Modulation of Ten-Eleven Translocation 1 (TET1), Isocitrate Dehydrogenase (IDH) Expression, α-Ketoglutarate (α-KG), and DNA Hydroxymethylation Levels by Interleukin-1β in Primary Human Chondrocytes

5-Hydroxymethylcytosine (5-hmC) generated by ten-eleven translocation 1–3 (TET1–3) enzymes is an epigenetic mark present in many tissues with different degrees of abundance. IL-1β and TNF-α are the two major cytokines present in arthritic joints that modulate the expression of many genes associated with cartilage degradation in osteoarthritis. In the present study, we investigated the global 5-hmC content, the effects of IL-1β and TNF-α on 5-hmC content, and the expression and activity of TETs and isocitrate dehydrogenases in primary human chondrocytes. The global 5-hmC content was found to be ∼0.1% of the total genome. There was a significant decrease in the levels of 5-hmC and the TET enzyme activity upon treatment of chondrocytes with IL-1β alone or in combination with TNF-α. We observed a dramatic (10–20-fold) decrease in the levels of TET1 mRNA expression and a small increase (2–3-fold) in TET3 expression in chondrocytes stimulated with IL-1β and TNF-α. IL-1β and TNF-α significantly suppressed the activity and expression of IDHs, which correlated with the reduced α-ketoglutarate levels. Whole genome profiling showed an erasure effect of IL-1β and TNF-α, resulting in a significant decrease in hydroxymethylation in a myriad of genes including many genes that are important in chondrocyte physiology. Our data demonstrate that DNA hydroxymethylation is modulated by pro-inflammatory cytokines via suppression of the cytosine hydroxymethylation machinery. These data point to new mechanisms of epigenetic control of gene expression by pro-inflammatory cytokines in human chondrocytes.     

A negative feedback loop involving NF-κB/TIR8 regulates IL-1β-induced epithelial- myofibroblast transdifferentiation in human tubular cells

Renal tubular epithelial-myofibroblast transdifferentiation (EMT) plays a central role in the development of renal interstitial fibrosis (RIF). The profibrotic cytokine interleukin (IL)-1 and the IL-1 receptor (IL-1R) also participate in RIF development, and Toll/IL-1R 8 (TIR8), a member of the Toll-like receptor superfamily, has been identified as a negative regulator of IL-1R signaling. However, the functions of TIR8 in IL-1-induced RIF remain unknown. Here, human embryonic kidney epithelial cells (HKC) and unilateral ureteric obstruction (UUO)-induced RIF models on SD rats were used to investigate the functions of TIR8 involving IL-1β-induced EMT. We showed that IL-1β primarily triggers TIR8 expression by activating nuclear factor-κB (NF-κB) in HKC cells. Conversely, high levels of TIR8 in HKC cells repress IL-1β-induced NF-κB activation and inhibit IL-1β-induced EMT. Moreover, in vitro and in vivo findings revealed that TIR8 downregulation facilitated IL-1β-induced NF-κB activation and contributed to TGF-β1-mediated EMT in renal tubular epithelial cells. These results suggested that TIR8 exerts a protective role in IL-1β-mediated EMT and potentially represents a new target for RIF treatment.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00620-8.     

Butyrate inhibits IL-1β-induced inflammatory gene expression by suppression of NF-κB activity in pancreatic beta cells

Cytokine-induced beta cell dysfunction is a hallmark of type 2 diabetes (T2D). Chronic exposure of beta cells to inflammatory cytokines affects gene expression and impairs insulin secretion. Thus, identification of anti-inflammatory factors that preserve beta cell function represents an opportunity to prevent or treat T2D. Butyrate is a gut microbial metabolite with anti-inflammatory properties for which we recently showed a role in preventing interleukin-1β (IL-1β)-induced beta cell dysfunction, but how prevention is accomplished is unclear. Here, we investigated the mechanisms by which butyrate exerts anti-inflammatory activity in beta cells. We exposed mouse islets and INS-1E cells to a low dose of IL-1β and/or butyrate and measured expression of inflammatory genes and nitric oxide (NO) production. Additionally, we explored the molecular mechanisms underlying butyrate activity by dissecting the activation of the nuclear factor-κB (NF-κB) pathway. We found that butyrate suppressed IL-1β-induced expression of inflammatory genes, such as Nos2, Cxcl1, and Ptgs2, and reduced NO production. Butyrate did not inhibit IκBα degradation nor NF-κB p65 nuclear translocation. Furthermore, butyrate did not affect binding of NF-κB p65 to target sequences in synthetic DNA but inhibited NF-κB p65 binding and RNA polymerase II recruitment to inflammatory gene promoters in the context of native DNA. We found this was concurrent with increased acetylation of NF-κB p65 and histone H4, suggesting butyrate affects NF-κB activity via inhibition of histone deacetylases. Together, our results show butyrate inhibits IL-1β-induced inflammatory gene expression and NO production through suppression of NF-κB activation and thereby possibly preserves beta cell function.     

Study on the Mechanism of BMSCs in Regulating NF-κB Signal Pathway by Targeting miR-449a to Improve the Inflammatory Response to Peripheral Nerve Injury

Objective:To evaluate the mechanism of Bone Marrow Mesenchymal Stem Cells (BMSCs) in regulating NF-κB signal pathway by targeting miR-449a.Methods:Stem cells were transfected by over-expressing and inhibiting miR-449a to detect the levels and viability of miR-449a in stem cells after transfection. Stem cells and neurons were co-cultured in vitro to evaluate the in vitro mechanism of stem cells over-expressing miR-449a on neurons.Results:After the addition of neurons, the neuronal activity of miR-449a over-expression group increased significantly, the expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, and the inflammatory cytokines (TNF-α and IL-1β) decreased significantly (P<0.05). In vivo experiments in rats also showed that rats were unresponsive, did not chirp or elude after being stimulated. After stem cell therapy, the weight and response of rats gradually returned to normal levels. miR-449a expression significantly increased in the stem cell + miR-449a over-expression group, expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, inflammatory cytokines (TNF-α and IL-1β) significantly decreased, and cell activity significantly increased (P<0.05).Conclusions:BMSCs can modulate NF-κB signaling pathway by targeting miR-449a, so as to reduce the inflammatory response to peripheral nerve injury and repair nerve injury.     

Abietic acid attenuates sepsis-induced lung injury by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway to inhibit M1 macrophage polarization

Lung injury is one of the leading causes of death in sepsis. Abietic acid (AA) has demonstrated anti-inflammatory and bacteriostatic properties. Herein, we established a mouse model of sepsis by cecal ligation and puncture, and intraperitoneally injected AA to treat. Lung injury was assessed by H&E staining and the inflammation in bronchoalveolar lavage fluid (BALF) were assessed by counting the number of inflammatory cells and detecting the content of inflammatory factors. Meanwhile, we also designed to study the effect of AA on lipopolysaccharide (LPS)-induced inflammatory response and macrophage marker gene expression in RAW264.7 cells in vitro. In this study, we found that AA inhibited LPS-induced secretion of inflammatory mediators (IL-1β, TNF-α, IL-6 and MIP-2), and decreased the expression of M1 macrophage e markers (CD16 and iNOS) and p-p65 protein, while increased the expression of M2 macrophage markers (CD206 and Arg-1) in RAW264.7 cells in vitro. In vivo, the therapy of AA not only rescued septic animals, but also attenuated lung injury in sepsis mice. Moreover, AA decreased the number of total cells, neutrophils and macrophages, the conceration of total protein, and the levels of inflammatory mediators in BALF of sepsis mice. Further, we found that AA inhibited M1 macrophage polarization and blocked nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in BALF of sepsis mice. In conclusion, Abietic acid attenuates sepsis-induced lung injury, and its mechanism may be related to reducing inflammation by inhibiting NF-κB signaling to inhibit M1 macrophage polarization.     

The relationship between osteoclastogenic and anti-osteoclastogenic pro-inflammatory cytokines differs in human osteoporotic and osteoarthritic bone tissues

Background

Pro-inflammatory cytokines possess osteoclastogenic or anti-osteoclastogenic activities. They influence osteoclasts directly or via the receptor activator of nuclear factor κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) system. Recent evidence suggests that inflammation may play a role in osteoporosis (OP) and osteoarthritis (OA). We aimed therefore to determine whether there is a difference between both groups: first, in the expression of the osteoclastogenic and anti-osteoclastogenic cytokines, second, in correlation of these cytokines with bone mineral density (BMD) and levels of bone turnover markers (BTM) and third, in correlation between the expression of these cytokines and osteoclast specific genes and RANK/RANKL/OPG genes.

Methods

Human bone samples from 54 age and sex matched patients with OP or OA were collected during hip arthroplasty surgery. The expression of 25 genes encoding pro-inflammatory cytokines, their receptors, osteoclast specific genes and RANK/RANKL/OPG genes was measured using quantitative real-time PCR. Total hip, femoral neck and lumbar spine BMD and BTM in blood samples were measured. The comparison between OP and OA was assessed using Student''s t-test or Mann-Whitney U test and correlations between gene expression, BMD and BTM were determined using nonparametric correlation.

Results

The results demonstrated a higher expression of interleukin (IL)-6 and IL-1α in OP, and interferon (IFN)-γ in OA (p < 0.0005). Negative correlations of total hip BMD with tumor necrosis factor-α (TNF-α) in OA and with RANKL/RANK in OP were found (p < 0.05). Significant correlations with BTM were shown for IL-1α and IFN-γ in OP (rho = 0.608 and -0.634) and for TNF-α, IL-6 and transforming growth factor-β1 (TGF-β1) in OA (rho = 0.591, -0.521 and 0.636). Results showed OP specific negative correlations (IFN-γ with ITGB3, IFN-β1 with CTSK, tartrate resistant acid phosphatase (TRAP), CALCR, RANK, RANKL, IL-1α with CTSK, OPG, IL-17A with CALCR) and positive (TGF-β1 with CTSK, TRAP, RANK), and OA specific negative (IL-1α with osteoclast associated immunoglobulin-like receptor (OSCAR), TNF-α with RANK, RANKL, OPG) and positive (IL-6 with RANK, RANKL, OPG) correlations.

Conclusions

Our results demonstrate that the relationship between osteoclastogenic and anti-osteoclastogenic pro-inflammatory cytokines differs in human OP and OA bone and could present an important factor for characteristics of OP and OA bone phenotypes.     

Indacaterol Inhibits Tumor Cell Invasiveness and MMP-9 Expression by Suppressing IKK/NF-κB Activation

The β₂ adrenergic receptor (ADRB2) is a G protein-coupled transmembrane receptor expressed in the human respiratory tract and widely recognized as a pharmacological target for treatments of asthma and chronic obstructive pulmonary disorder (COPD). Although a number of ADRB2 agonists have been developed for use in asthma therapy, indacaterol is the only ultra-long-acting inhaled β₂-agonist (LABA) approved by the FDA for relieving the symptoms in COPD patients.The precise molecular mechanism underlying the pharmacological effect of indacaterol, however, remains unclear. Here, we show that β-arrestin-2 mediates the internalization of ADRB2 following indacaterol treatment. Moreover, we demonstrate that indacaterol significantly inhibits tumor necrosis factor-α (TNF-α)-induced NF-κB activity by reducing levels of both phosphorylated-IKK and -IκBα, thereby decreasing NF-κB nuclear translocation and the expression of MMP-9, an NF-κB target gene. Subsequently, we show that indacaterol significantly inhibits TNF-α/NF-κB-induced cell invasiveness and migration in a human cancer cell line. In conclusion, we propose that indacaterol may inhibit NF-κB activity in a β-arrestin2-dependent manner, preventing further lung damage and improving lung function in COPD patients.     

TNF-α Suppresses Apelin Receptor Expression in Mouse Quadriceps Femoris-Derived Cells

Expression of the apelin receptor, APJ, in skeletal muscle (SM) is known to decrease with age, but the underlying mechanism remains unclear. Increased tumor necrosis factor (TNF)-α levels are observed in SM with age and are associated with muscle atrophy. To investigate the possible interconnection between TNF-α elevation and APJ reduction with aging, we investigated the effect of TNF-α on APJ expression in cells derived from the quadriceps femoris of C57BL/6J mice. Expression of Tnfa and Apj in the quadriceps femoris was compared between 4- (young) and 24-month-old (old) C57BL/6J mice (n = 10 each) using qPCR. Additionally, APJ-positive cells and TNF-α protein were analyzed by flow cytometry and Western blotting, respectively. Further, quadricep-derived cells were exposed to 0 (control) or 25 ng/mL TNF-α, and the effect on Apj expression was examined by qRT-PCR. Apj expression and the ratio of APJ-positive cells among quadricep cells were significantly lower in old compared to young mice. In contrast, levels of Tnfa mRNA and TNF-α protein were significantly elevated in old compared to young mice. Exposing young and old derived quadricep cells to TNF-α for 8 and 24 h caused Apj levels to significantly decrease. TNF-α suppresses APJ expression in muscle cells in vitro. The increase in TNF-α observed in SM with age may induce a decrease in APJ expression.     

The Relationship Between the Tumor Cell Expression of Hypoxic Markers and Survival in Patients With ER-positive Invasive Ductal Breast Cancer

The prognostic significance of hypoxia markers, hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), and carbonic anhydrase IX (CAIX), was investigated in estrogen receptor (ER)-positive breast cancer patients. Immunohistochemistry determined the expression of makers in two independent ductal ER-positive cohorts (Training set, n=373 and Validation set, n=285) and was related to clinicopathological parameters and disease-free survival (DFS). In the training cohort, nuclear HIF-1α (1) was independently associated with poorer DFS in luminal A tumors [hazard ratio (HR) = 0.53 95% confidence interval (CI): 0.30–0.94, p=0.030]. In the validation cohort, both HIF-1α (1) and CAIX were independently associated with decreased DFS in the entire cohort (HR = 1.85 95% CI: 1.10–3.11, p=0.019; HR = 1.74 95% CI: 1.08–2.82, p=0.023), in luminal A disease (HR = 1.98 95% CI: 1.02–3.83, p=0.042), and in luminal B disease (HR = 2.75 95% CI: 1.66–4.55, p<0.001), respectively. Taken together, elevated cytoplasmic HIF-1α (1) expression was an independent prognostic factor in luminal A disease, whereas CAIX was an independent prognostic factor in luminal B disease. Further work in large tissue cohorts is required.     

Circulating nuclear factor-kappa B mediates cancer-associated inflammation in human breast and colon cancer

BackgroundInflammation is recognized as a hallmark feature of cancer development and progression. The aim of our study was to investigate the significance of serum nuclear factor kappa-B (NF-κB) levels as a circulating marker in the monitoring of inflammation in breast and colon cancer; to show the relationship between NF-κB with inflammatory parameters as tumour necrosis factor-α (TNF-α), soluble TNF-related apoptosis-inducing ligand (sTRAIL), interleukin-6 (IL-6), pentraxin-3 (PTX-3), procalcitonin (PCT), and C-reactive protein (CRP) levels.MethodsSerum NF-κB, TNF-α, sTRAIL, IL-6, PTX-3, PCT, and serum CRP levels were measured using enzyme-linked immunosorbent assay (ELISA) in 40 patients with breast cancer, 40 patients with colon cancer and 30 healthy controls.ResultsThe serum NF-κB, TNF-α, IL-6, PTX-3, PCT, and serum CRP concentration was significantly higher, and the serum sTRAIL concentration was significantly lower in the patients with breast and colon cancer than in healthy controls. NF-κB was positively correlated with CRP and negatively correlated with sTRAIL.ConclusionsThese results suggest that increased NF-κB may decrease the clinical efficacy of sTRAIL in solid tumour cells. There is a relationship between inflammation and carcinogenesis so that the development of cancer occurs with chronic inflammation in breast and colon. The study results have shown that colon and breast cancer patients have increased systemic inflammation, as measured by increased circulating cytokines, and acute-phase proteins, or by abnormalities in circulating cells. NF-κB may combine with other markers of the systemic inflammatory response in prognostic scores in cancer. In addition to surgical resection of the tumour, and conventional radio and chemotherapy for cancer treatment, the use of sTRAIL or other agonists for cancer therapy appeared a new potential therapy.     

Melatonin reverses tumor necrosis factor-alpha-induced metabolic disturbance of human nucleus pulposus cells via MTNR1B/Gαi2/YAP signaling

Background: Intervertebral disc degeneration (IDD), the main cause of low back pain, is closely related to the inflammatory microenvironment in the nucleus pulposus (NP). Tumor necrosis factor-α (TNF-α) plays an important role in inflammation-related metabolic disturbance of NP cells. Melatonin has been proven to regulate the metabolism of NP cells, but whether it can protect NP cells from TNF-α-induced damage is still unclear. Therefore, this study aims to investigate the role and specific mechanism of melatonin on regulating the metabolism of NP cells in the inflammatory microenvironment.Methods: Western blotting, RT-qPCR and immunohistochemistry were used to detect the expression of melatonin membrane receptors (MTNR1A/B) and TNF-α in human NP tissues. In vitro, human primary NP cells were treated with or without vehicle, TNF-α and melatonin. And the metabolic markers were also detected by western blotting and RT-qPCR. The activity of NF-κB signaling and Hippo/YAP signaling were assessed by western blotting and immunofluorescence. Membrane receptors inhibitors, pathway inhibitors, lentiviral infection, plasmids transfection and immunoprecipitation were used to explore the specific mechanism of melatonin. In vivo, the rat IDD model was constructed and melatonin was injected intraperitoneally to evaluate its therapeutical effect on IDD.Results: The upregulation of TNF-α and downregulation of melatonin membrane receptors (MTNR1A/B) were observed in degenerative NP tissues. Then we demonstrated that melatonin could alleviate the development of IDD in a rat model and reverse TNF-α-impaired metabolism of NP cells in vitro. Further investigation revealed that the protective effects of melatonin on NP cells mainly rely on MTNR1B, which subsequently activates Gαi2 protein. The activation of Gαi2 could upregulate the yes-associated protein (YAP) level, resulting in anabolic enhancement of NP cells. In addition, melatonin-mediated YAP upregulation increased the expression of IκBα and suppressed the TNF-α-induced activation of the NF-κB pathway, thereby inhibiting the catabolism of NP cells.Conclusions: Our results revealed that melatonin can reverse TNF-α-impaired metabolism of NP cells via the MTNR1B/Gαi2/YAP axis and suggested that melatonin can be used as a potential therapeutic drug in the treatment of IDD.     

IKKalpha, a critical regulator of epidermal differentiation and a suppressor of skin cancer

IκB kinase α (IKKα), one of the two catalytic subunits of the IKK complex involved in nuclear factor κB (NF-κB) activation, also functions as a molecular switch that controls epidermal differentiation. This unexpected function requires IKKα nuclear translocation but does not depend on its kinase activity, and is independent of NF-κB signalling. Ikkα^–/– mice present with a hyperproliferative and undifferentiated epidermis characterized by complete absence of a granular layer and stratum corneum. Ikkα-deficient keratinocytes do not express terminal differentiation markers and continue to proliferate even when subjected to differentiation-inducing stimuli. This antiproliferative function of IKKα is also important for the suppression of squamous cell carcinogenesis. The exact mechanisms by which nuclear IKKα controls keratinocyte proliferation and differentiation remained mysterious for some time. Recent studies, however, have revealed that IKKα is a major cofactor in a TGFβ–Smad2/3 signalling pathway that is Smad4 independent. This pathway controls cell cycle withdrawal during keratinocyte terminal differentiation. Although these are not the only functions of nuclear IKKα, this multifunctional protein is a key regulator of keratinocyte and epidermal differentiation and a critical suppressor of skin cancer.