Endotoxin Tolerance in Abdominal Aortic Aneurysm Macrophages, In Vitro: A Case-Control Study.

Macrophages are implicated in the pathogenesis of abdominal aortic aneurysm (AAA). This study examined the environmentally conditioned responses of AAA macrophages to inflammatory stimuli. Plasma- and blood-derived monocytes were separated from the whole blood of patients with AAA (30-45 mm diameter; n = 33) and sex-matched control participants (n = 44). Increased concentrations of pro-inflammatory and pro-oxidant biomarkers were detected in the plasma of AAA patients, consistent with systemic inflammation and oxidative stress. However, in monocyte-derived macrophages, a suppressed cytokine response was observed in AAA compared to the control following stimulation with lipopolysaccharide (LPS) (tumor necrosis factor alpha (TNF-α) 26.9 ± 3.3 vs. 15.5 ± 3.2 ng/mL, p < 0.05; IL-6 3.2 ± 0.6 vs. 1.4 ± 0.3 ng/mL, p < 0.01). LPS-stimulated production of 8-isoprostane, a biomarker of oxidative stress, was also markedly lower in AAA compared to control participants. These findings are consistent with developed tolerance in human AAA macrophages. As Toll-like receptor 4 (TLR4) has been implicated in tolerance, macrophages were examined for changes in TLR4 expression and distribution. Although TLR4 mRNA and protein expression were unaltered in AAA, cytosolic internalization of receptors and lipid rafts was found. These findings suggest the inflamed, pro-oxidant AAA microenvironment favors macrophages with an endotoxin-tolerant-like phenotype characterized by a diminished capacity to produce pro-inflammatory mediators that enhance the immune response.

Omega-3 fatty acids decrease oxidative stress and inflammation in macrophages from patients with small abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is associated with inflammation and oxidative stress, the latter of which contributes to activation of macrophages, a prominent cell type in AAA. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to limit oxidative stress in animal models of AAA. The aim of this study was to evaluate the effect of the n-3 PUFA docosahexaenoic acid (DHA) on antioxidant defence in macrophages from patients with AAA. Cells were obtained from men with small AAA (diameter 3.0-4.5 cm, 75 ± 6 yr, n = 19) and age- matched male controls (72 ± 5 yr, n = 41) and incubated with DHA for 1 h before exposure to 0.1 µg/mL lipopolysaccharide (LPS) for 24 h. DHA supplementation decreased the concentration of tumour necrosis factor-α (TNF-α; control, 42.1 ± 13.6 to 5.1 ± 2.1 pg/ml, p < 0.01; AAA, 25.2 ± 9.8 to 1.9 ± 0.9 pg/ml, p < 0.01) and interleukin-6 (IL-6; control, 44.9 ± 7.7 to 5.9 ± 2.0 pg/ml, p < 0.001; AAA, 24.3 ± 5.2 to 0.5 ± 0.3 pg/ml, p < 0.001) in macrophage supernatants. DHA increased glutathione peroxidase activity (control, 3.2 ± 0.3 to 4.1 ± 0.2 nmol/min/ml/µg protein, p = 0.004; AAA, 2.3 ± 0.5 to 3.4 ± 0.5 nmol/min/ml/µg protein, p = 0.008) and heme oxygenase-1 mRNA expression (control, 1.5-fold increase, p < 0.001). The improvements in macrophage oxidative stress status serve as a stimulus for further investigation of DHA in patients with AAA.

A simple and effective method for the isolation and culture of human monocytes from small volumes of peripheral blood.

Innate immune cell defects contribute to severe autoimmunity and the pathogenesis of inflammatory disease. Monocyte-derived macrophages typically retain disease related signatures and represent an excellent in vitro model to uncover and validate mechanisms contributing to specific pathological states. Monocyte isolation procedures vary widely in terms of purity, yield, cost, degree of technical difficulty and volume of peripheral blood needed. This paper outlines a novel isolation method that yields monocytes through density gradient centrifugation (Ficoll® and hyperosmotic Percoll®). The protocol has been optimised for small volumes of blood (42 ml) and is simple, reproducible and inexpensive compared to other methods. Monocyte recovery is 70% (relative to monocyte numbers within the buffy coat) and the highly functional macrophages produced are characterised by excellent purity (98.6 ±â€¯0.6%) and intact activation and phagocytic capacities. The method is well suited to investigations involving patient populations where a particular subset of immune cells is known to contribute to the pathogenesis of a specific disease or is aberrant as a consequence of that disease.

n-3 PUFAs improve erythrocyte fatty acid profile in patients with small AAA: a randomized controlled trial.

Abdominal aortic aneurysm (AAA) is an important cause of death in older adults, which has no current drug therapy. Inflammation and abnormal redox status are believed to be key pathogenic mechanisms for AAA. In light of evidence correlating inflammation with aberrant fatty acid profiles, this study compared erythrocyte fatty acid content in 43 AAA patients (diameter 3.0-4.5 cm) and 52 healthy controls. In addition, the effect of omega-3 PUFA (n-3 PUFA) supplementation on erythrocyte fatty acid content was examined in a cohort of 30 AAA patients as part of a 12 week randomized placebo-controlled clinical trial. Blood analyses identified associations between AAA and decreased linoleic acid (LA), and AAA and increased Δ6-desaturase activity and biosynthesis of arachidonic acid (AA) from LA. Omega-3 PUFA supplementation (1.5 g DHA + 0.3 g EPA/day) decreased red blood cell distribution width (14.8 ± 0.4% to 13.8 ± 0.2%; P = 0.003) and levels of pro-inflammatory n-6 PUFAs (AA, 12.46 ± 0.23% to 10.14 ± 0.3%, P < 0.001; adrenic acid, 2.12 ± 0.13% to 1.23 ± 0.09%; P < 0.001). In addition, Δ-4 desaturase activity increased (DHA/docosapentaenoic acid ratio, 1.85 ± 0.14 to 3.93 ± 0.17; P < 0.001) and elongase 2/5 activity decreased (adrenic acid/AA ratio, 0.17 ± 0.01 to 0.12 ± 0.01; P < 0.01) following supplementation. The findings suggest that n-3 PUFAs improve fatty acid profiles and ameliorate factors associated with inflammation in AAA patients.

Cytokine Responses to Acute Exercise in Healthy Older Adults: The Effect of Cardiorespiratory Fitness.

Markers of chronic inflammation increase with aging, and are associated with cardiovascular disease prevalence and mortality. Increases in fitness with exercise training have been associated with lower circulating concentrations of cytokines known to have pro-inflammatory actions (such as interleukin-6 [IL-6]) and higher circulating concentrations of anti-inflammatory cytokines (interleukin-10 [IL-10]). However, the effect of cardiorespiratory fitness on acute cytokine responses to a single bout of exercise in healthy older individuals is unknown. We compared the response of plasma cytokines IL-6, tumor necrosis factor-alpha (TNF-α) and IL-10 to a bout of moderate-intensity continuous and higher-intensity interval exercise between older individuals with higher and lower levels of cardiorespiratory fitness. Sixteen lower-fit (VO2peak: 22.6±2.8 mL.kg-1.min-1) and fourteen higher-fit participants (VO2peak: 37.4±5.9 mL.kg-1.min-1) completed three 24 min experimental protocols in a randomized order: (1) moderate-intensity continuous exercise (40% of peak power output [PPO]); (2) higher-intensity interval exercise (12 × 1 min intervals at 70% PPO separated by 1 min periods at 10% PPO); or (3) non-exercise control. Plasma cytokines were measured at rest, immediately after, and during 90 min of recovery following exercise or control. Plasma IL-6 concentrations at baseline were greater in the higher-fit compared to the lower-fit group (P = 0.02), with no difference in plasma IL-10 or TNF-α concentrations at baseline between groups. Plasma IL-6 and IL-10 concentrations in both groups increased immediately after all protocols (IL-6: P = 0.02, IL-10: P < 0.01). However, there was no difference in the IL-6 and IL-10 response between the exercise and non-exercise (control) protocols. After all protocols, no changes in plasma TNF-α concentrations were observed in either the higher- or lower-fit groups. In this study, basal concentrations of circulating IL-6 were elevated in older individuals with higher levels of cardiorespiratory fitness. However, changes in plasma cytokine concentrations after exercise were not different to changes after non-exercise control in both the lower- and higher-fit groups.

Effects of acute exercise on endothelial function in patients with abdominal aortic aneurysm.

Endothelial dysfunction is observed in patients with abdominal aortic aneurysm (AAA), who have increased risk of cardiovascular events and mortality. This study aimed to assess the acute effects of moderate- and higher-intensity exercise on endothelial function, as assessed by flow-mediated dilation (FMD), in AAA patients (74 ± 6 yr old, n = 22) and healthy adults (72 ± 5 yr old, n = 22). Participants undertook three randomized visits, including moderate-intensity continuous exercise [40% peak power output (PPO)], higher-intensity interval exercise (70% PPO), and a no-exercise control. Brachial artery FMD was assessed at baseline and at 10 and 60 min after each condition. Baseline FMD was lower [by 1.10% (95% confidence interval: 0.72-.81), P = 0.044] in AAA patients than in healthy adults. There were no group differences in FMD responses after each condition ( P = 0.397). FMD did not change after no-exercise control but increased by 1.21% (95% confidence interval: 0.69-1.73, P < 0.001) 10 min after moderate-intensity continuous exercise in both groups and returned to baseline after 60 min. Conversely, FMD decreased by 0.93% (95% confidence interval: 0.41-1.44, P < 0.001) 10 min after higher-intensity interval exercise in both groups and remained decreased after 60 min. We found that the acute response of endothelial function to exercise is intensity-dependent and similar between AAA patients and healthy adults. Our findings provide evidence that regular exercise may improve vascular function in AAA patients, as it does in healthy adults. Improved FMD after moderate-intensity exercise may provide short-term benefit. Whether the decrease in FMD after higher-intensity exercise represents an additional risk and/or a greater stimulus for vascular adaptation remains to be elucidated. NEW & NOTEWORTHY Abdominal aortic aneurysm patients have vascular dysfunction. We observed a short-term increase in vascular function after moderate-intensity exercise. Conversely, higher-intensity exercise induced a prolonged reduction in vascular function, which may be associated with both short-term increases in cardiovascular risk and signaling for longer-term vascular adaptation in abdominal aortic aneurysm patients.