A in Human Platelets Regulates the Synthesis of Proinflammatory Cytokines by Monocytes in Aging

This information is current as Robert A. Campbell, Zechariah Franks, Anish Bhatnagar, of November 27, 2017. Jesse W. Rowley, Bhanu K. Manne, Mark A. Supiano, Hansjorg Schwertz, Andrew S. Weyrich and Matthew T. Rondina J Immunol published online 22 November 2017

http://www.jimmunol.org/content/early/2017/11/22/jimmun Downloaded from ol.1700885

Supplementary http://www.jimmunol.org/content/suppl/2017/11/22/jimmunol.170088 Material 5.DCSupplemental http://www.jimmunol.org/

Why The JI?

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on November 27, 2017 • Speedy Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 November 22, 2017, doi:10.4049/jimmunol.1700885 The Journal of Immunology

Granzyme A in Human Platelets Regulates the Synthesis of Proinflammatory Cytokines by Monocytes in Aging

Robert A. Campbell,*,† Zechariah Franks,* Anish Bhatnagar,* Jesse W. Rowley,*,‡ Bhanu K. Manne,* Mark A. Supiano,x,{ Hansjorg Schwertz,*,‖ Andrew S. Weyrich,*,‡ and Matthew T. Rondina*,†,x

Dysregulated inflammation is implicated in the pathobiology of aging, yet platelet–leukocyte interactions and downstream cytokine synthesis in aging remains poorly understood. Platelets and monocytes were isolated from healthy younger (age <45, n = 37) and older (age ‡65, n = 30) adults and incubated together under autologous and nonautologous conditions. Synthesis of inflammatory cytokines by monocytes, alone or in the presence of platelets, was examined. Next-generation RNA-sequencing allowed for unbiased profiling of the platelet transcriptome in aging. Basal IL-8 and MCP-1 synthesis by monocytes alone did not differ between older and younger adults. However, in the presence of autologous platelets, monocytes from older adults synthesized Downloaded from greater IL-8 (41 6 5 versus 9 6 2 ng/ml, p < 0.0001) and MCP-1 (867 6 150 versus 216 6 36 ng/ml, p < 0.0001) than younger adults. Platelets from older adults were sufficient for upregulating the synthesis of inflammatory cytokines by monocytes. Using RNA-sequencing of platelets followed by validation via RT-PCR and immunoblot, we discovered that (GrmA), a not previously identified in human platelets, increases with aging (∼9-fold versus younger adults, p < 0.05) and governs increased IL-8 and MCP-1 synthesis through TLR4 and caspase-1. Inhibiting GrmA reduced excessive IL-8 and MCP-1 synthesis in aging to levels similar to younger adults. In summary, human aging is associated with changes in the platelet http://www.jimmunol.org/ transcriptome and proteome. GrmA is present and bioactive in human platelets, is higher in older adults, and controls the synthesis of inflammatory cytokines by monocytes. Alterations in the platelet molecular signature and signaling to monocytes may contribute to dysregulated inflammatory syndromes in older adults. The Journal of Immunology, 2018, 200: 000–000.

latelets are anucleate cells with long-established roles functions in aging are thought to contribute to this heightened central to hemostasis initiation and vascular wall repair. thrombosis risk (6), but remain under studied. Thrombosis and Initially thought to be merely circulating cell fragments inflammation are centrally linked and injurious inflammation is P by guest on November 27, 2017 with a relatively fixed repertoire of functional responses, platelets central to the pathobiology of aging. For example, aging is asso- are increasingly recognized to be versatile effector cells that bridge ciated with elevated levels of IL-6, IL-8, and C-reactive protein thrombotic, inflammatory, and immune continuums (1, 2). Activated (7, 8). IL-6 has been implicated in mediating thrombosis during platelets stably adhere to and tether monocytes via P-selectin/P-selectin systemic inflammatory insults (9, 10). Increased levels of IL-6, IL-8, glycoprotein ligand 1 (PSGL-1) and, in parallel, secrete RANTES and MCP-1 during aging may contribute to adverse outcomes in older from platelet a granules. RANTES then binds to CCL5 on monocytes, adults (11–13). driving downstream synthesis of proinflammatory products by Whereas classic platelet hemostatic functions have been examined monocytes (3, 4). in aging, age-associated alterations in the platelet transcriptome and Thromboembolic events remain the most common cause of proteome and their effects on platelet–monocyte signaling events morbidity and mortality in older adults (5) and dysregulated platelet have not previously been examined. In this study, we examined

*Molecular Medicine Program, University of Utah, Salt Lake City, UT 84112; Award Number UL1TR001067. This work was also supported by the University of †Division of General Internal Medicine, Department of Internal Medicine, School Utah Flow Cytometry Facility in addition to the National Cancer Institute through of Medicine, University of Utah, Salt Lake City, UT 84132; ‡Division of Respiratory, Award Number 5P30CA042014-24. The contents of this article are the responsibility Critical Care, and Occupational Pulmonary Medicine, University of Utah, Salt Lake of the authors and do not necessarily represent the official views of the U.S. Depart- City, UT 84132; xGeorge E. Wahlen Veterans Affairs Medical Center, Geriatric ment of Veterans Affairs, the U.S. Government, or the National institutes of Health. Research, Education and Clinical Center, Salt Lake City, UT 84148; {Division of The sequences presented in this article have been submitted to Geriatrics, School of Medicine, University of Utah, Salt Lake City, UT 84132; and ‖ Omnibus under accession number SRA2779748. Fastq files have been submitted to Division of Vascular Surgery, School of Medicine, University of Utah, Salt Lake the Sequence Read Archive under accession numbers SRR5907423–SRR5907428. City, UT 84132 Address correspondence and reprint requests to Dr. Matthew T. Rondina, Molecular ORCIDs: 0000-0003-0027-694X (R.A.C.); 0000-0001-7078-6959 (A.B.); 0000- Medicine Program, University of Utah Health Sciences Center, Eccles Institute of 0001-6848-4484 (B.K.M.); 0000-0003-3455-3730 (M.T.R.). Human Genetics, Building 533, Room 4220A, 15 North 2030 East, Salt Lake City, Received for publication June 16, 2017. Accepted for publication October 30, 2017. UT 84112. E-mail address: [email protected] This work was supported by National Heart, Lung, and Blood Institute Grants The online version of this article contains supplemental material. HL112311, HL130541, HL126547 (all to A.S.W. and M.T.R.), and HL066277 (to Abbreviations used in this article: FPKM, fragment per kilobase of transcript per A.S.W.), National Institute on Aging Grant AG048022 (to M.T.R.), National Institute million mapped reads; GrmA, granzyme A; HSA, human serum albumin; PF4, plate- of Diabetes and Digestive and Kidney Diseases Grant GM103806 (to J.W.R.), and a let factor 4; PMA, platelet–monocyte aggregation; PSGL-1, P-selectin glycoprotein pilot award funded by the University of Utah Center on Aging (to M.T.R.). This ligand 1; qRT-PCR, quantitative RT-PCR; rhGrmA, recombinant human GrmA; material is the result of work supported with resources and the use of facilities at the RNA-seq, RNA-sequencing. George E. Wahlen Veterans Affairs Medical Center, Salt Lake City, UT. The research reported in this publication was supported (in part or in full) by the National Center Ó for Advancing Translational Sciences of the National Institutes of Health under Copyright 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700885 2 PLATELET GRANZYME A REGULATES CYTOKINE SYNTHESIS whether the platelet molecular signature was altered in older adults evaluated by flow cytometry (13, 18–20). All Abs were from BD Biosci- and dissected a previously unrecognized mechanism whereby platelet– ences. Isolated, unstimulated monocytes were costained for CD14 FITC monocyte interactions drive excessive inflammation in aging. and CD16 PE with appropriate isotype controls. Platelet surface P-selectin expression and the formation of PMAs were evaluated as before (13, 18–20). Briefly, whole blood was left alone (baseline) or stimulated with Materials and Methods the PAR1 agonist SFLLRN for 10 min at 37˚C (5 mM for P-selectin and Human subjects 15 mM for PMAs). Whole blood was then incubated with Abs against P-selectin (CD62p), CD14 FITC, or CD41 PE for 20 min at room tem- The University of Utah Institutional Review Board approved this study perature. Samples were then immediately fixed and run using a FACScan (# 00051506) and all subjects provided informed consent. Healthy younger (BD Biosciences) with appropriate isotype controls. Samples were ana- , (aged 45 y) and older (aged $65 y) adults were eligible for participation lyzed using FlowJo v9 (TreeStar, OR). For whole blood platelet activation and recruited through advertising flyers approved by the Institutional Re- studies, platelets were gated by forward and side scatter using a loga- view Board. All study procedures and data capture were done in accor- rithmic scale followed by specific platelet Ag staining compared with dance with ethical regulations and patient data were anonymized. The age isotype control. For monocyte studies, cells were gated by forward and cut-off for older adults was based on commonly accepted definitions of side scatter using a linear scale followed by specific monocyte Ag staining aging, and the age cut-off for younger adults was chosen to give sufficient compared with isotype controls. A gating strategy for measuring the per- age separation between groups. Age cut-offs were all chosen a priori. centage of platelets expressing surface P-selectin (CD62p) in whole blood Subjects were excluded from the study if they were pregnant (self- is shown in Supplemental Fig. 1C–E. reported), had received a blood transfusion within the last 30 d, or had a history of cardiopulmonary disease (including myocardial infarction, ar- Platelet and monocyte incubations rhythmia, chronic obstructive pulmonary disease, or asthma), infection 6 8 within the past 30 d, inherited platelet disorder, cancer (whether active or Monocytes (2 3 10 cells per ml, final concentration) and platelets (2 3 10 cells per ml, final concentration) were incubated either separately or together, in remission), venous or arterial thromboembolic disease, liver or renal Downloaded from disease, or diabetes. Subjects taking clopidogrel, dipyridamole, selective depending on experimental conditions, at 37˚C for 18 h, as before (3). serotonin reuptake inhibitors, and phosphodiesterase inhibitors at any dose Cell-free supernatants were then harvested by centrifugation. In select or frequency were excluded. Subjects refrained from taking nonsteroidal experiments, human recombinant granzyme A (GrmA) (R&D Systems, anti-inflammatory drugs for 4 wk prior to study participation. Aspirin was Minneapolis, MN) was added (100 nM, final concentration) to incubating not an exclusionary criterion as many older adults take aspirin for the cells. Platelets were preincubated with an anti-GrmA Ab (R&D Systems) or prevention of cardiovascular disease and stroke. When approved by sub- control IgG (10 mg/ml, final concentration) that specifically blocks GrmA for jects’ medical providers, aspirin was temporarily discontinued for 4 wk 1 h, before the addition of monocytes. To identify the cognate receptors prior to study participation. regulating cytokine synthesis, monocytes were preincubated with the specific http://www.jimmunol.org/ TLR4 inhibitor, CLI-095 (1 mM, final concentration; InvivoGen, San Diego, Platelet and monocyte isolation CA) or a specific WEHD-FMK caspase-1 inhibitor (1 mM, final concen- tration; R&D Systems) for 1 h. Human peripheral venous blood was drawn into acid citrate dextrose (1.4 ml per 8.6 ml blood) through standard venipuncture technique and used immediately Chemokine and cytokine protein expression upon collection. The whole blood was first centrifuged at 150 3 g for 20 min at 20˚C to separate platelet-rich plasma from RBCs and WBCs. From the Platelet chemokines (e.g., P-selectin, RANTES, platelet factor 4 (PF4)] and platelet-rich plasma, platelets were leukocyte reduced and isolated as pre- monocyte cytokines (IL-6, IL-8, and MCP-1) were measured in harvested viously described (14–17) to yield a highly purified population of cells. supernatants by commercially available ELISA (R&D Systems) per the Isolated platelets were resuspended in serum-free M199 (Lonza, Walkersville, manufacturer’s instructions. For determination of chemokines in cell ly- 8 MD) medium in round-bottom polypropylene tubes (Becton Dickinson, sates, platelets (2 3 10 cells per ml, final concentration) were lysed in by guest on November 27, 2017 Franklin Lakes, NJ). The purity of isolated platelets was confirmed by flow radioimmunoprecipitation assay buffer. P-selectin, RANTES, and PF4 cytometry and direct counting via a hemocytometer, where ,3 leukocytes were measured by ELISA (R&D Systems) per the manufacturer’s in- were observed in each preparation containing 1 3 107 platelets (.99.9% structions. purity, Supplemental Fig. 1A). The number of leukocytes in each preparation did not vary by age (data not shown). Next-generation RNA-sequencing and RNA expression For the isolation of monocytes, the RBC/WBC mixture was resuspended Highly purified platelets were isolated as described above. For next- with 0.9% sterile saline back to the original volume and layered over an equal generation RNA-sequencing (RNA-seq), isolated platelets were carefully volume of Ficoll-Paque Plus (GE Healthcare Biosciences, Piscataway, NJ). lysed in Trizol, and DNAse-treated total RNA was isolated, as previously The layered cells were then centrifuged for 30 min at 400 3 g at 20˚C. After described (14, 16, 21–23). An Agilent bioanalyzer was used for quality 30 min, the mononuclear leukocyte layer was removed and washed with control and to quantitate RNA. RNA Integrity Number scores were similar HBSS (Sigma-Aldrich, St. Louis, MO) with 1% human serum albumin between all samples (data not shown). RNA-seq libraries were prepared (HSA; University of Utah Hospital, Salt Lake City, UT), and centrifuged for with TruSeq V2 with oligo-dT selection (Illumina, San Diego, CA). Reads 3 g 10 min at 400 at 20˚C. The cell pellet was then resuspended in HSA and were aligned (Novoalign) to the reference genome GRCh37/hg19 and a CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were pseudo-transcriptome containing splice junctions. The Deseq2 analysis added for 15 min at 4˚C. The cells were then washed with HSA to remove package was used to assign reads to composite transcripts (one per gene) any free CD14 microbeads and then resuspended in HSA. The monocytes and quantitate fragments per kilobase of transcript per million mapped were then isolated by running the suspended cells through an autoMACs reads (FPKMs) as previously described (24). The expression of candi- cell separator (Miltenyi Biotec) using the PosselD2 program. The date transcripts identified by RNA-seq was further examined using quan- monocyteswerewashedwithHSAandresuspendedinM199(Bio- titative RT-PCR (qRT-PCR). Forward and reverse primers were as Whitaker, Walkersville, MD) and counted. The purity of monocytes was . follows, respectively: 1) GrmA:59-CATTGATTGATGTGGGGACA-39, confirmed to be 99.4% by flow cytometry (Supplemental Fig. 1B). 59-TCTGGGATTTCTGGTTCAGG-39;2)GrmH:59-GCCTTCCTGA- Flow cytometry GAAAATGCAG-39,59-GAGCAGCTGTCAGCACAAAG-39;3)GrmM: 59-AGCTGGACGGGAAAGTGAAG-39,59-CCAGAAGCGGCTGTTGT- The expression of monocyte surface markers, platelet surface adhesion TAC-39;4)Granulysin:59-GATGAGGCTGCTGAAAGGTC-39,59-GTG- molecules (e.g., P-selectin), and platelet–monocyte aggregation (PMA) was GAGGGAGTTTGGTGAGA-39.

Table I. Characteristics of study cohort

Young (n = 37) Old (n = 30) p Value Age, y 27.1 6 7.2 71.9 6 6.1 p , 0.001 Males, n (%) 17 (46%) 13 (43%) p = 0.85 Body mass index, kg/m2 23.7 6 4.0 28.7 6 5.5 p , 0.001 Aspirin use, n (%) 1 (3%) 10 (34%) p , 0.05 Data represent the mean 6 SD, unless otherwise indicated. The Journal of Immunology 3

layered onto Vectabond (Vector Laboratories, Burlingame, CA)-coated coverslips using a cytospin centrifuge (Shandon Cytospin; Thermo Fisher Scientific, Waltham, MA). Fluorescence microscopy was performed using an Olympus IX81, FV300 (Olympus, Melville, NY) confocal- scanning microscope equipped with a 603/1.42 NA oil objective for viewing platelets. An Olympus FVS-PSU/IX2-UCB camera and scanning unit and Olympus Fluoview FV 300 image acquisition software version 5.0 were used for recording. Protein expression studies All samples were normalized for starting cell concentrations. The samples were centrifuged at 13,000 3 g for 4 min and then resuspended in 0.5 ml of deionized water. The protein was pelleted by centrifugation at 13,000 3 g for 4 min. The protein pellets were solubilized in sample buffer containing 0.1 M Tris, 2% SDS, 1% (v/v) glycerol, 0.1% bromophenol blue, and 100 mM DTT then boiled for 10 min. Proteins were resolved by SDS polyacrylamide gels and transferred to nitrocellulose membrane (Whatman Protran). Membranes were blocked with Odyssey blocking buffer for 1 h at ambient temperature, incubated overnight at 4˚C with the desired pri- mary Ab for GrmA (Santa Cruz Biotechnology), and then washed four times with TBST. Membranes were then incubated with appropriate secondary infrared dye-labeled Ab for 60 min at room temperature and washed four times with TBST. Membranes were examined and quantified Downloaded from with a Li-Cor Odyssey infrared imaging system. In select experiments, platelets were stimulated with for the appropriate time under stirring conditions at 37˚C, and the reaction was stopped by the addition of 0.6 NHClO4. Statistical methods

For RNA-seq analysis, Deseq2 was used to identify differentially http://www.jimmunol.org/ expressed transcripts, as we have previously described (24). To focus on robustly expressed transcripts, only transcripts with per-group average .3 FPKM were included in the analysis. RNAs .2-fold differentially FIGURE 1. Aged platelets enhance IL-8 and MCP-1 synthesis. Mono- expressed between older and younger adults and with a nominal p value cytes and platelets were isolated from younger (age ,45 y, n = 27) or older ,0.05 (for discovery) were included in the analysis. To make these data adults (age $65 y, n = 27). (A and B) Monocytes alone were incubated in publicly available, a complete dataset of the RNA-seq studies was the presence or absence of thrombin (IIa, 0.1 U/ml) for 18 h. IL-8 and uploaded to Gene Expression Omnibus (SRA2779748). For relevant MCP-1 synthesis by monocytes alone did not significantly increase with studies, we calculated the mean 6 SEM and performed ANOVA to thrombin and was similar between younger and older adults. (C and D) identify differences among multiple experimental groups. If significant

Isolated monocytes were incubated with thrombin activated, autologous differences existed, a Student Newman–Keuls post hoc procedure was by guest on November 27, 2017 used to determine the location of the difference between groups. When platelets, or nonautologous (switch) platelets. IL-8 and MCP-1 synthesis single comparisons were performed, data were tested for normality and was significantly increased in the presence of platelets from older adults. In skewness and a Student t test or Wilcoxon rank sum was employed, comparison, in switch experiments, when monocytes from an older adult as appropriate. Statistical significance was set at a two-tailed p value were incubated with platelets from a younger adult, IL-8 and MCP-1 ,0.05. synthesis was significantly decreased. *p , 0.05. Results Immunocytochemistry Platelets from older adults enhance IL-8 and MCP-1 synthesis Freshly isolated platelets were fixed in suspension with paraformaldehyde (2% final concentration), placed in chamber slides, and subsequently in- Table I shows the clinical characteristics of the study cohort. The cubated with IgG or a specific Ab against GrmA (1:100 dilution, 60 min; two groups [younger (age ,45 y) and older (age $65 y)] were Santa Cruz Biotechnology, Dallas, TX). Fixed platelets were subsequently well balanced with regards to gender. Older adults had a higher

FIGURE 2. Aging does not alter monocyte CD14 or CD16 surface expression, platelet surface P-selectin expression, or the formation of platelet- monocyte aggregates. Whole blood was drawn from younger (age ,45 y) and older (age $65 y) subjects. Blood was stained for surface monocyte markers CD14 or CD16 [(A), n = 6 younger and n = 6 older], surface adhesion platelet P-selectin marker CD62p [(B), n = 23 younger and n =13 older], or platelet–monocyte aggregates as measured by double positivity for CD41 and CD14 [(C), n = 26 younger and n = 14 older]. Expression was adjusted for nonspecific staining by using appropriate isotype controls. In (B and C), whole blood was fixed immediately (baseline, BL) or activated with thrombin-receptor activating peptide (TRAP: 5 mM for CD41/CD14, 15 mM for P-selectin) prior to Ab staining and assessment by flow cytometry. *p , 0.05. 4 PLATELET GRANZYME A REGULATES CYTOKINE SYNTHESIS Downloaded from http://www.jimmunol.org/ by guest on November 27, 2017

FIGURE 3. Aging does not alter platelet expression or secretion of the adhesion molecule P-selectin, the signaling molecule RANTES, or the monocyte chemotactic molecule PF4. (A) Platelets were freshly isolated from younger (age ,45 y, n = 3) and older subjects (age $65 y, n = 3). Total RNA was isolated from platelets and the platelet transcriptome was assessed by next-generation RNA-seq. The total RNA expression of P-selectin, RANTES, and PF4 was examined in RNA-sequenced, highly purified platelet lysates. Shown are representative Integrative Genomics Viewer browser images of each transcript in platelets from a younger (top panels, blue) and older (bottom panels, red) adult with quantified transcript FKPM levels on the right (FPKM: n = 3 per group). The y-axis represents the relative expression of each mRNA, with higher peaks indicating increased expression. (B–D) To determine the protein expression, platelets were isolated from younger (age ,45 y, n = 11) and older subjects (age $65 y, n = 11). Platelets were either immediately lysed (baseline, BL) or activated with thrombin (IIa, 0.1 U/ml, t = 30 min). Platelet supernatants were harvested by centrifugation. Total P-selectin (B), RANTES (C), and PF4 (D) protein levels were measured in platelets lysates (for total protein) and in supernatants (Sups) by ELISA. body mass index and were taking aspirin more commonly (Table I). To determine whether platelets were the cellular drivers of the When monocytes were allowed to incubate alone (i.e., in the ab- increased synthesis of IL-8 and MCP-1 in older adults, we next sence of platelets), basal IL-8 and MCP-1 protein levels in the su- performed switch experiments. In these switch experiments, pernatant were negligible and did not differ between younger and monocytes from an older adult were coincubated with platelets older adults (Fig. 1A, 1B). In comparison, when monocytes were isolated from a gender-matched younger adult (e.g., nonautologous coincubated with autologous platelets (i.e., monocytes and platelets conditions). In parallel, monocytes from a younger adult were from the same subject), IL-8 and MCP-1 synthesis increased ro- coincubated with platelets from an older adult. In all these ex- bustly in both groups but was significantly higher (∼4-fold) in older periments, monocytes and platelets from younger or older adults adults compared with younger adults (Fig. 1C, 1D). In older sub- were isolated simultaneously and experiments performed on the jects, the synthesis of IL-8 and MCP-1 did not significantly differ same day in parallel. As shown in Fig. 1C and 1D, IL-8 and MCP-1 between aspirin users and nonusers (IL-8: 35.8 6 14.8 versus 24.9 synthesis by monocytes from older adults was rescued when 6 6.7 ng/ml, MCP-1: 1105 6 267 versus 710 6 309 ng/ml). We coincubated with platelets from younger adults. Conversely, IL-8 also observed increased IL-6 synthesis in older adults, when and MCP-1 synthesis by monocytes from younger adults was cocultured with autologous platelets, as compared with younger significantly enhanced when coincubated with platelets from older subjects, but the differences did not meet statistical significance adults. Synthesis of IL-8 by young monocytes was similar to that (Supplemental Fig. 2A). seen in older monocytes, when coincubated with aged platelets The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on November 27, 2017

FIGURE 4. GrmA is present in human platelets and its expression increases with aging. (A) Platelets were isolated from younger (n = 3) and older (n =3) adults. Total RNA was isolated from platelets and the platelet transcriptome was interrogated using next-generation RNA-seq. All transcripts identified by

RNA-seq were then filtered to identify only those that were differentially expressed (log2 fold-change $2). The heat map illustrates only those differentially expressed (log2 fold-change $2), upregulated (red), and downregulated (blue) transcripts that were identified in platelets from older adults as compared with younger adults. (B) Representative Integrative Genomics Viewer browser images of GrmA transcript expression in platelets isolated from a younger (top, blue) and older (bottom, red) adult. The y-axis represents the relative expression of GrmA, with higher peaks indicating increased total mRNA expression. On the bottom, the thick bars on the x-axis illustrate the exons and the 59 and 39 ends of the transcript are annotated (left and right, respectively). (C) GrmA expression was quantified by qRT-PCR in platelets isolated from older and younger adults. GrmA expression, but not GrmH, GrmM,or granulysin expression, was significantly increased in older adults. (D) Protein expression of GrmA was measured in isolated platelets (5 3 107 platelets equally loaded for each subject) by immunoblot and quantified by densitometry in older (n = 7) and younger (n = 7) adults. The left panel illustrates a representative immunoblot from n = 4 younger subjects and n = 3 older subjects with actin as a loading control in the bottom. The right panel shows normalized quantification of GrmA protein expression. *p , 0.05 versus younger adults.

(Fig. 1C). These results indicate that aged platelets are responsible We next examined whether aging was associated with changes in for triggering increased IL-8 and MCP-1 produced by monocytes the mRNA levels, intracellular expression, or secretion of proteins in older adults. required for platelet–monocyte interactions and downstream signal- dependent cytokine synthesis. In unstimulated platelets, there was The expression of cell-surface adhesion molecules and the no difference in the basal mRNA or intracellular protein expres- secretion of platelet chemokines that regulate IL-8 and MCP-1 sion of P-selectin and RANTES (required for platelet signaling to synthesis are not altered in aging monocytes and cytokine synthesis), or PF4 [a chemotactic factor Induction of IL-8 and MCP-1 synthesis by monocytes requires stable for monocytes (25)] between younger and older adults (Fig. 3). adhesion of platelets to monocytes, primarily through engagement of Similarly, the secretion of P-selectin, RANTES, or PF4 by acti- P-selectin on the platelet surface to PSGL-1 on monocytes (3). Thus, vated platelets was similar between younger and older adults we sought to determine whether the expression of monocyte and (Fig. 3). Together, these findings suggest that the increased IL-8 platelet surface adhesion or signaling molecules was increased in and MCP-1 synthesis by monocytes from older adults was not due our cohort of older adults. We did not identify any differences to enhanced expression of these platelet surface adhesion and in basal or activation-dependent expression of platelet surface signaling molecules. Accordingly, we turned our attention to P-selectin, platelet–monocyte aggregate formation, or the number heretofore unidentified proteins in human platelets with the ca- of monocytes positive for CD14+ or CD16+ between younger and pacity for regulating proinflammatory gene synthesis by mono- older adults (Fig. 2). cytes. 6 PLATELET GRANZYME A REGULATES CYTOKINE SYNTHESIS Downloaded from

FIGURE 5. GrmA is present in human platelets, secreted upon activation, and induces synthesis of IL-8 and MCP-1. (A) Platelets were isolated from an older adult (age $65) and stained with an anti-GrmA Ab (GrmA, magenta, right panels) or isotype IgG control (left panels). Platelets were costained with wheat germ agglutinin (WGA, green), which stains granules and membranes of platelets. The merged image and inset on the right illustrates the overlay of GrmA (yellow arrows) and WGA. Scale bar is shown on the lower left. Representative of n = 3 independent experiments. (B) Platelets were isolated from young (age ,45) or older adults (age $65) and either left alone or stimulated with thrombin (IIa, 0.1 U/ml; t = 30 min). The immunoblot shows that GrmA http://www.jimmunol.org/ is present at baseline in the platelet lysate and is secreted almost entirely into the platelet supernatant upon stimulation (WBC: WBC lysate, positive control). Levels of both intracellular and secreted GrmA were increased in older adults, as compared with younger adults. Actin is shown as a loading control (note that actin is absent from the supernatant, as expected). Representative of n = 3 independent experiments. (C and D) Platelets are necessary for GrmA-induced synthesis of IL-8 and MCP-1 by monocytes. Human monocytes and platelets were isolated as described in the Materials and Methods. Monocytes (2 3 106 cells per ml, final concentration) were left alone or incubated with rhGrmA (100 nM final, t = 18 h). Platelets (2 3 108 cells per ml, final concentration) were left alone or activated with thrombin (IIa, 0.1 U/ml, t = 30 min). Monocytes and platelets were also incubated together in the absence or presence of rhGrmA (100 nM final, t = 18 h). Supernatants were harvested by centrifugation. IL-8 (C) and MCP-1 (D) levels were measured by ELISA. n $ 3 independent experiments. *p , 0.05 versus all other conditions. by guest on November 27, 2017

GrmA is expressed in human platelets and increases with aging GrmA increases IL-8 and MCP-1 synthesis To globally interrogate the platelet transcriptome and identify other To examine the role of GrmA in potentiating IL-8 and MCP-1 molecules governing enhanced cytokine synthesis during aging, we synthesis by monocytes, we next incubated monocytes and next performed RNA-seq on isolated platelets from a subset of platelets with recombinant human GrmA (rhGrmA), at a con- younger (n = 3) and older (n = 3) adults. We identified numerous centration (100 nM) similar to that used by other investigators in (n = 514) transcripts significantly differentially expressed with prior publications (28, 29). When either monocytes or platelets aging, with most being increased in older adults (455 mRNAs were incubated alone in the presence or absence of rhGrmA, increased and 59 mRNAs decreased; Fig. 4A, Supplemental IL-8, and MCP-1 synthesis did not significantly increase Tables I, II). Among the differentially expressed candidates, we (Fig. 5C, 5D). These data indicate that under these experi- focused on GrmA, a serine protease that governs proinflammatory mental conditions and in the absence of platelets, GrmA is responses and protein synthesis by monocytes that, in some set- insufficient to trigger appreciable IL-8 and MCP-1 synthesis. tings, may be perforin independent (26, 27). By RNA-seq, GrmA However, when monocytes were coincubated with platelets in expression was robustly increased in platelets from older adults as the presence of rhGrmA, IL-8, and MCP-1 synthesis was sig- compared with younger adults (Fig. 4B). Subsequent interrogation nificantly upregulated (Fig. 5C, 5D). We did not identify GrmA of GrmA expression by qRT-PCR on a larger cohort confirmed that in resting human monocytes (Fig. 6A). As a positive control, platelet GrmA expression was significantly increased in older we confirmed that the TLR4 inhibitor blocked LPS-induced adults (Fig. 4C). The expression of GrmH, GrmM, and granulysin synthesis of IL-8 by resting monocytes (Fig. 6B). The addi- did not differ between younger and older adults (Fig. 4C). GrmA tion of polymyxin B, which blocks LPS-induced activation of protein expression was also significantly increased in platelets TLR4, did not prevent GrmA-induced cytokine synthesis, in- from older adults (Fig. 4D). Immunofluorescence studies con- dicating that LPS contamination was not causing the observed firmed the presence of GrmA protein within human platelets in increase in IL-8 and MCP-1 synthesis (Supplemental Fig. 2B, submembranous areas and in a granular pattern that did not ap- 2C). Inhibition of GrmA, using a specific anti-GrmA Ab, re- preciably colocalize with a granules (Fig. 5A). When platelets duced cytokine production to levels similar to conditions where from younger or older adults were activated with thrombin, GrmA GrmA was absent (Fig. 6C). Control IgG had no effect on IL-8 was almost entirely secreted into the extracellular milieu (Fig. 5B). or MCP-1 synthesis, indicating that this reduction of GrmA- Thus, GrmA in platelets is under signal-dependent secretion and induced synthesis of IL-8 and MCP-1 is independent of FcgR released extracellularly (where it may signal to monocytes). GrmA Ab binding (Fig. 6C). Taken together, these findings identify secretion was higher in platelets from older adults as compared with that platelet GrmA controls the synthesis of IL-8 and MCP-1 platelets from younger adults (Fig. 5B). protein by monocytes. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 6. GrmA is not present in resting human monocytes and blocking FcgR does not reduce GrmA-induced IL-8 or MCP-1 synthesis. (A) GrmA mRNA is not present in resting human monocytes. Human monocytes were isolated as described in the Materials and Methods. Isolated monocytes were interrogated by next-generation RNA-seq. On the bottom, the thick bars on the x-axis illustrate the exons with the 59 and 39 ends annotated (left and right, respectively). Shown are Integrative Genomics Viewer browser images from n = 3 independent experiments. (B) Blocking TLR4 on monocytes prevents IL-8 synthesis. Monocytes were isolated from younger adults. Monocytes were stimulated with LPS (100 ng/ml, t = 18 h) in the presence or absence of a TLR4 inhibitor. IL-8 was measured by ELISA in harvested supernatants. (C) Blocking FcgR does not inhibit GrmA-induced IL-8 or MCP-1 synthesis. Human platelets and monocytes were isolated as described in the Materials and Methods. Platelets (activated with thrombin, 0.1 U/ml) and monocytes were left alone (i.e., not treated [NT]) or stimulated with rhGrmA (100 nM). At the same time, either an IgG1 Ab, which blocks FcgR, or a specific Ab against GrmA

(anti-GrmA) was added to the incubating cells (t = 18 h). IL-8 and MCP-1 were measured by ELISA in harvested supernatants. n = 3 independent by guest on November 27, 2017 experiments. *p , 0.05.

Inhibiting GrmA in older adults rescues IL-8 and MCP-1 rapid, short-lived functions primarily for hemostasis and wound synthesis through TLR4 and caspase-1–dependent mechanisms repair (2). Emerging evidence indicates, however, that platelets We next sought to determine if inhibiting GrmA in platelets from possess a broad and dynamic repertoire of functions (2, 30–32). older adults would normalize (to levels seen in younger adults) IL-8 Platelets process precursor mRNAs in response to activating sig- and MCP-1 synthesis by monocytes. To establish this, platelets and nals, synthesize new proteins, and have activities that span in- monocytes from older adults were coincubated in the presence or flammatory and immune continuums (16, 32). absence of an anti-GrmA Ab. When GrmA was blocked, the Aging is associated with injurious thromboinflammation and synthesis of IL-8 and MCP-1 was reduced to levels similar to those alterations in platelet functions (33–35). In older adults with acute, seen in younger adults (Fig. 7A). systemic inflammatory syndromes, increased platelet activation To dissect the mechanisms of action whereby GrmA was in- correlates with increased circulating, proinflammatory cytokine ducing cytokine synthesis, we next measured GrmA-induced IL-8 levels, and adverse clinical outcomes (13). Circulating plasma and MCP-1 cytokine synthesis in the presence or absence of levels of IL-6 increase with age (11, 12) and monocytes from older specific inhibitors to TLR4 or caspase-1. In younger adults, GrmA- adults express greater intracellular IL-6 and IL-8 (8). MCP-1 is induced production of IL-8 and MCP-1 was blocked completely chemotactic for mononuclear leukocytes into inflamed vascular when TLR4 was inhibited (Fig. 7B). In contrast, when caspase-1 tissues and IL-8 orchestrates firm adhesion of monocytes to vas- was inhibited, we observed partial rescue of IL-8 whereas MCP-1 cular endothelium (36). As we and others have shown, the for- synthesis was completed blocked (Fig. 7B). Similar findings were mation of circulating platelet–monocyte aggregates is increased in observed in older adults (Fig. 7C). TL4 mRNA and protein ex- older adults with systemic inflammatory syndromes (13) and PMA pression in either isolated platelets or monocytes did not differ results in the synthesis of IL-8 and MCP-1 (3). Stable adhesion of between younger and older subjects (Fig. 7D–G). Consistent with platelets to monocytes requires the expression of P-selectin on the this, analyses also did not identify any differences platelet surface and subsequent engagement of PSGL-1 on the in the TLR pathway in platelets based on RNA-seq data (data not monocyte. The release of the platelet a granule protein RANTES shown). (through binding to CCR5 on the monocyte surface) causes translocation of NF-Kb into the nucleus of the monocyte and triggers the synthesis of IL-8 and MCP-1 (3). Discussion In this study, using both autologous and nonautologous mono- Platelets are circulating anucleate blood cells traditionally thought cytes (switch co-culture conditions), we demonstrate that aged to have a relatively fixed transcriptome and proteome, and have platelets drive excessive production of IL-6, IL-8, and MCP-1 by 8 PLATELET GRANZYME A REGULATES CYTOKINE SYNTHESIS Downloaded from FIGURE 7. Blocking endogenous GrmA inhibits IL-8 and MCP-1 synthesis in a TLR4- and caspase-1–dependent mechanism. (A) Monocytes (2 3 106 cells per ml, final concentration) and platelets (2 3 108 cells per ml, final concentration) were isolated from younger (age ,45 y, n = 5) or older adults (age $65 y, n = 5). Monocytes and platelets were coincubated together (t = 18 h) together in the presence of thrombin (IIa, 0.1 U/ml, t = 30 min) and an anti-GrmA blocking Ab or IgG control Ab (IgG). Supernatants were harvested by centrifugation. IL-8 and MCP-1 protein levels in supernatants were then measured by ELISA. Shown is the fold-change in IL-8 and MCP-1 synthesis in older adults (red) as compared with younger adults (blue). (B) Monocytes and platelets were isolated from younger adults (age ,45 y, n = 5). rhGrmA (100 nM final) was added to the cells in the presence of a TLR4 inhibitor or, separately, a caspase-1 inhibitor. Cells were allowed to incubate together for 18 h. Supernatants were harvested by centrifugation. IL-8 and MCP-1 protein http://www.jimmunol.org/ levels in supernatants were measured by ELISA. (C) Blocking endogenous GrmA in older adults reduces IL-8 and MCP-1 synthesis in a TLR4- and caspase-1–dependent mechanism. Monocytes (2 3 106 cells per ml, final concentration) and platelets (2 3 108 cells per ml, final concentration) were isolated from older adults (age $65 y, n = 5). rhGrmA (100 nM final) was added to the cells in the presence of a TLR4 inhibitor or, separately, a caspase-1 inhibitor. Platelets and monocytes were allowed to incubate together for 18 h in the presence of thrombin (0.1 U/ml). Supernatants were harvested by centrifugation. IL-8 and MCP-1 protein levels in supernatants were measured by ELISA. (D–G) TLR4 expression on platelets and monocytes does not differ between younger and older subjects. Human platelets and monocytes were isolated from younger (,45 y) and older ($65 y) subjects as described in the Materials and Methods. Platelet TLR4 mRNA expression was examined by RNA-seq [(D), n = 3 subjects per group] and by qRT-PCR [(E), n =3 subjects per group]. (F) Monocyte TLR4 mRNA expression was examined by qRT-PCR (n = 3 subjects per group). (G) TLR4 protein expression was determined in platelets and monocytes from younger (,45 y) and older ($65 y) subjects by immunoblot (n = 3–4 subjects per group). Actin, shown on the by guest on November 27, 2017 bottom of each blot, was used as a loading control. *p , 0.05. monocytes. We identify that, molecularly, platelet GrmA mediates reduced cytokine synthesis in response to GrmA. These findings the heightened synthesis of these proinflammatory cytokines and are consistent with prior reports showing that GrmA enhances that platelet GrmA is significantly enriched (at both the mRNA and activation of the inflammasome (a caspase-1–dependent event) and protein level) and bioactive in older adults. Inhibiting GrmA LPS-mediated signaling, which acts via TLR4 (29). In addition, in vitro in older adults reduced IL-8 and MCP-1 synthesis to levels mice globally deficient in GrmA exhibit better survival in response comparable to those in younger adults. Importantly, we confirmed to a lethal LPS challenge, as compared with wild-type mice where that our findings were not due to any inadvertent contamination by GrmA is endogenously present (29, 40). LPS. We could not find any prior studies that identified GrmA in The strengths of our study include the use of freshly isolated, human platelets. This is also the first evidence, to our knowledge, primary human cells (e.g., platelets and monocytes), our approxi- that shows where aging-associated increases in GrmA are sufficient mation of physiological conditions when incubating platelets and to drive proinflammatory gene synthesis by monocytes. monocytes together, and our rigorous validation of the expression and These findings build upon and extend our understanding of the activity of GrmA in younger and older human participants. We have cellular expression and function of GrmA. In humans, also made all the platelet RNA-seq data publicly available. Fastq files are a family of five structurally related serine proteases found have been submitted to the Sequence Read Archive so that readers ubiquitously in cytotoxic lymphocytes that differ in their substrate and investigators may query the data directly (National Center for specificity (27, 37). GrmA was initially identified within cyto- Biotechnology Information BioProject PRJNA397446, accession num- plasmic granules within cytotoxic T cells and NK cells (38). More bers SRR5907423–SRR5907428). The BioProject can be accessed at recently, GrmA has been found within other nucleated cells and in https://www.ncbi.nlm.nih.gov/bioproject/PRJNA397446. the extracellular space (39), been shown to activate macrophages, Nonetheless, whether GrmA is acting specifically and selectively monocytes, and mast cells, and induces inflammatory responses on monocytes, platelets, or both remains to be determined. Although independent of perforins (26, 27). human platelets are not known to express or synthesize IL-8 and Our studies also provide new evidence that GrmA functionally MCP-1 protein, they do express TLR4 on their surface and have a regulates the production of cytokines by monocytes. Although we functional inflammasome (22, 41–43). Thus, altered signaling focused on elucidating the mechanism of excessive cytokine through these pathways may influence how platelets interact production in aging, our data demonstrate that even in younger with monocytes, leading to increased monocyte-driven cytokine adults, modulating GrmA (either by addition of exogenous GrmA synthesis in older adults. Platelets are increasingly recognized as or by inhibiting endogenous GrmA) serves to control IL-8 and effector cells during systemic, injurious inflammatory responses. MCP-1 synthesis. Moreover, inhibition of TLR4 or caspase-1 Our data support established and emerging investigations examining The Journal of Immunology 9 whether antiplatelet therapies modulate cytokine synthesis (and thus 12. Donato, A. J., A. D. Black, K. L. Jablonski, L. B. Gano, and D. R. Seals. 2008. Aging is associated with greater nuclear NF kappa B, reduced I kappa B alpha, inflammation). For example, dipyridamole, but not aspirin, attenu- and increased expression of proinflammatory cytokines in vascular endothelial ates nuclear translocation of NF-kB and MCP-1 synthesis (44). This cells of healthy humans. Aging Cell 7: 805–812. may explain in part why the combination of aspirin plus extended- 13. Rondina, M. T., M. Carlisle, T. Fraughton, S. M. Brown, R. R. Miller, III, E. S. Harris, A. S. Weyrich, G. A. Zimmerman, M. A. Supiano, and release dipyridamole offered better secondary stroke risk reduction C. K. Grissom. 2015. Platelet-monocyte aggregate formation and mortality risk than aspirin alone in clinical trials (45, 46). Whether targeting in older patients with severe sepsis and septic shock. J. Gerontol. A Biol. Sci. platelets in other inflammatory diseases offers clinical benefits Med. Sci. 70: 225–231. 14. Rondina, M. T., M. Freitag, F. G. Pluthero, W. H. Kahr, J. W. Rowley, remains an active area of study. L. W. Kraiss, Z. Franks, G. A. Zimmerman, A. S. Weyrich, and H. Schwertz. Although not a central focus of our study, our findings also 2016. Non-genomic activities of retinoic acid receptor alpha control actin cy- toskeletal events in human platelets. J. Thromb. Haemost. 14: 1082–1094. demonstrate that the platelet transcriptome is altered in aging. We 15. Franks, Z., R. A. Campbell, A. Vieira de Abreu, J. T. Holloway, J. E. Marvin, identified numerous differentially expressed transcripts in platelets B. F. Kraemer, G. A. Zimmerman, A. S. Weyrich, and M. T. Rondina. 2013. isolated from older adults with enrichment of pathways implicated Methicillin-resistant Staphylococcus aureus-induced thrombo-inflammatory re- sponse is reduced with timely antibiotic administration. Thromb. Haemost. 109: in cell-cell signaling and inflammatory pathways (data not shown). 684–695. Our age-related changes in the platelet transcriptome were simi- 16. Rondina, M. T., H. Schwertz, E. S. Harris, B. F. Kraemer, R. A. Campbell, larly noted in a younger cohort of healthy patients aged 18–46 y, N. Mackman, C. K. Grissom, A. S. Weyrich, and G. A. Zimmerman. 2011. The septic milieu triggers expression of spliced tissue factor mRNA in human where more than 120 mRNAs and 15 microRNAs demonstrated platelets. J. Thromb. Haemost. 9: 748–758. age-dependent expression levels (47). We extend this published 17. Schwertz, H., N. D. Tolley, J. M. Foulks, M. M. Denis, B. W. Risenmay, M. Buerke, R. E. Tilley, M. T. Rondina, E. M. Harris, L. W. Kraiss, et al. 2006. work by offering, to our knowledge, the first human platelet Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombo- RNA-seq dataset comparing younger (age ,45 y) and older genicity of human platelets. J. Exp. Med. 203: 2433–2440. Downloaded from (age $65 y) individuals. We have made this dataset available and 18. Shih, L., D. Kaplan, L. W. Kraiss, T. C. Casper, R. C. Pendleton, C. L. Peters, M. A. Supiano, G. A. Zimmerman, A. S. Weyrich, and M. T. Rondina. 2016. hope it will serve as a discovery tool for investigators in the field. Platelet-monocyte aggregates and c-reactive protein are associated with vte in In conclusion, human aging is associated with changes in the older surgical patients. Sci. Rep. 6: 27478. platelet transcriptome and proteome. GrmA is present and bioactive 19. Rondina, M. T., C. K. Grissom, S. Men, E. S. Harris, H. Schwertz, G. A. Zimmerman, and A. S. Weyrich. 2012. Whole blood flow cytometry in human platelets, increases during aging, and regulates inflam- measurements of in vivo platelet activation in critically-Ill patients are influenced

matory gene synthesis by monocytes. Alterations in the platelet by variability in blood sampling techniques. Thromb. Res. 129: 729–735. http://www.jimmunol.org/ 20. Rondina, M. T., B. Brewster, C. K. Grissom, G. A. Zimmerman, molecular signature and downstream signaling to monocytes may D. H. Kastendieck, E. S. Harris, and A. S. Weyrich. 2012. In vivo platelet ac- contribute to dysregulated inflammatory syndromes and adverse tivation in critically ill patients with primary 2009 influenza A(H1N1). Chest outcomes in older adults. 141: 1490–1495. 21. Rowley, J. W., S. Chappaz, A. Corduan, M. M. Chong, R. Campbell, A. Khoury, B. K. Manne, J. G. Wurtzel, J. V. Michael, L. E. Goldfinger, et al. 2016. Dicer1- Acknowledgments mediated miRNA processing shapes the mRNA profile and function of murine We thank Diana Lim for creativity and excellent figure preparation, Kendra platelets. Blood 127: 1743–1751. 22. Rowley, J. W., A. J. Oler, N. D. Tolley, B. N. Hunter, E. N. Low, D. A. Nix, Richardson for editorial assistance, and Antoinette Blair for careful reading C. C. Yost, G. A. Zimmerman, and A. S. Weyrich. 2011. Genome-wide RNA-seq of the manuscript. analysis of human and mouse platelet transcriptomes. Blood 118: e101–e111.

23. Kahr, W. H., J. Hinckley, L. Li, H. Schwertz, H. Christensen, J. W. Rowley, by guest on November 27, 2017 F. G. Pluthero, D. Urban, S. Fabbro, B. Nixon, et al. 2011. Mutations in Disclosures NBEAL2, encoding a BEACH protein, cause gray platelet syndrome. Nat. The authors have no financial conflicts of interest. Genet. 43: 738–740. 24. Noetzli, L., R. W. Lo, A. B. Lee-Sherick, M. Callaghan, P. Noris, A. Savoia, M. Rajpurkar, K. Jones, K. Gowan, C. Balduini, et al. 2015. Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and pre- References disposition to lymphoblastic leukemia. Nat. Genet. 47: 535–538. 1. Thachil, J. 2015. Platelets in inflammatory disorders: a pathophysiological and 25. Deuel, T. F., R. M. Senior, D. Chang, G. L. Griffin, R. L. Heinrikson, and clinical perspective. Semin. Thromb. Hemost. 41: 572–581. E. T. Kaiser. 1981. Platelet factor 4 is chemotactic for neutrophils and mono- 2. Rondina, M. T., A. S. Weyrich, and G. A. Zimmerman. 2013. Platelets as cellular cytes. Proc. Natl. Acad. Sci. USA 78: 4584–4587. effectors of inflammation in vascular diseases. Circ. Res. 112: 1506–1519. 26. Hildebrand, D., K. A. Bode, D. Rieß, D. Cerny, A. Waldhuber, F. Ro¨mmler, 3. Weyrich, A. S., M. R. Elstad, R. P. McEver, T. M. McIntyre, K. L. Moore, J. Strack, S. Korten, J. H. Orth, T. Miethke, et al. 2014. Granzyme A produces J. H. Morrissey, S. M. Prescott, and G. A. Zimmerman. 1996. Activated platelets bioactive IL-1b through a nonapoptotic inflammasome-independent pathway. signal chemokine synthesis by human monocytes. J. Clin. Invest. 97: 1525–1534. Cell Reports 9: 910–917. 4. Dixon, D. A., N. D. Tolley, K. Bemis-Standoli, M. L. Martinez, A. S. Weyrich, 27. Wensink, A. C., C. E. Hack, and N. Bovenschen. 2015. Granzymes regulate J. D. Morrow, S. M. Prescott, and G. A. Zimmerman. 2006. Expression of COX- proinflammatory cytokine responses. J. Immunol. 194: 491–497. 2 in platelet-monocyte interactions occurs via combinatorial regulation involving 28. Sower, L. E., C. J. Froelich, N. Allegretto, P. M. Rose, W. D. Hanna, and adhesion and cytokine signaling. J. Clin. Invest. 116: 2727–2738. G. R. Klimpel. 1996. Extracellular activities of human granzyme A. Monocyte 5. Johnson, S. A., G. P. Eleazer, and M. T. Rondina. 2016. Pathogenesis, diagnosis, and activation by granzyme A versus alpha-thrombin. J. Immunol. 156: 2585–2590. treatment of venous thromboembolism in older adults. J. Am. Geriatr. Soc. 64: 1869–1878. 29. Metkar, S. S., C. Menaa, J. Pardo, B. Wang, R. Wallich, M. Freudenberg, S. Kim, 6. Mohebali, D., D. Kaplan, M. Carlisle, M. A. Supiano, and M. T. Rondina. 2014. S. M. Raja, L. Shi, M. M. Simon, and C. J. Froelich. 2008. Human and mouse Alterations in platelet function during aging: clinical correlations with throm- granzyme A induce a proinflammatory cytokine response. Immunity 29: 720–733. boinflammatory disease in older adults. J. Am. Geriatr. Soc. 62: 529–535. 30. Middleton, E. A., A. S. Weyrich, and G. A. Zimmerman. 2016. Platelets in 7. Wilkerson, W. R., and D. C. Sane. 2002. Aging and thrombosis. Semin. Thromb. pulmonary immune responses and inflammatory lung diseases. Physiol. Rev. 96: Hemost. 28: 555–568. 1211–1259. 8. de Pablo-Bernal, R. S., J. Can˜izares, I. Rosado, M. I. Galva´, A. I. Alvarez-Rı´os, 31. Herter, J. M., J. Rossaint, and A. Zarbock. 2014. Platelets in inflammation and A. Carrillo-Vico, S. Ferrando-Martı´nez, M. A. Mun˜oz-Ferna´ndez, M. Rafii-El-Idrissi immunity. J. Thromb. Haemost. 12: 1764–1775. Benhnia, Y. M. Pacheco, et al. 2016. Monocyte phenotype and polyfunctionality are 32. Semple, J. W., J. E. Italiano, Jr., and J. Freedman. 2011. Platelets and the im- associated with elevated soluble inflammatory markers, cytomegalovirus infection, and mune continuum. Nat. Rev. Immunol. 11: 264–274. . functional and cognitive decline in elderly adults. J.Gerontol.ABiol.Sci.Med.Sci.71: 33. Johnson, M., E. Ramey, and P. W. Ramwell. 1975. Sex and age differences in 610–618. human platelet aggregation. Nature 253: 355–357. 9. Hozumi, H., J. Russell, S. Vital, and D. N. Granger. 2016. Il-6 mediates the 34. Supiano, M. A., O. A. Linares, J. B. Halter, K. M. Reno, and S. G. Rosen. 1987. intestinal microvascular thrombosis associated with experimental colitis. Functional uncoupling of the platelet alpha 2-adrenergic receptor-adenylate Inflamm. Bowel Dis. 22: 560–568. cyclase complex in the elderly. J. Clin. Endocrinol. Metab. 64: 1160–1164. 10. Tang, Y. H., S. Vital, J. Russell, H. Seifert, and D. N. Granger. 2015. Interleukin- 35. Sie, P., J. Montagut, M. Blanc, B. Boneu, C. Caranobe, J. C. Cazard, and 6 mediates enhanced thrombus development in cerebral arterioles following a R. Bierme´. 1981. Evaluation of some platelet parameters in a group of elderly brief period of focal brain ischemia. Exp. Neurol. 271: 351–357. people. Thromb. Haemost. 45: 197–199. 11. Pararasa, C., J. Ikwuobe, S. Shigdar, A. Boukouvalas, I. T. Nabney, J. E. Brown, 36. Gerszten, R. E., E. A. Garcia-Zepeda, Y. C. Lim, M. Yoshida, H. A. Ding, A. Devitt, C. J. Bailey, S. J. Bennett, and H. R. Griffiths. 2016. Age-associated M. A. Gimbrone, Jr., A. D. Luster, F. W. Luscinskas, and A. Rosenzweig. 1999. changes in long-chain fatty acid profile during healthy aging promote pro- MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium inflammatory monocyte polarization via PPARg. Aging Cell 15: 128–139. under flow conditions. Nature 398: 718–723. 10 PLATELET GRANZYME A REGULATES CYTOKINE SYNTHESIS

37. Voskoboinik, I., J. C. Whisstock, and J. A. Trapani. 2015. Perforin and gran- 43. Hottz, E. D., J. F. Lopes, C. Freitas, R. Valls-de-Souza, M. F. Oliveira, zymes: function, dysfunction and human pathology. Nat. Rev. Immunol. 15: 388– M. T. Bozza, A. T. Da Poian, A. S. Weyrich, G. A. Zimmerman, F. A. Bozza, and 400. P. T. Bozza. 2013. Platelets mediate increased endothelium permeability in 38. Masson, D., M. Zamai, and J. Tschopp. 1986. Identification of granzyme A dengue through NLRP3-inflammasome activation. Blood 122: 3405–3414. isolated from cytotoxic T-lymphocyte-granules as one of the proteases encoded 44. Weyrich, A. S., M. M. Denis, J. R. Kuhlmann-Eyre, E. D. Spencer, D. A. Dixon, by CTL-specific . FEBS Lett. 208: 84–88. G. K. Marathe, T. M. McIntyre, G. A. Zimmerman, and S. M. Prescott. 2005. 39. Cullen, S. P., M. Brunet, and S. J. Martin. 2010. Granzymes in cancer and im- Dipyridamole selectively inhibits inflammatory gene expression in platelet- munity. Cell Death Differ. 17: 616–623. monocyte aggregates. Circulation 111: 633–642. 40. Anthony, D. A., D. M. Andrews, M. Chow, S. V. Watt, C. House, S. Akira, 45. Diener, H. C., L. Cunha, C. Forbes, J. Sivenius, P. Smets, and A. Lowenthal. P. I. Bird, J. A. Trapani, and M. J. Smyth. 2010. A role for granzyme M in TLR4- 1996. European stroke prevention study. 2. Dipyridamole and acetylsalicylic driven inflammation and endotoxicosis. J. Immunol. 185: 1794–1803. acid in the secondary prevention of stroke. J. Neurol. Sci. 143: 1–13. 41. Aslam, R., E. R. Speck, M. Kim, A. R. Crow, K. W. Bang, F. P. Nestel, H. Ni, 46. ESPRIT Study Group, P. H. Halkes, J. van Gijn, L. J. Kappelle, P. J. Koudstaal, and A. H. Lazarus, J. Freedman, and J. W. Semple. 2006. Platelet Toll-like receptor A. Algra. 2006. Aspirin plus dipyridamole versus aspirin alone after cerebral ischaemia expression modulates lipopolysaccharide-induced thrombocytopenia and tumor of arterial origin (ESPRIT): randomised controlled trial. Lancet 367: 1665–1673. necrosis factor-alpha production in vivo. Blood 107: 637–641. 47. Simon, L. M., L. C. Edelstein, S. Nagalla, A. B. Woodley, E. S. Chen, X. Kong, 42. Hottz, E. D., A. P. Monteiro, F. A. Bozza, and P. T. Bozza. 2015. Inflammasome L. Ma, P. Fortina, S. Kunapuli, M. Holinstat, et al. 2014. Human platelet in platelets: allying and inflammation in infectious and sterile dis- microRNA-mRNA networks associated with age and gender revealed by eases? Mediators Inflamm. 2015: 435783. integrated plateletomics. Blood 123: e37–e45. Downloaded from http://www.jimmunol.org/ by guest on November 27, 2017