Phorbol 12-myristate 13-acetate Research Articles

Generation of primary feline chimeric antigen receptor T cells.

The purpose of this study was to develop procedures to engineer feline chimeric antigen receptor (CAR) T cells. 6 healthy cats were used in this study. Blood was collected, and CD3+ primary T cells were enriched by magnetic activated cell sorting, expanded, and used to generate CAR T cells. Phorbol myristate acetate plus ionomycin and concanavalin A induced similar early proliferation of CD3-enriched feline CD4+ and CD8+ T cells but phorbol myristate acetate plus ionomycin induced greater expansion over 12 days. Chimeric antigen receptor T cells were engineered by transduction with an FIV-based lentiviral system to express a human CD19 CAR. Feline CD19 CAR T cells demonstrated specific cytotoxicity against human CD19+ target cells. Conditions were developed to polarize the T cells to THelper subsets. We generated functional and specific primary feline CAR T cells and demonstrated conditions to polarize the cells, which may be therapeutically advantageous for CAR T-cell use in a variety of disease contexts. CAR T therapy has been used with great success for human hematologic malignancies and is under development for use in canines. Our study is the first demonstration of functional feline CAR T cells and describes the procedures for their engineering. These findings lay the foundation for future development of CAR T therapy for multiple feline diseases.

Sesquiterpenoids from Inula britannica and their potential mechanism for immunomodulation

The immune system serves as a role of diseases, such as Parkinson's disease, and acute lung injury. An immunoregulatory activity-directed separation depended on phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Ion)-mediated Jurkat leukemic T cells was used for studying chemical constituents from Inula britannica L. in depth. Five previously undescribed aromatic sesquiterpenoid dimers inulabritanoids J−N (1−5) and a previously undescribed germacrane-type sesquiterpenoid britanicafanin F (6) were afforded from I. britannica as well as eight known sesquiterpenoids (7–14). Their structures were elucidated through 1D and 2D NMR, HRMS, and ECD spectra along with quantum chemical calculations. Immunomodulatory effects of compounds 1–14 were assayed in PMA plus Ion-mediated Jurkat cells, and indicated that compounds 8, 9, and 13 displayed significantly inhibitory effects toward IL-2 and IFN-γ. Further investigation of mechanism of action revealed that compound 13 inhibited phosphorylations of p38, ERK, and JNK to suppress c-Jun and c-Fos expressions, resulting in blocking the nuclear translocation of AP-1 (a complex of c-Jun and c-Fos) to regulate mRNA expressions of IL-2 and IFN-γ. Molecular docking analysis demonstrated that compound 13 could enter into the cavity of p38, ERK, and JNK, and from hydrogen bond interactions with Gly33, Lys53 Ser154, and Asp168 for p38, Lys54, Glu71, Ser153, and Asp167 for ERK, and Met149 and Asn152 for JNK, which supported the abovementioned results. These findings suggested that sesquiterpenoids from the genus Inula served as immunomodulators for treating diseases involved in immune and inflammatory responses.

MiR-214-3p inhibits LPS-induced macrophage inflammation and attenuates the progression of dry eye syndrome by regulating ferroptosis in cells.

Dry eye disease (DED) is an ocular illness caused by insufficient tear secretion or poor tear quality, and inflammation is a key factor in its pathogenesis. Previous studies have shown that miRNAs are important regulatory factors in DED. The purpose of this study was to explore the potential mechanism by which miR-214-3p influenced the DED process by regulating the macrophage inflammatory response. We induced THP-1 cells to differentiate into M0 macrophages with 100 ng/mL phorbol-12-myristate-13-acetate (PMA) and then added 15 ng/mL lipopolysaccharide (LPS) to induce inflammation. The expression of related genes and proteins was detected via RT‒qPCR, Western blotting, ELISA and immunofluorescence staining; cell viability was measured using the CCK-8 assay; and flow cytometry was used to detect ROS levels. In tear and serum samples from DED patients, the levels of miR-214-3p, IL-10, and Arg1 were decreased, and the levels of IL-6, TNF-α, IL-1β, and iNOS expression were increased. Moreover, the overexpression of miR-214-3p attenuated the effect of LPS and inhibited M1 polarization and inflammation in macrophages. Mechanistically, miR-214-3p inhibited macrophage ferroptosis by downregulating TFRC expression, thereby inhibiting macrophage M1 polarization and inflammation and alleviating the progression of DED. Our study indicated that the upregulation of miR-214-3p expression might be a new target for DED therapy.

Modified lipoprotein-induced sFlt1 production in human placental trophoblasts is mediated by protein kinase C

BackgroundPreeclampsia is prevalent in women with diabetes, but the mechanism is unclear. We previously found that oxidized, glycated lipoproteins robustly upregulated soluble fms-like tyrosine kinase-1 (sFlt1), a key mediator of preeclampsia. Here, we determined the role of protein kinase C (PKC) and its subtypes in sFlt1 regulation in placental trophoblasts, and whether this mechanism might mediate the effect of modified lipoproteins. MethodsCultured human HTR8/SVneo and BeWo trophoblasts were treated with the PKC activator phorbol-12-myristate-13-acetate (PMA) for 24h, ± PKC inhibitors GF109203X (general), Ro31-8220 (PKCα-selective), LY333531 (PKCβ-selective) and rottlerin (PKCδ-selective). The effect of ‘heavily oxidized, glycated’ low-density lipoproteins (HOG-LDL) vs. native LDL (N-LDL), ± high glucose (30 mM), was evaluated in HTR8/SVneo cells. sFlt1 secretion (ELISA), mRNA expression (RT-qPCR), and cellular PKC activity were measured. ResultsPMA stimulated robust sFlt1 release and mRNA expression in both cell lines; these effects were inhibited by GF109203X, Ro31-8220 and LY333531 in a concentration-dependent manner. Rottlerin inhibited sFlt1 in BeWo, but modestly enhanced it in HTR8/SVneo cells. HOG-LDL enhanced PKC activity vs. N-LDL in HTR8/SVneo cells. Also, HOG-LDL, but not high glucose, significantly increased sFlt1 secretion and mRNA expression; this response was inhibited by GF109203X, Ro31-8220 and LY333531 at concentrations comparable to those that blocked PMA induction of sFlt1. ConclusionModified lipoproteins upregulate sFlt1 in trophoblasts via a PKC-mediated mechanism, involving at least α and β isoforms. The data suggest potential therapeutic targets to reduce the risk of preeclampsia in women with diabetes.

Impact of antiplatelets, anticoagulants and cyclic nucleotide stimulators on neutrophil extracellular traps (NETs) and inflammatory markers during COVID-19.

While the association between coronavirus disease-19 (COVID-19) and neutrophils extracellular traps (NETs) is recognized, uncertainties remain regarding its precise onset, timing of resolution and target therapy. To assess changes in inflammatory and NET markers during the first week of COVID-19 hospitalization, and the association with disease severity. "In vitro" experiments investigated the effect of antiplatelets, anticoagulants, and cyclic nucleotide stimulators on NETs release. Prospective cohort study, changes in interleukin (IL)-6, IL-8, IL-17, TNF-α, RANTES, PF4, and citrullinated-H3 (citH3) levels within each outcome group was evaluated using ANOVA. Differences between moderately ill, critically ill, and non-survivors were determined using Kruskal-Wallis and logistic regression. Healthy neutrophils were stimulated with phorbol-12-myristate-13-acetate (PMA) or COVID-19 sera and treated with unfractionated heparin (UFH), low molecular weight heparin (LMWH), aspirin (ASA), ticagrelor, cinaciguat, sildenafil, and milrinone. The proportion of NETosis was assessed using IncuCyte Cell Imager. Of the 125 patients, 40.8% had moderate COVID-19, 40.8% had critical COVID-19 but recovered, and 18.4% died. From admission to hospitalization day 8, IL-6 levels decreased in moderately and critically ill, but not in non-survivors, while citH3 levels increased in critically ill and non-survivors. IL-6, IL-8, and TNF-α levels were associated with critical and fatal COVID-19. The release of NETs by neutrophils stimulated with PMA or COVID-19 sera was decreased in the presence of ASA, UFH, LMWH and cyclic nucleotide stimulators in a dose-dependent manner. In the first week of hospitalization, NET markers rose later than inflammatory markers in severe COVID-19 cases. Cyclic nucleotide stimulators, ASA and heparin may emerge as treatment approaches as they may modulate NETosis.

Persicaria minor (Huds.) Opiz Exhibits Antihypertensive Effects by Inhibiting the Angiotensin-Converting Enzyme/Angiotensin II Type 1 Receptor Pathway in Human Endothelial Cells.

Overactivation of the angiotensin-converting enzyme (ACE)/angiotensin II type 1 receptor (AT1R) pathway leads to vasoconstriction and elevated blood pressure. Persicaria minor (Huds.) Opiz is an herbal plant known for its antioxidant, anti-hyperlipidemic, and anti-atherosclerotic properties, with bioactive compounds that exhibit antihypertensive effects. Therefore, this study aimed to evaluate the antihypertensive effects of the standardized aqueous extract of P. minor leaf (AEPM) through the ACE/AT1R pathway in human umbilical vein endothelial cells (HUVECs) induced with phorbol 12-myristate 13-acetate (PMA). HUVECs were stimulated with PMA to induce ACE, with or without AEPM or captopril treatment, for 24 h. Subsequently, ACE mRNA expression, ACE protein levels, ACE activity, angiotensin II levels, and AT1R expression were measured. The results demonstrated that AEPM treatment significantly reduced ACE mRNA expression, ACE protein levels, ACE activity, angiotensin II levels, and AT1R expression in PMA-induced HUVECs. The modulatory effects of AEPM on the ACE/AT1R pathway were comparable to those of captopril. Ex vivo experiments further confirmed that AEPM reduced the contraction responses of rat aortic rings to PMA. In conclusion, P. minor effectively inhibits the ACE/AT1R pathway in PMA-induced HUVECs, suggesting its potential as a natural antihypertensive agent.

Oxysterols Suppress Release of DNA from Granulocytes into Extracellular Space After Stimulation with Phorbol Myristate Acetate.

Neutrophils eject their DNA strings and cellular proteins into the extracellular space upon treatment with various stimulants. In the present study, we examined the effects of four major oxidized cholesterol metabolites on DNA release from granulocytes. When oxysterols were added to HL-60-derived granulocytes stimulated with phorbol 12-myristate 13-acetate (PMA), they suppressed the release of DNA and myeloperoxidase from the cells. Among the four oxysterols tested, 7-ketocholesterol was the most effective. Addition of the same concentration of 7-ketocholesterol did not induce any cytotoxic effects, as evaluated based on the release of lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assays. DNA release from human peripheral blood neutrophils after PMA stimulation was also suppressed by 7-ketocholesterol. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis was used to quantify sterol content in the cells. The addition of oxysterols increased the cellular content of the corresponding compounds by more than 10-fold compared to those at baseline. Treatment of HL-60-derived granulocytes with methyl-β-cyclodextrin that removes sterol compounds from the membranes increased DNA release from the cells in a dose-dependent manner. These results suggest that oxysterols have suppressive effects on DNA release from granulocytes stimulated with PMA.

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.

Leukocyte Platelet-Rich Plasma-Derived Exosomes Restrained Macrophages Viability and Induced Apoptosis, NO Generation, and M1 Polarization.

Chronic refractory wounds refer to wounds that cannot be repaired timely. Platelet-rich plasma (PRP) has significant potential in chronic wound healing therapy. The exosomes isolated from PRP were proved to exhibit more effectiveness than PRP. However, the therapeutic potential of exosomes from PRP on chronic refractory wounds remained elusive. Hence, this study aimed to clarify the action of exosomes from PRP on chronic refractory wounds by evaluating the response of macrophages to exosomes. Pure platelet-rich plasma (P-PRP) and leukocyte platelet-rich plasma (L-PRP) were prepared from the fasting venous blood of healthy volunteers. Exosomes were extracted from P-PRP and L-PRP using ultracentrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot. Macrophages were obtained by inducing THP-1 cells with phorbol-12-myristate-13 acetate (PMA). The internalization of exosomes into macrophages was observed utilizing confocal laser scanning microscopy after being labeled with PKH67. Cell viability was determined by CCK-8 assay. Cell apoptosis was measured utilizing a flow cytometer. The polarization status of M1 and M2 macrophages were evaluated by detecting their markers. Nitric oxide (NO) detection was conducted using the commercial kit. Exosomes from P-PRP and L-PRP were absorbed by macrophages. Exosomes from L-PRP restrained viability and induced apoptosis of macrophages. Besides, exosomes from P-PRP promoted M2 polarization, and exosomes from L-PRP promoted M1 polarization. Furthermore, exosomes from L-PRP promoted NO generation of macrophages. Exosomes from L-PRP restrained viability, induced apoptosis and NO generation of macrophages, and promoted M1 polarization, while exosomes from P-PRP increased M2 polarization. The exosomes from L-PRP presented a more effective effect on macrophages than that from P-PRP, making it a promising strategy for chronic refractory wound management.

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.

Primary 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. The 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. We 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. Our 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.

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.

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.