Phorbol 12-myristate 13-acetate Research Articles

Inhibition of peptidyl arginine deiminase-4 ameliorated pulmonary fibrosis via modulating M1/M2 polarisation of macrophages.

Pulmonary fibrosis (PF) arises from dysregulated wound healing, leading to excessive extracellular matrix (ECM) deposition and impaired lung function. Macrophages exhibit high plasticity, polarizing to pro-inflammatory M1 during early inflammation and anti-inflammatory, fibrosis-inducing M2 during later stages of PF. Additionally, neutrophils and neutrophil extracellular traps (NETs) release mediated by peptidyl arginine deiminase (PAD-4), also play a key role in PF progression. PAD-4 inhibitor chloro-amidine (CLA) has shown anti-fibrotic effects in bleomycin (BLM) induced PF mouse model in our earlier study. Here, we have demonstrated that CLA also exhibited inhibition of macrophage polarisation in in-vitro in THP-1 monocytes and in-vivo in BLM induced PF. THP-1 monocytes were exposed to NETs isolated from phorbol 12-myristate-13-acetate (PMA) stimulated and PMA plus CLA treated differentiated HL-60 (dHL-60) cells. Monocytes exposed to stimulated NETs resulted in increased oxidative stress, disrupted mitochondrial membrane potential and increased M1 and M2 macrophage markers. These alterations were abrogated in THP-1 cells upon exposure to CLA treated NETs. Further, CLA treatment in BLM induced mice improved abnormal BALF, biochemical, and histological parameters in line with our previous findings. Additionally, CLA also reduced M1 and M2 markers time-dependently, as shown by immunofluorescence (IF), western blot, and RT-PCR analysis. CLA treatment led to decreased expression of PAD-4, M1-related pro-inflammatory cytokines and M2-related pro-fibrotic cytokines and mediators, as confirmed by western blot and ELISA analysis. Thus, it is established that inhibition of PAD-4 lead to mitigation of macrophage polarisation and a combined anti-fibrotic effect is achieved which can be explored further.

Neutrophil extracellular traps promote growth of lung adenocarcinoma by mediating the stability of m6A-mediated SLC2A3 mRNA-induced ferroptosis resistance and CD8(+) T cell inhibition.

To investigate the potential mechanisms underlying neutrophil extracellular traps (NETs) confer ferroptosis resistance and CD8(+) T cell inhibition in lung adenocarcinoma (LUAD). By the intravenous injection of LLC cells into the tail vein, a LUAD mouse model was created. Phorbol-12-myristate-13-acetate (PMA) stimulated neutrophils to facilitate NETs formation and combined with NETs inhibitor DNase I to explore NETs mechanism on LLC cell proliferation, migration, ferroptosis resistance, and CD8(+) T cell activity. CitH3, myeloperoxidase (MPO), cell-free DNA, and MPO-DNA levels in LUAD were increased, indicating an increase in NETs formation in LUAD. PMA promoted NETs formation in tumours of mice, increased the number of CD3(+)CD4(+) T cells, decreased perforin, granzyme A, granzyme B, IFNγ, and TNF-α levels, and promoted LUAD growth and the number of lung tumour nodules, indicating that PMA promoted NETs formation, reduced the activity of CD8(+)T cells, and promoted LUAD growth. DNase I partially reversed the effects of PMA. NETs promoted LLC cell proliferation and migration, while DNase I reversed NETs effects. Erastin inhibited LLC cell proliferation and migration and promoted ferroptosis. NETs partially reversed Erastin effects. Further results showed that NETs promoted LLC cell proliferation and migration and inhibited ferroptosis by promoting YTHDF2-mediated SLC2A3 mRNA degradation. Sh-YTHDF2 partially reversed the effect of NETs on LLC cells, whereas si-SLC2A3 partially reversed sh-YTHDF2 effects on LLC cells. In addition, NETs inhibited LLC cell ferroptosis by inhibiting CD8(+) T cell activity. Sh-YTHDF2 and DNase I inhibited NETs formation in tumours, increased the activity of CD8(+) T cells and inhibited LUAD growth. Our results suggested that NETs promoted the growth of LUAD through inhibiting ferroptosis and CD8(+) T cell activity by promoting YTHDF2-mediated SLC2A3 mRNA degradation.

In Vitro Infection of Human Macrophages with Porphyromonas gingivalis W83

This study aimed to investigate the innate immune response of human macrophages to Porphyromonas gingivalis W83 using a novel in vitro infection model. The growth kinetics of P. gingivalis W83 were analyzed, revealing an exponential growth phase at 8 h (optical density = 0.70). To establish a reliable macrophage model, the differentiation of THP-1 monocytes into macrophages was optimized using low concentrations of phorbol 12-myristate 13-acetate (PMA). This approach induced enhanced adherence and morphological changes, with full differentiation achieved after 48 h of PMA treatment followed by 24 h of rest. Polarization towards the pro-inflammatory M1 phenotype was successfully induced with interferon-γ (IFN-γ) and lipopolysaccharide (LPS), as confirmed using cytokine profiling. Cytokine analysis using Luminex® technology demonstrated significant increases in interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-6, indicating the effective activation of macrophages towards a pro-inflammatory phenotype. Building upon this macrophage model, this study investigated the interactions between macrophages and P. gingivalis W83 during its exponential growth phase. After a one-hour infection period, bacterial DNA quantification in supernatants and lysed macrophages revealed minimal levels of internalized or adherent bacteria, supporting the hypothesis that P. gingivalis effectively evades immune detection. These findings emphasize the utility of this model in uncovering the sophisticated immune evasion strategies employed by P. gingivalis, with significant implications for the development of targeted therapeutic interventions.

Calycosin extracted from Astragali Radix reduces NETs formation to improve renal fibrosis via TLR4/NF-κB pathway.

Astragali Radix (A. Radix) is the dry root of the leguminous plants Astragalus membranaceus (Fisch) Beg. var. mongholicus (Beg) Hsiao, and Astragalus membranaceus (Fisch) Bge., being used as a medicinal and edible resource. AR is used in traditional Chinese medicine prescriptions to treat chronic nephritis. Calycosin (CA), the primary active compound derived from Astragali Radix, shows significant antifibrotic effects in multiple organs, but the anti-renal fibrosis effect of CA is rarely reported, and the associated mechanism of action is still need to be elucidated. The objective of this study was to investigate the protective effects of CA on the kidney against renal fibrosis and the underlying molecular mechanisms. Evaluation of the effects of CA on renal fibrosis using unilateral ureteral obstruction (UUO) mice and transforming growth factor β1 (TGF β1)-induced cell fibrosis. The mechanism of action supporting the investigated anti-renal fibrosis effects were studied by a series of biochemical experiments. Our research demonstrated that CA reduced kidney cell fibrosis in mice with UUO and in TGF-β1-stimulated NRK-52E cells. Additionally, CA mitigated renal fibrosis via toll-like receptor 4 (TLR4)/ nuclear factor-kappa B (NF-κB) and had a synergistic effect with resatorvid (TAK-242). Our findings reveal an unobserved impact of CA in inhibiting neutrophil extracellular traps (NETs) formation in UUO mice and neutrophils activated by phorbol 12-myristate 13-acetate. Our findings revealed that calycosin reduces NETs production to alleviate renal fibrosis via TLR4 and NF-κB, supporting its potential as a strategy for treating renal fibrosis.

Akkermansia muciniphila relieves inflammatory response in DSS-induced ulcerative colitis in mice through regulating macrophage polarization via SCFAs-SLC52A2/FFAR2 pathway.

Ulcerative colitis (UC) remains an intractable and relapsing disease featured by intestinal inflammation. The anti-UC activity of Akkermansia muciniphila (AKK), an intestinal microorganism, has been widely investigated. The current work is to explore the impacts of AKK on UC and its possible reaction mechanism. In vivo UC model was induced by dextran sulfate sodium (DSS) and phorbol-12-myristate-13-acetate (PMA)-induced THP-1-M0 and raw264.7 macrophages were treated by lipopolysaccharide (LPS). H&E staining evaluated tissue damage. Inflammatory and oxidative stress levels were assessed by relevant kits. The high-throughput analysis of fatty acids was performed by the LC/MS method. RT-qPCR and Western blot detected related gene expression. Flow cytometry measured cell apoptosis and macrophage polarization. Energy metabolism was detected by ELISA, related assay kits, JC-1 staining, and Western blot. AKK reduced the pathological damage of mice colon tissues, alleviated oxidative stress and inflammatory response, upregulated the expression of Occludin-1 and SCFAs receptors, and stimulated M1 to M2 macrophage polarization in vivo. After FFAR2 was silenced, the promoting role of AKK in the viability and M1 to M2 macrophage polarization and the inhibitory role in oxidative stress, inflammation, apoptosis, energy metabolism disorder, necroptosis, and pyroptosis were both reverted. Conclusively, AKK might mediate SCFAs-SLC52A2/FFAR2 pathways to exert protective activities against intestinal inflammatory response in UC, suggesting that AKK might represent a novel and promising candidate for UC therapy.

Enniatin B alters bovine polymorphonuclear leukocytes phagocytosis and extracellular reactive oxygen species production in vitro.

Enniatins (ENNs) affect human and animal health. Different ENN analogs have been identified, but Enniatin B (ENN B) is the most detected in foods and feeds. This study investigated the effect of ENN B on bovine polymorphonuclear leukocytes (PMNs) challenged with increasing ENN B concentrations (0.625, 1.25, 2.5, 5, and 10μM). Bovine PMNs were isolated from the peripheral blood of dairy cows to evaluate the cell viability, chemotactic function, ability to phagocyte Gram+ and Gram- microorganisms, and extracellular Reactive Oxygens Species (ROS), with or without phorbol 12-myristate-13-acetate (PMA) as a pro-inflammatory challenge. Results demonstrated that ENN B did not affect bovine PMN viability and chemotactic activity at all concentrations (p=0.952; p=0.218, respectively). E. coli and S. aureus phagocytosis ability were reduced by ENN B at the highest concentrations (5 and 10μM) compared to the negative control (p≤0.001; p=0.001, respectively). Extracellular ROS production was increased by ENN B challenge under physiological and pro-inflammatory conditions (p=0.014; p<0.001, respectively). In conclusion, ENN B did not exert cytotoxic effects on bovine PMNs, while reduced phagocytic ability and increased the production of extracellular ROS, highlighting its potential role as an immunomodulator of the bovine innate immune response in vitro. IMPLICATIONS: Emerging mycotoxin Enniatin B is a common grain contaminant worldwide that can exert cytotoxic and immunotoxic effects in animal cells. We hypothesized that Enniatin B could in vitro affect the bovine immune response. In our study, Enniatin B did not affect bovine polymorphonuclear cell viability and chemotaxis, while a reduction of phagocytosis and a modulation of extracellular reactive oxygen species were observed. The present study shows that Enniatin B in vitro exerts a potential role as an immunomodulator of the bovine innate immune response putting animals at an increased risk of infection diseases.

Evaluating the effects of c-phycocyanin from cyanobacterium Spirulina platensis as an anti-inflammatory agent on stimulated human chondrocyte cells

ABSTRACT Inflammation is a critical defence mechanism in higher animals but uncontrolled inflammation is implicated in many diseases and can lead to chronic conditions, posing significant challenges to healthcare and the global economy. Conventional therapies often come with limitations and adverse effects, underscoring the need for safer and more effective alternatives. This study investigates the efficacy of C-Phycocyanin (C-PC), a pigment derived from Spirulina platensis cultivated under optimal light conditions, as an anti-inflammatory agent. This research focused on its effects on nitric oxide secretion, which supports the body’s immune system by killing pathogens, and interleukin-1 beta (IL-1β) gene expression as an inflammatory indicator, in phorbol 12-myristate 13-acetate (PMA)-stimulated human chondrocyte cells (C28/I2). The Methyl Thiazol Tetrazolium (MTT) assay was employed to evaluate the metabolic activity of cells and confirm the non-toxicity of C-PC. The results demonstrated that C-PC maintained 99.32% cell viability in the C28/I2 in vitro model after seven days. Notably, C-PC treatment prevented cell death and promoted cell proliferation, increasing the number of viable cells by 12.34% during the same period. Additionally, it significantly reduced IL-1β gene expression and nitric oxide secretion by 70.24% and 91.25%, respectively, effectively reducing levels to those observed in unstimulated conditions. Scanning electron microscopy (SEM) and crystal violet staining showed that C-PC treatment restored normal morphology and protected the cells from inflammation-induced changes.

Identification of Biomarkers Related to Liquid-Liquid Phase Separation for Ulcerative Colitis Based on Single-Cell and Bulk RNA Transcriptome Sequencing Data.

Liquid-Liquid Phase Separation (LLPS) is a process involved in the formation of established organelles and various condensates that lack membranes; however, the relationship between LLPS and Ulcerative Colitis (UC) remains unclear. This study aimed to comprehensively clarify the correlation between ulcerative colitis (UC) and liquid-liquid phase separation (LLPS). In this study, bioinformatics analyses and public databases were applied to screen and validate key genes associated with LLPS in UC. Furthermore, the roles of these key genes in UC were comprehensively analyzed. Based on the single-cell transcriptomic data of UC obtained from the Gene Expression Omnibus (GEO) database, differences between patients with UC and their controls were compared using the limma package. The single-cell data were then filtered and normalized by the 'Seurat' package and subjected to dimension reduction by the Uniform Manifold Approximation and Projection (UMAP) algorithm. The LLPS-related genes (LLPSRGs) were searched on the Dr- LLPS website to obtain cross-correlated genes, which were scored using the ssGSEA algorithm. Next, functional enrichment, interaction network, immune landscape, and diagnostic and drug prediction of the LLPSRGs were comprehensively explored. Finally, the results were validated using external datasets and quantitative real-time PCR (qRT-PCR). A total of eight cell types in UC were classified, namely, fibroblasts, macrophages, endothelial cells, neutrophils, NK cells, B cells, epithelial cells, and T cells. The intersection between differently expressed genes (DEGs) among the eight cell types identified 44 key genes, which were predominantly enriched in immune- and infection-related pathways. According to receiver operating characteristic (ROC) curves, PLA2G2A, GZMK, CD69, HSP90B1, and S100A11 reached an AUC value of 0.94, 0.95, 0.86, 0.89, and 0.93, respectively. Drug prediction revealed that decitabine, tetrachlorodibenzodioxin, tetradecanoylphorbol acetate, thapsigargin, and cisplatin were the potential small molecular compounds for PLA2G2A, GZMK, CD69, HSP90B1, and S100A11. Immune cell infiltration analysis demonstrated that the infiltration of CD4 memory T cell activation, macrophage M1, T macrophage M0, neutrophils, and mast cell activation was higher in the UC group than in the normal group. The LLPSRGs play crucial roles in UC and can be used as prognostic and diagnostic markers for UC. The current findings contribute to the management of UC.

Inhibition of CCR2 attenuates NLRP3-dependent pyroptosis after myocardial ischaemia-reperfusion in rats via the NF-kB pathway.

Myocardial infarction (MI) is a leading cause of mortality worldwide, contributing significantly to long-term cardiac dysfunction and heart failure. Effective therapeutic strategies are urgently needed to mitigate the extensive damage caused by MI and subsequent ischemia-reperfusion (I/R) injury. This study investigates the role of the Chemokine receptor 2 (CCR2) in regulating NLRP3-dependent cardiomyocyte pyroptosis following myocardial ischemia-reperfusion (MIR), elucidating its molecular mechanisms. A myocardial ischemia-reperfusion model was established using 124 Sprague-Dawley rats by ligating the left coronary artery, inducing 30min of ischemia. Following ischemia, RS504393, a selective CCR2 antagonist, was administered intraperitoneally one hour after reperfusion. To further explore the underlying mechanisms, the NF-κB pathway agonist Phorbol 12-myristate 13-acetate (PMA) was administered 1h post-MIR. The results showed a marked increase in CCR2 expression in the heart, peaking on the first day of reperfusion. Treatment with RS504393 significantly improved short-term cardiac function and reduced myocardial infarction size, decreased myocardial pyroptosis and suppressed the expression of NLRP3, GSDMD, Caspase-1, IL-1β, and IL-18 through inhibition of the NF-κB signaling pathway. This effect was reversed with the administration of PMA. In summary, the inhibition of CCR2 shows potential in mitigating myocardial injury following MIR by modulating the NF-κB signaling pathway. These findings highlight CCR2 as a promising therapeutic target for myocardial ischemia-reperfusion injury.

Activation of protein kinase C decreases equilibrative nucleobase transporter 1-mediated substrate uptake via phosphorylation of threonine 231.

Protein kinase C (PKC) signalling has been shown to be dysregulated in various cancers including acute lymphoblastic leukemia (ALL). We have previously determined that changes in the expression levels of SLC43A3-encoded equilibrative nucleobase transporter 1 (ENBT1) can significantly alter 6-mercaptopurine (6-MP) toxicity in ALL cells. 6-MP is a common drug used in ALL chemotherapy. Furthermore, it has been reported that activation of PKC by phorbol 12-myristate 13-acetate (PMA) impacts nucleobase uptake via an ENBT1-like transporter in Lilly Laboratories Culture-Porcine Kidney 1 (LLC-PK1) cells. We hypothesized that activation of PKC would also alter ENBT1-mediated uptake of nucleobases in leukemia cell models. Using MOLT-4, SUP-B15, and K562 cells, we incubated the cells with PMA or its inactive isoform 4α-PMA for 30min and determined changes to ENBT1-mediated substrate uptake. All of the cell lines tested showed decreased ENBT1-mediated substrate uptake when exposed PMA, relative to that observed using 4α-PMA. Pre-incubation with the broad-spectrum PKC inhibitor, Gö6983, reversed the decrease caused by PMA. Finally, to determine the residue responsible for this PKC-mediated effect, we transiently transfected HEK293 cells (which do not express endogenous ENBT1) with wild-type SLC43A3 transcript or constructs mutated to modify the predicted PKC sites in ENBT1. We found that the mutation of threonine 231 to alanine prevents the decrease in ENBT1-mediated uptake following incubation with PMA, suggesting its involvement. This study shows that activation of PKC decreases ENBT1-mediated uptake, suggesting that aberrant activation of PKC in ALL could decrease ENBT1-mediated 6-MP uptake potentially leading to decreased therapeutic efficacy.

Phase partitioning of the neutrophil oxidative burst is coordinated by accessory pathways of glucose metabolism and mitochondrial activity.

Neutrophils are a part of the innate immune system and produce reactive oxygen species (ROS) to extinguish pathogens. The major source of ROS in neutrophils is NADPH oxidase, which is fueled by NADPH generated via the pentose phosphate pathway; however, it is unclear how other accessory glucose metabolism pathways and mitochondrial activity influence the respiratory burst. We examined the temporal dynamics of the respiratory burst and delineated how metabolism changes over time after neutrophil activation. Bone marrow-derived neutrophils were stimulated with phorbol 12-myristate 13-acetate, and the respiratory burst was measured via extracellular flux analysis. Metabolomics experiments utilizing 13C6-glucose highlighted the activation of glycolysis as well as ancillary pathways of glucose metabolism in activated neutrophils. Phorbol 12-myristate 13-acetate stimulation acutely increased 13C enrichment into glycerol 3-phosphate (G3P) and citrate, whereas increases in 13C enrichment in the glycogen intermediate, UDP-hexose, and end products of the hexosamine and serine biosynthetic pathways occurred only during the late phase of the oxidative burst. Targeted inhibition of the G3P shuttle, glycogenolysis, serine biosynthesis, and mitochondrial respiration demonstrated that the G3P shuttle contributes to the general magnitude of ROS production; that glycogen contributes solely to the early respiratory burst; and that the serine biosynthetic pathway activity and complex III-driven mitochondrial activity influence respiratory burst duration. Collectively, these results show that the neutrophil oxidative burst is highly dynamic, with coordinated changes in metabolism that control the initiation, magnitude, and duration of ROS production.

Olive leaf extract (OLE) reduces mast cell-mediated allergic inflammation.

Mast cell-mediated reactions promote various allergic disease, including anaphylaxis, allergic rhinitis, asthma, and atopic dermatitis. Different data demonstrated an intricate relationship between the use of antihistaminic drugs, the onset of side effects, and the development of resistance, underscoring the importance to find novel therapeutic approaches to treat allergic diseases. Olive leaf extract (OLE), is a by-product of the olive tree rich in bioactive compounds, known for its numerous therapeutic properties, including antioxidant, anti-tumoral and antidiabetic effects. In this study, we investigated the effect of OLE on the mast-cell-mediated allergic inflammation using human mast cells HMC-1.2. OLE reduced histamine and β-Hexosaminidase release from HMC cells activated by phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI) through modulation of calcium signal. Moreover, OLE decreased the PMACI-stimulated gene expression of proinflammatory cytokines such as tumor necrosis factor-a (TNF-α), interleukin-8 (IL-8) and interleukin-6 (IL-6) in human mast cells. This result was confirmed by multiplex assay in which the pre-treatment with OLE reduced the effective secretion of TNF-α, IL-6 and IL-8. These effects were correlated to ROS reduction and modulation of both mitochondrial mass and membrane potential. Finally, the inhibitory effect of OLE was nuclear factor (NF)-kB dependent as demonstrated by both activity assay and Western Blot analysis. Taken together, our results demonstrated that OLE inhibits mast-cell-derived allergic inflammation modulating mast cells degranulation, proinflammatory cytokines release and NF-kB activation. Therefore, OLE could represent a novel potential therapeutic approach for the treatment of mast cell-associated disorders.

Topical application of magnolol ameliorates psoriasis-like dermatitis by inhibiting NLRP3/Caspase-1 pathway and regulating tryptophan metabolism.

Psoriasis (PSO) is a common inflammatory skin disease caused by multiple factors. Magnolia officinalis is an important medicinal plant in China, with various values such as ecology, medicine, food, and daily chemicals. However, its diverse application potential has not been fully explored. Magnolol (MGO) is the main active compound of Magnolia officinalis with significant anti-inflammatory effect. To investigate the application potential of MGO in inflammatory skin disease, the effects and underlying mechanisms of topical MGO treating psoriasis were explored in this study. Network pharmacology and molecular docking firstly predicted that topical MGO may treat psoriasis by regulating pyroptosis pathway and acting on caspase-1 (CASP1). In vitro experiments then demonstrated that MGO could inhibit the level of inflammatory cytokines and the key protein expression of NOD-like receptor protein 3 (NLRP3)/Caspase-1 pathway in lipopolysaccharide (LPS)-stimulated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells. Meanwhile, MGO could inhibit CuSO4-induced neutrophils migration in Tg (mpx:EGFP) zebrafish by suppressing inflammation and pyroptosis. This study further indicated that topical application of MGO ameliorated imiquimod (IMQ)-induced psoriasis-like dermatitis by reducing the release of inflammatory factors and decreasing the key protein expression of pyroptosis-related NLRP3/Caspase-1 pathway. Metabolomics analysis revealed that topical application of MGO could significantly regulate tryptophan metabolism and affect the level of tryptophan in skin lesions. Tryptophan could also regulate inflammation-related genes and inhibit pyroptosis-related NLRP3/Caspase-1 pathway in LPS-stimulated PMA-differentiated THP-1 cells. In conclusion, this study suggested that topical MGO may ameliorate psoriasis-like dermatitis by inhibiting NLRP3/Caspase-1 pathway and regulating tryptophan metabolism.

Influence of macrophages and neutrophilic granulocyte-like cells on crystalline silica-induced toxicity in human lung epithelial cells.

In many industrial activities, workers may be exposed by inhalation to particles that are aerosolized, To predict the human health hazard of these materials, we propose to develop a co-culture model (macrophages, granulocytes, and alveolar epithelial cells) designed to be more representative of the inflammatory pulmonary response occurring in vivo. Phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells were used as macrophages, All-trans retinoic acid (ATRA)-differentiated HL60 were used as granulocytes and A549 were used as epithelial alveolar type II cells. A crystalline silica sample DQ12 was used as a prototypical particle for its capabilities to induce DNA damage, inflammatory response, and oxidative stress in epithelial cells; its polyvinylpyridine-N-oxide (PVNO)-surface modified counterpart was also used as a negative particulate control. Cells in mono-, bi- or tri-culture were exposed to DQ12 or DQ12-PVNO for 24h. DQ12 but not DQ12-PVNO induced a significant increase in DNA damage in A549 cells. The presence of differentiated THP-1 reduced the genotoxic effects of this crystalline silica sample. The exposure of A549 to DQ12 but not DQ12-PVNO induced a significant change in interleukin-8 (IL-8) protein levels which was exacerbated when differentiated THP-1, and HL-60, were added. In addition, while no production of TNFα was detected in the A549 monoculture, elevated levels of this cytokine were observed in the co-culture systems. This work shows that a cell culture model that takes into consideration the complexity of the pulmonary inflammatory response might be more dependable to study the toxicological properties of particles than "simple" monoculture models.

A Disintegrin and Metalloproteinase 17 Disrupts Bovine Macrophage MER Proto-Oncogene Tyrosine Kinase Integrity to Impede Apoptotic Cell Clearance and Promote Inflammation in Clinical Mastitis.

In clinical mastitis of dairy cows, the abnormal accumulation of apoptotic cells (ACs) and subsequent secondary necrosis and inflammation pose significant concerns, with macrophage-mediated efferocytosis, crucial for ACs clearance, remaining unexplored in this context. In nonruminants, MER proto-oncogene tyrosine kinase (MERTK) receptors are essential for efferocytosis and A Disintegrin and Metalloproteinase 17 (ADAM17) is thought to play a role in regulating MERTK integrity. This study aimed to delineate the in situ role of efferocytosis in clinical mastitis, with a particular focus on the interaction between MERTK and ADAM17 in bovine macrophages. In mastitic mammary tissue, a significant accumulation of ACs was observed, along with active macrophage efferocytosis. Western blotting analysis revealed elevated expressions of MERTK and ADAM17, and immunofluorescence confirmed that MERTK is predominantly localized within CD163+ macrophages. Additionally, elevated levels of soluble MERTK (sol-MER) in serum indicated impaired integrity and functionality of MERTK. In vitro experiments with the bovine macrophage cell line Bo-mac cells selectively phagocytosed apoptotic MAC-T cells, a process associated with increased MERTK phosphorylation and an anti-inflammatory phenotype. The activation of ADAM17 by Phorbol 12-myristate 13-acetate (PMA) induced sol-Mer release and impaired efferocytosis, with these effects reversed by the ADAM17 inhibitor TAPI-1. Bo-mac efferocytosis was influenced by the presence and activation of MERTK. Silencing MERTK interrupted efferocytosis, a phenomenon also observed with the inhibition of MERTK phosphorylation by UNC2025, which concurrently suppressed anti-inflammatory cytokine production. These findings suggest that targeting the MERTK-ADAM17 axis could enhance inflammatory resolution, providing a novel therapeutic strategy for dairy cattle health management.

Suppression of FcεRI-evoked Degranulation in RBL-2H3 Cells on Gelatin Methacryloyl Hydrogel.

Cell-extracellular matrix (ECM) interactions play multiple roles in developmental, physiological, and pathological processes. ECM stiffness substantially affects cellular morphology, migration, and function. In this study, we investigated the effect of ECM comprising gelatin methacryloyl (GelMA) on the activation of rat basophilic leukemia (RBL-2H3) cells, a model mast cell line. Maintenance of intracellular Ca2+ concentration ([Ca2+]i) elevation and subsequent degranulation, evoked by crosslinking the high-affinity IgE receptors (FcεRI), were significantly suppressed in RBL-2H3 cells on collagen-coated GelMA hydrogel than those on collagen-coated glass dishes and plastic wells. Thapsigargin and phorbol myristate acetate caused sustained [Ca2+]i increase and degranulation to a similar extent in cells on both GelMA hydrogel and plastic wells/glass dishes. F-actin was clearly accumulated along the periphery of RBL-2H3 cells in plane attached to glass, but not GelMA hydrogel, suggesting that the loose actin cytoskeleton of RBL-2H3 cells on GelMA hydrogel caused suppressive degranulation through unstable FcεRI aggregation.

Rhaponticin Alleviates Collagen-induced Arthritis by Inhibiting NLRP3/GSDMD-mediated Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) play an important role in the inflammatory response and progressive joint destruction in rheumatoid arthritis (RA). Rhaponticin (Rha) is a stilbene glycoside compound with antioxidant and anti-inflammatory effects. This study aimed to investigate the therapeutic potential of Rha in RA, with a specific focus on its effects on NETs and on the underlying mechanisms of Rha. NETs formation induced by phorbol 12-myristate 13-acetate (PMA) and a collagen-induced arthritis (CIA) mouse model were implemented to evaluate the pharmacological effects of Rha in vitro and in vivo. The potential mechanism of Rha in improving RA was screened and verified using the SuperPred and DisGeNET databases. Disulfiram (a GSDMD inhibitor) and S100a8cre GSDMDfl/fl mice were used to confirm whether GSDMD is key to the role of Rha.The findings demonstrate that Rha significantly inhibited reactive oxygen species and NETs production in PMA-activated neutrophils. In vivo, Rha treatment significantly relieved joint symptoms in CIA mice and NETs production. Mechanistically, Rha reduced NETs production via inhibition of NLRP3/GSDMD activation. Neutrophil-specific GSDMD depletion eliminated the effects of Rha on NETs production in vitro. Disulfiram eliminated the effects of Rha on the inhibition of NETs production and alleviated joint inflammation in mice in vivo and in vitro. Overall, our results indicated that Rha exerts a protective effect against CIA by inhibiting NETs production through the NLRP3/GSDMD pathway. The results of this study provide new strategies for treating RA.

15-Deoxy-Δ-12,14-Prostaglandin J2 Represses Immune Escape of Lung Adenocarcinoma by Polarizing Macrophages Through Epidermal Growth Factor Receptor/Ras/Raf Pathway.

Lung adenocarcinoma (LUAD) is a widespread and deadly form of cancer. Prostaglandin 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) possesses antioxidant, anti-inflammatory, and anticancer properties. However, it is unclear whether this effect on LUAD progression stems from its ability to influence macrophage polarization. By utilizing 3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, colony formation, transwell assays, and enzyme linked immunosorbent assay (ELISA), we investigated how 15d-PGJ2 affects A549 cell viability, proliferation, apoptosis, and invasion, as well as levels of interleukin (IL)-4, IL-13, and IL-17. Human monocytic cell line THP-1 was induced into M2 macrophages using phorbol 12-myristate 13-acetate and IL-4/IL-13, followed by treatment with 15d-PGJ2. The study employed flow cytometry to observe the polarization of macrophages, quantitative reverse transcription polymerase chain reaction (qRT-PCR) to identify epidermal growth factor receptor (EGFR) expression, western blot for identifying expression of macrophage marker proteins, and examining EGFR/rat sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf) activation. In a coculture setup, CD8+ T cells were shown to have a proliferation capacity by carboxifluorescein diacetate succinimidyl ester (CFSE), a killing ability by lactate dehydrogenase, and an analysis of their interferon gamma and tumor necrosis factor alpha levels by ELISA. 15d-PGJ2 reduced invasion capacity and expression of inflammatory cytokines, lowered A549 cell viability in a dose-dependent way, and promoted apoptosis. 15d-PGJ2 facilitated the transition of M2 macrophages to the M1 type, inhibited Ras/Raf pathway activation, reduced EGFR expression in macrophages, and stimulated CD8+ T cells to enhance anti-tumor immunity. 15d-PGJ2 repressed M2 macrophage polarization and LUAD immune evasion by targeting the EGFR/Ras/Raf pathway in macrophages.

Nuclear Factor-κB Signaling Regulates the Nociceptin Receptor but Not Nociceptin Itself.

The nociceptin receptor (NOP) and nociceptin are involved in the pathways of pain and inflammation. The potent role of nuclear factor-κB (NFκB) in the modulation of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β on the nociceptin system in human THP-1 cells under inflammatory conditions were investigated. Cells were stimulated without/with phorbol-myristate-acetate (PMA), TNF-α, IL-1β, or PMA combined with individual cytokines. To examine NFκB's contribution to the regulation of the nociceptin system, PMA-stimulated cells were treated with NFκB inhibitor BAY 11-7082, JSH-23, or anacardic acid before culturing with TNF-α or IL-1β. NOP and prepronociceptin (ppNOC) mRNA were quantified by RT-qPCR; cell membrane NOP and intracellular nociceptin protein levels were measured by flow cytometry. Phosphorylation and localization of NFκB/p65 were determined using ImageStream. PMA + TNF-α decreased NOP mRNA compared to stimulation with PMA alone, while PMA + IL-1β did not. BAY 11-7082 and JSH-23 reversed the repression of NOP by PMA + TNF-α. TNF-α and IL-1β attenuated PMA's upregulating effects on ppNOC. None of the inhibitors preserved the upregulation of ppNOC in PMA + TNF-α and PMA + IL-1β cultures. TNF-α strongly mediated the nuclear translocation of NFκB/p65 in PMA-treated cells, while IL-1β did not. Proinflammatory cytokines suppressed NOP and ppNOC mRNA in PMA-induced human THP-1 cells. NFκB signaling seems to be an important regulator controlling the transcription of NOP. These findings suggest that the nociceptin system may play an anti-inflammatory role during immune responses.

Hospitalised older adults with community-acquired pneumonia and sepsis have dysregulated neutrophil function but preserved glycolysis

ObjectiveCommunity-acquired pneumonia (CAP) is a leading cause of hospitalisation in older adults and is associated with a high likelihood of adverse outcomes. Given the ageing population and lack of therapeutic...