Early Pep-13-induced immune responses are SERK3A/B-dependent in potato.

Potato plants treated with the pathogen-associated molecular pattern Pep-13 mount salicylic acid- and jasmonic acid-dependent defense responses, leading to enhanced resistance against Phytophthora infestans, the causal agent of late blight disease. Recognition of Pep-13 is assumed to occur by binding to a yet unknown plasma membrane-localized receptor kinase. The potato genes annotated to encode the co-receptor BAK1, StSERK3A and StSERK3B, are activated in response to Pep-13 treatment. Transgenic RNAi-potato plants with reduced expression of both SERK3A and SERK3B were generated. In response to Pep-13 treatment, the formation of reactive oxygen species and MAP kinase activation, observed in wild type plants, is highly reduced in StSERK3A/B-RNAi plants, suggesting that StSERK3A/B are required for perception of Pep-13 in potato. In contrast, defense gene expression is induced by Pep-13 in both control and StSERK3A/B-depleted plants. Altered morphology of StSERK3A/B-RNAi plants correlates with major shifts in metabolism, as determined by untargeted metabolite profiling. Enhanced levels of hydroxycinnamic acid amides, typical phytoalexins of potato, in StSERK3A/B-RNAi plants are accompanied by significantly decreased levels of flavonoids and steroidal glycoalkaloids. Thus, altered metabolism in StSERK3A/B-RNAi plants correlates with the ability of StSERK3A/B-depleted plants to mount defense, despite highly decreased early immune responses.

Obesity-associated extracellular mtDNA activates central TGFß pathway to cause blood pressure increase.

Hypothalamic inflammation was recently found to mediate obesity-related hypertension, but the responsible upstream mediators remain unexplored. In this study, we show that dietary obesity is associated with extracellular release of mitochondrial DNA (mtDNA) into the cerebrospinal fluid and that central delivery of mtDNA mimics transforming growth factor-ß (TGFß) excess to activate downstream signaling pathways. Physiological study reveals that central administration of mtDNA or TGFß is sufficient to cause hypertension in mice. Knockout of the TGFß receptor in proopiomelanocortin neurons counteracts the hypertensive effect of not only TGFß but also mtDNA excess, while the hypertensive action of central mtDNA can be blocked pharmacologically by a TGFß receptor antagonist or genetically by TGFß receptor knockout. Finally, we confirm that obesity-induced hypertension can be reversed through central treatment with TGFß receptor antagonist. In conclusion, circulating mtDNA in the brain employs neural TGFß pathway to mediate a central inflammatory mechanism of obesity-related hypertension.

Honokiol ameliorates endothelial dysfunction through suppression of PTX3 expression, a key mediator of IKK/IκB/NF-κB, in atherosclerotic cell model.

Pentraxin 3 (PTX3) was identified as a marker of the inflammatory response and overexpressed in various tissues and cells related to cardiovascular disease. Honokiol, an active component isolated from the Chinese medicinal herb Magnolia officinalis, was shown to have a variety of pharmacological activities. In the present study, we aimed to investigate the effects of honokiol on palmitic acid (PA)-induced dysfunction of human umbilical vein endothelial cells (HUVECs) and to elucidate potential regulatory mechanisms in this atherosclerotic cell model. Our results showed that PA significantly accelerated the expression of PTX3 in HUVECs through the IκB kinase (IKK)/IκB/nuclear factor-κB (NF-κB) pathway, reduced cell viability, induced cell apoptosis and triggered the inflammatory response. Knockdown of PTX3 supported cell growth and prevented apoptosis by blocking PA-inducted nitric oxide (NO) overproduction. Honokiol significantly suppressed the overexpression of PTX3 in PA-inducted HUVECs by inhibiting IκB phosphorylation and the expression of two NF-κB subunits (p50 and p65) in the IKK/IκB/NF-κB signaling pathway. Furthermore, honokiol reduced endothelial cell injury and apoptosis by regulating the expression of inducible NO synthase and endothelial NO synthase, as well as the generation of NO. Honokiol showed an anti-inflammatory effect in PA-inducted HUVECs by significantly inhibiting the generation of interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1. In summary, honokiol repaired endothelial dysfunction by suppressing PTX3 overexpression in an atherosclerotic cell model. PTX3 may be a potential therapeutic target for atherosclerosis.

Honokiol ameliorates endothelial dysfunction through suppression of PTX3 expression, a key mediator of IKK/IkappaB/NF-kappaB, in atherosclerotic cell model

Pentraxin 3 (PTX3) was identified as a marker of the inflammatory response and overexpressed in various tissues and cells related to cardiovascular disease. Honokiol, an active component isolated from the Chinese medicinal herb Magnolia officinalis, was shown to have a variety of pharmacological activities. In the present study, we aimed to investigate the effects of honokiol on palmitic acid (PA)-induced dysfunction of human umbilical vein endothelial cells (HUVECs) and to elucidate potential regulatory mechanisms in this atherosclerotic cell model. Our results showed that PA significantly accelerated the expression of PTX3 in HUVECs through the IkappaB kinase (IKK)/IkappaB/nuclear factor-kappaB (NF-kappaB) pathway, reduced cell viability, induced cell apoptosis and triggered the inflammatory response. Knockdown of PTX3 supported cell growth and prevented apoptosis by blocking PA-inducted nitric oxide (NO) overproduction. Honokiol significantly suppressed the overexpression of PTX3 in PA-inducted HUVECs by inhibiting IkappaB phosphorylation and the expression of two NF-kappaB subunits (p50 and p65) in the IKK/IkappaB/NF-kappaB signaling pathway. Furthermore, honokiol reduced endothelial cell injury and apoptosis by regulating the expression of inducible NO synthase and endothelial NO synthase, as well as the generation of NO. Honokiol showed an anti-inflammatory effect in PA-inducted HUVECs by significantly inhibiting the generation of interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1. In summary, honokiol repaired endothelial dysfunction by suppressing PTX3 overexpression in an atherosclerotic cell model. PTX3 may be a potential therapeutic target for atherosclerosis.

Stimulation of DDX3 expression by ginsenoside Rg3 through the Akt/p53 pathway activates the innate immune response via TBK1/IKKε/IRF3 signalling.

DEAD-box RNA helicase DDX3 is a well-known host factor that inhibits hepatitis B viral proliferation and boosts innate immune responses via TANK-binding kinase 1 (TBK1)/IKKε-mediated and/or interferon (IFN)-ß promoter stimulator-1 (IPS-1)-mediated IFN-ß induction. Previously, we demonstrated the anti-hepatitis B activity of Rg3 via stimulation of TRAF6/TAK1 degradation and inhibition of JNK/AP-1 signaling. To determine the effects of Rg3 on innate immunity, an IFN-ß promoter assay was performed. Rg3 ameliorated IFN-ß expression via upregulation of both the TBK1/IKKε pathway and DDX3 expression. In addition, Rg3 induced the phosphorylation of IRF3 and its translocation into nucleus, which is a key molecule to induction of IFN-ß expression. To evaluate the molecular mechanism of Rg3 on DDX3 expression, the DDX3 promoter (-1406/+105) was subjected to luciferase assay and ChIP analysis. p53 phosphorylation resulted in upregulation of DDX3 expression, which enhanced DDX3 promoter transactivation activity. Transient transfection with wild-type p53 increased DDX3 promoter activity in Hep3B cells which have null mutant of p53, whereas knockdown p53 by si-p53 reduced DDX3 promoter activity in HepG2.2.15 and HepG2 cells, respectively. Rg3- mediated phosphorylation of p53 resulted in inhibition of Akt phosphorylation, which in turn reduced MDM2-mediated p53 degradation. An Akt inhibitor augmented DDX3 promoter activity and reduced the secretion of hepatitis B surface antigen. Our data indicate that Rg3 enhances innate immunity by inducing IFN-ß expression through upregulation of DDX3 promoter activity via p53-mediated transactivation and activation of the TBK1/IKKε/IRF3 pathway.

Differential responses of hepatic endoplasmic reticulum stress and inflammation in diet-induced obese rats with high-fat diet rich in lard oil or soybean oil.

SCOPES: To investigate the effects of high-fat diet enriched with lard oil or soybean oil on liver endoplasmic reticulum (ER) stress and inflammation markers in diet-induced obese (DIO) rats and estimate the influence of following low-fat diet feeding. METHODS AND RESULTS: Male SD rats were fed with standard low-fat diet (LF, n = 10) and two isoenergentic high-fat diets enriched with lard (HL, n = 45) or soybean oil (HS, n = 45) respectively for 10 weeks. Then DIO rats from HL and HS were fed either high-fat diet continuously (HL/HL, HS/HS) or switched to low-fat diet (HL/LF, HS/LF) for another 8 weeks. Rats in control group were maintained with low-fat diet. Body fat, serum insulin level, HOMA-IR and ectopic lipid deposition in liver were increased in HL/HL and HS/HS compared to control, but increased to a greater extent in HL/HL compared to HS/HS. Markers of ER stress including PERK and CHOP protein expression and phosphorylation of eIF2α were significantly elevated in HL/HL group while phosphorylation of IRE1α and GRP78 protein expression were suppressed in both HL/HL and HS/HS. Besides, inflammatory signals (OPN, TLR2, TLR4 and TNF-α) expressions significantly increased in HL/HL compared to others. Switching to low-fat diet reduced liver fat deposition, HOMA-IR, mRNA expression of TLR4, TNF-α, PERK in both HL/LF and HS/LF, but only decreased protein expression of OPN, PERK and CHOP in HL/LF group. In addition, HL/LF and HS/LF exhibited decreased phosphorylation of eIF2α and increased phosphorylation of IRE1α and GRP78 protein expression when compared with HL/HL and HS/HS respectively. CONCLUSIONS: Lard oil was more deleterious in insulin resistance and hepatic steatosis via promoting ER stress and inflammation responses in DIO rats, which may be attributed to the enrichment of saturated fatty acid. Low-fat diet was confirmed to be useful in recovering from impaired insulin sensitivity and liver fat deposition in this study.

Elevated Tribbles homolog 2-specific antibody levels in narcolepsy patients.

Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness and attacks of muscle atonia triggered by strong emotions (cataplexy). Narcolepsy is caused by hypocretin (orexin) deficiency, paralleled by a dramatic loss in hypothalamic hypocretin-producing neurons. It is believed that narcolepsy is an autoimmune disorder, although definitive proof of this, such as the presence of autoantibodies, is still lacking. We engineered a transgenic mouse model to identify peptides enriched within hypocretin-producing neurons that could serve as potential autoimmune targets. Initial analysis indicated that the transcript encoding Tribbles homolog 2 (Trib2), previously identified as an autoantigen in autoimmune uveitis, was enriched in hypocretin neurons in these mice. ELISA analysis showed that sera from narcolepsy patients with cataplexy had higher Trib2-specific antibody titers compared with either normal controls or patients with idiopathic hypersomnia, multiple sclerosis, or other inflammatory neurological disorders. Trib2-specific antibody titers were highest early after narcolepsy onset, sharply decreased within 2-3 years, and then stabilized at levels substantially higher than that of controls for up to 30 years. High Trib2-specific antibody titers correlated with the severity of cataplexy. Serum of a patient showed specific immunoreactivity with over 86% of hypocretin neurons in the mouse hypothalamus. Thus, we have identified reactive autoantibodies in human narcolepsy, providing evidence that narcolepsy is an autoimmune disorder.

Aurora-A kinase: a novel target both for cellular immunotherapy and molecular target therapy against human leukemia.

BACKGROUND: Although cellular immunotherapy still remains in its infancy, it is one of the important treatment options against cancer. The marked improvement of its clinical efficacy requires a 'better' target antigen, which is well recognized by cancer-cell-specific cytotoxic T lymphocytes. We have recently demonstrated the potential of Aurora-A kinase (Aurora-A) as such a 'better' target for cellular immunotherapy against human leukemia. Aurora-A is a member of the serine/threonine kinase family that properly regulates the cell division process, and has recently been implicated in tumorigenesis. On the other hand, small-molecule inhibitors targeting Aurora-A have recently been developed and preliminary but promising observations from Phase I clinical trials have been reported. These facts highlight the attractiveness of Aurora-A as an important target of comprehensive cancer therapies. OBJECTIVE/METHODS: In this review, we cover Aurora-A in the areas of immunotherapy and small-molecule inhibitor therapy against cancers. RESULTS/CONCLUSIONS: Aurora-A kinase is an attractive molecule not only as a target for small-molecule inhibitors, but also as a potential target for immunotherapy against cancer.

Endoplasmic reticulum stress regulator XBP-1 contributes to effector CD8+ T cell differentiation during acute infection.

The transcription factor X-box-binding protein-1 (XBP-1) plays an essential role in activating the unfolded protein response in the endoplasmic reticulum (ER). Transcribed XBP-1 mRNA is converted to its active form by unconventional cytoplasmic splicing mediated by inositol-requiring enzyme-1 (IRE-1) upon ER stress. We report activation of the IRE-1/XBP-1 pathway in effector CD8(+) T cells during the response to acute infection. Transcription of unspliced XBP-1 mRNA is up-regulated by IL-2 signals, while its splicing is induced after TCR ligation. Splicing of XBP-1 mRNA was evident during the expansion of Ag-specific CD8(+) T cells in response to viral or bacterial infection. An XBP-1 splicing reporter revealed that splicing activity was enriched in terminal effector cells expressing high levels of killer cell lectin-like receptor G1 (KLRG1). Overexpression of the spliced form of XBP-1 in CD8(+) T cells enhanced KLRG1 expression during infection, whereas XBP-1(-/-) CD8(+) T cells or cells expressing a dominant-negative form of XBP-1 showed a decreased proportion of KLRG1(high) effector cells. These results suggest that, in the response to pathogen, activation of ER stress sensors and XBP-1 splicing contribute to the differentiation of end-stage effector CD8(+) T cells.

Immunomodulatory effect of combination therapy with lovastatin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside alleviates neurodegeneration in experimental autoimmune encephalomyelitis.

Combination therapy with multiple sclerosis (MS) therapeutics is gaining momentum over monotherapy for improving MS. Lovastatin, an HMG-CoA reductase inhibitor (statin), was immunomodulatory in an experimental autoimmune encephalomyelitis (EAE) model of MS. Lovastatin biases the immune response from Th1 to a protective Th2 response in EAE by a different mechanism than 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, an immunomodulating agent that activates AMP-activated protein kinase. Here we tested these agents in combination in an EAE model of MS. Suboptimal doses of these drugs in combination were additive in efficacy against the induction of EAE; clinical symptoms were delayed and severity and duration of disease was reduced. In the central nervous system, the cellular infiltration and proinflammatory immune response was decreased while the anti-inflammatory immune response was increased. Combination treatment biased the class of elicited myelin basic protein antibodies from IgG2a to IgG1 and IgG2b, suggesting a shift from Th1 to Th2 response. In addition, combination therapy lessened inflammation-associated neurodegeneration in the central nervous system of EAE animals. These effects were absent in EAE animals treated with either drug alone at the same dose. Thus, our data suggest that agents with different mechanisms of action such as lovastatin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, when used in combination, could improve therapy for central nervous system demyelinating diseases and provide a rationale for testing them in MS patients.

Identification of tribbles homolog 2 as an autoantigen in autoimmune uveitis by phage display.

Autoimmune uveitis is a group of ocular inflammatory disorders with unknown causes. As in other autoimmune diseases, identification of autoantigens from uveitis patients would markedly improve our understanding of the disease mechanism. Here, we report that a candidate autoantigen was identified by phage display in an unbiased fashion. A bacteriophage T7 display cDNA library was generated from human eye and characterized. Patient-specific phages were enriched by four rounds of phage display with purified patient IgG. Enriched phages demonstrated a 20-fold increase in binding specificity to the patient IgG compared with control IgG. Two clonal phages with particularly high relative binding specificities were isolated and characterized. The encoded genes, tribbles homolog 2 (TRB2) and an unknown protein, had 170- and 42-fold increases in their binding specificities to the patient IgG, respectively. The patient-specific immunoreactivities were further confirmed by Western blotting. Anti-TRB2 antibody activities were detected in several uveitis patients but not in control subjects, suggesting that TRB2 is a uveitis-associated candidate autoantigen. These results demonstrate that autoantigens can be identified by phage display using uveitis patient serum.

Inhibitors of mitogen-activated protein kinases differentially regulate eosinophil-activating cytokine release from human airway smooth muscle.

Airway smooth muscle (ASM) is a potential source of multiple proinflammatory cytokines during airway inflammation. In the present study, we examined a requirement for mitogen-activated protein (MAP) kinase activation for interleukin (IL)-1beta-stimulated GM-CSF, RANTES, and eotaxin release. IL-1beta induced concentration-dependent phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs), p38 MAP kinase, and c-Jun amino-terminal kinase (SAPK/JNK). p42/p44 ERK and p38 MAP kinase phosphorylation peaked at 15 min and remained elevated up to 4 h. SAPK/JNK phosphorylation also peaked at 15 min but fell to baseline within 60 min. SB 203580 selectively inhibited IL-1beta-stimulated activation of p38 MAP kinase; U 0126 was selective against p42/p44 ERK activity. SB 202474, an inactive analog, had no effect on p42/p44 ERK, p38 MAP kinase, or SAPK/JNK activation, or on eotaxin or RANTES release. Eotaxin release was inhibited by SB 203580 and U 0126, whereas RANTES release was prevented by U 0126 only. GM-CSF release was inhibited by U 0126 but enhanced by SB 203580. These data indicate that RANTES release is dependent on p42/p44 ERK activation but occurs independently of p38 MAP kinase activity. Eotaxin release, however, is dependent on both p38 MAP kinase- and p42/p44 ERK-dependent mechanisms. GM-CSF release is p42/p44 ERK dependent and is tonically suppressed by a mechanism that is partially dependent on p38 MAP kinase, though direct inhibition of cyclooxygenase (COX) activity due to poor inhibitor selectivity may also contribute.

RSK3 encodes a novel pp90rsk isoform with a unique N-terminal sequence: growth factor-stimulated kinase function and nuclear translocation.

A novel pp90rsk Ser/Thr kinase (referred to as RSK3) was cloned from a human cDNA library. The RSK3 cDNA encodes a predicted 733-amino-acid protein with a unique N-terminal region containing a putative nuclear localization signal. RSK3 mRNA was widely expressed (but was predominant in lung and skeletal muscle). By using fluorescence in situ hybridization, the human RSK3 gene was localized to band q27 of chromosome 6. Hemagglutinin epitope-tagged RSK3 was expressed in transiently transfected COS cells. Growth factors, serum, and phorbol ester stimulated autophosphorylation of recombinant RSK3 and its kinase activity toward several protein substrates known to be phosphorylated by RSKs. However, the relative substrate specificity of RSK3 differed from that reported for other isoforms. RSK3 also phosphorylated potential nuclear target proteins including c-Fos and histones. Furthermore, although RSK3 was inactivated by protein phosphatase 2A in vitro, the enzyme was not activated by ERK2/mitogen-activated protein (MAP) kinase. In contrast, the kinase activity of another epitope-tagged RSK isoform (RSK-1) was significantly increased by in vitro incubation with ERK2/MAP kinase. Finally, we used affinity-purified RSK3 antibodies to demonstrate by immunofluorescence that endogenous RSK3 undergoes serum-stimulated nuclear translocation in cultured HeLa cells. These results provide evidence that RSK3 is a third distinct isoform of pp90rsk which translocates to the cell nucleus, phosphorylates potential nuclear targets, and may have a unique upstream activator. RSK3 may therefore subserve a discrete physiologic role(s) that differs from those of the other two known mammalian RSK isoforms.