Tailored Modulation of Cellular Pro-inflammatory Responses With Disaccharide Lipid A Mimetics.

Pro-inflammatory signaling mediated by Toll-like receptor 4 (TLR4)/myeloid differentiation-2 (MD-2) complex plays a crucial role in the instantaneous protection against infectious challenge and largely contributes to recovery from Gram-negative infection. Activation of TLR4 also boosts the adaptive immunity which is implemented in the development of vaccine adjuvants by application of minimally toxic TLR4 activating ligands. The modulation of pro-inflammatory responses via the TLR4 signaling pathway was found beneficial for management of acute and chronic inflammatory disorders including asthma, allergy, arthritis, Alzheimer disease pathology, sepsis, and cancer. The TLR4/MD-2 complex can recognize the terminal motif of Gram-negative bacterial lipopolysaccharide (LPS)-a glycophospholipid lipid A. Although immense progress in understanding the molecular basis of LPS-induced TLR4-mediated signaling has been achieved, gradual, and predictable TLR4 activation by structurally defined ligands has not yet been attained. We report on controllable modulation of cellular pro-inflammatory responses by application of novel synthetic glycolipids-disaccharide-based lipid A mimetics (DLAMs) having picomolar affinity for TLR4/MD-2. Using crystal structure inspired design we have developed endotoxin mimetics where the inherently flexible ß(1 → 6)-linked diglucosamine backbone of lipid A is replaced by a conformationally restricted α,α-(1↔1)-linked disaccharide scaffold. The tertiary structure of the disaccharide skeleton of DLAMs mirrors the 3-dimensional shape of TLR4/MD-2 bound E. coli lipid A. Due to exceptional conformational rigidity of the sugar scaffold, the specific 3D organization of DLAM must be preserved upon interaction with proteins. These structural factors along with specific acylation and phosphorylation pattern can ensure picomolar affinity for TLR4 and permit efficient dimerization of TLR4/MD-2/DLAM complexes. Since the binding pose of lipid A in the binding pocket of MD-2 (±180°) is crucial for the expression of biological activity, the chemical structure of DLAMs was designed to permit a predefined binding orientation in the binding groove of MD-2, which ensured tailored and species-independent (human and mice) TLR4 activation. Manipulating phosphorylation and acylation pattern at the sugar moiety facing the secondary dimerization interface allowed for adjustable modulation of the TLR4-mediated signaling. Tailored modulation of cellular pro-inflammatory responses by distinct modifications of the molecular structure of DLAMs was attained in primary human and mouse immune cells, lung epithelial cells and TLR4 transfected HEK293 cells.

Cleavage-Mediated Regulation of Myd88 Signaling by Inflammasome-Activated Caspase-1.

Coordination among multiple signaling pathways ensures an appropriate immune response, where a signaling pathway may impair or augment another signaling pathway. Here, we report a negative feedback regulation of signaling through the key innate immune mediator MyD88 by inflammasome-activated caspase-1. NLRP3 inflammasome activation impaired agonist- or infection-induced TLR signaling and cytokine production through the proteolytic cleavage of MyD88 by caspase-1. Site-specific mutagenesis was used to identify caspase-1 cleavage site within MyD88 intermediary segment. Different cleavage site location within MyD88 defined the functional consequences of MyD88 cleavage between mouse and human cells. LPS/monosodium urate-induced mouse inflammation model corroborated the physiological role of this mechanism of regulation, that could be reversed by chemical inhibition of NLRP3. While Toll/interleukin-1 receptor (TIR) domain released by MyD88 cleavage additionally contributed to the inhibition of signaling, Waldenström's macroglobulinemia associated MyD88L265P mutation is able to evade the caspase-1-mediated inhibition of MyD88 signaling through the ability of its TIRL265P domain to recruit full length MyD88 and facilitate signaling. The characterization of this mechanism reveals an additional layer of innate immunity regulation.

O-GlcNAc protein modification in C2C12 myoblasts exposed to oxidative stress indicates parallels with endogenous antioxidant defense.

A growing body of evidence demonstrates the involvement of protein modification with O-linked ß-N-acetylglucosamine (O-GlcNAc) in the stress response and its beneficial effects on cell survival. Here we investigated protein O-GlcNAcylation in skeletal muscle cells exposed to oxidative stress and the crosstalk with endogenous antioxidant system. The study focused on antioxidant enzymes superoxide dismutase 2 (SOD2), catalase (CAT), and glutathione peroxidase 1 (GPX1), and transcriptional regulators proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and forkhead box protein O1 (FOXO1), which play important roles in oxidative stress response and are known to be O-GlcNAc-modified. C2C12 myoblasts were subjected to 24 h incubation with different reagents, including hydrogen peroxide, diethyl maleate, high glucose, and glucosamine, and the inhibitors of O-GlcNAc cycling enzymes. Surprisingly, O-GlcNAc levels were significantly increased only with glucosamine, whilst other treatments showed no effect. Significant changes at the mRNA level were observed with concomitant upregulation of the genes for O-GlcNAc enzymes and stress-related proteins with oxidizing agents and downregulation of these genes with agents promoting O-GlcNAcylation. Our findings suggest a role of O-GlcNAc in the stress response and indicate an inhibitory mechanism controlling O-GlcNAc levels in the muscle cells. This could represent an important homeostatic regulation of the cellular defense system.

Glutathione depletion and acute exercise increase O-GlcNAc protein modification in rat skeletal muscle.

Post-translational modification of intracellular proteins with O-linked ß-N-acetylglucosamine (O-GlcNAc) profoundly affects protein structure, function, and metabolism. Although many skeletal muscle proteins are O-GlcNAcylated, the modification has not been extensively studied in this tissue, especially in the context of exercise. This study investigated the effects of glutathione depletion and acute exercise on O-GlcNAc protein modification in rat skeletal muscle. Diethyl maleate (DEM) was used to deplete intracellular glutathione and rats were subjected to a treadmill run. White gastrocnemius and soleus muscles were analyzed for glutathione status, O-GlcNAc and O-GlcNAc transferase (OGT) protein levels, and mRNA expression of OGT, O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase. DEM and exercise both reduced intracellular glutathione and increased O-GlcNAc. DEM upregulated OGT protein expression. The effects of the interventions were significant 4 h after exercise (P < 0.05). The changes in the mRNA levels of O-GlcNAc enzymes were different in the two muscles, potentially resulting from different rates of oxidative stress and metabolic demands between the muscle types. These findings indicate that oxidative environment promotes O-GlcNAcylation in skeletal muscle and suggest an interrelationship between cellular redox state and O-GlcNAc protein modification. This could represent one mechanism underlying cellular adaptation to oxidative stress and health benefits of exercise.

Altering the redox state of skeletal muscle by glutathione depletion increases the exercise-activation of PGC-1α.

We investigated the relationship between markers of mitochondrial biogenesis, cell signaling, and antioxidant enzymes by depleting skeletal muscle glutathione with diethyl maleate (DEM) which resulted in a demonstrable increase in oxidative stress during exercise. Animals were divided into six groups: (1) sedentary control rats; (2) sedentary rats + DEM; (3) exercise control rats euthanized immediately after exercise; (4) exercise rats + DEM; (5) exercise control rats euthanized 4 h after exercise; and (6) exercise rats + DEM euthanized 4 h after exercise. Exercising animals ran on the treadmill at a 10% gradient at 20 m/min for the first 30 min. The speed was then increased every 10 min by 1.6 m/min until exhaustion. There was a reduction in total glutathione in the skeletal muscle of DEM treated animals compared to the control animals (P < 0.05). Within the control group, total glutathione was higher in the sedentary group compared to after exercise (P < 0.05). DEM treatment also significantly increased oxidative stress, as measured by increased plasma F2-isoprostanes (P < 0.05). Exercising animals given DEM showed a significantly greater increase in peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α) mRNA compared to the control animals that were exercised (P < 0.05). This study provides novel evidence that by lowering the endogenous antioxidant glutathione in skeletal muscle and inducing oxidative stress through exercise, PGC-1α gene expression was augmented. These findings further highlight the important role of exercise induced oxidative stress in the regulation of mitochondrial biogenesis.

Antioxidant supplementation during exercise training: beneficial or detrimental?

High levels of reactive oxygen species (ROS) produced in skeletal muscle during exercise have been associated with muscle damage and impaired muscle function. Supporting endogenous defence systems with additional oral doses of antioxidants has received much attention as a noninvasive strategy to prevent or reduce oxidative stress, decrease muscle damage and improve exercise performance. Over 150 articles have been published on this topic, with almost all of these being small-scale, low-quality studies. The consistent finding is that antioxidant supplementation attenuates exercise-induced oxidative stress. However, any physiological implications of this have yet to be consistently demonstrated, with most studies reporting no effects on exercise-induced muscle damage and performance. Moreover, a growing body of evidence indicates detrimental effects of antioxidant supplementation on the health and performance benefits of exercise training. Indeed, although ROS are associated with harmful biological events, they are also essential to the development and optimal function of every cell. The aim of this review is to present and discuss 23 studies that have shown that antioxidant supplementation interferes with exercise training-induced adaptations. The main findings of these studies are that, in certain situations, loading the cell with high doses of antioxidants leads to a blunting of the positive effects of exercise training and interferes with important ROS-mediated physiological processes, such as vasodilation and insulin signalling. More research is needed to produce evidence-based guidelines regarding the use of antioxidant supplementation during exercise training. We recommend that an adequate intake of vitamins and minerals through a varied and balanced diet remains the best approach to maintain the optimal antioxidant status in exercising individuals.