patients with aggressive forms of periodontal
disease ( 39 ). The lead SNP in our analysis as-
sociated with severe periodontitis (rs2465836)
is in strong linkage disequilibrium with the
lead marker (rs1247559) in a recent European
study of aggressive periodontitis ( 20 ). More-
over, the SNP (rs4252200) leading the signal
for highAacolonization is proximal to the
previously reported aggressive periodontitis
and cardiovascular disease–related polymor-
phism (rs4252120) ( 40 ). Future studies of the
functional role of common polymorphisms
in thePLGlocus are necessary to define
whether defective fibrinolysis underlies sus-
ceptibility and could be targeted in a subset
of nonsyndromic periodontitis patients. No-
tably, the pathway of fibrin-neutrophil–mediated
inflammation in periodontal disease uncov-
ered here identifies a putative new therapeutic
target in this disease. In support of this notion,
a monoclonal antibody that targets theaMb 2
binding site on fibrin,g377 to 395, recently
demonstrated therapeutic activity in an ani-
mal model of multiple sclerosis ( 41 ). In con-
clusion, this work demonstrates an essential
role for plasmin-mediated fibrinolysis in oral
mucosal homeostasis and disease and sug-
gests that targeting fibrin-neutrophil engage-
ment may be beneficial for the prevention or
treatment of the prevalent human disease
periodontitis.
Materials and methods
Mice
Plg−/−,PlgS743A/S743A,Plat−/−;Plau−/−,Itgam−/−
(aM−/−),Fga−/−,Fgg390-396A/390-396A, andEla−/−mice
have been previously described ( 10 , 11 , 15 , 42 – 46 ).
All studies used littermate controls. Mice were
on a C57BL/6J background, except forPlg−/−;
Fga−/−mice and theirPlg−/−;Fga+/−,Plg+/−;
Fga−/−, andPlg+/−;Fga+/−littermates, which
were on the mixed Black Swiss/C57BL/6J back-
ground. Mice were genotyped as previously
described ( 10 , 11 , 15 , 42 – 45 , 47 ). All experiments
were performed under approved protocols in
an Association for Assessment and Accredita-
tion of Laboratory Animal Care International-
certified vivarium and were approved by the
Institutional Animal Care and Use Commit-
tee before initiation of experimentation. All
mice were housed in a SPF facility under reg-
ular housing conditions (1 to 5 animals per
cage: 14 light–10 dark cycle with standard
chow and water ad libitum, temperature of
22°C and 30 to 70% humidity). Mice were pro-
vided with water pouches containing ultraviolet
(UV)–treated ultra-filtered water, which has
been chlorinated to 4 parts per million (ppm).
Mice are regularly fed NIH-07 Mouse/Rat Diet
(Envigo, cat. no. 7022; Madison, WI). Standard
environmental enrichment for all mouse cages
was 7099 TEK-Fresh bedding. Singly housed
mice also receive a paper Shepherd Shack.
Trained animal keepers together with veteri-
narians ensured the animals’welfare. GFPlg−/−
mice were generated by Taconic Biosciences,
Inc (Derwood, MD).Study design
Both male and female mice were evaluated to-
gether, as our preliminary data did not reveal
gender as a confounding factor in periodontitis
development. The number of mice used in each
experiment is indicated in figures and/or figure
legends. The experimental unit was a single
mouse. Experiments using genetically modified
mice were littermate-controlled. For pharma-
cological experiments, mice were randomly
assigned to control or treatment groups. Mice
were tagged in a manner so that the investi-
gator was blinded to genotype and experimen-
tal group designation.Bone loss measurements
The distance between the cementoenamel junc-
tion and alveolar bone crest (CEJ-ABC distance)
was measured at six predetermined sites after
defleshing and staining with methylene blue
as previously described ( 48 ). Results are dis-
played as means ± SEM.MicroÐcomputed tomography (micro-CT)
Mouse maxillae were dissected at 24 weeks
(six mice for each age group) for quantitative
analysis of the alveolar bone. Hemi-maxillae
were then examined by a micro-CT system (mCT
50, Scanco Medical AG, Bassersdorf, Switzerland)
as previously described ( 49 ). The values are
expressed as means ± SD.Fraser-Lendrum staining
Decalcification, paraffin embedding, and stain-
ing of 5-mm sections of mouse maxillae and
mandibles using the Fraser-Lendrum method
was performed by Histoserv Inc. (Germantown,
MD) ( 50 ).Immunofluorescence staining and quantification
Immunofluorescence confocal microscopy was
performed using 5-mm sections of decalcified
and paraffin-embedded maxillae and mandi-
bles (Histoserv Inc., Germantown, MD). Im-
munofluorescence staining was performed
as previously described ( 51 ). Primary antibodies
were used at 1:1000 dilution [anti-fibrin(ogen):
( 42 ); anti-Ly6G: BD Pharmingen 551459; anti-
MPO: ab65871; anti–Cit-H3: ab5103] O/N at
4°C. Secondary staining was performed with
Rhodamine Red-X anti-rat, JacksonImmuno
712-296-150 or Alexa Fluor 647-conjugated
AffiniPure F(ab′)2 anti-rabbit, JacksonImmuno
711-606-152; 1:500 (Jackson Immuno Research
Labs, West Grove, PA) and 4′,6-diamidino-2-
phenylindole (DAPI) (1:1000) for 1 hour at room
temperature. Inverted Nikon A1R+ confocal
microscope (20X-air or 60X-oil immersion)
using NIS-Elements software (Nikon, Tokyo,
Japan) was used to capture images.For fibrin quantification, the average fluo-
rescent intensity was measured using NIS-
Elements software. A fixed-area square region
of interest was drawn around the tissue and
the Time Measurement analysis tool was used
to measure the average fluorescent inten-
sity within the ROI for the AI555 channel.
Five individual fields were measured per
tissue and the mean fluorescence intensity
values were calculated in Microsoft Excel for
Mac (version 16.15). Results are displayed as
means ± SEM.
To quantify the MPO- or Cit-H3–stained
area in the gingiva, a statistical threshold (set
at two standard deviations above the mean
of the image histogram) was set. An investi-
gator-drawn region of interest determined
the appropriate area of the sample, and the
square area was reported. A similar analysis
was applied to immunofluorescent images
of Ly6G to determine the number of neutro-
phils in the gingival lesions. The area was
calculated and divided by the average area of
a neutrophil (determined here to be ~22.1mm^2 ,
n= 95) to estimate the number of invading
neutrophils. The analysis was performed with
MetaMorph and results were displayed as
means ± SEM.Antibiotics treatment
Plg−/−,Plat−/−;Plau−/−, andPlgS743A/S743Amice
were treated with vancomycin (0.5 mg/ml),
doripenem (0.25 mg/ml), and neomycin (5mg/ml)
in autoclaved drinking water from 5-6 to 12-17
weeks of age.Oral microbiome analysis
Oral murine swab samples were obtained and
subjected to DNA isolation, amplicon sequenc-
ing of the 16Sribosomal RNA (rRNA) V4 re-
gion and generated microbiome data were
analyzed as previously described ( 31 ).RNA-seq
Total RNA was extracted from experimental
tissue samples (extracted from M1 to M3
from the mandibles) and sequencing libra-
ries were prepared by the Illumina Nextera
XT method following manufacturer’s recom-
mendations (Illumina, San Diego, CA). The
multiplexed libraries were sequenced in a
NextSeq500 instrument in 150-bp paired-end
mode. Demultiplexed samples were mapped,
and transcripts quantified to the GRCm38.v11
mousegenomeusingtheSTARv2.5.2aligner.
Gene-level counts were filtered to remove low-
expressed genes (keeping genes with >5 counts
in at least one sample). The filtered gene ex-
pression matrix was analyzed by principal com-
ponent analysis to identify outlier samples
(none detected). Differential gene expression
was evaluated by three independent statis-
tical methods (DESeq2, edgeR, and Limma-
Voom). Differentially expressed genes [falseSilvaet al.,Science 374 , eabl5450 (2021) 24 December 2021 8 of 11
RESEARCH | RESEARCH ARTICLE