Low-frequency electromagnetic fields do not alter responses of inflammatory genes and proteins in human monocytes and immune cell lines.

The effects of low frequency electromagnetic fields (LF EMF) on human health are the subject of on-going research and serious public concern. These fields potentially elicit small effects that have been proposed to have consequences, either positive or negative, for biological systems. To reveal potentially weak but biologically relevant effects, we chose to extensively examine exposure of immune cells to two different signals, namely a complex multiple waveform field, and a 50 Hz sine wave. These immune cells are highly responsive and, in vivo, modulation of cytokine expression responses can result in systemic health effects. Using time course experiments, we determined kinetics of cytokine and other inflammation-related genes in a human monocytic leukemia cell line, THP-1, and primary monocytes and macrophages. Moreover, cytokine protein levels in THP-1 monocytes were determined. Exposure to either of the two signals did not result in a significant effect on gene and protein expression in the studied immune cells. Also, additional experiments using non-immune cells showed no effects of the signals on cytokine gene expression. We therefore conclude that these LF EMF exposure conditions are not expected to significantly modulate innate immune signaling.

Extremely low frequency electromagnetic field exposure does not modulate toll-like receptor signaling in human peripheral blood mononuclear cells.

The effects of extremely low frequency electromagnetic fields (ELF-EMF) on human health remain unclear. It has been reported that ELF-EMF may modulate the innate immune response to microorganisms in animal models and mammalian cell-lines. With the recently gained insight in innate immune signaling and the discovery of pattern recognition, we aim to study whether ELF-EMF modulates innate inflammatory signaling pathways. We used human peripheral blood mononuclear cells (PBMCs), isolated from blood from healthy volunteers, which we stimulated with specific TLR2 and TLR4 ligands, and with several microorganisms. The cells were subsequently exposed in B(dc)=3 µT to a highly controlled and standardized ELF-EMF signal (20-5000Hz, B(ac)=5 µT, 30 min) and cytokine production was measured at different time points after stimulation. No significant difference in immune response, as reflected by IL-1ß, IL-6, TNFα, IL-8 and IL-10 production, could be detected after stimulation with LPS (TLR4 ligand), Pam3Cys (TLR2 ligand) or a panel of heat killed microorganisms: Mycobacterium tuberculosis, Salmonella typhimurium, Candida albicans, Aspergillus fumigatus and Staphylococcus aureus (multiple TLR ligands). We therefore conclude that under our experimental conditions, ELF-EMF does not modulate the innate immune response of human primary cells after TLR stimulation in vitro.

Activation of peroxisome proliferator-activated receptor alpha in human peripheral blood mononuclear cells reveals an individual gene expression profile response.

BACKGROUND: Peripheral blood mononuclear cells (PBMCs) are relatively easily obtainable cells in humans. Gene expression profiles of PBMCs have been shown to reflect the pathological and physiological state of a person. Recently, we showed that the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) has a functional role in human PBMCs during fasting. However, the extent of the role of PPARalpha in human PBMCs remains unclear. In this study, we therefore performed gene expression profiling of PBMCs incubated with the specific PPARalpha ligand WY14,643. RESULTS: Incubation of PBMCs with WY14,643 for 12 hours resulted in a differential expression of 1,373 of the 13,080 genes expressed in the PBMCs. Gene expression profiles showed a clear individual response to PPARalpha activation between six healthy human blood donors. Pathway analysis showed that genes in fatty acid metabolism, primarily in beta-oxidation were up-regulated upon activation of PPARalpha with WY14,643, and genes in several amino acid metabolism pathways were down-regulated. CONCLUSION: This study shows that PPARalpha in human PBMCs regulates fatty acid and amino acid metabolism. In addition, PBMC gene expression profiles show individual responses to WY14,643 activation. We showed that PBMCs are a suitable model to study changes in PPARalpha activation in healthy humans.

Fasting induces changes in peripheral blood mononuclear cell gene expression profiles related to increases in fatty acid beta-oxidation: functional role of peroxisome proliferator activated receptor alpha in human peripheral blood mononuclear cells.

BACKGROUND: Peripheral blood mononuclear cells (PBMCs) are the only readily available cells in healthy humans. Various studies showed disease-characteristic gene expression patterns in PBMCs. However, little is known of nutritional effects on PBMC gene expression patterns. Fatty acids are nutrients that regulate gene expression by activating the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). PBMCs express PPARalpha, making these cells interesting to study FA-dependent gene expression. OBJECTIVE: The aim of this study was to elucidate whether PBMC gene expression profiles also reflect nutrition-related metabolic changes. Furthermore, we focused on the specific role of PPARalpha in regulation of PBMC gene expression during fasting, when plasma free fatty acids are elevated. DESIGN: Four healthy male volunteers fasted for 48 h. PBMC RNA was hybridized on Affymetrix whole genome microarrays. To elucidate the role of PPARalpha, PBMCs of 9 blood donors were incubated with the specific PPARalpha ligand Wy14643. RESULTS: After 24 and 48 h of fasting, 1200 and 1386 genes were changed >1.4-fold, respectively. Many of those genes were involved in fatty acid beta-oxidation and are known PPARalpha target genes. Incubation of PBMCs with Wy14643 resulted in up-regulation of genes that were also up-regulated during fasting. CONCLUSIONS: We conclude that PBMC gene expression profiles reflect nutrition-related metabolic changes such as fasting and that part of the fasting-induced changes are likely regulated by PPARalpha.

Measurements of low oxygen tension in vitro and response of macrophages to levels applicable to peri-and postoperative treatment of traumatic brain injury.

Established clinical guidelines for treatment of severe traumatic brain injury aim at maintaining intracranial and cerebral perfusion pressures. Recently, it has been shown that additional regulation of cerebral oxygen delivery helps to decrease patient mortality and leads to improved 6-month quality-of-life scores. However, eubaric oxygen-guided therapy is still controversial since it is well known that hyperoxia can cause unwanted secondary brain injury. Research studies are warranted to better understand the range of oxygen pressures that positively influence brain cell behavior. We perform such studies using a two-enzyme in vitro system that allows exposing tissue culture cells to various steady-state, or rapidly changing, oxygen pressures. Here, we present a mathematical model of the system and its validation by real-time monitoring of oxygen tensions. We additionally present preliminary evidence that human brain macrophages have a different oxygen tolerance compared to systemic macrophages and propose improvements to our in vitro system to make it applicable for data collection that aim at refining oxygen-guided therapy for patients with traumatic brain injury.

The effect of low oxygen with and without steady-state hydrogen peroxide on cytokine gene and protein expression of monocyte-derived macrophages - biomed 2011.

An early event during inflammation and infection is the migration of monocytes into tissues where they differentiate into macrophages. Such monocyte-derived macrophages face an unfavorable environment characterized by extremely low oxygen tension and accumulation of reactive oxygen species such as hydrogen peroxide. Previous experiments showed that a macrophage cell line cultured under these conditions responded to inflammatory stimulants with an increased respiratory burst compared to cells kept at regular ambient oxygen conditions. The objective of the present study was to test if this effect was accompanied by an increased cytokine production. Using cytokine protein arrays and real time gene expression analysis, we indeed found that low oxygen exposure increased expression of characteristic macrophage inflammatory cytokines such as IL-1, IL-6, and TNF-alpha. Interestingly, cells exposed to low oxygen and steady-state hydrogen peroxide levels did not show this increase, indicating different cell mechanisms might be involved in increasing macrophage hydrogen peroxide and cytokine release.

Postprandial dietary lipid-specific effects on human peripheral blood mononuclear cell gene expression profiles.

BACKGROUND: Dietary polyunsaturated fatty acids (PUFAs) have a variety of beneficial effects, and immune cells play an important role in these effects. The mechanisms of action of PUFAs are still not completely understood, but it is known that PUFAs can influence the expression of a broad set of genes. OBJECTIVE: The objective was to determine the postprandial effects of intake of different fatty acids on the gene expression profiles of peripheral blood mononuclear cells (PBMCs). DESIGN: In a single-blind crossover study, 21 healthy male volunteers consumed shakes enriched in PUFAs, monounsaturated fatty acids (MUFAs), or saturated fatty acids (SFAs) in random order. Blood samples were collected before and at several time points after intake. Whole-genome gene expression profiles of PBMCs were examined before and 6 h after intake of the PUFA and SFA shakes. In addition, ex vivo incubation of human PBMCs with different fatty acids was performed. RESULTS: Whole-genome expression analysis showed distinct differences between PUFA and SFA consumption. PUFA intake decreased the expression of genes in liver X receptor signaling, whereas SFA intake increased the expression of these genes. PUFA intake also increased the expression of genes related to cellular stress responses. MUFA intake had an intermediate effect on several genes. Ex vivo experiments showed a direct effect of free fatty acids on PBMC gene expression. CONCLUSION: This study showed that PBMCs can reveal fatty acid-specific gene expression profiles in young healthy men after the consumption of different fatty acids, as evidenced by the opposite effects of PUFA and SFA intakes on the expression of genes involved in liver X receptor signaling. This trial was registered at www.clinicaltrials.gov as NCT01000194.

Fish-oil supplementation induces antiinflammatory gene expression profiles in human blood mononuclear cells.

BACKGROUND: Polyunsaturated fatty acids can have beneficial effects on human immune cells, such as peripheral blood mononuclear cells (PBMCs). However, the mechanisms of action of polyunsaturated fatty acids on immune cells are still largely unknown. OBJECTIVE: The objective was to examine the effects of supplementation with the polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on whole-genome PBMC gene expression profiles, in healthy Dutch elderly subjects participating in a double-blind trial, by using whole-genome transcriptomics analysis. DESIGN: The subjects were randomly allocated to 1 of 3 groups: 1) consumption of 1.8 g EPA+DHA/d (n = 36), 2) consumption of 0.4 g EPA+DHA/d (n = 37), or 3) consumption of 4.0 g high-oleic acid sunflower oil (HOSF)/d (n = 38). All supplements were given in capsules. Before and after 26 wk of intervention, blood samples were collected. Microarray analysis was performed on PBMC RNA from 23 subjects who received 1.8 g EPA+DHA/d and 25 subjects who received HOSF capsules. Quantitative real-time polymerase chain reaction was performed in all 111 subjects. RESULTS: A high EPA+DHA intake changed the expression of 1040 genes, whereas HOSF intake changed the expression of only 298 genes. EPA+DHA intake resulted in a decreased expression of genes involved in inflammatory- and atherogenic-related pathways, such as nuclear transcription factor kappaB signaling, eicosanoid synthesis, scavenger receptor activity, adipogenesis, and hypoxia signaling. CONCLUSION: These results are the first to show that intake of EPA+DHA for 26 wk can alter the gene expression profiles of PBMCs to a more antiinflammatory and antiatherogenic status. This trial was registered at clinicaltrials.gov as NCT00124852.