Cytokine-induced Expression Research Articles

Novel trisaccharide based phospholipids as immunomodulators

A focused library of novel mannosylated glycophospholipids was synthesized employing imidate coupling and H-phosphate phosphorylation methods. All novel glycophospholipids were evaluated for their receptor interactions by molecular docking studies. Docking studies revealed dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) specific interaction of the glycophospholipid ligand P4 acts, which was further confirmed by in vitro DC-SIGN expression on monocyte-derived dendritic cells (MoDCs). Further, in vitro and in vivo immunomodulatory activity among the six compounds (P1-P6) examined, compound P4 displayed good immunopotentiation and adjuvant properties as indicated by the induced cytokine expression and enhanced ovalbumin (OVA) specific antibody (IgG) titers. Phosphatidylinositol mannosides (PIMs) analogues in the present study enforced the immunomodulatory properties, truncating parent PIMs or tailor-made of PIMs may bring the novel efficacious molecules, which will be useful in vaccine preparation against different diseases.

S100A8 acts as an autocrine priming signal for heme-induced human Mϕ pro-inflammatory responses in hemolytic inflammation.

Intravascular hemolysis, in addition to reducing red cell counts, incurs extensive vascular inflammation and oxidative stress. One product of hemolysis, heme, is a potent danger associated molecular pattern (DAMP), activating leukocytes and inducing cytokine expression and processing, among other pro-inflammatory effects. We explored pathways by which heme-induced inflammation may be amplified under sterile conditions. Incubation of human Mϕs, differentiated from CD14+ cells, with heme induced time- and concentration-dependent gene and protein expression of S100A8, a myeloid cell-derived alarmin. Human Mϕ stimulation with recombinant S100A8, in turn, induced robust pro-IL-1β expression that was dependent upon NF-κB activation, gene transcription, and partially dependent upon TLR4-mediated signaling. Moreover, heme itself stimulated significant Mϕ pro-IL-1β gene and protein expression via an S100A8-mediated mechanism and greatly amplified S100A8-driven NLRP3 inflammasome-mediated IL-1β secretion. In vivo, induction of acute intravascular hemolysis in mice induced a rapid elevation of plasma S100A8 that could be abolished by hemopexin, a heme scavenger. Finally, plasma S100A8 levels were found to be significantly elevated in patients with the inherited hemolytic anemia, sickle cell anemia, when compared with levels in healthy individuals. In conclusion, we demonstrate that hemolytic processes are associated with S100A8 generation and that some of the inflammatory effects of heme may be amplified by autocrine S100A8 production. Findings suggest a mechanism by which hemolytic inflammation could be propagated via leukocyte priming by endogenous proteins, even in sterile inflammatory environments such as those that occur in the hemolytic diseases. S100A8 may represent a therapeutic target for reducing inflammation in hemolytic disorders.

Trace levels of peptidoglycan in serum underlie the NOD-dependent cytokine response to endoplasmic reticulum stress

NOD1 and NOD2 are intracellular sensors of bacterial peptidoglycan that belong to the Nod-like receptor family of innate immune proteins. In addition to their role as direct bacterial sensors, it was proposed that the nucleotide-binding oligomerization domain (NOD) proteins could detect endoplasmic reticulum (ER) stress induced by thapsigargin, an inhibitor of the sarcoplasmic or endoplasmic reticulum calcium ATPase family that pumps Ca2+ into the ER, resulting in pro-inflammatory signaling. Here, we confirm that thapsigargin induces NOD-dependent pro-inflammatory signaling in epithelial cells. However, the effect was specific to thapsigargin, as tunicamycin and the subtilase cytotoxin SubAB from Shiga toxigenic Escherichia coli, which induce ER stress by other mechanisms, did not induce cytokine expression. The calcium ionophore A23187 also induced NOD-dependent signaling, and calcium chelators demonstrated a role for both intracellular and extracellular calcium in mediating thapsigargin-induced and NOD-dependent pro-inflammatory signaling, in part through the activation of plasma membrane-associated calcium release-activated channels. Moreover, our results demonstrate that both endocytosis and the addition of serum to the cell culture medium were required for thapsigargin-mediated NOD activation. Finally, we analyzed cell culture grade fetal calf serum as well as serum from laboratory mice using HPLC and MS identified the presence of various peptidoglycan fragments. We propose that cellular perturbations that affect intracellular Ca2+ can trigger internalization of peptidoglycan trace contaminants found in culture serum, thereby stimulating pro-inflammatory signaling. The presence of peptidoglycan in animal serum suggests that a homeostatic function of NOD signaling may have been previously overlooked.

Berberine hydrochloride protects against cytokine-induced inflammation through multiple pathways in undifferentiated C2C12 myoblast cells.

Obesity is associated with skeletal muscle insulin resistance and the development of metabolic syndrome. Undifferentiated skeletal muscle cells are sensitive to oxidative stress. Berberine hydrochloride (BBR) improves insulin resistance and exhibits anti-inflammatory properties. However, the underlying mechanism and the cell signaling pathways involved remain largely elusive. We therefore investigated the anti-inflammatory effects of BBR and the signaling pathways using skeletal C2C12 myoblast cells. Undifferentiated C2C12 myoblast cells were treated with interleukin-1β alone or in combination with tumor necrosis factor-α in the presence or absence of BBR. We found that BBR reduced the cytokine-induced expression of inducible nitric oxide synthase and stress-related kinases including p-38 mitogen-activated protein kinase, nuclear factor kappa B (NF-κB), and stress-activated protein kinases/Jun amino-terminal kinases (SAPK/JNK) in C2C12 myoblast cells. Furthermore, BBR reversed cytokine-mediated suppression of AMP-activated protein kinase (AMPKα), sirtuin-1 (SIRT-1), and PPAR-γ coactivator-1α (PGC-1α). In addition, cytokine-induced reduction of mitochondrial marker proteins and function were rescued after BBR treatment. Catalase, an antioxidant enzyme, was elevated after BBR treatment. Our results demonstrate that BBR ameliorates cytokine-induced inflammation. The anti-inflammatory effect of BBR in skeletal progenitor cells is mediated through pathways including activation of the AMPKα-SIRT-1-PGC-1α, inhibition of the mitogen-activated protein kinase 4 (MKK4)-SAPK/JNK-C-JUN, as well as protection of mitochondrial bioenergetics. BBR may be a potential medication for metabolic syndrome.

Macrophage Migration Inhibitory Factor Promotes Chemotaxis of Astrocytes through Regulation of Cholesterol 25-Hydroxylase Following Rat Spinal Cord Injury

Oxysterol derived from cholesterol metabolism is involved in the inflammatory activation, and consequently in development of major chronic diseases. Multiple cytokines have been found to induce the expression of cholesterol metabolism-related enzymes. Several studies have shown that the protein level of cholesterol-25-hydroxylase (CH25H) is remarkably increased in response to injury of central nervous system (CNS), but little is known about the mechanisms of cytokine-induced expression of CH25H in specific cell types, and the resultant effects. In the present study, we demonstrated that ch25h expression was significantly upregulated in the astrocytes of rat injured spinal cord, in parallel with those of MIF. Administration of MIF inhibitor 4-IPP in the lesion sites attenuated injury-induced ch25h expression. MIF facilitated ch25h expression of astrocytes through interaction with CD74 membrane receptor, which in turn promoted production of chemokines, as identified by transcriptome profiles. MIF-induced release of oxysterol 25-hydroxycholesterol (25-HC) from astrocytes affects cell migration, but inhibited cell viability in dose-dependent manner, suggesting that MIF aggravates progressive neuropathology through regulation of cholesterol metabolism following CNS injury. These results have provided a novel mechanism and a potential therapeutic strategy for injured CNS.

The Proton Pump Inhibitor Lansoprazole Has Hepatoprotective Effects in In Vitro and In Vivo Rat Models of Acute Liver Injury.

The proton pump inhibitor lansoprazole (LPZ) is clinically used to reduce gastric acid secretion, but little is known about its possible hepatoprotective effects. This study aimed to investigate the hepatoprotective effects of LPZ and its potential mechanisms using in vitro and in vivo rat models of liver injury. For the in vitro model of liver injury, primary cultured rat hepatocytes were treated with interleukin-1β in the presence or absence of LPZ. The influence of LPZ on inducible nitric oxide synthase (iNOS) induction and nitric oxide (NO) production and on the associated signaling pathways was analyzed. For the in vivo model, rats were treated with D-galactosamine (GalN) and lipopolysaccharide (LPS). The effects of LPZ on survival and proinflammatory mediator expression (including iNOS and tumor necrosis factor-α) in these rats were examined. LPZ inhibited iNOS induction partially through suppression of the nuclear factor-kappa B signaling pathway in hepatocytes, thereby reducing potential liver injury from excessive NO levels. Additionally, LPZ increased survival by 50% and decreased iNOS, tumor necrosis factor-α, and cytokine-induced neutrophil chemoattractant-1 mRNA expression in the livers of GalN/LPS-treated rats. LPZ also inhibited nuclear factor-kappa B activation by GalN/LPS. LPZ inhibits the induction of several inflammatory mediators (including cytokines, chemokines, and NO) partially through suppression of nuclear factor-kappa B, resulting in the prevention of fulminant liver failure. The therapeutic potential of LPZ for liver injuries warrants further investigation.

Recruitment of Brd3 and Brd4 to acetylated chromatin is essential for proinflammatory cytokine-induced matrix-degrading enzyme expression

BackgroundProinflammatory cytokines, which can upregulate the expression of matrix-degrading enzymes in chondrocytes, play important roles in the development of osteoarthritis. BET family proteins, acting as the “readers” of acetylated modifications on histones, have been linked to transcriptional regulation. And a BET protein inhibitor, I-BET151, has been shown to inhibit the induction of matrix-degrading enzymes by proinflammatory cytokines in chondrocytes. Our objective is to clarify the role and mechanism of BET proteins on matrix-degrading enzyme gene expression by using a human chondrosarcoma cell line (SW1353).MethodsWe pretreated SW1353 cells with I-BET151 prior to treatment with IL-1β or TNF-α and then checked the expression of four matrix-degrading enzyme genes (MMP1, MMP3, MMP13, and ADAMTS4). We performed knockdown of BET protein family members (BRD2, BRD3, and BRD4) with corresponding siRNAs in SW1353 cells prior to treatment with IL-1β or TNF-α and checked the expression of the matrix-degrading enzyme genes. We evaluated Brd-mediated transcriptional regulation on the matrix-degrading enzyme genes by ChIP assay.ResultsWe confirmed that I-BET151 could suppress the IL-1β- or TNF-α-induced expression of MMP1, MMP3, MMP13, and ADAMTS4 in SW1353 cells. Brd3 and Brd4 were required for the IL-1β- or TNF-α-induced expression of matrix-degrading enzyme genes in SW1353 cells. We revealed that inducible acetylation of H4k5/8/12 and the recruitment of Brd3, Brd4, and p-TEFb to chromatin were involved in IL-1β- or TNF-α-induced transcription.ConclusionsOur findings suggested that Brd3 and Brd4 were essential for the IL-1β- or TNF-α-induced transcription of matrix-degrading enzyme genes, and recruitment of Brd3 and Brd4 to chromatin of these genes played the main role in this process.

14-3-3 Protein of Neospora caninum Modulates Host Cell Innate Immunity Through the Activation of MAPK and NF-κB Pathways.

Neospora caninum is an obligate intracellular apicomplexan parasite, the etiologic agent of neosporosis, and a major cause of reproductive loss in cattle. There is still a lack of effective prevention and treatment measures. The 14-3-3 protein is a widely expressed acidic protein that spontaneously forms dimers within apicomplexan parasites. This protein has been isolated and sequenced in many parasites; however, there are few reports about the N. caninum 14-3-3 protein. Here, we successfully expressed and purified a recombinant fusion protein of Nc14-3-3 (rNc14-3-3) and prepared a polyclonal antibody. Immunofluorescence and immunogold electron microscopy studies of tachyzoites or N. caninum-infected cells suggested that 14-3-3 was localized in the cytosol and the membrane. Western blotting analysis indicated that rNc14-3-3 could be recognized by N. caninum-infected mouse sera, suggesting that 14-3-3 may be an infection-associated antigen that is involved in the host immune response. We demonstrated that rNc14-3-3 induced cytokine expression by activating the MAPK and AKT signaling pathways, and inhibitors of p38, ERK, JNK, and AKT could significantly decrease the production of IL-6, IL-12p40, and TNF-α. In addition, phosphorylated nuclear factor-κB (NF-κB/p65) was observed in wild-type peritoneal macrophages (PMs) treated with rNc14-3-3, and the protein level of NF-κB/p65 was reduced in the cytoplasm but increased correspondingly in the nucleus after 2 h of treatment. These results were also observed in deficient in TLR2-/- PMs. Taken together, our results indicated that the N. caninum 14-3-3 protein can induce effective immune responses and stimulate cytokine expression by activating the MAPK, AKT, and NF-κB signaling pathways but did not dependent TLR2, suggesting that Nc14-3-3 is a novel vaccine candidate against neosporosis.

Are thermogenic proteins and adipokine chemerin affected by monoclonal antibody therapy in asthma?

BACKGROUND: Irisin is a thermogenic protein that sources outgoing energy by converting white adipose tissue to brown adipose tissue. Chemerin is originally identified as a chemoattractant protein that mainly mediating the chemotaxis of dendritic cells and natural killer cells (NKCs). The aim of this study is to assess the potential impact of immune modulation-related chemerin and irisin concentrations together with cell surface markers (CSM) in allergic asthmatic patients under omalizumab treatment. MATERIALS AND METHODS: The study participants were age- and sex-matched 30 healthy controls (Group I) and consecutive patients who had severe persistent asthma disease (Group II). Asthma patients took omalizumab treatment for 12 months within every 2 weeks. Flow cytometry analysis was used to evaluate CSM, enzyme-linked immunosorbent assay (ELISA) for interleukin-1 (IL-1) β expression. In addition, NK activity (NKA) and induced cytokine expression (by bioassay and ELISA, respectively) before and after omalizumab therapy were evaluated. RESULTS: Chemerin, irisin, and IL-1 β concentrations were significantly higher in severe persistent asthma patients compared to controls in serum (P = 0.01; P = 0.03; and P = 0.008, respectively). IL-1 β level decreased with treatment and it was statistically significant. Although levels decreased, no statistically significant difference was observed for Irisin, CD80, and CD56/16 levels. Chemerin level kept rising after treatment, and this was significant statistically. CONCLUSIONS: This is the first study to assess NKA and adipokines in asthma patients and their relationship with CSM. We observed that the level of these molecules is higher in asthma and is influenced by omalizumab treatment. Since no obvious change was observed for NKCs, omalizumab may be considered safe against cancer development.

Intravenous Immunoglobulin Controls Th17 Cell-Mediated Osteoclastogenesis.

The purpose of this study was to determine the regulatory role of intravenous Ig (IVIg) in Th17 cytokine–induced RANK ligand (RANKL) expression and osteoclast (OC) differentiation from OC precursors (pre-OC). Human CD14+ monocytes were isolated and stimulated by Th17 cytokines (IL-17, IL-21, and IL-22) and RANKL expression was investigated using a real-time PCR. CD14+ monocytes were incubated with RANKL, Th17 cytokines, and M-CSF, with/without IVIg, and OC differentiation was determined by counting tartrate-resistant acid phosphatase-positive multinucleated cells. OC differentiation was investigated after monocytes were cocultured with Th17 cells in the presence of IVIg. Th17 cell differentiation was determined using enzyme-linked immunosorbent assay and flow cytometry after CD4+ T cells were cultured with IVIg under Th17 condition. Th17 cytokines stimulated monocytes to express RANKL and IVIg suppressed the Th17 cytokine-induced RANKL expression. OCs were differentiated when pre-OC were cocultured with RANKL or Th17 cytokines and IVIg reduced the osteoclastogenesis. IVIg also decreased osteoclastogenesis when pre-OC were cocultured with Th17 cells. IVIg decreased both Th17 and Th1 cell differentiation while it did not affect Treg cell differentiation. In summary, IVIg inhibited Th17 cytokine-induced RANKL expression and OC differentiation. IVIg reduced osteoclastogenesis when monocytes were cocultured with Th17 cells. IVIg also reduced Th17 polarization. IVIg could be a new therapeutic option for Th17 cell–mediated osteoclastogenesis.

RAGE-dependent VCAM-1 expression in the lung endothelium mediates IL-33-induced allergic airway inflammation.

The receptor for advanced glycation endproducts (RAGE) has been implicated as a critical molecule in the pathogenesis of experimental asthma/allergic airway inflammation (AAI). It has been previously shown that RAGE acts both upstream of interleukin-33 (IL-33) release and downstream of IL-33 release via RAGE-dependent IL-33-induced accumulation of type 2 innate lymphoid cells (ILC2s) in the lungs, which perpetuate type 2 inflammation and mucus metaplasia. However, the mechanism by which RAGE mediates downstream IL-33-induced type 2 inflammatory responses is unknown. This study tested the hypothesis that ILC2s are recruited to the lungs via RAGE-dependent vascular cell adhesion molecule 1 (VCAM-1) expression on lung endothelial cells. House dust mite extract, Alternaria alternata extract, or rIL-33 was used to induce AAI/VCAM-1 expression in wild-type (WT) and RAGE-knockout (RAGE-KO) mice. Intravenous (i.v.) anti-VCAM-1 or intraperitoneal (i.p.) β7 blocking antibody administration was used to determine the role of VCAM-1 in IL-33-induced AAI. Enhanced VCAM-1 expression in the lungs by HDM, AA, or rIL-33 exposure was found to be RAGE-dependent. In addition, stimulation of primary mouse lung endothelial cells with IL-33 induced VCAM-1 expression in WT, but not RAGE-KO cells. Administration of VCAM-1 and β7-integrin blocking antibodies reduced IL-33-induced eosinophilic inflammation, mucus metaplasia, and type 2 inflammatory responses. This study demonstrates that allergen- and cytokine-induced VCAM-1 expression is RAGE-dependent and contributes to lung ILC2 accumulation and downstream eosinophilic inflammation, mucus metaplasia, and type 2 inflammatory responses.

The protein synthesis inhibitor brusatol normalizes high‐fat diet‐induced glucose intolerance in male C57BL/6 mice: role of translation factor eIF5A hypusination

The naturally occurring quassinoid compound brusatol improves the survival of insulin-producing cells when exposed to the proinflammatory cytokines IL-1β and IFN-γ in vitro. The aim of the present study was to investigate whether brusatol also promotes beneficial effects in mice fed a high-fat diet (HFD), and if so, to study the mechanisms by which brusatol acts. In vivo, we observed that the impaired glucose tolerance of HFD-fed male C57BL/6 mice was counteracted by a 2 wk treatment with brusatol. Brusatol treatment improved both β-cell function and peripheral insulin sensitivity of HFD-fed mice. In vitro, brusatol inhibited β-cell total protein and proinsulin biosynthesis, with an ED50 of ∼40 nM. In line with this, brusatol blocked cytokine-induced iNOS protein expression via inhibition of iNOS mRNA translation. Brusatol may have affected protein synthesis, at least in part, via inhibition of eukaryotic initiation factor 5A (eIF5A) hypusination, as eIF5A spermidine association and hypusination in RIN-5AH cells was reduced in a dose- and time-dependent manner. The eIF5A hypusination inhibitor GC7 promoted a similar effect. Both brusatol and GC7 protected rat RIN-5AH cells against cytokine-induced cell death. Brusatol reduced eIF5A hypusination and cytokine-induced cell death in EndoC-βH1 cells as well. Finally, hypusinated eIF5A was reduced in vivo by brusatol in islet endocrine and endothelial islet cells of mice fed an HFD. The results of the present study suggest that brusatol improves glucose intolerance in mice fed an HFD, possibly by inhibiting protein biosynthesis and eIF5A hypusination.-Turpaev, K., Krizhanovskii, C., Wang, X., Sargsyan, E., Bergsten, P., Welsh, N. The protein synthesis inhibitor brusatol normalizes high-fat diet-induced glucose intolerance in male C57BL/6 mice: role of translation factor eIF5A hypusination.

PDL1 is expressed in the islets of people with type 1 diabetes and is up-regulated by interferons-α and-γ via IRF1 induction.

BackgroundAntibodies targeting PD-1 and its ligand PDL1 are used in cancer immunotherapy but may lead to autoimmune diseases, including type 1 diabetes (T1D). It remains unclear whether PDL1 is expressed in pancreatic islets of people with T1D and how is it regulated.MethodsThe expression of PDL1, IRF1, insulin and glucagon was evaluated in samples of T1D donors by immunofluorescence. Cytokine-induced PDL1 expression in the human beta cell line, EndoC-βH1, and in primary human pancreatic islets was determined by real-time RT-PCR, flow cytometry and Western blot. Specific and previously validated small interference RNAs were used to inhibit STAT1, STAT2, IRF1 and JAK1 signaling. Key results were validated using the JAK inhibitor Ruxolitinib.FindingsPDL1 was present in insulin-positive cells from twelve T1D individuals (6 living and 6 deceased donors) but absent from insulin-deficient islets or from the islets of six non-diabetic controls. Interferons-α and -γ, but not interleukin-1β, induced PDL1 expression in vitro in human islet cells and EndoC-βH1 cells. Silencing of STAT1 or STAT2 individually did not prevent interferon-α-induced PDL1, while blocking of JAKs – a proposed therapeutic strategy for T1D – or IRF1 prevented PDL1 induction.InterpretationThese findings indicate that PDL1 is expressed in beta cells from people with T1D, possibly to attenuate the autoimmune assault, and that it is induced by both type I and II interferons via IRF1.

Podocyte NADPH Oxidase 5 Promotes Renal Inflammation Regulated by the Toll-Like Receptor Pathway.

Oxidative stress associated with a proinflammatory state occurs in endothelial dysfunction, hypertension, chronic kidney disease, and diabetes. The NADPH oxidase (Nox) family of reactive oxygen species (ROS) generating enzymes is implicated in these processes, yet little information regarding the role of Nox5 is available. Our aim was to investigate the role of Nox5 in promoting renal inflammation and identify mechanisms regulating its activity. Mice with podocyte-specific Nox5 (Nox5pod+) expression demonstrated greater glomerular inflammation and increased expression of Toll-like receptors (TLRs) and proinflammatory cytokines. In a lipopolysaccharide (LPS) model of acute kidney injury, Nox5pod+ and control littermates exhibited increased TLR and Nox1 expression. Compared with control littermates, Nox5pod+ animals developed greater glomerular inflammation and ROS production. Immortalized human podocytes (hPODs) incubated with LPS demonstrated TLR induction, increased Nox5 expression, and enhanced ROS production. Inhibition of interleukin-1 receptor-associated kinases (IRAK)-1 and -4 that lie downstream of TLR inhibited LPS-induced ROS production. Interaction between IRAK1 and Nox5 was confirmed by coimmunoprecipitation. Furthermore, LPS treatment of hPODs resulted in phosphorylation of threonine residue(s) in Nox5 that was attenuated by an IRAK1/4 inhibitor. Innovation and Conclusion: These results are the first to demonstrate that Nox5 is a downstream target of the TLR pathway and that Nox5-derived ROS may be modulated by IRAK1/4 activity. Nox5-derived ROS in podocytes can promote a proinflammatory state in the kidney via induction of cytokine expression and upregulation of TLRs leading to a feed-forward loop in which TLR activation enhances Nox5-mediated ROS production.

Regulation of monoamine oxidase A (MAO-A) expression, activity, and function in IL-13–stimulated monocytes and A549 lung carcinoma cells

Monoamine oxidase A (MAO-A) is a mitochondrial flavoenzyme implicated in the pathogenesis of atherosclerosis and inflammation and also in many neurological disorders. MAO-A also has been reported as a potential therapeutic target in prostate cancer. However, the regulatory mechanisms controlling cytokine-induced MAO-A expression in immune or cancer cells remain to be identified. Here, we show that MAO-A expression is co-induced with 15-lipoxygenase (15-LO) in interleukin 13 (IL-13)-activated primary human monocytes and A549 non-small cell lung carcinoma cells. We present evidence that MAO-A gene expression and activity are regulated by signal transducer and activator of transcription 1, 3, and 6 (STAT1, STAT3, and STAT6), early growth response 1 (EGR1), and cAMP-responsive element-binding protein (CREB), the same transcription factors that control IL-13-dependent 15-LO expression. We further established that in both primary monocytes and in A549 cells, IL-13-stimulated MAO-A expression, activity, and function are directly governed by 15-LO. In contrast, IL-13-driven expression and activity of MAO-A was 15-LO-independent in U937 promonocytic cells. Furthermore, we demonstrate that the 15-LO-dependent transcriptional regulation of MAO-A in response to IL-13 stimulation in monocytes and in A549 cells is mediated by peroxisome proliferator-activated receptor γ (PPARγ) and that signal transducer and activator of transcription 6 (STAT6) plays a crucial role in facilitating the transcriptional activity of PPARγ. We further report that the IL-13-STAT6-15-LO-PPARγ axis is critical for MAO-A expression, activity, and function, including migration and reactive oxygen species generation. Altogether, these results have major implications for the resolution of inflammation and indicate that MAO-A may promote metastatic potential in lung cancer cells.

Abstract 514: Focal Adhesion Kinase Inhibition Prevents Prolonged Nuclear Factor-κB Activation and Reduces Atherosclerosis in ApoE-/- Mice

A key event in atherogenesis is the expression of cell adhesion molecules on endothelial cells (ECs) that recruit leukocytes to the vessel wall, where they accumulate over time and form atherosclerotic plaques filled with fatty lipids. Although lipid-lowering drugs have been beneficial in slowing down atherosclerosis, there is currently no treatment available to reduce the underlying triggers that promote EC activation. Focal adhesion kinase (FAK) is an integrin-associated protein tyrosine kinase that plays a key role in cytokine-induced pro-inflammatory molecule expression; however, the molecular mechanism has not been elucidated. We have revealed that a small molecule FAK inhibitor (PF-271) in human ECs reduces tumor necrosis factor-α (TNF-α)-induced sustained activation of nuclear factor-κB (NF-kB), a major inflammatory transcription factor. While NF-κB is normally bound to inhibitor of NF-κB α (IκBα) in the cytosol, TNF-α leads to rapid activation and nuclear localization of NF-κB after degradation of IκBα by activation of IκB kinase (IKK). Chronic exposure to cytokines leads to oscillations in NF-κB activation as IκBα is continually made by active NF-κB and degraded by active IKK. Surprisingly, we discovered that FAK inhibition blocks prolonged NF-κB activation through a time-dependent increase in IκBα protein stability, suggesting decreased IKK activity. By performing in vitro IKK kinase assays, we found that FAK inhibition did indeed reduce sustained IKK activity as seen by reduced serine phosphorylation of IκBα 3hr after TNF-α stimulation. Since both cell adhesion molecule expression and NF-κB in ECs is important for atherosclerotic lesion development, we tested the efficacy of FAK inhibition on atherogenesis in vivo . We found that oral delivery of PF-271 (twice daily, 8 weeks) significantly reduced atherosclerotic lesion area in ApoE-/- mice fed a western diet. Taken together, our data demonstrates that FAK inhibition potentially reduces atherogenesis by blocking sustained NF-κB signaling through decreased IKK activity.

Nrf2 Activation Provides Atheroprotection in Diabetic Mice Through Concerted Upregulation of Antioxidant, Anti-inflammatory, and Autophagy Mechanisms.

Interactive relationships between metabolism, inflammation, oxidative stress, and autophagy in the vascular system play a key role in the pathogenesis of diabetic cardiovascular disease. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a stress-sensitive guarantor of cellular homeostasis, which cytoprotective contributions extend beyond the antioxidant defense. We investigated the beneficial effects and underlying mechanisms of the Nrf2 inducer tert-butyl hydroquinone (tBHQ) on diabetes-driven atherosclerosis. In the experimental model of streptozotocin-induced diabetes in apolipoprotein E-deficient mice, treatment with tBHQ increased Nrf2 activity in macrophages and vascular smooth muscle cells within atherosclerotic lesions. Moreover, tBHQ significantly decreased the size, extension and lipid content of atheroma plaques, and attenuated inflammation by reducing lesional macrophages (total number and M1/M2 phenotype balance), foam cell size and chemokine expression. Atheroprotection was accompanied by both systemic and local antioxidant effects, characterized by lower levels of superoxide anion and oxidative DNA marker 8-hydroxy-2′-deoxyguanosine, reduced expression of NADPH oxidase subunits, and increased antioxidant capacity. Interestingly, tBHQ treatment upregulated the gene and protein expression of autophagy-related molecules and also enhanced autophagic flux in diabetic mouse aorta. In vitro, Nrf2 activation by tBHQ suppressed cytokine-induced expression of pro-inflammatory and oxidative stress genes, altered macrophage phenotypes, and promoted autophagic activity. Our results reinforce pharmacological Nrf2 activation as a promising atheroprotective approach in diabetes, according to the plethora of cytoprotective mechanisms involved in the resolution of inflammation and oxidative stress, and restoring autophagy.

Kujigamberol interferes with pro-inflammatory cytokine-induced expression of and N-glycan modifications to cell adhesion molecules at different stages in human umbilical vein endothelial cells

Kujigamberol is the norlabdane compound isolated from Kuji amber and has recently been shown to prevent Ca2+-signal transduction and exert anti-allergy effects in vitro and in vivo. However, the anti-inflammatory activities of kujigamberol remain unclear. In the present study, we investigated the biological activities of kujigamberol on cell adhesion molecules expressed on human umbilical vein endothelial cells (HUVEC) in response to pro-inflammatory cytokines. Kujigamberol decreased the molecular weight of intercellular adhesion molecule-1 (ICAM-1) by altering N-glycan modifications. In contrast to ICAM-1, kujigamberol reduced the interleukin-1α- or tumor necrosis factor α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin at the mRNA and protein levels. Kujigamberol B, but not kujiol A, decreased the molecular weight of the ICAM-1 protein. Kujigamberol moderately inhibited yeast α-glucosidases, whereas it was only weakly inhibited by kujigamberol B and more weakly by kujiol A. Three compounds did not inhibit Jack bean α-mannosidases. The present results reveal new biological activities of kujigamberol, which interfere with the pro-inflammatory cytokine-induced expression of and N-glycan modifications to cell adhesion molecules in HUVEC.

Canonical TGF-β Signaling Pathway Represses Human NK Cell Metabolism.

Cytokines stimulate rapid metabolic changes in human NK cells, including increases in both glycolysis and oxidative phosphorylation pathways. However, how these are subsequently regulated is not known. In this study, we demonstrate that TGF-β can inhibit many of these metabolic changes, including oxidative phosphorylation, glycolytic capacity, and respiratory capacity. TGF-β also inhibited cytokine-induced expression of the transferrin nutrient receptor CD71. In contrast to a recent report on murine NK cells, TGF-β-mediated suppression of these metabolic responses did not involve the inhibition of the metabolic regulator mTORC1. Inhibition of the canonical TGF-β signaling pathway was able to restore almost all metabolic and functional responses that were inhibited by TGF-β. These data suggest that pharmacological inhibition of TGF-β could provide a metabolic advantage to NK cells that is likely to result in improved functional responses. This has important implications for NK cell-based cancer immunotherapies.

IL-2 And IL-15 Induced NKG2D, CD158a and CD158b Expression on T, NKT- like and NK Cell Lymphocyte Subsets from Regional Lymph Nodes of Melanoma Patients.

Regional lymph nodes (LN)s represent important immunological barriers in spreading of malignant tumors. However, they are the most frequent early metastatic site in melanoma. Immunomodulatory agents including cytokines have been included in therapy of melanoma and have shown severe side effects and toxicity. In this sense, there is a growing need for bringing these agents to further in vitro testing that may enlighten aspects of their regional application. Therefore, the aim of this study was to investigate the effect of interleukin (IL)-2 and IL-15, the two cytokines with similar immune-enhancing effects, on the expression of activating NKG2D, inhibitory CD158a and CD158b receptors on CD8+ T, NKT-like and NK cell lymphocyte subsets from regional LNs of melanoma patients. In this study, we showed significant effects of IL-2 and IL-15 cytokine treatments on the expression of activating NKG2D and on inhibitory CD158a and CD158b receptors on lymphocytes, CD8+ T, NKT-like and NK cell lymphocyte subsets originating from regional LNs of melanoma patients. Furthermore, IL-2 and IL-15 by inducing the expression of NKG2D activating receptor on innate and on adaptive lymphocyte subsets and by augmenting NK cell antitumor cytotoxicity that correlated with the cytokine-induced NKG2D expression, increased antitumor potential of immune cells in regional LNs of melanoma patients irrespective of LN involvement. These findings indicate the importance of immune cell population from regional LNs of melanoma patients in the development of immune intervention strategies that may if applied locally increase antitumor potential to the level that controls tumor progressions.