discover rate (FDR) < 0.01] were considered for
further analyses, based on results from DESeq2.
Preparation of single-cell suspensions
Mousegingivaeweredissectedanddigestedfor
50 min at 37°C with Collagenase IV (GIBCO,
Gaithersburg, MD, USA) and DNase (Sigma,
St. Louis, MO, USA), as previously described ( 52 ).
Flow cytometry
Single-cell suspensions of gingiva were stained
withtheLIVE/DEADFixableAquaDeadCell
Stain Kit, for 405 nm excitation (L34966;
AmCyan; Invitrogen, Waltham, MA), and cell
surface markers were stained with the follow-
ing anti-mouse antibodies at a concentration
of 2mg/ml: anti-Ly6C (AL-21; BV711; BD Bio-
sciences, Franklin Lakes, NJ), anti-Ly6G (1A8;
PE-Cy7; BD Biosciences), anti-B220 (RA3-6B2;
BV785; BioLegend, San Diego, CA), anti-CD11B
(M1/70; PerCP-Cy5.5; BioLegend), anti-TCRb
(H57-597; APC-Cy7; BioLegend), anti-CD11C
(N418; FITC; eBioscience), and anti-CD45 (30.F11;
BV605; eBioscience). All samples were analyzed
using a FACS Fortessa cytometer (BD Biosci-
ences). Data analysis was performed using
FlowJo software (Tree Star). Results were dis-
played as means ± SEM.
Purification of fibrinogen
Wild-type andFgg390-396A/390-396Afibrinogen
was purified from citrated-plasma by ammo-
nium sulfate precipitation as described ( 53 ).
Neutrophil isolation from mouse bone marrow
Neutrophils were isolated as described pre-
viously ( 54 ). Briefly, bone marrow cells were
flushed out from femurs and tibiae of 8- to
12-week-old mice with RPMI 1640 supplemented
with 2 mM EDTA, 20% heat-inactivated fe-
tal bovine serum (FBS), and 1% penicillin–
streptomycin. Neutrophils were isolated from
total bone marrow cells by density gradient
centrifugation at 300gfor 30 min without
break on Histopaque 1119/Histopaque 1077. The
purity of the neutrophils was above 95% as
determined by flow cytometry analysis. Then
neutrophils were washed with phosphate-buffered
saline (PBS) and suspended in RPMI 1640 sup-
plemented with 2% heat-inactivated FBS.
Neutrophil isolation from human blood
Human neutrophils were isolated from hep-
arinized peripheral blood from the NIH blood
bank using density gradient centrifugation
at 300gfor 30 min without break on Ficoll-
Paque premium (GE Healthcare, Pittsburg,
PA) followed by removal of red blood cells
(RBCs) using dextran-promoted rosette for-
mation and hypotonic lysis of remaining RBC.
For ROS analysis, blood from four female and
11 male donors was used. For NET analyses,
blood from four female and three male donors
was used.
Cell-based assays
Fibrin coating of wells
Plates were coated with purified wild-type or
mutated fibrin (0.60mg/ml mouse fibrinogen +
5 U/ml mouse thrombin) at 37°C overnight in
a humidified incubator. Excess thrombin was
washed off with Dulbecco’s minimum essen-
tial medium (DMEM) and nonspecific bind-
ing sites were blocked using 1% nonfat dry milk
(1 hour) followed by three DMEM washes. Plates
were allowed to dry for 15 min before use.Cell adhesion assay
Cell adhesion assays were performed in 96-well
Blackm-Plates (ibidi USA, Inc., Fitchburg, WI).
Aliquots of cells (1 × 10^5 cells/ml) were added
to fibrin-coated wells, incubated at 37°C for
30 min, and subsequently washed with buf-
fered saline to remove nonadherent cells. The
number of adherent cells was determined by
counting DAPI-stained cells in multiple high-
powered (40X) fields. All analyses were per-
formed in triplicate.ROS production
ROS assays were performed in 96-well clear
plates (ThermoFisher Scientific). Aliquots (100ml)
of cells (3.3 × 10^6 cells/ml) were added to wells
and incubated at 37°C for 30 min. Control wells
without cells were used to negate any contam-
inant from fibrin. Cells were supplemented
with (25ml) cytochrome c (1800 nM) with or
without 15 nM PMA (phorbol 12-myristate
13-acetate) and/or superoxide dismutase (SOD;
60 U/ml), and absorbance was measured at
550 nm for 5 hour every 10 min. The absor-
bance values at 550 nm (differences in OD
between samples with and without SOD) were
converted to nanomoles of O 2 −based on the
extinction coefficient of cytochrome c: DE550=
21 × 10^3 M−^1 cm−^1. The results were expressed
as nanomoles of O 2 −per 1 × 10^6 cells.NETosis
NETosis assays were performed in eight-well
m-Slides (ibidi USA, Inc.). Aliquots (200ml) of
cells (2.5 × 10^4 cells/ml) and Hoechst (1mg/ml)
were added per well and incubated at 37°C
for 30 min in a humidified incubator. Wells
were washed with FluoroBrite DMEM (Thermo
Fisher Scientific) to remove nonadherent cells
and supplemented with 200ml of FluoroBrite
DMEM containing 0.2mM Sytox Green. Time-
lapse series were acquired every 5 min for
5 hours on an inverted Nikon TI-E microscope
using a Plan Fluor 20X (N.A. 0.5) objective and
a Flash-4 v3 sCMOS camera (Hamamatsu,
Japan). Excitation and emission were provided
by a Sola light engine (Lumencor, Beaverton,
OR) and standard DAPI and green fluorescent
protein (GFP) filter sets (Chroma) for Hoechst
and Sytox Green, respectively. Light inten-
sity and camera exposure times varied between
3 and 5% and 100 ms, respectively. An environ-mental chamber (Precision Plastics) kept sam-
ples humidified and at a constant 37°C with
5% CO 2. All hardware was controlled through
NikonElements.Afterthefirst10minofim-
aging, cells were supplemented with 200ml of
FluoroBrite DMEM containing 0.2mM Sytox
Green with or without 15 nM PMA and con-
tinued imaging for 5 hours.NET analysis and quantification
Analysis and quantification were performed
as published ( 55 ). For an example of the pro-
cessing and analysis, see fig. S7B. Briefly, to
distinguish cells undergoing apoptosis from
NETosis, a threshold was applied to each im-
age stack and measured using the“Analyze
Particles”plugin using different size filters to
exclude smaller cells (NETosis > 120; Apo-
ptosis > 52). The Hoechst channel was used
to determine live from dead cells. An un-
sharpen mask (radius = 15, 0.60 scaling) was
used to remove out of focus blur. The user
then visually determined if each was correct
and adjusted false positives before any fur-
ther statistical analysis.DNase I treatment
Plg−/−mice of 8 weeks of age were i.p. injected
with either 400 U of DNase I (Millipore Sigma,
Burlington, MA) in 100mlof0.9%NaClor
0.9% NaCl, twice a week (Tuesday and Friday
in the midafternoon) for 12 weeks. Heads
were harvested and bone loss measurements
were taken as described under“Bone loss
measurements.”siRNA-LNP preparation and analysis
siFbg or control siRNA targeting Luciferase
(siLuc), purchased from Integrated DNA Tech-
nologies (Coralville, IA), were encapsulated
in lipid nanoparticles (LNPs) and analyzed
as previously described ( 56 , 57 ). These LNPs
naturally accumulate in the liver and are up
taken into hepatocytes ( 58 ), where fibrinogen
is made. Briefly, an siRNA solution (amine:
phosphate ratio of 3 in 25 mM sodium acetate,
pH4) was mixed with a lipid solution con-
sisting of an ionizable lipid (DLin-MC3-DMA),
1,2-distearoyl-sn-glycero-3-phosphocholine
(DSPC), cholesterol, and dimyristoyl glycerol-
polyethyleneglycol (DMGPEG) (50:10:38.5:1.5
mol%, respectively, in ethanol) using a T-
junction mixer and dialyzed against PBS (pH
7.4). Total lipid content and nucleic acid en-
trapment of the resulting siRNA-LNP was
quantified using a Cholesterol E Assay Kit
(Wako Chemicals, Richmond, VA) and Ribo-
Green assay, respectively. We confirmed that
siFbg led to a significant decrease in both
fibrinogen mRNA in the liver and fibrinogen
levels in blood plasma using the mouse total
fibrinogen enzyme-linked immunosorbent
assay (ELISA) kit (Innovative Research Inc.,
Arlington, VA).Silvaet al.,Science 374 , eabl5450 (2021) 24 December 2021 9 of 11
RESEARCH | RESEARCH ARTICLE