IL-33 Is Involved in the Anti-Inflammatory Effects of Butyrate and Propionate on TNFα-Activated Endothelial Cells

Short-chain fatty acids (e.g., butyrate and propionate) are able to diminish endothelial cell activation. The aim of this study was to investigate whether intracellular IL-33 mediates the effects of butyrate and propionate on TNFα-induced IL-8 production and vascular cell adhesion molecule-1 (VCAM-1) expression. In addition, it was investigated whether regulating NF-κB and MAPK signaling pathways are involved. Intracellular IL-33 was measured in human endothelial cells (HUVECs) pre-incubated for 24 h with butyrate (0.1 mM or 5 mM), propionate (0.3 mM or 10 mM), or trichostatin A (TSA, 0.5 μM) prior to TNFα (1 ng/mL) stimulation (24 h). The effects of butyrate, propionate, and TSA on TNFα-induced IL-8, vascular cell adhesion molecule-1 (VCAM-1), NF-κB, and MAPK signaling pathways in normal HUVECs and IL-33 siRNA (siIL-33)-transfected HUVECs were compared to study the role of IL-33 in the protective effects of butyrate and propionate. Endogenous IL-33 was highly expressed in the perinuclear in HUVECs, which was significantly reduced by TNFα stimulation. The TNFα-induced reduction in IL-33 was prevented by pre-incubation with butyrate or propionate. Butyrate (0.1 mM), propionate (0.3 mM), and TSA inhibited the IL-8 production and activation of NF-κB. Interestingly, this effect was not observed in siIL-33-transfected HUVECs. The effects of butyrate (5 mM), propionate (10 mM), and TSA (0.5 μM) on VCAM-1 expression and activation of MAPK signaling pathways were not affected by siIL-33 transfection. In conclusion, we showed that the inhibitory effects of butyrate and propionate on TNFα-induced IL-8 production were mediated by the HDACs/IL-33/NF-κB pathway, while their effects on VCAM-1 expression might be associated with the HDACs/MAPK signaling pathway, independently of IL-33.     

Internalization of Neutrophil-Derived Microvesicles Modulates TNFα-Stimulated Proinflammatory Cytokine Production in Human Fibroblast-Like Synoviocytes

Neutrophil-derived microvesicles (NDMVs) have the potential to exert anti-inflammatory effects. Our study aimed to explore the effects of NDMVs on proinflammatory cytokines expressed by tumor necrosis factor α (TNFα)-stimulated fibroblast-like synoviocytes (FLS). FLS were isolated from the synovium of knee osteoarthritis (OA) patients undergoing surgery. NDMVs, isolated from TNFα-stimulated healthy neutrophils, were characterized by electron microscopy and nanoparticle tracking analysis. MTT and scratch wound healing assays were used to measure FLS viability and migration after treatment with NDMVs, while internalization of fluorescently labeled NDMVs was appraised by flow cytometry and confocal microscopy. Levels of proinflammatory cytokines in supernatants were quantified by the Bio-Plex system. Incubation of FLS with NDMVs at a vesicle/cell ratio of 100 resulted in a time-dependent uptake, with 35% of synoviocytes containing microvesicles over a 6–24 h time period, with no significant change in cell viability. TNFα stimulated the cytokine expression in FLS, and NDMVs down-regulated TNFα-induced expression of IL-5, IL-6, IL-8, MCP-1, IFNγ and MIP-1β. However, this down-regulation was selective, as NDMVs had no significant effects on TNFα-stimulated expression of IL-2 or IL-4. NDMVs were internalized by FLS to inhibit TNFα-stimulated broad-spectrum proinflammatory cytokine secretion. NDMVs, therefore, may exhibit an anti-inflammatory role in the regulation of the FLS function.     

Preconceptional Immunization Can Modulate Offspring Intrathymic IL-17-Producing γδT Cells with Epigenetic Implications Mediated by microRNAs

The mechanisms through which maternal immunization can modulate offspring thymic maturation of lymphocytes are not fully understood. Here, we aimed to evaluate whether maternal OVA-immunization can inhibit the maturation of IL-17-producing γδT cells in offspring thymus, and if this mechanism has epigenetic implications mediated by microRNAs (miRNAs) expression. Wild-type (WT) C57BL/6 females were immunized with OVA in Alum or Alum alone and were mated with normal WT males. Evaluating their offspring thymus at 3 or 20 days old (d.o.), we observed that maternal OVA immunization could inhibit the thymic frequency of offspring CD27- and IL-17⁺ γδT cells at the neonatal and until 20 days old. Furthermore, we evaluated the expression of function-related γ and δ variable γδTCR chains (Vγ1, Vγ2, Vγ3, Vδ4, and Vδ6.3), observing that maternal OVA-immunization inhibits Vγ2 chains expression. The small RNAs (sRNAs), particularly miRNAs, and messenger RNAs (mRNA) expression profiles by pools of thymus tissue samples (from 9 to 11 mice) from offspring OVA-immunized or Alum-immunized mothers were analyzed via Illumina sequencing platform and bioinformatics approaches. Using a fold change >4, our results showed that seven miRNAs (mmu-miR-126a-3p, 101a-3p, 744-3p,142-5p, 15a-5p, 532-5p, and 98-5p) were differentially expressed between both groups. Ten target genes were predicted to interact with the seven selected miRNAs. There were no enriched categories of gene ontology functional annotation and pathway enrichment analysis for the target genes. Interestingly, four of the identified miRNAs (mmu-miR-15a, mmu-miR-101 mmu-miR-126, and mmu-miR-142) are related to IL-17 production. Our data is of significance because we demonstrate that maternal immunization can modulate offspring thymic maturation of IL-17-producing γδT cells possibly by an epigenetic mechanism mediated by miRNAs.     

Atorvastatin Attenuates Programmed Death Ligand-1 (PD-L1) Induction in Human Hepatocellular Carcinoma Cells

Liver cancer is the sixth most common cancer worldwide with high morbidity and mortality. Programmed death ligand 1 (PD-L1) is a major ligand of programmed death 1 receptor (PD1), and PD1/PD-L1 checkpoint acts as a negative regulator of the immune system. Cancers evade the host’s immune defense via PD-L1 expression. This study aimed to investigate the effects of tumor-related cytokines, interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα) on PD-L1 expression in human hepatocellular carcinoma cells, HepG2. Furthermore, as atorvastatin, a cholesterol-lowering agent, is documented for its immunomodulatory properties, its effect on PD-L1 expression was investigated. In this study, through real-time RT-PCR, Western blot, and immunocytochemistry methods, PD-L1 expression in both mRNA and protein levels was found to be synergistically upregulated in HepG2 by a combination of IFNγ and TNFα, and STAT1 activation was mainly responsible for that synergistic effect. Next, atorvastatin can inhibit the induction of PD-L1 by either IFNγ alone or IFNγ/TNFα combination treatment in HepG2 cells. In conclusion, in HepG2 cells, expression of PD-L1 was augmented by cytokines in the tumor microenvironment, and the effect of atorvastatin on tumor immune response through inhibition of PD-L1 induction should be taken into consideration in cancer patients who have been prescribed atorvastatin.     

A Chimeric IL-15/IL-15Rα Molecule Expressed on NFκB-Activated Dendritic Cells Supports Their Capability to Activate Natural Killer Cells

Natural killer (NK) cells, members of the innate immune system, play an important role in the rejection of HLA class I negative tumor cells. Hence, a therapeutic vaccine, which can activate NK cells in addition to cells of the adaptive immune system might induce a more comprehensive cellular response, which could lead to increased tumor elimination. Dendritic cells (DCs) are capable of activating and expanding NK cells, especially when the NFκB pathway is activated in the DCs thereby leading to the secretion of the cytokine IL-12. Another prominent NK cell activator is IL-15, which can be bound by the IL-15 receptor alpha-chain (IL-15Rα) to be transpresented to the NK cells. However, monocyte-derived DCs do neither secrete IL-15, nor express the IL-15Rα. Hence, we designed a chimeric protein consisting of IL-15 and the IL-15Rα. Upon mRNA electroporation, the fusion protein was detectable on the surface of the DCs, and increased the potential of NFκB-activated, IL-12-producing DC to activate NK cells in an autologous cell culture system with ex vivo-generated cells from healthy donors. These data show that a chimeric IL-15/IL-15Rα molecule can be expressed by monocyte-derived DCs, is trafficked to the cell surface, and is functional regarding the activation of NK cells. These data represent an initial proof-of-concept for an additional possibility of further improving cellular DC-based immunotherapies of cancer.     

Generation and Characterization of a Zebrafish IL-2Rγc SCID Model

The IL-2 family of cytokines act via receptor complexes that share the interleukin-2 receptor gamma common (IL-2Rγc) chain to play key roles in lymphopoiesis. Inactivating IL-2Rγc mutations results in severe combined immunodeficiency (SCID) in humans and other species. This study sought to generate an equivalent zebrafish SCID model. The zebrafish il2rga gene was targeted for genome editing using TALENs and presumed loss-of-function alleles analyzed with respect to immune cell development and impacts on intestinal microbiota and tumor immunity. Knockout of zebrafish Il-2rγc.a resulted in a SCID phenotype, including a significant reduction in T cells, with NK cells also impacted. This resulted in dysregulated intestinal microbiota and defective immunity to tumor xenotransplants. Collectively, this establishes a useful zebrafish SCID model.     

TGF-β/IL-7 Chimeric Switch Receptor-Expressing CAR-T Cells Inhibit Recurrence of CD19-Positive B Cell Lymphoma

Chimeric antigen receptor (CAR)-T cells are effective in the treatment of hematologic malignancies but have shown limited efficacy against solid tumors. Here, we demonstrated an approach to inhibit recurrence of B cell lymphoma by co-expressing both a human anti-CD19-specific single-chain variable fragment (scFv) CAR (CD19 CAR) and a TGF-β/IL-7 chimeric switch receptor (tTRII-I7R) in T cells (CD19 CAR-tTRII-I7R-T cells). The tTRII-I7R was designed to convert immunosuppressive TGF-β signaling into immune-activating IL-7 signaling. The effect of TGF-β on CD19 CAR-tTRII-I7R-T cells was assessed by western blotting. Target-specific killing by CD19 CAR-tTRII-I7R-T cells was evaluated by Eu-TDA assay. Daudi tumor-bearing NSG (NOD/SCID/IL2Rγ^-/-) mice were treated with CD19 CAR-tTRII-I7R-T cells to analyze the in vivo anti-tumor effect. In vitro, CD19 CAR-tTRII-I7R-T cells had a lower level of phosphorylated SMAD2 and a higher level of target-specific cytotoxicity than controls in the presence of rhTGF-β1. In the animal model, the overall survival and recurrence-free survival of mice that received CD19 CAR-tTRII-I7R-T cells were significantly longer than in control mice. These findings strongly suggest that CD19 CAR-tTRII-I7R-T cell therapy provides a new strategy for long-lasting, TGF-β-resistant anti-tumor effects against B cell lymphoma, which may lead ultimately to increased clinical efficacy.     

The Dual Role of the β2-Adrenoreceptor in the Modulation of IL-17 and IFN-γ Production by T Cells in Multiple Sclerosis

Norepinephrine is a neurotransmitter that also has an immunomodulatory effect and is involved in multiple sclerosis (MS) pathogenesis. This study aimed to clarify the role of the β₂-adrenoreceptor in the norepinephrine-mediated modulation of interleukin-17 (IL-17) and interferon-γ (IFN-γ) production, which play a critical pathogenetic role in MS. CD4⁺ T cells obtained from twenty-five relapsing-remitting MS patients and sixteen healthy subjects were cultured ex vivo with norepinephrine and/or β₂-adrenoreceptor antagonist or agonist, followed by a cytokine production analysis using ELISA. Norepinephrine suppressed IL-17 and IFN-γ production by the anti-CD3/anti-CD28-microbead-stimulated CD4⁺ T cells in both groups. Blockade of the β₂-adrenoreceptor with the specific antagonist ICI 118.551 enhanced norepinephrine-mediated IL-17 suppression but decreased its inhibitory effect on IFN-γ production in MS patients. In contrast, the β₂-adrenoreceptor agonist formoterol did not influence norepinephrine’s inhibitory effect on cytokine production in both groups. The blockade of the β₂-adrenoreceptor, even in the absence of exogenous norepinephrine, suppressed IL-17 production but did not influence IFN-γ production in both groups. Conversely, β₂-adrenoreceptor activation by formoterol decreased IFN-γ production and did not affect IL-17 production in both groups. These data illustrate the inhibitory effect of norepinephrine on IL-17 and IFN-γ production by CD4⁺ T cells in MS. The inhibitory effect of norepinephrine on IFN-γ production by CD4⁺ T cells in MS could be mediated via β₂-adrenoreceptor activation.     

IFN-Gamma and TNF-Alpha as a Priming Strategy to Enhance the Immunomodulatory Capacity of Secretomes from Menstrual Blood-Derived Stromal Cells

Mesenchymal stromal cells isolated from menstrual blood (MenSCs) exhibit a potent pro-angiogenic and immunomodulatory capacity. Their therapeutic effect is mediated by paracrine mediators released by their secretomes. In this work, we aimed to evaluate the effect of a specific priming condition on the phenotype and secretome content of MenSCs. Our results revealed that the optimal condition for priming MenSCs was the combination of interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) that produced a synergistic and additive effect on IDO1 release and immune-related molecule expression. The analyses of MenSC-derived secretomes after IFNγ and TNFα priming also revealed an increase in EV release and in the differentially expressed miRNAs involved in the immune response and inflammation. Proliferation assays on lymphocyte subsets demonstrated a decrease in CD4+ T cells and CD8+ T cells co-cultured with secretomes, especially in the lymphocytes co-cultured with secretomes from primed cells. Additionally, the expression of immune checkpoints (PD-1 and CTLA-4) was increased in the CD4+ T cells co-cultured with MenSC-derived secretomes. These findings demonstrate that the combination of IFNγ and TNFα represents an excellent priming strategy to enhance the immunomodulatory capacity of MenSCs. Moreover, the secretome derived from primed MenSCs may be postulated as a therapeutic option for the regulation of adverse inflammatory reactions.     

Pro- and Anti-Inflammatory Cytokines in the Context of NK Cell–Trophoblast Interactions

During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFβ and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFβ, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFβ, their cytotoxicity increased. In the case of adding TGFβ to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFβ in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction.     

Cord Blood T Cells Expressing High and Low PKCζ Levels Develop into Cells with a Propensity to Display Th1 and Th9 Cytokine Profiles,Respectively

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA⁺ to CD45RO⁺ cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-β) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases.     

MAO-A Inhibition by Metaxalone Reverts IL-1β-Induced Inflammatory Phenotype in Microglial Cells

Experimental and clinical studies have suggested that several neurological disorders are associated with the occurrence of central nervous system neuroinflammation. Metaxalone is an FDA-approved muscle relaxant that has been reported to inhibit monoamine oxidase A (MAO-A). The aim of this study was to investigate whether metaxalone might exert antioxidant and anti-inflammatory effects in HMC3 microglial cells. An inflammatory phenotype was induced in HMC3 microglial cells through stimulation with interleukin-1β (IL-1β). Control cells and IL-1β-stimulated cells were subsequently treated with metaxalone (10, 20, and 40 µM) for six hours. IL-1β stimulated the release of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), but reduced the anti-inflammatory cytokine interleukin-13 (IL-13). The upstream signal consisted of an increased priming of nuclear factor-kB (NF-kB), blunted peroxisome proliferator-activated receptor gamma (PPARγ), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression. IL-1β also augmented MAO-A expression/activity and malondialdehyde levels and decreased Nrf2 mRNA expression and protein levels. Metaxalone decreased MAO-A activity and expression, reduced NF-kB, TNF-α, and IL-6, enhanced IL-13, and also increased PPARγ, PGC-1α, and Nrf2 expression. The present experimental study suggests that metaxalone has potential for the treatment of several neurological disorders associated with neuroinflammation.     

Deciphering Repertoire of B16 Melanoma Reactive TCRs by Immunization,In Vitro Restimulation and Sequencing of IFNγ-Secreting T Cells

Recent advances in cancer immunotherapy have great promise for the treatment of solid tumors. One of the key limiting factors that hamper the decoding of physiological responses to these therapies is the inability to distinguish between specific and nonspecific responses. The identification of tumor-specific lymphocytes is also the most challenging step in cancer cell therapies such as adoptive cell transfer and T cell receptor (TCR) cloning. Here, we have elaborated a protocol for the identification of tumor-specific T lymphocytes and the deciphering of their repertoires. B16 melanoma engraftment following anti-PD1 checkpoint therapy provides better antitumor immunity compared to repetitive immunization with heat-shocked tumor cells. We have also revealed that the most error-prone part of dendritic cell (DC) generation, i.e., their maturation step, can be omitted if DCs are cultured at a sufficiently high density. Using this optimized protocol, we have achieved a robust IFNγ response to B16F0 antigens, but only within CD4+ T helper cells. A comparison of the repertoires of IFNγ-positive and -negative cells shows a prominent enrichment of certain clones with putative tumor specificity among the IFNγ+ fraction. In summary, our optimized protocol and the data provided here will aid in the acquisition of broad statistical data and the creation of a meaningful database of B16-specific TCRs.     

CCL4 Stimulates Cell Migration in Human Osteosarcoma via the mir-3927-3p/Integrin αvβ3 Axis

Osteosarcoma is the most common type of primary malignant bone cancer, and it is associated with high rates of pulmonary metastasis. Integrin αvβ3 is critical for osteosarcoma cell migratory and invasive abilities. Chemokine (C-C motif) ligand 4 (CCL4) has diverse effects on different cancer cells through its interaction with its specific receptor, C-C chemokine receptor type 5 (CCR5). Analysis of mRNA expression in human osteosarcoma tissue identified upregulated levels of CCL4, integrin αv and β3 expression. Similarly, an analysis of records from the Gene Expression Omnibus (GEO) dataset showed that CCL4 was upregulated in human osteosarcoma tissue. Importantly, the expression of both CCL4 and integrin αvβ3 correlated positively with osteosarcoma clinical stages and lung metastasis. Analysis of osteosarcoma cell lines identified that CCL4 promotes integrin αvβ3 expression and cell migration by activating the focal adhesion kinase (FAK), protein kinase B (AKT), and hypoxia inducible factor 1 subunit alpha (HIF-1α) signaling pathways, which can downregulate microRNA-3927-3p expression. Pharmacological inhibition of CCR5 by maraviroc (MVC) prevented increases in integrin αvβ3 expression and cell migration. This study is the first to implicate CCL4 as a potential target in the treatment of metastatic osteosarcoma.     

Strategies to Improve the Antitumor Effect of γδ T Cell Immunotherapy for Clinical Application

Human γδ T cells show potent cytotoxicity against various types of cancer cells in a major histocompatibility complex unrestricted manner. Phosphoantigens and nitrogen-containing bisphosphonates (N-bis) stimulate γδ T cells via interaction between the γδ T cell receptor (TCR) and butyrophilin subfamily 3 member A1 (BTN3A1) expressed on target cells. γδ T cell immunotherapy is classified as either in vivo or ex vivo according to the method of activation. Immunotherapy with activated γδ T cells is well tolerated; however, the clinical benefits are unsatisfactory. Therefore, the antitumor effects need to be increased. Administration of γδ T cells into local cavities might improve antitumor effects by increasing the effector-to-target cell ratio. Some anticancer and molecularly targeted agents increase the cytotoxicity of γδ T cells via mechanisms involving natural killer group 2 member D (NKG2D)-mediated recognition of target cells. Both the tumor microenvironment and cancer stem cells exert immunosuppressive effects via mechanisms that include inhibitory immune checkpoint molecules. Therefore, co-immunotherapy with γδ T cells plus immune checkpoint inhibitors is a strategy that may improve cytotoxicity. The use of a bispecific antibody and chimeric antigen receptor might be effective to overcome current therapeutic limitations. Such strategies should be tested in a clinical research setting.     

Expression of IL-8, IL-6 and IL-1β in Tears as a Main Characteristic of the Immune Response in Human Microbial Keratitis

Corneal infections are frequent and potentially vision-threatening diseases, and despite the significance of the immunological response in animal models of microbial keratitis (MK), it remains unclear in humans. The aim of this study was to describe the cytokine profile of tears in patients with MK. Characteristics of ocular lesions such as size of the epithelial defect, stromal infiltration, and hypopyon were analyzed. Immunological evaluation included determination of interleukine (IL)-1β, IL-6, IL-8, IL-10, IL-12 and tumor necrosis factor (TNF)-α in tear samples obtained from infected eyes of 28 patients with MK and compared with their contralateral non-infected eyes. Additionally, frequency of CD4⁺, CD8⁺, CD19⁺ and CD3⁻CD56⁺ cells was also determined in peripheral blood mononuclear cells in patients with MK, and compared with 48 healthy controls. Non-significant differences were observed in the size of the epithelial defect, stromal infiltration, and hypopyon. Nevertheless, we found an immunological profile apparently related to MK etiology. IL-8 > IL-6 in patients with bacterial keratitis; IL-8 > IL-6 > IL-1β and increased frequency of circulating CD3⁻CD56⁺ NK cells in patients with gram-negative keratitis; and IL-8 = IL-6 > IL-1β in patients with fungal keratitis. Characterization of tear cytokines from patients with MK could aid our understanding of the immune pathophysiological mechanisms underlying corneal damage in humans.     

TNFα-Induced LDL Cholesterol Accumulation Involve Elevated LDLR Cell Surface Levels and SR-B1 Downregulation in Human Arterial Endothelial Cells

Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. Here, the role of tumor necrosis factor alpha (TNFα) in modulating the low-density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. TNFα promoted an up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased ¹²⁵I-LDL surface binding. Using the non-hydrolysable label, Dil, TNFα could induce a massive increase in Dil-LDL by over 200 folds. The elevated intracellular Dil-LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), or apolipoprotein (apoE) depletion. Moreover, the TNFα-induced increase of LDL-derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil-LDL accumulation was reduced by over 99%. The effects of TNFα included an LDLR cell surface increase of 138%, and very large increases in ICAM-1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR-B1) was reduced. Additionally, LDLR antibody bound rapidly in TNFα-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNFα on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by inhibitors against NF-κB, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on Transwell inserts, TNFα did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNFα promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation.     

Adipose-Derived Extract Suppresses IL-1β-Induced Inflammatory Signaling Pathways in Human Chondrocytes and Ameliorates the Cartilage Destruction of Experimental Osteoarthritis in Rats

We investigated the effects of adipose-derived extract (AE) on cultured chondrocytes and in vivo cartilage destruction. AE was prepared from human adipose tissues using a nonenzymatic approach. Cultured human chondrocytes were stimulated with interleukin-1 beta (IL-1β) with or without different concentrations of AE. The effects of co-treatment with AE on intracellular signaling pathways and their downstream gene and protein expressions were examined using real-time PCR, Western blotting, and immunofluorescence staining. Rat AE prepared from inguinal adipose tissues was intra-articularly delivered to the knee joints of rats with experimental osteoarthritis (OA), and the effect of AE on cartilage destruction was evaluated histologically. In vitro, co-treatment with IL-1β combined with AE reduced activation of the p38 and ERK mitogen-activated protein kinase (MAPK) pathway and nuclear translocation of the p65 subunit of nuclear factor-kappa B (NF-κB), and subsequently downregulated the expressions of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, IL-6, and IL-8, whereas it markedly upregulated the expression of IL-1 receptor type 2 (IL-1R2) in chondrocytes. Intra-articular injection of homologous AE significantly ameliorated cartilage destruction six weeks postoperatively in the rat OA model. These results suggested that AE may exert a chondroprotective effect, at least in part, through modulation of the IL-1β-induced inflammatory signaling pathway by upregulation of IL-1R2 expression.