Human Monocyte Subsets Are Transcriptionally and Functionally Altered in Aging in Response to Pattern Recognition Receptor Agonists This information is current as of September 26, 2021. Talibah U. Metcalf, Peter A. Wilkinson, Mark J. Cameron, Khader Ghneim, Cindy Chiang, Anne M. Wertheimer, John B. Hiscott, Janko Nikolich-Zugich and Elias K. Haddad J Immunol published online 10 July 2017 http://www.jimmunol.org/content/early/2017/07/07/jimmun Downloaded from ol.1700148 Supplementary http://www.jimmunol.org/content/suppl/2017/07/07/jimmunol.170014 http://www.jimmunol.org/ Material 8.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published July 10, 2017, doi:10.4049/jimmunol.1700148 The Journal of Immunology Human Monocyte Subsets Are Transcriptionally and Functionally Altered in Aging in Response to Pattern Recognition Receptor Agonists Talibah U. Metcalf,* Peter A. Wilkinson,† Mark J. Cameron,‡ Khader Ghneim,† Cindy Chiang,x Anne M. Wertheimer,{,‖ John B. Hiscott,# Janko Nikolich-Zugich,{,‖ and Elias K. Haddad* 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 Ag 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+CD162), intermediate (CD14+CD16+), and nonclassical (CD14dimCD16+) monocytes. Monocytes sorted Downloaded from from nonfrail healthy adults (21–40 y) and old (‡65 y) individuals were analyzed after stimulation with TLR4, TLR7/8, and retinoic acid–inducible gene I agonists. Our data showed that under nonstimulated 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 IFN-a, IFN-g, IL-1b, 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 http://www.jimmunol.org/ signaling and monocyte functions, and have implications for strategies to enhance the immune response in the context of infection and immunization. The Journal of Immunology, 2017, 199: 000–000. he world population is undergoing a rapid expansion of of the immune system, referred to as immunosenescence, has been older adults. It is estimated that by 2030, one in five linked to poor responses to vaccines (2) and higher incidence of T Americans will be $65 y old (1). Age-associated decline infections (including influenza and bacterial pneumonia), cancer, and neurodegenerative and cardiovascular diseases that contribute by guest on September 26, 2021 *Division of Infectious Diseases and HIV Medicine, Department of Medicine, Drexel to increased morbidity and mortality in the elderly (3, 4). Many University, Philadelphia, PA 19102; †Department of Pathology, Case Western Re- individuals will age without major health problems; however, serve University, Cleveland, OH 44106; ‡Department of Epidemiology and Biosta- x immunosenescence can be associated with chronic low-grade in- tistics, Case Western Reserve University, Cleveland, OH 44106; Department of Microbiology, University of Chicago, Chicago, IL 60637; {Department of Immunobi- flammation and a state of increased disease and frailty referred to ology, University of Arizona College of Medicine, Tucson, AZ 85724; ‖Arizona Center as inflammaging (5). Further studies are necessary to understand # on Aging, University of Arizona College of Medicine, Tucson, AZ 85724; and Labo- the mechanisms of immunosenescence leading to effective vac- ratorio Pasteur, Istituto Pasteur-Fondazione Cenci Bolognetti, 00161 Rome, Italy cines and improve health outcome for older adults. ORCIDs: 0000-0003-4768-4094 (M.J.C.); 0000-0002-6627-4974 (A.M.W.). Age-related dysfunctions of the immune system include altera- Received for publication January 30, 2017. Accepted for publication June 8, 2017. tions in the distribution and function of cells involved in the com- This work was supported by National Institutes of Health Contracts HHSN272201100017C munication between the innate and adaptive immune responses. Of (NIA/NIAID/N01-A1 00017) and HHSN272201400055C. the innate cells, peripheral blood monocytes, derived from the bone E.K.H., T.U.M., and J.N.-Z. conceived the idea; T.U.M. and E.K.H. designed exper- iments; C.C. and J.B.H. helped with RIG-I experimental design; T.U.M. performed marrow myeloid precursors, are the most abundant, representing and analyzed all experiments; M.J.C. performed the microarray analysis; P.A.W. 10% of circulating blood leukocytes in human. Monocytes via performed bioinformatics analysis and Gene Expression Omnibus submission; pattern recognition receptors (PRRs) such as TLRs, MDA5, reti- K.G. performed preliminary bioinformatics analysis; T.U.M. and E.K.H. wrote the manuscript; and A.M.W. and J.N.-Z. edited the manuscript. noic acid–inducible gene I (RIG-I)–like receptor, and NOD1 are The microarray data presented in this article have been submitted to the National involved in the innate response to a wide range of pathogens (6, Center for Biotechnology Information Gene Expression Omnibus (https://www.ncbi. 7). They initiate and support the adaptive immunity through virtue nlm.nih.gov/geo/query/acc.cgi?token=ubibcawuvxitbwb&acc=GSE94499) under ac- of their functions, including phagocytosis, production of proin- cession number GSE94499. flammatory and anti-inflammatory mediators, transport of Ags to Address correspondence and reprint requests to Dr. Elias K. Haddad, Division of Infectious Diseases and HIV Medicine, Department of Medicine, Drexel University, specialized sites of T cell priming, and differentiation into APCs 245 North 15th Street, NCB 6th Floor, MS 461, Room 6302, Philadelphia, PA 19102. such as macrophages and dendritic cells (DCs) (8–10). Monocytes E-mail address: [email protected] represent a heterogeneous population with three distinct subsets The online version of this article contains supplemental material. distinguished by expression of CD14 and CD16. Classical monocytes Abbreviations used in this article: DC, dendritic cell; DEG, differential expressed have high CD14 and no CD16 expression (CD14+CD162)andare gene; FC, fold-change; 2FC, negative FC; +FC, positive FC; GO, gene ontology; + 2 IPA, Ingenuity Pathway Analysis; Log2FC, log 2-FC; MDS, multidimensional scaling; the most abundant (90%). CD14 CD16 monocytes produce high 59pppRNA, 59 triphosphate double stranded RNA; PRR, pattern recognition receptor; levels of reactive oxygen species (ROS) and IL-6, IL-8, IL-10, and RIG-I, retinoic acid–inducible gene I; ROS, reactive oxygen species; SOD, superoxide CCL2 in response to pathogens (11). The minor population (10%) dismutase. is subdivided into two subsets: intermediate monocytes with high + + Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 CD14 and low CD16 (CD14 CD16 ), and nonclassical monocytes www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700148 2 AGE ALTERNATIONS IN MONOCYTES STIMULATED WITH AGONISTS with low CD14 and high CD16 (CD14dimCD16+). CD14+CD16+ were sorted directly from PBMCs. For stimulation microarray data, PBMCs monocytes are low ROS producers but produce higher levels of were washed and total monocytes were enriched by negative selection using reactive nitrogen species, IL-1b, and TNF-a, whereas CD14dim a human monocyte enrichment kit without CD16 depletion (STEMCELL, + Vancouver, BC, Canada). Monocytes were counted and resuspended at 50 CD16 monocytes are involved in patrolling the vascular endo- million/ml with sorting buffer (RPMI 1640 without phenol red, 2% FBS, thelium via CX3CR1-CX3CL1 interactions and produce TNF-a, and 1 M HEPES 1:40 [25 mM]) into 5-ml polypropylene tubes. Cells were IL-1b, and CCL3 in response to viruses and immune complexes incubated with TruStain FcR block (BioLegend, San Diego, CA) for 5 min via a proinflammatory TLR7-TLR8-MyD88-MEK pathway (11, 12). at room temperature. The Ab mixtures CD19 PE Cy7 (BioLegend), CD3 allophycocyanin Cy7 (BD Biosciences, San Jose, CA), CD16 Pacific Blue Studies on the impact of aging on the function of human mono- (BD Biosciences) or Alexa Fluor 700 (BioLegend), and CD14 PerCP cytes are limited in number and often yield conflicting results. (R&D Systems, Minneapolis, MN) were added, and cells were incubated Disparate