Human Monocyte Subsets Are Transcriptionally and Functionally

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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 experiments; 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 outcomes could be because of the use of total monocytes for 20 min at 37˚C in a 5% CO2-humidified environment. Dead cells were (instead of individual subsets), different enrollment criteria for identified using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit for flow subject selection (including varying definitions of frailty), and cytometry (Life Technologies, Carlsbad, CA). Cells were washed and resuspended in sorting buffer at 30 million/ml and filtered using a 5-ml experimental protocols. Using nonfrail healthy adults (21–40 y of polystyrene tube with cell strainer cap (BD Biosciences). Cells were sorted age) and old ($65 y of age) subjects, we have previously shown that at 4˚C using BD FACSAria cell sorter into 5-ml FACS tubes containing the frequency and absolute numbers of the three monocyte subsets 500 ml of sorting buffer and 500 ml of FBS. Cells were kept on ice before were unchanged with aging (13). The expression of TLR3, TLR4, and after sorting. and TLR7 on total monocytes was also comparable between age In vitro stimulation of monocytes groups. However, we observed after stimulation of PBMCs with Sorted monocyte subsets were transferred to 5-ml polypropylene tubes, Downloaded from PRR agonists that the gene signal corresponding to monocytes was spun down, and suspended in cultured RPMI medium (RPMI 1640 with strongly induced at an early time in PBMCs from adult subjects L-glutamine [Corning Cellgro, Manassas, VA] supplemented with 10% FBS compared with old individuals (13). We hypothesize that aging and 13 [50 U] penicillin-streptomycin [Invitrogen, Carlsbad, CA]). Cells could be impacting the function of monocytes at the transcriptional were plated at 30,000 cells/well in 96-well V-bottom plates. TLR and level, which remains an open question. In this study, we assessed the RIG-I agonists were added at the following concentrations: LPS-TLR4 (0.5 mg/ml) or CLO97-TLR7/8 (1 mg/ml) or 59 triphosphate double transcriptional programs and function of monocyte subsets in re- stranded RNA (59pppRNA)–RIG-I (500 ng/ml). Optimal concentrations of sponse to PRRs ligation. We analyzed transcriptome data, measured different TLR agonists were selected based on median production of IL-6 http://www.jimmunol.org/ cytokine and chemokine production, and measured surface expres- and IFN-a and a $85% survival rate of monocytes. All PRR ligands were sion of key chemokine receptors. This approach allowed us to purchased commercially (Invivogen, San Diego, CA) except for 59-pppRNA, which was custom synthesized, as previously described (13). Monocytes identify alternations in the PRR responses in monocyte subsets from cultured in medium alone were used as a control for LPS and CLO97. adults and old subjects. Our findings represent a comprehensive Stimulation by 59-pppRNA required the use of a cationic transfection agent analysis of the influence of human aging on PRR signaling and LyoVec (Invivogen), so medium plus LyoVec alone was used as a control. monocyte function, and have implications for strategies to enhance Monocytes were cultured for up to 24 h at 37˚C in a 5% CO2-humidified the immune response in the context of infection and immunization. environment. Microarray and bioinformatics analyses Materials and Methods by guest on September 26, 2021 Ethics statement Agonist-stimulated cells were washed twice with cold PBS and lysed with 100 ml of cold RLT buffer (Qiagen, Germany) supplemented with 1% This study was approved by the Institutional Review Board of the Martin 2-ME (Sigma, St. Louis, MO), and then transferred to 1.7-ml tubes with Health System (Stuart, FL). All participants in this study were .21 y of age. 250 ml of cold RLT/2-ME buffer and quickly stored at 280˚C. For ex vivo Written informed consent was obtained from all subjects. study, individual monocyte subsets were sorted directly into a 1.7-ml tube with 500 ml of cold RLT/2-ME buffer and placed on dry ice, then trans- Patient recruitment and PBMC isolation ferred to 280˚C. A total of 30,000–100,000 cells were sorted depending on the subset and donor. Reverse transcription reactions were performed Healthy community-dwelling subjects were enrolled from Martin Health to obtain cDNAs, which were hybridized to Illumina Human HT-12 V4 System in two groups: adults (24–36 y, n = 11) and nonfrail old individuals Expression BeadChips according to the manufacturer’s instruction and n (67–83 y, = 11). Using a screening questionnaire, participants were asked quantified using an Illumina iScan System. The data were collected using about lifestyle, clinical history, and medication usage. Subjects were ex- Illumina GenomeStudio software. Analysis of the microarray data was cluded who self-reported comorbid conditions including cancer (within the conducted using the R statistical language (17) and packages from the last 5 y for those $65 y), immunocompromising disorders, and steroid us- community repository of Bioconductor (https://www.bioconductor.org) age, whereas inclusion criteria included controlled hypertension, occasional/ (18). Microarrays displaying unusually low median intensity, low vari- tolerable aching joints from arthritis and not taking daily nonsteroidal anti- ability, or low correlation relative to the bulk of the arrays were removed as inflammatory drugs or acetaminophen, and controlled diabetes. Our defini- technical outliers from the rest of the analysis. The raw data were pre- tion of nonfrail subjects was based on Fried criteria (14). The Katz index of processed using Quantile normalization, followed by a log2 transforma- independence in activities of daily living was used to assess the functional tion using facilities from the LIMMA package (19). Subsequently, the impairment of subjects $65 y (15). The index ranks adequacy of perfor- LIMMA package was also used to fit a linear model (two-group analysis) mance in bathing, dressing, toileting, transferring, continence, and feeding. to each probe and to perform a (moderated) Student t test on various dif- Subjects with a score lower than six (indicating moderate-to-severe im- ferences of interest (20). In addition, the LIMMA package from Bio- pairment) were excluded from the study. Old subjects were also assessed for conductor was used to identify differentially expressed genes (DEGs) dementia using the Mini-Cog assessment instrument for dementia (16). The between treated versus controls (untreated or LyoVec only) or adult versus Mini-Cog combines two simple cognitive tasks (three-item word memory old subjects. For data mining and functional analyses, DEGs satisfied a false and clock drawing test) with an empirical algorithm for scoring. Subjects discovery rate of 5% with $ +1.3 or # 21.3 fold-change (FC). Probes that with a recall score of one to two words with a normal clock drawing test or a did not map to annotated RefSeq genes and control probes were removed. recall score of three words was considered nondemented. Demographic data The expected proportions of false positives (false discovery rate of 5%) were for the cohort used in this study are described in Table I. PBMCs were iso- estimated from the unadjusted p value using the Benjamini and Hochberg lated from leukapheresis using Ficoll-Paque (GE Healthcare) density gradient method (21). Gene set variation analysis was used for differential pathway media. PBMCs were frozen in 90% Gibco heat-inactivated bovine serum analysis, which converts gene expression to pathways using gene sets (22). (Thermo Fisher Scientific) and 10% DMSO. MsigDB gene sets (23) were employed and results tested with the same Sorting monocyte subsets LIMMA linear modeling as outlined above. To gain a deeper insight into the biological functions unique to each monocyte subset, we analyzed Total PBMCs isolated from adults and old subjects were treated with unique upregulated and downregulated DEGs separately for enrichment of benzonase nuclease (Millipore, Billerica, MA) for 30 min at 37˚C in a 5% gene ontology (GO) biological processes using the DAVID functional CO2-humidified environment. For ex vivo microarray data, monocytes annotation tool (24, 25). An innate gene filter, derived from GO query GO: The Journal of Immunology 3

0045087, was used to highlight innate immune-related genes within the from adults and old subjects (n = 9 per group) into three subsets: datasets. Some network analysis was performed with Ingenuity Pathway classical (CD14+CD162), intermediate (CD14+CD16+), and non- Analysis (IPA; Ingenuity systems). Illumina experimental data were imported classical (CD14dimCD16+) (Fig. 1A). Differential gene expression into IPA with gene symbol. DEGs (selected based on a nominal p # 0.05 and FC $ +1.3 or # 21.3) that were associated with a canonical pathway in between monocyte subsets was evaluated by microarray analysis Ingenuity’s Knowledge Base were used for pathway analysis. The signi- using the Illumina BeadChips platform (see Materials and Methods). ficance of the association between the dataset and the canonical pathway was A multidimensional scaling (MDS) plot was created to visualize measured in two ways: 1) a ratio of the number of genes from the dataset that the relationship among the three monocyte datasets and between map to the pathway divided by the total number of genes that map to the . canonical pathway was displayed; and 2) overrepresentation Fisher exact test adult and old individuals using the relative expression of 18,000 was used to calculate a p value determining the probability that the association transcripts (Fig. 1B). Each point on the graph represents a sample between the genes in the dataset and the canonical pathway was explained by from one subset indicated by color from either adults or old in- chance alone. The pathways were ranked by 2log p value. Microarray data dividuals. The relative proximity of the samples along the x- and are available at the National Center for Biotechnology Information Gene y-axis reflect the principle components that separate the datasets Expression Omnibus. based on gene expression intensities. It was evident that CD14+ Cytokine and chemokine analysis CD162 (red/yellow), CD14+CD16+ (blue/green), and CD14dim + Supernatants collected from stimulated monocytes were analyzed for CD16 (black/gray) monocytes occupied nonoverlapping spaces chemokine/cytokine levels using Bio-Plex Pro magnetic bead assays (Bio- indicating that sorted subsets are primarily distinct and have unique Rad, Hercules, CA). The following human chemokine premixed panels was transcriptional profiles (Fig. 1B). Using a hierarchical cluster heat used: I-309 (CCL1), MCP-1 (CCL2), MIP-1a (CCL3), MCP-3 (CCL7), map to analyze the top 100 discriminant genes also showed a MCP-2 (CCL8), Eotaxin (CCL11), MCP-4 (CCL13), MIP-1d (CCL15), TARC (CCL17), MIP-3b (CCL19), 6Ckine (CCL21), MIP-3a (CCL20), distinct separation of monocyte datasets (Fig. 1C). Downloaded from MDC (CCL22), MPIF-1 (CCL23), Eotaxin-2 (CCL24), TECK (CCL25), The developmental relationship between monocyte subsets re- Eotaxin-3 (CCL26), CTACK (CCL27), GM-CSF, GRO-a (CXCL1), GRO-b mains unclear, but CD16+ monocytes are considered to be in a more (CXCL2), ENA-78 (CXCL5), GCP-2 (CXCL6), MIG (CXCL9), IP-10 advanced stage of differentiation, with CD14+CD16+ monocytes (CXCL10), I-TAC (CXCL11), SDF-1A+b (CXCL12), BCA-1 (CXCL13), exhibiting an intermediate phenotype when compared with classical SCYB16 (CXCL16), Fractalkine (CX3CL1), MIF, IL-1b, IL-2, IL-4, IL-6, IL-8,IL-10,IL-16,TNF-a,andIFN-g. The manufacturer’s protocol was and nonclassical monocytes (26). Our clustering analysis showed + 2 + + followed. Data were acquired on a Bio-Plex 200 System (using bead re- that the CD14 CD16 and CD14 CD16 monocyte subsets were http://www.jimmunol.org/ gions defined in the Bio-Rad protocol) and analyzed with the Bio-Plex closely clustered and are distant from the CD14dimCD16+ subset Manager 6.1 software from Bio-Rad. Supernatants collected from stimu- (Fig. 1C). Interestingly, when looking at the intensity of gene ex- lated monocytes were analyzed for IFN-a production using an IFN-a ELISA (PBL Assay Science, Piscataway, NJ). pression (red depicts high gene expression, and blue depicts low gene expression), we observed that the intensity of gene expression CX3CR1 surface receptor expression by flow cytometry for the CD14+CD16+ monocyte subset was in between those of + 2 dim + + PBMCs were treated with and without agonists for 18 h. Cells were washed CD14 CD16 and CD14 CD16 . This suggests that the CD14 with PBS and then stained with the following Abs: Annexin V FITC, CD3 CD16+ monocyte subset might represent a transitional differentia- Alexa Fluor 700, CD19 Alexa Fluor 700, CD14 BV650, CD16 PE-Cy7, and tion state between CD14+CD162 and CD14dimCD16+. This gene CX3CR1 PE. All Abs were from BioLegend. Dead cells were identified expression pattern was previously noted in other monocyte profil- by guest on September 26, 2021 using LIVE/DEAD Fixable Aqua Dead cell stain kit. Cells were evaluated + + on a BD LSR II flow cytometer, and frequencies and mean fluorescent ing studies, further supporting the idea that CD14 CD16 mono- intensity were gated using FlowJo software. cytes have a more intermediate differentiated phenotype (27, 28). Overall, these results demonstrated that sorted ex vivo monocyte Statistical analysis subsets are transcriptionally distinct.

For cytokine production, significant differences between multiple com- 2 parisons (LPS- or CLO97-treated versus untreated monocytes, 59-pppRNA– Unique gene expression of ex vivo CD16+ and CD16 treated versus LyoVec-only–treated monocytes, and adult versus old monocytes subjects) were determined by two-way ANOVA test followed by Bonferroni correction for multiple comparisons (with a = 0.05, 95% confidence inter- Using the MDS and hierarchical cluster analysis (Fig. 1B, 1C), we val). A p value ,0.05 was considered significant. For surface receptor up- did not observe a tight aggregation in ex vivo unstimulated regulation analysis, p values were determined by two-sided parametric t test monocyte subsets based on age groups. Therefore, transcriptome with Welch correction (did not assume equal SDs). Data were analyzed and analysis was performed independent of age while focusing on the figures generated using GraphPad Prism 6 software. transcriptional difference between subsets. Complied microarray data from 18 subjects (n = 9 per age group) for one subset was Results contrasted against the remaining two subsets (e.g., CD14dimCD16+ Ex vivo sorted monocyte subsets revealed distinct versus CD14+ CD162 and CD14+ CD16+). A total of 18,615 transcriptional profiles transcripts was detected across the three contrasts (Supplemental Healthy nonfrail individuals enrolled into the study were arranged Table I). Significantly, DEGs were denoted with an adjusted into two groups: adults and old subjects (n = 11 per group). The p , 0.05. Profiles of DEGs consisted of 6204 distinct transcripts average age for adults was 30 y (range 24–36 y), whereas it was for CD14+CD162 monocytes, 5289 transcripts for CD14dim 73 y (range 67–83 y) for old individuals (Table I). Individuals with CD16+, and 1731 transcripts for CD14+CD16+ monocytes (Fig. comorbid conditions including cancer (within the last 5 y for those 2A). This lower expression of significant genes for CD14+CD16+ .65 y old), immunocompromising disorders, and steroid usage monocytes is consistent with our cluster analysis (Fig. 1C), were excluded, whereas inclusion criteria included controlled which showed a higher gene expression in nonclassical and clas- hypertension, occasional/tolerable aching joints from arthritis and sical subsets. not taking daily nonsteroidal anti-inflammatory drugs or acetamino- Using Venny, an interactive tool for comparing lists by Venn phen, and controlled diabetes (see Materials and Methods). Mono- diagrams (29), we compared profiles of DEGs for each subset cytes were first evaluated under healthy nonstimulus conditions to which identified unique and common transcripts (Fig. 2B). CD14+ determine transcriptional differences between the three subsets and CD162 monocytes had the most unique genes at 1413, whereas the effects of aging on gene expression. Using CD14- and CD16- CD14dimCD16+ and CD14+CD16+ had 862 and 180 unique genes, conjugated Abs, we sorted monocytes from frozen PBMCs isolated respectively (Supplemental Table I). We focused our analysis on 4 AGE ALTERNATIONS IN MONOCYTES STIMULATED WITH AGONISTS

Table I. Demographic data for the cohort used for gene array, cytokine/ ITGAL, superoxide dismutase (SOD) 1, RARA, IFITM1/2/3, CD115 chemokine production analysis, and surface phenotypic analysis (CSF1R), SIGLEC10, IFNG, and TNF (Table II). The expression of the TNF transcript indicates that CD16+ monocytes are more Adults Old Subjects proinflammatory compared with classical CD14+CD162 mono- (n = 11) (n = 11) cytes. Immune-related upregulated genes for CD162 monocytes Average age (range), y 30 (24–36) 73 (67–83) (but downregulated in CD16+ monocytes) included SELL (CD62L), Gender, male/female (female %) 4/7 (64) 5/6 (55) CD93 (C1qR1), CD36, CD114 (CSF3R), CCR1/2, IL4R, IL6R, Race, n White (non-Hispanic) 9 (82%) 11 (100%) IL1B, MyD88, FPR1/2, HIF1A, and TLR2/4/5/6/8 (Table II). The + White (Hispanic) 2 (18%) upregulation of multiple TLR transcripts indicates that CD14 Comorbidities, n 1 (9%) 9 (82%) CD162 monocytes could have a better ability to respond to various None 10 (91%) 2 (18%) pathogens. Genes shared between CD14+CD162 and CD14+CD16+ Arthritis 1 (9%) 5 (45%) dim + Hypertension 5 (45%) monocytes (but downregulated in CD14 CD16 ) included CD14, Stroke 1 (9%) ALDH2, CD1A/B, CD63, and MHC class II molecules HLA- Heart disease 1 (9%) DMA/DMB/DRA/DRB3. CD14+CD16+ monocytes also upregu- Medications lated class II molecules HLA-DOA, HLA-DPB1, HLA-DQB1, and Prescription 1 (9%) 6 (55%) HLA-DRB6, whereas CD14+CD162 monocytes upregulated MHC Over-the-counter 5 (45%) 8 (73%) class I molecules HLA-B, HLA-F, and HLA-H (Table II). This downregulation of MHC class II and I transcripts suggests CD14dim immune responses using a gene filter derived from GO, which CD16+ monocytes have less Ag presentation potential compared Downloaded from included cytokines and chemokines, costimulatory factors, tran- with classical and intermediate monocytes. The results pertaining to scriptional factors, adhesion molecules, and metabolism-related CD16+ and CD162 monocytes were in line with other published genes. Genes upregulated for CD16+ monocytes (but downregu- transcriptional profile data providing initial validation of microarray lated in CD162 monocytes) included CX3CR1, FCGR3A (CD16), analysis (26, 27, 30). For example, classical monocytes express http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Distinct transcriptional proﬁles of ex vivo sorted monocyte subsets. (A) Flow cytometry gating scheme used to sort CD14dimCD16+, CD14+ CD16+, and CD14+CD162 monocyte subsets. (B) MDS plot analysis was used to visualize the relationship among the three subsets using the relative expression of .18,000 transcripts. Each point represents a sample from one subset indicated by color: CD14+CD162 (red/yellow), CD14+CD16+ (blue/ green), and CD14dimCD16+ (black/gray) from adults or old subjects (n = 9 per age group). The relative proximity of the samples along the x- and y-axis reﬂect the principle components that separate the datasets based on gene expression intensities. (C) Hierarchical clustering analysis heat map of the top 100 discriminant genes (listed on the right) for each monocyte: CD14dimCD16+ (green), CD14+CD162 (purple), and CD14+CD16+ (blue) dataset (age groups were combined for each dataset). Scale is provided. Red denotes increased gene expression, and blue denotes decreased gene expression. The colored circles under the heat map correspond to colors used in the MDS plot to indicate adults and old subjects. The Journal of Immunology 5

FIGURE 2. DEGs of ex vivo monocyte subsets. Compiled microarray data analysis from 18 samples (n = 9 per age group) for one subset was contrasted against the remaining two subsets. A total of 18,615 transcripts was detected across the three contrasts. (A) Graph depicts the total number of DEGs (adjusted p , 0.05) subdivided into upregulated (+FC) Downloaded from and downregulated (2FC) for each subset. (B) Venn diagram analysis of total DEGs revealed unique and common DEGs for each monocyte subset. Listed for each subset are selected top GO biological processes related to unique upregulated and downregulated http://www.jimmunol.org/ DEGs. by guest on September 26, 2021

CD62L and CCR2, and migrate in response to CCL2, whereas and Methods). MDS was again used to visualize the relationship patrolling CD14dimCD16+ monocytes express CX3CR1 and migrate among the three monocyte datasets and between treatment and into tissues expressing CX3CL1. negative controls. Each point on the graph represents a treated To gain a deeper insight into the biological functions unique to subset indicated by color and shape: CLO97, LPS, and negative each monocyte subset, we analyzed unique upregulated and down- control (no treatment) in Fig. 3A, and 59pppRNA-LyoVec and regulated DEGs separately for enrichment of GO biological processes negative control (LyoVec only) in Fig. 3B. It was evident from using the DAVID functional annotation tool (Supplemental Table I) the analysis that each monocyte subset occupied nonoverlapping (24). We observed that top GO biological processes (with signifi- spaces and that stimulation with CLO97, LPS, and 59pppRNA cant p , 0.05) were related to transcription, cell cycle, metabolic produced unique transcriptional profiles compared with negative processes, and cell death, suggesting ex vivo monocyte subsets are controls. in a steady-state of differentiation (Fig. 2B). Interestingly, the non- We performed transcriptome analysis by normalizing LPS- and classical subset is known to have highly motile patrolling behavior CLO97-stimulated monocyte datasets to untreated monocytes, in vivo, which supports the unique expression of transcripts related whereas 59pppRNA/LyoVec was normalized to LyoVec-only–treated to cytoskeleton organization. Overall, our transcriptional profiling monocyte subsets. DEGs were selected based on FC $ +1.3 or of the three monocyte subsets revealed distinct functional charac- # 21.3 and p , 0.05 (Supplemental Table II). These criteria were teristics, and more importantly the transcriptional profiles of ex vivo chosen based on our own validation scheme used for other pub- unstimulated monocytes sorted from healthy non-frail old subjects lished studies (13, 31). Fig. 3C shows the number of upregulated appear not to be affected by aging. and downregulated DEGs induced after treatment for all three subsets sorted from adult and old subjects. In comparison with Transcriptional profiles of agonist-stimulated monocyte subsets 59pppRNA, LPS and CLO97 treatment of all three subsets induced We then determined the transcriptional responses of monocyte a higher number of total DEGs, with the majority of these tran- subsets after stimulation with various PRRs agonists. Monocyte scripts being downregulated in comparison with untreated mono- subsets were sorted from adult and old subjects (n = 8 per age cytes. Focusing on transcriptional profiles for adults, we assessed group) and stimulated for 24 h with LPS (TLR4), CLO97 (TLR7/8), the impact of each treatment on individual monocyte subsets by or 59pppRNA complexed with LyoVec (a cationic transfection re- evaluating the elicited chemokine and cytokine responses. Table III agent that facilitates intracellular delivery) (RIG-I) (see Materials shows the top 5 upregulated and downregulated chemokine and 6 AGE ALTERNATIONS IN MONOCYTES STIMULATED WITH AGONISTS

Table II. The expression pattern of selected monocyte-related DEGs transcript expression in subsets. LPS and CLO97 treatment did induce expression of the proinﬂammatory cytokine IL-6 in all three CD14+ CD14+ CD14dim subsets. Overall, these data revealed that treatment of individual CD162 CD16+ CD16+ Genes (Log2FC) (Log2FC) (Log2FC) monocyte subsets induced distinct transcriptional patterns of IFNs, cytokines, and chemokines because of the different array of PRRs CCR1 2.176 22.441 found on monocyte subsets. CCR2 1.462 20.496 20.967 CD14 3.180 0.676 23.856 Agonists elicited distinct transcription responses between CD163 3.133 23.191 CD1D 1.738 21.894 adults and old subjects CD36 3.177 22.961 We then sought to determine the impact of aging on PRR-induced 2 CD93 2.720 2.959 responses in sorted monocyte subsets. To assess the enrichment of CD97 20.417 CSF1R (CD115) 21.484 0.713 0.771 DEGs in both age groups, we contrasted transcriptional proﬁles for CSF3R 2.465 22.595 monocyte subsets from adults with old subjects (Supplemental CX3CR1 20.901 0.604 Table III). DEGs were selected based on FC $ +1.3 or # 21.3 FCGR1A (CD64) 1.148 0.465 21.613 and p , 0.05. Fig. 4A depicts total number of enriched DEGs for FCGR3A (CD16) 20.627 0.683 FPR1 2.239 22.433 each treated subset. Positive FC (+FC) values indicated DEGs HIF1A 1.667 20.520 21.146 enriched in adults, whereas negative FC (2FC) values indicate HLA-DMA 0.551 0.993 21.544 DEGs enriched in old subjects.

HLA-DMB 0.590 0.684 21.273 For adults, we observed the highest number of enriched DEGs Downloaded from HLA-DOA 20.342 1.078 20.736 + 2 2 for 59pppRNA-treated CD14 CD16 (508 DEGs) and CLO97- HLA-DRA 0.761 0.468 1.230 + 2 HLA-DRB3 0.631 0.620 21.250 treated CD14 CD16 (412 DEGs) monocytes, whereas for old + 2 ICAM3 0.425 20.439 subjects 59pppRNA-treated CD14 CD16 (413 DEGs) and LPS- ICAM2 21.339 0.696 treated CD14dimCD16+ (411 DEGs) monocytes had the highest ICAM4 21.617 1.802 enrichment of DEGs. We focused our analysis on immune genes ICAM5 0.149 2 and observed a wide range of immune-related transcripts for both http://www.jimmunol.org/ IFITM1 3.640 0.887 2.753 + IFITM2 20.996 0.421 0.575 age groups. For example, 59pppRNA-treated classical CD14 2 IFITM3 21.147 0.666 CD16 monocytes had a significant enrichment of IFN transcripts IFNG 20.554 0.773 IFNA1/8/10/14/16, IFNGR2, and IRF9 in adults compared with 2 ITGAL 1.262 1.054 old subjects. Also, adults had a higher enrichment of costimulatory LYN 20.315 LYZ 2.863 22.185 molecules (PDL1, CD80, and CD83), phagocytosis Fc receptor MAFB 1.434 21.037 (FCGR2A), TLR8, monocyte activation marker (CD48), and cy- MNDA 0.992 21.012 tokines (CCL19, CCL4, IL15, and IL24). Old subjects had an MPO 1.060 21.062 enrichment of chemokines (CCR2, CCL24, CXCR4, and CXCL5), 2 MYD88 0.614 0.446 by guest on September 26, 2021 PLBD1 2.882 23.045 TGFBR2, SOD2, and costimulatory molecule CD276 (B7-H3) RARA 20.521 0.390 (Fig. 4B). S100A12 3.614 23.377 Enriched DEGs for adult and old subjects were further evalu- S100A8 0.628 0.792 21.420 ated using the IPA software. Supplemental Table III lists the log 2- 2 S100A9 0.930 0.613 1.543 FC (Log2FC) of statistical significantly (p , 0.05) expressed path- SELL (CD62L) 2.795 20.761 22.034 SIGLEC10 21.811 0.658 1.153 ways for each treated monocyte subset. +FC values indicate SOD1 20.843 0.753 pathways significantly enriched in adults, whereas 2FC values in- SOD2 0.949 20.690 dicate pathways significantly enriched in old subjects. Focusing on TLR2 0.488 20.186 20.303 59pppRNA-treated CD14+CD162 monocytes, adults had a higher TLR4 0.669 20.558 TLR5 0.778 21.046 enrichment of immune response pathways compared with old TLR6 0.312 20.268 subjects, including the TLR signaling pathway, the RIG-I–like re- TLR7 20.549 ceptor signaling pathway, antigen processing and presentation, DC TLR8 0.454 20.537 maturation, regulation of IFNA signaling, and communication be- 2 2 TNF 1.248 0.537 0.711 tween innate and adaptive immune cells (Fig. 5A). CD14+CD16 TREM1 2.018 22.261 isolated from old individuals had enrichment of superoxide meta- Transcriptome analysis was generated by contrasting one ex vivo sorted subset bolic process, oxygen and ROS metabolic process, and response to with the remaining two subsets. +FC values (+Log2FC) indicate upregulated genes, whereas 2FC values (2Log2FC) indicate downregulated genes. If no value is given, oxidative stress pathways (Fig. 5A). These results demonstrate that 2 then genes were not significantly expressed for that subset. 59pppRNA-stimulated classical CD14+CD16 monocytes from adults display a qualitatively stronger antiviral transcriptional re- cytokine transcripts for each treatment condition. Interestingly, we sponse when compared with those from old individuals. observed that 59pppRNA induced a high upregulation of antiviral Another example of altered immune responses was LPS-treated IFN-a and IFN-b transcripts in CD14+CD162 and CD14+CD16+ CD14dimCD16+ monocytes. We observed that old subjects had a monocytes, whereas CLO97 and LPS induced a high upregulation higher enrichment of MHC class I and II transcripts, including of IFN-g transcript in CD14+CD16+ and CD14dimCD16+ mono- HLA-DRB4, HLA-DRB6, HLA-C, and HLA-G, which corresponded cytes (Table III). It is worth noting that the expression levels of to an enrichment of functional pathways related to communication IFN-a and IFN-b transcripts were 2-fold higher in CD14+CD162 such as antigen processing and presentation, and communication than in CD14+CD16+ monocytes, and IFN-g expression levels were between innate and adaptive immune cells (Figs. 4C, 5B). There 2-fold higher in CLO97-treated CD14+CD16+ monocytes. Other was also an enrichment of other immune-related functional examples of unique responses included the monocyte chemo- pathways, including PD1 signaling, superoxide metabolic process, attractant transcript CXCL10 was induced by 59pppRNA in all three myeloid leukocyte differentiation, and the role of PRRs in rec- subsets, whereas LPS and CLO97 treatment downregulated CXCL10 ognition of bacteria and viruses, whereas monocytes sorted from The Journal of Immunology 7

FIGURE 3. Distinct transcriptional proﬁles for agonist-stimulated monocyte subsets. MDS plot analysis was used to visualize the relationship among the three subsets treated with (A) CLO97 (circle), LPS (triangle), and negative control no treatment (square), and (B)59pppRNA-conjugated LyoVec (triangle) and negative control LyoVec only (circle). Color depicts individual subsets: CD14dimCD16+ (pink), CD14+CD16+ (green), and CD14+ CD162 (blue). (C) Transcriptional proﬁles were generated by normalizing individual treatments to their respective negative controls. Graph depicts the total number of DEGs (FC $ +1.3 or # 21.3 and p , 0.05) subdivided into upregulated (+FC) and downregulated (2FC) for adults Downloaded from (top) and old subjects (bottom). n =8 per age group.

adults had an enrichment of pathways related to transcription/ (8717 versus 5216 pg/ml for CD14+CD162 and 3116 pg/ml for dim + RNA metabolic processes and regulation of cell cycle. We ob- CD14 CD16 ) and by CLO97 (7699 pg/ml versus 5099 pg/ml for http://www.jimmunol.org/ served that untreated nonclassical CD14dimCD16+ monocytes CD14+CD162 and 2680 pg/ml for CD14dimCD16+) (Supplemental expressed low levels of MHC class I and II transcripts (Table II); Table IV), which is in line with CD14+CD16+ monocytes having therefore, the enrichment of these transcripts in response to LPS more of a proinflammatory phenotype. Differential expressed che- suggests a possible differentiation of nonclassical monocytes into mokine and cytokine transcripts were observed for 59pppRNA- macrophages or DCs. Overall, our data revealed a wide range of treated CD14dimCD16+ monocytes (Table III), which did not potential age-associated alternations in PRR signaling in indi- correlate with our multiplex analysis for both age groups. We did, vidual monocyte subsets sorted from both adults and old subjects, however, observe upregulation of IFN-g (LPS- and CLO97-treated indicating monocytes undergo complex cellular differentiations in CD14dimCD16+) and IFN-a (59pppRNA-treated CD14+CD162 response to different pathogens. and CD14+CD16+) transcripts (Table III) that corresponded to by guest on September 26, 2021 significant production in these subsets (Fig. 6). We also observed Stimulated monocytes from adults produced higher levels of the decreased production of CXCL10 after treatment with CLO97 inflammatory cytokines, IFNs, and chemokines and LPS, but increased production in response to 59pppRNA (Fig. To assess whether the observed transcriptional immune response 6) that related to CXCL10 transcript expression (Table III). Overall, in monocytes translated into functional production of IFNs, cyto- we observed distinct production of chemokines and cytokines for kines, and chemokines, we profiled supernatants harvested from each monocyte subset in response to PRR agonists. agonist-stimulated monocyte subsets from both age groups using A comparison of adults with old subjects revealed age-related multiplex immunoassays. Production of analytes was considered sig- alternations in the production of IFN-g that was higher in nificantly (p , 0.05) increased or decreased by comparing with neg- CD14dimCD16+ monocytes sorted from adults treated with LPS ative controls: no treatment (for LPS and CLO97) and LyoVec only (1359 versus 594 pg/ml, p , 0.001) or CLO97 (1412 versus (for 59pppRNA-LyoVec) (Supplemental Table IV). Focusing on adults 729 pg/ml, p , 0.001) for up to 24 h (Fig. 7A). CD14+CD162 at 24 h, we observed that LPS and CLO97 stimulation of all three monocytes from adults produced higher IL-1b in response to subsets elicited the most significant changes (compared with no CLO97 (21,295 versus 11,616 pg/ml, p , 0.05) and higher CCL20 treatment) in cytokines and chemokine production. Interestingly, in response to LPS (1666 versus 1163 pg/ml, p , 0.05) (Fig. 7B, 59pppRNA stimulation induced significant changes (compared 7C). Also, classical CD14+CD162 monocytes from adults pro- + 2 with LyoVec only) in analyte production only in CD14 CD16 duced higher IFN-a (34 versus 19 pg/ml, p , 0.01) and CCL8 + + and CD14 CD16 monocyte subsets. In response to LPS treat- (2271 versus 1237 pg/ml, p , 0.01) in response to 59pppRNA ment, distinct analytes were produced including CX3CL1, IL-1b, (Fig. 7D, 7E). The kinetics of expression or biological factors, + 2 and IL-6 for classical CD14 CD16 monocytes, CCL26 and IL-4 such as mRNA degradation, could affect the correlation between + + for CD14 CD16 monocytes, and IFN-g, CCL1, CCL11, CCL23, protein and transcript levels; however, we did observe that 59pppRNA dim + andCXCL13forCD14 CD16 monocytes (Fig. 6). CLO97 treat- elicited a higher IFN-a transcriptional response in classical ment of monocyte subsets elicited unique analytes CCL7, CXCL1, monocytes from adults compared with old subjects (Fig. 4B). + 2 CXCL5, and CXCL6 for classical CD14 CD16 monocytes; Overall, monocytes from old subjects had a decreased production + + CXCL10, CXCL11, and IL-10 for intermediate CD14 CD16 of antiviral IFNs and proinflammatory cytokines and chemokines. monocytes; and IFN-g, CXCL12, CCL22, and CXCL13 for CD14dimCD16+ monocytes. Unique analytes induced by 59pppRNA Expression of fractalkine receptor CX3CR1 on included CCL7 and CXCL6 for classical CD14+CD162 monocytes monocyte subsets and CXCL10, CXCL11, CCL24, and CXCL13 for intermediate CX3CR1 interaction with its ligand fractalkine CX3CL1 has been CD14+CD16+ monocytes (Fig. 6). Interestingly, we observed higher shown to be critical for monocyte survival by reducing ROS and production of TNF-a by CD14+CD16+ monocytes treated by LPS trafficking into CX3CL1 tissues including the gut and heart under 8 AGE ALTERNATIONS IN MONOCYTES STIMULATED WITH AGONISTS

Table III. The top 5 upregulated and downregulated chemokine and cytokine transcripts for each treatment condition

CD14+CD162 FC CD14+CD16+ FC CD14dimCD16+ FC 59pppRNA-LyoVec (versus LyoVec only) CXCL10a 12.80 IFNA14b 4.16 CCL8 4.43 IFNA14b 9.32 IFNA16b 4.12 CXCL10a 2.18 IFNA16b 8.49 IFNB1b 3.64 IL1RN 2.04 IFNA2b 8.30 CXCL10a 3.31 CCR7 2.01 IFNB1b 7.64 IL27 3.05 IFNB1 1.93 CCL24 21.92 CCL24 21.72 CCL24 21.50 IL1B 22.16 IL1A 21.92 CCL1 21.61 IL1A 22.32 IL1B 21.96 IL3RA 21.74 CXCL6 22.55 CXCL5 22.17 CXCL8 21.76 CXCL5 23.53 CCL1 22.67 IL1B 22.39 LPS (versus NT) IL23A 6.76 IL23A 12.98 IFNGb 12.59 CCL22 4.96 IFNGb 10.01 IL2RA 6.90 IL24 4.43 CCL20 6.55 IL6a 5.08 IL6a 4.22 IL24 6.17 IL1B 3.93 CCL3 3.30 IL6a 4.77 CCR7 3.66 IL3RA 23.13 CXCL16 24.15 IL18BP 28.19 CCL7 23.36 CCL13 25.41 CXCL10a 28.41 Downloaded from CCR1 24.33 CCL2 26.19 CXCL16 29.86 CCL8 27.99 CCL8 28.80 CCL8 210.88 CCL13 210.45 CXCL10a 29.39 IL13RA1 214.11 CLO97 (versus NT) CCL22 4.34 IFNGb 25.98 IFNGb 12.69 IL6a 3.80 IL18RAP 5.44 IL6a 10.39 CXCL2 3.09 IL6a 4.41 CCL19 6.08 http://www.jimmunol.org/ CCL20 2.58 CXCR4 3.57 CCR7 5.38 CCL3 2.54 IL36G 3.47 IL2RA 4.79 CCR1 22.78 IL7R 22.78 IL6R 23.59 CCL23 23.00 IL13RA1 22.93 IL3RA 23.70 IL18BP 23.48 IL18BP 22.96 IL18BP 24.77 CXCL10a 24.14 CCL13 23.03 CXCL16 25.30 CCL13 24.21 CCL23 27.18 IL13RA1 26.88 Profiles were generated by contrasting treatment against negative controls. +FC indicates upregulation, whereas 2FC indicates downregulation. aCXCL10 and IL-6 unique expression pattern in response to agonists. bUnique expression pattern of IFN-g and IFN-a in response to agonists. by guest on September 26, 2021 both steady-state and inflammatory conditions (32, 33). This significant age-related alternations; however, agonist-stimulated strongly suggests that higher expression of CX3CR1 in old sub- monocytes isolated from adults and old subjects did show alter- jects could lead to enhanced recruitment of monocytes to in- nations at transcriptional and functional levels. These included flammatory sites and might contribute to pathogenic effects in the genes and biological processes associated with defense mecha- heart and other organs observed in the elderly. We thus validate nisms against bacteria and viruses, as well as with regulation of the expression of this molecule at the protein level in our mono- adaptive immune response. These alterations could explain some cyte subsets. CD14dimCD16+ monocytes showed the highest ex- of the defects associated with the increased susceptibility to in- pression of CX3CR1 compared with intermediate and classical fections observed in the elderly population. monocytes (Fig. 8). Interestingly, we observed under nonstimu- Monocytes were isolated from healthy subjects selected using an lated conditions significantly (p , 0.05) higher expression of extensive screening procedure including medial history question- CX3CR1 on CD14+CD162 and CD14dimCD16+ monocytes from naire and testing of blood for immune cell activation and under- old subjects compared with adults (Fig. 8), indicating a possible lying viral infections. For this study, enrollment criteria for older higher number of CX3CR1+ monocytes in CX3CL1-expressing subjects included nonfrail, nondementia, and independent living tissue in old individuals. Overall, we observed age-related alter- with control hypertension and arthritis to circumvent older indi- nations in fractalkine receptor CX3CR1 that could be associated viduals with increased frailty that can be associated with increased with age-mediated pathogenesis in the elderly. comorbidities and medication usage. This in-depth screening process reduces the impact of cofounders in a smaller cohort influ- Discussion encing results. Our previous study (13) showed that a small cohort In this study, we have performed a comprehensive and detailed of healthy nonfrail subjects was able to provide an accurate immune analysis on the impact of aging on the three main monocyte subsets representative of age-associated alternations. (CD14+CD162, CD14dimCD16+, and CD14+CD16+) using geno- Contrasting the three monocyte subsets, we observed that CD14+ mics and functional assays. In particular, we determined whether CD16+ monocytes have a more intermediate transcriptional ex- aging would impact the ability of monocyte subsets to respond to pression profile compared with CD14+CD162 and CD14dimCD16+ TLR triggering with RIG-I agonist (59pppRNA), TLR4 agonist monocytes. The developmental relationship between monocyte (LPS), and TLR7/8 agonist (CL097). To our knowledge, this is the subsets remains an unanswered critical question. Using hierarchical first study to examine the impact of healthy human aging on ex clustering analysis, microarray studies have observed a close vivo and TLR-stimulated monocyte subsets at both the tran- proximity between intermediate (CD14+CD16+) and nonclassical scriptional and functional levels. Our data showed that under (CD14dimCD16+), whereas classical monocytes (CD14+CD162) healthy nonstimulus conditions, monocyte subsets did not reveal were the most distant (27, 28). In our hands, using a similar The Journal of Immunology 9

had an enrichment of genes known to be associated with phagocytosis, migration, and MHC class II molecules, indicating that they are also predisposed for Ag presentation and serving on the front line of defense against pathogens (34). In line with this, we observed that both CD14+CD162 and CD14+CD16+ monocytes in response to LPS produced a higher level of ROS than CD14dimCD16+ monocytes (data not shown). It is also likely that CD14+CD162 monocytes might be involved in replenishing monocytes in the blood and tissue by serving as the precursor to CD16+ monocytes, and studies have shown that CD14+CD162 monocytes are recruited in the tissue to become tissue-resident macrophages under homeo- static conditions (35). These data support our hierarchical analysis that showed a closer relationship between CD14+CD16+ and CD14+CD162 monocytes. Nonclassical CD14dimCD16+ monocytes upregulated a high level of transcripts involved in cytoskeleton organization that support their patrolling motile function in response to viral Ags Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Adults contrasted with old subjects revealed distinct enriched transcripts in response to agonists. Transcriptional profiles for monocyte subsets from adults were contrasted with old subjects (n = 8 per age group). DEGs were selected based on FC $ +1.3 or # 21.3 and p , 0.05. (A) Graph depicts the total number of DEGs for adults and old subjects for each treatment. (B and C) Tables show the FCs and p values for selected enriched immune-related genes for 59-pppRNA–treated CD14+CD162 (B) and LPS-treated CD14dimCD16+ (C) monocytes. +FC values (blue) indicate genes that are significantly enriched in the adults, whereas 2FC values (red) indicate genes that are significantly enriched in the old subjects. hierarchical analysis, we observed a closer relationship between CD14+CD16+ and CD14+CD162 monocytes, with the CD14dimCD16+ subset being the most distant. This shift in subset clustering could be because of the use of a larger sample number (18 subjects), whereas the earlier mentioned studies contained three to four subjects. Despite this discrepancy, all studies uniformly showed that CD14+CD16+ monocytes have an intermediate transcriptional expression profile. We observed under nonstimulated conditions that CD14+CD162 monocytes had an enrichment of MHC class I and II molecules, FIGURE 5. Adults contrasted with old subjects revealed altered bio- indicating this subset might have greater ability to trigger the logical functions in response to agonists. Tables depict significantly enriched activation of Ag-specific lymphocytes directly or by further dif- functional pathways (p , 0.05) for 59-pppRNA–treated CD14+CD162 ferentiation into Ag-presenting macrophages or DCs. The high monocytes (A) and LPS-treated CD14dimCD16+ monocytes (B). +FC enrichment of TLRs also supports the idea that this subset is most (Log2FC) values (blue) indicate pathways that are significantly enriched in likely the first line of innate immune defense against pathogens. adults, whereas negative Log2FC values (red) indicate pathways that are 2 Similar to CD14+CD16 monocytes, intermediate CD16+ monocytes significantly enriched in old subjects. 10 AGE ALTERNATIONS IN MONOCYTES STIMULATED WITH AGONISTS

FIGURE 6. Distinct chemokine and cytokine production by individual agonists. Table lists the significant production of chemokines and cytokines elicited from agonist-treated CD14+CD162 (A), CD14+CD16+ (B), and CD14dimCD16+ (C) monocytes sorted from adults. An upward arrow indicates Downloaded from significantly higher production compared with negative controls (NT or LyoVec), whereas a downward arrow indicates significantly lower production compared with negative controls. n = 8 per age group. ****p , 0.0001, ***p , 0.001, **p , 0.01, *p , 0.05, statistical significance between treatment and negative control using two-way ANOVA followed by Bonferroni correction for multiple comparisons.

(11). In line with this, we observed transcripts related to cyto- preliminary studies investigating infection rate of dengue virus http://www.jimmunol.org/ skeleton organization, cell adhesion, and locomotion. Cros et al. type 2 and IL-6 production of monocyte subsets, and observed (11) also observed that CD14dimCD16+ nonclassical monocytes similar levels between adults and old subjects (data not shown). It responded to viruses and immune complexes but poorly to surface- is possible that dengue virus does not influence the function of old associated TLR4 stimulation (LPS) compared with classical and monocytes similar to influenza virus, which causes a more virulent intermediate subsets. In our hands, we noted significant production infection in older individuals. of chemokines and cytokines by CD14dimCD16+ in response to In addition, we also observed that LPS- and CLO97-treated LPS, and other studies have observed significant production of CD14dimCD16+ “nonclassical” monocytes sorted from old sub- TNF-a and IL-1b (28, 36). This discrepancy in results could be jects also produced lower levels of IFN-g. The ability of mono- because of use of different concentrations and stocks of LPS, or the cytes to produce IFN-g suggests an important role of IFN-g in by guest on September 26, 2021 nature of anti-CD14 Ab used in sorting of individual subsets that linking innate and adaptive immune responses. IFN-g affects could block LPS activity. We also observed that CD14dimCD16+ many biological functions primarily related to host defense, cell monocytes expressed elevated levels of granzymes transcripts in- cycle, apoptosis, and inflammation. It enhances the upregulation cluding GZMA and GZMB. Granzymes have been shown to in- of MHC class I and II molecules and improves Ag presentation by activate viruses, cleave surface receptors and extracellular matrix professional APCs. In addition, IFN-g elicits leukocyte trafficking, proteins that facilitate leukocyte migration, and induce production maturation, and differentiation (including naive monocytes into of proinflammatory cytokines and phagocytosis by monocytes (37, DCs) (40). Overall, our data show that the IFN-mediated antiviral 38). This suggests that these proteases could play an essential role response in monocyte subsets was significantly impacted with in the immunoregulation by nonclassical monocytes. CD16+ NK aging, which supports the implied immune response to viruses cells are also known to express high levels of granzymes, and it is observed in old individuals. possible that the gating scheme used to sort ex vivo CD14dimCD16+ We also observed age-related alternations in IL-1b, CCL8, and monocytes could have included a small percentage of these cells. CCL20 production by CD14+CD162 monocytes from old subjects We did not observe significant age-related alternations in tran- treated with LPS, 59pppRNA, and CLO97, respectively. IL-1b is scriptional profiles of nonstimulated monocyte subsets. Therefore, produced as a result of pro–IL-1b transcriptional upregulation via we investigated whether aging would impact the ability of these TLR signaling and a second signal that activates the NLRP3 subsets to respond to TLR ligands as we have previously reported inflammasome; however, human monocytes are unique in their for PBMCs (13). In line with this, we observed alternations in ability to secrete IL-1b by an alternative inflammasome activation chemokine and cytokine production, expression in chemokine using a single LPS stimulation of TLR4 (41). IL-1b is an im- surface receptor, and the expression of multiple innate immune portant mediator for host defense against a wide variety of viruses genes. We observed that 59pppRNA-treated CD14+CD162 “clas- and bacteria species by eliciting proinflammatory signaling path- sical” monocytes sorted from old subjects produced significantly ways, increasing cell adhesion molecules on endothelial cells, and lower levels of IFN-a and had lower expression levels of IFN-a trigging of adaptive immunity such as Th17 response (42, 43). Our transcripts. These data are supported by work from Pillai et al. results are in line with other studies that have shown significantly (39) that recently showed type I IFN (IFN-b) responses to influenza lowered IL-1b production by LPS-treated monocytes from old A virus were significantly attenuated in older human monocytes. subjects (44). CCL8 and CCL20 levels are also reduced in aging They showed that monocytes and macrophages from old individuals subjects. CCL8 is recognized by receptors CCR1, CCR2, and had intact RIG-I signaling to elicit the production of proinflam- CCR5 that are expressed on different cell types including gran- matory cytokines and the inflammasome, but had impaired signal- ulocytes and mononuclear phagocytes and T lymphocytes (45, ing to induce type I IFNs. To determine whether the alternations 46), whereas CCL20/CCR6 mediates the recruitment of immature in antiviral responses translated to other viruses, we performed DCs and lymphocytes into mucosal-associated tissues (47). Overall, The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. Age-related alternations in cytokine and chemokine production. (A–C) Graphs depict the means 6 SEM of IFN-g, IL-1b, and CCL20 concentrations in response to stimulation with LPS and CLO97 for up to 24 h for adults and old subjects. (D and E) Graphs depict the means 6 SEM of IFN-a and CCL8 concentrations in response to stimulation with 59-pppRNA for up to 24 h for adults and old subjects. All concentrations were measured using the human chemokine 40-plex from Bio-Rad except for IFN-a measured by ELISA. n = 8 per age group. ****p , 0.0001, ***p , 0.001, **p , 0.01, *p , 0.05, statistical significance between adults and old subjects using two-way ANOVA followed by Bonferroni correction for multiple comparisons. decreased production of these mediators resulting in reduced mi- ROS and reactive nitrogen species are involved in the regula- gration of immune cells to sites of infection such as lung and gastric tion of diverse processes, including phagocytes, antibacteria, cell mucosa could explain why infections such as influenza and bacterial proliferation, and apoptosis (48, 49). The production of ROS pneumonia have worse outcomes in old subjects. serves as a link between cellular senescence and age-associated pathologies, and could be involved in accelerating immunosenescence (50). Our data revealed that agonist-stimulated monocyte subsets sorted from old subjects had an enrichment of transcripts related to ROS and reactive nitrogen species production. This included elevated levels of SOD2, CCS, NQO1, ARG2, CAT, DDAH2, and GCHFR transcripts, which corresponded to enrichment of the superoxide metabolic process and ROS metabolic pathways. We have observed comparable levels of ROS using fluorescent probes, as well as mitochondrial function/oxidative burst using extracellular flux analyzer (Seahorse Bioscience), between untreated and agonist-treated monocytes from adults and old subjects (data not shown). The impact of aging on monocyte mitochondrial function remains an open question, and our data FIGURE 8. Age-related alterations in CX3CR1 surface receptor ex- revealed, at the transcriptional level, an impact of aging on the pression. Graph depicts the mean fluorescent intensity (MFI) of CX3CR1 production of ROS. for untreated monocyte subsets from adults (black) and old (gray) subjects. At the transcriptional levels, we observed age-related alterations The p values (*p ,0.05) were calculated using unpaired t test with Welch in costimulatory receptors crucial in the development of an ef- correction. n = 8 per age group. fective immune response including 59pppRNA-induced higher 12 AGE ALTERNATIONS IN MONOCYTES STIMULATED WITH AGONISTS expression of CD80, PDL1, and CD83 (DCs maturation) tran- 8. Jakubzick, C., E. L. Gautier, S. L. Gibbings, D. K. Sojka, A. Schlitzer, scripts in CD14+CD162 monocytes from adults, whereas LPS T. E. Johnson, S. Ivanov, Q. Duan, S. Bala, T. Condon, et al. 2013. Minimal + + differentiation of classical monocytes as they survey steady-state tissues and induced higher CD40 and CD80 expression in CD14 CD16 transport antigen to lymph nodes. Immunity 39: 599–610. monocytes from old subjects. 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