TNF-α Stimulation Research Articles

Artificial Tertiary Lymphoid Structures: Exploring Mesenchymal Stromal Cells as a Platform for Immune Niche Formation

Constructing artificial tertiary lymphoid structures (TLSs) opens new avenues for advancing cancer immunotherapy and personalized medicine by creating controllable immune niches. Mesenchymal stromal cells (MSCs) offer an ideal stromal source for such constructs, given their potent immunomodulatory abilities and accessibility. In this study, we explored the potential of adipose-derived MSCs to adopt TLS-supportive phenotypes and facilitate lymphocyte organization. Single-cell RNA sequencing revealed a distinct subpopulation of MSCs expressing key fibroblastic reticular cell (FRC)-associated markers, including IL-7, PDPN, and IL-15, though lacking follicular dendritic cell (FDC) markers. TNF-α stimulation, but not LTα2β1, further enhanced FRC marker expression (IL-7, PDPN, and ICAM1). Notably, in 3D spheroid co-culture with lymphocytes, MSCs upregulated additional FRC markers, specifically CCL21. Upon implantation into adipose tissue, MSC-lymphocyte organoids maintained structural integrity and showed extensive T-cell infiltration and partial vascularization after 15 days in vivo, although organized B-cell follicles and FDC markers were still lacking. These findings highlight MSCs’ intrinsic ability to adopt an FRC-like phenotype that supports T-cell and HEV organization, suggesting that further optimization, including genetic modification, may be needed to achieve an FDC phenotype and replicate the full architectural and functional complexity of TLSs.

Decellularized adipose matrix hydrogel-based in situ delivery of antagomiR-150-5p for rat abdominal aortic aneurysm therapy

Abdominal aortic aneurysm (AAA) is a progressive aortic disease featured by inflammation, vascular smooth muscle cells (VSMCs) depletion, and elastin degradation. MicroRNAs were related to AAA formation, which bring the approach for precise and targeted drug therapy for AAA. We developed a new strategy based on decellularized adipose matrix (DAM) hydrogel immobilized on the adventitia to release antagomiR-150-5p for preventing the AAA development. In this study, Cacl2-induced and elastase-induced rat AAA models were established. We found that miR-150-5p was upregulated while Notch3 was downregulated in two rat AAA models. Then a mold was designed for shaping hydrogel for miR-150-5p delivery around the abdominal aorta. Interestingly, inhibition of miR-150-5p in AAA by local release of antagomiR-150-5p with DAM hydrogel significantly prevented aortic dilation and elastin degradation. Moreover, inflammatory cell infiltration, the expression of inflammatory cytokines (MCP-1, TNF-α, and NF-κB (p65)), and matrix metalloproteinases (MMP-2, MMP-9) were increased while Notch3 and α-SMA were decreased in rat AAA, which can be attenuated by antagomiR-150-5p treatment. In VSMCs with TNF-α stimulation, we further demonstrated that inhibition of miR-150-5p downregulated NF-κB (p65), MMP-2, and MMP-9 and upregulated elastin via Notch3. This work presents a translational potential strategy for AAA repair via DAM hydrogel sustained release of antagomiR-150-5p, and highlights the mechanism of miR-150-5p during AAA progression by regulating Notch3.

Adipose stem cell exosomes, stimulated by pro-inflammatory factors, enhance immune evasion in triple-negative breast cancer by modulating the HDAC6/STAT3/PD-L1 pathway through the transporter UCHL1

BackgroundTriple-negative breast cancer (TNBC) is characterized by high invasiveness and metastasis potential. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is strongly associated with breast cancer progression, although the underlying mechanisms are largely unknown.MethodsThe gene expression profiles of TNBC samples were downloaded from the TCGA database, and ubiquitination enzymes related to immune regulation were screened. UCHL1 expression in the TNBC tissues and in adipose-derived mesenchymal stem cells (ADSCs) stimulated in vitro with pro-inflammatory cytokines were analyzed. Exosomes were isolated from these stimulated ADSCs and transfected with scrambled (si-NC) or UCHL1-specific (si-UCHL1) siRNA constructs. TNBC cells were treated with the ADSCs-derived exosomes (ADSCs-Exos) and then co-cultured with macrophages or T cells. Finally, the tumorigenic potential of the ADSCs-Exos was evaluated by injecting the exosomes into mice bearing TNBC xenografts.ResultsUCHL1 was highly expressed in TNBC tissues and the stimulated ADSCs. The exosomes derived from stimulated ADSCs increased the viability and migration capacity of TNBC cells in vitro, and significantly increased Ki-67 expression through UCHL1. Furthermore, ADSCs-Exos induced M2 polarization of THP-1 monocytes by upregulating CD206 and Arg-1, and downregulating TNF-α and iNOS, and also decreased the proportion of CD3+CD8+ T cells. Mechanistically, UCHL1 regulated the STAT3 and PD-L1 signaling pathways through HDAC6. Exosomes derived from the control and cytokine-stimulated ADSCs also promoted tumor growth in vivo, and increased the expression of UCHL1, CD206, HDAC6, STAT3, and PD-L1. However, UCHL1 knockdown reversed the pro-tumorigenic effects of the ADSCs-derived exosomes in vivo and in vitro.ConclusionPro-inflammatory factors (IFN-γ + TNF-α) stimulating ADSCs-Exos enhance immune evasion in triple-negative breast cancer by regulating the HDAC6/STAT3/PD-L1 pathway via UCHL1 transporter. Thus, UCHL1 inhibition may enhance the response of TNBC to immunotherapy.Graphical

Abstract 4134539: Hybrid Cardiomyocyte Targeting Peptide Down-regulates NF-κB Pathway in Human Cardiomyocytes

Introduction: Role of NF-κB driven inflammation leading to heart failure with preserved ejection fraction has received little attention. Our previous in vivo work with cardiac targeting peptide (CTP) in guinea pigs identified suppression of NF- κB pathway by CTP. In the current work we sought to further study the effects of a hybrid version of CTP (hCTP: amino acids at positions 3 and 11 substituted with D-amino acids) on TNF-α mediated NF-κB activation, as well as expression of its downstream inflammatory markers IL-1β and IL-8. Methods: Human cardiomyocytes (CMCs) were transfected using Lipofectamine 3000 with a reporter luciferase plasmid carrying an NF-κB promoter site upstream of the luciferase gene. Cells were treated with various concentrations of hCTP for 24 hours, followed by a TNF-α challenge (20ng/mL), addition of luciferin as substrate, and measurement of luminescence. Cell viability in response to above manipulations was assessed by FACS using live-dead stain. CMCs were treated as above, and cell culture supernatants analyzed for IL-8 levels using human ELISA kit. In another set of experiments, CMCs were transfected with NF-κB reporter plasmid, and after treatment for 24 hours with various concentrations of hCTP, incubated with 10nM angiotensin 2 (Ang2) and 100nM phenylephrine (PE) for 24hrs to assess levels of IL-1β in the cell culture media using an ELISA assay. Results: hCTP inhibited TNF-α mediated NF-κB activation with a dose-response curve, with maximum inhibition with hCTP seen at 10µM concentrations (p<0.001). Treatment with hCTP inhibited the TNF-α induced IL-8 secretion with significant decreases seen even at the 1µM dose. Similarly, secretion of IL-1β production by CMCs in response to Ang2/PE stimulation was suppressed by hCTP even at the lowest tested dose (1µM; p<0.5). Conclusions: Our data suggests that hCTP inhibited NF-κB pathway transcriptional activity in response to TNF-α stimulation in a dose-dependent fashion in human CMCs. Furthermore, treatment with hCTP inhibited TNF-α induced IL-8 and IL-1β secretion even at doses as low as 1µM. The ability of hCTP to efficiently targeting this inflammatory pathway suggests that it has therapeutic potential in the treatment of heart failure. Further in vivo studies are ongoing to test this hypothesis.

Epigenetic BET inhibitor apabetalone counters inflammatory and fibrotic processes in activated cardiac fibroblasts providing insight into reduced hospitalizations for heart failure in BETonMACE trial

Abstract Background After myocardial infarction (MI), sustained crosstalk between monocytes and cardiac fibroblasts (CFs) promotes chronic inflammation and interstitial fibrosis that can contribute to heart failure (HF). Resident and myocardium-infiltrating immune cells produce proinflammatory cytokines IL-1β and TNFα that stimulate CFs to produce monocyte chemoattractants. Monocytes secrete TGF-β1 that transforms CFs into contractile myofibroblasts overproducing the extracellular matrix (ECM) responsible for cardiac fibrosis. Epigenetic BET inhibitors (BETi) reduce CF transdifferentiation to prevent cardiac fibrosis and HF in animal models. In patients with cardiovascular disease, type 2 diabetes and recent MI, administration of the BETi apabetalone (APA) resulted in less hospitalization due to HF vs. placebo (BETonMACE phase 3 trial; hazard ratio 0.59, p=0.03). Purpose To examine APA’s in vitro effects on inflammatory and profibrotic processes in CFs associated with cardiac fibrosis and HF. Methods Immortalized and primary human CFs were stimulated with cytokines (10ng/mL IL-1β, TNFα, TGF-β1) or with conditioned media from 10ng/mL IFNγ and 100ng/mL lipopolysaccharide-treated THP-1 macrophages ± 5μM APA on plastic or in 3D collagen gels. Gene expression was analyzed by PCR, protein levels by FACS or ELISA, collagen deposition with picrosirius red, THP-1 monocyte migration in Boyden chambers, and CF contraction with 3D collagen gels. Results IL-1β or TNFα stimulation of CFs upregulated monocyte chemoattractant 1 (CCL2) and vascular cell adhesion protein 1 (VCAM1) expression, promoting THP-1 cell migration and adhesion to CFs. APA treatment reduced cytokine-induced CCL2 (>20%) and VCAM1 (>70%) gene expression as well as THP-1 cell migration and adhesion to stimulated CFs (>60%). TGF-β1 treatment induced CF transdifferentiation into myofibroblasts as shown by increased expression of α smooth muscle actin (α-SMA) protein, and secretion of ECM proteins fibronectin and periostin. APA treatment reduced myofibroblast mRNA and protein expression of α-SMA (~30%), fibronectin (~30%) and periostin (97%). Myofibroblast-mediated collagen deposition was also reduced by APA by ~20%. α-SMA-dependent CF contraction can be visualized in attached 3D collagen gels. TGF-β1, IL-1β, TNFα or macrophage-conditioned media promoted CF-mediated gel contraction by 2.5, 1.4, 2.1, or 1.8-fold, respectively. APA treatment countered gel contraction by 30-60%, thereby reducing the myofibroblast-like behaviour in organotypic cultures. Conclusions In vitro, APA treatment reduced proinflammatory crosstalk between monocytes and CFs, resulting in less monocyte migration, monocyte - CF adhesion and CF contraction. APA also reduced signaling by TGF-β1, thus reducing profibrotic and contractile behaviour of CFs responsible for cardiac fibrosis. Since fibrosis contributes to HF, this data provides insight into the observed reduction in hospitalization due to HF in the BETonMACE trial.

Inflammatory profile of eosinophils in asthma-COPD overlap and eosinophilic COPD: a multi-omics study.

Elevated blood eosinophil levels in patients with chronic obstructive pulmonary disease (COPD) with or without asthma are linked to increased exacerbations and the effectiveness of inhaled corticosteroid treatment. This study aimed to delineate the inflammatory cellular properties of eosinophils in patients with asthma-COPD overlap (ACO) and eosinophilic COPD (eCOPD). Eosinophils were isolated from the peripheral blood of healthy volunteers, patients with non-eCOPD, and those with ACO/eCOPD. Multi-omics analysis involving transcriptomics, proteomics, and lipidomics was performed, followed by bioinformatic data analyses. In vitro experiments using eosinophils from healthy volunteers were conducted to investigate the molecular mechanisms underlying cellular alterations in eosinophils. Proteomics and transcriptomics analyses revealed cellular characteristics in overall COPD patients represented by viral infection (elevated expression of sterol regulatory element-binding protein-1) and inflammatory responses (elevated levels of IL1 receptor-like 1, Fc epsilon receptor Ig, and transmembrane protein 176B). Cholesterol metabolism enzymes were identified as ACO/eCOPD-related factors. Gene Ontology and pathway enrichment analyses demonstrated the key roles of antiviral responses, cholesterol metabolism, and inflammatory molecules-related signaling pathways in ACO/eCOPD. Lipidomics showed the impaired synthesis of cyclooxygenase-derived mediators including prostaglandin E2 (PGE2) in ACO/eCOPD. In vitro assessment confirmed that IL-33 or TNF-α stimulation combined with IL-5 and IFN-γ stimulation induced cellular signatures in eosinophils in ACO/eCOPD. Atorvastatin, dexamethasone, and PGE2 differentially modulated these inflammatory changes. ACO/eCOPD is associated with viral infection and an inflammatory milieu. Therapeutic strategies using statins and inhaled corticosteroids are recommended to control these pathogenic changes.

Regulation of voltage-gated sodium channels by TNF-α during herpes simplex virus latency establishment.

During lytic or latent infection of sensory neurons with herpes simplex virus type 1 (HSV-1) there are significant changes in the expression of voltage-gated Na+ channels, which may disrupt the transmission of pain information. HSV-1 infection can also evoke the secretion of various pro-inflammatory cytokines, including TNF-α and IL-6. In this work, we hypothesized that TNF-α regulates the expression of Na+ channels during HSV-1 latency establishment in ND7/23 sensory-like neurons. Latency establishment was mimicked by culturing HSV-1 infected ND7/23 cells in the presence of acyclovir (ACV) for 3 days. Changes in the functional expression of voltage-gated Na+ channels were assessed by whole-cell recordings. Our results demonstrate that infection of ND7/23 cells with the HSV-1 strain McKrae with GFP expression (M-GFP) causes a significant decrease in sodium currents during latency establishment. Exposure of ND7/23 cells to TNF-α during latency establishment reverses the effect of HSV-1, resulting in a significant increase in sodium current density. However, Na+ currents were not restored by 3day-treatment with IL-6. There were no changes in the pharmacological and biophysical properties of sodium currents promoted by TNF-α, including sensitivity to tetrodotoxin and the current-voltage relationship. TNF-α stimulation of ND7/23 cells increases p38 signaling. Inhibition of p38 signaling with SB203580 or SB202190 eliminates the stimulatory effect of TNF-α on sodium currents. These results indicate that TNF-α signaling in sensory neurons during latency establishment upregulates the expression of voltage-gated Na+ channels in order to maintain the transmission of pain information.

Construction of self-driving anti-αFR CAR-engineered NK cells based on IFN-γ and TNF-α synergistically induced high expression of CXCL10

IntroductionOvarian cancer is the most malignant gynecological tumor. Previous studies have demonstrated that chimeric antigen receptor (CAR)-engineered NK-92 cells targeting folate receptor α (αFR) (NK-92-αFR-CAR) can specifically kill αFR-positive ovarian cancer cells. However, the migration barrier restricts antitumor effects of CAR-engineered cells. ObjectivesTo elucidate the mechanism by which NK-92-αFR-CAR cells induce the secretion of chemokine CXCL10 during killing ovarian cancer cells. It is speculated that NK-92-αFR-CAR-CXCR3A can target αFR and have chemotaxis of CXCL10, and they may have stronger killing effect of ovarian cancer. MethodsStudy the mechanism of CXCL10 expression strongly induced by TNF-α and IFN-γ combined stimulation in ovarian cancer cells. Construct the fourth generation of NK-92-αFR-CAR-CXCR3A cells, which were co-expressed CXCR3A and αFR-CAR. Evaluate the killing and migration effects of NK-92-αFR-CAR-CXCR3A in vitro and in vivo. ResultsRNA sequencing (RNA-seq) first revealed that the expression level of the chemokine CXCL10 was most significantly increased in ovarian cancer cells co-cultured with NK-92-αFR-CAR. Secondly, cytokine stimulation experiments confirmed that IFN-γ and TNF-α secreted by NK-92-αFR-CAR synergistically induced high CXCL10 expression in ovarian cancer cells. Further signaling pathway experiments showed that IFN-γ and TNF-α enhanced the activation level of the IFN-γ-IFNGR-JAK1/2-STAT1-CXCL10 signaling axis. Cytotoxicity experiments showed that NK-92-αFR-CAR-CXCR3A cells could not only efficiently kill αFR-positive ovarian cancer cells in vitro but also secrete IFN-γ and TNF-α. Higher migration than that of NK-92-αFR-CAR was detected in NK-92-αFR-CAR-CXCR3A using transwell assay. NK-92-αFR-CAR-CXCR3A effectively killed tumor cells in different mouse xenograft models of ovarian cancer and increased infiltration into tumor tissue. ConclusionThis study confirmed that IFN-γ and TNF-α secreted by αFR-CAR-engineered NK cells can synergistically induce high expression of CXCL10 in ovarian cancer cells and constructed self-driving αFR-CAR-engineered NK cells that can break through migration barriers based on CXCL10, which may provide a new therapeutic weapon for ovarian cancer.

VCAM-1 mediates proximal tubule-immune cell cross talk in failed tubule recovery during AKI-to-CKD transition.

Studies in animal models have suggested a linkage between the inflammatory response to injury and subsequent nephron loss during the acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Failure of normal repair during the CKD transition correlates with de novo expression of vascular cell adhesion protein-1 (VCAM-1) by a subset of injured proximal tubule cells. This study identified the role of VCAM-1 expression in promoting the failed repair state. Single-cell transcriptome analysis of patients with AKI and CKD and whole kidney RNA and protein analyses of mouse models of CKD confirmed a marked increase of VCAM-1 expression in the proximal tubules of injured kidneys. In immortalized mouse proximal tubular cells and primary cultured renal cells (PCRCs), VCAM-1 expression was induced by proinflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Analyses of bulk RNA sequencing of TNF-α-treated primary cultured renal cells or pseudo-bulk RNA sequencing of biopsies from Kidney Precision Medicine Project datasets indicated activation of NF-κB and an enrichment of inflammatory response and cell adhesion pathways in VCAM-1-positive cells. Pharmacological inhibition of NF-κB signaling or genetic deletion of myeloid differentiation factor 88 and TIR domain-containing adapter-inducing interferon-β suppressed TNF-α- and IL-1β-induced VCAM-1 expression in vitro. TNF-α stimulation or overexpression of VCAM-1 significantly increased splenocyte adhesion to the mouse proximal tubular monolayer in culture. These results demonstrate that persistence of proinflammatory cytokines after AKI can induce NF-κB-dependent VCAM-1 expression by proximal tubule cells, mediating increased immune cell adhesion to the tubule and thus promoting further tubule injury and greater risk of progression from AKI to CKD.NEW & NOTEWORTHY We demonstrated the induction of VCAM-1 and its biological function in proximal tubules. We found that proinflammatory cytokines (TNF-α and IL-1β) significantly induced VCAM-1 expression via NF-κB signaling pathway. TNF-α treatment or overexpression of VCAM-1 in immortalized MPT cells increased CD45+ splenocyte adhesion. Pharmacological inhibition of NF-κB or genetic deletion of Vcam1 suppressed TNF-α-induced splenocyte adhesion in vitro, suggesting that VCAM-1 mediates proximal tubular-immune cell cross talk in failed tubule recovery during AKI-to-CKD transition.

SPHK2 Knockdown Inhibits the Proliferation and Migration of Fibroblast-Like Synoviocytes Through the IL-17 Signaling Pathway in Osteoarthritis.

Synovial inflammation is vital for the progression of osteoarthritis (OA). The objective of this study was to explore the effects and potential molecular mechanisms of sphingosine kinase 2 (SPHK2) on the proliferation and migration of fibroblast-like synoviocytes (FLS). A TNF-α-stimulated FLS model and a papain-induced OA rat model were constructed. The functions of SPHK2 knockdown in OA were explored by a series of in vivo and in vitro assays.Downstream target genes of SPHK2 were investigated using transcriptome sequencing and validated by reverse transcription quantitative PCR (RT-qPCR). The effects of the SPHK2/IL-17 signaling pathway on inflammation, proliferation, and migration of OA-FLS were investigated using the IL-17 pathway inhibitor (secukinumab) and the activator (rhIL-17A). TNF-α stimulation promoted SPHK2 expression at mRNA and protein levels in OA-FLS. SPHK2 knockdown reduced IL-1β, IL-6, MMP-2, MMP-9, cyclinD1, and PCNA levels and suppressed proliferation and migration of OA-FLS. SPHK2 knockdown alleviated cartilage damage and synovial inflammation in the OA rat model. LRRIQ3, H4C8, CXCL1, CABP4, COL23A1, and PROK2 expression levels were regulated by SPHK2. SPHK2 knockdown inhibited the protein levels of IL-17A, IL-17RA, and Act1. The IL-17 pathway inhibitor secukinumab enhanced the inhibitory effect of SPHK2 knockdown on the proliferation and migration of OA-FLS, while the IL-17 pathway activator rhIL-17A exerted the opposite effect. SPHK2 knockdown inhibits proliferation and migration of OA-FLS by blocking the IL-17 pathway, which provides a novel approach to the OA treatment.

TNFα-Induced Inflammation Model-Evaluation of Concentration and Passage-Dependent Effects on Bovine Chondrocytes.

Inflammation models are widely used in the in vitro investigation of new therapeutic approaches for osteoarthritis. TNFα (tumor necrosis factor alpha) plays an important role in the inflammatory process. Current inflammation models lack uniformity and make comparisons difficult. Therefore, this study aimed to systematically investigate whether the effects of TNFα are concentration-dependent and whether chondrocyte expansion has an effect on the inflammatory model. Bovine chondrocytes were enzymatically isolated, expanded to passages 1-3, and transferred into a 3D pellet culture. Chondrocyte pellets were stimulated with recombinant bovine TNFα at different concentrations for 48 h to induce inflammation. Gene expression of anabolic (collagen 2, aggrecan, cartilage oligomeric protein (COMP)), catabolic (matrix metalloproteinases (MMP3, MMP13)), dedifferentiation (collagen 1) markers, inflammation markers (interleukin-6 (IL-6), nuclear factor kappa B (NFkB), cyclooxygenase-2 (COX), prostaglandin-E-synthase-2 (PTGES2)), and the apoptosis marker caspase 3 was determined. At the protein level, concentrations of IL-6, nitric oxide (NO), and sulfated glycosaminoglycans (GAG) were evaluated. Statistical analysis was performed using the independent t-test, and significance was defined as p < 0.05. In general, TNFα caused a decrease in anabolic markers and an increase in the expression of catabolic and inflammatory markers. There was a concentration-dependent threshold of 10 ng/mL to induce significant inflammatory effects. Most of the markers analyzed showed TNFα concentration-dependent effects (COMP, PRG4, AGN, Col1, MMP3, and NFkB). There was a statistical influence of selected gene expression markers from different passages on the TNFα chondrocyte inflammation model, including Col2, MMP13, IL-6, NFkB, COX2, and PTGES2. Considering the expression of collagen 2 and MMP3, passage 3 chondrocytes showed a higher sensitivity to TNFα stimulation compared to passages 1 and 2. On the other hand, MMP13, IL-6, NFkB, and caspase 3 gene expression were lower in P3 chondrocytes compared to the other passages. On the protein level, inflammatory effects showed a similar pattern, with cytokine effects starting at 10 ng/mL and differences between the passages. TNFα had a detrimental effect on cartilage, with a clear threshold observed at 10 ng/mL. Although TNFα effects showed concentration-dependent patterns, this was not consistent for all markers. The selected passage showed a clear influence, especially on inflammation markers. Further experiments were warranted to explore the effects of TNFα concentration and passage in long-term stimulation.

Swertiamarin ameliorates 2, 4, 6-trinitrobenzenesulfonic acid-induced colitis in mice by inhibiting intestinal epithelial cell apoptosis

To investigate the mechanism by which swertiamarin (STM) ameliorates CD-like colitis in mice. A Caco-2 cell model of TNF-α-stimulated apoptosis was established and divided into three groups: Con, TNF-α and STM, and the effects of STM on apoptosis and barrier function were assessed by Tunel staining, western blotting, immunofluorescence, and transepithelial electric resistance (TEER). A mouse model of 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) -induced CD-like colitis was established to assess the effects of STM on colitis, intestinal barrier function and epithelial cell apoptosis. The regulatory role of the PI3K/AKT pathway in STM-induced resistance to intestinal epithelial cell apoptosis was investigated in both the cell model and mouse models. TUNEL staining showed that in Caco-2 cells with TNF-α stimulation, STM treatment significantly reduced the percentage of TUNEL-stained cells (P<0.05). STM obviously reduced TNF-α-induced enhancement of cleaved-caspase 3 and Bax expressions (P<0.05), increased Bcl-2 expression (P<0.05), protected intestinal barrier integrity and function by restoring transepithelial electrical resistance (TEER) of the cells, promoted normal localization and expressions of the tight junction proteins (ZO1 and claudin 1) (P<0.05), and inhibited the expression of pro-inflammatory factors (IL-6 and CCL3) (P<0.05) in TNF-α-stimulated Caco-2 cells. In the mouse models, STM significantly alleviated TNBS-induced CD-like colitis and intestinal barrier dysfunction (P<0.05) as shown by improved weight loss, lowered Disease Activity Index (DAI) score and inflammation score, reduction of IL-6 and CCL3 release, and restoration of intestinal barrier permeability, colonic TEER, bacterial translocation, and localization and expressions of the tight junction proteins. Mechanistically, STM inhibited the expressions of p-PI3K and p-AKT in both the cell model and mouse model(P<0.05), and treatment with 740Y-P (a PI3K/AKT pathway activator) significantly attenuated the inhibitory effect of STM on TNF-α-induced apoptosis in Caco-2 cells (P<0.05). STM inhibits intestinal epithelial cell apoptosis at least in part by suppressing activation of the PI3K/AKT pathway to ameliorate intestinal barrier dysfunction and colitis in mice.

Actin networks modulate heterogeneous NF-κB dynamics in response to TNFα.

The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in pancreatic ductal adenocardinoma (PDAC) tumours. In this study, we characterised previously unexplored endogenous RELA-GFP dynamics in PDAC cell lines through live single-cell imaging. Our observations revealed that TNFα stimulation induces rapid, sustained, and non-oscillatory nuclear translocation of RELA. Through Bayesian analysis of single-cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. RNA-seq analysis identified distinct clusters of RELA-regulated gene expression in PDAC cells, including TNFα-induced RELA upregulation of the actin regulators NUAK2 and ARHGAP31. Further, siRNA-mediated depletion of ARHGAP31 and NUAK2 altered TNFα-stimulated nuclear RELA dynamics in PDAC cells, establishing a novel negative feedback loop that regulates RELA activation by TNFα. Additionally, we characterised the NF-κB pathway in PDAC cells, identifying how NF-κB/IκB proteins genetically and physically interact with RELA in the absence or presence of TNFα. Taken together, we provide computational and experimental support for interdependence between the F-actin network and the NF-κB pathway with RELA translocation dynamics in PDAC.

Endothelium-Derived Extracellular Vesicles Expressing Intercellular Adhesion Molecules Reflect Endothelial Permeability and Sepsis Severity.

Currently, clinical indicators for evaluating endothelial permeability in sepsis are unavailable. Endothelium-derived extracellular vesicles (EDEVs) are emerging as biomarkers of endothelial injury. Platelet endothelial cell adhesion molecule (PECAM) and vascular endothelial (VE)-cadherin are constitutively expressed endothelial intercellular adhesion molecules that regulate intercellular adhesion and permeability. Herein, we investigated the possible association between EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) and endothelial permeability and sepsis severity. Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor alpha (TNF-α) directly or after pretreatment with permeability-modifying reagents such as angiopoietin-1, prostacyclin, or vascular endothelial growth factor (VEGF) to alter TNF-α-induced endothelial hyperpermeability. Endothelial permeability was measured using the dextran assay or transendothelial electrical resistance. Additionally, a prospective cross-sectional observational study was conducted to analyze circulating EDEV levels in patients with sepsis. EDEVs were examined in HUVEC culture supernatants or patient plasma (nonsepsis, n = 30; sepsis, n = 30; septic shock, n = 42) using flow cytometry. The Wilcoxon rank-sum test was used for comparisons between 2 groups. Comparisons among 3 or more groups were performed using the Steel-Dwass test. Spearman's test was used for correlation analysis. Statistical significance was set at P < .05. TNF-α stimulation of HUVECs significantly increased EDEV release and endothelial permeability. Pretreatment with angiopoietin-1 or prostacyclin suppressed the TNF-α-induced increase in endothelial permeability and inhibited the release of PECAM+ and VE-cadherin+ EDEVs. In contrast, pretreatment with VEGF increased TNF-α-induced endothelial permeability and the release of PECAM+ and VE-cadherin+ EDEVs. However, pretreatment with permeability-modifying reagents did not affect the release of EDEVs expressing inflammatory stimulus-inducible endothelial adhesion molecules such as E-selectin, intracellular adhesion molecule-1, or vascular cell adhesion molecule-1. The number of PECAM+ EDEVs on admission in the septic-shock group (232 [124, 590]/μL) was significantly higher (P = .043) than that in the sepsis group (138 [77,267]/μL), with an average treatment effect of 98/μL (95% confidence interval [CI], 2-270/μL), and the number of VE-cadherin+ EDEVs in the septic-shock group (173 [76,339]/μL) was also significantly higher (P = .004) than that in the sepsis group (81 [42,159]/μL), with an average treatment effect (ATE) of 79/μL (95% CI, 19-171/μL); these EDEV levels remained elevated until day 5. EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) may reflect increased endothelial permeability and could be valuable diagnostic and prognostic markers for sepsis.

Exploring cell death mechanisms in spheroid cultures using a novel application of the RIP3-caspase3-assay

This study explores the application of the RIP3-caspase3-assay in heterogeneous spheroid cultures to analyze cell death pathways, emphasizing the nuanced roles of apoptosis and necroptosis. By employing directly conjugated monoclonal antibodies, we provide detailed insights into the complex mechanisms of cell death. Our findings demonstrate the assay’s capability to differentiate between RIP1-independent apoptosis, necroptosis, and RIP1-dependent apoptosis, marking a significant advancement in organoid research. Additionally, we investigate the effects of TNFα on isolated intestinal epithelial cells, revealing a concentration-dependent response and an adaptive or threshold reaction to TNFα-induced stress. The results indicate a preference for RIP1-independent cell death pathways upon TNFα stimulation, with a notable increase in apoptosis and a secondary role of necroptosis. Our research underscores the importance of the RIP3-caspase3-assay in understanding cell death mechanisms in organoid cultures, offering valuable insights for disease modeling and the development of targeted therapies. The assay’s adaptability and robustness in spheroid cultures enhances its potential as a tool in personalized medicine and translational research.

Dynamic changes in the proximitome of neutral sphingomyelinase-2 (nSMase2) in TNFα stimulated Jurkat cells.

Ceramides generated by the activity of the neutral sphingomyelinase 2 (nSMase2) play a pivotal role in stress responses in mammalian cells. Dysregulation of sphingolipid metabolism has been implicated in numerous inflammation-related pathologies. However, its influence on inflammatory cytokine-induced signaling is yet incompletely understood. Here, we used proximity labeling to explore the plasma membrane proximal protein network of nSMase2 and TNFα-induced changes thereof. We established Jurkat cells stably expressing nSMase2 C-terminally fused to the engineered ascorbate peroxidase 2 (APEX2). Removal of excess biotin phenol substantially improved streptavidin-based affinity purification of biotinylated proteins. Using our optimized protocol, we determined nSMase2-proximal biotinylated proteins and their changes within the first 5min of TNFα stimulation by quantitative mass spectrometry. We observed significant dynamic changes in the nSMase2 microenvironment in response to TNFα stimulation consistent with rapid remodeling of protein networks. Our data confirmed known nSMase2 interactors and revealed that the recruitment of most proteins depended on nSMase2 enzymatic activity. We measured significant enrichment of proteins related to vesicle-mediated transport, including proteins of recycling endosomes, trans-Golgi network, and exocytic vesicles in the proximitome of enzymatically active nSMase2 within the first minutes of TNFα stimulation. Hence, the nSMase2 proximal network and its TNFα-induced changes provide a valuable resource for further investigations into the involvement of nSMase2 in the early signaling pathways triggered by TNFα.

Exopolysaccharides from Limosilactobacillus reuteri: their influence on in vitro activation of porcine monocyte-derived dendritic cells - brief report

The aim of this study was to evaluate the immunomodulatory potential of two α-D-glucans from Limosilactobacillus reuteri L26 Biocenol™ (EPS-L26) and L. reuteri DSM17938 (EPS-DSM17938), with respect to their influence on in vitro activation of porcine dendritic cells (DCs). We used immature DCs differentiated from porcine blood monocytes under in vitro conditions. Based on the surface expression of MHC II and costimulatory CD80/86 molecules, we showed that both used EPSs favour the maturation of monocyte-derived DCs (MoDCs) similarly to the commonly used stimulant tumour necrosis factor α (TNF-α). In contrast to TNF-α stimulation, MoDCs treated with both used EPSs significantly up-regulated the mRNA levels not only for interleukin (IL)-10 (P < 0.0001 for EPS-DSM17938; P = 0.0037 for EPS-L26), but also for IL-12 (P = 0.0176 for EPS-DSM17938; P = 0.0019 for EPS-L26). These cytokines are known to regulate T-cell kinetics and play a key role in maintaining immune homeostasis. Interestingly, only relatively linear α-D-glucan (EPS-DSM17938) significantly increased gene expression of the major pro-inflammatory cytokine IL-1β (P = 0.0011) and the “SOS” cytokine IL-6 (P = 0.0127). However, it is important to highlight the need for further studies aimed at cytokine kinetics in DCs, as well as a co-culture study with allogenic T-lymphocytes.

Macrophages modulate mesenchymal stem cell function via tumor necrosis factor alpha in tooth extraction model.

Mesenchymal stem cells (MSCs) and macrophages collaboratively contribute to bone regeneration after injury. However, detailed mechanisms underlying the interaction between MSCs and inflammatory macrophages (M1) remain unclear. A macrophage-depleted tooth extraction model was generated in 5-wk-old female C57BL/6J mice using clodronate liposome (12.5mg/kg/mouse, intraperitoneally) or saline injection (control) before maxillary first molar extraction. Mice were sacrificed on days 1, 3, 5, 7, and 10 after tooth extraction (n = 4). Regenerated bone volume evaluation of tooth extraction socket (TES) and histochemical analysis of CD80+M1, CD206+M2 (anti-inflammatory macrophages), PDGFRα+MSC, and TNF-α+ cells were performed. In vitro, isolated MSCs with or without TNF-α stimulation (10ng/mL, 24h, n = 3) were bulk RNA-sequenced (RNA-Seq) to identify TNF-α stimulation-specific MSC transcriptomes. Day 7 micro-CT and HE staining revealed significantly lower mean bone volume (clodronate vs control: 0.01mm3 vs 0.02mm3, p<.0001) and mean percentage of regenerated bone area per total TES in clodronate group (41.97% vs 54.03%, p<.0001). Clodronate group showed significant reduction in mean number of CD80+, TNF-α+, PDGFRα+, and CD80+TNF-α+ cells on day 5 (306.5 vs 558.8, p<.0001; 280.5 vs 543.8, p<.0001; 365.0 vs 633.0, p<.0001, 29.0 vs 42.5, p<.0001), while these cells recovered significantly on day 7 (493.3 vs 396.0, p=.0004; 479.3 vs 384.5, p=.0008; 593.0 vs 473.0, p=.0010, 41.0 vs 32.5, p=.0003). RNA-Seq analysis showed that 15 genes (|log2FC| > 5.0, log2TPM > 5) after TNF-α stimulation were candidates for regulating MSC's immunomodulatory capacity. In vivo, Clec4e and Gbp6 are involved in inflammation and bone formation. Clec4e, Gbp6, and Cxcl10 knockdown increased osteogenic differentiation of MSCs in vitro. Temporal reduction followed by apparent recovery of TNF-α-producing M1 macrophages and MSCs after temporal macrophage depletion suggests that TNF-α activated MSCs during TES healing. In vitro mimicking the effect of TNF-α on MSCs indicated that there are 15 candidate MSC genes for regulation of immunomodulatory capacity.

Increased mRNA expression of ADAMTS1 and ADAMTS4 in peripheral blood mononuclear cells of psoriasis patients developed psoriatic arthritis

Background and purpose: Psoriatic arthritis (PsA) is a chronic inflammatory disease associated with psoriasis (PsO) affecting both skin and joint. ADAMTS (A disintegrin-like and metalloproteinase domain with thrombospondin-1 repeats) is a large family of proteoglycanase enzymes and the expression levels of ADAMTS proteases are upregulated in arthritis. In this study, we aimed to determine mRNA expression levels of ADAMTS1, ADAMTS4 and ADAMTS5 and identifying the key signaling pathways involved in the regulation of these proteases in peripheral blood mononuclear cells (PBMCs) of patients with PsO who later developed PsA. Materials and methods: 25 PsA patients, 25 PsO patients and 25 healthy individuals were included in this study. PBMCs were isolated from venous blood and mRNA expression levels of ADAMTS1, ADAMTS4 and ADAMTS5 were measured through Real-time quantitative PCR (RTqPCR). Results: mRNA expression levels of ADAMTS1 and ADAMTS4 were found to be increased in PsA patients compared with control and PsO patients. In response to TNF-a stimulation, the expression of ADAMTS1 in PsA patients was determined to be reduced in a Erk1/2 activity dependent manner, whereas p38 and JNK activities were shown to induce the expression of ADAMTS4 in PsA patients. The reduced ADAMTS1 expression in PsA patients induced by IL-1b stimulation was revealed to be dependent on NFkB activity. Conclusions: mRNA expressions of ADAMTS1 and ADAMTS4 regulated by MAPKs and NFkB were increased in PBMCs of PsA patients. This study supports the hypothesis that ADAMTS1 and ADAMTS4 mRNA level might be diagnostic markers for identifying psoriatic patients who are more likely to develop PsA and a future drug target for PsA treatment.

Alleviation of preeclampsia-like symptoms through PlGF and eNOS regulation by hypoxia- and NF-κB-responsive miR-214-3p deletion

Preeclampsia is caused by placental hypoxia and systemic inflammation and is associated with reduced placental growth factor (PlGF) and endothelial nitric oxide synthase (eNOS) levels. The molecular signaling axes involved in this process may play a role in the pathogenesis of preeclampsia. Here, we found that hypoxic exposure increased hypoxia-inducible factor-1α (HIF-1α)/Twist1-mediated miR-214-3p biogenesis in trophoblasts, suppressing PlGF production and trophoblast invasion. TNF-α stimulation increased NF-κB-dependent miR-214-3p expression in endothelial cells, impairing eNOS expression and causing endothelial dysfunction. Synthetic miR-214-3p administration to pregnant mice decreased PlGF and eNOS expression, resulting in preeclampsia-like symptoms, including hypertension, proteinuria, and fetal growth restriction. Conversely, miR-214-3p deletion maintained the PlGF and eNOS levels in hypoxic pregnant mice, alleviating preeclampsia-like symptoms and signs. These findings provide new insights into the role of HIF-1/Twist1- and NF-κB-responsive miR-214-3p-dependent PlGF and eNOS downregulation in the pathogenesis of preeclampsia and establish miR-214-3p as a therapeutic or preventive target for preeclampsia and its complications.