NFAT5 Amplifies Antipathogen Responses by Enhancing Chromatin Accessibility, H3K27 Demethylation, and Transcription Factor Recruitment.

The ability of innate immune cells to respond to pathogen-associated molecular patterns across a wide range of intensities is fundamental to limit the spreading of infections. Studies on transcription responses to pathogen-activated TLRs have often used relatively high TLR ligand concentrations, and less is known about their regulation under mild stimulatory conditions. We had shown that the transcription factor NFAT5 facilitates expression of antipathogen genes under TLR stimulation conditions corresponding to low pathogen loads. In this study, we analyze how NFAT5 optimizes TLR-activated responses in mouse macrophages. We show that NFAT5 was required for effective recruitment of central effectors p65/NF-κB and c-Fos to specific proinflammatory target genes, such as Nos2, Il6, and Tnf in primary macrophages responding to low doses of the TLR4 ligand LPS. By contrast, NFAT5 was not required for p65/NF-κB recruitment in response to high LPS doses. Using the transposase-accessible chromatin with high-throughput sequencing assay, we show that NFAT5 facilitated chromatin accessibility mainly at promoter regions of multiple TLR4-responsive genes. Analysis of various histone marks that regulate gene expression in response to pathogens identified H3K27me3 demethylation as an early NFAT5-dependent mechanism that facilitates p65 recruitment to promoters of various TLR4-induced genes. Altogether, these results advance our understanding about specific mechanisms that optimize antipathogen responses to limit infections.

Palmitate Conditions Macrophages for Enhanced Responses toward Inflammatory Stimuli via JNK Activation.

Obesity is associated with low-grade inflammation and elevated levels of circulating saturated fatty acids, which trigger inflammatory responses by engaging pattern recognition receptors in macrophages. Because tissue homeostasis is maintained through an adequate balance of pro- and anti-inflammatory macrophages, we assessed the transcriptional and functional profile of M-CSF-dependent monocyte-derived human macrophages exposed to concentrations of saturated fatty acids found in obese individuals. We report that palmitate (C16:0, 200 µM) significantly modulates the macrophage gene signature, lowers the expression of transcription factors that positively regulate IL-10 expression (MAFB, AhR), and promotes a proinflammatory state whose acquisition requires JNK activation. Unlike LPS, palmitate exposure does not activate STAT1, and its transcriptional effects can be distinguished from those triggered by LPS, as both agents oppositely regulate the expression of CCL19 and TRIB3 Besides, palmitate conditions macrophages for exacerbated proinflammatory responses (lower IL-10 and CCL2, higher TNF-α, IL-6, and IL-1ß) toward pathogenic stimuli, a process also mediated by JNK activation. All of these effects of palmitate are fatty acid specific because oleate (C18:1, 200 µM) does not modify the macrophage transcriptional and functional profiles. Therefore, pathologic palmitate concentrations promote the acquisition of a specific polarization state in human macrophages and condition macrophages for enhanced responses toward inflammatory stimuli, with both effects being dependent on JNK activation. Our results provide further insight into the macrophage contribution to obesity-associated inflammation.

Reshaping of Human Macrophage Polarization through Modulation of Glucose Catabolic Pathways.

Macrophages integrate information from the tissue microenvironment and adjust their effector functions according to the prevalent extracellular stimuli. Therefore, macrophages can acquire a variety of activation (polarization) states, and this functional plasticity allows the adequate initiation, regulation, and resolution of inflammatory responses. Modulation of the glucose metabolism contributes to the macrophage adaptation to the surrounding cytokine milieu, as exemplified by the distinct glucose catabolism of macrophages exposed to LPS/IFN-γ or IL-4. To dissect the acquisition of macrophage effector functions in the absence of activating cytokines, we assessed the bioenergetic profile of macrophages generated in the presence of GM-CSF (GM-MØ) or M-CSF (M-MØ), which do not release pro- or anti-inflammatory cytokines unless subjected to additional activating stimuli. Compared to M-MØ, GM-MØ displayed higher oxygen consumption rate and aerobic glycolysis (extracellular acidification rate [ECAR]), as well as higher expression of genes encoding glycolytic enzymes. However, M-MØ exhibited a significantly higher oxygen consumption rate/ECAR ratio. Surprisingly, whereas aerobic glycolysis positively regulated IL1B, TNF, and INHBA mRNA expression in both macrophage subtypes, mitochondrial respiration negatively affected IL6, IL1B, TNF, and CXCL10 mRNA expression in M-MØ. The physiological significance of these results became evident under low oxygen tensions, as hypoxia enhanced ECAR in M-MØ via HIF-1α and HIF-2α, increased expression of glycolytic enzymes and GM-MØ-specific genes, and diminished expression of M-MØ-associated genes. Therefore, our data indicate that GM-MØ and M-MØ display distinct bioenergetic profiles, and that hypoxia triggers a transcriptomic switch in macrophages by promoting a HIF-1α/HIF-2α-dependent increase in ECAR.

Metformin Potentiates the Benefits of Dietary Restraint: A Metabolomic Study.

Prevention of the metabolic consequences of a chronic energy-dense/high-fat diet (HFD) represents a public health priority. Metformin is a strong candidate to be incorporated in alternative therapeutic approaches. We used a targeted metabolomic approach to assess changes related to the multi-faceted metabolic disturbances provoked by HFD. We evaluated the protective effects of metformin and explored how pro-inflammatory and metabolic changes respond when mice rendered obese, glucose-intolerant and hyperlipidemic were switched to diet reversal with or without metformin. Mice treated with metformin and diet-reversal showed a dramatically improved protection against HFD-induced hepatic steatosis, a beneficial effect that was accompanied by a lowering of liver-infiltrating pro-inflammatory macrophages and lower release of pro-inflammatory cytokines. Metformin combined with diet reversal promoted effective weight loss along with better glucose control, lowered levels of circulating cholesterol and triglycerides, and reduced adipose tissue content. Our findings underscored the ability of metformin to target the contribution of branched chain amino acids to adipose tissue metabolism while suppressing mitochondrial-dependent biosynthesis in hepatic tissue. The relationship between adipose tissue and liver might provide clinical potential for combining metformin and dietary modifications to protect against the metabolic damage occurring upon excessive dietary fat intake.

Understanding the role of circulating chemokine (C-C motif) ligand 2 in patients with chronic ischemia threatening the lower extremities.

The role of chemokine (C-C motif) ligand 2 (CCL2) in peripheral artery disease is unclear. We measured the difference between serum and plasma levels of CCL2 in patients with chronic ischemia threatening the lower extremities following the observation that atypical chemokine receptors in blood and tissue cells may prevent CCL2 from entering the circulation and consequently modulate its function of attracting monocytes to the site of lesion. To identify the influence of CCL2, we compared the patients' values to those in bio-banked samples from a control population. Further, we explored the association with the Asp42Gly polymorphism (rs12075) in Duffy antigen chemokine receptor; one of these atypical chemokine receptors. When possible, we evaluated in surgically excised normal and affected arteries the calcium burden as well as the expression of CCL2 and related receptors reflecting the inflammatory status. Our findings indicate that circulating CCL2 was significantly associated with the severity and presence of the disease (OR 0.966, 95% CI 0.944 to 0.988, p = 0.003). Circulating CCL2 was dependent on the rs12075 genotype (AA>AG>GG), which, probably, indicates a higher expression of chemokine receptor in the arteries of AA subjects. The associations with genetic variants and the over-expression of atypical chemokine receptors in diseased arteries may have potential implications and our data indicate that CCL2 may represent a previously unrecognized factor that needs to be considered in the screening of patients with risk factors for peripheral artery disease.

A possible role for CCR5 in the progression of atherosclerosis in HIV-infected patients: a cross-sectional study.

BACKGROUND: Chemokines can block viral entry by interfering with HIV co-receptors and are recognised mediators of atherosclerosis development. A number of experimental drugs that inhibit HIV entry arrest the development of atherosclerosis in animal models. We hypothesised that the expression of chemokine receptors in circulating leukocytes is associated with the rate of atherosclerosis progression in HIV-infected patients. METHODS: The increase in intima-media thickness during a 2-year follow-up was used to classify HIV-infected patients (n = 178) as progressors (n = 142) or non-progressors (n = 36) with respect to atherosclerosis. Logistic regression was used to assess variables associated with atherosclerosis progression. Mutations in the CCR5Δ32, CCR2 64I, and CX3CR1 (T280M and V249I) co-receptors as well as the levels of CCR5, CXCR4, CX3CR1, and CCR2 mRNA expression in circulating leukocytes were analysed as independent variables. RESULTS: Among the baseline variables, only genetic variants explained the dichotomous outcome. The expression of CCR2 and CXCR4 did not discriminate between progressors and non-progressors. Conversely, CCR5 and CX3CR1 expression was higher in not only progressors but also patients with detectable viral load. The logistic regression, however, demonstrated a significant role for CCR5 expression as a predictor of atherosclerosis progression (B = 2.1, OR = 8.1, p = 0.04) and a negligible effect for CXC3R1 and CCR2 expression. CONCLUSIONS: Available CCR5 antagonists should be investigated for their potential to delay the course of atherosclerosis in HIV-infected patients.

Mitochondrial dysfunction: a basic mechanism in inflammation-related non-communicable diseases and therapeutic opportunities.

Obesity is not necessarily a predisposing factor for disease. It is the handling of fat and/or excessive energy intake that encompasses the linkage of inflammation, oxidation, and metabolism to the deleterious effects associated with the continuous excess of food ingestion. The roles of cytokines and insulin resistance in excessive energy intake have been studied extensively. Tobacco use and obesity accompanied by an unhealthy diet and physical inactivity are the main factors that underlie noncommunicable diseases. The implication is that the management of energy or food intake, which is the main role of mitochondria, is involved in the most common diseases. In this study, we highlight the importance of mitochondrial dysfunction in the mutual relationships between causative conditions. Mitochondria are highly dynamic organelles that fuse and divide in response to environmental stimuli, developmental status, and energy requirements. These organelles act to supply the cell with ATP and to synthesise key molecules in the processes of inflammation, oxidation, and metabolism. Therefore, energy sensors and management effectors are determinants in the course and development of diseases. Regulating mitochondrial function may require a multifaceted approach that includes drugs and plant-derived phenolic compounds with antioxidant and anti-inflammatory activities that improve mitochondrial biogenesis and act to modulate the AMPK/mTOR pathway.

Ubiquitous transgenic overexpression of C-C chemokine ligand 2: a model to assess the combined effect of high energy intake and continuous low-grade inflammation.

Excessive energy management leads to low-grade, chronic inflammation, which is a significant factor predicting noncommunicable diseases. In turn, inflammation, oxidation, and metabolism are associated with the course of these diseases; mitochondrial dysfunction seems to be at the crossroads of mutual relationships. The migration of immune cells during inflammation is governed by the interaction between chemokines and chemokine receptors. Chemokines, especially C-C-chemokine ligand 2 (CCL2), have a variety of additional functions that are involved in the maintenance of normal metabolism. It is our hypothesis that a ubiquitous and continuous secretion of CCL2 may represent an animal model of low-grade chronic inflammation that, in the presence of an energy surplus, could help to ascertain the afore-mentioned relationships and/or to search for specific therapeutic approaches. Here, we present preliminary data on a mouse model created by using targeted gene knock-in technology to integrate an additional copy of the CCl2 gene in the Gt(ROSA)26Sor locus of the mouse genome via homologous recombination in embryonic stem cells. Short-term dietary manipulations were assessed and the findings include metabolic disturbances, premature death, and the manipulation of macrophage plasticity and autophagy. These results raise a number of mechanistic questions for future study.

Macrophage-specific MHCII expression is regulated by a remote Ciita enhancer controlled by NFAT5.

MHCII in antigen-presenting cells (APCs) is a key regulator of adaptive immune responses. Expression of MHCII genes is controlled by the transcription coactivator CIITA, itself regulated through cell type-specific promoters. Here we show that the transcription factor NFAT5 is needed for expression of Ciita and MHCII in macrophages, but not in dendritic cells and other APCs. NFAT5-deficient macrophages showed defective activation of MHCII-dependent responses in CD4+ T lymphocytes and attenuated capacity to elicit graft rejection in vivo. Ultrasequencing analysis of NFAT5-immunoprecipitated chromatin uncovered an NFAT5-regulated region distally upstream of Ciita This region was required for CIITA and hence MHCII expression, exhibited NFAT5-dependent characteristics of active enhancers such as H3K27 acetylation marks, and required NFAT5 to interact with Ciita myeloid promoter I. Our results uncover an NFAT5-regulated mechanism that maintains CIITA and MHCII expression in macrophages and thus modulates their T lymphocyte priming capacity.

Exploring the effects of the atherosclerosis progression and the choice of affected arteries in the design of experiments with Apolipoprotein E-deficient mice.

The objective of this study is to explore the longitudinal progression of atherosclerosis and the correlation between methods to measure the lesion in apolipoprotein E-deficient mice. Atherosclerosis progression was assessed by measurements of foam cell-rich depositions in their proximal aortas, and/or in surgically excised arteries, to assess the histological luminal narrowing. A longitudinal study was performed by comparing the values for carotid, aorta, and femoral and iliac arteries using common histological techniques. There were no significant differences in progression between different arteries, but correlation with the classical measurement of atherosclerosis in the aortic root was poor. Each laboratory requires specific standardization. Carotid arteries were sensitive to atherosclerosis in these mice, and progression was exponential. In conclusion, morphometric data show the importance of the choice of the duration of treatment, the appropriate controls, and the age at which to begin the experiments.

Metformin administration induces hepatotoxic effects in paraoxonase-1-deficient mice.

Metformin is the first-line pharmacological treatment of diabetes. In these patients, metformin reduces body weight and decreases the risk of diabetes-related complications such as cardiovascular disease. However, whether metformin elicits beneficial effects on liver histology is a controversial issue and, as yet, there is no consensus. Paraoxonase-1 (PON1), an enzyme synthesized mainly by the liver, degrades lipid peroxides and reduces oxidative stress. PON1 activities are decreased in chronic liver diseases. We evaluated the effects of metformin in the liver of PON1-deficient mice which, untreated, present a mild degree of liver steatosis. Metformin administration aggravated inflammation in animals given a standard mouse chow and in those fed a high-fat diet. Also, it was associated with a higher degree of steatosis in animals fed a standard chow diet. This report is a cautionary note regarding the prescription of metformin for the treatment of diabetes in patients with concomitant liver impairment.

Exploring the Process of Energy Generation in Pathophysiology by Targeted Metabolomics: Performance of a Simple and Quantitative Method.

Abnormalities in mitochondrial metabolism and regulation of energy balance contribute to human diseases. The consequences of high fat and other nutrient intake, and the resulting acquired mitochondrial dysfunction, are essential to fully understand common disorders, including obesity, cancer, and atherosclerosis. To simultaneously and noninvasively measure and quantify indirect markers of mitochondrial function, we have developed a method based on gas chromatography coupled to quadrupole-time of flight mass spectrometry and an electron ionization interface, and validated the system using plasma from patients with peripheral artery disease, human cancer cells, and mouse tissues. This approach was used to increase sensibility in the measurement of a wide dynamic range and chemical diversity of multiple intermediate metabolites used in energy metabolism. We demonstrate that our targeted metabolomics method allows for quick and accurate identification and quantification of molecules, including the measurement of small yet significant biological changes in experimental samples. The apparently low process variability required for its performance in plasma, cell lysates, and tissues allowed a rapid identification of correlations between interconnected pathways. Our results suggest that delineating the process of energy generation by targeted metabolomics can be a valid surrogate for predicting mitochondrial dysfunction in biological samples. Importantly, when used in plasma, targeted metabolomics should be viewed as a robust and noninvasive source of biomarkers in specific pathophysiological scenarios.

Paraoxonases and chemokine (C-C motif) ligand-2 in noncommunicable diseases.

Oxidative stress and inflammation underpin most diseases; their mechanisms are inextricably linked. Chronic inflammation is associated with oxidation, anti-inflammatory cascades are linked to decreased oxidation, increased oxidative stress triggers inflammation, and redox balance inhibits the inflammatory cellular response. Whether or not oxidative stress and inflammation represent the cause or consequence of cellular pathology, they contribute significantly to the pathogenesis of noncommunicable diseases (NCD). The incidence of obesity and other related metabolic disturbances are increasing, as are age-related diseases due to a progressively aging population. Relationships between oxidative stress, inflammatory signaling, and metabolism are, in the broad sense of energy transformation, being increasingly recognized as part of the problem in NCD. In this chapter, we summarize the pathologic consequences of an imbalance between circulating and cellular paraoxonases, the system for scavenging excessive reactive oxygen species and circulating chemokines. They act as inducers of migration and infiltration of immune cells in target tissues as well as in the pathogenesis of disease that perturbs normal metabolic function. This disruption involves pathways controlling lipid and glucose homeostasis as well as metabolically driven chronic inflammatory states that encompass several response pathways. Dysfunction in the endoplasmic reticulum and/or mitochondria represents an important feature of chronic disease linked to oxidation and inflammation seen as self-reinforcing in NCD. Therefore, correct management requires a thorough understanding of these relationships and precise interpretation of laboratory test results.

Xenohormetic and anti-aging activity of secoiridoid polyphenols present in extra virgin olive oil: a new family of gerosuppressant agents.

Aging can be viewed as a quasi-programmed phenomenon driven by the overactivation of the nutrient-sensing mTOR gerogene. mTOR-driven aging can be triggered or accelerated by a decline or loss of responsiveness to activation of the energy-sensing protein AMPK, a critical gerosuppressor of mTOR. The occurrence of age-related diseases, therefore, reflects the synergistic interaction between our evolutionary path to sedentarism, which chronically increases a number of mTOR activating gero-promoters (e.g., food, growth factors, cytokines and insulin) and the "defective design" of central metabolic integrators such as mTOR and AMPK. Our laboratories at the Bioactive Food Component Platform in Spain have initiated a systematic approach to molecularly elucidate and clinically explore whether the "xenohormesis hypothesis," which states that stress-induced synthesis of plant polyphenols and many other phytochemicals provides an environmental chemical signature that upregulates stress-resistance pathways in plant consumers, can be explained in terms of the reactivity of the AMPK/mTOR-axis to so-called xenohormetins. Here, we explore the AMPK/mTOR-xenohormetic nature of complex polyphenols naturally present in extra virgin olive oil (EVOO), a pivotal component of the Mediterranean style diet that has been repeatedly associated with a reduction in age-related morbid conditions and longer life expectancy. Using crude EVOO phenolic extracts highly enriched in the secoiridoids oleuropein aglycon and decarboxymethyl oleuropein aglycon, we show for the first time that (1) the anticancer activity of EVOO secoiridoids is related to the activation of anti-aging/cellular stress-like gene signatures, including endoplasmic reticulum (ER) stress and the unfolded protein response, spermidine and polyamine metabolism, sirtuin-1 (SIRT1) and NRF2 signaling; (2) EVOO secoiridoids activate AMPK and suppress crucial genes involved in the Warburg effect and the self-renewal capacity of "immortal" cancer stem cells; (3) EVOO secoiridoids prevent age-related changes in the cell size, morphological heterogeneity, arrayed cell arrangement and senescence-associated ß-galactosidase staining of normal diploid human fibroblasts at the end of their proliferative lifespans. EVOO secoiridoids, which provide an effective defense against plant attack by herbivores and pathogens, are bona fide xenohormetins that are able to activate the gerosuppressor AMPK and trigger numerous resveratrol-like anti-aging transcriptomic signatures. As such, EVOO secoiridoids constitute a new family of plant-produced gerosuppressant agents that molecularly "repair" the aimless (and harmful) AMPK/mTOR-driven quasi-program that leads to aging and aging-related diseases, including cancer.