Response Of Human Cells Research Articles

Abstract 1087: Synergistic mechanisms against pediatric bone sarcoma models: Trabectedin enhances oncolytic virotherapy intratumoral spread and antitumor immune activation

Abstract Osteosarcoma and Ewing sarcoma are the most common pediatric bone malignancies and, despite decades of research, efforts to treat patients with advanced disease remain dismal. To address this inadequacy, we leverage oncolytic herpes simplex virotherapy as a double-edged attack against tumors through 1) tumor cell-specific lysis and 2) antitumor immune stimulation. However, previous trials of oncolytic herpes simplex virotherapy in pediatric sarcomas displayed limited responses, likely due to the observed presence of immunosuppressive monocytes and macrophages. To improve efficacy, we combined oncolytic herpes simplex virotherapy with trabectedin, an FDA-approved DNA-binding agent known to disrupt gene expression and deplete monocytes and macrophages. Surprisingly, we found that the efficacy of this combination far surpassed our predictions, inducing complete and partial regressions in multiple models of Ewing sarcoma and osteosarcoma. To explore potential mechanisms driving this high degree of synergy, we performed single cell RNA sequencing on tumors of mice treated with monotherapy or combination therapy. Interestingly, trabectedin synergized with the oncolytic virotherapy through two unexpected mechanisms. Our analysis revealed that trabectedin increased intratumoral oncolytic virus spread by disrupting expression of the intrinsic antiviral response in human Ewing sarcoma cells. Additionally, trabectedin not only reduced immunosuppressive macrophages but also stimulated cytotoxic granzyme expression in infiltrating T and NK cells in immunocompetent osteosarcoma models. These effector NK and T cells were necessary for the regressions of osteosarcoma allografts treated with trabectedin and oncolytic virotherapy. Thus, trabectedin enhanced both the oncolytic effect of herpes simplex virotherapy and the effector function of cytotoxic lymphocytes that become activated in response to the virus. The combination of trabectedin and oncolytic herpes simplex virotherapy offers the potential to unite these mechanisms against bone tumors to achieve better outcomes for children. Citation Format: Emily Ringwalt, Mark Currier, Andrea Glaspell, Matthew V. Cannon, Chun-Yu Chen, Amy C. Gross, Maren Cam, Pin-Yi Wang, Ryan D. Roberts, Timothy P. Cripe. Synergistic mechanisms against pediatric bone sarcoma models: Trabectedin enhances oncolytic virotherapy intratumoral spread and antitumor immune activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1087.

Regulation of the AGEs-induced inflammatory response in human periodontal ligament cells via the AMPK/NF-κB/ NLRP3 signaling pathway

The heightened prevalence and accelerated progression of periodontitis in individuals with diabetes is primarily attributed to inflammatory responses in human periodontal ligament cells (HPDLCs). This study is aimed at delineating the regulatory mechanism of nucleotide-binding oligomerization domain-like receptors (NLRs) in mediating inflammation incited by muramyl dipeptide (MDP) in HPDLCs, under the influence of advanced glycation end products (AGEs), metabolic by-products associated with diabetes. We performed RNA-seq in HPDLCs induced by AGEs treatment and delineated activation markers for the receptor of AGEs (RAGE). It showed that advanced glycation end products modulate inflammatory responses in HPDLCs by activating NLRP1 and NLRP3 inflammasomes, which are further regulated through the NF-κB signaling pathway. Furthermore, AGEs synergize with NOD2, NLRP1, and NLRP3 inflammasomes to augment MDP-induced inflammation significantly.

Antimicrobial Peptide- and Dentin Matrix-Functionalized Hydrogel for Vital Pulp Therapy via Synergistic Bacteriostasis, Immunomodulation, and Dentinogenesis.

The preservation of vital pulps is crucial for maintaining the physiological functions of teeth; however, vital pulp therapy (VPT) of pulpitis teeth remains a substantial challenge due to uncontrolled infection, excessive inflammation, and limited regenerative potential. Current pulp capping agents have restricted effects in the infectious and inflammatory microenvironment. To address this, a multifunctional hydrogel (TGH/DM) with antibacterial, immunomodulatory, and mineralization-promoting effects is designed. The antimicrobial peptide (AMP) and demineralized dentin matrix are incorporated into the hydrogel, achieving sustainable delivery of AMP and a cocktail of growth factors. In vitro results show that TGH/DM could kill endodontic microbiota, ameliorate inflammatory responses of human dental pulp stem cells (hDPSCs), and prompt odontogenic differentiation of inflammatory hDPSCs via activation of peroxisome proliferator-activated receptor gamma. In vivo results suggest that TGH/DM is capable of inducing M2 phenotype transformation of macrophages in mice and fostering the regeneration of the dentin-pulp complex in inflamed pulps of beagle dogs. Overall, this study first proposes the synergistic regulation of AMP and tissue-specific extracellular matrix for the treatment of pulpitis, and the advanced hydrogel provides a facile and effective way for VPT.

Human Host Defense Peptide LL-37 Suppresses TNFα-Mediated Matrix Metalloproteinases MMP9 and MMP13 in Human Bronchial Epithelial Cells

Introduction: TNFα-inducible matrix metalloproteinases play a critical role in the process of airway remodeling in respiratory inflammatory disease including asthma. The cationic host defense peptide LL-37 is elevated in the lungs during airway inflammation. However, the impact of LL-37 on TNFα-driven processes is not well understood. Here, we examined the effect of LL-37 on TNFα-mediated responses in human bronchial epithelial cells (HBECs). Methods: We used a slow off-rate modified aptamer-based proteomics approach to define the HBEC proteome altered in response to TNFα. Abundance of selected protein candidates and signaling intermediates was examined using immunoassays, ELISA and Western blots, and mRNA abundance was examined by qRT-PCR. Results: Proteomics analysis revealed that 124 proteins were significantly altered, 12 proteins were enhanced by ≥2-fold compared to unstimulated cells, in response to TNFα. MMP9 was the topmost increased protein in response to TNFα, enhanced by ∼10-fold, and MMP13 was increased by ∼3-fold, compared to unstimulated cells. Furthermore, we demonstrated that LL-37 significantly suppressed TNFα-mediated MMP9 and MMP13 in HBEC. Mechanistic data revealed that TNFα-mediated MMP9 and MMP13 production is controlled by SRC kinase and that LL-37 enhances related upstream negative regulators, namely, phospho-AKT (T308) and TNFα-mediated TNFAIP3 or A20. Conclusions: The findings of this study suggest that LL-37 may play a role in intervening in the process of airway remodeling in chronic inflammatory respiratory disease such as asthma.

Metabolic alterations and mitochondrial dysfunction in human airway BEAS-2B cells exposed to vanadium pentoxide

Vanadium pentoxide (V+5) is a hazardous material that has drawn considerable attention due to its wide use in industrial sectors and increased release into environment from human activities. It poses potential adverse effects on animals and human health, with pronounced impact on lung physiology and functions. In this study, we investigated the metabolic response of human bronchial epithelial BEAS-2B cells to low-level V+5 exposure (0.01, 0.1, and 1 ppm) using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Exposure to V+5 caused extensive changes to cellular metabolism in BEAS-2B cells, including TCA cycle, glycolysis, fatty acids, amino acids, amino sugars, nucleotide sugar, sialic acid, vitamin D3, and drug metabolism, without causing cell death. Altered mitochondrial structure and function were observed with as low as 0.01 ppm (0.2 μM) V+5 exposure. In addition, decreased level of E-cadherin, the prototypical epithelial marker of epithelial-mesenchymal transition (EMT), was observed following V+5 treatment, supporting potential toxicity of V+5 at low levels. Taken together, the present study shows that V+5 has adverse effects on mitochondria and the metabolome which may result in EMT activation in the absence of cell death. Furthermore, results suggest that high-resolution metabolomics could serve as a powerful tool to investigate metal toxicity at levels which do not cause cell death.

ATR signaling controls the bystander responses of human chondrosarcoma cells by promoting RAD51-dependent DNA repair

Purpose Radiation-induced bystander effect (RIBE) frequently is seen as DNA damage in unirradiated bystander cells, but the repair processes initiated in response to that DNA damage are not well understood. RIBE-mediated formation of micronuclei (MN), a biomarker of persistent DNA damage, was previously observed in bystander normal fibroblast (AG01522) cells, but not in bystander human chondrosarcoma (HTB94) cells. The molecular mechanisms causing this disparity are not clear. Herein, we investigate the role of DNA repair in the bystander responses of the two cell lines. Methods Cells were irradiated with X-rays and immediately co-cultured with un-irradiated cells using a trans-well insert system in which they share the same medium. The activation of DNA damage response (DDR) proteins was detected by immunofluorescence staining or Western blotting. MN formation was examined by the cytokinesis-block MN assay, which is a robust method to detect persistent DNA damage. Results Immunofluorescent foci of γH2AX and 53BP1, biomarkers of DNA damage and repair, revealed a greater capacity for DNA repair in HTB94 cells than in AG01522 cells in both irradiated and bystander populations. Autophosphorylation of ATR at the threonine 1989 site was expressed at a greater level in HTB94 cells compared to AG01522 cells at the baseline and in response to hydroxyurea treatment or exposure to 1 Gy of X-rays. An inhibitor of ATR, but not of ATM, promoted MN formation in bystander HTB94 cells. In contrast, no effect of either inhibitor was observed in bystander AG01522 cells, indicating that ATR signaling might be a pivotal pathway to preventing the MN formation in bystander HTB94 cells. Supporting this idea, we found an ATR-dependent increase in the fractions of bystander HTB94 cells with pRPA2 S33 and RAD51 foci. A blocker of RAD51 facilitated MN formation in bystander HTB94 cells. Conclusion Our results indicate that HTB94 cells were likely more efficient in DNA repair than AG01522 cells, specifically via ATR signaling, which inhibited the bystander signal-induced MN formation. This study highlights the significance of DNA repair efficiency in bystander cell responses.

Involvement of IL-17 A/IL-17 Receptor A with Neutrophil Recruitment and the Severity of Coronary Arteritis in Kawasaki Disease

PurposeTo assess the role of the interleukin (IL)-17 A/IL-17 receptor A (IL-17RA) in Kawasaki disease (KD)-related coronary arteritis (CA).MethodsIn human study, the plasma levels of IL-17 A and coronary arteries were concurrently examined in acute KD patients. In vitro responses of human coronary endothelial cells to plasma stimulation were investigated with and without IL-17RA neutralization. A murine model of Lactobacillus casei cell-wall extract (LCWE)-induced CA using wild-type Balb/c and Il17ra-deficient mice were also inspected.ResultsThe plasma levels of IL-17 A were significantly higher in KD patients before intravenous immunoglobulin therapy, especially in those with coronary artery lesion. The pre-IVIG IL-17 A levels positively correlated with maximal z scores of coronary diameters and plasma-induced endothelial mRNA levels of chemokine (C-X-C motif) ligand-1, IL-8, and IL-17RA. IL-17RA blockade significantly reduced such endothelial upregulations of aforementioned three genes and inducible nitric oxide synthase, and neutrophil transmigration. IL-17RA expression was enhanced on peripheral blood mononuclear cells in pre-IVIG KD patients, and in the aortic rings and spleens of the LCWE-stimulated mice. LCWE-induced CA composed of dual-positive Ly6G- and IL-17 A-stained infiltrates. Il17ra-deficient mice showed reduced CA severity with the fewer number of neutrophils and lower early inducible nitric oxide synthase and chemokine (C-X-C motif) ligand-1 mRNA expressions than Il17ra+/+ littermates, and absent IL-17RA upregulation at aortic roots.ConclusionIL-17 A/IL-17RA axis may play a role in mediating aortic neutrophil chemoattraction, thus contributory to the severity of CA in both humans and mice. These findings may help to develop a new therapeutic strategy toward ameliorating KD-related CA.

Enhancing the angiogenesis and osteogenesis of low elastic modulus Ti-Nb-Zr-Sn alloy surface through grafting growth factors-containing gelatin nanoparticles

The study aimed to apply a multiple surface modification for low elastic modulus Ti-24 % Nb-4 % Zr-8 % Sn (Ti2448) alloy, to enhance its biological properties for bone implant applications. We employed a combined sandblasting/acid etching/alkaline etching process to modify the Ti2448 alloy surfaces with mixed nano/submicron/micron-scaled pits and super wettability. We prepared gelatin nanoparticles (GNPs) loaded with growth factors: vascular endothelial growth factor/platelet-derived growth factor (VEGF/PDGF) and/or bone morphogenetic protein-2 (BMP-2). The growth factors-containing GNPs were then sequentially grafted onto the surface-modified Ti2448 alloy in a layered (single layer: BMP-2 or VEGF/PDGF; double layer: inner BMP-2 and outer VEGF/PDGF) or mixed BMP-2/VEGF/PDGF manner. Surface characterizations, including morphology, roughness, surface energy, and cytotoxicity, of Ti2448 alloy were analyzed. For biological response, we investigated the tube formation ability of human umbilical vein endothelial cells (HUVECs) and the cell responses (adhesion, proliferation, and mineralization) of human bone marrow mesenchymal stem cells (hMSCs). The results demonstrated the successful preparation of GNPs with 200∼300 nm particle size and their successful loading with the growth factors using natural crosslinker genipin. The growth factor-containing GNPs were effectively grafted onto the surface-modified hydrophilic Ti2448 alloy without significantly reducing its wettability (water contact angle remained below 40°). The results of the study showed that the GNPs-grafted Ti2448 alloy surfaces, with the GNPs loaded with VEGF/PDGF and/or BMP-2, exhibited enhanced biological properties. The GNPs-grafted surfaces were found to be non-toxic and capable of promoting tube formation of HUVECs, particularly when single VEGF/PDGF layer or double (inner BMP-2/ outer VEGF/PDGF) layer was loaded on GNPs (enhancing the cumulative tube length approximately 1.4 times that of no GNPs-grafted surfaces). Moreover, the GNPs-grafted surfaces enhanced the adhesion-related proteins and extracellular matrix mineralization of hMSCs, particularly with the loading of single VEGF/PDGF or mixed VEGF/PDGF/BMP-2 on GNPs (enhancing αvβ3 integrin patch number > 3.5 times, vinculin patch number > 2 times, and mineralization approximately 1.4 times). Based on these findings, we concluded that the proposed Ti2448 alloy surfaces, when grafted with VEGF/PDGF- and/or BMP-2-containing GNPs, have the potential for bone implant applications. The surface modifications on the Ti2448 alloy may promote angiogenesis in the early stage of bone repair (through VEGF/PDGF) and osteogenesis in the late stage (through BMP-2), thereby accelerating the overall bone healing process.

Interferons are key cytokines acting on pancreatic islets in type 1 diabetes.

The proinflammatory cytokines IFN-α, IFN-γ, IL-1β and TNF-α may contribute to innate and adaptive immune responses during insulitis in type 1 diabetes and therefore represent attractive therapeutic targets to protect beta cells. However, the specific role of each of these cytokines individually on pancreatic beta cells remains unknown. We used deep RNA-seq analysis, followed by extensive confirmation experiments based on reverse transcription-quantitative PCR (RT-qPCR), western blot, histology and use of siRNAs, to characterise the response of human pancreatic beta cells to each cytokine individually and compared the signatures obtained with those present in islets of individuals affected by type 1 diabetes. IFN-α and IFN-γ had a greater impact on the beta cell transcriptome when compared with IL-1β and TNF-α. The IFN-induced gene signatures have a strong correlation with those observed in beta cells from individuals with type 1 diabetes, and the level of expression of specific IFN-stimulated genes is positively correlated with proteins present in islets of these individuals, regulating beta cell responses to 'danger signals' such as viral infections. Zinc finger NFX1-type containing 1 (ZNFX1), a double-stranded RNA sensor, was identified as highly induced by IFNs and shown to play a key role in the antiviral response in beta cells. These data suggest that IFN-α and IFN-γ are key cytokines at the islet level in human type 1 diabetes, contributing to the triggering and amplification of autoimmunity.

SIRT6-mediated vascular smooth muscle cells senescence participates in the pathogenesis of abdominal aortic aneurysm

Background and aimsIn this study, we carried out a clinical sample study, and in vivo and in vitro studies to evaluate the effect of SIRT6 and SIRT6-mediated vascular smooth muscle senescence on the development of abdominal aortic aneurysm (AAA). Method and ResultsAAA specimen showed an increased P16, P21 level and a decreased SIRT6 level compared with control aorta. Time curve study of Ang II infusion AAA model showed similar P16, P21 and SIRT6 changes at the early phase of AAA induction. The in vivo overexpression of SIRT6 significantly prevented AAA formation in Ang II infusion model. The expression of P16 and P21 was significantly reduced after SIRT6 overexpression. SIRT6 overexpression also attenuated chronic inflammation and neo-angiogenesis in Ang II infusion model. The overexpression of SIRT6 could attenuate premature senescence, inflammatory response and neo-angiogenesis in human aortic smooth muscle cells (HASMC) under Ang II stimulation. ConclusionsSIRT6 overexpression could limit AAA formation via attenuation of vascular smooth muscle senescence, chronic inflammation and neovascularity.

Dimethyloxallyl glycine-loaded mesoporous bioactive glass/poly(D,L-lactide) composite scaffolds with ultrasound stimulation for promoting bone repair.

Introduction: Bone tissue engineering is considered the ideal approach for bone repair. Mesoporous bioactive glass (MBG) possesses the characteristics of high drug-loading capacity and bioactivity. Low-intensity pulsed ultrasound contributes to promoting fracture healing and bone defect repair, and dimethyloxalyl glycine (DMOG) is a small molecular inhibitor that can suppress prolyl hydroxylase, reducing the degradation of hypoxia-inducible factor. Methods: In this study, we proposed to prepare DMOG-loaded MBG/poly(D,L-lactide) composite scaffolds (DMOG-MBG/PDLLA) for promoting bone repair. The effects of ultrasound stimulation and DMOG release on the cell responses of rat bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) and bone repair in vivo were investigated. Results and Discussion: The results showed that both ultrasound stimulation and DMOG release could promote the proliferation, adhesion and differentiation of BMSCs and HUVECs, respectively. After the implantation of scaffolds in rat cranial bone defect model for 8 weeks, the results indicated that the combined ultrasound stimulation and DMOG release contributed to the highest ability for promoting bone repair. Hence, the DMOG-MBG/PDLLA scaffolds with ultrasound stimulation are promising for application in bone repair.

A numerical study of a single isolated human ventricular cell response to a periodic pulse stimulus current using the Rogers–McCulloch model

Among the primary causes of cardiac arrhythmias are abnormalities in the generation of action potentials (APs) by the cardiac cells, which may be induced by alterations in the electrophysiological properties of the cells as well as the stimuli delivered to them. Through numerical simulations using the phenomenological model of Rogers–McCulloch, this study examined how external stimuli modulate the electrical activity of a single isolated human ventricular cell functioning under normal conditions. First, we investigated how the initial conditions of the two model state variables affect how the cell responds to a single rectangular current pulse. This allows for the determination of a critical curve, which separates the initial conditions that allow for AP activation from those that inhibit it. This critical curve can be used to predict AP activation not only when the cell is stimulated by a single current pulse but also by a periodic stimulus. Then, we examined the cell response to a periodic train of rectangular current pulses, with a focus on the effects of the stimulus pacing length and amplitude. Our findings demonstrated that, despite not considering any alteration in the dynamics of ionic currents through the cell membrane, the Rogers–McCulloch model was capable of reproducing some of the most common activation patterns that have been widely identified in several clinical, experimental, and numerical studies. Depending on the stimulus properties, the following activation patterns were identified: (1) AP activation in synchrony with the stimulus, with one AP activation per pulse; (2) quasi-periodic and periodic Wenckebach-like patterns, in which successive AP activations are interrupted by a skipped beat; (3) completely irregular patterns; (4) abnormal potential oscillations that interrupt the repolarization of a previously activated AP, followed or not by a skipped beat; and (5) abnormal potential oscillations that completely inhibit cell repolarization.

MicroRNA-320a enhances LRWD1 expression through the AGO2/FXR1-dependent pathway to affect cell behaviors and the oxidative stress response in human testicular embryonic carcinoma cells.

Testicular cancer is fairly rare but can affect fertility in adult males. Leucine-rich repeats- and WD repeat domain-containing protein 1 (LRWD1) is a sperm-specific marker that mainly affects sperm motility in reproduction. Our previous study demonstrated the impact of LRWD1 on testicular cancer development; however, the underlying mechanisms remain unclear. In this study, various plasmids associated with LRWD1 and miR-320a manipulation were used to explore the roles and regulatory effects of these molecules in NT2D1 cellular processes. A Dual-Glo luciferin-luciferase system was used to investigate LRWD1 transcriptional activity, and qRT-PCR and western blotting were used to determine gene and protein expression. The results suggested that miR-320a positively regulated LRWD1 and positively correlated with NT2D1 cell proliferation but negatively correlated with cell migration and invasion ability. In addition, the miRNA-ribonucleoprotein complex AGO2/FXR1 was shown to be essential in the mechanism by which miR-320a regulates LRWD1 mRNA expression. As miR-320a was required to regulate LRWD1 expression through the AGO2 and FXR1 complex, eEF2 and eLF4E were also found to be involved in miR-320a increasing LRWD1 expression. Furthermore, miR-320a and LRWD1 were responsive to oxidative stress, and NRF2 was affected by the presence of miR-320a in response to ROS stimulation. This is the first study showing the role of miR-320a in upregulating the testicular cancer-specific regulator LRWD1 and the importance of the AGO2/FXR1 complex in miR-320a-mediated upregulation of LRWD1 during testicular cancer progression.

Role for CCN1 in lysophosphatidic acid response in PC-3 human prostate cancer cells.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are bioactive phospholipids that act as mitogens in various cancers. Both LPA and S1P activate G-protein coupled receptors (GPCRs). We examined the role of CCN1/CYR61, an inducible matricellular protein, in LPA-induced signal transduction in PC-3 human prostate cancer cells. We found that both LPA and S1P induced expression of CCN1 and CCN2 within 2-4h. CCN1 was induced by 18:1-LPA, but not by 18:0-, 18:2-, or 18:3-LPAs. A free fatty acid receptor-4 agonist inhibited LPA-induced CCN1 induction. CCN1 appeared in the ECM within 2h after LPA addition. LPA caused biphasic activation of Erk MAPK, with an initial peak at 10-20min followed by a later phase after 6h. LPA increased adhesion of PC-3 cells to culture substrates (standard culture plates, fibronectin, or extracellular matrix) at 2h, an intermediate event between early and late LPA signals. Knockdown of CCN1 suppressed LPA-induced adhesion to ECM or fibronectin. ECM from CCN1 knockdown cells was a poor substrate for adhesion, as compared to ECM from control cells. These results suggest that CCN1 contributes to LPA responses in the tumor microenvironment. The LPA-CCN1 axis holds promise for the development of novel therapeutic strategies in cancer.

Exploring the therapeutic potential of Rab11: A comprehensive study on its effectiveness in alleviating rotenone-induced molecular pathogenesis of Parkinson's disease in SH-SY5Y cells and its synergistic application with L-DOPA in Drosophila models

Dysfunctional mitophagy contributes to Parkinson's disease (PD) by affecting dopamine-producing neurons. Mutations in parkin and pink1 genes, linked to familial PD, impede the removal of damaged mitochondria. Previous studies suggested Rab11's involvement in mitophagy alongside Parkin and Pink1. Additionally, mitochondria-endoplasmic reticulum contact sites (MERCS) regulate cellular functions, including mitochondrial quality control and calcium regulation. Our study explored whether activating mitophagy triggers the unfolded protein response and ER stress pathway in SH-SY5Y human cells. We induced a PD-like state by exposing undifferentiated SH-SY5Y cells to rotenone, an established PD-inducing agent. This led to reduced Rab11 and PERK- expression while increasing ATP5a, a mitochondrial marker, when Rab11 was overexpressed. Our findings suggest that enhancing endosomal trafficking can mitigate ER stress by regulating mitochondria, rescuing cells from apoptosis. Furthermore, we assessed the therapeutic potential of Rab11, both alone and in combination with L-Dopa, in a Drosophila PD model. In summary, our research underscores the role of mitophagy dysfunction in PD pathogenesis, highlighting Rab11's importance in alleviating ER stress and preserving mitochondrial function. It also provides insights into potential PD management strategies, including the synergistic use of Rab11 and L-Dopa.

Ginger-derived bioactive compounds attenuate the Toll-like receptor mediated responses of human dendritic cells

Ginger has been used for thousands of years for the treatment of many illnesses, from nausea to migraines. Recently, an interest has grown in ginger compounds in the context of autoimmune and inflammatory diseases due to their significant anti-inflammatory effects. Nevertheless, the effects and mechanism of action of these phytochemicals in human immune cells, particularly in dendritic cells (DCs) are unclear. In the present study, we investigated the effects of 6-gingerol and 6-shogaol, the major compounds found in ginger rhizome, on the functionality of primary human monocyte-derived DCs (moDCs). Here we report for the first time that 6-gingerol and 6-shogaol dampen the immunogenicity of human DCs by inhibiting their activation, cytokine production and T cell stimulatory ability. In particular, the bioactive compounds of ginger dose-dependently inhibited the upregulation of activation markers, and the production of different cytokines in response to synthetic Toll-like receptor (TLR) ligands. Moreover, both compounds could significantly reduce the Escherichia coli-triggered cytokine production and T cell stimulatory capacity of moDCs. We also provide evidence that the ginger-derived compounds attenuate DC functionality via inhibiting the nuclear factor-κB (NF-kB), mitogen activated protein kinase (MAPK), and mammalian target of rapamycin (mTOR) signaling cascades. Further, 6-shogaol but not 6-gingerol activates the AMP-activated protein kinase (AMPK) and nuclear factor erythroid 2-related factor 2 (NRF2) pathways that might contribute to its anti-inflammatory action. Altogether, our results indicate that ginger-derived phytochemicals exert their anti-inflammatory activities via multiple mechanisms and suggest that 6-shogaol is more potent in its ability to suppress DC functionality than 6-gingerol.

Diverse modulatory effects of bibenzyls from Dendrobium species on human immune cell responses under inflammatory conditions.

Dendrobium plants are widely used in traditional Chinese medicine. Their secondary metabolites such as bibenzyls and phenanthrenes show various pharmacological benefits such as immunomodulation and inhibitory effects on cancer cell growth. However, our previous study also showed that some of these promising compounds (i.e., gigantol and cypripedin) also induced the expression of inflammatory cytokines including TNF in human monocytes, and thus raising concerns about the use of these compounds in clinical application. Furthermore, the effects of these compounds on other immune cell populations, apart from monocytes, remain to be investigated. In this study, we evaluated immunomodulatory effects of seven known bibenzyl compounds purified from Dendrobium species in human peripheral blood mononuclear cells (PBMCs) that were stimulated with lipopolysaccharide (LPS). Firstly, using flow cytometry, moscatilin (3) and crepidatin (4) showed the most promising dose-dependent immunomodulatory effects among all seven bibenzyls, determined by significant reduction of TNF expression in LPS-stimulated CD14+ monocytes. Only crepidatin at the concentration of 20 μM showed a significant cytotoxicity, i.e., an increased cell death in late apoptotic state. In addition, deep immune profiling using high-dimensional single-cell mass cytometry (CyTOF) revealed broad effects of Dendrobium compounds on diverse immune cell types. Our findings suggest that to precisely evaluate therapeutic as well as adverse effects of active natural compounds, a multi-parameter immune profiling targeting diverse immune cell population is required.

Source differences in the components and cytotoxicity of PM2.5 from automobile exhaust, coal combustion, and biomass burning contributing to urban aerosol toxicity

Abstract. Although air quality guidelines generally use the atmospheric concentration of fine particulate matter (PM2.5) as a metric for air pollution evaluation and management, the fact cannot be ignored that different particle toxicities are unequal and significantly related to their sources and chemical compositions. Therefore, judging the most harmful source and identifying the toxic component would be helpful for optimizing air quality standards and prioritizing targeted PM2.5 control strategies to protect public health more effectively. Since the combustions of fuels, including oil, coal, and biomass, are the main anthropogenic sources of environmental PM2.5, their discrepant contributions to health risks of mixed ambient aerosol pollution dominated by the respective emission intensity and unequal toxicity of chemical components need to be identified. In order to quantify the differences between these combustion primary emissions, 10 types of PM2.5 from each typical source group, i.e., vehicle exhaust, coal combustion, and plant biomass (domestic biofuel) burning, were collected for comparative study with toxicological mechanisms. In total, 30 types of individual combustion samples were intercompared with representative urban ambient air PM2.5 samples, whose chemical characteristics and biological effects were investigated by component analysis (carbon, metals, soluble ions) and in vitro toxicity assays (cell viability, oxidative stress, inflammatory response) of human lung adenocarcinoma epithelial cells (A549). Carbonaceous fractions were plenteous in automobile exhaust and biomass burning, while heavy metals were more plentiful in PM2.5 from coal combustion and automobile exhaust. The overall ranking of mass-normalized cytotoxicity for source-specific PM2.5 was automobile exhaust > coal combustion > domestic plant biomass burning > ambient urban air, possibly with differential toxicity triggers, and showed that the carbonaceous fractions (organic carbon, OC; elemental carbon, EC) and redox-active transition metals (V, Ni, Cr) assisted by water-soluble ions (Ca2+, Mg2+, F−, Cl−) might play important roles in inducing cellular reactive organic species (ROS) production, causing oxidative stress and inflammation, resulting in cell injury and apoptosis, and thus damaging human health. Coupled with the source apportionment results of typical urban ambient air PM2.5 in eastern China, reducing toxic PM2.5 from these anthropogenic combustions will be greatly beneficial to public health. In addition to the air pollution control measures that have been implemented, like strengthening the vehicle emission standards, switching energy from coal to gas and electricity, and controlling the open incineration of agricultural straws, further methods could be considered, especially by preferentially reducing the diesel exhaust, lessening the coal combustion by replacement with low-ash clean coals, and depressing the rural crop straw biomass burning emissions.

P150 Ulcerative Colitis Patients’ Luminal Zinc Induces Clostridioides difficile Virulence and Exacerbates Inflammatory Responses

Abstract Background The refractory acute severe UC patients are often complicated by C. difficile infection (CDI), resulting in higher surgery and mortality rates. However, few studies have observed C. difficile’s pathogenicity in real UC patients’ gastrointestinal microenvironments, and the actual role of C. difficile in UC development remains unknown. We found zinc concentrations among UC patients’ feces were significantly higher than those among Crohn’s disease patients and healthy subjects. Additionally, we previously reported that excessive zinc would promote the growth and virulence of C. difficile. Therefore, we hypothesized that the increased UC patients’ luminal zinc may up-regulate C. difficile virulence and promote intestinal inflammatory responses, leading to an exacerbated UC progression. Methods UC patients’ stool samples (n=40) were collected and analyzed by qPCR and ICP-MS to determine the presence of C. difficile and the fecal zinc concentrations. C. difficile was cultured in BHIs with or without zinc (ZnSO4) in an anaerobic chamber. Expression of virulence genes was analyzed using RT-qPCR, transcriptome sequencing, and western blot. Human colonic organoids and Caco-2 cells were co-cultured with C. difficile or zinc-treated C. difficile, and cytotoxicity, permeability, and inflammatory cytokine release were assessed. TLR5 knock-out Caco-2 cells were constructed using sgRNA-guided Cas9 nuclease and co-cultured with C. difficile or zinc-treated C. difficile to verify whether zinc-induced C. difficile’s pathogenicity was mainly through flagellar formation. Results C. difficile was detected in ~35% of UC patients’ fecal samples. Luminal zinc concentrations (~915μM, p<0.01) were significantly higher among UC patients than CD patients or healthy subjects. The UC patients’ luminal concentration of zinc promoted C. difficile growth, suppressed its exotoxins release, and induced higher expression of its flagella-related genes, resulting in higher cytotoxicity and inflammatory responses in human colonic organoids and Caco-2 cells. However, zinc-treated C. difficile did not induce higher inflammation and pathogenicity in TLR5 knock-out Caco-2 cells. Conclusion The association between C. difficile and UC aggravation may partially stem from UC patients’ excess luminal zinc promoting C. difficile flagellin expression, inducing inflammatory responses, and worsening UC progression.

Human Coronary Artery Endothelial Cell Response to Porphyromonas gingivalis W83 in a Collagen Three-Dimensional Culture Model.

P. gingivalis has been reported to be an endothelial cell inflammatory response inducer that can lead to endothelial dysfunction processes related to atherosclerosis; however, these studies have been carried out in vitro in cell culture models on two-dimensional (2D) plastic surfaces that do not simulate the natural environment where pathology develops. This work aimed to evaluate the pro-inflammatory response of human coronary artery endothelial cells (HCAECs) to P. gingivalis in a 3D cell culture model compared with a 2D cell culture. HCAECs were cultured for 7 days on type I collagen matrices in both cultures and were stimulated at an MOI of 1 or 100 with live P. gingivalis W83 for 24 h. The expression of the genes COX-2, eNOS, and vWF and the levels of the pro-inflammatory cytokines thromboxane A2 (TXA-2) and prostaglandin I2 (PGI2) were evaluated. P. gingivalis W83 in the 2D cell culture increased IL-8 levels at MOI 100 and decreased MCP-1 levels at both MOI 100 and MOI 1. In contrast, the 3D cell culture induced an increased gene expression of COX-2 at both MOIs and reduced MCP-1 levels at MOI 100, whereas the gene expression of eNOS, vWF, and IL-8 and the levels of TXA2 and PGI2 showed no significant changes. These data suggest that in the collagen 3D culture model, P. gingivalis W83 induces a weak endothelial inflammatory response.