Extracellular Polyphosphate Promotes Macrophage and Fibrocyte

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Extracellular Polyphosphate Promotes Macrophage and Fibrocyte Differentiation, Inhibits Leukocyte Proliferation, and Acts as a Chemotactic Agent for Neutrophils This information is current as of October 1, 2021. Patrick M. Suess, Luis E. Chinea, Darrell Pilling and Richard H. Gomer J Immunol published online 3 June 2019 http://www.jimmunol.org/content/early/2019/05/31/jimmun

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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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 3, 2019, doi:10.4049/jimmunol.1801559 The Journal of Immunology

Extracellular Polyphosphate Promotes Macrophage and Fibrocyte Differentiation, Inhibits Leukocyte Proliferation, and Acts as a Chemotactic Agent for Neutrophils

Patrick M. Suess,1 Luis E. Chinea, Darrell Pilling, and Richard H. Gomer

Fibrocytes are monocyte-derived fibroblast like cells that participate in wound healing, but little is known about what initiates fibrocyte differentiation. Blood platelets contain 60–100-mer polymers of phosphate groups called polyphosphate, and when activated, platelets induce blood clotting (the first step in wound healing) in part by the release of polyphosphate. We find that activated platelets release a factor that promotes fibrocyte differentiation. The factor is abolished by treating the crude platelet factor with the polyphosphate-degrading enzyme polyphosphatase, and polyphosphate promotes fibrocyte differentiation. Mac- rophages and recruited neutrophils also potentiate wound healing, and polyphosphate also promotes macrophage differentiation

and induces chemoattraction of neutrophils. In support of the hypothesis that polyphosphate is a signal that affects leukocytes, we Downloaded from observe saturable binding of polyphosphate to these cells. Polyphosphate also inhibits leukocyte proliferation and proteasome activity. These results suggest new roles for extracellular polyphosphate as a mediator of wound healing and inﬂammation and also provide a potential link between platelet activation and the progression of ﬁbrosing diseases. The Journal of Immunology, 2019, 203: 000–000.

ibrocytes are monocyte-derived cells that have properties recruitment, activation, and proliferation of a variety of leuko- http://www.jimmunol.org/ of both inflammatory macrophages and tissue remodeling cytes including neutrophils, lymphocytes, and monocyte-derived F fibroblasts (1). Fibrocytes participate in wound healing by macrophages to a site of tissue damage, and these events are secreting inflammatory cytokines and ECM proteins (2). Fibro- important steps in the process of tissue repair (12–14). cytes are also found at fibrotic lesions and have been implicated in In this article, we report that polyphosphate has multiple effects the progression of fibrosing diseases (2). What initiates fibrocyte on leukocyte function, including inducing monocytes to differ- differentiation in a wound remains poorly understood (3–5). entiate into fibrocytes and macrophages, inhibits leukocyte pro- Blood clotting is an early and critical component of wound liferation and proteasome activity, and acting as a chemoattractant healing, which is induced upon the activation of platelets at the for neutrophils. We found that the incubation of PBMC with ac- site of tissue damage (6). Numerous factors released from acti- tivated releasates from platelets also induces fibrocyte differenti- by guest on October 1, 2021 vated platelets interact with leukocytes and elicit various func- ation, and this activity is lost upon treatment of releasates with the tions (7, 8); however, the role of platelet-derived factors on polyphosphate-degrading enzyme exopolyphosphatase, suggesting fibrocyte differentiation have not been well investigated. One a new role for polyphosphate as a mediator of innate immunity and factor released by activated platelets is polyphosphate, a highly wound healing. anionic, linear polymer of phosphates bound by phosphoanhydride bonds (9). Platelet-derived extracellular polyphosphate contrib- Materials and Methods utes to wound healing and inflammation by binding to and en- Platelet, neutrophil, and PBMC isolation and culture hancing activation of the plasma protease factor XII, which Human peripheral blood was collected from healthy volunteers who gave triggers a response leading to fibrin and bradykinin generation written consent, with specific approval from the institutional review board (10). Topical application of polyphosphate enhances wound of Texas A&M University. Whole blood was collected in vacutainers healing in vivo, leading to an increase in collagen and a-smooth containing acid–citrate–dextrose to prevent clotting. Blood was gently muscle actin (11). The inflammatory response also drives the transferred to 15-ml conical tubes using plastic transfer pipettes and clarified by centrifugation at 200 3 g for 20 min. The upper two thirds of the straw-colored platelet-rich plasma was transferred to a fresh tube con- Department of Biology, Texas A&M University, College Station, TX 77843 taining HEP buffer (140 mM NaCl, 2.7 mM KCL, 3.8 mM HEPES, 5 mM 1 EGTA, pH 7.4) containing 1 mM PG E1 (Sigma-Aldrich, St Louis, MO) at Current address: University of Michigan Medical School, Ann Arbor, MI. a 1:1 ratio. Samples were mixed gently and clarified by centrifugation at ORCIDs: 0000-0002-9805-9005 (P.M.S.); 0000-0003-3511-2830 (L.E.C.); 0000- 100 3 g for 20 min. The supernatant was transferred to a new tube, and 0002-7413-2773 (D.P.). platelets were collected by centrifugation at 800 3 g for 20 min. The Received for publication November 28, 2018. Accepted for publication May 8, 2019. platelet pellet was washed two times in platelet wash buffer (10 mM so- dium citrate, 150 mM NaCl, 1 mM EDTA, 1% [w/v] dextrose, pH 7.4) This work was supported by National Institutes of Health Grant R01 GM102280. without resuspension. The platelet pellet was gently resuspended in Address correspondence and reprint requests to Dr. Richard H. Gomer, Department Tyrode’s buffer (134 mM NaCl, 12 mM NaHCO3, 2.9 mM KCl, 0.34 mM of Biology, Texas A&M University, ILSB, 301 Old Main Drive, College Station, TX Na2HPO4, 1 mM MgCl2, 10 mM HEPES, pH 7.4) containing 5 mM glu- 77843-3474. E-mail address: [email protected] cose and 3 mg/ml BSA (Amresco, Solon, OH). Platelets were counted with The online version of this article contains supplemental material. a hemocytometer, and the platelet concentration was adjusted in Tyrode’s Abbreviations used in this article: FMI, forward migration index; FSC, forward buffer. To activate platelets, samples were incubated with 1.2 U/ml of scatter; PPX1, yeast exopolyphosphatase; RPMI-BSA, RPMI 1640 containing 2% human thrombin (Sigma-Aldrich) for 1 h, and secreted material was iso- BSA; SFM, serum-free medium. lated by passage through a 0.22-mm type 25-243 syringe filter (Genesee Scientific, San Diego, CA). Exopolyphosphatase treatment of platelet Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 releasates was performed as previously described (15). To remove

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801559 2 EXTRACELLULAR POLYPHOSPHATE EFFECT ON LEUKOCYTES thrombin, exopolyphosphatase, and large proteins, samples were clarified filter as described above. After centrifugation, 10 ml of the diluted by centrifugation for 10 min at 14,000 3 g through a 30-kDa cutoff Spin-X thrombin, flow through, or Tyrode’s buffer were mixed with 90 mlof UF size exclusion filter (Corning, Tewksbury, MA). Platelets were also 0.2 mM D-FPIPR-pNA chromogenic thrombin substrate (Molecular In- activated by incubation on ice or at 37˚C with continual mixing (16, 17). novations, Novi, MI) in PBS in the well of a 96-well plate, and the ab- Platelets at 37˚C were also activated in the presence of 200 mg human sorbance at 405 nM was read every 30 s for 10 min. Fits to a line were used collagen-I–coated beads (SphereCol; Advanced BioMatrix, Carlsbad, CA) to determine the change in absorbance with time. per 1 3 108 platelets (16). After 60 min, platelet releasates were clarified by centrifugation for 10 min at 14,000 3 g. Releasates from platelets Statistics incubated with collagen beads were further clarified by centrifugation for Statistical analysis was performed using Prism (GraphPad, San Diego, CA). 10 min at 14,000 3 g through a 30-kDa cutoff Spin-X UF size exclusion Differences between two groups were assessed by Student t test. Differ- filter. ences between multiple groups were assessed by one-way ANOVA using PBMC and neutrophils were isolated as previously described (18, 19), Tukey posttest. Significance was defined as p , 0.05. with the exception that neutrophils were isolated using Polymorphprep gradients (Axis-Shield Diagnostics, Oslo, Norway), following the manu- facturer’s instructions, and centrifugation of gradients was done for Results 40 min. Neutrophils were then resuspended in RPMI 1640 (Lonza, Activated platelets induce fibrocyte differentiation, and this Wakersville, MD) containing 2% BSA (RPMI-BSA). PBMC were cultured in either an 8-well or 96-well culture plate with 200 ml/well at 5 3 105 effect requires polyphosphate cells per ml in RPMI 1640 (Lonza)–defined SFM or RPMI 1640 with 10% Fibrocytes participate in the formation of granulation tissue and bovine calf serum (VWR Life Science Seradigm, Randor, PA) as previ- new connective tissue matrix in both wound healing and fibrosis ously described (20, 21) at 37˚C in a humidified incubator with 5% CO2. PBMC were treated in the presence or absence of polyphosphate (Spec- (2). Platelet activation and blood clotting is the first step in trum Chemical, New Brunswick, NJ) at the indicated concentrations for wound healing, and platelet trapping, activation, and reactivity 6 d. Fibrocytes differentiation was assessed as previously described (22). with monocytes has been observed in fibrotic diseases (29–31). Downloaded from Fibrocytes were identified after fixation as adherent cells with an elongated Platelet-rich plasma and platelet releasates enhance wound spindle-shaped morphology, being distinct from small lymphocytes or adherent monocytes (Supplemental Fig. 1). Macrophage differentiation healing by promoting the development of granulation tissue and was assessed as previously described (21). For proliferation and pro- new connective tissue matrix (32–35). To test the possibility that teasome experiments, PBMC at 0.5 3 106 cells/ml were stimulated platelets affect fibrocyte differentiation, we incubated platelet with 2 mg/ml PHA (Sigma-Aldrich) and 10 ng/ml IL-2 (BioLegend, releasates (clarified by passing through 30-kDa cutoff spin fil-

San Diego, CA) in RPMI 1640/10% bovine calf serum, 100 U/ml http://www.jimmunol.org/ penicillin, 100 mg/ml streptomycin, and 2 mM glutamine (all from ters) from activated and nonactivated platelets with PBMC from Lonza) for 3 d before the addition of polyphosphate. Cells were healthy human donors and assessed fibrocyte differentiation. As counted using a hemocytometer. previously observed (3, 36, 37), fibrocyte counts in the controls ranged from 220 to 1300 per 105 PBMC for the seven blood Immunohistochemistry, flow cytometry, and polyphosphate donors used in this study, so all fibrocyte counts were expressed binding assay as a percentage of the control count for each donor. PBMC and Immunohistochemistry and flow analysis of macrophages were performed platelets were isolated from healthy donors, and for each donor, as previously described (18, 20, 23). Macrophages were stained with 5 mg/ml their PBMC were incubated with the ,30-kDa filtrate from their Abs against CD54 (BioLegend), CD68 (BioLegend), or CD206 (BioLegend) platelets. Filtrates from unactivated platelets inhibited fibrocyte or with mouse IgG1 (BioLegend) as a control. Polyphosphate binding to by guest on October 1, 2021 PBMC was measured using biotinylated polyphosphate (Kerafast, Boston, differentiation at concentrations corresponding to 0.09, 11.25, MA) as previously described (15). PBMC were analyzed by flow cytometry and 22.5 3 107 platelets/ml (Fig. 1A). A total of 4.5 3 108 as described previously (20, 24, 25). Anti-CD3, -CD4, -CD14, and -CD16 platelets/ml were activated by incubation with 1.2 U/ml thrombin Abs were from BioLegend. Briefly, PBMC were incubated with 5 mg/ml (9). We previously found that 3–75 ng/ml (0.003–0.075 U/ml) primary Abs or biotinylated polyphosphate diluted in TBS containing 2% BSA on ice for 30 min. PBMC were washed twice in ice-cold TBS, then thrombin increases fibrocyte differentiation (38). Using a throm- incubated on ice for 30 min with donkey F(ab9)2 anti-mouse IgG PE bin activity assay to detect thrombin, we observed that no de- preadsorbed secondary Ab (Jackson ImmunoResearch, West Grove, PA), tectable thrombin activity passed through the 30-kDa spin filters, or Alexa Fluor 647 streptavidin (BioLegend) in TBS containing 2% BSA. and with the sensitivity of the assay, we determined that there was PBMC were analyzed by flow cytometry using an Accuri C6 cytometer ,0.0074 6 0.0010 U/ml (mean 6 SEM, n = 4) of thrombin in (Accuri, BD Biosciences, San Jose, CA). the flow through. Filtered releasates from platelets activated by Neutrophil chemotaxis analysis incubation with thrombin increased fibrocyte differentiation (Fig. 1A, 1B). Each donor had fibrocyte-inducing activity over Chemotaxis was assessed using Insall chambers (26) to generate gradients of polyphosphate and quantify neutrophil migration on glass coverslips a narrow concentration range of activated platelet releasates coated with 10 mg/ml human plasma fibronectin (Corning, Bedford, MA), (Fig. 1A, Supplemental Fig. 1A–G). The concentrations of filtered as previously described (18, 19, 27). The cells were allotted at least 30 min exudate from activated platelets that increased fibrocyte differ- to adhere to the coverslip. After placing coverslips with adhered neutro- entiation ranged from 4.0 3 105 to 2.8 3 107 platelets/ml for phils on the Insall chamber, we waited 22 min for gradients to form before tracking neutrophils as previously described (18, 19, 27). Polyphosphate the various donors, corresponding to thrombin concentrations of 26 24 was resuspended in RPMI-BSA prior to adding to the Insall chamber. A ,6.2 6 0.8 3 10 –4.6 6 0.6 3 10 U/ml after 30-kDa fil- minimum of 10 neutrophils were tracked over a 38-min interval per video. tration, suggesting that there were differences in platelet contents For each donor, neutrophils were tracked in a control no-gradient RPMI- and/or monocyte sensitivity to platelet releasates between the BSA condition. We used two Insall chamber/microscope/camera setups in different donors and that there was insufficient thrombin at these parallel, allowing simultaneous filming for two experimental conditions at the same time for up to eight experimental conditions to be measured per dilutions to increase fibrocyte differentiation. When only the peak set of donor neutrophils. Neutrophils were used within 5 h of isolation. The activity for each donor was assessed, a significant increase in results are expressed as the mean 6 SEM of the movement of neutrophils fibrocyte differentiation was observed compared with either un- from five or more different donors. We never used the same donor twice for treated PBMC or PBMC cultured with releasates from unstimu- a given experiment. lated platelets (Fig. 1B). No significant differences were observed Proteasome activity and thrombin assays between male and female donors (Supplemental Fig. 1H). Proteasome activity was assessed as previously described (28). To measure To further ensure that the fibrocyte differentiating activity was not the retention of thrombin by spin filters, thrombin from a 1 U/ml stock was due to the presence of residual thrombin, we incubated platelets on ice diluted to 1.2 U/ml in Tyrode’s buffer and placed in a 30-kDa cutoff spin or at 37˚C in the presence or absence of collagen-coated beads. The Journal of Immunology 3

FIGURE 1. Platelet releasates have fibrocyte-inducing activity. (A) Releasates from platelets that were unactivated, activated, or activated and treated with exopolyphosphatase were incubated with PBMC, and fibrocyte-inducing activity was assessed after 96 h. *p , 0.05, **p , 0.01, ***p , 0.001 compared with no added releasate, with the color of the * corresponding to the color of the data series (paired t tests). (B) The concentration of platelets that had the highest fibrocyte-inducing activity (peak activity) for activated platelet releasates for each individual donor from (A) were plotted. PPX1 indicates polyphosphatase. All values are mean 6 SEM (n = 7, four male donors and three female donors). **p , 0.01, ***p , 0.001 (paired t tests). ns, not significant. Downloaded from

Platelet releasates generated from collagen- or temperature-activated with resolution of inflammation (Supplemental Fig. 2A). The platelets showed similar fibrocyte-inducing activity (Supplemental concentrations of polyphosphate that affect monocyte dif- Fig. 1I–K), suggesting that thrombin was not the fibrocyte differen- ferentiation did not induce proliferation of the PBMC pop- tiating activity. ulation (Supplemental Fig. 2B). The ability of polyphosphate to Platelets contain polyphosphate, activated platelets release enhance fibrocyte and macrophage differentiation suggests that http://www.jimmunol.org/ polyphosphate (9), and polyphosphate enhances wound healing polyphosphate may affect wound healing and inflammation. both in vitro and in vivo (11). To determine if polyphosphate is Polyphosphate shows saturable binding to PBMC required for the activity released by activated platelets that potentiates fibrocyte differentiation, after activation, platelets were Polyphosphate shows saturable binding to the cell surface of incubated with yeast exopolyphosphatase (PPX1), an enzyme that Dictyostelium (15) and in higher eukaryotes has a synergistic degrades polyphosphate (39), and then filtered. PPX1 treatment effect with ligands including histone H4, high mobility group abrogated the effect of the 30-kDa filtrate of activated platelets on box 1, and fibroblast growth factor 1, enhancing their binding fibrocyte differentiation for both male and female donors (Fig. 1, to their cell surface receptors (44–46). To determine if poly-

Supplemental Fig. 1). Like the releasates from unactivated plate- phosphate binds the surface of PBMC, we incubated human by guest on October 1, 2021 lets, several concentrations of the PPX1-treated activated platelet PBMC with biotinylated polyphosphate. Biotinylated poly- releasates also reduced fibrocyte differentiation. Together, these phosphate showed saturable binding to the surface of PBMC, with a 6 6 data suggest that activated platelets release a factor that promotes KD of 25 6 mM (mean SEM, n = 5) (Fig. 3). Fits to binding monocytes to differentiate into fibrocytes and that this factor re- curves with a variable Hill coefficient indicated that the binding quires polyphosphate. is cooperative, with a Hill coefficient of 3.5 6 1.3 (mean 6 SEM, n = 5). F tests comparing data fit to a model with a single class Polyphosphate directly potentiates fibrocyte and of binding sites to models with multiple binding sites indicated macrophage differentiation a single class of binding site. This suggests that one or more To directly test the effect of polyphosphate on fibrocyte differ- cell types in the PBMC population may have cell surface entiation, we added polyphosphate to PBMC cultures. The effect of polyphosphate on fibrocyte differentiation was assessed in serum-free medium (SFM), as the serum protein SAP potently inhibits fibrocyte differentiation (22). One to two picomolars polyphosphate promoted monocytes to differentiate into fibrocytes, whereas 30 and 125 pM polyphosphate appeared to de- crease fibrocyte differentiation (Fig. 2). In addition to fibrocytes, macrophages potentiate wound healing (40, 41), and factors released from platelets, such as IL-1, MIP-1a, CCL5, and TGF-b, affect macrophage recruitment, adhesion, activity, and cytokine release (42, 43). One hundred and twenty five to five hundred picomolars polyphosphate increased macrophage differentiation in the presence of serum, and 125–250 pM polyphosphate increased macrophage differentiation in the absence of serum. The polyphosphate effect on macrophage differentia- FIGURE 2. Polyphosphate has fibrocyte- and macrophage-inducing activity. The indicated concentrations of polyphosphate were incubated tion appeared to be more potent in the presence of serum, sug- with PBMC for 96 h, and fibrocytes and macrophages were counted. For gesting that polyphosphate may act synergistically with a each donor, counts were then normalized to the count for no added pol- serum factor (Fig. 2). In the absence of serum, polyphosphate yphosphate. All values are mean 6 SEM for the normalized counts caused a slight increase in the percent of cells expressing CD206, (n $ 4). *p , 0.05, **p , 0.01 compared with the no polyphosphate an anti-inflammatory proresolving marker that binds mannose and control, with the color of the * corresponding to the color of the data series N-acetylglucosamine residues found on bacteria, and is associated (paired t tests). 4 EXTRACELLULAR POLYPHOSPHATE EFFECT ON LEUKOCYTES

high FSC cells bound polyphosphate, and 26 6 3% (mean 6 SEM, n = 4) of the low FSC cells bound polyphosphate, indicating that some cells in the PBMC population do not show appreciable polyphosphate binding.

Polyphosphate affects the direction of neutrophil movement Neutrophils are recruited to sites of wound healing and inflammation (12, 13). In mice, disruption of inositol hexakisphosphate kinase 1 (Ip6k1) genetically or pharmacologically results in reduced platelet polyphosphate levels as well as reduced neutrophil recruitment during inflammation (48). Ip6k1 uses IP6 as a substrate to generate the inositol pyrophosphates IP7 and IP8 (49), and has been implicated in regulating cellular polyphosphate levels in yeast, Dictyostelium, and mice (15, 50, 51). To investigate if polyphosphate affects neutrophil migration, FIGURE 3. Polyphosphate binding to PBMC. PBMC were incubated we incubated human neutrophils in polyphosphate gradients. with the indicated concentrations of biotinylated polyphosphate, and Using forward migration index (FMI; the distance moved binding was determined using a streptavidin conjugated fluorophore and along the gradient divided by the total path length, with a

a flow cytometer. Values were normalized so that no polyphosphate was positive FMI indicating repulsion) as a metric, we found that Downloaded from 0% and the binding at 100 mM polyphosphate was 100%. All values are 0–10 pM polyphosphate gradients caused neutrophil chemo- mean 6 SEM (n $ 5). The curve is a nonlinear regression fit to saturable attraction, whereas 0–100 pM polyphosphate gradients caused binding with a variable Hill coefficient. neutrophil chemorepulsion (Fig. 4A–D). Polyphosphate gradients of 0–0.1, 0–1, and 0–1000 pM had no significant effect receptors for polyphosphate. To determine which cell types on neutrophil FMIs. Polyphosphate slightly increased neu- polyphosphate was interacting with in the PBMC population, trophil speed at 1, 100 pM, and 1 nM but not at 10 pM http://www.jimmunol.org/ we compared the population that showed polyphosphate bind- (Supplemental Fig. 4A). Polyphosphate had no significant ef- ing against known cell populations. As previously observed fect on the directness of neutrophil movement (distance from (25, 47), cells positive for the T cell marker CD3, the T cell start to end of a cell’s movement during the tracking, divided marker CD4, and the NK (and a subset of monocytes) marker by the path length) (Supplemental Fig. 4B). There were no CD16 had low forward scatter (FSC) values, whereas cells significant differences between the effect of polyphosphate on positive for the monocyte marker CD14 had relatively high the FMI from cells derived from male and female blood do- FSC values (Supplemental Fig. 3A–G). Cells with both low and nors (Supplemental Fig. 4C). Cells from males moved faster high FSC values bound polyphosphate, suggesting that poly- than females in 0–1 pM polyphosphate gradients, and in all

phosphate interacts with multiple cell types (Supplemental gradients, there were no sex-linked differences in directness by guest on October 1, 2021 Fig. 3H). We observed that 53 6 3(mean6 SEM, n =4)of (Supplemental Fig. 4C, 4D).

FIGURE 4. Polyphosphate impacts neutrophil chemotaxis. (A–D) Human neutrophils were incubated in a gradient of 0 to the indicated concentration of polyphosphate, and cell movement was tracked. In (A)–(C), the red dot indicates the ﬁnal average end point of the cells. All values are mean 6 SEM (n = 6 different donors). *p , 0.05, ***p , 0.001 compared with the no polyphosphate control (paired t tests). The Journal of Immunology 5

Polyphosphate inhibits proliferation and proteasome activity of it is activated, at 0.74 nmol poly P per 108 platelets, 2 3 1010 human PBMC at concentrations similar to those that platelets is 200 3 0.74 nmol = 148 nmol, so 2 3 1010 platelets/l is affect Dictyostelium 148 nmol/l or 148 nM polyphosphate, far above the 1–2 pM We previously found that extracellular polyphosphate inhibits polyphosphate that induces fibrocyte differentiation. Thus, either proliferation of Dictyostelium cells and four human leukemia cell platelets release only a fraction of their polyphosphate when they lines (15, 28). To determine if polyphosphate also inhibits pro- are activated, or platelets release something that inhibits fibrocyte liferation of human PBMC, we incubated PHA-stimulated PBMC differentiation and counteracts the polyphosphate. In support of with polyphosphate. One hundred micromolars and higher con- the latter possibility, platelets release many factors, including IL-2 centrations of polyphosphate significantly inhibited proliferation and IFN-g (52, 53), IL-2 and IFN-g are relatively small proteins of PBMC (Fig. 5A). Intriguingly, this proliferation inhibition (13 and 17 kDa, respectively) that would pass through a 30-kDa curve is remarkably similar to the effect of polyphosphate on cutoff filter, and both inhibit fibrocyte differentiation (25). Dictyostelium cells (15). Polyphosphate inhibits proteasome ac- Unactivated platelets appear to secrete a factor that inhibits tivity to inhibit Dictyostelium proliferation and also inhibits the fibrocyte differentiation. The polyphosphatase-treated exudate proteasome activity of some leukemia cell lines (15, 28). One from activated platelets also inhibits fibrocyte differentiation, hundred and fifty micromolars polyphosphate inhibited PBMC suggesting that platelets release the fibrocyte differentiation in- proteasome activity by ∼55% (Fig. 5B), again nearly identical to hibitor constitutively. A number of factors that inhibit fibrocyte the inhibition observed in Dictyostelium (28). These data suggest differentiation have been identified, including serum amyloid that the effects of polyphosphate on proliferation and proteasome P, Slit2, IFN-g, IL-12, galectin-3 binding protein, and 2 3 106 Da

inhibition is conserved from Dictyostelium to human leukocytes. hyaluronic acid (22, 25, 54–56). Of these, as discussed above, Downloaded from Although it is unlikely leukocytes would encounter such high IFN-g and IL-12 are present in platelets and would pass through a concentrations of extracellular polyphosphate in most circum- 30-kDa cutoff ﬁlter, and thus could potentially be some of the stances, concentrations where there is extensive localized platelet factors released by unactivated platelets that inhibit ﬁbrocyte activation, such as in thrombi, could be very high. differentiation. It is unclear if polyphosphate is directly eliciting these effects or

Discussion is acting synergistically with other extracellular molecules, as has http://www.jimmunol.org/ We found that a factor released by activated human platelets in- been shown previously (44, 46). However, polyphosphate shows duces human fibrocyte differentiation and that this factor appears saturable binding in the micromolar range to some cells in the to be polyphosphate. Polyphosphate also induces human macro- PBMC population, suggesting that these cells may have poly- phage differentiation, and, at very high concentrations, it inhibits phosphate receptors. Polyphosphate shows binding to PBMC with leukocyte proliferation. Polyphosphate induces fibrocyte differ- a Hill coefficient of 3.5, suggesting that the binding of poly- entiation at 1-2 pM, at 0–10 pM polyphosphate gradients cause phosphate to the cell surface is enhanced when other poly- neutrophil chemoattraction, at 0–100 pM polyphosphate gradients phosphate molecules are already present. cause neutrophil chemorepulsion, and at 100–500 pM poly- The molecular mechanisms used for chemotaxis and phagocy- by guest on October 1, 2021 phosphate induces macrophage differentiation (Figs. 2, 4). In the tosis are highly conserved between Dictyostelium and human absence of other factors released by platelets, research would immune cells, as appears to be the case for the effects of extra- suggest that moving away from where the platelets degranulate, cellular polyphosphate. Polyphosphate inhibits proliferation and macrophage differentiation from monocytes is potentiated, and proteasome activity of human PBMC at concentrations in the neutrophils are repelled. Neutrophils farther away are attracted, micromolar range, which is also true for Dictyostelium cells and and then, when even farther away, fibrocyte differentiation from human leukemia cell lines (15, 28). As calculated by Morrissey monocytes is potentiated. However, as discussed below, this sce- et al. (57), 1–3 mM polyphosphate would be contained in hu- nario is almost certainly modified by other factors released by man blood after full platelet activation; however, in platelet-rich platelets. thrombi, it is possible that this concentration could be orders of Platelet extracts yield ∼0.74 nmol polyphosphate per 108 magnitude higher and thus high enough to inhibit PBMC prolif- platelets (9). We observed peak fibrocyte differentiation at eration and proteasome activity. ∼2 3 107 platelets/ml (Fig. 1A), which would then be ∼2 3 1010 A link between fibrosis, platelets, and leukocytes has long been platelets/l. If all the polyphosphate in a platelet is released when observed (9, 29, 30). In this study, we find that a factor released

FIGURE 5. High concentrations of polyphosphate inhibit proteasome activity and proliferation of PBMC. (A) PBMC were stimulated for 3 d with PHA and IL-2 and then incubated with the indicated amounts of polyphosphate for an additional 96 h, and percentage of proliferation was assessed. Values were normalized to no polyphosphate controls. Red dotted lines indicate 0% and 100% proliferation. Values are mean 6 SEM (n $ 3). (B) PBMC were incubated with or without 150 mM polyphosphate for 96 h, and proteasome activity levels were determined. Values were normalized to no polyphosphate controls. Values are mean 6 SEM (n = 5). ***p , 0.001 (paired t test). 6 EXTRACELLULAR POLYPHOSPHATE EFFECT ON LEUKOCYTES from activated platelets promotes the differentiation of monocytes 21. Pilling, D., E. Galvis-Carvajal, T. R. Karhadkar, N. Cox, and R. H. Gomer. 2017. Monocyte differentiation and macrophage priming are regulated differentially by into profibrotic and proinflammatory cells and can lead to the pentraxins and their ligands. BMC Immunol. 18: 30. recruitment of neutrophils. These effects may contribute to the 22. Pilling, D., C. D. Buckley, M. Salmon, and R. H. Gomer. 2003. Inhibition of pathogenesis of fibrosis. If platelets are continually recruited and fibrocyte differentiation by serum amyloid P. J. Immunol. 171: 5537–5546. 23. Pilling, D., and R. H. Gomer. 2014. Persistent lung inflammation and fibrosis in activated, as has been observed in some forms of fibrosis (29, 31, serum amyloid P component (APCs-/-) knockout mice. PLoS One 9: e93730. 58, 59), then polyphosphate may contribute to a chronic inflam- 24. Pilling, D., T. Fan, D. Huang, B. Kaul, and R. H. Gomer. 2009. Identification of matory response, ultimately leading to fibrosis. Platelet trapping markers that distinguish monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts. PLoS One 4: e7475. and increased reactivity with monocytes has been observed at fi- 25. Shao, D. D., R. Suresh, V. Vakil, R. H. Gomer, and D. Pilling. 2008. Pivotal brotic lesions (29, 30). Polyphosphate may thus be a signal from advance: Th-1 cytokines inhibit, and Th-2 cytokines promote fibrocyte differentiation. J. Leukoc. Biol. 83: 1323–1333. platelets that promotes the progression of fibrotic lesions. 26. Muinonen-Martin, A. J., D. M. Veltman, G. Kalna, and R. H. Insall. 2010. An improved chamber for direct visualisation of chemotaxis. PLoS One 5: e15309. 27. Phillips, J. E., and R. H. Gomer. 2012. A secreted protein is an endogenous Acknowledgments chemorepellant in Dictyostelium discoideum. Proc. Natl. Acad. Sci. USA 109: We thank Michael Gray for the expression plasmid for purifying yeast 10990–10995. PPX1. 28. Suess, P. M., J. Watson, W. Chen, and R. H. 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1500 Unactivated 250 250 250 Unactivated

l 1250 l Unactivated Unactivated

o Activated o Activated

r 1000 r 200 t 200

t t 200 Activated

t Activated ,

, Activated ,

n n Activated

s s 750

o Activated

e Activated e

+PPX1 e

t +PPX1

c 500

150

y +PPX1

250 o

r r 100 100 100 n

200 b

F c 150

r r 50 50 50

e e 100

p 50 p 0 0 0 0 .1 1 0 0 1 1 0 0 1 1 0 0 1 1 0 0 0 1 0 . 1 0 . 1 0 . 1 0 1 0 1 0 1 0 1 releasates from n x 107 platelets / ml releasates from n x 107 platelets / ml releasates from n x 107 platelets / ml releasates from n x 107 platelets / ml E F G H ns

f Unactivated Unactivated y Unactivated 300 t

o 300 i

300

l Activated

i l Activated 1000

o 250 250 r r Activated ns

c 250 Activated e

Activated t

s +PPX1

s n +PPX1

s +PPX1

o ns

e 200

e 200 k

e 200

o 150 o 150 o 150

t 100

i e 100

e 100

r 50 50 50 o

i 0 0 0 o 10

c .1 1 0 0 .1 1 0 0 1 1 0 0 e e e e e 0 1 0 0 1 0 . 1 0 l l le l l l 1 1 0 1 a a a a a a 7 7 m m m m m releasates from n x 10 platelets / ml releasates from n x 10 platelets / ml 7 e m e e releasates from n x 10 platelets / ml d f d f 1 f te d e d X 1 a e t e P v t a t P X ti a iv a P c iv t iv + P a t c t d + n c a c e u a a t d n a te u iv a t v c ti a c I J K a

2 5 0 T y ro d e 's T ry o d e 's o n ly A c tiv a te d re le a s a te s 4 o C 2 2 5 * * * *

3 7 o C 3 0 0

l f

2 0 0 o y

o o

3 7 C + B e a d s i

t v

t 2 5 0 ,

1 7 5 i

t n

n 3 0 k D a F T

t c

1 5 0 r

2 0 0

a o

1 2 5 p

r p

s 1 5 0

1 0 0 t

y l r 7 5

c 1 0 0

t p

5 0 r

b i

o 5 0 F 2 5 c

0 0 l 0 1 1 .1 1 0 o o C o C s T 0 .0 0 1 r d F .0 0 t 4 7 a 0 n 3 e a o D B k 7 C + o C 0 re le a s a te s fro m n x 1 0 p la te le ts / m l 3 7 3

Figure S1. Individual donor responses to platelet releasates on fibrocyte inducing activity.

A-G) Fibrocyte inducing activity for each individual donor from Figure 1A. H) Peak fibrocyte inducing activity from activated platelets for each individual donor for males and females; ns indicates not significant. I) Platelets were activated at 4°C, 37°C, or 37°C with collagen- conjugated beads in Tyrode’s buffer. PBMCs were then incubated for 96 hours in buffer, activated releasates, or activated releasates in which the collagen-conjugated beads were removed, and fibrocyte inducing activity was assessed. J) The concentration of platelets that had the highest fibrocyte inducing activity (peak activity) for activated platelet releasates for each individual donor from I were plotted. K) PBMC incubated with platelet releasates from the equivalent (v:v) of

0.3125 x 107 platelets per ml treated with Tyrode’s buffer alone or collagen-beads were air-dried, fixed in methanol, and then stained with methylene blue. Solid arrow points to a fibrocyte. Bar is 100 µm. All values are mean ± SEM, n = 4. * indicates p <0.05 compared to the no polyphosphate control (1-way ANOVA, Dunnett’s test for).

F ig u r e S 2

A B

s 150

e 150

BCS Control l

, 125

h BCS +PolyP

o d 100 *** SFM Control 100

r n SFM +PolyP

a o 75

50 o 50

e 25

p e 0 0

P IgG1 CD54 CD68 CD206 0 5 0 5 0 0 0 0 0 0 . . . . . 0 0 0 0 0 0 0 1 1 2 1 2 3 4 5 Polyphosphate, pM

Figure S2. Effect of polyphosphate on cell surface markers and induced proliferation. A)

PBMC were incubated with 125 µM polyphosphate for 96 hours and stained with the indicated antibodies, and percent positive macrophages were assessed. B) PBMC were incubated with the indicated amounts of polyphosphate for 96 hours and counts were taken using a hemocytometer.

Values were normalized to no polyphosphate controls. All values are mean ± SEM, n > 4. *** p

<0.001 compared to SFM control (paired t-test).

F ig u r e S 3

A M o u s e Ig G B C D 3 + C C D 4 + D C D 1 6 +

E F L 4 -A C e lls o n ly F S A -6 4 7 G C D 1 4 + H + P o ly P

Figure S3. Polyphosphate binds multiple cell types in the PBMC population. PBMC were incubated with the indicated antibodies or 100 µM biotinylated polyphosphate, then incubated with fluorophore conjugated secondary antibodies or streptavidin-conjugated fluorophore. Binding populations were assessed by flow cytometry. FSC-A indicates forward scatter. Panel A is the control for the fluorophore (FL2-A) used in B, C, and D; Panel E is the cells only control and panel

F is the SA-647 only control for the fluorophore (FL4-A) used in G and H. Plots are representative of 3-4 individual experiments.

F ig u r e S 4

A B C

1 nM PolyP Female 1 nM PolyP Male 100 pM PolyP Female 1 nM PolyP * 1 nM PolyP 100 pM PolyP Male 100 pM PolyP 100 pM PolyP 10 pM PolyP Female * 10 pM PolyP Male 10 pM PolyP 10 pM PolyP 1 pM PolyP Female 1 pM PolyP Male 1 pM PolyP 1 pM PolyP ** 0.1 pM PolyP Female 0.1 pM PolyP 0.1 pM PolyP 0.1 pM PolyP Male Control Female Control Control Control Male 0 5 10 15 20 0.0 0.2 0.4 0.6 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 Directness Speed, m/min FMI

D E

1 nM PolyP Female 1 nM PolyP Female 1 nM PolyP Male 1 nM PolyP Male 100 pM PolyP Female 100 pM PolyP Female 100 pM PolyP Male 100 pM PolyP Male 10 pM PolyP Female 10 pM PolyP Female 10 pM PolyP Male 10 pM PolyP Male 1 pM PolyP Female 1 pM PolyP Female 1 pM PolyP Male ** 1 pM PolyP Male 0.1 pM PolyP Female 0.1 pM PolyP Female 0.1 pM PolyP Male 0.1 pM PolyP Male Control Female Control Female Control Male Control Male 0 5 10 15 20 0.0 0.2 0.4 0.6 0.8 Directness Speed, m/min

Figure S4. Polyphosphate alters the speed of neutrophil chemotaxis, but not directness. A-B)

The speed and directness of movement was assessed for the neutrophils followed in Figure 4.

Values are mean ± SEM, n = 6 different donors. * indicates p < 0.05, ** p <0.01 compared to the no-polyphosphate control (paired t-tests). C-E) The forward migration index, speed, and directness of neutrophil movement from Figure 4 was plotted for male and female donors. values are mean

± SEM, n = 3 different male donors. and 3 different female donors. ** indicates p <0.01 (t-test).