Human monocyte subsets are transcriptionally and functionally altered in aging in response to pattern recognition receptor agonists [InVitro]

Age-related alterations in immunity have been linked to increased incidence of infections and decreased responses to vaccines in the aging population. Human peripheral blood monocytes are known to promote antigen presentation and antiviral activities; however, the impact of aging on monocyte functions remains an open question. We present an in-depth global analysis examining the impact of aging on classical (CD14+CD16-), intermediate (CD14+CD16+), and non-classical (CD14dimCD16+) monocytes. Monocytes sorted from non-frail healthy adults (18-40 yrs) and OLD (≥ 65 yrs) individuals were analyzed after stimulation with TLR4, TLR7/8, and RIG-I agonists. Our data showed under non-stimulated conditions, monocyte subsets did not reveal significant age-related alternations; however, agonist stimulated-monocytes from adults and OLD subjects did show differences at the transcriptional and functional levels. These alternations in many immune-related transcripts and biological processes resulted in reduced production of IFNα, IFN, IL-1β, CCL20, and CCL8, and higher expression of CX3CR1 in monocytes from OLD subjects. Our findings represent a comprehensive analysis of the influence of human aging on pattern recognition receptors signaling and monocyte functions, and have implications for strategies to enhance the immune response in the context of infection and immunization. Healthy non-frail individuals enrolled into the study were arranged into two groups: adults (YNG) and old (OLD) subjects (n=11 per group). The average age for adults were 30 yrs (range 24-36 yrs), whereas old individuals were 73 yrs (range 67-83 yrs). Individuals with comorbid conditions including cancer (within the last 5 yrs for those greater than 65 yrs), immunocompromising disorders, and steroid usage were excluded; whereas inclusion criteria included controlled hypertension, occasional/tolerable “aching joints” from arthritis and not taking daily non-steroidal anti-inflammatory drugs (NSAIDS) or acetaminophen, and controlled diabetes (see Methods). Monocytes were first evaluated under healthy non-stimulus conditions to determine transcriptional differences between the three subsets, and the effects of aging on gene expression. Using CD14 and CD16 conjugated antibodies, monocytes were sorted from frozen PBMCs isolated from adults and old subjects (n=9 per group) into three subsets: (1) classical (CD14+CD16-), (2) intermediate (CD14+CD16+), and (3) non-classical (CD14dimCD16+). We then determined the transcriptional responses of monocyte subsets following stimulation with various PRRs agonists. Monocyte subsets were sorted from adult and old subjects (n=8/age group) and stimulated for 24 hours with LPS (TLR4), CLO97 (TLR7/8), or 5’-pppRNA complexed with Lyovec (a cationic transfection reagent that facilitates intracellular delivery) (RIG-I) (see Methods).