TNF-α Receptor Research Articles

Comprehensive metabolite and biological profile of “Sulmona Red Garlic” ecotype’s aerial bulbils

“Sulmona Red Garlic” is a well-known Italian traditional product. Bulbs, used for culinary purposes, have been largely investigated for their medicinal properties whereas aerial bulbils are usually removed as waste material. Here, for the first time, chemical composition and biological properties of the hydroalcoholic extract from aerial bulbils were investigated. Complementary information on metabolite composition were obtained using both NMR based untargeted and HPLC-DAD targeted methodologies. The NMR analysis revealed the presence of sugars, organic acids, amino acids, organosulphur compounds (methiin, alliin, allicin and cycloalliin), and other secondary metabolites. In particular, methiin and alliin were identified for the first time in the NMR spectra of aerial bulbil garlic extracts. Polyphenol content was determined by HPLC-DAD analysis: catechin, chlorogenic acid, and gallic acid turned out to be the most abundant phenolics.Hydroalcoholic extract blocked cell proliferation of colon cancer cell line HCT116 with an IC50 of 352.07 µg/mL, while it was non-toxic to myoblast cell line C2C12. In addition, it caused seedling germination reduction of two edible and herbaceous dicotyledon species, namely Cichorium intybus and C. endivia. Moreover, the same extract reduced the gene expression of TNF-α (tumor necrosis factor), HIF1-α (hypoxia-inducible factor), VEGFA (vascular endothelial growth factor), and transient receptor potential (TRP) M8 (TRPM8) indicating the ability to contrast cancer development through the angiogenic pathway. Final, in silico experiments were also carried out supporting the biological effects of organosulphur compounds, particularly alliin, which may directly interact with TRPM8.The results here reported suggest the potential use of garlic aerial bulbils often considered a waste product as a source in phytotherapeutic remedies.

Ethanol Extracts from the Aerial Parts of Inula japonica and Potentilla chinensis Alleviate Airway Inflammation in Mice That Inhaled Particulate Matter 10 and Diesel Particulate Matter.

Air pollution causes various airway diseases. However, many commonly used treatments can have high risks of side effects or are costly. To examine the anti-inflammatory properties of Inula japonica Thunb. and Potentilla chinensis Ser., a mouse model was generated via inhalation of both particulate matter 10 and diesel particulate matter, and 30% ethanol extracts of either I. japonica (IJ) or P. chinensis (PC) and a mixture of both ethanol extracts (IP) were orally administered to BALB/c mice for 12 days. IJ, PC, and IP inhibited immune cell numbers and their regulation in both the bronchoalveolar lavage fluid (BALF) and lungs. These agents suppressed the levels of interleukin (IL)-1α, IL-17, tumor necrosis factor (TNF)-α, C-X-C motif chemokine ligand (CXCL)-1, and CXCL-2 in BALF, and also inhibited F4/80 and IL-1 receptor-associated kinase (IRAK)-1 in lungs. They reduced the gene expression of TNF-α, CXCL-1, inducible NOS, COX-2, Mucin 5AC, and transient receptor potential cation channel subfamily V member 1 in lungs. These extracts also reduced histopathological changes and inflammatory progression, manifested as decreased cell infiltration, collagen deposition, and respiratory epithelial cell thickness. I. japonica and P. chinensis show potential for development as pharmaceuticals that suppress inflammatory progression and alleviate airway inflammation diseases caused by air pollutants.

Extracellular SOD modulates canonical TNFα signaling and α5β1 integrin transactivation in vascular smooth muscle cells

TNFα activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs). The extracellular superoxide anion (O2•-) produced is essential for the pro-inflammatory effects of the cytokine but the specific contributions of O2•- to signal transduction remain obscure. Extracellular superoxide dismutase (ecSOD, SOD3 gene) is a secreted protein that binds to cell surface heparin sulfate proteoglycans or to Fibulin-5 (Fib-5, FBLN5 gene), an extracellular matrix protein that also associates with elastin and integrins. ecSOD converts O2•- to hydrogen peroxide (H2O2) which prevents NO• inactivation, limits generation of hydroxyl radical (OH•), and creates high local concentrations of H2O2. We hypothesized that ecSOD modifies TNFα signaling in VSMCs. Knockdown of ecSOD (siSOD3) suppressed downstream TNFα signals including MAPK (JNK and ERK phosphorylation) and NF-κB activation (luciferase reporter and IκB phosphorylation), interleukin-6 (IL-6) secretion, iNOS and VCAM expression, and proliferation (Sulforhodamine B assay, PCNA western blot). These effects were associated with significant reductions in the expression of both Type1 and 2 TNFα receptors. Reduced Fib-5 expression (siFBLN5) similarly impaired NF-κB activation by TNFα, but potentiated FAK phosphorylation at Y925. siSOD3 also increased both resting and TNFα-induced phosphorylation of FAK and of glycogen synthase kinase-3β (GSK3β), a downstream target of integrin linked kinase (ILK). These effects were dependent upon α5β1 integrins and siSOD3 increased resting sulfenylation (oxidation) of both integrin subunits, while preventing TNFα-induced increases in sulfenylation. To determine how ecSOD modified TNFα-induced inflammation in intact blood vessels, mesenteric arteries from VSMC-specific ecSOD knockout (KO) mice were exposed to TNFα (10 ng/ml) in culture for 48 h. Relaxation to acetylcholine and sodium nitroprusside was impaired in WT but not ecSOD KO vessels. Thus, ecSOD association with Fib-5 supports pro-inflammatory TNFα signaling while tonically inhibiting α5β1 integrin activation.

Investigation of corticosteroid-NSAID prodrugs for TNF-α and IL6 receptors, their characterization, single crystal XRD and DFT studies: A combined experimental and theoretical approach

In continuation of our research towards the synthesis of steroidal derivatives, we have synthesized corticosteroids-NSAIDs derivatives 3i-3p as potent anti-inflammatory agents. The products obtained have been characterized using elemental analysis, spectroscopic techniques (FT-IR, 1H NMR, 13C NMR, UV–Vis and mass spectrometry) and product 3k has also been characterized with the help of single crystal X-ray crystallography. The inhibition of pro-inflammatory cytokines is a key approach to abate severe inflammation. In this work a series of corticosteroids-NSAIDs derivatives are reported whose activity were performed on RAW264.7 cell line and have been identified as potential TNF alpha and IL6 inhibitors. Our work demonstrates that these corticosteroid derivatives may be useful for the development of anti-inflammatory agents. Among these derivatives, 11,17-dihydroxy-3,20-diketo-pregn-1,4-diene-21-yl-2-(1,8‑diethyl-1,3,4,9-tetrahydropyrano[3,4-β]indol-1-yl)acetate (3j) was identified as the most potent anti-inflammatory agent, which showed significant anti-inflammatory activity by inhibiting the LPS-induced pro-inflammatory mediator TNF-α and IL6 in a dose-dependent manner without any cytotoxicity.

Effect of Ligustrazine on Lipopolysaccharide-induced Inflammatory Response of Vascular Endothelial Cells and its Possible Mechanism

Background Endothelial cell (EC) inflammation plays a crucial role in the development of several cardiovascular disorders (CD), including atherosclerosis and sepsis. Ligustrazine (Lig), a bioactive constituent derived from Traditional Chinese Medicine Ligusticum chuanxiong Hort, has exhibited in vivo anti-inflammatory properties. Despite the observed positive outcomes, the exact mechanisms underlying these beneficial effects remain unidentified. Aim The goal of this research is to investigate the influence and potential mechanism of Lig on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs). Introduction These experiments investigate the effectiveness of Lig in preventing LPS-induced damage in HUVECs, with the goal of elucidating the underlying processes at work. Materials and Methods To evaluate HUVECs’ viability, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was conducted. Enzyme-linked immunosorbent assay (ELISA) was employed to measure changes in ICAM-1, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein (MCP-1) levels. Real-time polymerase chain reaction (RT-PCR) was used to determine the levels of ICAM-1, IL-6, TNF-α, MCP-1, and toll-like receptors (TLR4) mRNA. Additionally, we performed WB analysis to assess the levels of nuclear factor-κB (NF-κB) p65, TLR4, IκBα, and p-IκBα. Results The findings demonstrated significantly suppressed cell viability due to LPS treatment, while Lig treatment increased cell viability in a concentration-dependent manner. Lig also effectively reduced the mRNA levels of ICAM-1, TNF-α, IL-6, and MCP-1. Furthermore, Lig pretreatment led to downregulation of TLR4, p-IκBα, and NF-κB p65 in HUVECs. Discussion The findings indicate that Lig reduces LPS-induced inflammation in HUVECs, and that the TLR4/NF-κB pathway is critical in increasing cell survival and minimizing inflammatory damage. This provides possible anti-inflammatory techniques for treating CD. Conclusion In conclusion, our work demonstrates Lig’s anti-inflammatory actions on LPS-stimulated HUVECs. The data suggest that Lig lowers inflammation via regulating the TLR4/NF-B pathway, boosting cell survival, and decreasing inflammatory responses.

Siraitia grosvenorii Extract Attenuates Airway Inflammation in a Mouse Model of Respiratory Disease Induced by Particulate Matter 10 Plus Diesel Exhaust Particles.

Exposure to particulate matter (PM) causes considerable breathing-related health risks. Siraitia grosvenorii fruit is a traditional remedial plant used in Korea and China to treat respiratory diseases. Our recently published study showed that S. grosvenorii extract (SGE) ameliorated airway inflammation in lipopolysaccharide- and cigarette-smoke-induced chronic obstructive pulmonary disease in mice. Thus, we aimed to assess the inhibitory effects of SGE on airway inflammation in mice exposed to a fine dust mixture of PM10 (PM diameter < 10 mm) and diesel exhaust particles (DEPs) known as PM10D. The mice (BALB/c) were treated with PM10D via intranasal injection three times over a period of 12 days, and SGE 70% ethanolic extract (50 or 100 mg/kg) was orally administered daily for 12 days. SGE attenuated neutrophil accumulation and the number of immune B and T cells from the lung tissue and bronchoalveolar lavage fluid (BALF) of the PM10D-exposed mice. SGE reduced the secretion of cytokines and chemokines, including interleukin (IL)-1α, tumor necrosis factor (TNF)-α, IL-17, C-X-C motif chemokine ligand (CXCL)1, and macrophage inflammatory protein (MIP)-2 in the BALF. Airway inflammation, infiltration of inflammatory cells, and collagen fibrosis in the lung after PM10D exposure were investigated via histopathological analysis, and SGE treatment ameliorated these symptoms. SGE decreased the mRNA expression of mucin 5AC (MUC5AC), CXCL1, TNF-α, MIP-2, and transient receptor potential ion channels in the lung tissues. Furthermore, SGE ameliorated the activation of mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) signaling by PM10D in the lungs. We conclude that SGE attenuated PM10D-induced neutrophilic airway inflammation by inhibiting MAPK/NF-κB activation. These results show that SGE may be a candidate for the treatment of inflammatory respiratory diseases.

Abstract P183: Porcupine Protects The Kidney By Suppressing Tubular Inflammation And Preserving Mitochondrial Integrity

Chronic Kidney Disease (CKD) progression may be slowed or stopped with early diagnosis and intervention, so developing novel therapies is paramount. The Wnt acyl transferase porcupine (PORCN) regulates the secretion of all 19 Wnt ligand isoforms that propagate β-catenin signaling. We find that mice with deletion of PORCN from kidney epithelia (PORCN iKKO) had augmented kidney injury in the nephrotoxic serum nephritis (NTS) model of CKD, with higher BUNs (202 ± 12 vs. 101 ± 38, mg/dL; p&lt;0.01), and renal mRNA levels for NGAL (6.1±1.14 vs. 1.0±0.35 au, p=0.0013), KIM-1(2.5±0.65 vs. 1.0±0.26 au, p=0.046), collagen I (2.4±0.31 vs. 1.0±0.38 au, p=0.025), and fibronectin (2.5±0.35 vs. 1.0±0.27 au, p=0.006) compared to wild-type (WT) controls. PORCN iKKOs also had upregulated renal protein levels for collagen I (1.5 ± 0.2 vs. 1.0 ± 0.1 au; p=0.01) and fibronectin (1.8 ± 0.3 vs. 1.0 ± 0.1 au; p&lt;0.01), recapitulating the mRNA patterns. We also detected marked TNFα mRNA upregulation in the PORCN iKKO kidneys vs WTs during CKD (2.6±0.67 vs. 1.0±0.30 au, p=0.036). TNFα is a potent driver of renal injury and fibrosis, acting via TNFα receptor 1(TNFR1) to promote ROS generation. At day 14 of NTS, PORCN iKKO kidneys contained higher levels of the ROS metabolite, 8-isoprostane (2348 ± 718 vs. 486 ± 77, pg/mg kidney; p=0.02), and expressed higher levels of mitochondrial dysfunction markers, including Drp1 (1.26 ± 0.06 vs. 1.0 ± 0.04 au; p&lt;0.01) and p62 (1.3 ± 0.09 vs. 1.0 ± 0.07 au; p=0.07). TNFR1 inhibition (R-7050, 12mg/kg, IP, every other day) reduced BUNs (94.13 ± 3 vs. 90.26 ± 4.1, mg/dL; p=0.46) and renal Drp1 protein levels (1.4 ± 0.17 vs. 1.0 ± 0.15 au; p=0.11) in the PORCN iKKO cohort to WT levels. In primary renal tubular cells (RTCs), TNFα treatment (50ng/ml, 12hrs) induced 8-isoprostane generation (387 ± 119 vs. 121 ± 39, pg/ml in medium; p=0.07), corroborating our in vivo studies. Moreover, treatment of the RTCs with the Drp1 inhibitor mdivi-1 (MD1, 10μm) for 12 hours attenuated TNFα-induced accumulation of fibronectin and collagen I. Our studies suggest that PORCN in renal epithelia protects against kidney damage by limiting TNFα induction, thereby diminishing Drp1-mediated mitochondrial dysfunction and ROS generation.

Mechanism of Bugansan Decoction in ameliorating learning and memory impairment in D-galactose-induced aging rats based on AGEs/RAGE/NF-κB pathway

ObjectiveTo investigate the underlying mechanism of the compound Bugansan Decoction (补肝散, BGSD) in intervening learning and memory in D-galactose (D-gal)-induced aging rats. MethodsA total of 40 rats were randomly assigned to four groups: control, model, BGSD [14.06 g/(kg·d)], and piracetam [0.4 g/(kg·d)] groups, with 10 rats in each group. D-gal [400 mg/(kg·d)] was injected intraperitoneally to establish the aging rat model. The rats' body weight, water intake, food intake, and gripping strength were recorded each week. The eight-arm maze and step-down test were used to measure the rats' capacity for learning and memory. Liver, thymus, spleen, and brain tissues were weighed to calculate the corresponding organ indices; serum malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were measured. Hematoxylin and eosin (HE) staining was adopted to observe the pathological changes of the hippocampus; enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in the hippocampus. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of receptors for advanced glycation end products (RAGE), nuclear factor-κB (NF-κB), TNF-α, IL-6, and IL-1β mRNA in the hippocampus. Western blot (WB) was employed to detect the expression levels of advanced glycation end products (AGEs), RAGE, and NF-κB protein in the hippocampus. ResultsIn D-gal-induced aging rats, BGSD significantly increased food intake, water intake, body weight, gripping strength, and organ indices (P < 0.05), and significantly decreased working memory error (WME), reference memory error (RME), and total memory errors (TE) in an eight-arm maze (P < 0.05). In the step-down test, step-down latency was prolonged and the frequency of errors dropped (P < 0.05). Additionally, BGSD could lessen the harm done to hippocampus neurons, increase serum SOD activity, lower MDA levels, and down-regulate the expression levels of the pro-inflammatory molecules TNF-α, IL-6, and IL-1β (P < 0.05). Further findings showed that BGSD significantly decreased hippocampal AGEs, RAGE, and NF-κB expression (P < 0.05). ConclusionBy blocking the AGEs/RAGE/NF-κB signaling pathway, BGSD may regulate the neuroinflammatory damage in D-gal-induced aging rats, and thus improve learning and memory.

Abstract P310: Transcriptomic Profiling Of Sars-cov-2 Spike Protein S1 Subunit Mediated Changes In Human Vascular -pericytes, -endothelial Cells (ECs), -smooth Muscle Cells And Lymphatic ECs: Evidence For Differential Gene Regulation

SARS-CoV-2 S1 spike protein (S1-SP) is increased in subjects with myocarditis after mRNA vaccine. However the underlying mechanism(s) of its vascular action are unclear. Using microarrays we assessed the effects of S1-SP on genes of vascular -ECs, -pericytes (PCs), -SMCs and LECs. Transcriptomic profiling of genes revealed that treatment of PCs, VECs, LECs and SMCs with 10ng/ml S1-SP for 36 hours upregulated 3935, 2512, 1422, 19 genes, respectively; and downregulated 3935, 3223, 1170, 37 genes, respectively. S1-SP significantly modulated genes in PCs, LECs and VECs, but not in SMCs. Pathway enrichment analysis highlighted modulation of TGF-β linked extracellular matrix, focal adhesion in PCS, VECs and LECs; whereas coronavirus disease, ribosome, cytoplasmic ribosomal protein, translation, protein metabolism, influenza infection, disease pathways were regulated in PCs and VECs, but not in LECs. A significant modulation in IL-2, cell senescence, IL4, TNFα and T-cell receptor associated apoptosis, Oncostatin M, p53 signaling was observed in LECs. Transcriptomic analysis showed induction of key COVID associated pro-inflammatory, coagulatory and matrix/adhesion molecules in PCs (PECAM1, CDH5, S1PR1, VWF, ESM1, EDN1, EFEMP1, ERG, ICAM2), but down regulation in VECs. Interestingly top genes that were down regulated in PCs (MYCT1, MMRN1, SEMA5A, GREM1, CEMIP, THBS2, BICC1, MYOCD, COL3A1, COL1A1, IGFBP3, PLPPR4, ITGA11, ADAMTS12, COL11A1, POSTN) were up regulated in VECs. Of all the genes regulated by S1-SP in PCs, VECs and LECs only two (FAP and P3H2) and eight (TMEM200A,MMP1, PLPP4, RGS5, TENM3, SULF1, COL12A1, SPOCK1) were up- and down regulated, respectively. DRG analysis highlighted contrasting modulation of genes associated with: myocarditis, metabolism (Glycolysis, one carbon metabolism, urea cycle and oxidative phosphorylation) and inflammation in PCs and VECs. No change in DRGs was observed in SMCs, whereas compared to PCs mixed regulation was observed in LECs. Also, Proteome array generated cytokine profile mimicked the impact of S1-SP on pro-inflammatory genes in PCs, VECs and LECs. Our findings provide first evidence that S1-SP differentially modulates genes and cytokines in PCs, LECs, VECs.

CD36 genetic polymorphism and salivary cues are associated with oleic acid sensitivity and dietary fat intake.

There is a lack of research on the combined effects of genetic variations (specifically CD36 SNPs-rs1761667 and rs1527483), dietary food habits (vegetarian or not), and the salivary environment on obesity and taste sensitivity, especially in the Indian population. The current study aims to better understand the relationship between impaired taste perception, fat consumption, higher BMI and obesity development by examining the combined association between CD36 SNPs, oleic acid (OA) detection threshold, and food habits among Indian participants. Furthermore, the relationship between oral fatty acid (FAs) sensitivity and taste physiology factors linked to inflammation and salivary proteins was considered. Participants with the minor allele (AA/AG) of CD36 (in both rs1527483 and rs1761667) consumed more fat, particularly saturated FAs (p = 0.0351). Salivary lipopolysaccharide, which causes inflammation, was significantly greater in non-vegetarians with a higher BMI (p < 0.05), and it exhibited a negative correlation (r = -0.232 and p < 0.05) with Ki67 gene expression, a marker for taste progenitor cells. A positive correlation (r = 0.474, p = 0.04) between TLR4 mRNA levels and the OA detection threshold was also observed. Participants with BMI > 25 kg/m2 had substantially higher TNF-α and IL-6 receptor mRNA expression levels, but there were no significant differences between the vegetarian and non-vegetarian groups. However, salivary CA-VI, which has a buffering capability on the oral environment, was lower in non-vegetarian adults with BMI >25. Thus, it was shown that non-vegetarians with overweight and obesity in India were in at-risk groups for the CD36 SNP (AA/AG at rs1761667 and rs1527483) and had higher levels of inflammatory markers, which exacerbated alterations in food behaviour and physiological changes, indicating their relevance in the development of obesity.

Genetically Engineered Biomimetic Nanoparticles for Targeted Delivery of mRNA to Treat Rheumatoid Arthritis.

In addition to inhibiting persistent inflammation, phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is known as an important therapeutic target for alleviating rheumatoid arthritis (RA) symptoms. Modulation of PTEN gene expression in synovial tissue using messenger RNA (mRNA) is a promising approach to combat RA. However, mRNA therapeutics are often hampered by unsatisfactory stability and inefficient localization in synovial tissue. In this study, a genetically engineered biomimetic membrane-coated mRNA (MR@P-mPTEN) carrier that effectively delivers mRNA-PTEN (mPTEN) directly to the RA joint is presented. By overexpressing tumor necrosis factor (TNF-α) receptors on macrophage biomimetic membranes via plasmid transfection, decoys that reduce inflammatory pathway activation are prepared for TNF-α. The resulting construct, MR@P-mPTEN, shows good stability and RA targeting based on in vivo fluorescence imaging. It is also found that MR@P-mPTEN competitively binds TNF-α and activates the PTEN pathway in vitro and in vivo, thereby inhibiting synovitis and joint damage. Clinical micro-computed tomography and histological analyses confirm the treatment effects. These results suggest that the genetically engineered biomimetic therapeutic platform MR@P-mPTEN both inhibits pro-inflammatory cytokines and upregulates PTEN protein expression to alleviate RA damage, providing a new a new combination strategy for RA treatment.

A novel therapeutic vaccine targeting the soluble TNFα receptor II to limit the progression of cardiovascular disease: AtheroVax™.

The burden of atherosclerotic cardiovascular disease contributes to a large proportion of morbidity and mortality, globally. Vaccination against atherosclerosis has been proposed for over 20 years targeting different mediators of atherothrombosis; however, these have not been adequately evaluated in human clinical trials to assess safety and efficacy. Inflammation is a driver of atherosclerosis, but inflammatory mediators are essential components of the immune response. Only pathogenic forms of sTNFR2 are acted upon while preserving the membrane-bound (wild-type) TNFR2 contributions to a non-pathogenic immune response. We hypothesize that the inhibition of sTNRF2 will be more specific and offer long-term treatment options. Here we describe pre-clinical findings of an sTNFR2-targeting peptide vaccine (AtheroVax™) in a mouse model. The multiple pathways to synthesis of the soluble TNFRII receptor (sTNFRII) were identified as sTNFRII(PC), sTNFRII(Δ7), and sTNFRII(Δ7,9). The sTNFRII(Δ7) peptide, NH2-DFALPVEKPLCLQR-COOH is specific to sTNFR2 based on an mRNA splice-variant in which exon 6 is joined to exon 8. The role of sTNFRII(Δ7) as a mediator of prolonged TNFα activity by preventing degradation and clearance was investigated. Inflammation is a critical driver of onset, progression and expansion of atherosclerosis. The TNFα ligand represents a driver of inflammation that is mediated by a splice variant of TNFR2, referred to as sTNFRII(Δ7). The multiple forms of TNFRII, both membrane bound and soluble, are associated with distinctly different phenotypes. sTNFRII(PC) and sTNFRII(Δ7) are not equivalent to etanercept because they lack a clearance mechanism. The unique peptide associated with sTNFRII(Δ7) contains a linear B-cell epitope with amino acids from both exon 6 and exon 8 supporting the vaccine design. Animal studies to evaluate the vaccine are ongoing, and results will be forthcoming. We describe a peptide vaccine targeting sTNFR2 in limiting the progression of atherosclerosis. A therapeutic vaccine limiting the progression of atherosclerosis will greatly contribute to the reduction in morbidity and mortality from cardiovascular disease. It is likely the vaccine will be used in combination with the current standards of care and lifestyle modifications.

Synthetic Peptide Antibodies as TNF-α Inhibitors: Molecularly Imprinted Polymer Nanogels Neutralize the Inflammatory Activity of TNF-α in THP-1 Derived Macrophages.

Tumor Necrosis Factor-α (TNF-α) is a cytokine that is normally produced by immune cells when fighting an infection. But, when too much TNF-α is produced as in autoimmune diseases, this leads to unwanted and persistent inflammation. Anti-TNF-α monoclonal antibodies have revolutionized the therapy of these disorders by blocking TNF-α and preventing its binding to TNF-α receptors, thus suppressing the inflammation. Herein, we propose an alternative in the form of molecularly imprinted polymer nanogels (MIP-NGs). MIP-NGs are synthetic antibodies obtained by nanomoulding the 3-dimensional shape and chemical functionalities of a desired target in a synthetic polymer. Using an in-house developed in silico rational approach, epitope peptides of TNF-α were generated and 'synthetic peptide antibodies' were prepared. The resultant MIP-NGs bind the template peptide and recombinant TNF-α with high affinity and selectivity, and can block the binding of TNF-α to its receptor. Consequently they were applied to neutralize pro-inflammatory TNF-α in the supernatant of human THP-1 macrophages, leading to a downregulation of the secretion of pro-inflammatory cytokines. Our results suggest that MIP-NGs, which are thermally and biochemically more stable and easier to manufacture than antibodies, and cost-effective, are very promising as next generation TNF-α inhibitors for the treatment of inflammatory diseases.

Role of tumor necrosis factor-alpha in the central nervous system: a focus on autoimmune disorders.

Tumor necrosis factor-alpha (TNF-α) is a pleiotropic immune cytokine that belongs to the TNF superfamily of receptor ligands. The cytokine exists as either a transmembrane or a soluble molecule, and targets two distinct receptors, TNF-α receptor 1 (TNFR1) and TNF-α receptor 2 (TNFR2), which activate different signaling cascades and downstream genes. TNF-α cellular responses depend on its molecular form, targeted receptor, and concentration levels. TNF-α plays a multifaceted role in normal physiology that is highly relevant to human health and disease. In the central nervous system (CNS), this cytokine regulates homeostatic functions, such as neurogenesis, myelination, blood-brain barrier permeability and synaptic plasticity. However, it can also potentiate neuronal excitotoxicity and CNS inflammation. The pleiotropism of TNF-α and its various roles in the CNS, whether homeostatic or deleterious, only emphasizes the functional complexity of this cytokine. Anti-TNF-α therapy has demonstrated effectiveness in treating various autoimmune inflammatory diseases and has emerged as a significant treatment option for CNS autoimmune diseases. Nevertheless, it is crucial to recognize that the effects of this therapeutic target are diverse and complex. Contrary to initial expectations, anti-TNF-α therapy has been found to have detrimental effects in multiple sclerosis. This article focuses on describing the various roles, both physiological and pathological, of TNF-α in the CNS. Additionally, it discusses the specific disease processes that are dependent or regulated by TNF-α and the rationale of its use as a therapeutic target.

Reparative Dentin Formation Following Dental Pulp Capping is Mediated by TNFR1 In Vivo

IntroductionTumor necrosis factor (TNF)-α is a pro-inflammatory cytokine that promotes biomineralization in vitro in dental pulp cells. However, the role of TNF-α-TNF receptor 1 (TNFR1) signaling in reparative dentin formation and related inflammatory pathways is not known. Therefore, the aim of this study was to evaluate the role of the TNF-α-TNFR1 axis in dental pulp repair following pulp capping in vivo. MethodsDental pulp repair response of genetically deficient TNF-α receptor-1 mice (TNFR1−/−; n = 20) was compared with that of C57Bl6 mice (wild type [WT]; n = 20). Pulp capping was performed with mineral trioxide aggregate on the mandibular first molars of mice. After 7 and 70 days, tissues were collected and stained with hematoxylin and eosin for histopathological and histometric evaluation, and assessed by the Brown and Brenn methods for histomicrobiological analysis and by immunohistochemistry to localize TNF-α, Runt-related transcription factor 2, Dentin Sialoprotein (DSP) and Osteopontin (OPN) expression. ResultsCompared with WT mice, TNFR1−/− mice showed significantly decreased reparative dentin formation with a lower mineralized tissue area (P < .0001). Unlike WT mice, TNFR1−/− mice also exhibited significant dental pulp necrosis, neutrophil recruitment, and apical periodontitis formation (P < .0001) without bacterial tissue invasion. TNFR1−/− animals further exhibited decreased TNF-α, DSP, and OPN expression (P < .0001), whereas Runt-related transcription factor 2 expression was unchanged (P > .05). ConclusionThe TNF-α-TNFR1 axis is involved in reparative dentin formation following dental pulp capping in vivo. Genetic ablation of TNFR1 modified the inflammatory process and inhibited the expression of the DSP and OPN mineralization proteins, which culminated in dental pulp necrosis and development of apical periodontitis.

TSG101 Physically Interacts with Linear Ubiquitin Chain Assembly Complex (LUBAC) and Upregulates the TNFα-Induced NF-κB Activation.

Linear ubiquitin chain assembly complex (LUBAC) is a ubiquitin E3 ligase complex composed of HOIP, HOIL-1L, and SHARPIN that catalyzes the formation of linear/M1- linked ubiquitin chain. It has been shown to play a pivotal role in the nuclear factor (NF)-κB signaling induced by proinflammatory stimuli. Here, we found that tumor susceptibility gene (TSG101) physically interacts with HOIP, a catalytic component of LUBAC, and potentiates LUBAC activity. Depletion of TSG101 expression by RNA interference decreased TNFα-induced linear ubiquitination and the formation of TNFα receptor 1 signaling complex (TNFRSC). Furthermore, TSG101 facilitated the TNFα-induced stimulation of the NF-κB pathway. Thus, we suggest that TSG101 functions as a positive modulator of HOIP that mediates TNFα-induced NF-κB signaling pathway.

Effects of Modified Glucosamine on the Chondrogenic Potential of Circulating Stem Cells under Experimental Inflammation.

Glucosamine (GlcN) is a glycosaminoglycan (GAGs) constituent in connective tissues. It is naturally produced by our body or consumed from diets. In the last decade, in vitro and in vivo trials have demonstrated that the administration of GlcN or its derivates has a protective effect on cartilage when the balance between catabolic and anabolic processes is disrupted and cells are no longer able to fully compensate for the loss of collagen and proteoglycans. To date, these benefits are still controversial because the mechanism of action of GlcN is not yet well clarified. In this study, we have characterized the biological activities of an amino acid (AA) derivate of GlcN, called DCF001, in the growth and chondrogenic induction of circulating multipotent stem cells (CMCs) after priming with tumor necrosis factor-alpha (TNFα), a pleiotropic cytokine commonly expressed in chronic inflammatory joint diseases. In the present work, stem cells were isolated from the human peripheral blood of healthy donors. After priming with TNFα (10 ng/mL) for 3 h, cultures were treated for 24 h with DCF001 (1 μg/mL) dissolved in a proliferative (PM) or chondrogenic (CM) medium. Cell proliferation was analyzed using a Corning® Cell Counter and trypan blue exclusion technique. To evaluate the potentialities of DCF001 in counteracting the inflammatory response to TNFα, we measured the amount of extracellular ATP (eATP) and the expression of adenosine-generating enzymes CD39/CD73, TNFα receptors, and NF-κB inhibitor IκBα using flow cytometry. Finally, total RNA was extracted to perform a gene expression study of some chondrogenic differentiation markers (COL2A1, RUNX2, and MMP13). Our analysis has shed light on the ability of DCF001 to (a) regulate the expression of CD39, CD73, and TNF receptors; (b) modulate eATP under differentiative induction; (c) enhance the inhibitory activity of IκBα, reducing its phosphorylation after TNFα stimulation; and (d) preserve the chondrogenic potentialities of stem cells. Although preliminary, these results suggest that DCF001 could be a valuable supplement for ameliorating the outcome of cartilage repair interventions, enhancing the efficacy of endogenous stem cells under inflammatory stimuli.

Disruption of TNF-α signaling improves osteoblastic differentiation of adipose-derived mesenchymal stem cells and bone repair.

Adipose tissue is an attractive source of mesenchymal stem cells (at-MSCs), but their low osteogenic potential limits their use in bone regeneration. Adipose tissue plays a role in pro-inflammatory diseases by releasing cytokines with a catabolic effect on bone, such as tumor necrosis factor-alpha (TNF-α). Thus, we hypothesized that endogenous TNF-α could have a negative effect on at-MSC differentiation into osteoblasts. Short interfering RNAs (siRNAs) targeting TNF-α receptors (siR1, siR2, and si1R/R2) were transfected into at-MSCs, and cell differentiation was assessed by measuring the expression of bone markers, ALP activity, and mineralized matrix. Scrambled was used as Control. Knockout at-MSCs (KOR1/R2) was injected in mice calvaria defects, and bone formation was evaluated by microtomography and histological analysis. Data were compared by Kruskal-Wallis or analysis of variance (5%). The expression of bone markers confirmed that at-MSCs differentiate less than bone marrow MSCs. In silenced cells, the expression of Alp, Runx2, and Opn was generally higher compared to Control. ALP, RUNX2, and OPN were expressed at elevated levels in silenced groups, most notably at-MSCs-siR1/R2. ALP was detected at high levels in at-MSCs-siR1/R2 and in-MSCs-siR1, followed by an increase in mineralized nodules in at-MSCs-siR1/R2. As the morphometric parameters increased, the groups treated with KOR1/R2 exhibited slight bone formation near the edges of the defects. Endogenous TNF-α inhibits osteoblast differentiation and activity in at-MSCs, and its disruption increases bone formation. While opening a path of investigation, that may lead to the development of new treatments for bone regeneration using at-MSC-based therapies.

TNF-α activates RELA expression via TNFRSF1B to upregulate OPA1 expression and inhibit chondrogenic differentiation of human adipose stem cells

BackgroundTumor necrosis factor-alpha (TNF-α), one of the pro-inflammatory cytokines mediating the local inflammatory process in joints, inhibits cartilage formation and has a detrimental effect on stem cell-based cartilage regeneration for the treatment of osteoarthritis (OA). However, the mechanisms behind this inhibitory effect are still poorly understood. Mitochondrial morphological changes mediated by mitochondrial fusion and fission are highly plastic, are quite sensitive to environmental stimuli and play a crucial role in maintaining cell structure and function. In our study, chondrogenic differentiated human adipose stem cells (hADSCs) were exposed to TNF-α and the effect of TNF-α on the ability of hADSCs to chondrogenic differentiate and on mitochondrial fusion and fission was observed and analyzed. The aim was to investigate the role and mechanisms of mitochondrial fusion and fission regulation in the chondrogenic differentiation of hADSCs under normal conditions and under exposure to TNF-α.MethodsWe used flow cytometry to identify hADSCs immunophenotypes CD29, CD44, CD34, CD45, and HLA-DR. Alcian blue staining and Sirius red staining were used to observe the formation of proteoglycans and collagen during the chondrogenic differentiation of hADSCs, respectively. The mRNA and protein expression levels of the cartilage formation marker SOX9, type II collagen (COL2A1), and Aggrecan were measured by real-time fluorescent quantitative PCR (RT-qPCR) and western blot, respectively. The fluorescent probes MitoTracker® Red CMXRos and JC-1 were used to visualize mitochondria morphology and detect mitochondrial membrane electricity (MMP). Affymetrix PrimeView™ chips were used for gene expression profiling.ResultsThe results showed that the chondrogenic differentiation of hADSCs was inhibited in the presence of TNF-α that optic atrophy 1 (OPA1) expression was significantly upregulated and mitochondria were prolonged and interconnected during this process. Gene microarray and RT-qPCR data showed that the presence of TNF-α led to increased expression of TNFα receptor 2 (TNFRSF1B) and RELA during chondrogenic differentiation of hADSCs.ConclusionsTNF-α inhibits chondrogenic differentiation of human adipose stem cells by activating RELA expression through TNFRSF1B upregulating OPA1 expression thereby increasing mitochondrial fusion.

Features of changes in the expression level of TNFα receptors and the functional response of cell lines upon stimulation with various doses of cytokine

TNFa is a pro-inflammatory cytokine that is signaled through type 1 (TNFR1) and type 2 (TNFR2) receptors. TNFR1 normally mediates apoptosis, cell survival, and cytokine secretion, while TNFR2 selectively mediates cell survival and cytokine secretion. But in some cases, when receptors are activated, the functional response of cells changes to the opposite. Activation of signaling pathways has its own triggers, which differ in the interaction between different forms of cytokine and different forms of receptor complexes, as well as changes in the ratio of different types of receptors. The study of the mechanisms of regulation in the ligand-receptor system is a priority task for many studies. This work shows the dose-dependent effect of TNFa on the expression of cytokine receptors and changes in the functional response of tumor cell lines of various origins. For this, a comparative assessment of the expression and co-expression of receptors, cell cycle phases and apoptosis of cell lines without stimulation and stimulated with TNFa at concentrations of 5 and 50 ng/mL was carried out. It was found that the K562 cell line was characterized by more pronounced changes in receptor co-expression, which were observed at a TNFa concentration of 50 ng/mL compared to both the control group and the 5 ng/mL group. The decrease in the relative content of cells expressing only TNFR1 was combined with a decrease in the percentage of cells in apoptosis, which confirms the literature data on the role of this receptor in the development of apoptosis. At the same time, no changes in expression density were observed for this cell line. For the ZR75-1 cell line, the largest number of effects was also found for a TNFa concentration of 50 ng/mL. An increase in the relative content of cells expressing only TNFR2 was combined with an increase in apoptosis; however, the expression density of this type of receptor was low, which could affect the switching of signaling pathways towards proapoptotic ones. Thus, our study allowed us to reveal the features of changes in the expression and co-expression of TNFa receptors characteristic of cell lines of various origins, as well as changes in the functional response of cells in response to stimulation with different doses of cytokine. All this allows us to expand our understanding of the regulatory mechanisms in the cytokine-receptor system.