Aging Impairs Alveolar and Increases Influenza-Induced Mortality in Mice

This information is current as Christine K. Wong, Candice A. Smith, Koji Sakamoto, of September 28, 2021. Naftali Kaminski, Jonathan L. Koff and Daniel R. Goldstein J Immunol published online 23 June 2017 http://www.jimmunol.org/content/early/2017/06/23/jimmun ol.1700397 Downloaded from

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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 June 23, 2017, doi:10.4049/jimmunol.1700397 The Journal of Immunology

Aging Impairs Alveolar Macrophage Phagocytosis and Increases Influenza-Induced Mortality in Mice

Christine K. Wong,*,†,1 Candice A. Smith,‡,1 Koji Sakamoto,* Naftali Kaminski,* Jonathan L. Koff,* and Daniel R. Goldstein*,†,‡,x

Influenza viral infections often lead to increased mortality in older people. However, the mechanisms by which aging impacts im- munity to influenza infection remain unclear. We employed a murine model of influenza infection to identify these mechanisms. With aging, we found reduced numbers of alveolar , cells essential for lung homeostasis. We also determined that these macrophages are critical for influenza-induced mortality with aging. Furthermore, aging vastly alters the transcriptional profile and specifically downregulates cell cycling pathways in alveolar macrophages. Aging impairs the ability of alveolar macrophages to limit lung damage during influenza infection. Moreover, aging decreases alveolar macrophage phagocytosis of apoptotic ,

downregulates the scavenging receptor CD204, and induces retention of neutrophils during influenza infection. Thus, aging induces Downloaded from defective phagocytosis by alveolar macrophages and increases lung damage. These findings indicate that therapies that enhance the function of alveolar macrophages may improve outcomes in older people infected with respiratory viruses. The Journal of Immunology, 2017, 199: 000–000.

lder people exhibit increased morbidity and mortality and reduced inflammasome activity (4–8). Despite these reports,

in response to respiratory viral infections (1, 2). As the there remains controversy as to the impact of aging on the suscep- http://www.jimmunol.org/ O number of older adults increases, the incidence of morbid tibility to influenza viral infections in murine models. On one hand, complications from respiratory infections also grows. Indeed, prior reports indicate that aged mice (age range 18–26 mo) exhibit during the years 1988–2002, people .65 y of age exhibit a 20% higher mortality, morbidity, and lower viral clearance than do young increase in hospitalization rates for community acquired pneumonia mice during influenza viral lung infection (4, 6, 7, 9). On the other (3). Hence, discerning how aging modifies immunity to influenza hand, a recent study documented that aged (24–26 mo old) mice virus on a molecular basis is critical to developing novel therapies infected with influenza virus exhibit significantly lower mortality and treatments that will protect older adults from respiratory and higher viral clearance than do young mice (10). Additionally, the infection. critical cellular mechanisms by which aging alters the host response Experimental studies have documented several age-associated to influenza viral lung infection remains unclear. by guest on September 28, 2021 alterations during influenza viral infection that include reduced In this study, we used dose titrating aliquots of influenza virus to antiviral CD8+ T cell responses, decreased NK cell function, al- evaluate how aging affects mortality and lung damage during in- tered PG production, increased expansion of regulatory T cells, fluenza viral infection. We identified alveolar macrophages (AM), key tissue resident macrophages for lung homeostasis (11), as critical for mortality during influenza viral lung infection with *Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06520; aging. The transcriptional signatures of AM from young and ad- †Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520; ‡Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109; vanced aged mice were substantially different: we found that 3545 and xInstitute of Gerontology, University of Michigan, Ann Arbor, MI 48109 genes were significantly altered with aging and that cell cycle 1C.K.W. and C.A.S. contributed equally to this work. pathways were markedly downregulated. Prior to and during in- ORCIDs: 0000-0001-7440-1809 (C.A.S.); 0000-0003-4567-7771 (D.R.G.). fection, advanced aged mice exhibited 2-fold lower concentrations Received for publication March 16, 2017. Accepted for publication May 24, 2017. of AM. Functionally, AM from advanced aged mice were im- This work was supported by National Institutes of Health Grant AG028082 and in paired in scavenging apoptotic neutrophils, displayed selective part by National Institutes of Health Grant HL130669 (to D.R.G.). C.A.S. is sup- downregulation of a key scavenging receptor, CD204, and exhibited ported by National Institutes of Health Grant T32-HL007853. defects in limiting lung damage. Therefore, our study has found that C.K.W., J.L.K., C.A.S., and D.R.G. designed experiments; C.K.W. and C.A.S. con- aging impairs the intrinsic function of AM to limit lung damage ducted the in vivo and in vitro experiments; C.K.W., C.A.S., and D.R.G. analyzed data; K.S. and N.K. conducted and analyzed microarrays and performed the micro- during influenza viral lung infection. array statistical analyses; D.R.G. and C.A.S. wrote the paper; C.K.W. and J.L.K. read and edited the paper; and D.R.G. procured funding. Materials and Methods The microarray data presented in this article have been submitted to the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo/) under ac- Mice and in vivo viral infection cession number GSE84901. C57BL/6 mice of 2–4 mo (representative weight, 26 6 2 g [SD]), 16 mo Address correspondence and reprint requests to Dr. Daniel R. Goldstein, University (36 6 3.7 g), and 22–24 mo of age (33 6 2.5 g) were obtained from the of Michigan, NCRC B020-209W, 2800 Plymouth Road, Ann Arbor, MI 48104. National Institute of Aging rodent facility. Mice were infected with puri- E-mail address: [email protected] fied human influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) (Advanced The online version of this article contains supplemental material. Biotechnologies, Eldersburg, MD) as previously reported (12). Mice were Abbreviations used in this article: AM, alveolar macrophage; BAL, bronchoalveolar anesthetized with isoflurane and instilled intranasally (i.n.) with 50 mlof lavage; DPI, day postinfection; FDR, false discovery rate; i.n., intranasal(ly); LDH, PBS containing the indicated dose of PR8 virus or 50 ml of PBS control. lactate dehydrogenase; MPO, myeloperoxidase; RSV, respiratory syncytial virus. Following infection, mice were monitored daily for changes in weight, clinical scores, and mortality. Clinical scores were determined by ruffled Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 fur, activity, hunched back, and mortality (Table I), specifically mice were

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700397 2 AGING AND INFLUENZA VIRAL INFECTION euthanized when 30% of their original weight was lost, which was re- F4/80, and CD11c as previously reported (13, 14). The Siglec-F Ab was corded as death in survival experiments. No animals were used in the study purchased from BD Biosciences. All other Abs were purchased from when they displayed evidence of infection or other illnesses prior to PR8 eBioscience. Flow cytometry data were acquired on an LSR II (BD Bio- infection. Both the Yale University and University of Michigan Institu- sciences) and analyzed with FlowJo (Ashland, OR) software. tional Animal Care and Use Committees approved the use of animals in To assess the AM proliferation rate, single-cell suspensions were prepared this study. Prior to viral infection, all mice were kept in pathogen-free and stained as described above followed by intracellular Ki67 (allophyco- conditions. cyanin) (eBioscience) staining according to the manufacturer’s protocol. For sorting of AM, single-cell suspensions were prepared as above from Sample collection and preparation lung homogenates and stained with CD45 (allophycocyanin-Cy7), CD11c (FITC), F4/80 (Pacific Blue) and Siglec-F (PE). Cells were sorted in PBS Tissues were harvested from PBS-instilled or PR8-infected mice (n = 3–5 containing 1% BSA and 2 mM EDTA on a FACSAria (BD Biosciences) per time point for each experiment) at 1, 3, 6, and 9 d postinfection (DPI). and collected into HBSS supplemented with 1% BSA and 2 mM EDTA for Each time point was repeated at least three times independently. After adoptive transfer or TRIzol (Invitrogen, Carlsbad, CA) for RNA extraction euthanasia, the bronchoalveolar lavage (BAL) was obtained by washing (see below). the twice with 1 ml of cold sterile 13 PBS. To harvest lungs, the chest cavity was opened and the lungs were removed and flash frozen in AM adoptive transfer liquid nitrogen. FACS-sorted AM were centrifuged at 5000 rpm for 5 min, resuspended in Assessment of tissue injury PBS, and 3 3 105 cells were transferred into recipient mice via i.n. route. Mice were subsequently infected with 104 PFU of PR8 i.n. 1 d following Lung injury was assessed by measuring total protein and lactate dehy- drogenase (LDH) levels in the BAL fluid. Total protein in BAL was adoptive transfer, and lungs were harvested on 3 DPI as described above. measured using the BCA protein assay kit (Thermo Fisher Scientific, AM in vivo phagocytosis test Waltham, MA) following the manufacturer’s instructions. LDH activity Downloaded from was measured with an LDH assay kit (Roche, New York, NY) according to Alexa Fluor 488 beads (Invitrogen) were resuspended in PBS and sonicated the manufacturer’s instructions. Myeloperoxidase (MPO) levels in the at room temperature for 30 min prior to administration into recipient mice. BAL were measured with kits from (eBioscience, San Diego, CA), After i.n. instillation of 1 3 107 beads in 40 ml of PBS, mice were allowed according to the manufacturer’s instructions. 1 h to recover with food and water before harvesting the lungs. Lung tissues were prepared and cells were obtained and stained as Viral load measurement described above. Fluorescent data were recorded by an LSR II flow cytometer or Beckman MoFlo Astrios and analyzed using FlowJo software.

Plaque assay. Lung viral titers were determined by plaque assay using http://www.jimmunol.org/ Madin–Darby canine kidney cells (provided by Dr. A. Iwasaki, Yale Neutrophils were purified from the bone marrow of young mice using School of Medicine) as previously reported (12). Madin–Darby canine EasySep mouse enrichment kits from Stemcell Technologies kidney cells were cultured in six-well plates overnight until the cell (Vancouver, BC, Canada) as specified in their protocol. The neutrophils 3 6 monolayer reached 100% confluence. Lung supernatants were thawed at were diluted to a final concentration of 1 10 /ml and rendered apoptotic room temperature and diluted to different dilutions (101–103) with 1% by incubation in RPMI 1640 with 0.5% FBS in 5% CO2 overnight at 37˚C. BSA in 13 PBS. Cells were washed twice with 13 PBS followed by 1 h Following overnight incubation, Molecular Probes Vybrant cell-labeling incubation with 200 ml of lung supernatants at 37˚C with frequent shaking. stain DiI (designated as Dil) was added in a volume of 5 ml per every 3 6 Cells were washed twice with 13 PBS to remove excess virus and were 1 10 cells to the neutrophils in the incubator for 20 min. The neutro- 3 overlaid with 2 ml of agarose mixture containing 50% 23 MEM, 17.35% phils were then centrifuged at 1420 g for 5 min a 4˚C. The supernatant was then discarded and the remaining neutrophils were resuspended in double distilled H2O, 1% DEAE-dextran, 7.5% NaHCO3, 0.35% acety- 6

3 3 by guest on September 28, 2021 lated trypsin (1 mg/ml), and 2% agarose. Plates were incubated at 37˚C for sterile 1 PBS at a concentration of 3 10 neutrophils per 50 ml. From 3 6 48 h. The agarose was removed from the plates and the plates stained with this suspension, 3 10 neutrophils were immediately instilled i.n. into 0.1% crystal violet. Virus plaques were counted and the PFU per milliliter mice that had been anesthetized by isoflurane. The mice were allowed to were calculated using the formula: number of plaques 3 dilution factor 3 regain consciousness and rest for 2 h before they were euthanized and BAL 5. All dilutions of each sample were run in duplicate. was harvested from the lungs. BAL was stained as described above, except Siglec- F (PE) was substituted for Siglec-F (Alexa Fluor 647), and fluo- RT-PCR. RNA from whole-tissue samples was extracted as described below. rescent data were recorded and analyzed using FlowJo software. During reverse transcription, RNA was transcribed with a PAN influenza Neutrophil apoptosis and purity assessment were performed with sam- primer (59-TCTAACCGAGGTCGAAACGTA-39). RT-PCR was run as ples of neutrophils before and after staining using the above-mentioned DiI described below using the influenza-specific primers (59-AAGACCAAT- cell stain. The aliquots of neutrophils were diluted to a concentration of CCTGTCACCTCTGA-39,59-CCTGACGTCGCATCTGCGAAAC-39). For 1 3 106 neutrophils/ml for further analysis to determine the level of apo- other genes measured by RT-PCR the following primers were used: c-Maf, ptosis and extent of staining with DiI cell stain, annexin V (BV421), CD11c 59-ACTGAACCGCAGCTGCGCGGGGTCAG-39,59-CTTCTCGTATTTCTCCTTG- (FITC), Ly6G (Alexa Fluor 647), and Thermo Fisher Live/Dead fixable dye TAGGCGTCC-39;andMafB,59-TCCACCTCTTGCTACGTGTG-39, in near infrared. The enriched samples of neutrophils were ∼80–82% pure for 59-CGTTAGTTGCCAATGTGTGG-39. each test, and of the cell membrane–stained neutrophils, 81.1–89.2% were For each group, data were normalized to b-actin expression (59-CCG- positively stained with DiI. To determine the extent of apoptosis, the flow CCCTAGGCACCAGGGTG-39,59-CCGCCCTAGGCACCAGGGTG-39)and plots of annexin V were compared with those of the Live/Dead. In experi- then compared among the groups. Data were analyzed using the Bio-Rad ment 1, 40% of the neutrophils were apoptotic, 50% were dead, and 10% Laboratories (Hercules CA) built-in system. were healthy. In experiment 2, 91.1% of the neutrophils were apoptotic, 7.3% Flow cytometry and cell sorting were dead, and 1.62% were healthy. To obtain single-cell suspensions, lungs were harvested from animals, Apoptosis assessment minced, and then digested with 1 mg/ml collagenase D (Roche) and 100 U/ml Apoptosis in AM or in neutrophils was measured by annexin V staining DNAse (DN25; Sigma-Aldrich, St. Louis, MO) in PBS without calcium and (BioLegend) according to the manufacturer’s protocol. magnesium for 45 min at 37˚C. After digestion, lung tissue was disrupted into single-cell suspension by passage through a 100-mm sieve (Fish- Cytospin and H&E staining erbrand; Thermo Fisher Scientific). To remove RBCs, cells were spun for 5 min at 1400 rpm and RBCs were lysed by resuspension of the pellets for BAL from healthy and infected mice was obtained as described above. After 3 min on ice in RBC lysis buffer (BioLegend, San Diego, CA). Lysed cells centrifugation, cell pellets were suspended with 13 RBC lysis buffer (BD were spun and resuspended with 1:50 FcgR blocker (BioLegend) in HBSS Biosciences) and incubated at room temperature for 3 min. Cells were containing 1% BSA and 2 mM EDTA. Cells were then incubated on ice for washed with PBS twice followed by resuspension with 100 mlof13 PBS. 10 min and stained with the indicated fluorescent Abs for 30 min on ice. Cells were counted using an automated hematology analyzer. Cells (1 3 105) Cells in the BAL were collected, spun down, and blocked 1:50 with FcgR were then spun down using a cytospin machine (Thermo Scientific) for 8 blocker on ice for 10 min followed by staining with the indicated fluo- min at room temperature. Slides were allowed to air dry before H&E rescent Abs. For all infected samples, cells were fixed with fixation buffer staining with a Hemacolor staining kit (EMD Millipore, Darmstadt, Ger- (BD Biosciences, San Diego, CA) for 15 min on ice after staining with many) following the manufacturer’s protocol. After staining, slides were indicated fluorescent Abs. AM were characterized according to their for- allowed to air dry overnight before mounting with Vectashield (Vector ward and side scatter profiles, and by high surface expression of Siglec-F, Laboratories, Burlingame, CA) and coverslip. Slides were then analyzed The Journal of Immunology 3

Table I. Clinical score RNA extraction, real-time PCR, and microarray RNA was extracted from the upper left lung using an RNA extraction kit Clinical Score Score Symptom(s) (Qiagen, Bergisch Gladbach, Germany) and was used as a template to generate 0 Normal cDNA with a cDNA synthesis kit (Clontech Laboratories, Mountain View, 1 Slightly ruffled fur CA). To extract RNA from purified AM, AM were placed into 200 mlof 2 Ruffled fur TRIzol followed by RNA extraction according to the manufacturer’s rec- 3 Ruffled fur and inactive ommendations. RNA was then transcribed into cDNA as described above. 4 Hunched back or moribund Real-time PCR was run using the CFX96 Touch real-time PCR detection 5 Dead system (Bio-Rad Laboratories) using the following cycles: 1) 95˚C for 10 s; 2) 94˚C for 10 s, 60˚C for 30 s, 72˚C for 20 s (repeat 39 times); 3) 95˚C for 10 s, 65˚C for 5 s, and 95˚C for 5 s. Results were analyzed using the Bio-Rad Laboratories built-in software or the D cycle threshold method. For micro- with a microscope and neutrophils were identified and counted based on arrays, RNA quantity was determined by NanoDrop at 260 nm and RNA their nuclear appearance. integrity was assessed by a 2200 TapeStation system with RNA screen tape Clodronate treatment (Agilent Technologies, Santa Clara, CA). Labeling was performed using the Agilent Technologies Low Input Quick Amp Labeling kit. In brief, the first To deplete AM, ready-made clodronate liposomes and control liposomes strand cDNA synthesis was performed using an oligo(dT) primer containing a (FormuMax Scientific, Sunnyvale, CA) were administered to mice using the T7 RNA polymerase promoter site. The cDNA was used as a template to manufacturer’s recommended dose via i.n. instillation 1 d before and 1 d generate Cy3-labeled cRNA that was used for hybridization. After purifica- after PR8 infection. On 6 DPI, clodronate-treated and control mice were tion and fragmentation, aliquots of each sample were hybridized to SurePrint euthanized and lung samples were harvested. For survival experiments, G3 Mouse Gene Expression v2 8360K microarrays (Agilent Technologies).

mice were monitored on a daily basis as described above. After hybridization, each array was sequentially washed and scanned by an Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Aging increases mortality, morbidity, and lung inflammation during influenza viral lung infection. (A) Young (n = 71), aged (n = 24), and advanced aged mice (n = 69) were infected i.n. with 1 3 104 PFU of influenza virus and mortality was recorded. Groups of mice that were given PBS control are shown (young, n = 12; aged, n = 7; and advanced aged, n = 12). *p , 0.05, **p , 0.01 (log rank). (B and C) Body weight (B) and clinical score (C) for young and advanced aged mice are shown during influenza viral infection described in (A). *p , 0.05, ****p # 0.0001. (D–F) Young and advanced aged mice were infected with influenza virus as stated in (A), LDH in BAL (D), protein concentration in BAL (E), and viral load as measured by plaque assay (F). (G) Histological assessment (H&E staining) of lungs from young and advanced aged mice. The lungs from noninfected mice treated with PBS control are shown. The lungs from mice at 6 DPI are shown and were assessed for histological inflammation. The lungs from advanced aged mice exhibited increased cellular infiltration in contrast to lungs from young mice at six DPI. Original magnification 310; scale bars, 200 mm. (H) Survival of advanced aged mice with various dose of PR8 infection. PBS, n =3;102 PFU, n =5;103 PFU, n = 15; 104/106 PFU, n = 10. *p , 0.05, **p , 0.001 (log rank). (D–F) Samples sizes were four to five mice per time point, and data shown are representative of five independent experiments. 4 AGING AND INFLUENZA VIRAL INFECTION

Agilent Technologies microarray scanner. Arrays were visually inspected Error bars are presented as mean with SEM. Data shown are representative individually for hybridization defects and quality control procedures were of at least three independent experiments unless otherwise stated. applied. Intensity information from captured array images and the annotation information from the microarray experiments were determined using Agilent Results Feature Extraction 12.0.0 software. Probes with annotations for “accession” were extracted, and interquartile normalization was applied to normalize the Aging induces higher morbidity and mortality during influenza gene expression signals by BRB-ArrayTools v4.5.0 (http://brb.nci.nih.gov/ viral infection BRB-ArrayTools/). In case of redundant probes, we took the one with the To study how aging impacts the clinical response to influenza viral highest interquartile range from the samples representing the gene expression levels. To assess genes impacted by aging, differentially expressed genes were lung infection, we first assessed the morbidity and mortality of in- identified using significant analysis of microarrays (http://www-stat.stanford. fluenza viral infection (A/PR/8/34, H1N1) in advanced aged (22–24 edu/∼tibs/SAM). A false discovery rate (FDR) of 5% was set as the threshold mo), aged (16–18 mo), and young mice (2–4 mo). One week after for significance. Data were visualized by generating heat maps with Java influenza viral lung infection (dose, 1 3 104 PFU), 80% of advanced TreeView. The microarray data have been deposited at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? aged mice, 50% of aged mice, and 25% of young mice died token=yvctemmwrtyttgj&acc=GSE84901) under accession number GSE84901. (Fig. 1A). Further comparisons between young and advanced aged mice revealed higher morbidity (Fig. 1B, 1C), increased lung dam- Gene enrichment analysis age (Fig. 1D, 1E), impaired viral clearance (Fig. 1F), and increased Gene set enrichment analysis was performed using the MetaCore software histological evidence of inflammation (Fig. 1G) than did young in- suite (http://www.genego.com/metacore.php). The gene list containing all fected mice. However, there was no difference in viral titers in serum the genes that were differentially expressed between young and advanced or evidence of extrapulmonary organ damage between the age groups aged macrophages (with statistical significance by significant analysis of (e.g., kidney and liver; data not shown). This finding suggests that Downloaded from microarrays test, FDR , 5%) was analyzed using the Pathway Maps tool in the MetaCore suite, which maps the listed genes to defined signaling advanced aged mice do not die of systemic organ failure during pathways that have been experimentally validated and are widely accepted. influenza viral infection in this model, but instead succumb to All reported enriched pathways are listed according to 2log (p values) and morbid complications of influenza viral lung infection. with an FDR , 0.05%. To further test the susceptibility of advanced aged mice to influenza infection, we infected advanced aged mice with dose ranges of in- Statistical analysis 2 6 fluenza virus (i.e., 1 3 10 to 1 3 10 PFU). We found that at 1 3 http://www.jimmunol.org/ A Mann–Whitney U test was used to calculate statistical significance for all 103 PFU, advanced aged mice exhibited a similar mortality as did comparisons besides survival analysis. The log-rank (Mantel–Cox) test was young mice infected with 1 3 104 PFU (compare Fig. 1A and 1H). used to calculate survival statistical significance. Statistical significance was 2 calculated using Prism 7 (GraphPad Software). A p value ,0.05 was con- No mortality was observed at 1 3 10 PFU dose in advanced sidered significant (*p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001). aged mice, whereas mortality could not be significantly increased at

FIGURE 2. AM are critical for mortality in both by guest on September 28, 2021 young and advanced aged mice. (A and B) Survival of young (A)at13 104 PFU of influenza virus and advanced aged mice at 1 3 103 PFU of influenza virus (B) with clodronate liposome or control lipo- some treatment during infection with. *p , 0.05, ** = p , 0.001 (log rank). (C) Lungs were obtained from noninfected young (2–4 mo) and advanced aged (22–24 mo) C57BL/6 mice. The BAL was harvested and a single suspension was obtained. The cells were stained with relevant fluorescently tagged mAbs and analyzed via flow cytometry. Quantifica- tion of number and frequency of AM is based on staining of frequency of F4/80+CD11chi cells in Siglec-F+ cells. Plots were gated on single CD45+ population from young and advanced aged mice. (D) Lungs were obtained from noninfected young (2–4 mo of age) and advanced aged (i.e., 22–24 mo of age) BALB/c mice and quantified as above. (C) and (D) are representative of an experiment repeated five times. *p , 0.05. (E) Quantification of the absolute numbers of AM in BAL at day 6 after influenza infection. Absolute numbers of AM are normalized to grams of lung tissue. n = 4–5 mice per time point. ***p , 0.001. (F) Lungs were obtained from young (2–4 mo of age) and advanced aged (i.e., 22–24 mo of age) C57BL/6 mice before (open symbols) or day 3 after infection (closed symbols). The lung ho- mogenates were harvested and a single suspension was obtained as described in Materials and Methods. Flow cytometric plots were gated on AM (i.e., CD45+Siglec-F+F4/80+CD11chi) and were stained with annexin V, an apoptosis marker. The Journal of Immunology 5

1 3 106 PFU dose in advanced aged mice as compared with the 1 3 the lungs of advanced aged mice than in young mice (Fig. 2C). A 104 PFU-infected group (Fig. 1H). Overall, these data demonstrate that similar effect was found in advanced aged and young BALB/c mice aging increases mortality and morbidity to influenza viral infection. (Fig. 2D), indicating that with aging, AM concentration decreases and also that these findings are not restricted to one murine strain. AM are critical for mortality with aging with reduced (Note that C57BL/6 mice were used for the remainder of this study). numbers of AM Although after influenza viral lung infection, the numbers of AM As AM exhibit antiviral properties that are critical for host defense decreased in both advanced aged and young mice log-fold; for to RNA viruses (e.g., influenza virus and respiratory syncytial advanced aged mice this reduction was more pronounced than for viruses [RSV]) (15–17), we assessed the role of AM in host de- young mice infected with the influenza virus (Fig. 2E). When we fense to influenza virus in both young (2–4 mo) and advanced assessed the degree of apoptosis in the aged cohorts prior to and aged (22–24 mo) C57BL/6 mice. after influenza infection, we found no significant alteration in the Clodronate liposomes have been employed in the lung to deplete degree of apoptosis between the groups either before or after in- myeloid cells, which include AM (17), during RNA viral infections fection (Fig. 2F), indicating that increased apoptosis is not likely a (15, 17). Therefore, we administered clodronate liposomes to young mechanism for reduced numbers of AM with aging before or after andadvancedagedmiceduringinfluenzaviralinfectiontotest influenza infection. the dependency of AM for mortality with aging. We specifically employed influenza doses that induced ,50% mortality in young Aging induces widespread transcriptional changes in AM mice (i.e., 1 3 104 PFU) and in advanced aged mice (i.e., 1 3 103 To investigate the global impact of age on AM, we next investigated

PFU), based on our dose titration experiments (Fig. 1A, 1H). In either the broad gene transcription profile of advanced aged and young Downloaded from case, clodronate liposomes significantly enhanced mortality (Fig. 2A, AM via microarray. We determined that the gene signatures of 2B). These data demonstrate that AM are critical for host defense in FACS-purified AM from young and advanced aged mice were both young and advanced aged mice during influenza viral infection. vastly different. The difference extended to 3545 genes that were We next quantified AM (defined as F4/80+ CD11chi, Siglec-F+ significantly different between the age groups (Fig. 3A, Sup- cells) in young and advanced aged mice before infection via flow plemental Table I). Among these altered genes, bioinformatics

cytometry. We found a 40–50% lower concentration of AM in analyses revealed that pathways involved in the cell cycle were http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Aging induces a downregulation of pathways enriched in cell cycle pathways in AM. FACS-purified AM from the lungs of noninfected young or advanced aged C57BL/6 mice were obtained, RNA was harvested from the lungs, and a microarray was performed. (A) An overall heat map of the array profile in AM from young and advanced aged mice. In total, 3545 genes were significantly altered between cohorts; n = 6 biological replicates per group. (B and C) Heat map of the two most downregulated pathways with aging: cell cycle metaphase checkpoint (B) and cell cycle initiation of mitosis (C). Gene names are shown adjacent to heat maps; n = 6 biological replicates per group were employed. (D) Lungs were obtained from young (2–4 mo of age) and advanced aged (i.e., 22–24 mo of age) C57BL/6 mice prior to infection. The lung homogenates were harvested and a single suspension was obtained as described in Materials and Methods. Flow cytometric plots were gated on AM (i.e., CD45+Siglec-F+F4/80+CD11chi) and stained for Ki67 (a proliferative marker) as described in Materials and Methods and analyzed via flow cytometry. **p , 0.01. Data are representative of an independent experiment repeated three times. (E and F) mRNA was harvested from FACS-purified AM from young and advanced aged mice and gene expression was measured for genes that repress self-renewal Maf-b (E) and c-Maf (F). **p , 0.01. 6 AGING AND INFLUENZA VIRAL INFECTION

FIGURE 4. Adoptive transfer of young AM into advanced aged mice reduces tissue injury caused by influenza virus. (A) Representative flow cytometry plot showing FACS-sorted AM from young donor C57BL/6 CD45.1+ mice that were transferred i.n. into the lungs of advanced aged (noninfected) CD45.2+ mice. At 4 d after adoptive transfer, the lungs and BAL were ob- tained and stained with indicated fluorescent Abs and analyzed via flow cytometry. (B)FACS-purifiedAM from CD45.2+ young and advanced aged mice were adoptively transferred via i.n. route into young, non- infected C57BL/6 CD45.1+ mice. At 4 d after transfer, the lungs were obtained and stained as described in (A). Gate shows frequency of donor cells. (C)FACS-puri- fied AM from young or advanced aged CD45.2+ mice were adoptively transferred into advanced aged mice 1 d prior to influenza infection. At 3 DPI, the BAL was obtained. The level of tissue injury was quantified by LDH level in the BAL. **p , 0.01. Pooled data from two independent experiments. (D) Total viral titer in the Downloaded from lung quantified by quantitative PCR.

most downregulated with aging (Fig. 3B, 3C, Supplemental We next transferred 3 3 105 AM from either young donor Table II). Some of these pathways included metaphase check- mice or advanced aged donor mice into advanced aged recipient 4 point, initiation of mitosis, spindle assembly, and chromosome mice. One day after transfer, mice were infected with 1 3 10 http://www.jimmunol.org/ separation. Pathways that were upregulated with aging included PFU of influenza virus. Three days after infection, we found that inflammatory pathways involved with substance P, PGE2, mac- advanced aged mice engrafted with young AM had less lung rophage inhibitory factor, oxidative burst and IL-8, and vascular damage than did advanced aged mice engrafted with age- endothelial growth factor signaling (Supplemental Table III). matched AM (Fig. 4C). The viral titers in these advanced aged Thus, at baseline aging has a large impact on the transcriptome of engrafted mice were comparable to those of advanced aged mice AM and leads to specific downregulation in pathways involved in that received AM from either young or advanced aged donors cell cycling but upregulation of certain inflammatory pathways. (Fig. 4D), indicating that defects with aging in AM did not As AM self-renew within the lung with minimal contribution impair viral control in this model. Interestingly, advanced aged from circulating (14, 18), we hypothesized that the mice that were infected with influenza virus but did not receive by guest on September 28, 2021 reduced transcriptional signature of proliferation of AM with adoptive AM transfer exhibited a similar degree of lung damage aging is due to impaired cell turnover. In support of this, we found as advanced aged mice that received adoptive transfer of AM that aged AM exhibited lower proliferation than did AM from purified from advanced aged mice (Fig. 4C). Overall, these data young mice, as demonstrated by Ki67 staining (Fig. 3D). We next show that aging impairs the intrinsic ability of AM to limit lung measured the gene expression of the repressors of self-renewal: damage during influenza viral infection. MafB and c-Maf (19, 20). We found that AM from advanced aged Aging impairs the ability of AM to phagocytose in vivo mice exhibited higher gene expression of c-Maf but not of Mafb as compared with AM from young mice (Fig. 3E, 3F). Overall, these To define the cell-intrinsic mechanism of aging on AM, we ex- data indicate that impaired cell proliferation likely contributes to amined the effect of aging on AM phagocytosis in vivo. As reduced AM numbers with aging. clearance of apoptotic neutrophils has been shown to contribute to inflammation resolution (21), we examined the impact of aging Adoptive transfer of young AM reduces lung damage in on the ability of AM to phagocytose apoptotic neutrophils advanced aged mice infected with influenza virus in vivo. For this purpose, we instilled apoptotic neutrophils i.n. We examined the functional impact of aging on AM responses into young and advanced aged mice and found that AM in ad- during influenza viral infection via adoptive transfer. Specifically, vanced aged mice exhibited a lower ability to phagacytose ap- we used an established approach to adoptively transfer AM into optotic neutrophils than did young mice (Fig. 5A, 5B). We also the lungs of mice (16) to examine whether adoptive transfer of found that AM in advanced aged mice exhibited a significantly AM from young mice into advanced aged mice could modify lower ability to bind and internalized florescent particles than AM lung damage to influenza viral infection. First, we i.n. transferred in young mice (Fig. 5C), which is consistent with the results of a 3 3 105 FACS-purified AM from young C57BL/6 CD45.1+ prior in vitro study (22). donor mice into noninfected, advanced aged C57BL/6 CD45.2+ When we enumerated neutrophil influx into the lungs of young mice. The transferred cells were detected in the BAL 4 d after andadvancedagedmiceduringinfluenza viral infection, we transfer, indicating that the aged lung permitted engraftment of found a similar peak of neutrophil influx between the groups AM (Fig. 4A). We then purified AM from young and from ad- (Fig. 5D), similar to what has been previously reported in young vancedagedC57BL/6CD45.2+ mice and i.n. transferred them infected mice (23). However, neutrophils were retained within into young C57BL/6 CD45.1+ recipients. Four days after trans- the lungs of the advanced aged mice but not in the young in- fer, we found similar numbers of young and advanced aged AM fected mice (Fig. 5D). Additionally, by the end of influenza in- in the lungs of young recipient mice (Fig. 4B), indicating that fection advanced aged mice exhibited 2-fold higher MPO levels, a aging did not impair the cell-intrinsic ability of AM to engraft marker of neutrophil activation, than did young mice (Fig. 5E). into the lung. Finally, we measured MPO levels in the BAL of clodronate- The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. Aging impairs the ability of AM to bind and internalize particles in vivo. (A and B) Young and advanced aged mice were instilled with 3 3 106 apoptotic neutrophils i.n. The lung tissue was subsequently harvested to obtain a cellular suspension, which was analyzed via flow cytometry. Representative flow cytometry plots in young and advanced aged mice are shown. Samples are gated on AM as per Fig. 1. (B) Quantification of data (t test). *p , 0.05. (C) Young and advanced aged mice were instilled with Alexa Fluor 488 fluorescent beads i.n. Lung tissue and gating as per (A). **p , 0.01. (B and C) Pooled data from two independent experiments. (D and E) Young and advanced aged mice were infected with influenza virus as per Fig. 1; by guest on September 28, 2021 neutrophils were quantified (via enumeration via cytospin) (D) and MPO level in BAL via ELISA (E). (F) Advanced aged mice were infected with 1 3 103 PFU of influenza virus. They were then administered clodronate liposomes or control liposomes as outlined in Fig. 2B. At day 6 postinfection, BAL was obtained and MPO levels were measured. *p , 0.05, **p , 0.01, **** p # 0.0001. treated advanced aged mice infected with 1 3 103 PFU of virus, Discussion a dose that we found accelerates mortality during influenza viral Our study has demonstrated that aging increases morbidity and infection (Fig. 2B). As compared with control treated and in- mortality during influenza viral lung infection in mice. These results fected advanced aged mice, clodronate-treated and infected are compatible with clinical studies that show that as people age they advanced aged mice exhibited a significant, 2-fold increase in exhibit increasing morbidity and mortality during influenza viral MPO levels (Fig. 5F). These data link AM to increased neu- infection (1–3). Our results are also consistent with two prior studies trophil activation within the lungs during influenza infection in aged mice (6, 7) but sharply contrast with a recent study that with aging. Overall, these data demonstrate that with aging AM found in a small cohort of mice (n = 5 per group) that mice aged exhibit a defect phagocytosing apoptotic neutrophils. The data .24 mo exhibited a significant longer survival during influenza viral also show that during influenza viral infection with aging, neutro- lung infection than did young mice (10). In our study, we employed phils are retained in the lung, which could contribute to neutrophil dose-titrating aliquots of virus, and demonstrated that advanced aged activation and lung damage. mice exhibit an increased susceptibility to influenza viral lung in- fection than that in young mice. Thus, our study resolves a recent Aging impairs the upregulation of CD204, a scavenging receptor controversy concerning the impact of aging on the susceptibility to Owing to the phagocytosis defect in AM with aging, we next influenza lung infection in mice (4, 6, 7, 9, 10). determined the cellular pathway by which aging impairs scav- Although prior studies have identified that signaling pathways enging of debris. For this purpose, we assessed the expression of (e.g., inflammasomes) (6) and altered inflammatory mediators scavenging receptors on AM from young and advanced aged (e.g., PGD2) contribute to age-enhanced mortality during influ- mice. Under basal conditions, we found that AM from advanced enza viral lung infection (7), the critical cells that enhance mor- aged mice exhibit a significant reduction in the scavenging tality to influenza viral lung infection with aging remain to be receptor CD204 (also known as macrophage scavenging receptor-A) elucidated. To identify the critical cell for age-enhanced mortality (24) compared with AM from young mice (Fig. 6A). However, during influenza viral infection, we identified AM as critical for expressions of CD44, CD36, Axl, and CD206 were not different mortality with aging. We documented that with aging AM exhibit between the groups (Fig. 6B–E), indicating that aging selec- an impaired ability to limit lung damage during influenza viral tively impairs the expression of a key phagocytic receptor CD204 infection. AM are known to maintain lung homeostasis by clear- on AM. ing debris (11, 25). Prior in vitro studies have found that with 8 AGING AND INFLUENZA VIRAL INFECTION Downloaded from

FIGURE 6. Aging downregulates CD204 on AM. (A–E) AM from young and advanced aged mice were stained with indicated fluo- rescently tagged mAbs and data were acquired via flow cytometry. Fluorescent intensity level of each surface scavenger receptor shown as http://www.jimmunol.org/ change in median fluorescent intensity (MFI, arbitrary units) from that obtained from iso- type control staining is shown as a histogram. Adjacent figure: representative flow plots: y-axis, percentage of maximum; x-axis, fluorescence. *p , 0.05. FMO, fluorescence minus one control. by guest on September 28, 2021

aging, AM exhibit reduced inflammatory responses to either neutrophil retention than do young infected mice. As excessive Streptococcus pneumoniae or RSV (26, 27). Our study has defined neutrophil numbers increase lung damage during influenza viral the impact of aging on AM function in vivo. Specifically, we show, infection (23), our study indicates that the retention of neutro- via adoptive transfer, that aging impairs the ability of AM to limit phils in the lung with aging contributes to increase lung damage. lung damage without altering viral clearance during influenza viral We also determined that aging selectively downregulates CD204, a infection. key scavenging receptor that has been found to be critical for To identify a cellular mechanism by which aging impairs the binding and internalization of apoptotic cells in vitro (28). ability of AM to limit lung damage during influenza viral in- CD204 is also important for limiting noninfectious lung damage fection, we documented that aging impairs the ability of AM to (29), host defense to systemic herpes viral infection (30), and bind and internalize apoptotic neutrophils and found that during for bacterial pneumonia (31). Why aging selectively down- influenza viral infection, advanced aged mice display more regulates this receptor is not clear from our study. Possibilities The Journal of Immunology 9 include that the basal inflammatory milieu is increased in the 7. Zhao, J., J. Zhao, K. Legge, and S. Perlman. 2011. Age-related increases in aging lung and that this increased inflammatory response may PGD2 expression impair respiratory DC migration, resulting in diminished T cell responses upon respiratory virus infection in mice. J. Clin. Invest. 121: 4921– downregulate CD204 (24), given that aging and senescence are 4930. associated with an increased inflammation generally (32). 8. Jiang, J., E. M. Fisher, and D. M. Murasko. 2011. CD8 T cell responses to in- fluenza virus infection in aged mice. Ageing Res. Rev. 10: 422–427. Clearly, future studies are required to examine these possibilities 9. Po, J. L., E. M. Gardner, F. Anaraki, P. D. Katsikis, and D. M. Murasko. 2002. in more detail. Age-associated decrease in virus-specific CD8+ T lymphocytes during primary AM are not the only immune cells within the lung that exhibit influenza infection. Mech. Ageing Dev. 123: 1167–1181. 10. Pillai, P. S., R. D. Molony, K. Martinod, H. Dong, I. K. Pang, M. C. Tal, phagocytic functions. Dendritic cells and recruited monocytes also A. G. Solis, P. Bielecki, S. Mohanty, M. Trentalange, et al. 2016. Mx1 reveals act as . However, AM orchestrate the recruitment of innate pathways to antiviral resistance and lethal influenza disease. Science 352: inflammatory monocytes during RSV infection via the production 463–466. 11. Hussell, T., and T. J. Bell. 2014. Alveolar macrophages: plasticity in a tissue- of type I IFNs (16). AM may function in a similar fashion in specific context. Nat. Rev. Immunol. 14: 81–93. response to influenza viral infection. Also, the impaired viral 12. Ueki, I. F., G. Min-Oo, A. Kalinowski, E. Ballon-Landa, L. L. Lanier, control that we noted with aging is probably not due to a de- J. A. Nadel, and J. L. Koff. 2013. Respiratory virus-induced EGFR activation suppresses IRF1-dependent interferon l and antiviral defense in airway epithe- creased ability of aged AM to clear virus, as adoptive transfer of lium. J. Exp. Med. 210: 1929–1936. young donor AM into advanced aged mice did not enhance viral 13. Kopf, M., C. Schneider, and S. P. Nobs. 2015. The development and function of clearance as compared with advanced aged mice that received lung-resident macrophages and dendritic cells. Nat. Immunol. 16: 36–44. 14. Guilliams, M., I. De Kleer, S. Henri, S. Post, L. Vanhoutte, S. De Prijck, advanced aged donor AM. Moreover, defects in AM phagocytic K. Deswarte, B. Malissen, H. Hammad, and B. N. Lambrecht. 2013. Alveolar function with aging may have secondary effects on the clearance macrophages develop from fetal monocytes that differentiate into long-lived of apoptotic cells or other debris during respiratory viral infec- cells in the first week of life via GM-CSF. J. Exp. Med. 210: 1977–1992. Downloaded from 15. Kumagai, Y., O. Takeuchi, H. Kato, H. Kumar, K. Matsui, E. Morii, K. Aozasa, tions. It remains to be seen in future studies how aging impacts T. Kawai, and S. Akira. 2007. Alveolar macrophages are the primary interferon- both viral clearance and the influx of monocytes into the lung a producer in pulmonary infection with RNA viruses. Immunity 27: 240–252. 16. Goritzka, M., S. Makris, F. Kausar, L. R. Durant, C. Pereira, Y. Kumagai, during respiratory viral infection. F. J. Culley, M. Mack, S. Akira, and C. Johansson. 2015. Alveolar macrophage- We found that aging leads to a reduction in both the number and derived type I interferons orchestrate innate immunity to RSV through recruit- the proliferative capacity of AM prior to infection. Our gene ment of antiviral monocytes. J. Exp. Med. 212: 699–714. 17. Tate, M. D., D. L. Pickett, N. van Rooijen, A. G. Brooks, and P. C. Reading. expression studies revealed that aging downregulates several 2010. Critical role of airway macrophages in modulating disease severity during http://www.jimmunol.org/ pathways involved in cell cycle regulation, and it upregulates a influenza virus infection of mice. J. Virol. 84: 7569–7580. repressor of self-renewal, C-Maf. Emerging evidence has now 18. Hashimoto, D., A. Chow, C. Noizat, P. Teo, M. B. Beasley, M. Leboeuf, C. D. Becker, P. See, J. Price, D. Lucas, et al. 2013. Tissue-resident macrophages established that AM, similar to other tissue-resident macrophages, self-maintain locally throughout adult life with minimal contribution from cir- self-renew in situ rather than being replenished from infiltrating culating monocytes. Immunity 38: 792–804. monocytes (14, 18). Our results imply that with aging the self- 19. Aziz, A., E. Soucie, S. Sarrazin, and M. H. Sieweke. 2009. MafB/c-Maf defi- ciency enables self-renewal of differentiated functional macrophages. Science renewal capacity of AM decreases, leading to a reduced pop- 326: 867–871. ulation size. The reduced population size along with the intrinsic 20. Soucie, E. L., Z. Weng, L. Geirsdo´ttir, K. Molawi, J. Maurizio, R. Fenouil, alteration in phagocytosis that our study reveals could both con- N. Mossadegh-Keller, G. Gimenez, L. VanHille, M. Beniazza, et al. 2016. Lineage-specific enhancers activate self-renewal genes in macrophages and by guest on September 28, 2021 tribute to enhanced mortality with aging during influenza viral embryonic stem cells. Science 351: aad5510. infection. 21. Serhan, C. N., N. Chiang, J. Dalli, and B. D. Levy. 2014. Lipid mediators in the In conclusion, our study has revealed a novel mechanism by which resolution of inflammation. Cold Spring Harb. Perspect. Biol. 7: a016311. 22. Higashimoto, Y., Y. Fukuchi, Y. Shimada, K. Ishida, M. Ohata, T. Furuse, C. Shu, aging impairs phagocytosis by AM of apoptotic neutrophils to S. Teramoto, T. Matsuse, E. Sudo, and H. Orimo. 1993. The effects of aging on contribute to an increased morbidity during influenza viral infection. the function of alveolar macrophages in mice. Mech. Ageing Dev. 69: 207–217. Future therapeutics aimed at improving the function of AM may 23. Narasaraju, T., E. Yang, R. P. Samy, H. H. Ng, W. P. Poh, A. A. Liew, M. C. Phoon, N. van Rooijen, and V. T. Chow. 2011. Excessive neutrophils and improve outcomes in older people infected with respiratory viruses. neutrophil extracellular traps contribute to acute lung injury of influenza pneu- monitis. Am. J. Pathol. 179: 199–210. 24. Kelley, J. L., T. R. Ozment, C. Li, J. B. Schweitzer, and D. L. Williams. 2014. Acknowledgments Scavenger receptor-A (CD204): a two-edged sword in health and disease. Crit. Rev. Immunol. 34: 241–261. We thank Dr. Bethany Moore and Dr. Jeffrey Curtis (both University 25. Litvack, M. L., T. J. Wigle, J. Lee, J. Wang, C. Ackerley, E. Grunebaum, and of Michigan) for their careful critique of the manuscript. M. Post. 2016. Alveolar-like stem cell-derived Myb2 macrophages promote recovery and survival in airway disease. Am. J. Respir. Crit. Care Med. 193: 1219–1229. Disclosures 26. Boyd, A. R., P. Shivshankar, S. Jiang, M. T. Berton, and C. J. Orihuela. 2012. Age-related defects in TLR2 signaling diminish the cytokine response by al- The authors have no financial conflicts of interest. veolar macrophages during murine pneumococcal pneumonia. Exp. Gerontol. 47: 507–518. 27. Wong, T. M., S. Boyapalle, V. Sampayo, H. D. Nguyen, R. Bedi, S. G. Kamath, References M. L. Moore, S. Mohapatra, and S. S. Mohapatra. 2014. Respiratory syncytial 1. Thompson, W. W., D. K. Shay, E. Weintraub, L. Brammer, C. B. Bridges, virus (RSV) infection in elderly mice results in altered antiviral gene expression N. J. Cox, and K. Fukuda. 2004. Influenza-associated hospitalizations in the and enhanced pathology. PLoS One 9: e88764. United States. JAMA 292: 1333–1340. 28. Todt, J. C., B. Hu, and J. L. Curtis. 2008. The scavenger receptor SR-A I/II 2. Thompson, W. W., D. K. Shay, E. Weintraub, L. Brammer, N. Cox, (CD204) signals via the receptor tyrosine kinase Mertk during apoptotic cell L. J. Anderson, and K. Fukuda. 2003. Mortality associated with influenza and uptake by murine macrophages. J. Leukoc. Biol. 84: 510–518. respiratory syncytial virus in the United States. JAMA 289: 179–186. 29. Arredouani, M. S., F. Franco, A. Imrich, A. Fedulov, X. Lu, D. Perkins, 3. Fry, A. M., D. K. Shay, R. C. Holman, A. T. Curns, and L. J. Anderson. 2005. R. Soininen, K. Tryggvason, S. D. Shapiro, and L. Kobzik. 2007. Scavenger Trends in hospitalizations for pneumonia among persons aged 65 years or older receptors SR-AI/II and MARCO limit pulmonary migration and in the United States, 1988–2002. JAMA 294: 2712–2719. allergic airway inflammation. J. Immunol. 178: 5912–5920. 4. Toapanta, F. R., and T. M. Ross. 2009. Impaired immune responses in the lungs 30. Suzuki, H., Y. Kurihara, M. Takeya, N. Kamada,M.Kataoka,K.Jishage,O.Ueda, of aged mice following influenza infection. Respir. Res. 10: 112. H. Sakaguchi, T. Higashi, T. Suzuki, et al. 1997. A role for macrophage scavenger 5. Nogusa, S., B. W. Ritz, S. H. Kassim, S. R. Jennings, and E. M. Gardner. 2008. receptors in atherosclerosis and susceptibility to infection. Nature 386: 292–296. Characterization of age-related changes in natural killer cells during primary 31. Arredouani, M. S., Z. Yang, A. Imrich, Y. Ning, G. Qin, and L. Kobzik. 2006. influenza infection in mice. Mech. Ageing Dev. 129: 223–230. The macrophage scavenger receptor SR-AI/II and lung defense against pneu- 6. Stout-Delgado, H. W., S. E. Vaughan, A. C. Shirali, R. J. Jaramillo, and mococci and particles. Am. J. Respir. Cell Mol. Biol. 35: 474–478. K. S. Harrod. 2012. Impaired NLRP3 inflammasome function in elderly mice 32. Tchkonia, T., Y. Zhu, J. van Deursen, J. Campisi, and J. L. Kirkland. 2013. during influenza infection is rescued by treatment with nigericin. J. Immunol. Cellular senescence and the senescent secretory phenotype: therapeutic oppor- 188: 2815–2824. tunities. J. Clin. Invest. 123: 966–972. Supplemental Table 2 Top 10 enriched pathways of down-regulated genes in

AM with aging at baseline. Note, all pathways listed reached p < 1x10-3 and

FDR< 0.05%

Rank Pathway Associated Network Objects MAD1 (mitotic checkpoint), CENP-B, Survivin, CENP-A, SPBC24, CDC20, MAD2a, CENP-H, BUBR1, INCENP, 1 Cell cycle_The metaphase checkpoint PMF1, Rod, DSN1, Aurora-B, BUB1, Aurora-A, PLK1, CDCA1, CENP-F, Zwilch, NSL1, AF15q14 Cyclin H, Wee1, Cyclin B2, CDC25C, CAK complex, FOXM1, Kinase MYT1, Lamin B, 2 Cell cycle_Initiation of mitosis KNSL1, AKT(PKB), Cyclin B1, USF1, c- Myc, PLK1, MAT1, Histone H1, CDK1 (p34), CDK7 CDC18L (CDC6), Cyclin B, CKS1, CDC20, MAD2a, BUBR1, CDH1, Tome-1, Cyclin A,

3 Cell cycle_Role of APC in cell cycle regulation Aurora-B, Kid, CDC25A, BUB1, Geminin, Emi1, Aurora-A, PLK1, CDK1 (p34), CDK2 CRM1, Cyclin D3, CDK4, Cyclin E, Cyclin D, Importin (karyopherin)-alpha, Cyclin A, 4 Cell cycle_Nucleocytoplasmic transport of CDK/Cyclins Cyclin B1, ERK1 (MAPK3), CDK1 (p34), CDK2 Ubiquitin, Chk1, UBE1, Wee1, CKS1, CDK4, Cyclin E, NEDD8, CDC25A, RING- 5 Cell cycle_Role of SCF complex in cell cycle regulation box protein 1, Cdt1, Emi1, PLK1, CDK1 (p34), CDK2 Cyclin B, INCENP, CAP-H/H2, Cyclin A, CNAP1, CAP-D2/D3, Aurora-B, TOP2, 6 Cell cycle_Chromosome condensation in prometaphase BRRN1, Aurora-A, Histone H1, CDK1 (p34) CDC18L (CDC6), MCM3, MCM5, RPA3, MCM4, Cyclin E, MCM10, ORC6L, RPA1, 7 Cell cycle_Start of DNA replication in early S phase MCM4/6/7 complex, MCM2, Cdt1, Geminin, Histone H1, CDK2 Ubiquitin, MAD1 (mitotic checkpoint), Tubulin alpha, Cyclin B, CDC20, MAD2a, 8 Cell cycle_Spindle assembly and chromosome separation Importin (karyopherin)-alpha, KNSL1, Aurora-B, Kid, TPX2, Aurora-A, Ran, CDK1 (p34), Tubulin (in microtubules) BAD, MEK1(MAP2K1), Bax, Calcineurin A (catalytic), H-Ras, PP1-cat alpha, ERK1/2, 14-3-3, IGF-1 receptor, G-protein 9 Apoptosis and survival_BAD phosphorylation beta/gamma, SOS, PP2C, AKT(PKB), Cytochrome c, p70 S6 kinase2, CDK1 (p34) Ubiquitin, PCNA, Brca1, TOP2 alpha, Ribonuclease H1, Cyclin A, Brca1/Bard1, 10 Cell cycle_Transition and termination of DNA replication MCM2, TOP2, POLD cat (p125), Bard1, CDK1 (p34), CDK2

38 Supplemental Table 3 Top 10 enriched pathways of up-regulated genes in AM

with aging. Note, all pathways listed reached p < 1x10-3 and FDR< 0.05%

Rank Pathway Associated Network Objects CCL3L1, GRO-2, MIP-1-beta, I-kB, CCL13, Immune response_Substance P-stimulated expression 1 AKT(PKB), CCL2, PKC-alpha, PDK

of proinflammatory cytokines via MAPKs (PDPK1), GRO-3, p38 MAPK, IP3 receptor COX-1 (PTGS1), Amphiregulin, GSK3 beta, NURR1, G-protein alpha-s, 2 PGE2 pathways in cancer AKT(PKB), Beta-arrestin1, PDK (PDPK1), HIF1A, Tcf(Lef), Adenylate cyclase, PKA- cat (cAMP-dependent), TCF7L2 (TCF4) GSK3 alpha/beta, IL-1 beta, RAP-1A, MIP- PDE4 regulation of cyto/chemokine expression in 1-beta, I-kB, CBP, AKT(PKB), CUX1, 3 arthritis CCL2, PDE4, Rap1GAP1, PKA-cat (cAMP- dependent) PKC-beta, AKT(PKB), IL8RA, PKC-alpha, Oxidative stress_Role of IL-8 signaling pathway in 4 p22-phox, p38 MAPK, PKC-beta2, p47- phox, IP3 receptor, IL8RB HMGI/Y, CBP, cPKC (conventional), MHC Immune response_MIF - the neuroendocrine- class II, CD74, G-protein alpha-s, PKC, 5 macrophage connector PLA2, PKA-cat (cAMP-dependent), IP3 receptor I-kB, PKC-beta, COX-1 (PTGS1), GSK3 beta, PKC, AKT(PKB), HSP90, CCL2, Development_VEGF signaling via VEGFR2 - generic 6 Vinculin, PKC-alpha, PDK (PDPK1), p38 cascades MAPK, PLAU (UPA), TCF7L2 (TCF4), IP3 receptor, FAK1 STAT3, IL-1 beta, PTAFR, AKT(PKB), Beta-arrestin1, CCL2, Calcineurin B Immune response_Platelet activating factor/ PTAFR 7 (regulatory), p38 MAPK, Adenylate cyclase, pathway signaling PKA-cat (cAMP-dependent), NFKBIA, IP3 receptor CBP, HSP90, SUMO-1, HSP70, E2I, PAI1, 8 Development_Glucocorticoid receptor signaling NFKBIA, TGF-beta receptor type I Acid sphingomyelinase, CYLD, HSP90 Apoptosis and survival_TNF-alpha-induced Caspase-8 9 alpha, AKT1, ErbB2, AKT(PKB), HSP90, c- signaling Cbl, ADAM17, Cathepsin B Talin, PIPKI gamma, GSK3 beta, Filamin A, eIF4G1/3, Collagen IV, PTEN, Vinculin, 10 Cytoskeleton remodeling_Cytoskeleton remodeling eIF4G1, Tcf(Lef), eIF4G2, p38 MAPK, PAI1, eIF4G3, PLAU (UPA), eIF4A, DOCK1, TGF-beta receptor type I, FAK1

39