65, p-p65, IκBa, and p-ΙκBa was determined by western blotting. BL cells overexpressing miR-525-5p were treated with phorbol 12-myristate 13-acetate (PMA), and Hoechst 33258 staining and Calcein AM/EthD-I staining were used to analyze the changes in chemotherapy sensitivity of BL cells to doxorubicin (DOX). Compared with reactive lymphoid hyperplasia, miR-525-5p was dramatically downregulated in BL tissues, while the rate of MyD88 protein positivity was significantly increased. Upregulation of miR-525-5p suppressed cell proliferation, colony-forming, and migration, induced cell cycle arrest and apoptosis, and enhanced the chemosensitivity to DOX in BL cells. MiR-525-5p targeted MyD88 to inhibit the activation of NF-κB signaling pathway. PMA treatment reactivated the NF-κB pathway and reversed apoptosis mediated by miR-525-5p overexpression. These findings revealed that miR-525-5p acts as a tumor suppressor, targeting MyD88 to modulate proliferation, cell cycle progression, and apoptosis in BL cells by regulation of NF-κB signaling pathway.

Combination of Methotrexate and Resveratrol Reduces Pro-Inflammatory Chemokines in Human THP-1 Cells.

Methotrexate (MTX) is a widely used anti-metabolite drug in cancer therapy, but its efficacy is often hindered by reactive oxygen species (ROS)-induced cellular toxicity. Resveratrol, a natural polyphenol, possesses antioxidant and anticancer properties. Therefore, this in vitro study aimed to investigate the synergistic anti-proliferative and anti-inflammatory effects of MTX and resveratrol in human THP-1 cells. THP-1 cells were differentiated into macrophage-like cells using phorbol 12-myristate 13-acetate. In vitro experiments assessed the impact of various concentrations of MTX and resveratrol on cell viability and proliferation using the MTT assay. Concentration-effect curves were generated to elucidate their relationship. Gene expression was analyzed by RT-qPCR, while chemokine expression was measured via ELISA. Phagocytic and migratory activities were also evaluated. Differentiated THP-1 cells were treated with MTX and resveratrol and stimulated with dimethyl sulfoxide (DMSO) and lipopolysaccharide (LPS) as inflammatory stimuli. The combination of MTX and resveratrol exhibited an anti-proliferative effect in THP-1 cells (p = 0.03). The concentration-effect curve revealed IC50 values of 49.15 µg at 24hours (R = 0.8236) and 2.029e-005 µg at 48hours (R = 0.97) for MTX, and 20.17 µg at 48hours (R = 1.000) and 55.38 µg at 96hours (R = 0.9666) for resveratrol. Co-treatment with MTX and resveratrol significantly reduced mRNA and chemokine expression of CCL2, CCL3, CCL4, CCL5, and CXCL10 (p < 0.05). Moreover, decreased phagocytic and migratory activities were confirmed by phagocytosis and migration assays (p < 0.05). The combination of MTX and resveratrol effectively attenuated pro-inflammatory activity in THP-1 cells, as evidenced by the downregulation of mRNA and chemokine expression. These findings suggest that the synergistic effects of MTX and resveratrol hold promise for enhancing cancer therapeutics.

Parecoxib inhibits tumorigenesis and angiogenesis in hepatocellular carcinoma through ERK-VEGF/MMPs signaling pathway.

Parecoxib, a well-recognized nonsteroidal anti-inflammatory drug, has been reported to possess anticancer properties in various tumor types. In this work, we aimed to investigate the potential anticancer effects of parecoxib on hepatocellular carcinoma (HCC) cells. To assess the impact of parecoxib on HCC cell proliferation, we employed Cell Counting Kit-8, colony formation, and 5-ethynyl-2'-deoxyuridine assays. Hoechst/propidium iodide (PI) double staining and flow cytometry were performed to evaluate apoptosis and cell cycle analysis. Wound healing and transwell assays were utilized to assess cell migration and invasion. Tube formation assay was employed to analyze angiogenesis. Protein levels were determined using western blotting, and mRNA expression levels were assessed using quantitative real-time polymerase chain reaction (PCR). A xenograft mouse model was used to confirm the antitumor effects of parecoxib on HCC tumors in vivo. Our data demonstrated that parecoxib effectively inhibited the proliferation of HCC cells in a dose- and time-dependent manner. In addition, parecoxib induced cell cycle arrest in the G2 phase and promoted apoptosis. Moreover, parecoxib hindered tumor migration and invasion by impeding the epithelial-mesenchymal transition process. Further investigation showed that parecoxib could significantly suppress angiogenesis through the inhibition of extracellular signal-regulated kinase (ERK)-vascular endothelial growth factor (VEGF) axis. Notably, treatment with the ERK activator phorbol myristate acetate upregulated the expression of matrix metalloproteinase (MMP)-2, MMP-9, and VEGF and reversed the function of parecoxib in HCC cells. Besides, parecoxib displayed its antitumor efficacy in vivo. Collectively, our results suggest that parecoxib ameliorates HCC progression by regulating proliferation, cell cycle, apoptosis, migration, invasion, and angiogenesis through the ERK-VEGF/MMPs signaling pathway.

Protein Kinase C Isozyme Immaturity/Deficiency in Cord Blood Monocytes and Neutrophils

Reduced/deficient expression of Protein Kinase C (PKC)ζ in Cord blood (CB) T cells is associated with allergy development in children and a propensity to maintain an immature T-helper (Th)2 cytokine profile. In addition, other PKC isozymes are also low in CBTCs. Since previous studies have reported that cord blood/neonatal monocyte and neutrophil functions are significantly lower than cells from adults, it was of interest to see if the CBTC PKC levels were reflected in CB monocytes and neutrophils. Compared to adult blood, CB expresses low levels of PKCα, β2, ε, θ, μ, ζ and λ/ι in monocytes and PKCα, β2, η, θ, μ, ζ and λ/ι in neutrophils. The T-cell PKCζ levels were positively correlated with levels in CB monocytes but not in neutrophils. However, neither the monocytes nor the neutrophil PKCζ were associated with T-cell development towards a Th1 or Th2 cytokine propensity, based on the production of interferon-gamma and interleukin-4 in response to phytohemagglutinin and phorbol myristate acetate. The results demonstrate that some newborn babies display a deficiency in PKC isozymes in monocytes and neutrophils, as reported for T cells. However, unlike T cells, the PKCζ levels of the phagocytes did not correlate with regulation of development towards a Th1 or Th2 cytokine phenotype.

HIV-1 latency reversal agent boosting is not limited by opioid use.

Opioid use may impact the HIV-1 reservoir and its reversal from latency. We studied forty-seven virally suppressed people with HIV (PWH) and observed that lower concentration of HIV-1 latency reversal agents (LRA), used in combination with small molecules that did not reverse latency, synergistically increased the magnitude of HIV-1 re-activation ex vivo, regardless of opioid use. This LRA boosting, which combined a Smac mimetic or low-dose protein kinase C agonist with histone deacetylase inhibitors, generated significantly more unspliced HIV-1 transcription than phorbol 12-myristate 13-acetate (PMA) with ionomycin (PMAi), the maximal known HIV-1 reactivator. LRA boosting associated with greater histone acetylation, modulated surface activation-induced markers, and altered T cell production of TNFα, IL-2, and IFNγ. HIV-1 reservoirs in PWH contained unspliced and polyadenylated (polyA) virus mRNA, the ratios of which were greater in resting than total CD4+ T cells and correct to 1:1 with PMAi exposure. We characterized treated suppressed HIV-1 infection as a period of inefficient, not absent, virus transcription. Multiply spliced HIV-1 transcripts and virion production did not consistently increase with LRA boosting, suggesting the presence of a persistent post-transcriptional block. LRA boosting can be leveraged to probe mechanisms of an effective cellular HIV-1 latency reversal program.

Toll-like receptor 2-mediated ERK activation significantly upregulates interleukin-6 expression in M2-polarized macrophages

Abstract Objectives M2-polarized macrophages and interleukin (IL)-6 significantly alter the tumor microenvironment and promote the malignant behaviors of tumor cells. This study aimed to establish M2-type macrophages from THP-1 cells, which are human leukemia monocytes, and investigate the significance of toll-like receptor 2 (TLR2) signaling in IL-6 production. Methods THP-1 cells were treated with phorbol 12-myristate 13-acetate, IL-4, and IL-13 to stimulate their differentiation into M2 macrophages. Cell differentiation was confirmed by cytokine production, marker expression, and morphological alterations. Treatment with TLR agonists induced TLR stimulation in M2 macrophages. Subsequently, secretion and expression levels of IL-6 in M2 macrophages were measured using enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, and western blotting. Results Myeloid differentiation factor 88, tumor necrosis factor-associated factor 6, and IL-1 receptor-associated kinase-1/4 signaling pathways contributed to IL-6 production upon TLR2 activation in M2 macrophages. While both TLR2 and TLR4 activated NF-κB in M2 macrophages, IL-6 production was mainly dependent on TLR2, not TLR4, suggesting the involvement of major mechanisms other than NF-κB in IL-6 production. Notably, TLR2-stimulated extracellular signal-regulated kinase (ERK) was necessary for abundant IL-6 production, indicating that TLR2-mediated ERK signaling plays an essential role in M2 macrophages. Conclusions These results highlight the significance of TLR2 signaling in IL-6 production by M2 macrophages and provide insights into the underlying regulatory mechanisms.

In Situ Imaging of Cellular Inflammatory Response to Antibiotic Exposure with a DNAzyme Nanorobot.

Antibiotic-induced inflammation involves the release of myeloperoxidase (MPO), an enzyme whose expression in tissues is associated with the inflammatory pathway. However, existing methods for detecting MPO in cells are limited. In this study, a DNAzyme nanorobot was developed using a scaffold of gold nanoparticles (AuNPs) decorated with functional DNAzyme strands and their fluorophore-labeled substrate strands. The DNAzyme remains inactive due to a self-assembled hairpin structure, with a phosphorothioate (PT) modification inserted into the stem domain. When MPO is present, it triggers a halogenation process that generates hypochlorous acid (HClO). HClO specifically catalyzes the cleavage of the PT-site, releasing free DNAzyme strands to cleave their substrates and generating an increasing fluorescent signal. The detection limit for MPO and its primary product, HClO, were determined to be 0.038 μg/mL and 0.013 μM, respectively. The DNAzyme nanorobot can be readily introduced into cells and function autonomously to differentiate increased MPO/HClO levels caused by antibiotics. This approach was applied to image RAW264.7 cells exposed to four prevalent antibiotics found in the environment (phorbol 12-myristate 13-acetate, erythromycin, penicillin, and tetracycline) as well as antibiotic production wastewater. This nanorobot offers novel strategies for monitoring inflammation to evaluate the health impacts of antibiotic exposure.

Understanding the complex chromatin dynamics in primary human neutrophils during PMA-induced NET formation

BackgroundPrimary human neutrophils play a pivotal role in innate immunity, mainly through the formation of neutrophil extracellular traps (NETs) in a process known as NETosis. This cell-death pathway is crucial for combating infections but is also implicated in many inflammatory diseases, such as sepsis, systemic lupus erythematosus, and rheumatoid arthritis.MethodsThe study presented here investigates chromatin dynamics during NET formation by stimulating primary human neutrophils with phorbol 12-myristate 13-acetate (PMA). We adapt the ATAC-Seq (assay for transposase-accessible chromatin using sequencing) method to isolated neutrophils and characterize a time-dependent chromatin response.ResultsWe found that chromatin accessibility patterns are consistent across individual donors and most chromatin changes occur within 30 min, with many continuing across the 90 min assessed in this study. Regulatory regions gaining accessibility were associated with the activity of pathways that have been implicated in NOX-dependent NET formation.ConclusionsOur findings increase the understanding of the chromatin changes underlying NET formation and also identify potential early-acting targets for modulating this process in inflammatory diseases.

IL-27 attenuated macrophage injury and inflammation induced by Mycobacterium tuberculosis by activating autophagy.

Interleukin-27 (IL-27) is a cytokine that is reported to be highly expressed in the peripheral blood of patients with pulmonary tuberculosis (PTB). IL-27-mediated signaling pathways, which exhibit anti- Mycobacterium tuberculosis (Mtb) properties, have also been demonstrated in macrophages infected with Mtb. However, the exact mechanism remains unclear. This study aimed to clarify the potential molecular mechanisms through which IL-27 enhances macrophage resistance to Mtb infection.Both normal and PTB patients provided bronchoalveolar lavage fluid (BALF). Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals and stimulated with 50ng/mL macrophage-colony stimulating factor (M-CSF) to obtain monocyte-derived macrophages (MDMs). Using 100ng/mL phorbol 12-myristate 13-acetate (PMA), THP-1 cells were induced to differentiate into THP-1-derived macrophage-like cells (TDMs). Both MDMs and TDMs were subsequently infected with the Mtb strain H37Rv and treated with 50ng/mL IL-27 prior to infection. The damage and inflammation of macrophages were examined using flow cytometry, enzyme-linked immunosorbent assay (ELISA), and Western blotting.Patients with PTB had elevated levels of IL-27 in their BALF. Preconditioning with IL-27 was shown to reduce H37Rv-induced MDMs and TDMs apoptosis while also decreasing the levels of Cleaved Caspase-3, Bax and the proinflammatory cytokines TNF-α, IL-1β, and IL-6, promoting the expression of Bcl-2 and the anti-inflammatory factors IL-10 and IL-4. Silencing of the IL-27 receptor IL-27Ra increased macrophage damage and inflammation triggered by H37Rv. Mechanistically, IL-27 activates autophagy by inhibiting TLR4/NF-κB signaling and activating the PI3K/AKT signaling pathway, thereby inhibiting H37Rv-induced macrophage apoptosis and the inflammatory response.Our study suggests that IL-27 alleviates H37Rv-induced macrophage injury and the inflammatory response by activating autophagy and that IL-27 may be a new target for the treatment of PTB.

MLKL promotes extracellular trap releasing in Vibrio splendidus AJ01-challenged coelomocytes of sea cucumber Apostichopus japonicus

Extracellular traps (ETs) is a novel host-defense mechanism for the immobilization and killing of invading microorganisms, of which pseudokinase mixed lineage kinase-like protein (MLKL) is necessary for ET formation. Here, we aimed to investigate the effects of various stimulations (including classic ET inducers and pathogen) on ET formation in the coelomocytes of invertebrate sea cucumber Apostichopus japonicus. Our results show that stimulation of sea cucumber coelomocytes with phorbol 12-myristate 13-acetate (PMA), zymosan, Vibrio splendidus AJ01, and AJ01 flagella all led to the formation of typical ET fibers, which can be further digested by DNase I. This finding suggests that DNA is an essential component of coelomocyte ETs. Meanwhile, the signals of histones H2A and H2B transferred to extracellular spaces and colocalized with AJ01, which indicates that histones are also crucial components of coelomocyte ETs. Scanning electron microscopy observation and antimicrobial activity analysis successfully revealed that AJ01 was trapped and killed by ET fibers, and this finding implies the antimicrobial role of sea cucumber coelomocyte ETs. More importantly, silencing and inhibition of AjMLKL after AJ01 infection not only considerably depressed ET generation but also increased the survival rate of AJ01. All our results indicate that AjMLKL mediates sea cucumber ET formation and plays critical roles during pathogen infection.

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists.

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in a process referred to as NETosis. However, in the past two decades, it has become clear that the accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases. Therefore, interest in the development of NETosis antagonists has risen. Variable and non-standardized methods to detect and analyze NETosis were developed concomitantly, each with its own advantages and limitations. Here, we describe a real-time microscopy method for the quantification of human NET release, allowing to study NETosis as well as NET inhibition in a high-throughput manner. The surface area-based semi-automated analysis recognizes NETs and distinguishes them from non-netting activated neutrophils. We demonstrate that the non-physiological NETosis inducers, calcium ionophore and phorbol-12-myristate-13-acetate (PMA), trigger the release of NETs with different characteristics and kinetics. Furthermore, we show that this approach allows studying NET release in response to disease-relevant stimuli, including immune complexes, N-Formylmethionine-leucyl-phenylalanine (fMLF), monosodium urate crystals, and calcium pyrophosphate crystals. To exemplify the utility of this method to study NETosis antagonists, we used CIT-013, a first-in-class monoclonal antibody inhibitor of NET release. CIT-013 targets citrullinated histone H2A and H4 and efficiently inhibits NET release with an IC50 of 4.6 nM. Other anti-histone antibodies tested lacked this NETosis-inhibitory capacity. Altogether, we demonstrate that this protocol enables specific, reliable, and reproducible high-throughput quantification of NETs, enhancing the study of NET release characteristics, kinetics, and pharmacology of NETosis antagonists.

Abstract A051: Bitter agonists increase macrophage phagocytosis of head and neck squamous cell carcinoma cells

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 7th most commonly diagnosed cancer worldwide and is expected to increase in incidence by 30% by 2030. The HNSCC microenvironment is characterized by substantial macrophage infiltration. However, tumor-associated macrophages often cultivate an immunosuppressive environment that aids tumor progression. Previous work has shown that macrophages express bitter taste receptors (T2Rs), a class of GPCRs that can be activated with bitter compounds. Previous work has also demonstrated that stimulation of T2Rs in macrophages with bitter agonists, such as denatonium benzoate, increases phagocytosis of bacteria, but the impact of bitterants on macrophage phagocytosis of tumor cells is unknown. The goal of this study was to assess the impact of bitter compounds on macrophage phagocytosis of HNSCC cells in vitro. Human peripheral blood mononuclear cells obtained from healthy donors were used to generate monocyte-derived macrophages (MDMs). To investigate the effects of bitter agonist stimulation on macrophage phagocytosis, CFSE-labeled MDMs and DAPI-labeled HNSCC cell line SCC47, were co-stimulated with denatonium benzoate, and macrophage phagocytosis was quantified. This revealed that treatment with denatonium benzoate led to a 70% increase in macrophage phagocytosis compared to controls. Additional analysis revealed a 1.8-fold increase in F-actin in MDMs in response to denatonium benzoate measured via CellMask™ Actin staining, and that macrophages had generated F-actin positive tunneling nanotubes (TNTs). Further characterization with live cell imaging showed that these TNTs were being utilized as a means of mitochondrial transfer as observed by MitoTracker™ and CellMask™ Actin staining. Furthermore, there was a 2.5-fold decrease in mitochondrial ATP production in MDMs stimulated with denatonium benzoate, possibly indicating metabolic reprogramming. Timelapse imaging of MDMs demonstrated a reduction in mitochondrial ATP production, with a concomitant increase in F-actin production. These observations were confirmed in Phorbol 12-myristate 13-acetate differentiated THP-1 cells. Taken together, these results indicate that bitter taste receptor stimulation may contribute to a more activated macrophage phenotype denoted by a decrease in mitochondrial ATP production, increased F-actin staining, and increased phagocytosis of tumor cells. Citation Format: Brianna L Hill, Sarah M Sywanycz, Zoey A Miller, Robert J Lee, Ryan M Carey. Bitter agonists increase macrophage phagocytosis of head and neck squamous cell carcinoma cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A051.

Neutrophil Diversity (Immature, Aged, and Low-Density Neutrophils) and Functional Plasticity: Possible Impacts of Iron Overload in β-Thalassemia.

Neutrophil dysfunction is a form of immune suppression in patients with β-thalassemia (Beta-thal), although data on this are limited. In this study, blood from patients and healthy volunteers was analyzed. Flow cytometry analysis demonstrated an increase in immature neutrophils (CD16- CD62L+) and aged (senescent) neutrophils (CD16+ CD62L-) in Beta-thal patients compared to healthy volunteers. The Beta-thal neutrophils demonstrated less prominent chemotaxis and phagocytosis than healthy neutrophils at the baseline. With phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) stimulations, some of the indicators, including the flow cytometry markers (CD11b, CD62L, CD66b, CD63, apoptosis, and reactive oxygen species) and neutrophil extracellular traps (NETs; detected by anti-citrullinated histone 3 immunofluorescence), were lower than the control. Additionally, low-density neutrophils (LDNs), which are found in the peripheral blood mononuclear cell (PBMC) fraction, were observed in Beta-thal patients but not in the control group. The expression of CD11b, CD66b, CD63, arginase I, and ROS in LDNs was higher than the regular normal-density neutrophils (NDNs). The proliferation rate of CD3+ T cells isolated from the PBMC fraction of healthy volunteers was higher than that of the cells from patients with Beta-thal. The incubation of red blood cell (RBC) lysate plus ferric ions with healthy NDNs transformed the NDNs into the aged neutrophils (decreased CD62L) and LDNs. In conclusion, iron overload induces neutrophil diversity along with some dysfunctions.

Protein Kinase C and Matrix Metalloproteinases Expression Using Phorbol Myristate Acetate in Degenerative Intervertebral Disc Cells.

Degeneration of nucleus pulposus (NP) cells involves multiple factors. The relationship between the canonical Wnt/β-catenin signaling pathway and matrix metalloproteinases (MMPs) is important in cellular senescence. Protein kinase C (PKC), an intermediate of the non-canonical Wnt pathway stimulated by phorbol myristate acetate (PMA), possibly prevents NP cell senescence, although not yet demonstrated in human-based studies. This study aimed to investigate the effect of PMA stimulation on the non-canonical and canonical Wnt pathways and MMP expression in human NP cells to ascertain its inhibitory effects on the senescence of NP cells. Human disc tissues of Pfirrmann grades 1 and 2 were collected from patients during spinal surgery and subsequently cultured. Protein and ribonucleic acid (RNA) were isolated from NP cells treated with PMA (400 nM) for 24 hours. Expression of MMP1, MMP13, tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), transient receptor potential vanilloid 4 (TRPV4), interleukin-6 (IL-6), and β-catenin were detected using western blot analysis. Messenger RNA (mRNA) expression of type II collagen and glycosaminoglycan (GAG) were analyzed using reverse transcription polymerase chain reaction. IL-6 and prostaglandin E2 (PGE2) levels were measured using enzyme-linked immunosorbent assay. Expression of PKC-δ (intermediate of the non-canonical Wnt pathway) and β-catenin (intermediate of the canonical Wnt pathway) was increased by PMA treatment. The mRNA levels of type II collagen and GAG increased; however, their protein levels were not altered. PMA treatment increased the expression of MMP1, TIMP1, ADAMTS5, IL-6, PGE2, and TRPV4; however, the expression of MMP13 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was unaltered. PMA activated PKC-δ, affecting the non-canonical Wnt pathway; however, its effect on β-catenin in the canonical Wnt pathway was limited. β-catenin activation through the TRPV4 channel led to increased expression of MMP1 and ADAMTS5 and that of IL-6 and PGE2 owing to NF-κB expression. Consequently, the degeneration of NP cells was not prevented.

Effect of JMJD3-IRF4 Signaling Pathway-Mediated Macrophage Polarization on the Malignant Biological Behavior of Multiple Myeloma Cells

To investigate the effect of macrophage polarization mediated by Jumonji domain containing-3 (JMJD3)-interferon regulatory factor 4 (IRF4) signaling pathway on the malignant biological behavior of multiple myeloma (MM) cells. THP-1 monocytes were induced to differentiate into macrophages by phorbol myristate acetate (PMA). THP-1 macrophages were divided into control group (normal culture), M2 induction group ［added recombinant human interleukin (IL) -4, IL-13 proteins］, M2+JMJD3 protein group (added recombinant human IL-4, IL-13 and JMJD3 proteins) and M2+JMJD3 inhibitor group (added recombinant human IL-4, IL-13 proteins and JMJD3 inhibitor), the proportion of CD206+ cells was detected by flow cytometry, the levels of IL-10 and transforming growth factor-β (TGF-β) in the culture supernatant were detected by ELISA assay, the expression levels of arginase-1 (Arg-1), JMJD3 and IRF4 mRNA were detected by real-time quantitative PCR (qRT-PCR), and the expression levels of Arg-1, JMJD3 and IRF4 proteins were detected by Western blot. Correspondingly, human MM cells U266 were cultured with THP-1 macrophage culture supernatant of each group, Methyl thiazolyl tetrazolium (MTT) method and plate colony formation assay were used to detect cell proliferation, cell apoptosis was detected by flow cytometry, Western blot was used to detect the expression levels of apoptosis-promoting protein Bcl-2-associated X protein (Bax) and cleaved caspase-3 in cells, and Transwell assay was used to detect cell migration and invasion. Compared with the control group, the proportion of CD206+ cells in THP-1 macrophages, the mRNA and protein expression levels of Arg-1, JMJD3 and IRF4, and the levels of IL-10 and TGF-β in the cell culture supernatant in M2 induction group were significantly increased (P <0.001), meanwhile, the proliferation activity and the number of clones of U266 cells were significantly increased (P <0.01), the apoptosis rate and the expression levels of apoptosis-promoting protein Bax and cleaved caspase-3 were significantly decreased (P <0.001), the numbers of migrated cells and invasive cells were increased (P <0.001). Compared with M2 induction group, the proportion of CD206+ cells in THP-1 macrophages, the mRNA and protein expression levels of Arg-1, JMJD3 and IRF4, and the levels of IL-10 and TGF-β in the cell culture supernatant in M2+JMJD3 protein group were further increased (P <0.01), meanwhile, the proliferation activity and the number of clones of U266 cells were further increased (P <0.05), the apoptosis rate and the expression levels of apoptosis-promoting protein Bax and cleaved caspase-3 were further decreased (P <0.01), the numbers of migrated cells and invasive cells were further increased (P <0.001); However, the change trends of the above indexes in M2+JMJD3 inhibitor group were opposite to those in M2+JMJD3 protein group. M2 polarization of macrophages mediated by JMJD3-IRF4 signaling pathway can promote the proliferation, migration and invasion of MM cells, and inhibit cell apoptosis.

Tangeretin inhibits airway inflammatory responses by reducing early growth response 1 (EGR1) expression in mice exposed to cigarette smoke and lipopolysaccharide

BackgroundTangeretin, a natural polymethoxyflavone compound, possesses potent anti-inflammatory activity that improves respiratory inflammation in chronic obstructive pulmonary disease (COPD). However, the molecular mechanisms underlying the anti-COPD effects of tangeretin remain unclear. In this study, we aimed to investigate the key molecular mechanisms by which tangeretin suppresses COPD-related inflammatory responses. MethodsWe conducted the investigation in phorbol-12-myristate-13-acetate (PMA)-stimulated human airway epithelial cells (in vitro) and cigarette smoke (CS)/lipopolysaccharide (LPS)-exposed mice (in vivo). ResultsTangeretin decreased the release of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and mucin 5AC (MUC5AC), by suppressing early growth response 1 (EGR1) expression in vitro. Tangeretin and EGR1 small interfering ribonucleic acid (siRNA) combination showed a synergistic reduction in MUC5AC and TNF-α secretion. Tangeretin administration significantly inhibited the levels of reactive oxygen species (ROS) production, elastase activity, TNF-α, IL-6, and monocyte chemoattractant protein-1 (MCP-1) secretion, and macrophage and neutrophil numbers in the bronchoalveolar lavage fluid of CS/LPS-exposed mice. Tangeretin also prevented CS/LPS-induced abnormal pathological changes and excessive MUC5AC and EGR1 expression in lung tissue. ConclusionComprehensively, tangeretin inhibits the lung inflammatory response associated with COPD by reducing EGR1 expression in PMA-induced human epithelial cells and in a CS/LPS-stimulated mouse model. This study shows that tangeretin has anti-COPD properties and can be a promising alternative (or complementary) treatment for inflammatory lung disease.

High glucose promotes lipopolysaccharide-induced macrophage pyroptosis through GSDME O-GlcNAcylation.

The high glucose (HG) environment in diabetic periodontitis aggravates the damage of periodontal tissue. Pyroptosis has been shown to be positively correlated with the severity of periodontitis, including macrophage pyroptosis. O-GlcNAcylation is a posttranslational modification that is involved in the pathogenesis of periodontitis. However, whether HG regulates macrophage pyroptosis through O-GlcNAcylation remains uncertain. This study aimed to investigate the effect of HG on the O-GlcNAcylation level of a pyroptosis regulator GSDME in macrophages to further probe the mechanisms of diabetic periodontitis. Blood samples were collected from patients with diabetic periodontitis. THP-1 monocytes were induced to differentiate into macrophages by phorbol 12-myristate 13-acetate and then treated with HG to simulate periodontitis invitro. GSDME expression of blood samples and macrophages was measured by quantitative real-time PCR. Pyroptosis was assessed by propidium iodide staining, measurement of cell viability, cytotoxicity, protein levels of inflammation factors, and pyroptosis-related proteins. O-GlcNAcylation of GSDME was analyzed using co-immunoprecipitation (co-IP), IP, and western blot. The results showed that GSDME expression was elevated in patients with periodontitis and HG-treated macrophages. HG inhibited cell viability but increased LDH content, levels of IL-1β, IL-18, TNF-α, NLRP3, GSDMD, and Caspase-1, indicating that HG promoted pyroptosis of macrophages, which was reversed by GSDME knockdown. HG treatment increased O-GlcNAcylation in macrophages. Mechanically, GSDME interacted with OGT, and OGT knockdown suppressed O-GlcNAcylation of GSDME at Ser (S)339 site. Knockdown of OGT inhibited pyroptosis in HG-treated macrophages, while GSDME overexpression partially reversed this inhibition. HG treatment enhanced OGT-mediated GSDME O-GlcNAcylation, thereby augmenting pyroptosis in LPS-induced macrophages. These results may provide a novel sight for the treatment of periodontitis.

Evidence for a Pro-Inflammatory State of Macrophages from Non-Obese Type-2 Diabetic Goto-Kakizaki Rats

Obesity causes insulin resistance (IR) through systemic low-grade inflammation and can lead to type 2 diabetes mellitus (T2DM). However, the mechanisms that cause IR and T2DM in non-obese individuals are unclear. The Goto-Kakizaki (GK) rat develops IR spontaneously and is a model of non-obese T2DM. These rats exhibit hyperglycemia beginning at weaning and exhibit lower body mass than control Wistar rats. Herein, we tested the hypothesis that macrophages of GK rats are permanently in a pro-inflammatory state, which may be associated with a systemic inflammation condition that mimics the pathogenesis of obesity-induced T2DM. Using eighteen-week-old GK and control Wistar rats, we investigated the proportions of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages isolated from the peritoneal cavity. Additionally, the production of inflammatory cytokines and reactive oxygen species (ROS) in cultured macrophages under basal and stimulated conditions was assessed. It was found that phorbol myristate acetate (PMA) stimulation increased GK rat macrophage ROS production 90-fold compared to basal levels. This response was also three times more pronounced than in control cells (36-fold). The production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), tended to be upregulated in cultured macrophages from GK rats under basal conditions. Macrophages from GK rats produced 1.6 times more granulocyte-macrophage colony-stimulating factor (GM-CSF), 1.5 times more monocyte chemoattractant protein-1 (MCP-1) and 3.3 times more TNF-α than control cells when stimulated with lipopolysaccharide (LPS) (p = 0.0033; p = 0.049; p = 0.002, respectively). Moreover, compared to control cells, GK rats had 60% more M1 (p = 0.0008) and 23% less M2 (p = 0.038) macrophages. This study is the first to report macrophage inflammatory reprogramming towards a pro-inflammatory state in GK rats.

Comparison the impact of mesenchymal stromal cells, their microvesicles and plasma enriched with soluble platelet factors on survival and apoptosis of rat spleen lymphocytes &lt;i&gt;in vitro&lt;/i&gt;

BACKGROUND: The trophic function is one of the important but insufficiently studied features of mesenchymal stromal cells (MSCs), their microvesicles (MVs), and plasma enriched with soluble platelet factors (PRPs), which ensure the viability of target cells. AIM: To analyze the capability of MSCs, MVs, and PRP to affect the viability and spontaneous and activation-induced apoptosis of rat spleen lymphocytes during culturing in vitro. MATERIALS AND METHODS: MSCs were isolated from the mononuclear cell fraction of the femoral bone marrow of Wistar rats using the plastic adhesion method. MVs were obtained from the conditioned medium of MSCs by centrifugation at 14,500 g. PRP was prepared by freezing/thawing rat peripheral blood platelet concentrate. Rat spleen lymphocytes were isolated on a density gradient of 1.077 g/cm3. The viability and degree of lymphocyte apoptosis in vitro were assessed by flow cytometry for 7-AAD incorporation and binding to annexin V. RESULTS: The presence of MSCs at 10 and 20% concentrations caused an increase in the number of living intact and PMA-activated lymphocytes by the end of 3-day in vitro cultivation. Compared with controls, the amount of necrotic cells decreased 8.3–13.5 times, and the number of apoptotic cells decreased 2.3–4.0 times, mainly due to lymphocytes at the late stage of apoptosis. MVs of MSCs at the indicated concentrations did not significantly affect the viability of lymphocytes cultured in vitro but reduced the level of apoptosis of intact and PMA-activated lymphocytes by 3.6 (р=0.03) and 4.8–5.2 (р=0.048; р=0.03) times respectively. Studies have shown that 1.25% rat PRP has a growth-stimulatory activity against MSCs but not against lymphocytes cultured in vitro. In the culture of intact lymphocytes, PRPs did not significantly affect cell viability with a slight decrease (2.7–2.9 fold, p 0.05) in the number of necrotic cells. In the culture of PMA-activated lymphocytes, 1.25–2.50% PRP increased the number of living cells by 1.6–2.2 times (р=0.002; р=0.01) and the number of necrotic cells by two times (р=0.02). CONCLUSION: MSCs, MVs of MSCs, and PRP, in decreasing order, enhanced the viability of rat spleen lymphocytes and suppressed late apoptosis during in vitro cultivation. Lymphocytes activated by phorbol myristate acetate are more sensitive to their vital action compared with resting cells.