Investigating the Pathogen-Associated Molecular Pattern Activation Effects on Human Peripheral Blood Progenitor and Monocytes in Aging

Abstract

Aging has become increasingly prevalent worldwide as the population of individuals aged 60 years and older surpasses that of individuals 18 years of age and younger, a trend that is projected to continue in the coming decades. Aging has not been characterized as a disease due to being a natural process of life. Yet, aging has been recognized to be a significant biological risk factor for diseases. Aging is influenced by intrinsic factors, which refer to internal changes at the cellular and organ levels, and extrinsic factors, which include external influences such as environmental pollutants, ultraviolet radiation, infectious agents, dietary habits, and physical activity. Altogether, aging alters the immune system composition and functionality of the cells which makes these individuals more susceptible to primary and secondary infections. The research questions addressed in this dissertation required the use of primary human immune cells because important differences exist between murine and human toll-like receptor (TLR) biology and antiviral signaling pathways. Variations in receptor expression, cellular distribution, ligand responsiveness, and downstream type I interferon signaling have been reported across species, making murine models insufficient for fully characterizing the mechanisms of innate immune sensing examined in this study. Therefore, there is a critical need to conduct human studies to directly characterize age-associated changes in the immune system. Such studies are essential for identifying clinically relevant therapeutic targets to improve immune function in aged individuals. Those targets will likely also be important for improving general immunity of all people during novel infectious events. To identify potential therapeutic targets, it is first necessary to characterize how pattern-recognition receptor (PRR) signaling pathways are altered during immune cell activation in different immune cell populations from young and aged donors. Our laboratory aims to investigate how pathogen-associated molecular patterns (PAMPs) influence progenitor developmental decisions down the myeloid pathway and how those mature innate immune cell respond to PAMPs by comparing cell expansion, apoptosis, cell cycle progression, cytokine production, and other functional responses between healthy young and aged donors. To uncover age-associated differences, we utilized two types of immune cells in young and aged individuals. The first perspective focused on hematopoietic stem and progenitor cells (HSPCs). Although these cells are not the primary mediators of pathogen clearance through cytokine production, they replenish the immune cell compartment and can be influenced by exposure to pathogen-associated molecular patterns (PAMPs).Therefore, uncovering some deficient developmental pathways to specific PAMPs can lead to understanding the immune system from progenitor to mature cells. Subsets of HSPCs were evaluated for toll-like receptors (TLRs) protein expression associated with the type 1 interferon (1-IFN) production by flow cytometry, which shows no differences between young and old individuals from our cohort. This does not address the functionality of the TLRs though in our cohort of relatively healthy volunteers, rather than the more frail of the aged human population. Therefore, we stimulated enriched HSPCs from peripheral blood mononuclear cells (PBMCs) with PAMPs associated with the 1-IFN production. We evaluated the expansion of stimulated HSPCs through viability cell counts and placed the stimulated HSPCs into a culture medium to evaluate their differentiation capacity into different myeloid cells. Interestingly, the only PAMP that reduced HSPC expansion in aged donors compared to young donors in our group was poly I:C. Polyinosinic:polycytidylic acid (poly I:C) is a synthetic double-stranded RNA analog that mimics viral double-stranded RNA produced during the replication of many RNA viruses, including reoviruses, flaviviruses, picornaviruses, and coronaviruses. It activates antiviral innate immune responses primarily through endosomal TLR3 and cytosolic RIG-I and MDA5 signaling pathways. These viruses are known to have increased susceptibility in aged individuals. Further, our results suggest that HSPCs from aged individuals in our cohort have defective monocyte associated colony development upon poly I:C stimulation. This led us to investigate PAMP activation, specifically poly I:C, from pan monocytes as the second perspective of the immune cells. We isolated pan monocytes from PBMCs with a bead-based enrichment kit and stimulated pan monocytes with poly I:C. We evaluated expansion, inflammatory and interferon cytokine production, and cellular processes such as apoptosis and cycling with stimulated monocytes comparing young and aged donors. Our data suggests an altered interferon response that is less robust than 1-IFNs responses in aged monocytes compared to young upon poly I:C stimulation. In combination with the altered interferon responses to poly I:C, the increased production of specific inflammatory cytokines reveals links to respiratory infections. Therefore, these findings warrant future tissue-specific investigations focused on the links between altered interferon and increased inflammatory cytokine responses and alveolar macrophages in aged individuals. Altogether, this study advances our understanding of how aging alters poly(I:C)-mediated responses in hematopoietic progenitor and myeloid cell populations. These findings provide a foundation for future studies aimed at identifying therapeutic targets to enhance immune responses to respiratory viral infections in older adults.