Phorbol 12-myristate 13-acetate Research Articles

#5607 EFFECTS OF COMMON OSMOLYTES ON THE EXPRESSION OF FOXP3 SPLICE VARIANTS IN HUMAN PBMCS

Abstract Background and Aims Regulatory T cells modulate autoimmune and allogenic responses in glomerulonephritis and transplantation. The two most abundant fork-head box P3 (FOXP3) splice variants, full-length FOXP3 (FOXP3-FL) and FOXP3 lacking exon 2 (FOXP3-Δ 2), confer suppressive ability to regulatory T cells. Some studies showed a weaker suppressive ability of FOXP3-Δ 2. Renal organic osmolytes, urea and Trimethylamine N-oxide (TMAO), play critical roles in the hyperosmotic renal environment with high concentrations of Na+ and glucose. However, the effects of these osmolytes on FOXP3 splice variants in peripheral blood mononuclear cells (PBMCs) remain unclear. The aim of this study was to evaluate the effects of common osmolytes on the expression of FOXP3 splice variants in human PBMCs. Method PBMCs were isolated from the blood of healthy donors using Ficoll density gradients and cultured in RPMI-1640 with or without additional osmolytes at 37°C. A part of PBMCs were activated with 25ng/ml Phorbol-12-myristate-13-acetate (PMA) and 1ug/ml ionomycin (PMA+IONO). FOXP3-FL and FOXP3-Δ 2 mRNA were detected by qPCR after 4hrs’ incubation, using specific primers. The relative expression value was calculated by the formula of (1+E0)∧ Ct0(β-actin)/(1+E)∧ Ct (target gene). Values are given as the mean±S.E.M. Statistical significance was determined by One-Way ANOVA with Post Hoc Test, using Graphpad Prism 9. A P-value <0.05 was considered significant. Results The cell viabilities after 4 hours’ incubation were all above 90%. PMA+IONO significantly increased the relative expression of both FOXP3-Δ 2 (from 0.001051 to 0.001609, P = 0.0289, n = 5) and FOXP3-FL (from 0.001024 to 0.001684, P = 0.0101, n = 5) (Figure 1). However, increased concentrations of urea (from 5mM to 50mM) had no effect on either of the two FOXP3 splice variants (P > 0.05, n = 5, Figure 1). TMAO of 4um significantly reduced FOXP3-FL in the PMA+IONO-activated PBMCs from 0.001605 to 0.0007208 (P = 0.0051, Figure 2). However, TMAO didn’t affect FOXP3-FL expression in non-activated PBMCs and had little effect on FOXP3-Δ 2 expression either (P > 0.05, n = 4). On the other hand, compared with control group, the high concentration of glucose (25mM) significantly increased the expression of FOXP3-Δ 2 from 0.002815 to 0.003461 (P = 0.0375, n = 3) in non-activated PBMCs (Figure 2). The high concentration of NaCl (147mM) had no significant effect on either of the FOXP3 splice variants (P > 0.05, n = 3, Figure 2). Furthermore, the equal osmolarity of mannitol didn’t affect the mRNA expressions of FOXP3 splice variants, either (P > 0.05, n = 3, Figure 2). But compared with glucose group (0.003461), the expression of FOXP3-Δ 2 was significantly lower in mannitol group (0.002785) (P = 0.0479, n = 3, Figure 2). It suggested that the effect of high glucose in non-activated PBMCs was independent of the osmolarity change. Conclusion In isolated human PBMCs, PMA+IONO increases both FOXP3-Δ 2 and FOXP3-FL expressions. PMA+IONO-induced FOXP3-FL increase can be inhibited by TMAO. Urea and NaCl had little effect on FOXP3 splice expressions. High concentration of glucose increases FOXP3-Δ 2 mRNA in non-activated PBMCs, which was independent of the osmolarity change.

Neutrophil extracellular traps: features of their formation in rheumatoid arthritis and osteoarthritis

The similarity of clinical manifestations and some pathogenetic mechanisms in rheumatoid arthritis (RA) and osteoarthritis (OA) is of particular interest in studying the features of extracellular neutrophil traps (NETs) formation in these musculoskeletal diseases. Our objective was to evaluate circulating neutrophil extracellular trap formation related to autoimmune inflammation in RA and reactive inflammation in OA.39 RA patients comprised the main group; 35 OA patients, control group; 33 healthy individuals were included into the reference group. Circulating neutrophils were isolated by the one-step centrifugation in double layer of iohexol density gradient. NETs were induced by phorbol-12-myristate-13–acetate (PMA) being visualized by fluorescence microscopy.The RA disease activity at the inclusion period should not exceed 2.6 DAS28 points. ОA patients were in clinical remission at the inclusion timepoint. In 17 RA patients, an increase in DAS28 exceeded 3.2 points; in 25 OA patients, an exacerbation was diagnosed during the study. The mean percentage of spontaneous and induced NETs in reference group was 3.8% (2.6-5.0) and 12.2% (9.0-15.4), respectively. Appropriate values for the patients with inactive RA were 5.9% (5.6-6.2) and 26% (23.9-28.1); 16.6% (16.1-17.1) and 38.0% (36.6-39.4) in active RA cases, respectively; 5.4% (5.2-5.6) and 20.3% (18.3-22.3) in OA without synovitis; 13.1% (12.6-18.1) and 28.3% (6. 4-30.2) in OA with synovitis, respectively. Spontaneous increase of NETs during RA activation was 181.4%; induced increase in NETs was 46.2%; and in cases of OA exacerbation these values were 142.6% and 39.4%, respectively. In RA patients, the rates of spontaneous NETs formation were 3.9 times higher than for induced values. Appropriate index was 3.6 times higher among the OA patients. The increase in spontaneous -to-induced NETs formation was more pronounced in active RA than in OA with synovitis. The main difference in NETs composition for active RA patients (in 88% of cases) and OA synovitis (in 50% of cases) is related to the contents of citrulline epitopes (p = 0.03).Transition of RA from the remission state to active inflammation, as well as OA exacerbation are accompanied by a significant increase in NETs, especially, spontaneous formation. The degree of increase in RA was higher than in OA, thus, probably, indicating higher involvement of neutrophils in generation of NETs during autoimmune inflammation rather than in reactive inflammation. A sufficient role of citrullinated epitopes in NETs demonstrates their influence upon induction and maintenance of autoimmune response to RA-specific autoantigens.

Short-Term Thermal Stress Affects Immune Cell Features in the Sea Urchin Paracentrotus lividus.

Due to global warming, animals are experiencing heat stress (HS), affecting many organic functions and species' survival. In this line, some characteristics of immune cells in sea urchins subjected to short-term HS were evaluated. Paracentrotus lividus adult females were randomly divided into three groups and housed in tanks at 17 °C. In two of these tanks, the temperatures were gradually increased up to 23 and 28 °C. Celomatic fluid was collected after 3 and 7 days. The coelomocytes were morphologically typed and evaluated for their mitochondrial membrane potential (MMP), lipoperoxidation extent (LPO), and hydrogen peroxide content (H2O2). Respiratory burst was induced by treatment with phorbol 12-myristate 13-acetate (PMA). HS caused a significant change in the coelomocytes' type distribution. MMP increased in the 23 °C-group and decreased in the 28 °C-group at both 3 and 7 days. LPO only increased in the 28 °C-group at 7 days. H2O2 progressively decreased together with the temperature increase. Respiratory burst was detected in all groups, but it was higher in the 17 °C group. In conclusion, the increase in temperature above the comfort zone for this animal species affects their immune cells with possible impairment of their functions.

T cell immunity in interstitial lung disease with non-small cell lung cancer patients

ObjectivesLimited treatment options are available for non-small cell lung cancer (NSCLC) patients with interstitial lung disease (ILD). The rationale for immunotherapy and its adverse events for NSCLC with ILD remains unclear. In this study, we examined T cell profiles and functions in the lung tissues of NSCLC patients with or without ILD to provide evidence for the potential mechanism of immune checkpoint inhibitor (ICI)-related pneumonitis in NSCLC patients with ILD. Material and MethodsWe investigated T cell immunity in the lung tissues of NSCLC patients with ILD to support the application of immunotherapy for these patients. We analyzed T cell profiles and functions in surgically resected lung tissues from NSCLC patients with and without ILD. The T cell profiles of infiltrating cells in lung tissues were analyzed by flow cytometry. T cell functions were measured based on cytokine production by T cells stimulated with phorbol 12-myristate 13-acetate and ionomycin. ResultsThe percentages of CD4+ T cells expressing immune checkpoint molecules (Tim-3, ICOS, and 4-1BB), CD103+CD8+ T cells, and regulatory T (Treg) cells were higher in NSCLC patients with than in those without ILD. A functional analysis of T cells in lung tissues indicated that CD103+CD8+ T cells positively correlated with IFNγ production, whereas Treg cells negatively correlated with IFNγ and TNFα production. Cytokine production by CD4+ and CD8+ T cells did not significantly differ between NSCLC patients with and without ILD, except for TNFα production by CD4+ T cells being lower in the former than in the latter. ConclusionIn NSCLC patients with ILD stable for surgery, T cells were active participants and balanced in part by Treg cells in lung tissues, suggesting the potential development of ICI-related pneumonitis in NSCLC patients with ILD.

Predominantly defective CD8+ T cell immunity to SARS-CoV-2 mRNA vaccination in lung transplant recipients

BackgroundAlthough mRNA vaccines have overall efficacy preventing morbidity/mortality from SARS-CoV-2 infection, immunocompromised persons remain at risk. Antibodies mostly prevent early symptomatic infection, but cellular immunity, particularly the virus-specific CD8+ T cell response, is protective against disease. Defects in T cell responses to vaccination have not been well characterized in immunocompromised hosts; persons with lung transplantation are particularly vulnerable to vaccine failure with severe illness.MethodsComparison groups included persons with lung transplantation and no history of COVID-19 (21 and 19 persons after initial mRNA vaccination and a third booster vaccination respectively), 8 lung transplantation participants recovered from COVID-19, and 22 non-immunocompromised healthy control individuals after initial mRNA vaccination (without history of COVID-19). Anti-spike T cell responses were assayed by stimulating peripheral blood mononuclear cells (PBMCs) with pooled small overlapping peptides spanning the SARS-CoV-2 spike protein, followed by intracellular cytokine staining (ICS) and flow cytometry for release of cytokines in response to stimulation, including negative controls (no peptide stimulation) and positive controls (phorbol myristate acetate [PMA] and ionomycin stimulation). To evaluate for low frequency memory responses, PBMCs were cultured in the presence of the mRNA-1273 vaccine for 14 days before this evaluation.ResultsIonophore stimulation of PBMCs revealed a less inflammatory milieu in terms of interleukin (IL)-2, IL-4, and IL-10 profiling in lung transplantation individuals, reflecting the effect of immunosuppressive treatments. Similar to what we previously reported in healthy vaccinees, spike-specific responses in lung transplantation recipients were undetectable (< 0.01%) when tested 2 weeks after vaccination or later, but were detectable after in vitro culture of PBMCs with mRNA-1273 vaccine to enrich memory T cell responses. This was also seen in COVID-19-recovered lung transplantation recipients. Comparison of their enriched memory responses to controls revealed relatively similar CD4+ T cell memory, but markedly reduced CD8+ T cell memory both after primary vaccination or a booster dose. These responses were not correlated to age or time after transplantation. The vaccine-induced CD4+ and CD8+ responses correlated well in the healthy control group, but poorly in the transplantation groups.ConclusionsThese results reveal a specific defect in CD8+ T cells, which have key roles both in transplanted organ rejection but also antiviral effector responses. Overcoming this defect will require strategies to enhance vaccine immunogenicity in immunocompromised persons.

Black carbon nanoparticles activate the crosstalk mechanism between necroptosis and macrophage extracellular traps to change macrophages fate

PM2.5 still poses a threat to public health even at very low levels. Black carbon (BC) is a key component of PM2.5. Macrophage extracellular traps (METs) are a means by which macrophages capture and destroy invading pathogens. Necroptosis is an inflammatory programmed cell death. However, there is no research on the crosstalk mechanism between necroptosis and METs after BC exposure. In our study, fluorescence labeling, fluorescent probes, qPCR, and immunofluorescence were applied. Our research found that under normal physiological conditions, when macrophages receive external stimuli (in our experiment, phorbol 12-myristate 13-acetate (PMA)), they will form METs, thus exhibiting innate immune function. However, exposure to BC can cause necroptosis in macrophages accompanied by increased levels of ROS and cytosolic calcium ions as well as altered expression of inflammatory factors and chemokines that prevent the formation of METs, and weakening innate immune function. Notably, inhibition of necroptosis restored the formation of METs, indicating that necroptosis inhibits the formation of METs. Our experiment will enrich the understanding of the mechanism of macrophage injury caused by BC exposure, provide a new direction for studying harmful atmospheric particle toxicity, and propose new therapeutic insights for diseases caused by atmospheric particulate matter. This study is the first to explore the crosstalk mechanism between necroptosis and METs after BC exposure.

Ambient NO2 hinders neutrophil extracellular trap formation in rats: Assessment of the role of neutrophil autophagy

NO2 has been known to impair immunity and exacerbate susceptibility to infectious diseases. However, scant notice has been taken of the effect of NO2 on neutrophils. Neutrophil extracellular traps (NETs) formation is necessary for NETosis development by neutrophils as an immune system against pathogens. By analyzing the morphology and signature components of NETs, we focused for the first time on finding that 10 ppm of NO2 exposure for 15 consecutive days can hinder the formation of NETs. Next, we used NO2 in vivo derivatives to probe the mechanism for NETs formation in vitro. Our findings showed that NO2 suppression of respiratory burst levels and mitogen-activated protein kinase (MAPK)/Phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling was related to NO2 reduction in NETs formation. Inhibition of phorbol myristate acetate (PMA)-induced NETs formation by NO2 hindered autophagy, as evidenced by increased mTOR protein expression, decreased LC3 protein expression, and reduced autophagic vesicles. By activating mTOR-mediated autophagy, rapamycin (Rapa) reduced the inhibition of PMA-induced NETs by NO2. This study will provide valuable insights into the mechanisms of immunotoxicity of NO2, new insights into the etiology of diseases linked to NETs formation, and a theoretical basis for protection against such illnesses.

The roles of different forms of IL-15 in human melanoma progression.

Melanoma is a lethal skin cancer, and the risk of developing it is increased by exposure to ultraviolet (UV) radiation. The production of cytokines such as interleukin-15 (IL-15), induced by the exposure of skin cells to UV rays, could also promote melanoma development. The aim of this study is to investigate the possible role of Interleukin-15/Interleukin-15 Receptor α (IL-15/IL-15Rα) complexes in melanoma development. The expression of IL-15/IL-15Rα complexes by melanoma cells was evaluated both ex vivo and in vitro by tissue microarray, PCR, and flow cytometry. The presence of the soluble complex (sIL-15/IL-15Rα) in the plasma of metastatic melanoma patients was detected using an ELISA assay. Subsequently, we investigated the impact of natural killer (NK) cell activation after rIL-2 starvation followed by exposure to the sIL-15/IL-15Rα complex. Finally, by analyzing public datasets, we studied the correlation between IL-15 and IL-15Rα expressions and melanoma stage, NK and T-cell markers, and overall survival (OS). Analysis of a melanoma tissue microarray shows a significant increase in the number of IL-15+ tumor cells from the benign nevi to metastatic melanoma stages. Metastatic melanoma cell lines express a phorbol-12-myristate-13-acetate (PMA)-cleavable membrane-bound IL-15 (mbIL-15), whereas cultures from primary melanomas express a PMA-resistant isoform. Further analysis revealed that 26% of metastatic patients present with consistently high plasmatic levels of sIL-15/IL-15Rα. When the recombinant soluble human IL-15/IL-15Rα complex is added to briefly starved rIL-2-expanded NK cells, these cells exhibit strongly reduced proliferation and levels of cytotoxic activity against K-562 and NALM-18 target cells. The analysis of public gene expression datasets revealed that high IL-15 and IL-15Rα intra-tumoral production correlates with the high levels of expression of CD5+ and NKp46+ (T and NK markers) and significantly correlates with a better OS in stages II and III, but not in stage IV. Membrane-bound and secreted IL-15/IL-15Rα complexes are continuously present during progression in melanoma. It is notable that, although IL-15/IL-15Rα initially promoted the production of cytotoxic T and NK cells, at stage IV promotion of the development of anergic and dysfunctional cytotoxic NK cells was observed. In a subgroup of melanoma metastatic patients, the continuous secretion of high amounts of the soluble complex could represent a novel NK cell immune escape mechanism.

Endoplasmic reticulum-targetable selenium-doped carbon nanodots with redox-responsive fluorescence for in situ free-radical scavenging in cells and mice

Endoplasmic reticulum, the largest cellular organelle where protein synthesis, folding, transportation, and the maintenance of Ca2+ homeostasis occurred, is sensitive to the imbalance of its inner environment. Particularly, overproduction of reactive oxygen species in endoplasmic reticulum region can initiate endoplasmic reticulum stress and is tightly related to many metabolic diseases. In this respect, to retain normal function of endoplasmic reticulum, it is highly important to synthesize antioxidants with endoplasmic reticulum-targeting capability for precisely clearing excessively generated reactive oxygen species in endoplasmic reticulum. Herein, we demonstrate the preparation of endoplasmic reticulum-targeting Se-doped carbon nanodots and assays with HeLa cells as well as in vivo tests with mice have shown that the prepared carbon nanodots can significantly eliminate both •OH and O2•−. Experimental results declared that the fabricated carbon nanodots with powerful scavenging capabilities toward •OH and O2•-, low cytotoxicity and endoplasmic reticulum targetability can be utilized to protect cells against raised levels of reactive oxygen species in endoplasmic reticulum. What’s more, the fabricated carbon nanodots effectively alleviated phorbol 12-myristate 13-acetate induced ear inflammation in live mice.

Effects of interleukin-23 on the activation of mucosal-associated invariant T cells from oral lichen planus.

Oral lichen planus (OLP) is a T cell-mediated inflammatory condition in the oral cavity. Mucosal-associated invariant T (MAIT) cells are gaining more relevance in immune diseases because they can be activated by cytokines without T cell receptor stimulation. Herein, we tested the effect of interleukin-23 (IL-23) on the activation status of OLP MAIT cells. Peripheral blood mononuclear cells (PBMCs) isolated from OLP patients were stimulated by IL-23 in the absence or presence of phorbol myristate acetate (PMA) and ionomycin. The activation status of MAIT cells was analyzed by flow cytometry after staining for CD3, CD4, CD8, CD161, TCR Vα7.2, and CD69. The fraction of MAIT cells in OLP peripheral blood was approximately 0.38% to 3.97%, and CD8+ subpopulations overwhelmed CD4+ cells. The mean percentages of OLP MAIT cells in PBMCs and CD8+MAIT cells in MAIT cells were approximately 40%. PMA and ionomycin significantly increased CD69 expression on OLP T cells, MAIT cells, and CD8+MAIT cells. Cells with enhanced activation had different responsiveness to exogenous IL-23, showing increased CD69 expression on OLP T cells, decreased CD69 on OLP CD8+MAIT cells, and no significant change on OLP MAIT cells. IL-23 showed different effects on the activation status of OLP MAIT cells and CD8+MAIT cells.

Antioxidant and Antitumorigenic Activities of the Standardized Water Extract From Fruit of Terminalia chebula Retz. var. chebula

Background Terminalia chebula Retz. var. chebula ( T chebula) has been traditionally used as a crude drug for treating various diseases, including skin disorders. This study aimed to investigate the potential antioxidant effects and antitumorigenic activity of an aqueous extract from the fruit of T chebula. Methods The extract's total phenolic content and antioxidant activity were measured using Folin-Ciocalteu, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) tests, respectively. The 2 ´,7 ´-dichlorodihydrofluorescin diacetate assay was employed to evaluate the extract's ability to reduce intracellular reactive oxygen species (ROS) in U-937 human monocytic cell lines which had been treated with hydrogen peroxide for 30 min. Male ICR mice ( n = 30, 5 groups) were used to investigate the influence of T chebula extract on dimethylbenz[a]anthracene (DMBA)/12- O-tetradecanoylphorbol 13-acetate (TPA)-induced carcinogenesis. After 20 weeks of treatment with T chebula extract, tumor incidence, size, and number were assessed. The major phenolic compounds in the extract were determined using high-performance liquid chromatography. Statistical analysis was performed using ANOVA followed by post hoc least significant difference test. Results T chebula extract effectively reduced intracellular ROS and scavenged antioxidants in vitro. The IC50 values of the extract as measured by DPPH and FRAP assays were 109.0 ± 14.5 μg/mL and 2.39 ± 0.17 μg/mL, respectively. Moreover, 4 mg of T chebula extract significantly decreased the incidence, volume, and number of tumors in DMBA/TPA-induced skin tumorigenesis in mice. Conclusion T chebula extract effectively reduced intracellular ROS, and significantly decreased the incidence, volume, and number of tumors in DMBA/TPA-induced skin tumorigenesis in mice. These findings suggest that T chebula extract could be a promising therapeutic agent for skin tumors and other oxidative stress-related diseases.

The Role of Macrophage Phenotype in the Vascularization of Prevascularized Human Bone Marrow Mesenchymal Stem Cell Sheets.

With the development of tissue engineering and regenerative medicine, prevascularized marrow mesenchymal stem cell (BMSC) sheets have been regarded as a promising method for tissue regeneration. Furthermore, the inflammatory response is one of the main regulators of vascularization and the restoration of engineered tissue function; among them, macrophages and cytokines produced by them are considered to be the decisive factors of the downstream outcomes. This study investigated the effect of macrophages on the formation of microvascular-like structures of human umbilical vein endothelial cells (HUVECs) in BMSC sheets. First, a human monocytic leukaemia cell line (THP-1 cells) was differentiated into derived macrophages (M0) with phorbol 12-myristate 13-acetate (PMA) and further activated into proinflammatory macrophages (M1 macrophages) with IFN-γ and LPS or anti-inflammatory macrophages (M2 macrophages) with IL-4. Then, HUVECs and prevascularized sheets were treated with conditioned media (CM) from different macrophages, and the impact of macrophage phenotypes on vascularized network formation in prevascularized cell sheets was examined by HE staining, CD31 immunofluorescence staining and ELISA. Our study showed that macrophages may guide the arrangement of endothelial cells through a paracrine pathway. Cell sheets that were cultured in the conditioned media from M2 macrophages were thinner than those cultured in other media. At various time points, the levels of TNF-α and VEGF in prevascularized sheets cultured with CM(M1) was higher than that in sheets cultured with other media; however, the levels of PDGF-BB in prevascularized sheets cultured with CM(M2) was higher than that in sheets cultured with other media. These findings suggest that the paracrine effect of macrophages can influence the formation of microvascular networks in prevascularized sheets by regulating the arrangement of cells, the thickness of the cell sheet and the secretion of cytokines related to angiogenesis. Macrophages with different phenotypes have unique effects on prevascularized sheets.

P-Hydroxylcinnamaldehyde induces tumor-associated macrophage polarization toward the M1 type by regulating the proteome and inhibits ESCC in vivo and in vitro.

P-Hydroxylcinnamaldehyde (CMSP) was firstly isolated from Chinese medicine Cochinchinnamomordica seed (CMS) by our team and has been verified to have growth-inhibiting abilities in malignant tumors including esophageal squamous cell carcinoma (ESCC). However, the detailed mechanism of its function is still unclear. Tumor-associated macrophages (TAMs) are an essential component of the tumor microenvironment (TME), playing important roles in tumor growth, metastasis, angiogenesis, and epithelial-mesenchymal transition (EMT). In the present study, we found that the percentage of M1-like macrophages was significantly increased in TME of ESCC cell derivedxenograft tumor model after CMSP treatment, while the ratios of other immune cells showed relatively low variation. To confirm these results, we further examined the effect of CMSP on macrophage polarization in vitro. The results revealed that CMSP also could induce phorbol-12-myristate-13-acetate (PMA)-induced M0 macrophages from THP-1 and mouse peritoneal macrophages toward the M1-like macrophages. Furthermore, CMSP could exert anti-tumor effect through TAMs in vitro co-culture model, in addition, the growth inhibition effect of CMSP was partly abolished in macrophage depletion model. To determine the potential pathway of CMSP induced polarization, we used quantitative proteomics (label-free) technology to explore the proteomic changes under CMSP treatment. The results revealed that immune-activating protein and M1 macrophage biomarkers were significantly increased after CMSP treatment. More importantly, CMSP stimulated pathways related to M1 macrophage polarization, such as the NF-κB signaling pathway and Toll-like receptor pathway, indicating that CMSP might induce M1-type macrophage polarization through these pathways. In conclusion, CMSP can regulate immune microenvironment in vivo and induce TAM polarization toward the M1 type by promoting proteomic changes, and exert anti-tumor effect through TAMs.

Inhibitory Effect of Cinobufotalin on Macrophage Inflammatory Factor Storm and Its Mechanism

To investigate the inflammatory effects of Cinobufotalin on monocytes in resting state and macrophages in activated state and its molecular mechanism. THP-1 cells were stimulated with Phorbol 12-myristate 13-acetate to induce differentiation into macrophages. Lipopolysaccharides was added to activate macrophages in order to establish macrophage activation model. Cinobufotalin was added to the inflammatory cell model for 24 h as a treatment. CCK-8 was used to detect cell proliferation, Annexin V /PI double staining flow cytometry was used to detect cell apoptosis, flow cytometry was used to detect macrophage activation, and cytometric bead array was used to detect cytokines. Transcriptome sequencing was used to explore the gene expression profile regulated by Cinobufotalin. Changes in the significantly regulated molecules were verified by real-time quantitative polymerase chain reaction and Western blot. 1∶25 concentration of Cinobufotalin significantly inhibited the proliferation of resting monocytes(P<0.01), and induced apoptosis(P<0.01), especially the activated macrophages(P<0.001, P<0.001). Cinobufotalin significantly inhibited the activation of macrophages, and significantly down-regulated the inflammatory cytokines(IL-6, TNF-α, IL-1β, IL-8) released by activated macrophages(P＜0.001). Its mechanism was achieved by inhibiting TLR4/MYD88/P-IκBa signaling pathway. Cinobufotalin can inhibit the inflammatory factors produced by the over-activation of macrophages through TLR4/MYD88/P-IκBa pathway, which is expected to be applied to the treatment and research of diseases related to the over-release of inflammatory factors.

Regulation of the Gene Expression of Airway MUC5AC Mucin through NF-κB Signaling Pathway by Artesunate, an Antimalarial Agent.

In this study, artesunate, an antimalarial agent, was investigated for its potential effect on the gene expression of airway MUC5AC mucin. The human pulmonary epithelial NCI-H292 cells were pretreated with artesunate for 30 min and then stimulated with phorbol 12-myristate 13-acetate (PMA), for the following 24 h. The effect of artesunate on PMA-induced nuclear factor kappa B (NF-kB) signaling pathway was also examined. Artesunate inhibited the glycoprotein production and mRNA expression of MUC5AC mucins, induced by PMA through the inhibition of degradation of inhibitory kappa Bα (IkBα) and NF-kB p65 nuclear translocation. These results suggest artesunate suppresses the gene expression of mucin through regulation of NF-kB signaling pathway, in human pulmonary epithelial cells.

Potentiated effects of lactate receptor GPR81 on immune microenvironment in breast cancer.

G protein-coupled receptor (GPR81), as lactate receptor, is an upstart in immune regulation, however, its mechanisms involved in tumor escape have not been fully elucidated. In this study, we explored the effects of GPR81 activation on triple-negative breast cancer (TNBC) cells and macrophages. The expression and relationship with immune infiltration of GPR81 were analyzed with TCGA database. Checkpoints and cytokines were evaluated with flow cytometry or ELISA. The TCGA-based data showed a marked decrease of GPR81 in breast cancer (BRCA) compared with normal breast, especially in the basal-like subtype. In normal mammary tissues, GPR81 had negative correlation with various immune checkpoints, nevertheless, this trend weakened accompanied with the reduction of GPR81. GPR81 stimulation had a significantly inhibitory influence on PD-L1 exposure in BT-549 and MDA-MB-231 cell lines, but not in MDA-MB-453 cell line. The pretreatment of siGPR81 to knockdown GPR81 expression resulted in a remitting of PD-L1 reduction when MDA-MB-231 cells were treated with GPR81 agonist 1. However, little effect of GPR81 activation was observed on the expression of PD-L1 on phorbol-12-myristate-13-acetate (PMA)-induced THP-1 cells. Furthermore, GPR81 agonist 1 exerted no significant impact on the secretion of cytokines in THP-1 cells. In general, it is suggested that GPR81 may facilitate immune monitoring via the reduction of PD-L1 in TNBC with glycolytic phenotype. Our results not only provide a novel insight into the effects of GPR81 on immune evasion but a potential therapy targeting GPR81 in BRCA.

Optimizing THP-1 Macrophage Culture for an Immune-Responsive Human Intestinal Model.

Previously established immune-responsive co-culture models with macrophages have limitations due to the dedifferentiation of macrophages in long-term cultures. This study is the first report of a long-term (21-day) triple co-culture of THP-1 macrophages (THP-1m) with Caco-2 intestinal epithelial cells and HT-29-methotrexate (MTX) goblet cells. We demonstrated that high-density seeded THP-1 cells treated with 100 ng/mL phorbol 12-myristate 13-acetate for 48 h differentiated stably and could be cultured for up to 21 days. THP-1m were identified by their adherent morphology and lysosome expansion. In the triple co-culture immune-responsive model, cytokine secretions during lipopolysaccharide-induced inflammation were confirmed. Tumor necrosis factor-alpha and interleukin 6 levels were elevated in the inflamed state, reaching 824.7 ± 130.0 pg/mL and 609.7 ± 139.5 pg/mL, respectively. Intestinal membrane integrity was maintained with a transepithelial electrical resistance value of 336.4 ± 18.0 Ω·cm2. Overall, our findings suggest that THP-1m can be effectively employed in models of long-term immune responses in both normal and chronic inflammatory states of the intestinal epithelium, making them a valuable tool for future research on the association between the immune system and gut health.

Development of the observation of membrane fusion with label-free liposomes by calcium imaging

Liposomes are artificial vesicles composed of lipid bilayers that have enabled drugs to be encapsulated and delivered to tumor tissue. Membrane-fusogenic liposomes fuse with the plasma membranes of cells to deliver encapsulated drugs directly to the cytosol, which makes it a promising method for rapid and highly efficient drug delivery. In a previous study, liposomal lipid bilayers were labeled with fluorescent probes, and colocalization of labeled lipids with plasma membrane was observed under a microscope. However, there was concern that fluorescent labeling would affect lipid dynamics and cause liposomes to acquire membrane fusogenic ability. In addition, encapsulation of hydrophilic fluorescent substances in the inner aqueous phase sometimes requires an additional step of removing unencapsulated substances after preparation, and there is a risk of leakage. Herein, we propose a new method to observe cell interaction with liposomes without labeling. Our laboratory has developed two types of liposomes with different cellular internalization pathways, i.e., endocytosis and membrane fusion. We found that cytosolic calcium influx would be triggered following the internalization of cationic liposomes, and different cell entry routes led to different calcium responses. Thus, the correlation between cell entry routes and calcium responses could be utilized to study liposome-cell interactions without fluorescent labeling lipids. Briefly, liposomes were added to phorbol 12-myristate 13-acetate (PMA)-primed THP-1 cells, and calcium influx was measured by time-lapse imaging using a fluorescent indicator (Fura 2-AM). Liposomes with high membrane fusogenic ability elicited a strong transient calcium response immediately after adding liposomes, whereas those taken up mainly by endocytosis elicited multiple weak calcium responses. In order to verify the cell entry routes, we also tracked the intracellular distribution of fluorescent-labeled liposomes in PMA-primed THP-1 cells using a confocal laser scanning microscope. It was shown that for fusogenic liposomes, colocalization with plasma membrane occurred at the same time as calcium elevation, whereas for liposomes with a high endocytosis potential, fluorescent dots were observed in the cytoplasm, suggesting the cell internalization by endocytosis. These results suggested that the calcium response patterns correspond to cell entry routes, and membrane fusion can be observed by calcium imaging.

Oxymatrine blocks the NLRP3 inflammasome pathway, partly downregulating the inflammatory responses of M1 macrophages differentiated from THP-1 monocytes

Many chronic inflammatory diseases, such as autoimmune inflammation, are associated with M1 macrophages, and the key to their treatment is blocking inflammation. Oxymatrine (OMT), a traditional Chinese medicine, has a marked anti-inflammatory effect. However, its anti-inflammatory target and mechanism in M1 cells remain unclear, which limits its clinical application. In this study, we investigated the anti-inflammatory effects of oxymatrine (OMT) on the M1 inflammatory response. We also determined the relationship between OMT treatment and the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway with OMT treatment. To this end, we induced the differentiation of human peripheral blood monocytes (THP-1) into M1 cells. THP-1 cells were induced with a phorbol ester (phorbol-12-myristate-13-acetate (PMA)) and differentiated into naïve M0 macrophages. M0 cells were induced into M1 cells using lipopolysaccharide (LPS). The experimental groups were divided into the M0 macrophage group (NC), M1 inflammatory response group (LPS group), and M1 group treated with different concentrations of OMT (LPS + OMT-L, LPS + OMT-M, LPS + OMT-H). The cells in the OMT-treated groups were treated with OMT for 6 h, followed by LPS for 24 h, and the LPS group was treated with LPS only. The resulting supernatants and cells were collected. The secretion levels of NO were detected by the Griess method and the secretion levels of TNF-α and IL-1β in the supernatants were detected by the ELISA method. The secretion levels of these inflammatory factors were reduced in every OMT-treated group compared to the LPS group (P < 0.01), and the most significant reductions were found in the OMT-H group (P < 0.0001). By western blotting, the protein expression levels of TLR4, NF-κB, NLRP3, and Caspase-1 were all found to be downregulated in the cells of OMT-treated groups compared to the LPS group (P < 0.0001). In situ changes in NLRP3 expression were observed using immunofluorescence. The fluorescence intensity of NLRP3 in M1 cells was weaker in all OMT intervention groups than in the LPS group (P < 0.001). In conclusion, OMT has significant anti-inflammatory effects on the M1 inflammatory responses, and the TLR4/NF-κB/NLRP3 pathway was blocked proportional to the concentration of OMT.

NET Proteome in Established Type 1 Diabetes Is Enriched in Metabolic Proteins.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by a T-cell-mediated destruction of the pancreatic insulin-producing beta cells. A growing body of evidence suggests that abnormalities in neutrophils and neutrophil extracellular trap (NET) formation (NETosis) are associated with T1D pathophysiology. However, little information is available on whether these changes are primary neutrophil defects or related to the environmental signals encountered during active disease. In the present work, the NET proteome (NETome) of phorbol 12-myristate 13-acetate (PMA)- and ionomycin-stimulated neutrophils from people with established T1D compared to healthy controls (HC) was studied by proteomic analysis. Levels of NETosis, in addition to plasma levels of pro-inflammatory cytokines and NET markers, were comparable between T1D and HC subjects. However, the T1D NETome was distinct from that of HC in response to both stimuli. Quantitative analysis revealed that the T1D NETome was enriched in proteins belonging to metabolic pathways (i.e., phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and UTP-glucose-1-phosphate uridylyltransferase). Complementary metabolic profiling revealed that the rate of extracellular acidification, an approximate measure for glycolysis, and mitochondrial respiration were similar between T1D and HC neutrophils in response to both stimuli. The NETome of people with established T1D was enriched in metabolic proteins without an apparent alteration in the bio-energetic profile or dysregulated NETosis. This may reflect an adaptation mechanism employed by activated T1D neutrophils to avoid impaired glycolysis and consequently excessive or suboptimal NETosis, pivotal in innate immune defence and the resolution of inflammation.