We evaluated Toll-like receptor (TLR) function in primary human dendritic cells from 104 young (age 21C30) and older ( 65 years) individuals. dysregulation of cytokine production that may limit further activation by TLR engagement. Our results provide evidence for immunosenescence in dendritic cells; notably, defects in cytokine production were strongly associated with poor antibody response to influenza immunization, a functional consequence of impaired TLR function in the aging innate immune response. Introduction Aging is associated with a progressive decline in immune function (immunosenescence) resulting in increased susceptibility to viral and bacterial infections and decreased response to vaccines (1C3). Age-associated perturbations in the humoral and cell-mediated arms of the adaptive immune system are well documented (3, 4); however, the impact of aging on the innate immune system is less well defined. Age related deficiencies of the innate immune system are incompletely understood but include reduced recruitment, U-10858 phagocytosis, granule release and microbial activity by PMN or macrophages, suggesting an age-related dysfunction in signal transduction as a manifestation of immunosenescence (5C9). Toll-like receptors (TLRs) are pattern recognition receptors that recognize conserved molecular patterns on microbes and are key to triggering antimicrobial host defense responses (10). Recognition of microbial components by TLRs initiates MyD88 or TRIF-dependent signal transduction pathways that culminate in the elaboration of type I interferons and proinflammatory cytokines that facilitate the linkage of innate to adaptive immune responses control innate immune responses (11). Deficiencies in human TLR signaling lead to increased severity of several diseases, including sepsis, immunodeficiencies, atherosclerosis U-10858 and asthma (12). We previously evaluated TLR function in monocytes and macrophages in the context of human aging, and observed an age-associated deficit in TLR1/2-induced cytokine production associated with reduced surface expression of TLR1 in monocytes and dysregulation of TLR3 expression in macrophages (13, 14). Monocytes from older individuals also demonstrated a generalized age-associated defect in TLR-induced expression of the CD80 costimulatory molecule associated with reduced protective antibody responses to influenza immunization (15). Similar reduced levels of TLRs have been noted in whole blood samples and colonic biopsies from older individuals (16), and in macrophages and pDCs from aged mice (17C19); other studies in different inbred mouse strains have implicated signaling differences in the absence of age-associated alterations in TLR expression (20, 21). Dendritic cells (DCs) are professional antigen-presenting cells (APC) that play a pivotal role in the linkage between innate and adaptive immunity (22). Human blood DCs, classified as myeloid DC (mDC) or plasmacytoid DC (pDC), have distinct functional activities. mDCs produce large amounts of IL-12 and induce strong T helper type1 (TH1) and cytotoxic T lymphocyte (CTL) responses (23). In contrast, pDCs produce large amounts of type I IFN in response to viral and bacterial stimuli. Stimulation of DCs and production of effector mechanisms involves phagocytosis, the upregulation of costimulatory and MHC molecules, a switch in chemokine receptor expression, the secretion of cytokines and chemokines, and the presentation of antigens by DCs (24). The consequences of aging on TLR function in human mDC and pDC populations remain incompletely understood. Here, we report on age-associated alterations in TLR function in such cells, and the consequences of age-associated alterations in innate immunity for vaccine responsiveness in humans. Materials and Methods Study Participants We recruited participants at vaccination clinics organized by the Yale University Health Services in October and November of 2007. Heparinized blood from healthy volunteers was obtained with informed consent under a protocol approved by the Human Investigations Committee of the Yale University School of Medicine. Older (age65) or young (21C30 years) participants with no history of acute illness in the two weeks prior to enrollment were evaluated. Immunocompromised individuals were excluded as described previously U-10858 (14). After informed consent was obtained, participants were evaluated using a screening questionnaire, and self-reported information was collected which included demographic information, height, weight, medications, and comorbid conditions (with specific inquiries regarding coronary artery disease, congestive heart failure, peripheral vascular disease, stroke, diabetes, hypertension, peptic ulcer disease, kidney disease, and chronic lung disease). Isolation of human peripheral blood dendritic cells Human peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque (GE Healthcare, NJ) gradient centrifugation. For FACS assays, DC populations were identified in PBMCs by staining with fluorescent antibodies to specific surface markers. Circulating DC numbers were calculated by multiplying the percentage of DCs by the absolute count of PBMCs in the cell populations gated by Rabbit polyclonal to Caspase 4. light scatter in flow cytometry. For PCR assays, DCs were purified by sorting on FACSAria (Becton Dickinson, CA). Purified mDC and.