SOLUBLE CD93 IS an APOPTOTIC CELL OPSONIN RECOGNIZED by the Αxβ2

bioRxiv preprint doi: https://doi.org/10.1101/341933; this version posted June 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

SOLUBLE CD93 IS AN APOPTOTIC CELL OPSONIN RECOGNIZED BY THE αxβ2

INTEGRIN.

Jack W.D. Blackburn*, Darius H.C. Lau*, Jessica Ellins, Angela Kipp, Emily N. Pawlak, Jimmy D.

Dikeakos, and Bryan Heit†

Department of Microbiology and Immunology, and the Center for Human Immunology, The University

of Western Ontario, London, Ontario, Canada.

*These authors contributed equally to this paper

†Corresponding Author: [email protected]

Running Head: sCD93 Efferocytosis

Keywords: CD93, Efferocytosis, Phagocytosis, Opsonin, Integrins

Abbreviations: ADAM - a disintegrin and metalloproteinase. BSA – bovine serum albumin. CHO –

Chinese hamster ovary cells. CTLD – C-type lectin-like domain. FBS – fetal bovine serum. MMP –

matrix metalloproteinase. PBMC – peripheral blood mononuclear cells. PFA – paraformaldehyde.

sCD93 – soluble CD93.

ABSTRACT

Efferocytosis – the phagocytic removal of apoptotic cells – is essential for the maintenance of

homeostasis and prevention of the inflammatory and autoimmune diseases which can follow the lysis of

uncleared apoptotic cells. CD93 is a transmembrane glycoprotein previously implicated in efferocytosis

and angiogenesis, and upon mutation, results in the onset of efferocytosis-associated diseases such as

atherosclerosis and rheumatoid arthritis. CD93 is produced as a cell surface protein which is shed as

soluble CD93, but it is unknown how CD93 mediates efferocytosis or whether its efferocytic activity is

mediated by the soluble or membrane-bound form. Herein, we demonstrate that the membrane bound

form of CD93 has no phagocytic, efferocytic, or tethering activity, whereas soluble CD93 potently

opsonizes apoptotic cells but not a broad range of Gram-Negative, Gram-Positive or fungal

microorganisms. Using mass spectrometry, we identified the αxβ2 integrin as the receptor required for

soluble CD93-mediated efferocytosis, and via deletion mutagenesis determined that soluble CD93 binds

to apoptotic cells via its C-Type Lectin-Like domain, and to αxβ2 by its EGF-like repeats. This bridging

of apoptotic cells to the αxβ2 integrin markedly enhanced efferocytosis by macrophages, and could be

abrogated by knockdown of αxβ2 integrin. Combined, these data elucidate the mechanism by which CD93

regulates efferocytosis and identify a previously unreported opsonin-receptor system utilized by the

immune system for the efferocytic clearance of apoptotic cells.

INTRODUCTION

CD93 is a 120kDa, type-1 transmembrane glycoprotein expressed on the surface of endothelial cells,

stem cells, platelets, circulating myeloid cells, and on maturing B cells (1–6). Part of the group XIV C-

type lectin family, CD93 shares a common structure with the other group XIV family proteins

thrombomodulin, CLEC14A and CD248: an N-terminal C-type lectin-like domain (CTLD), five EGF-

like repeats, a heavily glycosylated mucin domain, a transmembrane helix, and a small and highly

cationic cytosolic domain (7). A soluble form of CD93 (sCD93) comprised of the CTLD, EGF-like

domain and a portion of the mucin domain, can be detected in human plasma (4). The function of sCD93

remains poorly elucidated, however sCD93 serum levels are increased during inflammatory conditions

such as atherosclerosis and in autoimmune disorders such as rheumatoid arthritis (8, 9). CD93 was

originally identified as a receptor for C1q (7, 10), based on observations that CD93 enhanced

phagocytosis in a C1q-dependent manner (10–14). These results are controversial, as subsequent studies

demonstrated that CD93 neither interacts with C1q, nor contributes to the C1q dependent enhancement

of phagocytosis; findings later confirmed in knockout mice (15, 16). Furthermore, single nucleotide

polymorphisms in CD93 are strongly associated with diseases characterized by impaired apoptotic cell

clearance, such as atherosclerosis and systemic lupus erythematosus (17–19), suggesting that CD93 may

contribute to efferocytosis – the phagocytic removal of apoptotic cells. Indeed, CD93 was first identified

by thiohydantoin C-terminal sequencing of a protein immunoprecipitated by an antibody known to block

the efferocytic activity of monocyte-like U937 cells (11). Moreover, CD93-/- mice have significant

defects in apoptotic cell clearance compared to strain matched controls, and CD93 polymorphisms

predispose mice and humans to autoimmune and inflammatory disorders characterized by failed

efferocytosis (7, 16–18). As one example, patients homozygous for a P541S missense mutation in the

CD93 mucin domain have a 26% increase in their risk of coronary heart disease; a disease resulting from

the accumulation of uncleared apoptotic cells beneath the cardiac vasculature (17, 20).

Although the above studies indicate that CD93 is involved in efferocytosis, it remains unclear how CD93

mediates this process. While some studies have suggested that CD93 functions as a membrane-bound

receptor which directly induces the engulfment of apoptotic cells (18, 19, 21), CD93 lacks the ITAM

signaling motifs normally associated with phagocytic/efferocytic receptors. Instead, CD93 has a short

intracellular domain enriched in the cationic amino acids lysine and arginine, and associates with moesin

and GIPC – signaling molecules not normally associated with phagocytosis (22, 23). Although CD93

does not interact with classical mediators of phagocytosis, moesin-CD93 interactions are regulated by

phosphatidylinositol-3-kinase signaling similar to that present during phagocytosis. Moreover, the

intracellular domain of CD93 can be phosphorylated via a dystroglycan-dependent pathway that results

in rearrangement of the cell cytoskeleton (23, 24). Similarly, GIPC is involved in receptor-mediated

endocytosis, and therefore may be co-opted for the uptake of larger cargos (25). While not conclusively

demonstrated, it is possible that these pathways may represent a novel signaling pathway that can drive

the cytoskeletal rearrangements required for the engulfment of apoptotic cells. A second possibility is

that CD93 acts as a tethering receptor – e.g. CD93 stabilizes interactions between phagocytes and

apoptotic cells, thereby prolonging contact with other efferocytic receptors. Indeed, CD93 is recognized

to play a role in intercellular adhesion (3, 15, 24, 26, 27), consistent with a tethering function. In conflict

with these two models is the observation that sCD93-rich peritoneal lavage fluid from wild-type, but not

CD93-/- mice, greatly enhanced efferocytosis by bone-marrow derived CD93-/- macrophages (28). This

data indicates that the efferocytic capacity of CD93 is localized to the soluble form, thereby supporting

a third hypothesis – that CD93 does not act directly as an efferocytic receptor, and instead acts as a

soluble opsonin.

Clearly, there is data supporting three distinct and mutually exclusive models of CD93 efferocytic

function. In this study, we directly asses these three models of CD93 function, using in vitro models of

binding, efferocytosis and opsonization. These experiments disfavor the efferocytic receptor and

tethering receptor models of CD93 function, and instead demonstrate a strong opsonic effect of sCD93

against apoptotic cells but not against a panel of microorganisms. Using mass-spectrometry we then

identified αxβ2 integrin as the efferocytic receptor which recognizes sCD93 opsonized targets, with

apoptotic cells “bridged” by the CD93 CTLD domain to αxβ2 integrin on phagocytes via the CD93 EGF-

like domain. This represents a previously undescribed opsonin-receptor system which aids in the

efferocytosis of apoptotic cell by phagocytes such as macrophages.

MATERIALS AND METHODS

Materials

DH5a Escherichia coli was a gift from Dr. John McCormick (University of Western Ontario),

Rhodotorula minuta and Candida glabrate were gifts from Dr. Andre Lachance (University of Western

Ontario). Escherichia coli K29+ and K29-, and Burkholderia cenocepacia were gifts from Dr. Susan

Koval (University of Western Ontario), Micrococcus luteus T-18 and Staphylococcus pyrogenes 71-679

were gifts from Dr. Jeremy Burton (University of Western Ontario), Staphylococcus aureus UWO256

and Pseudomonas aeruginosa UWO533 were from the University of Western Ontario microbiology

teaching bank. The 12CA5 hybridoma (mouse-anti-HA) and THP-1 monocytes were gifts from Dr. Joe

Mymryk (University of Western Ontario). FcγRI and TIM4 expression constructs were a gift from Dr.

Sergio Grinstein (Hospital for Sick Children, Toronto). Saccharomyces cerevesea WLP001 was

purchased from White Labs (San Diego, CA), Jurkat, J774.2 and CHO cells, and all fluorescent

secondary antibodies were purchased from Cedarlane (Mississauga, Canada). Goat-anti-Human CD93

polyclonal serum was purchased from R&D Systems (Oakville, Canada). Roswell Park Memorial

(RPMI), Dulbecco’s Modified Eagle Medium (DMEM), serum-free CHO-100 medium, serum-free

hybridoma medium, Trypsin-EDTA, and Fetal Bovine Serum (FBS) were purchased from Wisent (Saint-

Jean-Baptiste, Canada). #1.5 thickness round cover slips, glass slides and 16% paraformaldehyde (PFA)

were purchased from Electron Microscopy Sciences (Hatfield, Pennsylvania). DTT, human IgG and

cysteine was purchased from Sigma-Aldrich (Oakville, Canada). GenJet Plus, Polyjet and all DNA

purification kits were purchased from Frogga Bio (North York, Canada). DAPI, permafluor mounting

medium, 30 kDa cut-off spin concentrators, FITC anti-human CD3, and Phusion DNA polymerase, and

N-Ethyl-N'-(3-dimethylaminopropyl)carbodiimide were purchased from Thermo Scientific

(Mississauga, Canada). Restriction enzymes and T4 DNA ligase was purchased from New England

Biolabs (Whitby, Canada). Magnetic 1 µm diameter carboxy-reactive beads, 3 µm diameter silica beads,

3 µm diameter P(S/DVB) polystyrene beads and 1 μm diameter Promag carboxy magnetic microspheres

were purchased from Bangs Laboratories (Fishers, Indiana). Phosphatidylcholine, phosphatidylserine

and biotin-phosphatidylethanolamine were purchased from Avanti Polar Lipids, Inc. (Alabaster,

Alabama). Anti-β1 (clone AIIB2), anti-β1 (clone H52) and anti-CD44 (clone H4C4) were purchased from

the Developmental Studies Hybridoma Bank (University of Iowa). Anti-αx (clone BU15) and all

immunoblotting supplies were purchased from Bio-Rad Canada (Montreal, Canada). All other chemicals

and bacterial culture media were purchased from Canada BioShop (Mississauga, Canada). Matlab

software was purchased from MathWorks (Natick, Massachusetts). Prism software was purchased from

Graphpad (La Jolla, California). FIJI is just ImageJ (FIJI) was downloaded from https://fiji.sc/ (29).

Cell Culture

CHO and HEK 293T cells were cultured in Ham’s F12 medium supplemented with 10% FBS, and split

upon reaching confluency by washing once with phosphate-buffered saline (PBS, 137 mM NaCl, 10 mM

Na2HPO4) and detached by the addition of Trypsin-EDTA. J774.2 cells were grown in DMEM

supplemented with 10% FBS, and split by detaching cells with a cell scraper. Detached CHO, HEK 293T,

and RAW264.7 cells were then passaged by diluting 1:10 into fresh media. THP-1, Jurkat and 12CA5

cells were grown in RPMI supplemented with 10% FBS and DMEM supplemented with 10% hybridoma

grade FBS respectively, and split by diluting 1:10 upon reaching a density of ~2 x 106 cells/ml. For

phagocytosis/efferocytosis experiments, ~1 × 105 J774.2 or CHO cells were split onto #1.5 thickness, 18

mm diameter circular coverslips placed into the wells of a 12-well plate. If required, 18-24 hours later

the cells were transfected with 1 μg DNA/well using FugeneHD (J774.2) or GenJet (CHO) as per

manufacturers’ instruction. To generate THP-1 derived macrophages, 1 × 105 THP-1 cells were placed

into the well of a 12-well plate, and the media supplemented with 10 ng/mL PMA, and the resulting

macrophages used 72 hours later. To generate apoptotic cells, 5 ml of Jurkat cell culture at ~2 x 106

cells/mL was resuspended in 1.5 mL of serum-free RPMI supplemented with 10 μM staurosporine

following a 350×g/5 min centrifugation, and incubated overnight at 37°C. Apoptotic cells were

suspended in PBS using a 1,500×g/1 min centrifugation, labeled for 20 min with a 1:500 dilution of Cell

Proliferation Dye eFluor 670, washed 2× with PBS, and suspended in serum-free RPMI for immediate

use.

Primary Human Macrophage Culture

PBMCs were suspended in RPMI-1640 supplemented with 10% FBS and 1% antibiotic–antimycotic

solution at ~2 × 106 cells/mL. Monocytes were separated by adhesion to glass by placing 200 μl or 1.0 ml

of the suspension on sterile 18 mm coverslips or on 35 mm diameter gelatin-coated 6-well tissue culture

plates. Non-adherent cells were removed after a 1 h/37 °C incubation with two PBS washes. Monocytes

were differentiated into M0, M1 or M2 polarized macrophages as per our published protocols (30, 31).

Briefly, adherent monocytes were cultured for 5 days in 10 ng/ml M-CSF (M0 and M2) or 20 ng/ml GM-

CSF (M1), followed by 2 days culture in DMEM supplemented with 10% FBS with 10ng/ml M-CSF

(M0), 20 ng/ml GM-CSF supplemented with 250 ng/ml Salmonella lipopolysaccharide and 10 ng/ml

INFγ (M1), or 10 ng/ml M-CSF and 10 ng/ml IL-4 (M2).

Antibody and Fab Preparation

12CA5 cells were grown to ~2 x 106 cells/mL, collected with a 350×g/10 min centrifugation, suspended

in 60 ml of serum-free hybridoma media, and cultured for an additional 4 days. Cells and particulates

were removed by a 3,000×g/30 min centrifugation, and the antibodies concentrated and media exchanged

to TBS (10mM tris + 150mM NaCl), using a 10 kDa cutoff spin concentrator. For full-length antibody,

the resulting concentrate was diluted to 1 mg/ml in TBS with 20% glycerol and stored at -20°C. Fab’s

were prepared by diluting the concentrate to 5 mg/ml in TBS with 20mM cysteine-HCl at pH = 7.0; 0.5

ml of immobilized papain was added per 1 ml of antibody, incubated overnight on a rotator at 37°C, the

reaction terminated by the addition of an equal volume of TBS pH = 7.5, and the papain removed by a

21,000×g/5 min centrifugation. Fc fragments were depleted by the addition of 0.5 ml of immobilized

protein A, followed by a 4 hr room-temperature incubation, and separation of the Fc fragments by a

21,000×g/5 min centrifugation. Fabs were then purified to high purity (>90%) by FPLC using an Enrich

SEC650 size-exclusion column and a BioRad NGC FPLC platform. Purified Fab fragments were

suspended at 0.5 mg/ml in TBS with 20% glycerol and stored at -20°C.

Preparation of CD93 Expression and shRNA Vectors and Recombinant sCD93

The human CD93 ORF was ordered from the Harvard PlasmID Database. Expression vectors were

generated using either restriction digest or Gibson assembly using the primers and enzymes indicated in

Supplemental Table 1. All PCR reactions were performed using Phusion DNA polymerase with 35-40

cycles of 96°C/30 sec melting, 65-63°C/30 sec annealing and 72°C/90 sec elongation. Restriction digests

and ligations were performed for an hour at 37°C and 20°C, respectively. Gibson assembly was

performed as per manufacturer’s instructions. shRNAs were designed using the Hanon Lab shRNA

design webserver (http://cancan.cshl.edu/RNAi_central). shRNA inserts were ordered as complementary

single-stranded oligonucleotides which were annealed by mixing the oligos in equimolar amounts in

ddH2O, heating to 95°C, and then cooling at 2°C/min to 20°C. shRNA inserts and pGFP-C-shLenti

vectors were digested with AgeI/BamHI and ligated with T4 DNA ligase. All vector sequences were

confirmed with Sanger sequencing. The sCD93 and shRNA vectors were packaged into a VSV-G

pseudotyped lentiviral vector by triple transfection of HEK 293T cells with the VSV-G envelope-

encoding pMD2.G (2 ng, Addgene #12259), pCMV-DR8.2 (5 ng, Addgene #12263) and 2.5 µg of the

lentiviral vector of interest using PolyJet as per the manufacturer’s protocol. Seventy-two hour post

transfection, lentivirus was collected in 20% FBS and purified with a 0.2 µm filter, as described

previously (32). The resulting virions were stored at -80°C until needed. Virions were used to prepare

stably transfected CHO or THP-1 cell lines, using zsGreen or puromycin as a selectable marker. sCD93

proteins were collected by growing the transfected cell lines to confluency in a T75 flask (sCD93) or in

2 wells of a 6-well plate (sCD93-FLAG and deletion mutants), followed by exchanging the medium for

60 ml serum-free CHO100 medium followed by an additional 5 days of culture. The resulting

supernatants were collected, cleared with a 3,000×g/30 min centrifugation, sCD93 concentrated and

media exchanged to TBS (10mM tris + 150mM NaCl), using a 20 kDa cutoff spin concentrator. sCD93

was then purified to high purity (>95%) by FPLC using an Enrich SEC650 size-exclusion column and a

BioRad NGC FPLC platform, suspended at 1 mg/ml in TBS + 20% glycerol and stored at -20°C.

CD93 Shedding from Human PBMCs

The collection of blood from healthy donors was approved by the Health Science Research Ethics Board

of the University of Western Ontario and venipuncture was performed in accordance with the guidelines

of the Tri-Council Policy Statement on human research. Blood was collected in heparinized vacuum

collection tubes, and 5 ml of blood layered on an equal volume of Lympholyte-poly in a 15 ml centrifuge

tube, followed by centrifugation at 300 × g for 30 min at 20 °C. The top band of PBMCs was collected

with a sterile transfer pipette, washed once (300 × g, 6 min, 20 °C) with phosphate-buffered saline (PBS,

137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), and then suspended in DMEM

supplemented with 10% FBS for future experiments. For CD93 shedding experiments, PBMCs or

neutrophils were suspended at 1 × 106 cells/mL in serum-free DMEM supplemented with 10 ng/ml

GM6001 or 100 ng/ml TAPI-2, and 10 ng/ml PMA, 10 ng/ml LPS or 100 ng/ml TNFα added. Cells were

incubated for 60 min at 37°C; for time-courses, 100 μl samples were withdrawn from this culture at

desired time-points. Shedding was stopped by cooling the cells on ice and fixating for 20 min with 4%

PFA in PBS, and shedding quantified by immunostaining. Shedding following apoptosis was quantified

by suspending PBMCs at 1 x 106 cells/mL in serum-free DMEM supplemented with 100 ng/ml TAPI-2

and staurosprorine. A portion of the PBMCs were immediately fixed and immunostained for CD93, and

the remaining population incubated for 24 hours at 37°C, followed by fixation and immunostaining for

CD93.

Immmunostaining & Microscopy

Immunolabeling was performed on unfixed cells, or on cells fixed with 4% paraformaldehyde (PFA) in

PBS for 20 min at room temperature. For intracellular staining, PFA-fixed cells were permeabilized and

blocked with PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA) for 1 hr at room

temperature. Primary antibodies were added to the cells in PBS containing 1% BSA at an appropriate

dilution (1 μg/ml full-length 12CA5, 0.5 μg/ml 12CA5 Fab or 1:1,000 anti-CD93 polyclonal sera), for

20 min (non-permeabilized) or 1 hr (permeabilized cells) with gentle agitation. Samples were then

washed 3 × 15 min in PBS and fluorescently-labeled secondary Fab’s added at a 1:5,000 dilution in PBS

containing 1% BSA for 20 min (non-permeabilized) or 1 hr (permeabilized cells) with gentle agitation.

If required, cells were counter-stained with 1 μg/ml Hoechst 33342. Samples were then washed 3 × 15

min in PBS and mounted on glass slides with Permafluor mounting media. Samples were imaged on a

Leica DMI6000B microscope equipped with 63×/1.40NA and 100×/1.40 NA objectives, photometrics

Evolve-512 delta EM-CCD camera, Chroma Sedat Quad filter set and the LAS-X software platform. Z-

stacks with 0.19 μm spacing were taken of 30-50 cells/condition, and the resulted images deconvolved

using iterative blinded deconvolution in LAS-X, and the resulting images exported for analysis using

FIJI (29).

Bacterial and Fungal Cell Culture & Preparation

E. coli was grown in LB medium, P. aeruginosa, B. cenocepacia, M. luteus, S. pyrogenes, and S. aureus

were grown in BHI medium. R. minuta, C. glabrate, and S. cerevisiae were grown in YPD medium. All

microorganisms were grown overnight to stationary phase in 5 ml cultures on a shaking incubator at

37°C (bacteria) or 32°C (fungi). 1 × 107 cells were washed in PBS using 6,000×g 1 min centrifugation,

fixed with 4% PFA in PBS for 20 min, and labeled by the addition of a 1:500 dilution of Cell Proliferation

Dye eFluor 670 and a 1:500 dilution of NHS-biotin for 30 min at room temperature. After 30 min, the

labeling reaction was quenched with LB medium, the organisms washed 3 times with PBS, diluted to

1×107/ml in serum-free DMEM and stored at 4°C until needed.

Preparation of Apoptotic Cell and Pathogen Mimics

Bead-based mimics were prepared as per our published protocols (30, 31). Briefly, Fab, antibody, BSA,

cell supernatant, and recombinant sCD93 functionalized beads were prepared by washing 10 μl of 3 µm

diameter P(S/DVB) polystyrene bead 2× in 1 ml of PBS using a 6,000×g/1 min centrifugation. Beads

were then suspended in 100 μl of cell-conditioned medium or 100 μl PBS + 10 μl of one of 12CA5 Fab,

50 mg/ml human IgG, 5% BSA, or recombinant sCD93, and incubated at room temperature with rotation

for 30 min. Beads were then washed as above and suspended in 100 μl of PBS or serum-free DMEM.

Apoptotic cell mimics were produced by preparing 4 μmol of a 20% phosphatidylserine, 79.9%

phosphatidylcholine, 0.1% biotin-phosphatidylethanolamine mixture in chloroform. Subsequently, 10 μl

of 3 µm diameter silica beads were added, the mixture vortexed for 1 min, and the chloroform evaporated

under a stream of nitrogen for 1 hr. Beads were resuspended in 1 mL of PBS, vortexed, and washed as

above. sCD93 covalently linked beads were prepared by washing 10 µL promag carboxy magnetic

microspheres in 1 mL of MES at pH 7.2, and then suspended in 100 µL of the same buffer. To this, 1 mg

of N-Ethyl-N'-(3-dimethylaminopropyl)carbodiimide was added and the beads incubated for 15 min on

a rotator. The beads were washed twice in PBS and resuspended in 500 µL PBS to which 10 μL of 1%

BSA or 10 μL of recombinant sCD93 were added and incubated for 4 hrs on a rotator at room

temperature. Beads were washed and resuspended in 1 mL quenching solution (PBS with 40 mM glycine

and 1% BSA), followed by a wash and resuspension in 100 μL storage buffer (0.1% BSA with PBS). All

beads were stored at 4ºC until used.

Efferocytosis, Phagocytosis and Endocytosis Assays

These assays were performed as per our published methods (30, 31, 33). Briefly, for bead-based targets,

10 μL of the bead suspensions (~10 beads:macrophage) were added to each well of macrophages in a 12-

well plate. Apoptotic cells or bacterial targets were first opsonized by incubating 100 μl of the cell

suspension with 10 μL of recombinant sCD93 or an equimolar concentration of BSA for 30 min at 37°C,

followed by addition of the full 110 μL volume to a well of macrophages in a 12-well plate (~10 apoptotic

cells:macrophage, ~50 microorganisms:macrophage). Once the targets were added, the samples were

centrifuged at 200×g for 1 min to force contact between the macrophages and the targets, and the plate

incubated at 37°C/5% CO2 for 30 min unless otherwise indicated. At the end of the experiment the media

was aspirated, the samples washed vigorously 3 times with PBS to remove any unbound targets, and the

cells fixed in 4% PFA for 20 min. If targets were biotinylated, a 1:500 dilution of fluorescent streptavidin

was then added to detect uninternalized targets; for apoptotic Jurkat cells a 1:1000 dilution of FITC-

conjugated anti-human CD3 was added to detect uninternalized targets. Cells were then washed 3 times

with PBS, mounted on slides with Permafluor, and imaged as described above.

Efferocytosis/phagocytosis was then quantified – for streptavidin-labeled samples the efferocytic index

was quantified as the number of engulfed targets per macrophage, or as beads/cell if streptavidin staining

was not available. For endocytosis assays, HA-CD93 expressing CHO cells were cooled to 10°C and

labeled with a 1:100 dilution of 12CA5 Fab for 20 min. The cells were washed three times with PBS and

then labeled for 20 min at 10°C with a 1:500 dilution of goat-anti-mouse Cy3-labeled Fab (non-

crosslinking) or goat-anti-mouse Cy3-488 labeled F(ab’)2 (cross-linking). The cells were then washed

with warmed serum-free RPMI, and placed in a 37°C incubator for 60 min. The cells were then fixed

with PFA, any non-internalized CD93 labeled for 20 min with a 1:500 dilution of donkey-anti-goat

Alexa488-labeled Fab, the samples washed 3 times in PBS, mounted on slides with Permafluor, and

imaged as described above. Endocytosis was quantified using FIJI as the fraction of the Cy3-CD93

staining that lacked Alexa-488 colocalization.

Suspension Efferocytosis Assay

Wild-type and shRNA expressing THP-1 monocytes were mixed in a 1:1 ratio, and 1 × 105 of the mixed

cells placed in a microfuge tube along with 1 × 106 sCD93-coated beads. The mixture centrifuged at 300

× g/2 min to force contact between the cell and beads, and the tubes incubated for 20 min at 37°C. The

supernatants were then removed and the cells fixed for 20 min with 4% PFA. The fixed cells were then

placed into an imaging chamber and the number of beads bound by untransduced (zsGreen-) versus

shRNA-transduced (zsGreen+) cells quantified, with at least 100 cells quantified per condition in each

experiment.

Recovery and Identification of the sCD93 Receptor

5×106 THP-1 monocytes were deposited into each well of a 6-well tissue culture plate and differentiated

into macrophages as described above. The cells were cooled to 10°C and to 3 wells 100 μl/well of BSA-

conjugated promag carboxy magnetic microspheres were added, with the other 3 wells receiving 100

μl/well sCD93-conjugated microspheres. The samples were then centrifuged for 1 min at 200×g to force

contact between cells and spheres, and the plate incubated for 10 min at 10 °C. Beads were then reversibly

crosslinked to putative sCD93 receptors using the ReCLIP method (31, 34); briefly, the samples were

washed with PBS and then PBS with 0.5 mM dithiobis[succinimidyl propionate] and 0.5 mM dithio-

bismaleimidoethane added to the cells for 1 min at 10°C. The solution was then aspirated and the reaction

quenched for 10 min with 5 mM L-cysteine and 20 mM Tris-Cl, pH = 7.4. The cells were then disrupted

with RIPA buffer and the beads recovered with a magnetic column. The beads were then washed 3 times

with RIPA buffer, vortexing the beads for 1 min between washes, and recovering the beads with a

magnetic column. After the final wash the beads were suspended in 40 μl of Laemmli buffer with 50 mM

DTT, boiled for 10 min, resolved on a 4%-20% SDS-PAGE gel for 4 hrs at 100V, and stained using a

colloidal coomassie stain. Bands unique to the sCD93 beads were then excised using an Ettan Robotic

Spot-Picker. Gel slices were destained with 50 mM ammonium bicarbonate and 50% acetonitrile, treated

with 10 mM DTT and 55 mM iodoacetamide, and digested with trypsin. Peptides were extracted by a

solution of 1% formic acid and 2% acetonitrile and freeze dried. Dried peptide samples were then

suspended in a solution of 10% acetonitrile and 0.1% trifluoroacetic acid. Mass spectrometry analysis of

each sample was performed using an AB Sciex 5800 TOF/TOF System. Raw mass spectrometry data

analysis was performed using MASCOT database search (http://www.matrixscience.com).

Immunoblotting

1 × 105 shRNA-expressing THP-1 cells were removed from culture, washed once with PBS, and lysed

in Laemmli’s buffer containing 10% β-mercaptoethanol and Halt protease inhibitor cocktail, boiled

briefly, and then loaded onto a 10% SDS-PAGE gel. Gels were run for 30 min at 50 V, and then the

voltage increased to 100 V until maximal migration. The separated proteins were then transferred to

nitrocellulose membrane overnight at 40V and blocked for two hours with TBS-T containing 2.5% milk

powder. Primary antibodies were added at a 1:250 dilution (CD44, β1, β2), or at 1:1000 (αx, GAPDH) for

2 hrs in TBS-T containing 2.5% milk powder, washed 3 × 15 min in TBS-T, and then an appropriate

Dylight-700 labeled secondary antibody added at a 1:10,000 for 1 hr in TBS-T containing 2.5% milk

powder. After 3 × 15 min washes in TBS-T the blots were imaged on an Odyssey CLx imaging system.

Statistics and Data Analysis

All datasets were tested for normal distribution using a Shapiro-Wilk test. Normally distributed data is

presented as mean ± SEM and analyzed using a Students T-test or ANOVA with Tukey correction. Non-

parametric data is presented as median ± interquartile range and analyzed using a Kruskal-Wallis test

with Dunn correction. All statistical analyses were performed using Graphpad Prism. All image analysis

was performed using FIJI (29).

RESULTS

CD93 Does Not Act as an Efferocytic or Tethering Receptor

Previous studies have suggested that CD93 acts as an efferocytic or tethering receptor, engaging targets

either via the opsonin C1q, or directly (7, 10–14, 16, 35, 36). These claims are controversial, with studies

indicating that C1q is not a CD93 ligand, and that the soluble form – not the membrane-bound form – of

CD93 enhances efferocytosis (15, 16). As such, using multiple approaches we sought to directly assess

whether the membrane-bound form of CD93 acts as an efferocytic or tethering receptor. First, CD93 was

ectopically expressed in CHO cells, which have the molecular machinery required for phagocytosis and

efferocytosis, but lack expression of phagocytic or efferocytic receptors (37). Transfection of CHO cells

with the phagocytic receptor FcγRI rendered CHO cells highly phagocytic to beads opsonized with IgG,

while transfection with the efferocytic receptor TIM4 rendered CHO cells efferocytic against apoptotic

cells, serum-opsonized apoptotic cells, and apoptotic cell mimics (Figure 1A). In marked contrast,

transfection with CD93 did not impart phagocytic or efferocytic activity against any of these targets

(Figure 1A), indicating that CD93 is unlikely to function as an efferocytic or tethering receptor of either

unopsonized or serum-opsonized apoptotic cells. We further explored the possibility that CD93 mediates

efferocytosis via an opsonin by mimicking opsonin-mediated receptor cross-linking by oligomerizing

HA-CD93 with anti-HA functionalized beads. No internalization of these bead was observed above the

level of control beads coated with an irrelevant IgG (Figure 1B). Because CHO cells are not natively

phagocytic or efferocytic, and may therefore be lacking a co-receptor or signaling molecule required by

CD93, we repeated this experiment using macrophages which have previously been reported to be

capable of CD93-dependent efferocytosis (16). To avoid Fc receptor activation, beads were

functionalized with anti-HA Fab fragments or Fab fragments generated from an irrelevant IgG, rather

than full-length antibodies. Anti-HA functionalized beads produced an accumulation of CD93 around

the beads reminiscent of a phagocytic cup (Figure 1C), but did not induce bead engulfment (Figure 1D).

Next, we quantified CD93 endocytosis following antibody-mediated cross-linking of CD93, as previous

reports have demonstrated that cross-linking is sufficient to activate and induce the endocytosis of

phagocytic and efferocytic receptors via the same signaling pathways triggered by bona fide

phagocytic/efferocytic targets (33, 38–40). Cell-surface HA-CD93 was labeled under cross-linking (Cy3-

F(ab’)2) or non-crosslinking (Cy3-Fab) conditions, endocytosis allowed to occur for 30 min, and residual

surface Fab/F(ab’)2 labeled with Alexa-488. Under these conditions, endocytosed CD93 is labeled only

with Cy3, while non-endocyotsed CD93 is labeled with both Cy3 and Alexa-488. Although cross-linking

resulted in significant clustering of CD93 on the cell surface (Figure 1E), CD93 was endocytosed at an

equal rate in non-crosslinked and cross-linked samples (Figure 1F). Finally, no tethering of apoptotic

Jurkat cells was observed by non-efferocytic HeLa cells ectopically expressing CD93 (Figure 1G).

Combined, these data demonstrate that the transmembrane form of CD93 is not an efferocytic or tethering

receptor.

CD93 Is Shed During Inflammation and Macrophage Differentiation

Soluble CD93 has been identified in human serum and is produced following stimulation of human and

murine myeloid cells with inflammatory stimuli (4, 28). CD93 is expressed on circulating myeloid cells,

but not on circulating lymphoid cells, and consistent with this we found that ~60% of human PBMCs

expressed CD93 (Figure 2A). This CD93 was shed upon stimulation with PMA, with permeabilization

prior to immunostaining confirming that PMA stimulation induced shedding rather than endocytosis

(Figure 2A). As PMA is a non-physiological stimulus, we assessed CD93 shedding by human monocytes

following LPS and TNFα stimulation, with both ligands inducing a time-dependent shedding of CD93

(Figure 2B). In murine cells CD93 is shed by an uncharacterized matrix metalloproteinase (MMP) (4).

Unexpectedly, the pan-MMP inhibitor GM6001 did not inhibit PMA-induced CD93 shedding by human

PBMCs, whereas TAPI2 – which inhibits both MMP and a disintegrin and metalloproteinase (ADAM)

family proteases – prevented PMA-induced CD93 cleavage (Figure 2C). Next, we assessed CD93

expression during differentiation of primary human monocytes into non-inflammatory (M0 and M2) and

inflammatory (M1) macrophages. CD93 was downregulated early during macrophage differentiation

(Figure 2D), and was absent from fully matured M0, M1, or M2 macrophages (Figure 2E). Lastly, we

assessed the shedding of CD93 following apoptosis, as both ADAMs and MMPs are active during

apoptosis (41, 42), with staurosporine-induced apoptosis of PBMC’s resulting in the TAPI2-inhibitable

shedding of CD93 (Figure 2F). Combined, these data further argue against CD93 acting in a membrane-

bound efferocytic or phagocytic receptor, as receptor shedding during infiltration of inflamed sites, and

during macrophage differentiation, would deprive myeloid cells of an efferocytic receptor in

environments where they would be expected to encounter apoptotic cells. As such, it is most probable

that CD93 mediates efferocytosis in its soluble form.

sCD93 Functions as an Opsonin

Based on previous studies which demonstrate that sCD93 enhances efferocytosis, and our data indicating

that membrane-bound CD93 does not directly function as an efferocytic or tethering receptor (Figure 1),

we next assessed whether sCD93 functions as an opsonin. First, we coated 3 µm diameter beads with

supernatants from unstimulated versus PMA-stimulated monocytes, and then assessed the phagocytic

activity of J774.2 macrophages against these targets. Mimics opsonized with supernatants from PMA-

stimulated cells were engulfed at a higher rate than beads opsonized with media from unstimulated cells,

with depletion of sCD93 abrogating this effect (Figure 3A). Next, apoptotic cell mimics (30) comprised

of silica beads coated in a lipid mixture which mimics the plasma membrane of apoptotic cells (20%

phosphatidylserine, 80% phosphatidylcholine) were coated with purified recombinant sCD93 or a control

protein (BSA), with sCD93 modestly increasing bead binding by macrophages (Figure 3B). As sCD93

appeared to be functioning as an apoptotic cell opsonin, we next assessed the capability of recombinant

sCD93 to opsonize apoptotic Jurkat cells. sCD93 significantly increased both the fraction of

macrophages engulfing at least one apoptotic cell (Figure 3C), and the number of apoptotic cells engulfed

per macrophage (Figure 3D), resulting in a net 4-fold increase in efferocytosis. No uptake of BSA- or

sCD93-opsonized non-apoptotic cells was observed (data not shown). Finally, the opsonic effect of

sCD93 was assessed against a range of microorganisms, using a panel of organisms representing a broad

range of microbial diversity and capsulation states. No opsonic effect was observed with any of the tested

Gram-negative bacteria (Figure 3E), Gram-positive bacteria (Figure 3F) or fungi (Figure 3G),

demonstrating that sCD93 functions as an apoptotic cell-specific opsonin.

Identification of the sCD93 Receptor

We next used a ligand-precipitation approach to identify the sCD93 receptor on macrophages. Briefly,

sCD93 was covalently conjugated to magnetic beads, these beads centrifuged onto macrophages, a

reversible cross-linking reagent used to stabilize the sCD93-receptor interaction, and the CD93-receptor

complex recovered with a magnetic column (31, 34). sCD93-coated beads precipitated two unique bands

not observed in the control, both of which were much larger than sCD93 (68 kDa, Figure 4A). These

bands were excised from the gel and the constituent proteins identified by liquid-chromatography/mass

spectrometry. While many proteins were non-specifically cross-linked to the sCD93 beads, filtering of

the dataset for high abundance proteins found in at least 3 of 4 independent experiments identified only

8 proteins as putative sCD93 receptor components (Table 1). These included three phagocytic integrin

subunits (αx, β2 and β1), CD44 which regulates integrin-mediated phagocytosis, and several integrin-

associated signaling molecules, indicating that the sCD93 receptor is likely an integrin (43–46). To

determine which of these proteins function as the sCD93 receptor, αx, β1, β2, and CD44 deficient THP-1

cell lines were generated using lentiviral-shRNAs (Figure 4B). Efferocytosis assays were then performed

using sCD93 coated beads and THP-1 cells in suspension, as differentiation of THP-1 cells to adherent

macrophages selected for cells with partial integrin knockdown (data not shown). shRNAs against αx and

β2 decreased both the fraction of macrophages engaging in efferocytosis and the number of beads

efferocytosed per cell (Figure 4C-D), with either shRNA producing a net decrease in efferocytosis of

~88%. Knockdown of β1 or CD44 did not impair sCD93-mediated efferocytosis, thus identifying αxβ2

integrin as the receptor for sCD93.

Identification of the Ligand and Receptor Binding Domains in sCD93

The identification of an integrin as the sCD93 receptor was unexpected, as human CD93 lacks a canonical

RGD or other integrin-recognition motif (47). As such, we identified the sCD93 receptor-binding and

apoptotic cell-binding domains of CD93 using deletion mutagenesis (Figure 5A), preparing purified

FLAG-tagged sCD93 mutants lacking the CTLD (CD93ΔCTLD), the EGF-like repeats (CD93ΔEGF), or the

mucin domain (CD93ΔMucin). First, efferocytosis assays were performed using beads non-specifically

coated in these mutant proteins, allowing us to probe sCD93 binding by macrophages independently of

sCD93’s ligand-binding activity. These experiments demonstrated that the EGF-like domains are

required for sCD93 binding by THP-1-derived macrophages, indicating this domain is recognized by

αxβ2 (Figure 5B). Next, efferocytosis of apoptotic cells opsonized with these mutant proteins was

assessed; this confirmed the requirement for the EGF-like domains, and in addition, demonstrated that

the opsonic effect of sCD93 was lost when the CTLD domain was deleted (Figure 5C). Combined, these

results indicate that CD93 acts as a bipartite opsonin; binding to apoptotic cells via its CTLD, and then

bridging the apoptotic cell to αxβ2 integrin on phagocytes via its EGF-like domains.

DISCUSSION

In this study, we have demonstrated that soluble CD93 is rapidly released from monocytes by proteolytic

cleavage in response to inflammatory stimuli and during monocyte-to-macrophage differentiation. The

soluble fragment then acts as an apoptotic cell opsonin, binding to apoptotic cells via its CTLD domain

and bridging the apoptotic cell to αxβ2 integrins on phagocytes via its EGF-like domain. This opsonic

activity greatly enhances the uptake of apoptotic cells by phagocytes, to an extent similar to that observed

for other apoptotic cell opsonins (48–52), and could be abrogated by knockdown of the αxβ2 integrin, or

by deletion of the apoptotic cell-binding or integrin-binding domains of CD93. Combined, these data are

consistent with a model in which CD93 is shed during inflammation – both following the extravasation

of leukocytes into the inflamed sites, and during the differentiation of monocytes into macrophages –

thus providing the localized delivery of an apoptotic cell opsonin to environments typified by high levels

of cell death.

CD93 is a member of the group XIV C-type lectin family, which also includes thrombomodulin,

CLEC14A and CD248 (53). While all members of this family contain a C-type lectin-like domain,

carbohydrate binding has not been reported for any of these receptors. Rather, all known ligands for this

gene family are proteins – thrombomodulin binds thrombin (54), while the extracellular matrix protein

multimerin-2 was recently shown to be a ligand for CLEC14A, CD248 and CD93 (55). The CD93-

multimerin-2 interaction has been characterized in depth, with CD93 binding a coiled-coiled domain in

multimerin-2 via its CTLD (56). Consistent with this, we observed minimal opsonization of beads coated

in a lipid mixture that mimics the plasma membrane of an apoptotic cell, while robust opsonization of

apoptotic cells was observed. This would suggest that CD93 is not recognizing the common “eat-me”

signal phosphatidylserine, and instead is recognizing another – potentially proteinaceous – “eat me”

signal. Multimerin-2 is unlikely to be this ligand, as multimerin-2 is not expressed by the target cell type

used in this study, nor is it upregulated during apoptosis (57–59). Previous studies have suggested that

C1q and related lectins (MBL, SPA) may also be CD93 ligands (7, 13, 14, 35, 36), and C1q is known to

opsonize apoptotic cells (60), suggesting that C1q may represent a putative ligand for CD93 on apoptotic

cells. This, however, is unlikely as the reported interaction between CD93 and C1q is thought to be

artefactual with CD93-deficient macrophages retaining normal phagocytic activity to C1q-opsonized

targets, and attempts to directly measure C1q-CD93 interactions failing to identify an interaction between

these proteins (15, 16). Consistent with these studies, we observed sCD93-dependant opsonization in the

absence of serum, further suggesting that C1q is not a ligand for sCD93. Several proteinaceous “eat me”

signals have been reported in the literature that may act as sCD93 ligands. These include normally

intracellular proteins which are trafficked to the cell surface or secreted upon induction of apoptosis (e.g.

calreticulin, tubby and TULP1), serum-derived and macrophage-secreted pattern recognition receptors

(e.g. PTX3, Gal3), and alterations in post-transcriptional glycosylation that may expose protein

interaction motifs normally masked by carbohydrate groups (51, 52, 61–64). Finally, it is possible that

the CTLD of CD93 retains some carbohydrate-binding capability, and thus CD93 may directly recognize

the aberrant glycosylation patterns found on apoptotic cells (63).

αxβ2 (also known as complement receptor 4, and as CD11c/CD18) has a well-established role as a

phagocytic receptor that mediates the engulfment of iC3b-opsonized bacteria and apoptotic cells (65,

66). The signaling and function of this integrin is well understood, and indeed, our precipitation of the

sCD93 receptor identified many of the proteins known to regulate αxβ2-mediated phagocytosis as part of

the sCD93 receptor complex. Talin induces integrin conformational changes from a low-affinity to high-

affinity conformation (67), focal adhesion kinase mediates the outside-in signaling of integrins (68, 69),

while PAK2 is activated by focal adhesion kinase to mediate cytoskeletal rearrangements downstream

of integrin activation (70, 71). Moreover, the presence of β1 integrin and CD44 in our

immunoprecipitates, despite their lack of activity as a sCD93 receptor, is likely a result of the structuring

of integrins and their co-receptors into focal contacts; pre-formed assemblies of multiple integrins, their

co-receptors, and signaling molecules (72, 73). This clustering enhances integrin function through

increased avidity and expands the diversity of ligands that can be engaged by an activated cluster;

however, this pre-clustering also renders all proteins in the cluster to become susceptible to the

proximity-based crosslinking method used in this study (34, 74–76). The identification of an integrin as

the sCD93 receptor was initially unexpected due to the absence of a canonical integrin binding motif in

CD93. However, αxβ2 is known to bind proteins lacking these motifs, often by binding EGF-like domains

in the target protein, consistent with our identification of the EGF-like domains of sCD93 as the αxβ2

binding site (77, 78).

Previous investigations into the role of CD93 in leukocyte function have identified several seemingly

disparate roles for sCD93 which may be explained by our identification of αxβ2 integrin as the sCD93

receptor. Jeon et al. demonstrated that sCD93 induced monocyte-to-macrophage differentiation and

increased TNFα synthesis following LPS exposure (9). Engagement of β2 integrins is known to induce

monocyte-to-macrophage differentiation through PCK-dependent suppression of the transcriptional

repressor Foxp1 and concurrent upregulation of the M-CSF receptor (79, 80). While this phenomenon

was observed downstream of αmβ2, all β2-integrins activate the same PKC signaling pathway via their

common β2 chain – and thus αxβ2 would be expected to activate the same macrophage differentiation

program as αmβ2 (81–83). Similarly, β2-integrins enhance TNFα release following LPS stimulation.

Endocytosis of integrins – a likely outcome following engaging a soluble ligand such as sCD93 –

enhances LPS responses by co-endocytosing TLR4, thus increasing TLR4 signaling from endosomes

(84–86). Moreover, TNFα release is further enhanced through the cooperative signaling of the integrin

signaling molecule FAK and the TLR signaling molecule MyD88, an interaction which has been

previously shown to synergistically increase TNFα release following LPS stimulation (87). In another

report, CD93-deficiency was associated with enhanced leukocyte recruitment in a model of peritonitis,

with recruitment reduced to normal levels in CD93 chimeras (27). αxβ2 is known to be a potent regulator

of monocyte adhesion to endothelial-expressed ICAM-1, ICAM-4 and VCAM-1, and is required for

monocyte recruitment to the inflamed peritoneum (88, 89). It is possible that CD93 may act as a

competitive inhibitor of αxβ2, thus decreasing intravascular adhesion and limiting leukocyte infiltration

into inflamed tissues.

Developing B cells represent another immune cell population which express CD93. CD93 expression, as

detected by the AA4.1 and 493 antibodies, is a classical marker of developing B cells (90) and demarcates

hematopoietic stem cells, B cell/myeloid bipotential progenitors, and all stages of B cell development up

to the transitional B cells which migrate from the bone marrow to become naive B cells in peripheral

lymphoid tissues (90, 91). Although the role of CD93 in B cell development is unclear, it is established

that CD93-deficiency does not prevent B cell development (16), but interestingly, diabetes-susceptible

NOD mice bear a mutation in CD93 that limits CD93 expression during B cell lymphopoiesis (18, 92).

B cell development is characterized by high levels of apoptosis, with ~90% of lymphoid progenitors that

commit to the B cell lineage undergoing apoptosis prior to developing into naïve B cells (93). While the

receptors involved in removing apoptotic cells from the bone marrow have not been well characterized,

our data suggest that CD93 released from apoptosing B cells may enhance efferocytosis in the bone

marrow in an autocrine or paracrine manner. Poor clearance of these cells may promote autoimmunity,

as failed efferocytosis of apoptotic B cells has been suggested to increase the stimulation of immature

self-reactive B cells, thereby allowing them to undergo sustained receptor editing or to bypass central

tolerance (94). Moreover, CD93 is re-expressed during plasma cell differentiation, which is another

phase of the B cell lifecycle typified by high levels of apoptosis (95), further consistent with a model in

which B cells release sCD93 to ensure their timely removal during developmental stages where B cell

apoptosis is common. Consistent with this hypothesis, MMP and ADAM proteases are known to be

activated during apoptosis (41, 42), and we observed shedding of CD93 in response to staurosporine-

induced apoptosis of PBMCs.

In addition to its multifaceted effects on myeloid cells and opsonic activity, CD93 also has an established

role as a potent angiogenic factor (96). The mechanism by which CD93 promotes angiogenesis has not

been fully elucidated, with evidence supporting the presence of two distinct pathways. The first putative

pathway is regulated by sCD93, with sCD93 directly inducing endothelial cell division, migration and

tubule formation with a potency similar to that of VEGF (96). The endothelial receptor which recognizes

sCD93 is unknown, but is unlikely to be αxβ2 as this integrin is not normally expressed by endothelium

(97, 98). The second angiogenic pathway enhances endothelial cell migration, is driven by full-length

(membrane-bound) CD93 on endothelium, and requires interactions between membrane-bound CD93

and dystroglycan or multimerin-2 in the extracellular matrix (24, 55, 56). The signaling downstream of

the CD93-multimerin-2 interaction has not been characterized, however, CD93-dystroglycan interactions

induce phosphorylation of the intracellular tail of CD93, with this phosphorylation required for actin

reorganization and endothelial cell migration (24). How CD93 induces cell migration remains unknown,

but CD93 is known to interact with moesin and GIPC, suggesting these proteins may play a role in

mediating signaling and cell migration through full-length CD93 (22, 23). The dual-role of sCD93 as an

angiogenic factor and apoptotic cell opsonin suggests that CD93 plays an important role in the resolution

of inflammation, as the removal of apoptotic cells and revascularization of tissues are key steps during

the resolution of inflammation, and are required to return the tissue to a healthy state.

We conclude that CD93 is shed from myeloid cells under inflammatory conditions, with the resulting

sCD93 opsonizing apoptotic cells and enabling efferocytosis via αxβ2, while simultaneously enhancing

angiogenesis within the inflamed site. This mechanism would ensure the concentrated, localized delivery

of sCD93 to the inflamed environment – thus enabling the ready removal of cells killed during

inflammatory responses, and preparing the inflamed site for revascularization following resolution of

inflammation. More generally, CD93 may be shed by multiple cell types in response to cell death, thus

allowing these cells to ensure their removal through autocrine or paracrine opsonization.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Carole Creuzenet and Jocelynn Peters (University of Western

Ontario) for culturing and preparing the Campylobacter jejuni and Helicobacter pylori used in this study.

This study was funded by Canadian Institutes of Health Research Operating Grant MOP-123419, Natural

Sciences and Engineering Research Counsel of Canada Discovery Grant 418194, and an Ontario

Ministry of Research and Innovation Early Research Award to BH. JDD was funded by a Canadian

Institutes of Health Research Operating Grant MOP- 286719 whereas ENP was funded by an Alexander

Graham Bell Doctoral Canada Graduate Scholarship from the Natural Sciences and Engineering Council.

The funding agencies had no role in study design, data collection and analysis, decision to publish, or

preparation of the manuscript.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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TABLES

Table 1: MS/MS Identification of Putative Members of the sCD93 Receptor Complex Protein Symbol Peptides* Coverage (%)** Integrins Integrin αx ITGAX (CD11c) 6 10.8 Integrin β1 ITGB1 (CD29) 12 24.3 Integrin β2 ITGB2 (CD18) 19 31.1 Integrin-Associated Phagocytic Receptors CD44 CD44 3 4.6 Integrin-Associated Signaling Molecules Talin TLN1 48 29.3 Focal Adhesion Kinase 2 FAK2 27 33.2 PAK2 PAK2 8 20.4 Moesin MOES 49 88.2 * Peptides = total number of unique peptides from each protein identified across four independent experiments.

** % Coverage = portion of protein sequence covered by the identified peptides.

FIGURE CAPTIONS

Figure 1: CD93 is not a phagocytic or efferocytic receptor. A) Total bound and engulfed control beads

(BSA), phagocytic targets (IgG coated beads/IgG), apoptotic cell mimics (PtdSer), apoptotic cells (AC)

and human-serum opsonized apoptotic cells (AC+HS) by CHO cells ectopically expressing human

CD93, FcγRI and TIM4. B) Phagocytosis of beads functionalized with anti-HA or an irrelevant IgG by

CHO cells ectopically expressing HA-tagged CD93. C) Accumulation of CD93 beneath anti-HA Fab

functionalized beads in RAW 264.7 macrophages ectopically expressing HA-CD93-GFP. D) Uptake of

beads functionalized with anti-HA Fabs or an irrelevant IgG by RAW 264.7 cells ectopically expressing

HA-tagged CD93. E-F) Images (E) and quantification (F) of non-crosslinked (Fab) and crosslinked

(F(ab’)2) HA-CD93 ectopically expressed in CHO cells. Cell-surface CD93 is labeled before (Pre, red)

and after (Post, green) endocytosis. G) Number of tethered apoptotic Jurkat cells by mock-transfected

and CD93-transfected HeLa cells. Data is representative of, or quantifies as mean ± SEM, a minimum of

50 images acquired over a minimum of 3 independent experiments. * = p < 0.05, ns = p > 0.05, ANOVA

with Tukey correction (A, compared to BSA for the same receptor), or Students t-test (B,D,F). Scale bars

are 5 µm.

Figure 2: CD93 is shed during inflammation and macrophage differentiation. CD93 cleavage and

expression was quantified in primary human monocytes and macrophages using immunofluorescence

microscopy. A) Cell surface (Surface) and whole-cell (Permeabilized) CD93 immunostaining on PBMCs

left untreated (UT) or stimulated with 100 ng/mL PMA for 1 hr. Cells are counter-stained with DAPI. B)

Time-course of CD93 cleavage from monocytes following stimulation with 100 ng/mL PMA, 10 ng/ml

LPS, or 100 ng/ml TNFα. C) Effect of the MMP inhibitor GM6001 and MMP/ADAM inhibitor TAPI2

on PMA-induced CD93 cleavage by human monocytes. D) CD93 surface expression on human PBMC’s

at day 0, 2 and 5 of PBMC-to-M0 macrophage differentiation. E) Intensity of CD93 staining on M0, M1

and M2 macrophages, measured relative to undifferentiated PBMCs. F) CD93 shedding following 24

hrs treatment of PBMC’s with staurosporine (STS) ± TAPI-2, relative to CD93 on freshly isolated

PBMCs (0 hrs). Data is representative of (A) or quantified as mean ± SEM (B–F), ≥17 cells imaged over

3 independent experiments. * = p < 0.05 compared to untreated (C), PBMC (E) and 0 hrs (F), ns = p >

0.05, ANOVA with Tukey correction. Scale bar is 10 μm.

Figure 3: CD93 is an apoptotic cell opsonin. A) Number of supernatant-opsonized apoptotic cell

mimics engulfed by J774.2 macrophages (Mθ), using mimics opsonized with cell culture media (Media),

monocyte-conditioned medium (Unshed), supernatants from PMA-activated monocytes (Shed), or

CD93-depleated supernatants from PMA-activated monocytes (αCD93). B) Total number of

recombinant sCD93-coated versus BSA-coated apoptotic cell mimics bound or engulfed by J774.2

macrophages. C) Fraction of macrophages that engulfed one or more BSA-opsonized or sCD93-

opsonized apoptotic cells. D) Average number of BSA-opsonized versus sCD93-opsonized apoptotic

cells (AC) engulfed per J774 macrophage over 1 hr. E-G) Phagocytosis of unopsonized and sCD93

opsonized (E) Gram-negative bacteria: capsulated (cap) and acapsulated (acap) Escherichia. coli,

capsulated Campylobacter jejuni and Heliobacter pylori, and acapsular Pseudomonas aeruginosa; (F)

Gram-positive bacteria: capsular Streptococcus pyogenes and Staphylococcus aureus, and acapsular

Micrococcus luteus; and (G) fungi: capsular Rhodotorula mucilaginosa, and acapsular Sacchoarmyces

cerevesea and Candida glabrata. n = 3, mean ± SEM. * p < 0.05 compared to unopsonized, † p < 0.05

compared to sCD93 opsonized, ns = p > 0.05 compared to non-opsonized; ANOVA with Tukey

correction (A), Students t-test (B-D), or paired t-test comparing the same organism ± sCD93 (E-G).

Figure 4: Identification of the sCD93 receptor. A) Representative blot demonstrating the precipitation

of putative sCD93 receptors with sCD93-functionalized versus BSA-functionalized (Control) magnetic

beads and reversible cross-linking. Arrows indicate protein bands uniquely precipitated by the sCD93-

functionalized beads. B) Confirmation of shRNA knockdown of putative CD93 receptors in THP-1

macrophages. Knockdown levels are expressed as fold-knockdown after normalization to GAPDH

staining. Representative immunoblots are shown below the graph. Scrm = THP-1 expressing scrambled

(control) shRNA, sh = shRNA-expressing THP-1 cells. Dashed line indicates the expression level in

Scrm-siRNA expressing cells. C) Effect of shRNA knockdown of putative sCD93 receptors on the

fraction of macrophages engulfing one or more sCD93-opsonized beads. D) Effect of shRNA knockdown

on the number of sCD93-opsonized beads engulfed per macrophage. Data is representative of (A) or

quantifies (B-D) four independent experiments. Data is presented as mean ± SEM. * p < 0.05, ANOVA

with Tukey correction.

Figure 5: Identification of sCD93-receptor and apoptotic cell binding domains in sCD93. A)

Structural diagrams of FLAG-tagged full-length and CTLD-deficient (sCD93ΔCTLD), EGFL-deficient

(sCD93ΔEGF) and Mucin-deficient (sCD93ΔMucin) mutants of sCD93. SP = signal peptide, CTLD = C-

Type Lectin-Like Domain, EGFL = EGF-like, TM = transmembrane domain. B) Binding of beads

opsonized with full-length and mutant sCD93 by THP-1 macrophages. C) Binding of apoptotic cells

(AC) by full-length and mutant sCD93. Data quantifies three independent experiments. Data is presented

as mean ± SEM, * p < 0.05, Kruskal-Wallis with Dun Correction (B) or ANOVA with Tukey Correction

(C).

)A 12 )B 2.0 BSA * 10 * * IgG ns PtdSer 1.5 8 * AC 6 AC+HS 1.0 4 0.5 2 ns 0 0.0 FcγRI TIM4 CD93 Anti-HA IgG C) Bead Ex D) 1.2 ns CD93 DAPI Bead 0.8

CD93 0.4

Ex 0.0 Anti-HA IgG E) Fab F(ab’) F) 3 2 ns Pre Post 2

0 Fab F(ab)' 2 G) 0.6 ns

0.4

0.2

0.0 Mock CD93

Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/341933; this version posted June 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

)B 1.2 C) 1.2 )D 1.0 ns UT 1.0 1.0 0.8 0.8 TAPI2 0.8 UT A) Surface Permeabilized PMA 0.6 0.6 0.6 LPS TNFα 0.4 * 0.4 0.4 0.2 * ns 0.2 ns 0.2 0 UT PMA - + + + 0.0 0.0 0 20 40 60 GM6001 - - + - 0 2 5 Time (min) TAPI2 - - - + Days E) 1.2 F) 1.5 0 hrs 24 hrs 1.0 1.0 PMA 0.8 CD93 0.2 0.5 (rel. 0 hrs) 0 (rel. * Staining Su rface rface Staining Su rface DAPI PBMC) (rel. 0.1 * * *

93 CD 93 0.0 93 CD 93 0.0 STS - - - + + PBMC M0 M1 M2 TAPI2 - - + - + Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/341933; this version posted June 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

D) A) 3 B) 2.5 C) 1.0 * 3 * * 2.0 * 0.8 2 2 Mθ † 1.5 0.6

1.0 0.4 1 AC/ Mθ 1 0.5 0.2

0 0.0 Eerocytic Mθ (fraction) 0.0 0 Mimics Unshed Shed αC D93 BSA sCD93 BSA sCD93 BSA sCD93

E) 4 F) 2.5 ns G) 3 ns BSA BSA BSA ns 3 sCD93 2.0 sCD93 sCD93 2 ns 1.5 ns 2 ns ns 1.0 ns 1 * 1 0.5 ns ns 0 0.0 0

Gram-Negative Gram-Positive Fungi Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/341933; this version posted June 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

A) Ladder Control sCD93 B) 1.0 kDa 245 0.5 180 130 0.08 100 75 63 0.04 48 35 0.00 α β β CD44 25 x 2 1 Scrm sh Scrm sh Scrm sh Scrm sh 17 Target 11 GAPDH

)C 0.6 )D 2.5 2.0 0.4 Mθ 1.5 * 1.0 * 0.2 ** 0.5 0.0 0.0 E erocytic Mθ (fraction) UT Scm αx β2 β1 CD44 UT Scm αx β2 β1 CD44

Figure 4 bioRxiv preprint doi: https://doi.org/10.1101/341933; this version posted June 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Figure 5 bioRxiv preprint doi: https://doi.org/10.1101/341933; this version posted June 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Supplemental Table 1: Enzymes and cloning approaches used to prepare all expression vectors used in this study. FWD = forward primer, REV = reverse primer. Cloning Strategy & Construct Backbone Vector Primers Enzymes FWD: AAAAA AAAAA GAATT CATGG CCACC TCCAT GGGCC TGCT Restriction digest, CD93 pEGFP-N1 REV: GGTGG CGACC GGTTC CGCAG EcoRI/AgeI TCTGT CCCAG GTGTC G FWD: AAAAA AAAAA GAATT CATGG CCACC TCCAT GGGCC TGCT Restriction digest, CD93-GFP pEGFP-N1 REV: AAAAA AAAAA GAATT CATGG EcoRI/AgeI CCACC TCCAT GGGCC TGCT FWD: CCAGA TTACG CTCCC AGCCG CCTCC CCAAG TGGT Blunt-end ligation of PCR- HA-CD93 CD93 REV: AACAT CGTAT GGGTA AAGGA linearized vector GCGGC TGGGA CAGGT CCAG FWD: AAAAA AAAAA GAATT CATGG pLXV-EF1α-zsGreen CCACC TCCAT GGGCC TGCT Restriction digest, sCD93 or REV: TTTTT TTTTT GGATC CTTAC EcoRI/BamHI pLXV-EF1α-puro TTTTG CCCGT CAGTG CCATC GTTG CAAAA CAACG ATGGC ACTGA CGGGC AAAAG GATTA CAAGG BamHI restriction digest ACCAT GACGG AGATT ACAAA and ligation to form a sCD93-FLAG sCD93 GATCA TGATA TAGAT TATAA linear product, followed by GGATG ATGAC GATAA ATAAG Gibson assembly to GATCC CGCCC CTCTC CCTCC recircularize. CCCCC C Fragment 1: FWD: GTGGT CTGCA TTGAG GGCTT CGTGT GCAAG REV: ΔCTLD: AGCCC TCAAT GCAGA CCACC GCCTC CGTG TCA ΔEGF: TTGGG GCCAG GTCGA ACACA TCGGG GGCCT T PCR amplification of sCD93-FLAG, amplifying ΔMucin: GGATG CTGGG GGTGC the vector from either side ΔCTLD, AAGAG ACCCC ATTTG G of the deletion site to a site ΔEGF, sCD93-FLAG in the backbone opposite ΔMucin Fragment 2: sCD93. The resulting 2 FWD: GGAGT CAGGC AACTA TGGAT PCR fragments were then GAACG AAA reassembled with Gibson REV: assembly ΔCTLD: GGTGG TCTGC ATTGA GGGCT TCGTG TGCAA G ΔEGF: TGTGT TCGAC CTGGC CCCAA ATGGG GTCTC T ΔMucin: CTCTT GCACC CCCAG CATCC ATCAC GCCAC A

Supplemental Table 2: shRNA vector sequences. Target Gene Oligonucleotide GGAAA GGACG GGATC CCGAT TGTTC CATGC CTCAT ATCTC GAGAT ATGAG Integrin α x GCATG GAACA ATCGT TTTTTA CCGGT GAAGC TTGTC GGAAA GGACG GGATC CGCAC CCTGA TAAGC TGCGA AACTC GAGTT TCGCA Integrin β 2 GCTTA TCAGG GTGCT TTTTT ACCGG TGAAG CTTGTC GGAAA GGACG GGATC CGCCT TGCAT TACTG CTGAT ATCTC GAGAT ATCAG Integrin β 1 CAGTA ATGCA AGGCT TTTTT ACCGG TGAAG CTTGT C GGAAA GGACG GGATC CCGCT ATGTC CAGAA AGGAG AACTC GAGTT CTCCT CD44 TTCTG GACAT AGCGT TTTTT ACCGG TGAAG CTTGT C