Reconstituted high-density lipoprotein modulates activation of human leukocytes

An anti-inflammatory effect of reconstituted High Density Lipoprotein (rHDL) has been demonstrated in atherosclerosis and in sepsis models. An increase of adhesion molecules as well as tissue factor expression on endothelial cells in response to inflammatory or danger signals are attenuated by the treatment with rHDL. Here we show the inhibitory effect of rHDL on the activation of human leukocytes in a whole blood assay as well as on monocyte-derived human dendritic cells (DC). Multiplex analysis of human whole blood showed that phytohaemagglutinin (PHA)-induced secretion of the cytokines IL-1β, IL-1RA, IL-2R, IL-6, IL-7, IL-12(p40), IL-15 and IFN-α was inhibited. Furthermore, an inhibitory effect on the production of the chemokines CCL-2, CCL-4, CCL-5, CXCL-9 and CXCL-10 was observed. Activation of granulocytes and CD14+ monocytes by PHA is inhibited dose-dependently by rHDL shown as decreased up-regulation of ICAM-1 surface expression. In addition, we found a strong inhibitory effect of rHDL on toll-like receptor 2 (TLR2)- and TLR4-mediated maturation of DC. Treatment of DC with rHDL prevented the up-regulation of cell surface molecules CD80, CD83 and CD86 and it inhibited the TLR-driven activation of inflammatory transcription factor NF-κB. These findings suggest that rHDL prevents activation of crucial cellular players of cellular immunity and could therefore be a useful reagent to impede inflammation as well as the link between innate and adaptive immunity.

Myogenin modulates exercise endurance by altering skeletal muscle metabolism

The function of myogenic regulatory factors (MRFs) during adult life is not well understood. The requirement of one of these MRFs, myogenin (Myog), during embryonic muscle development suggests an equally important role in adult muscle. In this study, we have determined the function of myogenin during adult life using a conditional allele of Myog. In contrast to embryonic development, myogenin is not required for adult viability, and Myog-deleted mice exhibited no remarkable phenotypic changes during sedentary life. Remarkably, sedentary Myog-deleted mice demonstrated enhanced exercise endurance during involuntary treadmill running. Altered blood glucose and lactate levels in sedentary Myog-deleted mice after exhaustion suggest an enhanced glycolytic metabolism and an ability to excessively deplete muscle and liver glycogen stores. Traditional changes associated with enhanced exercise endurance, such as fiber type switching, and increased oxidative potential, were not detected in sedentary Myog-deleted mice. After long-term voluntary exercise, trained Myog-deleted mice demonstrated an enhanced adaptive response to exercise. Trained Myog-deleted mice exhibited superior exercise endurance associated with an increased proportion of slow-twitch fibers and increased oxidative capacity. In a parallel experiment, dystrophin-deficient young adult mice showed attenuated muscle fatigue following the deletion of Myog. These results demonstrate a novel and unexpected role for myogenin in modulating skeletal muscle metabolism.

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

CROSSTALK BETWEEN R1175 METHYLATION AND Y1173 PHOSPHORYLATION NEGATIVELY MODULATES EGFR-MEDIATED ERK ACTIVATION

Post-translational protein modifications are critical regulators of protein functions as they expand the signaling potentials of the modified proteins, leading to diverse physiological consequences. Currently, increasing evidence suggests that protein methylation is as important as other post-translational modifications in the regulation of various biological processes. This drives us to ask whether methylation is involved in the EGFR (epidermal growth factor receptor) signaling, a biological process extensively regulated by multiple post-translational modifications including phosphorylation, glycosylation and ubiquitination. We found that EGFR R1175 is methylated by a protein arginine methyltransferase named PRMT5. During EGFR activation, PRMT5-mediated R1175 methylation specifically enhances EGF-induced EGFR autophosphorylation at Y1173 residue. This novel modification crosstalk increases SHP1 recruitment to EGFR and suppresses EGFR-mediated ERK activation, resulting in inhibition of cell proliferation, migration, and invasion of EGFR-expressing cells. Based on these findings, we provide the first link between arginine methylation and tyrosine phosphorylation and identify R1175 methylation as an inhibitory modification specifically against EGFR-mediated ERK activation.

CD40 activation induces NREM sleep and modulates genes associated with sleep homeostasis

The T-cell derived cytokine CD40 ligand is overexpressed in patients with autoimmune diseases. Through activation of its receptor, CD40 ligand leads to a tumor necrosis factor (TNF) receptor 1 (TNFR1) dependent impairment of locomotor activity in mice. Here we report that this effect is explained through a promotion of sleep, which was specific to non-rapid eye movement (NREM) sleep while REM sleep was suppressed. The increase in NREM sleep was accompanied by a decrease in EEG delta power during NREM sleep and by a decrease in the expression of transcripts in the cerebral cortex known to be associated with homeostatic sleep drive, such as Homer1a, Early growth response 2, Neuronal pentraxin 2, and Fos-like antigen 2. The effect of CD40 activation was mimicked by peripheral TNF injection and prevented by the TNF blocker etanercept. Our study indicates that sleep-wake dysregulation in autoimmune diseases may result from CD40 induced TNF:TNFR1 mediated alterations of molecular pathways, which regulate sleep-wake behavior.

Language context modulates reading route: an electrical neuroimaging study.

INTRODUCTION The orthographic depth hypothesis (Katz and Feldman, 1983) posits that different reading routes are engaged depending on the type of grapheme/phoneme correspondence of the language being read. Shallow orthographies with consistent grapheme/phoneme correspondences favor encoding via non-lexical pathways, where each grapheme is sequentially mapped to its corresponding phoneme. In contrast, deep orthographies with inconsistent grapheme/phoneme correspondences favor lexical pathways, where phonemes are retrieved from specialized memory structures. This hypothesis, however, lacks compelling empirical support. The aim of the present study was to investigate the impact of orthographic depth on reading route selection using a within-subject design. METHOD We presented the same pseudowords (PWs) to highly proficient bilinguals and manipulated the orthographic depth of PW reading by embedding them among two separated German or French language contexts, implicating respectively, shallow or deep orthography. High density electroencephalography was recorded during the task. RESULTS The topography of the ERPs to identical PWs differed 300-360 ms post-stimulus onset when the PWs were read in different orthographic depth context, indicating distinct brain networks engaged in reading during this time window. The brain sources underlying these topographic effects were located within left inferior frontal (German > French), parietal (French > German) and cingular areas (German > French). CONCLUSION Reading in a shallow context favors non-lexical pathways, reflected in a stronger engagement of frontal phonological areas in the shallow versus the deep orthographic context. In contrast, reading PW in a deep orthographic context recruits less routine non-lexical pathways, reflected in a stronger engagement of visuo-attentional parietal areas in the deep versus shallow orthographic context. These collective results support a modulation of reading route by orthographic depth.

Biomechanical loading modulates proinflammatory and bone resorptive mediators in bacterial-stimulated PDL cells.

The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL) cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS) of low (CTSL) and high (CTSH) magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2) and prostaglandin E2 (PGE2) was evaluated by ELISA. Gene expression and protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were evaluated by quantitative RT-PCR and ELISA, respectively. F. nucleatum increased the production of COX2 and PGE2, which was further increased by CTS. F. nucleatum-induced increase of PGE2 synthesis was significantly (P < 0.05) increased when CTSH was applied at 1 and 3 days. In addition, CTSH inhibited the F. nucleatum-induced upregulation of OPG at 1 and 3 days, thereby increasing the RANKL/OPG ratio. OPG and RANKL mRNA results correlated with the protein results. In summary, our findings provide original evidence that CTS can enhance bacterial-induced syntheses of molecules associated with inflammation and bone resorption by PDL cells. Therefore, biomechanical, such as orthodontic or occlusal, loading may enhance the bacterial-induced inflammation and destruction in periodontitis.

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation.

INTRODUCTION Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. METHODS Bone marrow-derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4(+) T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. RESULTS The frequency of PS particle-positive CD11c(+)/CD11b(+) BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4(+) T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. CONCLUSION These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4(+) T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles.

SIRT2 deficiency modulates macrophage polarization and susceptibility to experimental colitis

BACKGROUND SIRT2 belongs to a highly conserved family of NAD+-dependent deacylases, consisting of seven members (SIRT1-SIRT7), which vary in subcellular localizations and have substrates ranging from histones to transcription factors and enzymes. Recently SIRT2 was revealed to play an important role in inflammation, directly binding, deacetylating, and inhibiting the p65 subunit of NF-κB. METHODS A Sirt2 deficient mouse line (Sirt2-/-) was generated by deleting exons 5-7, encoding part of the SIRT2 deacetylase domain, by homologous recombination. Age- and sex-matched Sirt2-/- and Sirt2+/+ littermate mice were subjected to dextran sulfate sodium (DSS)-induced colitis and analyzed for colitis susceptibility. RESULTS Sirt2-/- mice displayed more severe clinical and histological manifestations after DSS colitis compared to wild type littermates. Notably, under basal condition, Sirt2 deficiency does not affect the basal phenotype and intestinal morphology Sirt2 deficiency, however, affects macrophage polarization, creating a pro-inflammatory milieu in the immune cells compartment. CONCLUSION These data confirm a protective role for SIRT2 against the development of inflammatory processes, pointing out a potential role for this sirtuin as a suppressor of colitis. In fact, SIRT2 deletion promotes inflammatory responses by increasing NF-κB acetylation and by reducing the M2-associated anti-inflammatory pathway. Finally, we speculate that the activation of SIRT2 may be a potential approach for the treatment of inflammatory bowel disease.

Developmental oestrogen exposure differentially modulates IGF-I and TNF-α expression levels in immune organs of Yersinia ruckeri-challenged young adult rainbow trout (Oncorhynchus mykiss).

Intensified aquaculture has strong impact on fish health by stress and infectious diseases and has stimulated the interest in the orchestration of cytokines and growth factors, particularly their influence by environmental factors, however, only scarce data are available on the GH/IGF-system, central physiological system for development and tissue shaping. Most recently, the capability of the host to cope with tissue damage has been postulated as critical for survival. Thus, the present study assessed the combined impacts of estrogens and bacterial infection on the insulin-like growth factors (IGF) and tumor-necrosis factor (TNF)-α. Juvenile rainbow trout were exposed to 2 different concentrations of 17β-estradiol (E2) and infected with Yersinia ruckeri. Gene expressions of IGF-I, IGF-II and TNF-α were measured in liver, head kidney and spleen and all 4 estrogen receptors (ERα1, ERα2, ERβ1 and ERβ2) known in rainbow trout were measured in liver. After 5 weeks of E2 treatment, hepatic up-regulation of ERα1 and ERα2, but down-regulation of ERß1 and ERß2 were observed in those groups receiving E2-enriched food. In liver, the results further indicate a suppressive effect of Yersinia-infection regardless of E2-treatment on day 3, but not of E2-treatment on IGF-I whilst TNF-α gene expression was not influenced by Yersinia-infection but was reduced after 5 weeks of E2-treatment. In spleen, the results show a stimulatory effect of Yersinia-infection, but not of E2-treatment on both, IGF-I and TNF-α gene expressions. In head kidney, E2 strongly suppressed both, IGF-I and TNF-α. To summarise, the treatment effects were tissue- and treatment-specific and point to a relevant role of IGF-I in infection.

Anticipatory cognitive stress appraisal modulates suppression of wound-induced macrophage activation by acute psychosocial stress

Anticipatory cognitive stress appraisal (ACSA) can affect the stress-induced release of stress hormones, which, in turn, can modulate microbicidal potential of macrophages. This study examines whether ACSA modulates wound-induced activation of macrophage microbicidal potential in 22 acutely stressed compared to 17 nonstressed healthy men. After catheter-induced wound infliction and completing the ACSA questionnaire, the stress group underwent an acute mental stress task, while the nonstressed group did not. Macrophage microbicidal potential and stress hormones were repeatedly measured. In acutely stressed men, but not in nonstressed men, higher scores in ACSA related to lower macrophage microbicidal potential. This association was statistically mediated by the norepinephrine (NE) stress response. Our data suggest that ACSA modulates stress-induced suppression of wound-induced macrophage activation and that the NE stress response underlies this effect.