Inflammatory Target Gene Expression Research Articles

Circulating MicroRNAs in Extracellular Vesicles as Potential Biomarkers of Alcohol-Induced Neuroinflammation in Adolescence: Gender Differences.

Current studies evidence the role of miRNAs in extracellular vesicles (EVs) as key regulators of pathological processes, including neuroinflammation and neurodegeneration. As EVs can cross the blood–brain barrier, and EV miRNAs are very stable in peripheral circulation, we evaluated the potential gender differences in inflammatory-regulated miRNAs levels in human and murine plasma EVs derived from alcohol-intoxicated female and male adolescents, and whether these miRNAs could be used as biomarkers of neuroinflammation. We demonstrated that while alcohol intoxication lowers anti-inflammatory miRNA (mir-146a-5p, mir-21-5p, mir-182-5p) levels in plasma EVs from human and mice female adolescents, these EV miRNAs increased in males. In mice brain cortices, ethanol treatment lowers mir-146a-5p and mir-21-5p levels, while triggering a higher expression of inflammatory target genes (Traf6, Stat3, and Camk2a) in adolescent female mice. These results indicate, for the first time, that female and male adolescents differ as regards the ethanol effects associated with the inflammatory-related plasma miRNAs EVs profile, and suggest that female adolescents are more vulnerable than males to the inflammatory effects of binge alcohol drinking. These findings also support the view that circulating miRNAs in EVs could be useful biomarkers for screening ethanol-induced neuroinflammation and brain damage in adolescence.

2012 – EXCESSIVE R-LOOPS TRIGGER AN INFLAMMATORY CASCADE LEADING TO ABERRANT HEMATOPOIETIC STEM AND PROGENITOR CELL EXPANSION

Aberrant expansion of hematopoietic stem and progenitor cells (HSPCs) is a major hallmark of myelodysplastic syndromes (MDS), although the underlying molecular regulation remains poorly understood. DEAD-box Helicase 41 (DDX41) is an understudied MDS-mutated factor whose function in hematopoiesis is unknown. Using an in vivo zebrafish model of Ddx41-deficiency, we unveiled a critical role for this helicase in regulating HSPCs. Mutants for ddx41 have more HSPCs than their siblings as measured by in situ hybridization and quantification by flow cytometry. We determined that in vivo Ddx41 suppresses the accumulation of R-loops, nucleic acid structures consisting of RNA:DNA hybrids and ssDNAs whose equilibrium is essential for cellular homeostasis. Using both global and hematovascular-restricted expression of RNASEH1-GFP to deplete R-loops, we revealed that excess R-loops in ddx41 mutants promote HSPC expansion. To uncover potential mechanisms driving HSPC expansion in ddx41 mutants, we identified elevated inflammatory target gene expression in HSPCs from ddx41 mutants compared to sibling controls using RNA-seq analysis. We established that excess R-loop levels induced by ddx41 deficiency triggered TBK1 phosphorylation and NFκB activation via the cGAS-STING pathway. Moreover, R-loop-triggered HSPC expansion was cGAS/STING-dependent. In human cells with diminished DDX41, blast counts, R-loop levels, and inflammatory signaling were elevated, suggesting a conservation of mechanism. These findings delineate a functional consequence for R-loop imbalance in HSPC biology and establish an in vivo link between R-loops, inflammation, and hematopoiesis.

Excessive R-Loops Trigger an Inflammatory Cascade Leading to Aberrant Hematopoietic Stem and Progenitor Cell Expansion

Mutations in DEAD-box Helicase 41 (DDX41) are observed in patients with myelodysplastic syndromes (MDS) with inferior overall survival, implicating this factor in disease pathogenesis. DDX41 is an understudied factor with links to inflammation and RNA processing, but whose function in hematopoiesis is unknown. Using a novel zebrafish model of Ddx41-deficiency, we unveiled a critical role for Ddx41 in regulating hematopoietic stem and progenitor cells (HSPCs). Mutants for ddx41 have more HSPCs than their siblings as measured by in situ hybridization for the HSPC marker runx1 as well as quantification of cd41:gfp+ and runx1:mcherry+ HSPCs by flow cytometry. To uncover potential mechanisms driving HSPC expansion in ddx41 mutants, we performed RNA-seq analysis of purified HSPCs from ddx41 mutants and sibling controls. Expression of inflammatory target genes was significantly elevated in ddx41 mutants including those regulated by the transcription factor NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells). As inflammatory signaling and NFκB signaling in particular is elevated in MDS, we next explored Ddx41 regulation of NFκB signaling. Using an in vivonfkb:gfp reporter, we demonstrated that NFκB signaling was indeed elevated in ddx41 mutants. DDX41 is implicated in the STING (STimulator of INterferon Genes) and TBK1 (TANK binding kinase 1) pathway that mediates type I interferon production in response to foreign or damaged endogenous DNAs. By lowering pathway activity via knockdown of sting or pharmacological inhibition of TBK1, we determined that the STING/TBK1 pathway triggered excessive NFκB activation in ddx41 mutants. Moreover, the elevated inflammatory signaling was detrimental to normal HSPC homeostasis as lowering STING/TBK1 pathway activation significantly reduced HSPC expansion in ddx41 mutants, as measured by runx1in situ hybridization and cd41:gfp flow cytometric quantification. R-loops, nucleic acid structures consisting of RNA:DNA hybrids and displaced ssDNAs, can activate the STING/TBK1 pathway via the nucleic acid sensor cGAS (cyclic GMP-AMP synthase). Recent proteomic analysis showed that DDX41 can bind R-loops. Via immunofluorescence quantification of RNA:DNA hybrid levels in ddx41 mutants versus sibling controls, we revealed that Ddx41 acts as a suppressor of R-loop accumulation. Using a RNASEH1-GFP transgenic zebrafish line that permits inducible depletion of R-loops, we revealed that excess R-loops in ddx41 mutants promote HSPC expansion as measured by runx1in situ hybridization and runx1:mcherry flow cytometric quantification. Moreover, using RT-qPCR analysis of NFκB target genes, we determined that R-loop depletion dampened NFκB activation in ddx41 mutants, suggesting the R-loop-mediated effects on HSPC numbers was via inflammatory signaling. We deciphered cGAS activity was critical for this effect as both nfkb:gfp reporter activity and HSPC levels in ddx41 mutants were diminished following treatment with a pharmacological inhibitor of cGAS. Our data demonstrate that DDX41 insufficiency triggers an R-loop-mediated inflammatory cascade leading to aberrant HSPC expansion and is the first study to delineate a functional consequence for R-loops in HSPC biology. Furthermore, recent studies showed R-loop accumulation in other common forms of MDS suggesting our novel in vivo findings are more broadly applicable to the MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.

Identification and functional characterization of IRAK-4 in grass carp (Ctenopharyngodon idellus)

IL-1R-associated kinase 4 (IRAK4), a central TIR signaling mediator in innate immunity, can initiate a cascade of signaling events and lead to induction of inflammatory target gene expression eventually. In the present study, we cloned and characterized an IRAK4 orthologue from grass carp (Ctenopharyngodon idella). The full length cDNA of CiIRAK4 was 2057 bp with an ORF of 1422 bp encoding a polypeptide of 472 amino acids. Multiple alignments showed that IRAK4s were highly conserved among different species. Phylogenetic tree analysis revealed that CiIRAK4 shared high homologous with zebra fish IRAK4. Expression analysis indicated that CiIRAK4 was widely expressed in all tested tissues. It was significantly up-regulated after treatment with poly I:C, especially obvious in liver and spleen. Also, CiIRAK4 could be induced by poly I:C and LPS in CIK cells. Fluorescence microscopy assays showed that CiIRAK4 localized in the cytoplasm. RNAi-mediated knockdown and overexpression assays indicated that CiIRAK4 might have little effect on NF-kappa B p65 translocation from cytoplasm to nucleus, indicating that CiIRAK4 was dispensable for activation of NF-kappa B p65. In addition, IRAK4 promoted IRF5 nuclear translocation, which has nothing to do with the interaction between IRAK4 and IRF5. It suggested that fish IRAK4 kinase regulated IRF5 activity through indirect ways.

Eicosapentaenoic acid suppresses TRIF-dependent signaling pathway of TLRs by targeting TBK1

Toll-like receptors (TLRs) induce an innate immune system. In general, there are two main pathways in TLRs: the myeloid differentiation primary response protein 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF)-dependent pathways. In this study, it has been investigated whether eicosapentaenoic acid (EPA), a polyunsaturated fatty acid (PUFA), and arachidic acid (ACA), a saturated fatty acid (SFA), can modulate the TLR signaling pathways. EPA suppressed the activation of interferon regulatory factor 3 (IRF3) and the expression of Interferon gamma-induced protein (IP-10) induced by Toll-like receptor 3 (TLR3) or TLR4 agonists by targeting TANK-binding kinase 1 (TBK1); however, ACA did not. These results demonstrate that EPA inhibits the TRIF-dependent signaling in the TLR3 and TLR4 pathways. The results raise the possibility that certain dietary PUFAs can modulate TLR-derived signaling and inflammatory target gene expression and can alter the susceptibility to microbial infection and chronic inflammatory diseases. Practical application Eicosapentaenoic acid (EPA) is a bioactive lipid that modulates inflammation and immunity. TLRs play a central role as initiators of the innate immune responses. EPA regulates TRIF-dependent pathways of TLRs by targeting TBK1. EPA may be a useful strategy to understand the mechanism of antiinflammatory activities.

In vitro modulation of inflammatory target gene expression by a polyphenol-enriched fraction of rose oil distillation waste water

Classical production of rose oil is based on water steam distillation from the flowers of Rosa damascena. During this process, large quantities of waste water accrue which are discharged to the environment, causing severe pollution of both, groundwater and surface water due to a high content of polyphenols. We recently developed a strategy to purify the waste water into a polyphenol-depleted and a polyphenol-enriched fraction RF20-(SP-207). RF20-(SP-207) and sub-fraction F(IV) significantly inhibited cell proliferation and migration of HaCaT cells. Since there is a close interplay between these actions and inflammatory processes, here we focused on the fractions' influence on pro-inflammatory biomarkers. HaCaT keratinocytes were treated with RF20-(SP-207), F(IV) (both at 50μg/mL) and ellagic acid (10μM) for 24h under TNF-α (20ng/mL) stimulated and non-stimulated conditions. Gene expression of IL-1β, IL-6, IL-8, RANTES and MCP-1 was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and cellular protein secretion of IL-8, RANTES and MCP-1 was determined by ELISA based assays. RF20-(SP-207) and F(IV) significantly decreased the expression and cellular protein secretion of IL-1β, IL-6, IL-8, RANTES and MCP-1. The diminishing effects on inflammatory target gene expression were slightly less pronounced under TNF-α stimulated conditions. In conclusion, the recovered polyphenol fraction RF20-(SP-207) from rose oil distillation waste water markedly modified inflammatory target gene expression in vitro, and, therefore, could be further developed as alternative treatment of acute and chronic inflammation.

MALT1 Protease Activity Controls the Expression of Inflammatory Genes in Keratinocytes upon Zymosan Stimulation

The protease activity of the paracaspase mucosa-associated lymphoid tissue lymphoma translocation gene 1(MALT1) plays an important role in antigen receptor-mediated lymphocyte activation by controlling the activity of the transcription factor nuclear factor-κB and is thus essential for the expression of inflammatory target genes. MALT1 is not only present in cells of the hematopoietic lineage, but is ubiquitously expressed. Here we report that stimulation with zymosan or Staphylococcus aureus induced MALT1 protease activity in human primary keratinocytes. Inhibition of the Src family of kinases or novel protein kinase C isoforms as wellas silencing of CARMA2 or BCL10 interfered with activation of MALT1 protease. Silencing or inhibition of MALT1 protease strongly decreased the expression of important inflammatory genes such as TNFα, IL-17C, CXCL8 and HBD-2. MALT1-inhibited cells were unable to mount an antimicrobial response upon zymosan stimulation or phorbolester/ionomycin treatment, demonstrating a central role of MALT1 protease activity in keratinocyte immunity and suggesting MALT1 as a potential target in inflammatory skin diseases.

PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression.

JNK pathway is involved in the inhibition of inflammatory target gene expression and NF-kappaB activation by melittin

BackgroundBee venom therapy has been used to treat inflammatory diseases including rheumatoid arthritis in humans and in experimental animals. We previously found that bee venom and melittin (a major component of bee venom) have anti-inflammatory effect by reacting with the sulfhydryl group of p50 of nuclear factor-kappa B (NF-κB) and IκB kinases (IKKs). Since mitogen activated protein (MAP) kinase family is implicated in the NF-κB activation and inflammatory reaction, we further investigated whether activation of MAP kinase may be also involved in the anti-inflammatory effect of melittin and bee venom.MethodsThe anti-inflammatory effects of melittin and bee venom were investigated in cultured Raw 264.7 cells, THP-1 human monocytic cells and Synoviocytes. The activation of NF-κB was investigated by electrophoretic mobility shift assay. Nitric oxide (NO) and prostaglandin E2 (PGE2) were determined either by Enzyme Linked Immuno Sorbent Assay or by biochemical assay. Expression of IκB, p50, p65, inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2) as well as phosphorylation of MAP kinase family was determined by Western blot.ResultsMelittin (0.5–5 μg/ml) and bee venom (5 and 10 μg/ml) inhibited lipopolysaccharide (LPS, 1 μg/ml) and sodium nitroprusside (SNP, 200 μM)-induced activation of c-Jun NH2-terminal kinase (JNK) in RAW 264.7 cells in a dose dependent manner. However, JNK inhibitor, anthra [1,9-cd]pyrazole-6 (2H)-one (SP600215, 10–50 μM) dose dependently suppressed the inhibitory effects of melittin and bee venom on NF-κB dependent luciferase and DNA binding activity via suppression of the inhibitory effect of melittin and bee venom on the LPS and SNP-induced translocation of p65 and p50 into nucleus as well as cytosolic release of IκB. Moreover, JNK inhibitor suppressed the inhibitory effects of melittin and bee venom on iNOS and COX-2 expression, and on NO and PGE2 generation.ConclusionThese data show that melittin and bee venom prevent LPS and SNP-induced NO and PGE2 production via JNK pathway dependent inactivation of NF-κB, and suggest that inactivation of JNK pathways may also contribute to the anti-inflammatory and anti-arthritis effects of melittin and bee venom.

Differential modulation of toll‐like receptor signaling pathways by selenium

Toll-like receptors (TLRs) play role in recognition of microbial components and induction of innate immunity. The microbial components activate signaling pathways of TLRs; MyD88- and TRIF-dependent pathways leading to activation of NF-κB and IRF3, respectively. Selenium is an essential trace element nutrient and is normal component of diets. Here, we attempted to identify the molecular targets of selenium in TLR signaling pathways. Selenium inhibited NF-κB activation induced by LPS, Poly[I:C] or overexpression of MyD88 or IKKβ because selenium was shown to inhibit the activity of IKKβ which is the key kinase in MyD88-dependent pathway of TLRs. However, it is not known whether selenium inhibits TRIF-dependent pathway. Selenium inhibited the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C], or the overexpression of TRIF or TBK1. These results suggest that selenium can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs. Therefore, the results raise the possibility that selenium can modulate TLR-derived signaling and inflammatory target gene expression, and alter susceptibility to microbial infection and chronic inflammatory diseases.

Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IκB kinase

We previously found that bee venom (BV) and melittin (a major component of BV) has anti-inflammatory effect by reacting with the sulfhydryl group of p50 of NF-κB. Since the sulfhydryl group is present in IκB kinase (IKKα and IKKβ), anti-inflammatory effect of melittin via interaction with IKKs was investigated. We first examined binding of melittin to IKKs using surface plasmon resonance analyzer. Melittin binds to IKKα (Kd=1.34×10−9M) and IKKβ (Kd=1.01×10−9M). Consistent with the high binding affinity, melittin (5 and 10μg/ml) and BV (0.5, 1 and 5μg/ml) suppressed sodium nitroprusside, TNF-α and LPS induced-IKKβ and IKKβ activities, IκB release, and NF-κB activity as well as the expressions of iNOS and COX-2, and the generation of nitric oxide (NO) and prostaglandin E2 (PGE2) in Raw 264.7 mouse macrophages and synoviocytes obtained from rheumatoid arthritis patients. The binding affinities of melittin to mutant IKKs, was reduced, and the inhibitory effect of melittin on IKK and NF-κB activities, and NO and PGE2 generation were abrogated by the reducing agents or in Raw 264.7 transfected with mutant plasmid IKKα (C178A) or IKKβ (C179A). These results suggest that melittin binding to the sulfhydryl group of IKKs resulted in reduced IKK activities, IκB release, NF-κB activity and generation of inflammatory mediators, indicating that IKKs may be also anti-inflammatory targets of BV.

Suppression of MyD88- and TRIF-dependent signaling pathways of toll-like receptor by (−)-epigallocatechin-3-gallate, a polyphenol component of green tea

Toll-like receptors (TLRs) play an important role in recognition of microbial components and induction of innate immunity. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and/or TRIF-dependent pathways leading to activation of NF-κB. (−)-Epigallocatechin-3-gallate (EGCG), a flavonoid found in green tea, is known to inhibit NF-κB activation induced by many pro-inflammatory stimuli. EGCG was shown to inhibit the activity of IKKβ which is the key kinase in the canonical pathway for NF-κB activation in MyD88-dependent pathway of TLRs. However, it is not known whether EGCG inhibits TRIF-dependent pathway through which more than 70% of lipopolysaccharide (LPS)-induced genes are regulated. Therefore, we attempted to identify the molecular target of EGCG in TRIF-dependent pathways of TLR3 and TLR4. EGCG inhibited the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C], or the overexpression of TRIF. The inhibition of IRF3 activation by EGCG was mediated through the suppression of the kinase activity of TBK1. However, EGCG did not inhibit activation of IRF3 induced by overexpression of constitutively active IRF3. These results suggest that the molecular target of EGCG is TBK1 in TRIF-dependent signaling pathways of TLR3 and TLR4. Therefore, our results suggest that green tea flavonoids can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs and subsequent inflammatory target gene expression.

Effect of rottlerin, a PKC-δ inhibitor, on TLR-4-dependent activation of murine microglia

In microglia, Toll-like receptors have been shown to recognize pathogen-associated molecular patterns and initiate innate immune responses upon interaction with infectious agents. The effect of rottlerin, a PKC-δ specific inhibitor, on TLR-4-mediated signaling was investigated in murine microglia stimulated with lipopolysaccharide and taxol. Pretreatment of microglia cells with rottlerin decreased LPS- and taxol-induced nitric oxide production in a concentration-dependent manner (IC 50 = 99.1 ± 1.5 nM). Through MTT and FACS analysis, we found that the inhibition effect of rottlerin was not due to microglial cell death. Rottlerin pretreatment also attenuated LPS-induced phosphorylation of IκB-α, nuclear translocation of NF-κB, and expression of type II nitric oxide synthase. In addition, microglial phagocytosis in response to TLR-4 activation was diminished in which rottlerin was pretreated. Together, these data raise the possibility that certain PKC-δ specific inhibitors can modulate TLR-4-derived signaling and inflammatory target gene expression, and can alter susceptibility to microbial infection and chronic inflammatory diseases in central nervous system.

The regulation of the expression of inducible nitric oxide synthase by Src-family tyrosine kinases mediated through MyD88-independent signaling pathways of Toll-like receptor 4

Bacterial lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4) leading to the expression of inflammatory gene products. Src-family tyrosine kinases (STKs) are known to be activated by LPS in monocytes/macrophages. Therefore, we determined the role of STKs in TLR4 signaling pathways and target gene expression in macrophages. The activation of NFκB, and p38 MAPK, and the expression of inducible nitric oxide synthase (iNOS) induced by LPS were not affected in macrophages deficient in three STKs (Lyn, Hck, and Fgr). These results suggest that the deletion of the three STKs among possibly nine STKs is not sufficient to abolish total activity of STKs possibly due to the functional redundancy of other STKs present in macrophages. However, two structurally unrelated pan-inhibitors of STKs, PP1 and SU6656, suppressed LPS-induced iNOS expression in MyD88-knockout as well as wild-type macrophages. The suppression of iNOS expression by the inhibitors was correlated with the downregulation of IFNβ (a MyD88-independent gene) expression and subsequent decrease in STAT1 phosphorylation. Moreover, PP1 suppressed the expression of IFNβ and iNOS induced by TRIF, a MyD88-independent adaptor of TLR4. PP1 suppressed STAT1 phosphorylation induced by LPS, but not by IFNβ suggesting that STKs are involved in the primary downstream signaling pathways of TLR4, but not the secondary signaling pathways downstream of IFNβ receptor. Together, these results demonstrate that STKs play a positive regulatory role in TLR4-mediated iNOS expression in a MyD88-independent (TRIF-dependent) manner. These results provide new insight in understanding the role of STKs in TLR4 signaling pathways and inflammatory target gene expression.

Specific Inhibition of MyD88-Independent Signaling Pathways of TLR3 and TLR4 by Resveratrol: Molecular Targets Are TBK1 and RIP1 in TRIF Complex

TLRs can activate two distinct branches of downstream signaling pathways. MyD88 and Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF) pathways lead to the expression of proinflammatory cytokines and type I IFN genes, respectively. Numerous reports have demonstrated that resveratrol, a phytoalexin with anti-inflammatory effects, inhibits NF-kappaB activation and other downstream signaling pathways leading to the suppression of target gene expression. However, the direct targets of resveratrol have not been identified. In this study, we attempted to identify the molecular target for resveratrol in TLR-mediated signaling pathways. Resveratrol suppressed NF-kappaB activation and cyclooxygenase-2 expression in RAW264.7 cells following TLR3 and TLR4 stimulation, but not TLR2 or TLR9. Further, resveratrol inhibited NF-kappaB activation induced by TRIF, but not by MyD88. The activation of IFN regulatory factor 3 and the expression of IFN-beta induced by LPS, poly(I:C), or TRIF were also suppressed by resveratrol. The suppressive effect of resveratrol on LPS-induced NF-kappaB activation was abolished in TRIF-deficient mouse embryonic fibroblasts, whereas LPS-induced degradation of IkappaBalpha and expression of cyclooxygenase-2 and inducible NO synthase were still inhibited in MyD88-deficient macrophages. Furthermore, resveratrol inhibited the kinase activity of TANK-binding kinase 1 and the NF-kappaB activation induced by RIP1 in RAW264.7 cells. Together, these results demonstrate that resveratrol specifically inhibits TRIF signaling in the TLR3 and TLR4 pathway by targeting TANK-binding kinase 1 and RIP1 in TRIF complex. The results raise the possibility that certain dietary phytochemicals can modulate TLR-derived signaling and inflammatory target gene expression and can alter susceptibility to microbial infection and chronic inflammatory diseases.