Fibrinogen Is a Specific Trigger for Cytolytic Eosinophil Degranulation Mackenzie E

Fibrinogen Is a Specific Trigger for Cytolytic Eosinophil Degranulation Mackenzie E. Coden, Lucas F. Loffredo, Matthew T. Walker, Brian M. Jeong, Kiwon Nam, Bruce S. Bochner, This information is current as Hiam Abdala-Valencia and Sergejs Berdnikovs of September 27, 2021. J Immunol 2020; 204:438-448; Prepublished online 9 December 2019; doi: 10.4049/jimmunol.1900932

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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Fibrinogen Is a Speciﬁc Trigger for Cytolytic Eosinophil Degranulation

Mackenzie E. Coden,* Lucas F. Loffredo,* Matthew T. Walker,* Brian M. Jeong,* Kiwon Nam,* Bruce S. Bochner,* Hiam Abdala-Valencia,† and Sergejs Berdnikovs*

In inflamed human tissues, we often find intact eosinophilic granules, but not eosinophils themselves. Eosinophils, tissue-dwelling granulocytes with several homeostatic roles, have a surprising association with fibrinogen and tissue remodeling. Fibrinogen is a complex glycoprotein with regulatory roles in hemostasis, tumor development, wound healing, and atherogenesis. Despite its significance, the functional link between eosinophils and fibrinogen is not understood. We tested IL-5–primed mouse bone marrow–derived and human blood–sorted eosinophil activity against FITC-linked fibrinogen substrates. The interactions between these scaffolds and adhering eosinophils were quantified using three-dimensional laser spectral, confocal, and transmission electron microscopy. Eosin- ophils were labeled with major basic protein (MBP) Ab to visualize granules and assessed by flow cytometry. Both mouse and human Downloaded from eosinophils showed firm adhesion and degraded up to 27 6 3.1% of the substrate area. This co-occurred with active MBP-positive granule release and the expression of integrin CD11b. Mass spectrometry analysis of fibrinogen proteolytic reactions detected the presence of eosinophil peroxidase, MBP, and fibrin a-, b-, and g-chains. Eosinophil activity was adhesion dependent, as a blocking Ab against CD11b significantly reduced adhesion, degranulation, and fibrinogenolysis. Although adhered, eosinophils exhibited no proteolytic activity on collagen matrices. Cytolytic degranulation was defined by loss of membrane integrity, cell death, and presence of cell-free granules. From transmission electron microscopy images, we observed only fibrinogen-exposed eosinophils undergoing http://www.jimmunol.org/ this process. To our knowledge, this is the first report to show that fibrinogen is a specific trigger for cytolytic eosinophil degranulation with implications in human disease. The Journal of Immunology, 2020, 204: 438–448.

ibrinogen is a complex glycoprotein with multiple regu- and atherogenesis (1). Normally, most fibrinogen is produced by latory functions in blood clotting, wound healing, cell– the liver and found in plasma. It is also known that, in cases of F matrix interactions, inflammation, tumor development, epithelial damage, plasma exudate to the area introduces fibrinogen. It is less appreciated that epithelial cells are also a source of local fibrinogen production and release. Mechanically damaged by guest on September 27, 2021 *Division of Allergy and Immunology, Department of Medicine, Northwestern † bronchial epithelial cells have been shown to release fibrinogen University Feinberg School of Medicine, Chicago, IL 60611; and Division of Pulmonary and Critical Care, Department of Medicine, Northwestern University independently of plasma proteins (2), and alveolar epithelial cells Feinberg School of Medicine, Chicago, IL 60611 were found to synthesize and release fibrinogen de novo when ORCID: 0000-0002-9411-6009 (B.S.B.). stimulated with proinflammatory mediators (3). Received for publication August 5, 2019. Accepted for publication November 11, Eosinophils are multifunctional granulocytes that are virtually 2019. absent in morphogenetically quiescent tissues but infiltrate tissues This work was supported by National Institutes of Health/National Institute of Allergy in significant numbers during morphogenesis (normal development and Infectious Diseases Grants R21AI115055 and R01AI127783 (to S.B.). Additionally, this study was supported by the Ernest S. Bazley Foundation. Proteomics services were or pathologic remodeling) and type 2 immune responses (4). performed by the Northwestern Proteomics Core Facility, generously supported by Na- Multiple reports have shown that eosinophils interact with fibrino- tional Cancer Institute (NCI) Cancer Center Support Grant (CCSG) P30 CA060553 gen in different contexts. Canine bone marrow and tissue-activated awarded to the Robert H. Lurie Comprehensive Cancer Center and the National Resource for Translational and Developmental Proteomics (supported by Grant P41 GM108569). eosinophils are specifically attracted to fibrinogen-enriched scaf- Imaging work was performed at the Northwestern University Center for Advanced folds during wound repair (5). Human airway and IL-5 stimulated Microscopy generously supported by NCI CCSG Grant P30 CA060553 awarded to blood eosinophils in asthma exhibit a hyperadhesive phenotype the Robert H. Lurie Comprehensive Cancer Center. toward fibrinogen (6). Furthermore, fibrinogen has been suggested S.B. conceived and designed the study. M.E.C., L.F.L., B.M.J., M.T.W., and S.B. performed experiments and contributed intellectually. H.A.-V. and K.N. performed as a chemoattractant for eosinophils into areas of inflammation next-generation sequencing experiments. B.S.B. provided human blood eosinophils (5). Multiple reports have shown eosinophils to interact with fi- and contributed intellectually. M.E.C. and S.B. performed bioinformatics analysis. brinogen through receptor CD11b (a b , Mac-1), which is known M.E.C., L.F.L., M.T.W., B.M.J., and S.B. analyzed data. M.E.C., L.F.L., and S.B. M 2 prepared the figures. M.E.C., L.F.L., B.M.J., and S.B. interpreted the results. The to be a central mediator of leukocyte adhesion, migration, and manuscript was written by S.B. and M.E.C. The final version of the manuscript was activation. Numerous studies have highlighted that the CD11b approved by S.B. integrin is essential for neutrophil (7), monocyte, and macrophage Address correspondence and reprint requests to Dr. Sergejs Berdnikovs, Division binding to fibrinogen, and that inhibiting the CD11b receptor will of Allergy and Immunology, Northwestern University Feinberg School of Med- icine, 240 East Huron Street, McGaw M-317, Chicago, IL 60611. E-mail address: largely prevent adhesion (8). Eosinophils have also been shown to [email protected] adhere to fibrinogen via receptor CD11b (9). The online version of this article contains supplemental material. Eosinophils are thought to contribute to disease pathological Abbreviations used in this article: ECM, extracellular matrix; GO, gene ontology; condition by releasing proinflammatory cytokines and cytotoxic MBP, major basic protein 1; MMP, metalloproteinase; PI, propidium iodide; RNA-seq, granule proteins (10). Human eosinophils contain granules rich in RNA sequencing; TEM, transmission electron microscopy; TF, tissue factor. major basic protein 1 (MBP), eosinophil cationic protein (ECP), Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 eosinophil-derived neurotoxin (EDN), and eosinophil peroxidase www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900932 The Journal of Immunology 439

(EPX). Mouse eosinophils also contain MBP and EPX (11). Al- Fibrinogen interaction assay though it is well known that human eosinophils degranulate upon FITC-labeled fibrinogen, murine (FB01; Oxford Biomedical Research) and stimulation and in inflammatory settings, mouse eosinophil de- porcine (FB04; Oxford Biomedical Research), nonlabeled murine fibrin- granulation is hard to achieve and is rarely reported in in vitro and ogen (MCI-5150; Haematologic Technologies), FITC-labeled type 1 bovine in vivo inflammation models (12). Although studies have shown collagen (C4361; Sigma-Aldrich), and nonlabeled type 1 murine collagen 3 some amount of mouse eosinophil piecemeal degranulation in (2150-1425; Bio-Rad Laboratories) were diluted to 100 mg/ml in 1 TBS, (pH 8.0) (46-012-CM; Corning). One hundred microliters per well of response to stimulation by PAF (13) and PMA (14), the evidence fibrinogen or collagen was plated on a 96-well plate (655090; Greiner Bio- of granule protein release is minor (15), and the concentration of One), which was left lightly shaking overnight at 4˚C. The following day, stimulus needed to trigger degranulation is much higher than what solutions were aspirated, and 100 ml FBS was added to each well. The is needed to degranulate human eosinophils. Cytolytic eosinophil plate was incubated for 1.5–2 h at 37˚C. Meanwhile, eosinophils were prepared in media suspensions at 1000 cells/ml in fresh media with IL-5 degranulation (a form of cytolytic cell death) occurs when eo- (10 ng/ml). Cells were incubated for 30 min with either 10 mg/ml of anti- sinophils lose membrane integrity and cell-free granules are left mouse/human CD11b clone M1/70 (101201; BioLegend) or rat IgG2b k behind (11). This form of degranulation is often found in human isotype control (400602; BioLegend) or were left untreated. After the plate allergic airway and skin disease (16–19). was incubated, FBS was aspirated, and 100 ml of eosinophil suspension Thus far, no studies have attempted to demonstrate the direct link was added to each well. After 4 h at 37˚C, cell suspensions were removed, and wells were washed two to three times with 13 TBS to remove between fibrinogen deposition and eosinophil degranulation, in nonadherent eosinophils. One hundred microliters of 2% paraformalde- part, because of poor understanding of eosinophil–tissue envi- hyde (28908; Thermo Fisher Scientific) diluted in PBS was added to each ronment interactions. This report 1) demonstrates the specific well. Recombinant MBP was from LSBio (LS-G14093) and was tested cytolytic degranulation and proteolytic activity of mouse and at 1 mg/ml in fibrinogen interaction assays. Downloaded from human IL-5–primed eosinophils adhering to fibrinogen (but not Imaging collagen) substrates from different species, 2) shows that eosino- 3 phils necessitate CD11b upregulation to adhere and degranulate Plates were imaged using fluorescent confocal microscopy at both 10 and 203 on Olympus Disk Scanning Unit Confocal Microscope (phase con- upon exposure to fibrinogen, 3) shows that fibrinogen (and, to trast, FITC channel) or a Keyence BZ-X800 Microscope, laser spectral some extent, type I collagen) triggers the expression of proin- microscopy at 203 on Nikon A1R Laser Scanning Confocal microscope

flammatory and remodeling (IL-13, IL1a, IL-4, Tnf, Ccl24, and (Nikon Imaging Core, Northwestern University), and/or transmission http://www.jimmunol.org/ versican) mediators from murine eosinophils, 4) provides evi- electron microscopy (TEM) on the FEI Tecnai Spirit G2 (Nikon Imaging Center, Northwestern University, Chicago, IL). At least two to four rep- dence that fibrinogen elicits higher metabolic activity (NADH resentative field images were taken per well to assess fibrinogen degra- production) in eosinophils compared with collagen substrates, dation and eosinophil granule dispersion. For MBP immunofluorescence and 5) provides a methodological platform for further studies staining, plates were fixed with 2% paraformaldehyde for 15 min at room aimed at manipulating, visualizing, and quantifying eosinophil– temperature. Samples were then blocked with 5% goat serum and 0.3% 3 matrix interactions. Triton X-100 in 1 PBS for 1 h before incubating with primary Ab diluted in Ab dilution buffer (1% BSA and 0.3% Triton X-100 in 13 PBS). Af- terward, samples were incubated with secondary Ab diluted in the same buffer. Primary staining was with rat anti-mouse MBP (the Lee labo-

Materials and Methods by guest on September 27, 2021 ratories, Mayo Clinic, Phoenix, AZ) at a 1:500 dilution, and secondary Mouse eosinophils staining was with goat anti-rat IgG Texas Red (T6392; Life Technologies) Murine eosinophils were cultured from bone marrow according to a pub- at a 1:500 dilution. DRAQ5 (BioLegend) staining was done in at a 1:200 3 lished protocol (20). In brief, bone marrow was harvested from 10–12-wk-old dilution in 1 PBS for 15 min prior to assay. DAPI (4083; Cell Signaling 3 wild-type BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) by Technologies) staining was performed on fixed cells at 1 mg/ml in 1 PBS flushing femurs and tibiae using RPMI 1640 medium with L-glutamine for 5 min. For TEM, samples were fixed with 2% paraformaldehyde and (10-040-CV; Corning). Cells were cultured in media containing RPMI 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer prior to embed- 1640 with L-glutamine, 1% Penicillin/Streptomycin (SV30010; HyClone), ding in a resin mixture of EMbed 812 kit. Samples were then sectioned on 1% L-glutamine (SH30034.01; HyClone), 1% MEM Nonessential Amino a Leica Ultracut UC6 Ultramicrotome and collected on 200 mesh copper Acids (25-025-CI; Corning), 1% Sodium Pyruvate (25-000-CI; Corning), grids and stained with 3% uranyl acetate and Reynold’s lead citrate. 2.5% HEPES (25-060-CI; Corning), 20% heat-inactivated FBS (35-016-CV; Corning), and 0.0000035% 2-ME (M6250; Sigma-Aldrich). Cell media was Quantification of fibrinogen degradation supplemented with murine 100 ng/ml recombinant stem cell factor (250-03; ImageJ (22) was used to quantify degraded fibrinogen area on two- PeproTech) and 100 ng/ml rFLT3-L (250-31L; PeproTech) from days 0 to 4. dimensional images taken with fluorescent confocal microscopy. Images On day 4, media was replaced with fresh media containing 10 ng/ml murine were converted to greyscale and cropped to the lower right quadrant rIL-5 (215-15; PeproTech). Media containing IL-5 was changed every other (250 3 250 pixels). Threshold was adjusted such that only the degraded day until day 14, when cells were used for experimentation. Flow cytometry area pixels were selected, and the particle analysis tool was used to cal- and cytospins were used to confirm maturation and purity of day 14 cells culate both the total percentage selected and the diameter of each selected using a Diff-Quik Staining Kit (2609650; Electron Microscopy Sciences). area. Areas touching the field edges were excluded. Human eosinophils Flow cytometry Human eosinophils were obtained from the laboratory of Dr. B. Bochner Cell suspensions from culture or cells from fibrinogen interaction assays (Northwestern University Institutional Review Board), which were isolated [cells from supernatants combined with cells detached from fibrinogen following a published protocol (21). Briefly, 60 ml of 0.1 M EDTA- surface with Trypsin/EDTA (CC-5012; Lonza)] were stained with a Zombie anticoagulated peripheral blood was obtained from healthy donors. 3 Aqua Fixable Viability Kit (423102; BioLegend) for 20 min at room Blood was diluted with two volumes of 1 PBS, and eosinophils were temperature, blocked with anti-CD16/CD32 (553142; BD Biosciences) for isolated by Percoll density gradient centrifugation using 40 ml of diluted 10 min in flow-staining buffer at 4˚C, and incubated with either APC–Cy7- blood and 10 ml of room temperature Percoll PLUS (density 1.090) 3 conjugated Siglec-F (561039; BD Biosciences) or AF647-conjugated (17544502; GE Healthcare), centrifuging for 20 min at 300 g at room CD11b (562680; BD Biosciences) in flow-staining buffer for 30 min at temperature, which concentrated and removed mononuclear cells (lym- 4˚C. Cells were washed in 13 PBS and fixed in 2% paraformaldehyde. phocytes and monocytes) and basophils at the plasma–Percoll interface. Samples were run on an LSR II flow cytometer (BD Biosciences), and data In the remaining pellets, erythrocytes were removed by hypotonic lysis in were analyzed on FlowJo v10 software (Tree Star, Ashland, OR). ice-cold water, and neutrophils were removed by immunomagnetic se- lection using a CD16 MicroBead Kit (130-045-701; Miltenyi Biotec). Mass spectrometry Resulting eosinophil purity was confirmed by Shandon Kwik-Diff Staining (9990700; Thermo Fisher Scientific). Once purified, eosinophils were Supernatants from fibrinogen interaction assays were removed from plates, primed with 30 ng/ml of IL-5 (R&D Systems) overnight prior to assay. and cells were spun out at 300 3 g for 5 min. Proteins were purified by 440 FIBRINOGEN-TRIGGERED DEGRANULATION OF EOSINOPHILS acetone/TCA precipitation, reduced, and alkylated. Digested peptides were and segmented nuclear morphology (Fig. 1A). These cells desalted on C18 spin columns prior to analysis. MS1 peptide intensity was were .99% pure on day 14, as determined by Siglec-F expres- measured by searching the data against a mouse database using MaxQuant sion (Fig. 1C). Wells coated with FITC-linked fibrinogen substrate database search engine and label-free quantification pipeline. Proteomic analysis was performed by the Northwestern University Proteomics Core could be visualized using fluorescent confocal microscopy and Facility (Robert H. Lurie Comprehensive Cancer Center). maintained a homogeneous coating in the absence of eosinophils (Fig. 1D). The addition of eosinophils resulted in significant fi- Quantitative PCR brinogen degradation (Fig. 1E, dark areas). Three-dimensional im- RNA was isolated from cells using the QIAGEN RNeasy Mini Kit (74136; aging confirmed the complete fibrinogen scaffold loss in these QIAGEN). cDNA was synthesized using a qScript cDNA Synthesis Kit areas (Fig. 1F). Furthermore, MBP staining shows that eosinophils (95047; Quanta BioSciences) and analyzed by real-time PCR on a 7500 Real-Time PCR System (Applied Biosystems) using primers/probes from and eosinophil granules are localized to areas of fibrinogen loss Integrated DNATechnologies and PrimeTime Gene Expression Master Mix (Fig. 1F). This fibrinogen degradation was dependent on eosino- (1055771; Integrated DNA Technologies). phil intracellular activity, as cells that had been fixed with 2% RNA sequencing paraformaldehyde prior to assay did not degrade the substrate (Supplemental Fig. 1). All cells were lysed, and RNA was extracted using the RNeasy Plus Mini Extraction Kit (74136; QIAGEN). RNA quality was assessed using an Fibrinogen degradation occurs in a species-specific manner Agilent High Sensitivity RNA ScreenTape system (Agilent Technologies). and increases with cell density The NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) was used for full-length cDNA synthesis and library preparation. We developed an image analysis protocol to quantify the degree to

The sequencing of cDNA libraries was done on an Illumina NextSeq which eosinophils degrade the fibrinogen substrate under different Downloaded from ∼ 500 instrument (Illumina) at a target read depth of 10 million aligned reads experimental conditions. By using ImageJ, we first converted the per sample. The pool was denatured and diluted to create a 2.5 pM DNA solution. The PhiX control was spiked at 1%. The pool was then sequenced fluorescent image to black and white and then quantified the total by 1 3 5 cycles using the NextSeq 500 High-Output Kit (Illumina). degraded area, represented as the percentage of the image that was degraded (black) relative to that which remained intact (white) Eosinophil viability testing (Fig. 2A). Using this technique, we were able to quantify eosin-

Characterization of eosinophils as live or necrotic was determined by FITC ophil proteolytic activity as percent area of degraded fibrinogen. http://www.jimmunol.org/ Annexin V Apoptosis Detection Kit with Propidium Iodide (PI; 640914, The percent area degraded increased significantly with number BioLegend) for the nonadherent fraction or trypan blue dye exclusion test of cells plated (Fig. 2B, 2C). Fibrinogenolysis occurred in a (T10282, Invitrogen) for the adherent fraction. After 4 h of assay, nonadherent eosinophils were removed and stained with annexin V and PI and species-specific manner (Fig. 3). Mouse eosinophil fibrinogeno- analyzed via flow cytometry. Annexin V(2)PI(2) eosinophil populations lytic activity was the highest on mouse fibrinogen substrate, and (live cells) were determined by nonstained controls. Adherent eosinophils human eosinophils had higher activity on porcine fibrinogen than were stained in situ with trypan blue dye diluted 1:1 with cell media. mouse eosinophils. However, the data also showed significant Samples were visualized on a Life Technologies EVOS XL Core microscope at 103. Multiple fields of view within each well were counted to species cross-reactivity, revealing the conserved nature of this determine the number of cells that stained blue (dead) relative to those eosinophil–fibrinogen interaction (Fig. 3). with intact membranes that did not take up the dye (live). by guest on September 27, 2021 Fibrinogen induces cytolytic eosinophil degranulation Biolog metabolism assay The majority of eosinophil-degraded fibrinogen areas were ,9 mm To determine metabolic differences, 20,000 eosinophils per well were in diameter, and only a small percentage corresponded to areas seeded onto blank 96-well Biolog plates (30311; Biolog) coated with between 9 and 12 mm, or the size of a single intact eosinophil fibrinogen or collagen in media made with phenol red-free RPMI 1640 (17-105-CV; Corning). Biolog Redox Dye MB (74352; Biolog) was (Fig. 4A). This suggested that something other than the cell itself, added, and cells were then incubated in an OmniLog automated incubator/ possibly eosinophil granule proteins, was creating the smaller reader (12111; Biolog, Hayward, CA) at 37˚C. Dye reduction (measuring holes. Further evidence for eosinophil degranulation can be seen NAD[P]H-dependent oxidoreductase activity) was measured at 590 nm in the detection of MBP-positive staining (red) in all degraded absorbance. Kinetic background values were manually subtracted, and initial rates were measured between the beginning of each run and when areas, suggesting that MBP-rich granule release from cells is the uptake reached its maximum (between 0.25 and 1.25 h). followed by substrate degradation (Fig. 4B). As additional con- firmation of degranulation, we performed proteomic analysis of the Bioinformatical and statistical analysis products released in the fibrinogen assay supernatants (after re- RNA-sequencing (RNA-seq) reads were demultiplexed using bcl2fastq moving the cellular fraction). Using untargeted mass spectrometry (v 2.17.1.14). Quality control was performed using FastQC. Low-quality analysis, we detected significant levels of degranulation products reads were discarded using trimmomatic (v 0.33). Reads were aligned MBP and EPX in assay supernatants, confirming that eosinophils using Spliced Transcripts Alignment to a Reference. Read counts were generated using HTSeq. Differential expression analysis was done using are degranulating upon exposure to fibrinogen (Fig. 4C left, also the DESeq2 R/Bioconductor package (23). All computational analysis was Supplemental Table I). Moreover, proteome analysis also showed performed on genomic nodes of Quest, Northwestern’s High Performance the presence of fibrin monomers when eosinophils were allowed to Computing Cluster. Gene ontology (GO; biological process) analysis was interact with fibrinogen, showing that fibrinogen is being broken performed in GOrilla (24). Statistical significance of all data were determined by t tests or ANOVA tests, followed by Tukey post hoc pairwise down into its fibrin subunits (a-, b-, and g-chains) (Fig. 4C right) testing, or Mann–Whitney or Kruskal–Wallis tests, followed by Dunn and supporting eosinophil-mediated fibrinogenolysis. multiple comparisons test whenever data did not fit parametric test criteria. We performed TEM on eosinophils exposed to either fibrinogen All data are represented as mean 6 SEM. Statistical analysis was per- or a control collagen substrate to ensure that substrate degradation formed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA). An by eosinophils and cytolytic degranulation was specific to fibrin- a level of 0.05 was used as a significance cutoff. ogen and not a general property of adhering eosinophils. When activated eosinophils were exposed to collagen substrates, they Results retained typical eosinophil morphology (Fig. 4D). These cells Eosinophils degrade fibrinogen substrate appeared intact with visible granules inside the cells (Fig. 4D After the 14 d culture protocol (Fig. 1A, 1B), eosinophils showed arrows). Eosinophils exposed to fibrinogen had a different ap- a mature morphology, as identified by red eosin–stained granules pearance than the collagen-exposed eosinophils. One form we The Journal of Immunology 441 Downloaded from http://www.jimmunol.org/

FIGURE 1. Bone marrow–derived murine eosinophils degrade fibrinogen substrates. (A) Day 14 IL-5–cultured eosinophils stained with a Diff-Quik Staining Kit and photographed with a 603 oil objective. Note mature red granular morphology. (B) Schematic showing timeline of bone marrow–derived murine eosinophil culture protocol. (C) Purity of cultured eosinophils, all cells were .99% Siglec-F positive (right) by nonstained control (left). by guest on September 27, 2021 (D) FITC-linked fibrinogen substrate (green) without eosinophil addition visualized using fluorescent confocal microscopy (original magnification 310). The substrate maintains a homogenous composition when eosinophils are not added, as is seen in this image. (E) Dark spots represent areas of degraded fibrinogen after addition of eosinophils (original magnification 310). (F) Dark spots depict areas of total fibrinogen degradation, as visualized by using three-dimensional (3D) stack images. Anti-MBP–stained cells (red) localize to degraded areas and completely penetrate fibrinogen (original magnification 320). Bottom two images are top images magnified; arrows point to anti-MBP–stained cells penetrating the fibrinogen substrate. Note that cells tend to be smaller than the surrounding empty areas. observed was intact cells that were exceptionally granule packed two different species, murine and bovine, were similarly inactive (Fig. 4E, arrows pointing to granules). The other form we ob- relative to fibrinogen exposure (Supplemental Fig. 2). We found served was eosinophils with fragmented membranes and cell- that, unlike in the fibrinogen interaction, eosinophils exposed to free granules (Fig. 4F, 4Giii). We were able to observe eosino- collagen will adhere to, but do not degrade, the substrate. This phils in various stages of degranulation on the fibrinogen sub- shows that it is a fibrinogen-specific process (Fig. 5A). Further- strate, as demonstrated in the image sequence in Fig. 4G. From more, bright field images showed that eosinophils adherent to left to right, we visualized the following: 1) intact eosinophils collagen have a rounded cell-intact phenotype as opposed to those packed with granules leaving behind areas of visibly degraded adhered to fibrinogen (Fig. 5B). This adds evidence supporting substrate (Fig. 4Gi arrow), 2) followed by initial loss of membrane that eosinophils adherent on collagen remain fully intact and are integrity beginning from the center of the cell (Fig. 4Gii arrow). 3) not degranulating. Eosinophils more readily adhere to fibrinogen Next, we note the presence of cell-free granules, often with circular than collagen, as seen by the larger percentage of cells recovered patterns in the middle (Fig. 4Giii red arrow). 4) Finally, we would (the nonadherent cell fraction) after 4 h of incubation (Fig. 5B). observe a “suction-cup” imprint of degranulated eosinophils on the In analyzing the two fractions, adherent and nonadherent, we fibrinogen substrate with few cell remnants, again with a circular observed no difference in the viability of the nonadherent fraction pattern in the middle (Fig. 4Giv arrow). By all indications, eosin- after 4 h of substrate exposure, but eosinophils exposed to fibrino- ophils exposed to fibrinogen substrate in our assays undergo cyto- gen showed lower viability after 4 h than eosinophils exposed to lytic degranulation. collagen (Fig. 5C). Although eosinophils constitutively express CD11b, those ex- Eosinophil degranulation and proteolytic activity are specific posed to fibrinogen display a significant upregulation of this to fibrinogen and are CD11b dependent integrin (Fig. 5D). Blocking CD11b receptors on eosinophils To further understand the interaction between eosinophils and prior to adding them to fibrinogen resulted in a significantly di- fibrinogen, we compared eosinophil exposure to collagen and minished amount of substrate degradation compared with eosin- fibrinogen substrates. Eosinophils exposed to collagen from ophils treated with isotype control Ab (Fig. 5E). Interestingly, 442 FIBRINOGEN-TRIGGERED DEGRANULATION OF EOSINOPHILS

FIGURE 2. Quantification of fibrinogen degradation by eosinophils. (A) Original image used for quantification using ImageJ (left), thresholded version of image within ImageJ (middle), and quantification view of the image within ImageJ (right). Black outlines demarcate areas where degradation occurred, red Downloaded from numbers quantify number of areas of degradation. (B) Fibrinogen substrate (green) exposed to increasing numbers of eosinophils. Images are representative of at least two independent experiments. (C) Quantification of images from part (B) using ImageJ strategy from (A). Fields from three replicates were counted per condition from two independent experiments. **p , 0.01 by http://www.jimmunol.org/ Kruskal–Wallis. by guest on September 27, 2021

anti-CD11b–treated eosinophils appear to be capable of remov- in faint dark spots on the FITC-linked substrate as opposed to the ing ﬁbrinogen, but to a much lesser extent than isotype control much darker spots seen in the isotype control condition. Faint spots eosinophils. This can be seen, as the anti-CD11b condition resulted represent the shallow removal rather than complete penetration of

FIGURE 3. Eosinophils process fibrinogen in a species-specific manner. (A) Human IL-5–primed blood eosinophils on porcine fibrinogen substrate. (B)Murine bone marrow–derived eosinophils on porcine fibrinogen. (C) Murine bone marrow–derived eosinophils on murine fibrinogen. (D) Quantification of percentage area of fibrinogen degradation across different species. Six fields were counted per condition from at least two independent experiments for mouse eosinophils. One experiment was conducted using human eosinophils in triplicate. ****p , 0.0001 by ANOVA. HEos, human eosinophil; MEos, mouse eosinophil; MFg, mouse fibrinogen; PFg, porcine fibrinogen. The Journal of Immunology 443 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Cytolytic degranulation of eosinophils adhering to fibrinogen. (A) Diameter of fibrinogen-degraded areas versus their frequency. Red interval represents the diameter of a single intact eosinophil (9–12 mm); blue corresponds to smaller areas fitting the diameter of eosinophil granules (,9 mm). Most degraded areas on images correspond to the size of eosinophil granules. (B) MBP-stained murine eosinophils interacting with fibrinogen scaffold. Large red spots are intact eosinophils; smaller red dots are eosinophil granules. The arrow points to a spot the size of an intact eosinophil. Images are representative of at least two independent experiments. (C) Eosinophil degranulation products detected in mass spectrometry analysis of assay supernatants (left) and fibrinogen degradation products present in assay supernatants (right). Samples were analyzed in duplicate. Fg alone, fibrinogen substrate without eosinophils added to assay; Fg + Eos, fibrinogen substrate with eosinophils added to assay. (D–F) TEM images of eosinophils. Green arrows point out compact rod-like granules with electron lucent crystalline cores. Blue arrows denote representative eosinophil granules lacking a detectible crystalline core. Red arrows denote loss of membrane integrity beginning at the center of the eosinophil. (D)Rep- resentative image of an activated eosinophil on collagen control substrate with an intact cytoplasmic membrane. (E) Activated eosinophil on fibrinogen substrate. Note the increase in number and size of granules. (F) Cell-free granules on fibrinogen substrate. This image shows loss of membrane integrity characteristic of cytolytic degranulation. (G) Degranulation of eosinophils on fibrinogen substrates via cytolytic degranulation. (i) Intact eosinophil with a trail of degraded substrate behind it, noted by the black arrow. (ii) Granule-packed eosinophil with a center hole be- ginningtoforminthemembrane.(iii) Cell-free granules with membrane fragments exhibiting a circular pattern in the center. (iv) Suction-cup imprint left on the fibrinogen substrate with a circular pattern in the center.

fibrinogen in those areas (Fig. 5E). Furthermore, eosinophils some part, responsible for eosinophil adherence to fibrinogen and treated with anti-CD11b do not adhere to fibrinogen as readily as that activation of the CD11b receptor by fibrinogen is necessary controls. This supports the notion that CD11b is, at least, in for complete eosinophil degranulation in this system. 444 FIBRINOGEN-TRIGGERED DEGRANULATION OF EOSINOPHILS

FIGURE 5. Eosinophil proteolytic activity is specific to fibrinogen and is CD11b dependent. (A) Fluorescent images of FITC- linked type I collagen substrate remaining intact and fibrinogen substrate being degraded (green, substrate; black, absence of substrate). Eosinophils can be visualized by DRAQ5 staining (red). Images are representative of at least six independent experiments. Quantification of area degraded on collagen and fibrinogen substrates (right). ****p , 0.0001 by ANOVA. (B) Repre- sentative brightfield images of eosinophils adhered to collagen control substrate and fibrinogen substrate after washing. Bar graph on right quantifies percentage of cells recovered after washing on collagen and fibrinogen. Images and graph are representative of three separate experiments. ****p , 0.0001 by t test. (C) Viability of eosinophils that are nonadherent (left) or adherent (right) Downloaded from after 4 h of incubation on fibrinogen or collagen. Graphs are representative of four separate experiments. **p , 0.01 by t test. (D) Histogram quantifying CD11b receptor levels on eosinophils exposed to substrates by flow cytometry (left), and bar graphs http://www.jimmunol.org/ showing the median fluorescence intensity of cells exposed to substrates (right). Graphs are representative of two independent experiments. *p , 0.05 by Kruskal– Wallis. (E) Images of fibrinogen substrate when eosinophils were blocked with anti- CD11b or isotype control Ab (left). Arrows point to degraded areas. Eosinophils can be visualized by DAPI staining (blue). Images are representative of three independent ex- by guest on September 27, 2021 periments. First bar graph quantifies degraded area between the two treatments (middle). Second bar graph quantifies percentage of cells recovered after washing on fibrinogen with different Ab treatments (right). *p , 0.05, **p , 0.01 by Kruskal– Wallis, ****p , 0.0001 by Mann–Whitney U test.

Shared and unique eosinophil gene expression profiles elicited dominated by each substrate and found that fibrinogen-exposed by adhesion to fibrinogen versus type I collagen cells were downregulating processes related to primary metabolic To further interrogate the biological processes taking place in processes and cell migration, whereas those exposed to collagen eosinophils exposed to fibrinogen versus collagen, we performed were downregulating processes related to biosynthetic process, RNA-seq analysis on eosinophils isolated from our assays. Two cellular metabolic process, translation, and cell cycle transi- tions. We performed similar analysis for upregulated genes hundred and seventy-six eosinophil genes were differentially and found that 50.1% genes were exclusive to fibrinogen and regulated between cells exposed to fibrinogen and those exposed to 38.8% exclusive to collagen (Fig. 6C). However, the consensus- collagen (113 upregulated and 163 downregulated) (Fig. 6A). upregulated signature, at only 11.1% of the overall gene signature, When eosinophils adhered to fibrinogen were compared with eo- showed a striking alignment in inflammatory response genes, sinophils without substrate exposure, 746 genes were significantly such as the upregulation of Il-4, Il-13, Cxcl2, and Cxcl3. differentially expressed (471 upregulated and 275 downregulated). Furthermore, specific collagens, Col11a2 and Col20a1, and Interestingly, the same was true for eosinophils interacting with Serpinb2 showed consensus upregulation with fibrinogen and type I collagen: 770 genes were significantly differently expressed collagen interaction (Fig. 6D). To further examine differences in (545 upregulated and 225 downregulated) compared with eosin- metabolic processes from the GO analysis, we performed a live ophil no-substrate controls. We further compared these two dif- cell–functional Biolog metabolism assay on eosinophils and ferential expression lists (no substrate versus fibrinogen and no found that eosinophils interacting with fibrinogen had signifi- substrate versus collagen) and found that 26.5% downregulated cantly higher metabolic activity than those interacting with genes were unique to fibrinogen, and 36.3% downregulated genes collagen (Fig. 6E), confirming that there is a substantial meta- were unique to collagen. The remaining 37.2% were shared by the bolic difference triggered by substrate exposure. To validate two (Fig. 6B). We examined which GO biological processes were genes from the RNA-seq analysis, quantitative PCR analysis was The Journal of Immunology 445 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. Differential gene expression in eosinophils interacting with fibrinogen versus collagen. (A) Volcano plot showing differential gene expression analysis of RNA-seq data comparing eosinophils exposed to collagen versus those exposed to fibrinogen. Red represents genes with adjusted p values ,0.05. (B) Venn diagram comparing the downregulated gene signatures from eosinophils without substrate exposure to those exposed to either collagen or fibrinogen (center). Bar graphs on the left and right show GO biological processes dominated by fibrinogen (blue) or collagen (yellow). (C) Venn diagram and GO biological processes of the upregulated genes from the data shown in (B). (D) Heat map showing differential gene regulation of the consensus-upregulated signature from part (C)thatcorrespondwithgenesrelatedtoinflammatory response (left) and select remodeling genes identified in the bar graphs on the right. n = 4 replicates for no substrate exposure; n = 3 replicates for collagen and fibrinogen exposure. ***p , 0.001, ****p , 0001. C, collagen substrate; E, eosinophils without substrate; F, fibrinogen substrate. (E) Bar graph showing initial rate of reaction in a Biolog metabolic assay of eosinophils exposed to fibrinogen versus collagen. ***p , 0.001 by t test. 446 FIBRINOGEN-TRIGGERED DEGRANULATION OF EOSINOPHILS run on four separate experiments. In each experiment, two to Eosinophil activity in our assays was entirely adhesion dependent. four replicates per treatment were averaged to determine DDCT This is in line with previous reports showing that human eosino- values. This analysis validated the significant expression of in- phils exhibit a CD11b-dependent hyperadhesive phenotype toward flammatory genes in eosinophils activated by fibrinogen. Ex- fibrinogen in asthma (6) and degranulate in a CD11b-adhesion- pression of these genes triggered by collagen showed a similar dependent manner (25, 26). In humans, ECP and EDN granule trend, although at a lower fold change level and not reaching product release is also adhesion dependent and has been demon- statistical significance in most cases (Fig. 7). strated to increase upon binding to fibrinogen with or without IgG (27, 28). Likewise, in mice, we detected MBP and EPX Discussion release in the supernatants of cultures in which eosinophils were Unlike human eosinophils, murine eosinophils are notoriously allowed to adhere to fibrinogen. Similarly, a previous report difficult to degranulate in vitro and show only piecemeal de- showed that eosinophils from mice will not degranulate until granulation in response to inflammatory stimuli (15). Higher they have adhered to the extracellular matrix (ECM) (14). In concentrations of exogenous stimuli are typically necessary to keeping with these findings on the necessity of adhesion, our induce even piecemeal degranulation of mouse eosinophils when results show that the ability of murine eosinophils to degranulate compared with studies of human cells. To our knowledge, this is and degrade fibrinogen also acts through a CD11b-adhesion- the first report to demonstrate both visually and quantitatively that dependent manner. When eosinophils were treated with anti- adhesion to fibrinogen is sufficient to trigger potent cytolytic de- CD11b Ab prior to assay, we observed a significant decrease granulation of murine IL-5–primed eosinophils. This confirms the in percentage of adherent cells and fibrinogen degradation rel- necessity of a “two-hit” system: the presence of both immune and ative to nonblocked controls. Interestingly, fibrinogen degrada- Downloaded from specific tissue adhesive cues to trigger degranulation. tion was not entirely ablated by treatment with anti-CD11b, as http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 7. Quantitative PCR (qPCR) validation of select RNA-seq genes. Data presented as DDCT of gene values compared with no substrate controls. TBP was used as a housekeeping gene. Cells were either exposed to fibrinogen, collagen, or no substrate. Values were calculated as fold change relative to no substrate control. Each data point represents an average expression value of two to four replicates within a single experiment. Three to four independent experiments are summarized in each graph. *p , 0.05, **p , 0.01, ***p , 0.001 by one-tailed t tests. The Journal of Immunology 447 faint dark spots were left on fibrinogen and some smaller per- of factors are activated, leading to the conversion of fibrinogen centage of eosinophils did still remain adherent to the substrate to fibrin by activated thrombin. Tissue factor (TF), a molecule (Fig. 5E). They may have been able to dock via a different released in response to tissue damage, is released upstream of integrin, such as CD11c, which has also been shown to adhere thrombin activation. Work by Moosbauer et al. (35) showed that eosinophils to fibrinogen (9). blood eosinophils produce and can release TF, which suggests Interestingly, in our RNA-seq analysis, we did not find ex- that eosinophils play an upstream role in thrombin activa- pression of metalloproteinases (MMPs) or other proteases in eo- tion. Furthermore, eosinophils can generate thrombin in a sinophils interacting with fibrinogen substrates. MMPs are known TF-dependent manner when exposed to a procoagulant phos- to be produced by eosinophils (12) and may promote pathologic pholipid surface enriched in 12/15 lipoxygenase-derived oxi- changes in asthmatic airways by degrading the ECM. Specif- dized phospholipids (36). ically, MMP-9 has been shown to be overexpressed by eosino- There could be other modalities of eosinophil–fibrinogen in- phils from asthmatic patients (29), and MMP-9 levels are teraction, as studies have shown that fibrinogen can be deposited significantly increased in individuals with severe asthma relative in the ECM without proteolytic processing in nonvascular injury to those with mild or moderate asthma (30). It is worth noting contexts (37, 38). In particular, extrahepatic fibrinogen produced that this protein, when produced in eosinophils, has been found by the lung and intestinal epithelium during inflammation is to localize to perinuclear spaces, but not granules (29). Not thought to play a critical role during localized repair and remod- finding protease expression in our assays gives credence to the eling to restore tissue homeostasis (39–41). Disordered coagula- fact that the fibrinogen degradation seen in this study is not due tion and fibrinogen and thrombin accumulation in the airway is a to de novo synthesis of MMPs or other ECM modulatory pro- feature of human disease and can contribute to airway hyper- Downloaded from teins, such as cathepsins or ADAMs. We also conclude that responsiveness, remodeling, and decline in lung function (42–44). free MBP is not sufficient to trigger the potent fibrinogenolysis Compartmentalization of eosinophil recruitment and degranula- observed in our assays (Supplemental Fig. 3). It is possible tion in human and mouse models of airway disease may provide that another prestored proteolytic mediator, likely released in a additional clues to the existence and significance of this tissue- contact-dependent manner (visualized by TEM) at lower con- driven interaction. After segmental airway challenge in human

centrations, may account for substrate removal prior to or during asthma, only airway (but not blood) eosinophils show the hyper- http://www.jimmunol.org/ degranulation. adhesive phenotype for fibrinogen and other ECM proteins (6). There are three different major modes of release of eosinophil The same is true for mouse models of asthma (14). In support, we granule content: classic exocytotic degranulation, piecemeal de- previously found that the integrin profile of eosinophils in a mouse granulation (previously reported in mice), and cytolysis with the model of asthma changes upon trans-epithelial transition to the release of cell-free granules (11). In this report, we confirmed the airway, including upregulating CD11c and CD11b, receptors for presence of cell-free granules from eosinophils exposed to fi- fibrinogen (9). Moreover, airway eosinophils in asthma patients brinogen via electron microscopy. Cell-free granules have been with increased hyperresponsiveness express urokinase-type plas- reported to retain an intact outer membrane and can secrete their minogen activator receptor (uPAR; CD87) (45), which may also granular content after activation by other stimuli (17, 31). Re- support a role for eosinophils in tissue fibrinogen/fibrin processing by guest on September 27, 2021 markably, TEM indicated that several events precede cytolytic in disease. Interestingly, plasminogen-deficient mice show a dra- degranulation: 1) movement of eosinophils around substrate ac- matic reduction in the number of airway eosinophils in a murine companied with some level of substrate removal, 2) initiation of model of asthma (46). Although fibrinogen and type I collagen loss of membrane integrity from the center of the adhered eo- deposition both take place in allergic asthma (47, 48), we found sinophil, 3) complete loss of cellular membrane integrity and that the responses to two different substrates can be different. deposition of cell-free granules, and 4) typical suction-cup imprint Eosinophils degranulate only when adhering to fibrinogen, al- of adhered eosinophil on substrate supporting necessity for focal though both substrates may induce eosinophil inflammatory re- adhesion. This suggests that the induction of cytolytic degranu- sponses to various degrees. This could also be associated with the lation by fibrinogen is a tightly regulated process. Additionally, we spatiotemporal compartmentalization of these interactions in tissues. detected MBP and EPX granular protein release in assay super- Specific substrates could be deposited in different tissue compart- natants. Interestingly, this very closely resembles a novel form of ments (fibrinogen in the airway and collagen in the basement cell death and degranulation termed pyroptosis, which is charac- membrane) or represent different stages of remodeling. terized as caspase-1–mediated cell death with accompanying me- In summary, our data show that both mouse and human eo- diator release. This mode of degranulation was recently reported for sinophils promote fibrinogenolysis via CD11b-dependent adhe- mouse eosinophils infiltrating liver tissue in response to necrotic sion to fibrinogen. Moreover, we demonstrated that fibrinogen is, liver injury (32). Although the degranulation trigger was not so far, the only known trigger of cytolytic degranulation of murine identified in that study, fibrinogen is a likely candidate given that eosinophils, which features the release of free eosinophil granules the liver is a major and constitutive source of this protein (33). and eosinophil granular proteins. Eosinophil activity toward fi- This manner of free eosinophil granule deposition and subsequent brinogen in our assays is consistent with eosinophil biology in content release is a prominent feature of human allergic airway wound healing and the tissue remodeling environment of a and skin disease (16–19). Therefore, research elucidating the chronic inflammatory disease. The participation of eosinophils in triggers of cell-free granule release was identified as a high pri- fibrinogen processing and tissue remodeling warrants further ority by the National Institutes of Health Taskforce on the Re- investigation, as this has the significant potential to advance our search of Eosinophil-Associated Diseases (34). understanding of tissue eosinophil function and heterogeneity in Such dramatic engagement of eosinophils by fibrinogen raises health and disease. several important questions. Where do such interactions take place in an organism? Which biological processes necessitate fibrinogen Acknowledgments processing by eosinophils in health and disease? There is evidence We thank Yun Cao (Northwestern University) for help in preparing human that eosinophils have the ability to produce various molecules eosinophils. We also thank Dr. Dina Arvanitis, manager of the Nikon Im- associated with coagulation. In the coagulation cascade, a series aging Center (Northwestern University) and Dr. Farida Korobova, 448 FIBRINOGEN-TRIGGERED DEGRANULATION OF EOSINOPHILS microscopy specialist in the Nikon Imaging Center (Northwestern Univer- 23. Love, M. I., W. Huber, and S. Anders. 2014. Moderated estimation of fold sity) for assistance in visualizing fibrinogen–eosinophil interactions. change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15: 550. 24. Eden, E., R. Navon, I. Steinfeld, D. Lipson, and Z. Yakhini. 2009. GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. 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Airway fibrinogenolysis and the initiation of allergic in high purity from normal mouse bone marrow. J. Immunol. 181: 4004–4009. inflammation. Ann. Am. Thorac. Soc. 11(Suppl. 5): S277–S283. 21. Akuthota, P., R. Shamri, and P. F. Weller. 2012. Isolation of human eosinophils. 48. Nomura, A., Y. Uchida, T. Sakamoto, Y. Ishii, K. Masuyama, Y. Morishima, Curr. Protoc. Immunol. 98: 7.31.1–7.31.7. K. Hirano, and K. Sekizawa. 2002. Increases in collagen type I synthesis in 22. Schneider, C. A., W. S. Rasband, and K. W. Eliceiri. 2012. NIH Image to asthma: the role of eosinophils and transforming growth factor-beta. Clin. Exp. ImageJ: 25 years of image analysis. Nat. Methods 9: 671–675. Allergy 32: 860–865. Figure S1. Images comparing lack of substrate degradation when fixed eosinophils were added to fibrinogen substrate for 4 hours (left) with substrate degradation from live eosinophils (right) Figure S2. A) Bar graphs showing gene expression comparison between bovine and murine collagen relative to murine fibrinogen. *p<0.5, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA. B) Biolog metabolic assay comparing metabolic activity of eosinophils exposed to murine fibrinogen vs eosinophils exposed to murine collagen. **p<0.01 by t-test. Figure S3. A) Bar graph showing fluorescence intensity of FITC-linked fibrinogen exposed only to cell media control vs. cell media plus 1 μg/mL rMBP. p=0.0628 by t- test, n=4. B) Visualization of addition of eosinophils with no treatment (left) as compared to those treated with 1 μg/mL rMBP (right). Images representative of 3 replicates, from which at least 4 fields were imaged.

Mass spectrometry data comparing fibrinogen background control (no eosinophils added) to fibrinogen plus eosinophils Fibrinogen only control (no eosinophils added) Fibrinogen plus eosinophils Protein IDs Protein names Gene names Peptides Unique peptides Sequence coverage [%] Mol. weight [kDa] LFQ intensity NoEos LFQ intensity NoEos LFQ intensity Eos LFQ intensity Eos C0HKE9;C0HKE8;C0HKE7;C0HKE6;CHistone H2A type 1-H;Histone H2AHist1h2ah;H2afj;Hist2h2ac;Hist1h2 2 2 21.5 14.135 0 0 0 37034000 E9PV24 Fibrinogen alpha chain;Fibrinopep Fga 4 4 7.9 87.428 70775000 67089000 60481000 87700000 P18760;P45591 Cofilin-1;Cofilin-2 Cfl1;Cfl2 2 2 23.5 18.559 0 0 27905000 42121000 P49290 Eosinophil peroxidase;Eosinophil pEpx 4 4 6.6 81.379 0 0 81637000 95396000 P63260;P60710;P63268;P68134;P6Actin, cytoplasmic 2;Actin, cytoplaActg1;Actb;Actg2;Acta1;Actc1;Acta 4 4 14.7 41.792 74386000 102690000 285130000 718610000 Q61878 Bone marrow proteoglycan;EosinoPrg2 2 2 14.3 24.255 0 0 61286000 84531000 Q8K0E8;CON__P02676 Fibrinogen beta chain;Fibrinopept Fgb 3 3 10.2 54.752 0 0 31581000 42767000 Q8VCM7 Fibrinogen gamma chain Fgg 2 2 7.1 49.391 24179000 25068000 28231000 36111000

Table S1. Mass spectrometry data comparing fibrinogen background control (no eosinophils added) to fibrinogen plus eosinophils. LFQ = label free quantification, NoEos = fibrinogen only background control, Eos = fibrinogen plus eosinophils.