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BioRxiv : the Preprint Server For... Oct 2023Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their...
Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their recruitment in inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin and assess whether keratinocyte-derived signals impact neutrophil recruitment. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12h and resolves within 24h. A second TPA treatment or a UVB challenge, when applied at 24h but not 48h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of neutrophil chemoattractants by stressed keratinocytes. We show that K17 binds RACK1, a scaffold essential for PKCα activity. Finally, analyses of RNAseq data reveal the presence of a transcriptomic signature consistent with TAR and PKCα activation in chronic inflammatory skin diseases. These findings uncover a novel, transient, and keratin-dependent mechanism that amplifies neutrophil recruitment to the skin under stress, with direct implications for inflammatory skin disorders.
PubMed: 37873256
DOI: 10.1101/2023.10.11.561954 -
Frontiers in Immunology 2023The infusion of -generated regulatory B cells may represent a promising novel therapeutic approach for a variety of autoimmune and hyperinflammatory conditions including...
INTRODUCTION
The infusion of -generated regulatory B cells may represent a promising novel therapeutic approach for a variety of autoimmune and hyperinflammatory conditions including graft-versus-host disease.
METHODS
Previously, we developed a protocol for the generation of a novel population of regulatory B cells, which are characterized by secretion of enzymatically active granzyme B (). This protocol uses recombinant interleukin 21 (IL-21) and goat-derived F(ab)'2 fragments against the human B cell receptor (anti-BCR). Generally, the use of xenogeneic material for the manufacturing of advanced therapy medicinal products should be avoided to prevent adverse immune reactions as well as potential transmission of so far unknown diseases.
RESULTS
In the present work we demonstrated that phorbol-12-myristate-13-acetate (PMA/TPA), a phorbol ester with a particular analogy to the second messenger diacylglycerol (DAG), is a potent enhancer of IL-21-induced differentiation of pre-activated B cells into . The percentage of after stimulation of pre-activated B cells with IL-21 and PMA/TPA was not significantly lower compared to stimulation with IL-21 and anti-BCR.
DISCUSSION
Given that PMA/TPA has already undergone encouraging clinical testing in patients with certain haematological diseases, our results suggest that PMA/TPA may be a safe and feasible alternative for manufacturing of .
Topics: Humans; B-Lymphocytes, Regulatory; Granzymes; Tetradecanoylphorbol Acetate
PubMed: 37588597
DOI: 10.3389/fimmu.2023.1194880 -
Scientific Reports Jul 2023Jatropha curcas is an oilseed crop with biorefinery applications. Whilst cake generated following oil extraction offers potential as a protein source for animal feed,...
Jatropha curcas is an oilseed crop with biorefinery applications. Whilst cake generated following oil extraction offers potential as a protein source for animal feed, inactivation of toxic phorbol esters present in the material is necessary. Pleurotus pulmonarius is a detoxifying agent for jatropha cake with additional potential as animal feed, edible mushroom and for enzyme production. For the characterization of fungal genes involved in phorbol ester degradation, together with other industrial applications, reverse transcription-quantitative PCR (RT-qPCR) is a tool that enables accurate quantification of gene expression. For this, reliable analysis requires reference genes for normalization of mRNA levels validated under conditions employed for target genes. The stability of potential reference genes β-TUB, ACTIN, GAPDH, PHOS, EF1α, TRPHO, LAC, MNP3, MYP and VP were evaluated following growth of P. pulmonarius on toxic, non-toxic jatropha cake and a combined treatment, respectively. NormFinder and geNorm algorithms for expression stability analysis identified PHOS, EF1α and MNP3 as appropriate for normalizing gene expression. Reference gene combinations contrasting in ranking were compared following normalization of relative expression of the CHU_2040 gene, encoding an esterase enzyme potentially involved in phorbol ester degradation. The reference genes for P. pulmonarius will facilitate the elucidation of mechanisms involved in detoxification of phorbol esters as well as analysis of target genes for application in biorefinery models.
Topics: Animals; Reverse Transcription; Pleurotus; Agaricales; Animal Feed; Jatropha
PubMed: 37516784
DOI: 10.1038/s41598-023-39115-4 -
Biomolecules & Therapeutics May 2024In this study, we investigated the efficacy of kaempferol (a flavonoid found in plants and plant-derived foods such as kale, beans, tea, spinach and broccoli) on...
In this study, we investigated the efficacy of kaempferol (a flavonoid found in plants and plant-derived foods such as kale, beans, tea, spinach and broccoli) on vascular contractibility and aimed to clarify the detailed mechanism underlying the relaxation. Isometric contractions of divested muscles were stored and linked with western blot analysis which was carried out to estimate the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to estimate the effect of kaempferol on the RhoA/ROCK/CPI-17 pathway. Kaempferol conspicuously impeded phorbol ester-, fluoride- and a thromboxane mimetic-derived contractions regardless of endothelial nitric oxide synthesis, indicating its direct effect on smooth muscles. It also conspicuously impeded the fluoride-derived elevation in phospho-MYPT1 rather than phospho-CPI-17 levels and phorbol 12,13-dibutyrate-derived increase in phospho-CPI-17 and phospho-ERK1/2 levels, suggesting the depression of PKC and MEK activities and subsequent phosphorylation of CPI-17 and ERK1/2. Taken together, these outcomes suggest that kaempferol-derived relaxation incorporates myosin phosphatase retrieval and calcium desensitization, which appear to be modulated by CPI-17 dephosphorylation mainly through PKC inactivation.
PubMed: 38589300
DOI: 10.4062/biomolther.2023.186 -
Zhong Nan Da Xue Xue Bao. Yi Xue Ban =... Aug 2023The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one of the main signaling pathways related to autophagy....
OBJECTIVES
The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one of the main signaling pathways related to autophagy. Autophagy plays a key role in the formation of silicosis fibrosis. The phenotypic transformation of lung fibroblasts into myofibroblasts is a hallmark of the transition from the inflammatory phase to the fibrotic phase in silicosis. This study aims to investigate whether the PI3K/Akt/mTOR pathway affects the phenotypic transformation of silicosis-induced lung fibroblasts into myofibroblasts via mediating macrophage autophagy.
METHODS
The human monocytic leukemia cell line THP-1 cells were differentiated into macrophages by treating with 100 ng/mL of phorbol ester for 24 h. Macrophages were exposed to different concentrations (0, 25, 50, 100, 200, 400 μg/mL) and different times (0, 6, 12, 24, 48 h) of SiO dust suspension. The survival rate of macrophages was measured by cell counting kit-8 (CCK-8) method. Enzyme linked immunosorbent assay (ELISA) was used to measure the contents of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) in the cell supernatant. The co-culture system of macrophages and HFL-1 cells was established by transwell. A blank control group, a SiO group, a LY294002 group, a SC79 group, a LY294002+SiO group, and a SC79+SiO group were set up in this experiment. Macrophages in the LY294002+SiO group were pretreated with LY294002 (PI3K inhibitor) for 18 hours, and macrophages in the SC79+SiO group were pretreated with SC79 (Akt activator) for 24 hours, and then exposed to SiO (100 μg/mL) dust suspension for 12 hours. The expression of microtubule-associated protein 1 light chain 3 (LC3) protein in macrophages was detected by the immunofluorescence method. The protein expressions of PI3K, Akt, mTOR, Beclin-1, LC3 in macrophages, and collagen III (Col III), α-smooth muscle actin (α-SMA), fibronectin (FN), matrix metalloproteinase-1 (MMP-1), tissue metalloproteinase inhibitor-1 (TIMP-1) in HFL-1 cells were measured by Western blotting.
RESULTS
After the macrophages were exposed to SiO dust suspension of different concentrations for 12 h, the survival rates of macrophages were gradually decreased with the increase of SiO concentration. Compared with the 0 μg/mL group, the survival rates of macrophages in the 100, 200, and 400 μg/mL groups were significantly decreased, and the concentrations of TGF-β1 and TNF-α in the cell supernatant were obviously increased (all <0.05). When 100 μg/mL SiO dust suspension was applied to macrophages, the survival rates of macrophages were decreased with the prolonged exposure time. Compared with the 0 h group, the survival rates of macrophages were significantly decreased (all <0.05), the concentrations of TGF-β1 and TNF-α in the cell supernatant were significantly increased, and the protein expression levels of Beclin-1 and LC3II were increased markedly in the 6, 12, 24, and 48 h groups (all <0.05). Immunofluorescence results demonstrated that after exposure to SiO (100 μg/mL) dust for 12 h, LC3 exhibited punctate aggregation and significantly higher fluorescence intensity compared to the blank control group (<0.05). Compared with the blank control group, the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated in the SiO group (all <0.05). Compared with the SiO group, the protein expressions of PI3K, Akt, and mTOR were down-regulated and the protein expressions of LC3II and Beclin-1 were up-regulated in macrophages (all <0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were decreased (both <0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were down-regulated (all <0.05) in the LY294002+SiO group. Compared with the SiO group, the protein expressions of PI3K, Akt, and mTOR were up-regulated and the protein expressions of LC3II and Beclin-1 were down-regulated in macrophages (all <0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were increased (both <0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated (all <0.05) in the SC79+SiO group.
CONCLUSIONS
Silica dust exposure inhibits the PI3K/Akt/mTOR pathway, increases autophagy and concentration of inflammatory factors in macrophages, and promotes the phenotype transformation of HFL-1 cells into myofibroblasts. The regulation of the PI3K/Akt/mTOR pathway can affect the autophagy induction and the concentration of inflammatory factors of macrophages by silica dust exposure, and then affect the phenotype transformation of HFL-1 cells into myofibroblasts induced by silica dust exposure.
Topics: Humans; Proto-Oncogene Proteins c-akt; Transforming Growth Factor beta1; Silicon Dioxide; Phosphatidylinositol 3-Kinases; Matrix Metalloproteinase 1; Tissue Inhibitor of Metalloproteinase-1; Sirolimus; Beclin-1; Tumor Necrosis Factor-alpha; Dust; TOR Serine-Threonine Kinases; Lung; Fibroblasts; Silicosis; Macrophages; Autophagy
PubMed: 37875355
DOI: 10.11817/j.issn.1672-7347.2023.220581 -
Anais Da Academia Brasileira de Ciencias 2023Dermatitis is defined as a set of inflammatory diseases that affect the skin, with varied causes. Among the different types of dermatitis, contact dermatitis is the most...
Dermatitis is defined as a set of inflammatory diseases that affect the skin, with varied causes. Among the different types of dermatitis, contact dermatitis is the most prevalent. Although the current therapy is often effective, it is associated with adverse effects and the possibility of drug tolerance. N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline is a L-proline amino acid derivative found in the leaves of Sideroxylon obtusifolium, a species traditionally used to treat inflammatory diseases. The aim of this study was to investigate the topical anti-inflammatory effect of N-methyl-(2S, 4R)-trans-4-hydroxy-L-proline (NMP) in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritant contact dermatitis in mice. Topically administered NMP, at doses of 0.03 - 0.50 mg/ear, reduced TPA-induced ear edema and neutrophil migration, as evidenced by low tissue myeloperoxidase activity and verified by histological examination. In addition, NMP (0.06 mg/ear) reduced tissue levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, INF-γ and MCP-1) and of the anti-inflammatory cytokine IL-10, and reduced gene expression of TNF-α, IL-6 and IL-1β increased by TPA. The data suggest that N-methyl-(2S, 4R)-trans-4-hydroxy-L-proline acts as a topical anti-inflammatory agent that decreases the expression of inflammatory cytokines, making it useful for the treatment of skin inflammation. Further investigations are necessary for its development as a therapeutic agent.
Topics: Mice; Animals; Tetradecanoylphorbol Acetate; Irritants; Tumor Necrosis Factor-alpha; Interleukin-6; Dermatitis, Contact; Anti-Inflammatory Agents; Dermatitis; Cytokines; Sapotaceae
PubMed: 37909544
DOI: 10.1590/0001-3765202320220919 -
Journal of Virology Aug 2023Transcription of the human papillomavirus (HPV) oncogenes, and , is regulated by the long control region (LCR) of the viral genome. Although various transcription...
Transcription of the human papillomavirus (HPV) oncogenes, and , is regulated by the long control region (LCR) of the viral genome. Although various transcription factors have been reported to bind to the LCR, little is known about the transcriptional cofactors that modulate HPV oncogene expression in association with these transcription factors. Here, we performed DNA-pulldown purification of nuclear proteins in cervical cancer cells, followed by proteomic analyses to identify transcriptional cofactors that bind to the HPV16 LCR via the transcription factor TEAD1. We detected the proinflammatory cytokine S100A9 that localized to the nucleus of cervical cancer cells and associated with the LCR via direct interaction with TEAD1. Nuclear S100A9 levels and its association with the LCR were increased in cervical cancer cells by treatment with a proinflammatory phorbol ester. Knockdown of S100A9 decreased HPV oncogene expression and reduced the growth of cervical cancer cells and their susceptibility to cisplatin, whereas forced nuclear expression of S100A9 using nuclear localization signals exerted opposite effects. Thus, we conclude that nuclear S100A9 binds to the HPV LCR via TEAD1 and enhances viral oncogene expression by acting as a transcriptional coactivator. IMPORTANCE Human papillomavirus (HPV) infection is the primary cause of cervical cancer, and the viral oncogenes and play crucial roles in carcinogenesis. Although cervical inflammation contributes to the development of cervical cancer, the molecular mechanisms underlying the role of these inflammatory responses in HPV carcinogenesis are not fully understood. Our study shows that S100A9, a proinflammatory cytokine, is induced in the nucleus of cervical cancer cells by inflammatory stimuli, and it enhances HPV oncogene expression by acting as a transcriptional coactivator of TEAD1. These findings provide new molecular insights into the relationship between inflammation and viral carcinogenesis.
Topics: Female; Humans; Carcinogenesis; Human Papillomavirus Viruses; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Papillomavirus Infections; Proteomics; TEA Domain Transcription Factors; Transcription Factors; Uterine Cervical Neoplasms; Calgranulin B
PubMed: 37578237
DOI: 10.1128/jvi.00815-23 -
International Journal of Molecular... Nov 2023The function of the α-adrenergic receptor phosphorylation sites previously detected by mass spectrometry was evaluated by employing mutants, substituting them with...
The function of the α-adrenergic receptor phosphorylation sites previously detected by mass spectrometry was evaluated by employing mutants, substituting them with non-phosphorylatable amino acids. Substitution of the intracellular loop 3 (IL3) sites did not alter baseline or stimulated receptor phosphorylation, whereas substitution of phosphorylation sites in the carboxyl terminus (Ctail) or both domains (IL3/Ctail) markedly decreased receptor phosphorylation. Cells expressing the IL3 or Ctail receptor mutants exhibited a noradrenaline-induced calcium-maximal response similar to those expressing the wild-type receptor, and a shift to the left in the concentration-response curve to noradrenaline was also noticed. Cells expressing the IL3/Ctail mutant exhibited higher apparent potency and increased maximal response to noradrenaline than those expressing the wild-type receptor. Phorbol ester-induced desensitization of the calcium response to noradrenaline was reduced in cells expressing the IL3 mutant and abolished in cells in which the Ctail or the IL3/Ctail were modified. In contrast, desensitization in response to preincubation with noradrenaline was unaffected in cells expressing the distinct receptor mutants. Noradrenaline-induced ERK phosphorylation was surprisingly increased in cells expressing IL3-modified receptors but not in those expressing receptors with the Ctail or IL3/Ctail substitutions. Our data indicate that phosphorylation sites in the IL3 and Ctail domains mediate and regulate α-adrenergic receptor function. Phorbol ester-induced desensitization seems to be closely associated with receptor phosphorylation, whereas noradrenaline-induced desensitization likely involves other elements.
Topics: Phosphorylation; Calcium; Norepinephrine; Phorbol Esters; Receptors, Adrenergic
PubMed: 38069285
DOI: 10.3390/ijms242316963 -
BMC Complementary Medicine and Therapies Sep 2023Allergy is an inflammatory disorder affecting around 20% of the global population. The adverse effects of current conventional treatments give rise to the increased...
BACKGROUND
Allergy is an inflammatory disorder affecting around 20% of the global population. The adverse effects of current conventional treatments give rise to the increased popularity of using natural food products as complementary and alternative medicine against allergic diseases. Stingless bee honey, commonly known as Kelulut honey (KH) in Malaysia, has been used locally as a traditional remedy to relieve cough and asthma. This study evaluated the anti-allergic potential of KH collected from four different botanical sources on phorbol ester 12-myristate-3-acetate and calcium ionophore-activated human mast cells.
METHODS
The present study examined the inhibitory effects of all collected honey on the release of selected inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-4, IL-6, IL-8, histamine, and β-hexosaminidase in an activated HMC. Besides that, all honey's total phenolic content (TPC) was also examined, followed by using liquid chromatography with tandem mass spectrometry (LC-MS/MS) to identify the phytochemicals in the honey. Further examination of the identified phytochemicals on their potential interaction with selected signaling molecules in an activated mast cell was conducted using computational methods.
RESULTS
The results indicated that there were significant inhibitory effects on all selected inflammatory mediators' release by KH sourced from bamboo (BH) and rubber tree (RH) at 0.5% and 1%, but not KH sourced from mango (AH) and noni (EH). BH and RH were found to have higher TPC values and were rich in their phytochemical profiles based on the LC-MS/MS results. Computational studies were employed to determine the possible molecular target of KH through molecular docking using HADDOCK and PRODIGY web servers.
CONCLUSIONS
In short, the results indicated that KH possesses anti-allergic effects towards an activated HMC, possibly by targeting downstream MAPKs. However, their anti-allergic effects may vary according to their botanical sources. Nevertheless, the present study has provided insight into the potential application of stingless bee honey as a complementary and alternative medicine to treat various allergic diseases.
Topics: Humans; Bees; Animals; Honey; Anti-Allergic Agents; Mast Cells; Cell Degranulation; Chromatography, Liquid; Molecular Docking Simulation; Tandem Mass Spectrometry; Hypersensitivity
PubMed: 37667314
DOI: 10.1186/s12906-023-04129-y -
Medicina (Kaunas, Lithuania) Jun 2024: As modulators of the tumor microenvironment, macrophages have been extensively studied for their potential in developing anticancer strategies, particularly in...
: As modulators of the tumor microenvironment, macrophages have been extensively studied for their potential in developing anticancer strategies, particularly in regulating macrophage polarization towards an antitumorigenic (M1) phenotype rather than a protumorigenic (M2) one in various experimental models. Here, we evaluated the effect of PD98059, a mitogen-activated protein kinase kinase MAPKK MEK1-linked pathway inhibitor, on the differentiation and polarization of THP-1 monocytes in response to phorbol-12-myristate-13-acetate (PMA) under various culture conditions for tumor microenvironmental application. : Differentiation and polarization of THP-1 were analyzed by flow cytometry and RT-PCR. Polarized THP-1 subsets with different treatment were compared by motility, phagocytosis, and so on. : Clearly, PMA induced THP-1 differentiation occurs in adherent culture conditions more than nonadherent culture conditions by increasing CD11b expression up to 90%, which was not affected by PD98059 when cells were exposed to PMA first (post-PD) but inhibited when PD98059 was treated prior to PMA treatment (pre-PD). CD11b THP-1 cells treated with PMA and PMA-post-PD were categorized into M0 (HLA-DR and CD206), M1 (HLA-DR and CD206), and M2 (HLA-DR and CD206), resulting in an increased population of M1 macrophages. The transcription levels of markers of macrophage differentiation and polarization confirmed the increased M1 polarization of THP-1 cells with post-PD treatment rather than with PMA-only treatment. The motility and cytotoxicity of THP-1 cells with post-PD treatment were higher than THP-1 cells with PMA, suggesting that post-PD treatment enhanced the anti-tumorigenicity of THP-1 cells. Confocal microscopy and flow cytometry showed the effect of post-PD treatment on phagocytosis by THP-1 cells. : We have developed an experimental model of macrophage polarization with THP-1 cells which will be useful for further studies related to the tumor microenvironment.
Topics: Humans; Macrophages; Tetradecanoylphorbol Acetate; Flavonoids; THP-1 Cells; Cell Differentiation; Monocytes; Flow Cytometry; Phagocytosis
PubMed: 38929626
DOI: 10.3390/medicina60061009