Supplementary Materials

Listing of the CAPSys Study group.

Fig. S1. ADAM8 is primarily expressed and released by neutrophils during acute inflammation.

Fig. S2. Functional analysis of Adam8+/+ and Adam8-/- neutrophils.

Fig. S3. Rheologic parameters of cremaster venules analyzed in Figure 1A-D.

Fig. S4. ADAM8 levels during LPS-induced lung inflammation are dependent on neutrophils.

Fig. S5. Effect of ADAM8 ectodomain inhibition using a monoclonal antibody.

Fig. S6. SH3 domain inhibition attenuates the ADAM8-Myo1f interaction.

Fig. S7. Schematic model of drug actions on ADAM8 and Myo1f.

Fig. S8. Effect of ADAM8 inhibition on MPO release and phagocytosis in neutrophils.

Fig. S9. Proteolytic profiling of human neutrophils.

Fig. S10. PEPDab013 cleavage and ADAM8 protein levels in lung fluids of ARDS patients and healthy
controls.

Fig. S11: PEPDab014 cleavage in lung fluids of ARDS patients and healthy controls.

Fig. S12. Lower power field magnifications of histologic sections.

Table S1. Surrogate endpoint scoring system for assessing disease severity in mice.

Table S2. Summary of clinical data of patients with ARDS due to pneumonia.

Table S3. Summary of clinical data of patients with ARDS due to Primary Graft Dysfunction (PGD).

Table S4. Table of Key Resources.

Video S1. Representative intravital microscopy sequences of the mouse cremaster muscle of ADAM8+/+
and ADAM8-/- mice 2.5h after TNFα injection. Scale bar, 25µm.

Video S2. Representative 2-photon lung intravital microscopy of MRP8-Cre x mTmG mice. Neutrophils
(MRP8+, green) were tracked over 30 Minutes in the lung (red) at baseline, or in mice treated with either
the ADAM8 inhibitor BK-1361 or a control peptide during acute LPS-induced lung inflammation. Scale
bar, 50µm.
Listing of the CAPSys Study group

The following individuals and institutions constitute the CAPSys Study Group:

Petra Creutz, Aline leClaire, Agata Mikolajewska, Markus Vogtmann, Michaela Niebank, the ICU-Team,

Carmen Garcia, Norbert Suttorp, Martin Witzenrath, Charité Universitätsmedizin Berlin, Medizinische

Klinik m. S. Infektiologie und Pneumologie, Charitéplatz 1, 10117 Berlin; Sven Gläser, Henning Kahnert,

Vivantes Klinikum Spandau, Kard., Pneum. und kons. Intensivmedizin, Neue Bergstraße 6, 13585 Berlin;

Felix Rosenow, Universitätsklinikum Münster, Kardiologie 1, Albert-Schweitzer-Campus 1, 48149

Münster; Markus Scholz, Universität Leipzig, Institut für Medizinische Informatik, Statistik und

Epidemiologie, Haertelstraße 16-18, 04107 Leipzig; Julio Vera-González, Universitätsklinikum Erlangen,

Hautklinik, Hartmannstraße 14, 91052 Erlangen; Bernd Thomas Schmeck, Universitätsklinikum Gießen

und Marburg, Standort Marburg, Klinik für Pneumologie, Hans-Meerwein-Straße 2, 35043 Marburg;

Markus Löffler, Universität Leipzig, Institut für Medizinische Informatik, Statistik und Epidemiologie,

Haertelstraße 16-18, 04107 Leipzig; Uwe Völker, Sven Hammerschmidt, Ernst-Moritz-Arndt-Universität

Greifswald, Interfakultäres Institut für Genetik und Funktionelle Genomforschung, Friedrich-Ludwig-

Jahnstraße 15a, 17475 Greifswald; Michael Bauer, Universitätsklinikum Jena, Klinik für Anästhesiologie

und Intensivtherapie, Erlanger Allee 101, 07747 Jena; Michael Kiehntopf, Universitätsklinikum Jena,

Integrierte Biobank, Erlanger Allee 101, 07747 Jena; Trinad Chakraborty, Justus-Liebig-Universität

Gießen, Institut für Klinische Mikrobiologie, Frankfurter Straße 107, 35392 Gießen.
 A B D E
PMN PBMC PMN PBMC
100 15

A8/total protein (ng/µg)

Blood
***

A8 release (pg/ml)
Spleen 80 ns
* 10
Lung 60
Lower GIT ***
Granulocytes 40 ns
Adipose Tissue Monocytes ns 5
Blood Vessel T-cells 20 ns ns
B-cells
Nerve Dendritic cells 0 0
Liver NK cells 0 2 4 6 0 2 4 6
Kidney time [h] time [h]
C F G
Breast PMN PBMC PMN PBMC
Skin 2.0 8
**

A8 surface (x-fold)
Upper GIT

A8 mRNA (x-fold)
1.5 **
6
Pancreas ns * ns
Brain 1.0 ns 4
Heart Immune cells
Endothelial cells ns ns
Muscle Fibroblasts 0.5 2 ns ns
Epithelial cells
-5 0 5 10 15 0.0 0
log2 (Adam8 TPM+1) 0 2 4 6 0 2 4 6
time [h] time [h]

Figure S1: ADAM8 is primarily expressed and released by neutrophils during acute inflammation.
(A) Expression pattern of ADAM8 across healthy tissues based on the GTex human biospecimen
reference database (GTEx Analysis Release V8, ENSG00000151651.15). ADAM8 transcript levels are
highest in whole blood (mean transcripts per million (TPM): 304.4), followed by the spleen (mean TPM:
89.03) and the lung (mean TPM: 49.03). (B) Among the cells in the blood, ADAM8 was most enriched in
granulocytes. (C) Within normal lung tissue, ADAM8 is primarily expressed in immune cells, including
macrophages, granulocytes and lymphocytes. To compare the ADAM8 levels among the main immune
effectors during inflammatory activation, we isolated human PMNs and PBMCs, and assessed (D)
ADAM8 release, (E) whole cell protein, (F) surface and (G) mRNA expression over time at baseline and
following stimulation with TNFa. Data are given as mean ± SEM of 4 biological replicates;*, p < 0.05;
**, p < 0.01; ***, p < 0.001; two-way ANOVA followed by Sidak’s multiple comparison test. Activation
of PMNs resulted in the rapid release of soluble ADAM8 into the supernatant, with a corresponding
decline in cell-associated ADAM8. In contrast, TNFα-induced ADAM8 secretion by monocytic immune
cells was not significant. The surface expression of ADAM8 remained stable in PMNs, whereas PBMCs
showed an increase only at 6 hours after TNFα stimulation. ADAM8 mRNA expression was induced
early after stimulation in neutrophils and showed no significant change in PBMCs. Together, these data
suggest that PMNs are the major source of ADAM8 following acute immune activation, while
mononuclear cells exhibit a stable expression pattern in the early inflammatory phase.

Figure S1
A B C D
Adam8+/+ Adam8-/- 80 n=4 60 250 ns
* n=3 ** n=3

3D transmigration [%]

Phagocytosis [FLU]
60 200

TEndM [%]
40
150
40
ns 100
20 ns ns
20
Bone marrow PMNs 50
0 0 0
- + CXCL-1 - + CXCL-1 - + Pathogen

E F G H
Adam8+/+ Adam8-/- 200
400 15

Eucl. Distance [µm]

Acc. Distance [µm]
150

Velocity [µm/min]
300 * * 150 *
100 10
200 100
50
5
0 100 50
-50 0 0 0
[μm] -50 0 50 -50 0 50

Ad 8 +/+
8 -/-
Ad 8 +/+
8 -/-
Ad 8 +/+
8 -/-

am
am
am

am
am
am

Ad
Ad
Ad

Figure S2: Functional analysis of Adam8+/+ and Adam8-/- neutrophils. To investigate the role
of ADAM8 in neutrophil migration, we used transwell and 3D gel-matrix based in vitro assays.
(A) Bone marrow neutrophils from Adam8 deficient mice (Adam8-/-, white bars) or littermate
controls (Adam8+/+, black bars) were isolated and investigated in vitro. (B) Transmigration of
Adam8+/+ (black bars) and Adam8-/- (white bars) neutrophils across an endothelial monolayer
(TEndM) or (C) 3D transmigration across matrigel-coated transwells stimulated by CXCL-1
(100 ng/mL). Percentage of transmigrated cells for (B-C) was calculated based on the number
of neutrophils added to the transwell. Transmigration defects of Adam8-/- neutrophils indicate
that ADAM8 is instrumental for the passage of neutrophils through tissue barriers. (D)
Phagocytosis of pathogen particles (E. coli) labeled with pH-sensitive pHrodo (FLU,
fluorescent units). Data for (A-D) are means ± SD; *, p < 0.01; **, p < 0.005; two-way ANOVA
and Sidak’s multiple comparison test. (E-H) To investigate motility in interstitial spaces, 3D
chemotaxis assays on microslides (µ-Slides) were performed and the velocities of
Adam8+/+ and Adam8-/- neutrophils towards a CXCL-1 gradient in matrigel were analyzed. (E)
3D chemotactic migration (matrigel 200µg/ml) of Adam8+/+ and Adam8−/− neutrophils towards
a CXCL-1 (100 ng/mL) gradient using chemotaxis μ-slides. Representative trajectory plots are
shown, the triangle indicates the orientation of the gradient. (F) Violin plots of mean
accumulated distance (in µm), (G) velocity (µm/min) and (H) Euclidian distance (in µm) of
Adam8+/+ and Adam8−/− neutrophils. Data correspond to 3 independent experiments, 30 cells
per experiment were analyzed. *, p < 0.01, Student’s t-test.

Figure S2
 Mice Venules Diameter [µm] Vcenterline [µm s-1] Shear rate [s-1] WBC [µl-1 blood]
Adam8 +/+ 5 19 29 ± 4 3.8 ± 0.26 2.4 ± 0.16 4734 ± 678
Adam8-/- 6 22 31 ± 6 3.8 ± 0.16 2.4 ± 0.10 5541 ± 935
p-value ns (0.13) ns (0.90) ns (0.90) ns (0.12)

Figure S3: Rheologic parameters of cremaster venules analyzed in Figure 1A-D.

Figure S3
 A B C
20 1×107 PBS LPS
BAL/ lung neutrophils
8×106

ADAM8 ng/ml [BAL]

i.t. LPS collection macrophages
15

Lung
# cells/ml [BAL]
6×106
10 4×106
d0 d1 d2 d3 d4 d5 d6 d7 2×106
5

BAL
activation 1×106
resolution 0 0
0 1 2 3 4 5 6 7
ADAM8
i.t. LPS [days]

Figure S4: ADAM8 levels during LPS-induced lung inflammation are dependent on neutrophils.
(A) To determine ADAM8 expression during acute pulmonary inflammation in vivo, we challenged
mice with intratracheal (i.t) instillations of lipopolysaccharide (LPS) and collected BAL samples across
the activation and resolution phases of inflammation. Mice were sacrificed on days (d) 1, 3, 5, and 7
after LPS challenge (grey bar, grey triangles). (B) Soluble ADAM8 levels (grey) were temporally
associated with neutrophil accumulation (violet) in the BAL during the course of LPS-induced lung
inflammation (r=0.8236, p<0.0001), whereas no relationship was detected between ADAM8 and
macrophage (blue) recruitment (r=0.1824, p=0.3529), n=4-6 per timepoint. (C) Leukocytes recruited
into the alveolar space following LPS instillation in mice are immunopositive for ADAM8, whereas the
lung epithelium and endothelium lacked ADAM8 staining. Upper panel, lung tissue: lower panel, BAL
cytospin. Scale bar, 25 µm.

Figure S4
 (A) Inhibition of ADAM8
ectodomain (MAB1031)

(B) Peptidomimetic
inhibition of ADAM8 DI
domains (BK-1361)

Actin
(C) SH3 domain blockade

Prodomain (Pro)
Metalloprotease domain (MP) Motor

Myo1f
IQ Motif
ADAM8

Disintegrin domain (DI)

Cystein-rich domain (CYS) TH1 domain
EGF-like domain (EGF) TH2 domain
Transmembrane domain (TM) SH3 domain
Cytoplasmic domain (CD)

Figure S5: Schematic model of drug actions on ADAM8 and Myo1f. (A) The
monoclonal antibody MAB1031 (R&D) is directed against the ectodomain of
ADAM8 (aa 201-497) and has been shown to inhibit the catalytic activity of
ADAM8 previously (48). (B) The cyclic peptide BK-1361 is a small
peptidomimetic that interacts with the ‘KDX’ motif in the integrin-binding-loop of
human and mouse ADAM8, which efficiently blocks ADAM8 multimerization
and subsequent auto-activation (33). (C) Blocking of SH3 domains (49) could
prevent the intracellular interaction of ADAM8 and Myo1f. Protein domains of
ADAM8 and Myo1f (lower panels).

Figure S5
 A B C Coloc Map
D E
MAB1031(1) IgG(2)

200
Input 100 ** 80 **

3D transmigration [%]
+ IgG
Co-IP

IgG

IgG

Relative Myo1f binding

A8

A8
(1) (2) 1.5 80

150
60

TEndM [%]
< 150 ns

(pixel intesity)
IB: MYO1F 60
(125 kD) 1.0 40
< 100

100
40

+ MAB1031
< 100
IB: ADAM8 < 75
0.5 20 20

50
(60/ 90 kD)
< 50 0 0
0.0 - + + fMLP - + + fMLP

IgG
MAB1031
hPMN + TNFα

MAB1031
IgG

MAB1031
IgG
F G H I
+ IgG Ctrl + MAB1031 400 600 50

Eucl. Distance [µm]

ns ns

Acc. Distance [µm]

ns

Velocity [µm/min]
300 500
300 40
200 400
200 30
100 300
200 20
0 100
100 10
-100 0
[μm] -100 0 100 -100 0 100
IgG 0 0
MAB1031

IgG
MAB1031

IgG
MAB1031
Figure S6: Effect of ADAM8 ectodomain inhibition using a monoclonal antibody. (A) Co-IP of
activated human neutrophils treated with either an ADAM8 activity blocking antibody (MAB1031)
or control IgG (IgG) during TNFα stimulation. (B) Relative pixel intensity of Myo1f co-precipitated
with ADAM8 normalized to input using ImageJ Software, ns, not significant, Student’s t-test. (C)
Colocalization map (Coloc Map) of human neutrophils treated with MAB1031 or IgG as described
in D. Scale bar, 10 µM. (D) Effect of MAB1031 on fMLP-induced transendothelial migration of
human neutrophils (n=4 donors) and (E) migration across a 3D gel matrix (matrigel 200µg/ml),
respectively (n=3 donors). Data are given as mean ± SD, stars indicate **, p < 0.002; Student’s t-
test. (F) 3D chemotactic migration (matrigel 200µg/ml) of human neutrophils preincubated with
MAB1031 or control (IgG) toward an IL-8 (1 µg/mL) gradient using chemotaxis μ-slides.
Representative trajectory plots are shown, triangles indicate orientation of the gradient. Violin plots
of (G) mean Euclidian distance, (H) accumulated distance and (I) migration velocity of human
neutrophils preincubated with MAB1031 (grey) or IgG control (black). Data correspond to 3
independent experiments, 30 cells per experiment were analyzed. ns, not significant; Student’s t-test.

Figure S6
 A B C

Input A8 IgG Co-IP

Relative Myo1f binding

DMSO
DMSO
1.2

cpd_B
cpd_A

cpd_B
cpd_A
DMSO
cpd_B
cpd_A
1.0 ns

(pixel intesity)
< 150
IB: MYO1F 0.8
(125 kD) < 100 0.6
*
< 100 0.4
IB: ADAM8 < 75 0.2
(60/90 kD)
< 50 0.0

cpd_B
cpd_A
DMSO
1179
1173
hPMN + TNFα

D E 120 ns
100 ns
+ DMSO + cpd_B + cpd_A

Viability [%]
80
Coloc Map

60
40
20
0
S SO d9_
B

_A
50 100 150 200
PB DM c11p7 173

d
cp
1

Figure S7: SH3 domain inhibition attenuates the ADAM8-Myo1f interaction. (A)
Chemical structure of two inhibitors with the potential to block SH3 domains [cpd_A,
compound A, cpd_B, compound B, described previously (49)]. (B) Human neutrophils were
treated with 10µM cpd_A, cpd_B or DMSO as vehicle control during TNFα stimulation;
representative immunoblot of ADAM8/Myo1f Co-IP is shown. (C) Corresponding
densitometric analysis of Myo1f binding to ADAM8 normalized to input using ImageJ
Software. * p < 0.05, ns; not significant, one-way ANOVA followed by Tukey’s multiple
comparison test. (D) Colocalization map of human neutrophils treated with SH3
inhibitors/vehicle as described in (A) stained for Myo1f and ADAM8. (E) MTT viability
assay of primary human neutrophils incubated with 10µM cpd_A, cpd_B, vehicle control
(DMSO) or PBS for the duration of functional assays, n=3. ns; not significant, one-way
ANOVA followed by Tukey’s multiple comparison test.

Figure S7
A B C
CP IgG
BK1361 ns MAB1031 ns ns
MPO release (x-fold) 15 ns 15 300

MPO release (x-fold)

ns

Phagocytosis (FLU)
ns 250
ns
10 ns 10 200
ns 150
5 ns 5 100
ns ns
50
0 0 0
time [min] 0 30 120 240 360 time [min] 0 30 120 240 360 - + + + E. coli

+ BK1361
+ MAB1031
control
Figure S8: Effect of ADAM8 inhibition on MPO release and phagocytosis in
neutrophils. MPO release by primary human neutrophils stimulated with TNFα over
time quantified by ELISA. Pharmacological ADAM8 inhibition with (A) BK-1361 (white
bars) or (B) MAB1031 (grey bars) was not altered compared to respective controls (black
bars). n=3, ns, not significant; 2-way ANOVA followed by Sidak’s multiple comparison
test. (C) Phagocytic activity of PMNs measured by the internalization of pHrodo-
conjugated E.coli bioparticles (FLU, fluorescent units), respectively. n=3, ns, not
significant; 1-way ANOVA followed by Sidak’s multiple comparison test.

Figure S8
A B C
Catalytic efficiency [s-1 M-1] kcat/KM Cleavage rate [s-1] x10-3
homologous MMP-8 106 005
peptide # cleavage sequence 15

FRET-Substrate
cleavage site 008
MMP-9 105
005 LAQAPhe(homo)RSK Pro-TNFα 010
ADAM8 011 10
008 PChaGC(Me)HAK - 104
ADAM10 013
010 SPLAQAVRSSK Pro-TNFα ADAM17 103 5
014
011 GPLGMRGK - ADAM9 052
102
013 HGDQMAQKSK CD23 #1 #2 #3 #1 #2 #3

052
011
013
014
005
008
010
014 EHADLLAVVAK Pro-TGFα baseline + TNFα
FRET-Substrate
052 APFEMSAK -

D
PEPDab 005 PEPDab 008 PEPDab 010 PEPDab 011 PEPDab 013 PEPDab 014 PEPDab 052
1.5×104 8×103 8×104 1×104 1.5×104 5×104 3×104
TNFα
Δ fluorescence (FLU)

baseline 6×103 6×104 8×103 4×104

1.0×104 1.0×104 2×104
6×103 3×104
4×103 4×104
5.0×103 4×103 2×104
5.0×103 1×104
2×103 2×104 2×103 1×104
0.0 0 0 0 0.0 0 0
0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60
time [min] time [min] time [min] time [min] time [min] time [min] time [min]

E
PEPDab 005 PEPDab 008 PEPDab 010 PEPDab 011 PEPDab 013 PEPDab 014 PEPDab 052
5×103 1.5×104 5×104 5×103 8×103 4×104 2.0×104
TNFα
Δ fluorescence (FLU)

3
4×10 4×104 4×103
baseline 6×103 3×104 1.5×104
1.0×104
3×103 3×104 3×103
4×103 2×104 1.0×104
2×103 2×104 2×103
5.0×103
1×104 2×103 1×104 5.0×103
1×103 1×103
0.0 0 0 0 0 0.0
0 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60
0 20 40 60
time [min] time [min] time [min] time [min] time [min] time [min]
time [min]

F
PEPDab 005 PEPDab 008 PEPDab 010 PEPDab 011 PEPDab 013 PEPDab 014 PEPDab 052
1×104 1×104 8×104 1×104 1×104 8×104 4×104
TNFα
Δ fluorescence (FLU)

8×103 baseline 8×103 8×103 8×103

6×104 6×104 3×104
6×103 6×103 6×103 6×103
4×104 4×104 2×104
4×103 4×103 4×103 4×103

2×103 2×104 2×103 2×104 1×104

2×103 2×103

0 0 0 0 0 0 0
0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60
time [min] time [min] time [min] time [min] time [min] time [min] time [min]

G
PEPDab 005 PEPDab 008 PEPDab 010 PEPDab 011 PEPDab 013 PEPDab 014 PEPDab 052
1×104 1×104 8×104 1×104 1×104 8×104 4×104
Δ fluorescence (FLU)

8×103 8×103 8×103 8×103

6×104 6×10 4
3×104
6×103 6×103 6×103 6×103
4×104 4×104
4×103 2×104
4×103 4×103 4×103
2×103 2×104 2×104 1×104
2×103 2×103 2×103
0 0 0 0
0 20 40 60 0 20 40 60 0 20 40 60 0 0 0
0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60
time [min] time [min] time [min] time [min] time [min] time [min] time [min]
TNFα
TNFα + BB94
TNFα + PI

Figure S9: Proteolytic profiling of human neutrophils. (A) Cleavage sequences of fluorescent
substrates and, if applicable, homologous cleavage sites in endogenous substrates. (B) Standard
table of catalytic efficiencies of recombinant MMP-8, MMP-9, ADAM8, ADAM9, ADAM10 and
ADAM17 for FRET-based peptide substrates 005, 008, 010, 011, 013, 014 and 052, as described
previously (Miller et al., 2011). (C) Substrate cleavage patterns of isolated human neutrophils
obtained from three donors at baseline and after stimulation with TNFα (20ng/ml) were calculated
from (D-F) using a non-linear kinetic model. (D-F) Time-lapse fluorimetry (fluorescence change of
time) of unstimulated (black) and stimulated (red) human neutrophils using a panel of seven FRET-
based peptide substrates (PEPDab 005, 008, 010, 013, 014 and 052, Biozyme Inc., Apex, NC) to
deduce a profile of specific MMP and ADAM proteolytic activities. (G) Proteolytic activities were
reduced by ~90% using cOmplete Protease Inhibitor (green) with broad inhibitory specificity
against serine, cysteine, and metalloproteases, and blocked by ~70-90% using the broad-spectrum
metalloproteinase inhibitor batimastat BB-94 (blue). Representative time-lapse data are plotted for
TNFα stimulated neutrophils from (F) and respective inhibitors treatments.

Figure S9
 A 1000
C_01
1500
C_02 1500
C_03 1500
C_04
1000 1000 1000

Δ fluorescence (FLU)
500
500 500 500
0
0 0 0
-500
-500 -500 -500
-1000 -1000 -1000 -1000
-1500 -1500
0 20 40 60 80 0 20 40 60 80 -15000 20 40 60 80
-1500
0 20 40 60 80
time [min] time [min] time [min] time [min]

B ARDS_01 ARDS_02 ARDS_03 ARDS_04 ARDS_05

1500 1500 1500 800 2500
Δ fluorescence (FLU)

600 2000
1000 1000 1000
400 1500

200 1000
500 500 500
0 500

0 0 0 -200 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
C time [min] time [min] time [min] time [min] time [min]

ARDS_06 ARDS_07 ARDS_08 ARDS_09 ARDS_10 ARDS_11

4000 10000 5000 15000
4000 6000
Δ fluorescence (FLU)

8000 4000
3000 3000
10000
4000 6000 3000
2000 2000
4000 2000
2000 5000
1000 1000
2000 1000

0 0 0 0 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
time [min] time [min] time [min] time [min] time [min] time [min]

D Control
ARDS survivors
ARDS non-survivors
500
ADAM8 pg/ml [BAL]

400
300
200
100
nd nd nd nd nd nd nd nd nd
0
1 2 3 4 01 02 03 04 05 06 07 08 09 10 11
C0 C0 C0 C0 DS DS DS DS DS DS DS DS DS DS DS
R R R R R R R R
AR AR A A A A A A AR A A
Patient-ID

Figure S10: PEPDab013 cleavage and ADAM8 protein levels in lung fluids of
ARDS patients and healthy controls. Time-lapse fluorimetry (fluorescence
change over time) of (A) control BAL (grey), (B) BAL of ARDS survivors (black)
and (C) BAL of ARDS non-survivors (red) using the moderately ADAM8 specific
substrate PEPDab 013. (D) Detection of soluble ADAM8 in BAL samples of
control (white), ARDS survivors (black) and ARDS non-survivors using ELISA,
respectively. Dotted grey line, sensitivity cut-off 50pg/ml; nd, not detectable.

Figure S10
 A
C_01 C_02 C_03 C_04
2000 1500 2000 1500

Δ fluorescence (FLU)
1500 1000 1500 1000

1000 500 1000 500

500 0 500 0

0 -500 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 -500
0 20 40 60 80
time [min] time [min] time [min] time [min]
B ARDS_01 ARDS_02 ARDS_03 ARDS_04
7500 800 6000 1500
Δ fluorescence (FLU)

600
5000 4000 1000
400

200
2500 2000 500
0

0 -200 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
C time [min] time [min] time [min] time [min]

ARDS_06 ARDS_07 ARDS_08 ARDS_09 ARDS_10

1500 2000 25000 2000 5000

4000
Δ fluorescence (FLU)

20000
1500 1500
1000 3000
15000
1000 1000
10000 2000
500
500 500 1000
5000

0 0 0 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
time [min] time [min] time [min] time [min] time [min]

Figure S11: PEPDab014 cleavage in lung fluids of ARDS patients and healthy
controls. Time-lapse fluorimetry (fluorescence change over time) of (A) control
BAL (grey), (B) BAL of ARDS survivors (black) and (C) BAL of ARDS non-
survivors (red) using the moderately ADAM17 specific substrate PEPDab014.

Figure S11
 Lung tissue (P.a., Adam8-/-)

100μm
H&E

Lung tissue (P.a.,Adam8+/+)

100μm
H&E

Supplemental Figure 12. Lower power field

magnification of Figure 3G.
 Lung tissue (P.a. + BK1361)

100μm
100μm
H&E

Lung tissue (P.a. + CP)

100μm
H&E

Lower powerfield magnification of Figure 3R.

Control

100μm
ADAM8 (AF1031)

ARDS

100μm
ADAM8 (AF1031)

Lower powerfield magnification of Figure 4c.

 Lung tissue (PBS) Lung tissue (LPS)

100μm 100μm
ADAM8 (orb4376) ADAM8 (orb4376)

50μm 50μm
ADAM8 (orb4376) ADAM8 (orb4376)

Lower powerfield magnification of Figure S4c.

 BAL (PBS) BAL (LPS)

50μm 50μm
ADAM8 (orb4376) ADAM8 (orb4376)

25μm 25μm
ADAM8 (orb4376) ADAM8 (orb4376)

Lower powerfield magnification of Figure S4c.

 Mouse Clinical Assessment Score for Sepsis (M-CASS)
Score 1 2 3 4
Fur Aspect Normal fur Slightly ruffled fur Ruffled fur Ruffled fur and piloerection

Activity Normal Reduced Only when provoked Little or none with provocation

Posture Normal Hunched, moving Hunched, strained or stiff Hunched, little or no

freely movement movement

Behavior Normal Slow Abnormal when disturbed Abnormal, no relocation

or provoked
Chest Movements Normal Mild dyspnea Moderate dyspnea Severe dyspnea

Eyelids Normal, open Opened when Partially closed, even Mostly or completely closed,
disturbed when disturbed even when provoked

Table S1: Surrogate endpoint scoring systems for assessing disease severity in mice. Mouse Clinical
Assessment Score for Sepsis (M-CASS).

Table S1
Act ADAM8 [nM] Sex Age ARDS etiology BAL cells Outcome
0.830 F 61 Pneumonia 78% PMN non-survivor
(bacterial) 14% Macrophages
8% Lymphocytes
0.634 M 55 Pneumonia 62% PMN non-survivor
(viral) 11% Macrophages
7% Lymphocytes
0.516 M 47 Pneumonia 82% PMN non-survivor
(bacterial, viral) 13% Macrophages
5% Lymphocytes
0.472 F 52 Pneumonia 87% PMN non-survivor
(bacterial, viral, fungal) 11% Macrophages
7% Lymphocytes
0.403 F 51 Pneumonia 85% PMN non-survivor
(viral) 4% Macrophages
9% Lymphocytes
0.399 F 56 Pneumonia 73% PMN non-survivor
(bacterial, viral) 26% Macrophages
1% Lymphocytes
0.368 F 30 Pneumonia 71% PMN survivor
(bacterial, viral) 19 % Macrophages
10% Lymphocytes
0.340 M 40 Pneumonia N/A survivor
(bacterial, viral)

0.298 M 50 Pneumonia 51% PMN survivor, required

(bacterial, viral) 42% Macrophages lung transplantation
pre-existing lung fibrosis 7% Lymphocytes
0.211 M 53 Pneumonia 19% PMN survivor
(bacterial, viral) 49% Macrophages
30% Lymphocytes
0.177 F 24 Healthy N/A control

0.139 M 20 Pneumonia 71% PMN survivor

(bacterial, viral) 19% Macrophages
7% Lymphocytes
0.139 M 27 Healthy N/A control

0.095 M 23 Healthy N/A control

0.077 F 23 Healthy N/A control

Table S2: Summary of clinical data of patients with ARDS due to pneumonia.

Table S2
Act ADAM8 [nM] PGD Sex Age Transplant indication BAL cells MV [d] ICU stay [d]
1.310 Severe F 63 Non-IPF ILD 87% PMN 33 42
13% Macrophages
0% Lymphocytes
1.15 Severe F 21 Chronic transplant 0% PMN N/A 28
rejection 100% Macrophages
0% Lymphocytes
0.897 Severe M 68 IPF 90% PMN 2 8
6% Macrophages
4% Lymphocytes
0.698 Severe M 67 Non-IPF ILD 52% PMN 46 50
48% Macrophages
0% Lymphocytes
0.560 Mild F 54 Non-IPF ILD 55% PMN 5 13
45% Macrophages
0% Lymphocytes
0.558 Severe M 70 IPF N/A N/A N/A
0.497 Severe M 66 NSIP 21% PMN 41 31
78% Macrophages
1% Lymphocytes
0.447 Severe F 32 CTD-ILD 72% PMN 40 40
27% Macrophages
1% Lymphocytes
0.444 Severe M 61 COPD 47% PMN 28 35
53% Macrophages
0% Lymphocytes
0.332 Severe F 60 Non-IPF ILD 74% PMN 57 48
21% Macrophages
5% Lymphocytes
0.324 Mild M 63 CTD-ILD N/A 2 4
0.282 Mild 51 Non-IPF ILD 48% PMN 3 6
49% Macrophages
3% Lymphocytes
0.278 Severe M 66 IPF 75% PMN 4 6
23% Macrophages
2% Lymphocytes
0.264 Mild F 50 CF 30% PMN 1 3
68% Macrophages
1% Lymphocytes
0.248 Severe F 33 PAH (idiopathic) N/A N/A 18

0.214 Mild M 69 IPF N/A 1 5

0.195 Severe F 74 Non-IPF ILD 81% PMN 1 5

17% Macrophages
2% Lymphocytes
0.194 Severe M 68 IPF 88% PMN N/A 14
11% Macrophages
1% Lymphocytes
0.153 Mild F 41 CF 54% PMN 1 4
44% Macrophages
2% Lymphocytes
0.15 Mild F 65 IPF 71% PMN 1 5
24% Macrophages
5% Lymphocytes
0.145 Mild M 57 Non-IPF ILD 20% PMN 1 7
75% Macrophages
5% Lymphocytes
0.141 Severe M 33 NSIP 79% PMN 2 7
21% Macrophages
3% Lymphocytes
0.141 Mild M 70 COPD N/A 0 7

0.121 Mild F 31 CF 55% PMN 4 8

43% Macrophages
4% Lymphocytes

Table S3
 0.114 Mild M 70 Hypersensitivity 53% PMN 2 4
Pneumonitis 45% Macrophages
2% Lymphocytes
0.085 Mild M 69 IPF 80% PMN 4 6
18% Macrophages
2% Lymphocytes
0.0797 Severe M 49 CTD-ILD 77% PMN 3 7
21% Macrophages
3% Lymphocytes
0.0751 Mild M 68 Sarcoidosis N/A 2 5

0.067 Severe M 48 Chronic transplant 86% PMN 2 12

rejection 13% Macrophages
1% Lymphocytes
0.0653 Mild F 61 COPD N/A N/A 5
0.047 Mild M 49 CF N/A 1 5
0.019 Mild M 42 Non-IPF ILD N/A 21 N/A

Table S3: Summary of clinical data of patients with ARDS due to Primary Graft Dysfunction (PGD). IPF,
idiopathic fibrosis; ILD, Interstitial Lung Disease; NSIP, Non-specific interstial pneumonia; CTD;
Connective Tissue Disease; COPD, Chronic obstructive pulmonary disease; CF, Cystic fibrosis; PAH,
Pulmonary arterial hypertension.

Table S3
Supplemental Table 4. Table of Key Resources

REAGENT/RESOURCE SOURCE IDENTIFIER

Antibodies
eFluor450-conjugated Mouse CD11c (Clone: 418) ThermoFisher Cat # 48-0114-82, RRID: AB_464940
FITC-conjugated Mouse CD11b (Clone: M1/70) ThermoFisher Cat # 14-0112-82, RRID: AB_467108
PE-conjugated Mouse CD86 (Clone: B7-2) ThermoFisher Cat # 12-0862-82, RRID: AB_465768
APC-conjugated Mouse Ly6G (Clone: 1A8) ThermoFisher Cat # 17-9668-82, RRID: AB_2573307
PerCP-Cy5.5-conjugated Mouse CD45 (Clone: 30-F11) BioLegend Cat # 103131, RRID: AB_893344
PE-Cy7-conjugated Mouse F4/80 (Clone: BM8) ThermoFisher Cat # 25-4801-82, RRID: AB_469653
Human ADAM8 ectodomain (Clone: 143303) R&D Systems Cat # MAB1031, RRID: AB_2305036
Human ADAM8 Polyclonal Ectodomain Antibody R&D Systems Cat # AF1031, RRID: AB_354549
Human ADAM8 Polyclonal Cytoplasmic Domain Antibody LSBio Cat # LS-B4068-50, RRID: AB_10718869
Mouse IgG2B Isotype Control (Clone: 20116) R&D Systems Cat # MAB004, RRID: AB_357346
Human Myo1f Polyclonal Antibody Biorbyt Cat # orb221550
Anti-Goat IgG H&L (Alexa Fluor® 568) Abcam Cat # ab175474, RRID: AB_2636995
Anti-Rabbit IgG H&L (Alexa Fluor® 488) Abcam Cat # ab150077, RRID: AB_2630356
Anti-Rabbit IgG, HRP-linked Antibody Cell Signaling Cat # 7074, RRID: AB_2099233
Anti-Mouse IgG, HRP-linked Antibody Cell Signaling Cat # 7076, RRID: AB_330924
Anti-Goat IgG, HRP-linked Antibody ThermoFisher Cat # PA1-28664, RRID: AB_10990162
Mouse Myeloperoxidase Polyclonal R&D Systems Cat # AF3667, RRID: AB_2250866
Mouse ADAM8 Polyclonal R&D Systems Cat # orb4376, RRID: AB_10948754
Mouse Ly6G (Clone: 1A8) Bio X Cell Cat # BE0075-1, RRID: AB_1107721
Mouse S100A8 R&D Systems Cat # AF3059, RRID: AB_2184254
Anti-Rat IgG, Cy3 linked Antibody Jackson ImmunoResearch Cat # 712-165-153, RRID: AB_2340667
Anti-Goat IgG, AF488 linked Antibody Jackson ImmunoResearch Cat # 705-545-003, RRID: AB_2340428
Hoechst 33342 Solution ThermoFisher Cat # 62249
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) ThermoFisher Cat # D1306
Fixable Yellow Dead Cell Stain ThermoFisher Cat # L34967

Bacterial and Virus Strains

Pseudomonas aeruginosa (Strain PA103) ATCC ATCC 29260

Biological Samples
Lung fluids of patients with ARDS due to pneumonia Universities of Giessen and UGMLC/DLZ, Az 58/15 / Az 87/12
Marburg Lung Center,
Pneumonia Biorepository
Lung fluids of healthy volunteers Universities of Giessen and UGMLC/DLZ, Az 58/15 / Az 87/12
Marburg Lung Center
Lung fluids of patients with ARDS due to PGD University of California, San IRB # 13-10738
Francisco, Lung Transplant
Biorepository
Lung tissue from ARDS and non-ARDS patients University of Texas Health and IRB # HSC-MS-08-0354
Science Center, Houston
Chemicals, Peptides, and Recombinant Proteins
N-Formyl-Met-Leu-Phe (fMLP) Sigma Cat # F3506
Lipopolysaccharide (O111:B4) Sigma Cat # L4391
Mouse TNFa GenScript Cat # Z03333
Mouse Cxcl-1/KC GenScript Cat # Z02899
Human TNFa GenScript Cat # Z01001
Human IL-8 GenScript Cat # Z03061
Batimastat (BB-94) Sigma Cat # SML0041
cOmplete Protease Inhibitor Roche Cat # 11697498001
TRIzol Reagent ThermoFisher Cat # 15596026
Matrigel Corning Cat # 356237

Critical Commercial Assays

Mouse Myeloperoxidase ELISA R&D Systems Cat # DY3667
Mouse Cxcl-1 ELISA R&D Systems Cat # DY453
Mouse TNFa ELISA R&D Systems Cat # DY410
Mouse ADAM8 ELISA Biomatik Cat # EKU02052
Mouse Albumin ELISA Bethyl Laboratories Cat # E90-134
Human ADAM8 ELISA R&D Systems Cat # DY1031
Human Myeloperoxidase ELISA R&D Systems Cat # DY3174
Pierce BCA Protein Assay Kit ThermoFisher Cat # 23225
Dynabeads Antibody Coupling Kit ThermoFisher Cat # 14311D
VECTASTAIN Elite ABC-HRP Kit, Peroxidase (goat IgG) Vector Laboratories Cat # PK-6105
VECTASTAIN Elite ABC-HRP Kit, Peroxidase (Rabbit IgG) Vector Laboratories Cat # PK-6101
DAB Substrate Kit Vector Laboratories Cat # SK-4100
pHrodo Green E. coli BioParticles ThermoFisher Cat # P35366
High Capacity cDNA Reverse Transcription Kit ThermoFisher Cat # 4368814
Power SYBR Green PCR Master Mix ThermoFisher Cat # 4368706
TaqMan Fast Advanced Master Mix ThermoFisher Cat # 4444556
µ-Slide Chemotaxis Ibidi Cat # 80326
6.5mm Transwell Insert, 8µm Pore Size Corning Cat # 3422
Deposited Data
GTEx Analysis Release V8 (dbGaP Accession GTexPortal https://gtexportal.org/home/datasets
phs000424.v8.p2), ENSG00000151651.15
Experimental Models: Cell Lines
Mouse: Brain-derived Endothelial Cells (b.End3), p5 ATCC ATCC CRL-2299
Human: Microvascular Endothelial Cells (HMEC-1), p9 ATCC ATCC CRL-3243
Human: Leukocyte Cell Line (HL-60), p0 ATCC ATCC CRL-240, LOT: 64048671

Experimental Models: Organisms/Strains

Mouse: (WT) C57BL/6 Jackson Laboratory Cat# 000664; RRID: IMSR_JAX:000664
Mouse: (Adam8 ko), Adam8-/- A.J.P. Docherty, UCB, Slough, N/A
UK
Mouse: B6.Cg-Tg(S100A8-cre,-EGFP)1Ilw/J Jackson Laboratory Cat# 021614; RRID: IMSR_JAX:021614
Mouse: D2.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,- Jackson Laboratory Cat# 026862, RRID: IMSR_JAX:026862
EGFP)Luo/SjJ
Oligonucleotides
Human ADAM8 KiCqStart Primers Sigma Cat # H_ADAM8_1
Human ACTB KiCqStart Primers Sigma Cat # H_ACTB_1
Human GAPDH KiCqStart Primers Sigma Cat # H_GAPDH_1
Mouse IL1b TaqMan Gene Expression Assay (FAM) Thermo Fisher Cat # 4331182, Mm00434228_m1
Mouse TNFa TaqMan Gene Expression Assay (FAM) Thermo Fisher Cat # 4331182, Mm00443258_m1
Mouse GAPDH TaqMan Gene Expression Assay (FAM) Thermo Fisher Cat # 4331182, Mm99999915_g1
Eukaryotic 18S TaqMan Gene Expression Assay (FAM) ThermoFisher Cat # 4331182, Hs03003631_g1

Software and Algorithms

ImageJ Software NIH https://imagej.nih.gov/ij/
FlowJo Software TreeStar https://www.flowjo.com/
Imaris Oxford Instruments https://imaris.oxinst.com/
GraphPad Prism V8 Software GraphPad https://www.graphpad.com/
Matlab 2020b Software MathWorks https://www.mathworks.com