Cardiovascular risk factors: The effects of ageing and smoking on the immune system, an observational clinical study.

Currently immunomodulatory compounds are under investigation for use in patients with cardiovascular disease, caused by atherosclerosis. These trials, using recurrent cardiovascular events as endpoint, require enrollment of large patient groups. We investigated the effect of key risk factors for atherosclerosis development, ageing and smoking, on the immune system, with the objective to identify biomarkers differentiating between human populations, and potentially serving as endpoints for future phase 1B trials with immunomodulatory compounds. Blood was collected from young healthy volunteers (aged 18-25 years, n=30), young smokers (18-25 years, n=20), elderly healthy volunteers (>60 years, n=20), heavy smokers (>45 years, 15 packyears, n=11) and patients with stable coronary artery disease (CAD) (>60 years, n=27). Circulating immune cell subsets were characterized by flow cytometry, and collected plasma was evaluated by proteomics (Olink). Clear ageing effects were observed, mostly illustrated by a lower level in CD8+ and naïve CD4+ and CD8+ T cells, with an increase in CD4+ and CD8+ effector memory T cells in elderly healthy volunteers compared to young healthy volunteers. Heavy smokers showed a more inflammatory cellular phenotype, especially a shift in Th1/Th2 ratio: higher Th1 and lower Th2 percentages compared to young healthy volunteers. A significant decrease in circulating atheroprotective oxLDL-specific IgM was found in patients with CAD compared to young healthy volunteers. Elevated pro-inflammatory and chemotactic proteins TREM1 and CCL11 were observed in elderly volunteers compared to young volunteers. In addition, heavy smokers had an increase in pro-inflammatory cytokine IL-6 and lysosomal protein LAMP3. These data show that ageing and smoking are associated with an inflammatory immunophenotype, and that heavy smokers or aged individuals may serve as potential populations for future clinical trials investigating immunomodulatory drugs targeted for cardiovascular disease.

Sample Aging Profoundly Reduces Monocyte Responses in Human Whole Blood Cultures.

Human whole blood cultures are widely used for the investigation of physiological pathways and drug effects in vitro. Detailed information on the effect of "sample aging" (the time span between blood collection and experimental start) on the experimental outcome is not readily available in the public domain. We studied the effect of sample aging on the ability of immune cells to respond to cell-specific immune triggers (LPS, PMA/ionomycin, and SEB). Sample aging at room temperature profoundly inhibited the LPS-induced monocytic cytokine release in minimally diluted whole blood cultures. The reduction ranged from 20-50% after 30 minutes to 80-100% after 10 hours and differed between cytokines (IL-1ß, IL-2, IL-6, IFNγ, and TNFα). Sample storage at 4°C or 37°C even worsened this. PMA/ionomycin- and SEB-induced cytokine release, both mainly T-cell-driven, were also reduced by sample aging but to a lesser extent (20-50% after 24 hours). Intracellular cytokine staining revealed that the number of LPS-responding cells was not impacted by sample aging and reduced LPS responsivity could also not be explained by apoptosis or downregulated TLR4 expression. Thus, we speculate that sample aging induces an inhibitory pathway downstream from TLR4 in monocytes. These results underline the importance of quick sample handling when investigating innate immune responses in whole blood, especially for monocyte responses.