Fibrin(ogen) depletion in vivo
Plg−/−mice of 8 weeks of age were i.v. in-
jected with either siFbg or siLuc at 2 mg/kg
in 100ml of PBS once every 3 weeks (in the
morning) for 12 weeks.
Human oral mucosal samples
Gingival biopsies were harvested from healthy
volunteers and patients with severe untreated
periodontal disease. All patients were enrolled
in an IRB-approved clinical study at the NIH
hospital (NCT no. 01568697) and provided in-
formed written consent. Human biopsies were
stainedforMPOasfollowsandfibrin(Fraser-
Lendrum stain) as described above. Antigen
retrieval in 10 mM citric acid buffer was used,
and sections were blocked with 5% bovine
serum albumin in PBS for 1 hour. Sections
were incubated with MPO antibodies (ab9535)
at 1:500 dilution O/N, followed by secondary
anti-rabbit antibody (1:500; ImmPRESS HRP
Reagentkit,VectorLaboratories)for30min.
Sections were then washed three times in PBS
followed by the addition of developing solu-
tion (ImmPACT DAB, Vector Laboratories). The
reaction was stopped with water and slides
counterstained with hematoxylin QS (Vector
Laboratories Inc., Burlingame, CA).
Human genetic associations with clinical and
microbiological periodontal disease parameters
To examine the association of polymorphisms
in thePLGgene locus with the presence and
severity of human periodontal disease in an
independent cohort, we used existing clinical,
microbiological, and genotype information
from the Atherosclerosis Risk in Communi-
ties (ARIC) study ( 59 ). ARIC is a prospective
cohort study of atherosclerosis and cardiovas-
cular disease risk factors which included a
Dental ARIC ancillary study with comprehen-
sive oral examinations in a subset of ARIC
participants [n= 4504 European Americans
(EA); mean age = 62 years, females = 52%].
The analysis of existing, deidentified geno-
type and clinical periodontal data was ap-
provedasnonhumansubjectsresearch(NHSR)
bytheUNC-ChapelHillIRB(no.11-1035).All
participants of the parent ARIC study and
ancillary studies provided written informed
consent. Oral microbiome samples were col-
lected from subgingival tooth surfaces in a sub-
set of Dental ARIC participants (n= 1020 EA)
andweresubsequentlyanalyzedforthepres-
ence and abundance of eight common peri-
odontal pathogens. In this study, we based
oral disease diagnoses on the recently intro-
duced P^3 classification system ( 60 ), separat-
ing the population into seven distinct oral
health categories. Four categories focused on
the severity of periodontal disease [mild dis-
ease, severe inflammation (inflammation with-
out periodontal bone loss), localized disease,
and severe periodontitis (severe periodontal
bone loss)]. Two categories reflected oral dis-
ease burden not specific to periodontitis (tooth
loss and severe tooth loss), and the final cat-
egory was designated for periodontal health
( 61 , 62 ). Microbiome-specific categories were
assigned for patients with high [i.e., in the top
quintile ( 63 )] detection of eitherPorphyromonas
gingivalis(Pg, associated with the most com-
mon, chronic types of periodontitis) ( 64 ) or
Aggregatibacter actinomycetemcomitans(Aa,
associated with aggressive periodontitis) ( 39 ).
We used existing genotype data that were ini-
tially generated using the Affymetrix Genome-
Wide Human SNP Array 6.0 chip (n= ~900,000
markers for SNPs) and then imputed to the
HapMap Phase II CEU build 36 panel result-
ing in a final analysis set of 2.1 million SNPs
with minor allele frequency of >5% ( 59 ). Ge-
netic association models for single markers
(i.e., SNPs) were based on logistic regression
(i.e.,“disease”versus“healthy”periodontal
groups and“high”versus“lower”pathogen
colonization), assuming log-additive allelic
effects, and including adjustment terms of
study design, 10 principal components for
ancestry, age, and sex.
To evaluatePLGgenetic association with
clinical and microbiological parameters of peri-
odontal disease, first we defined an extended
gene region around it, 110 kb upstream to 40 kb
downstream of the gene boundaries, harbor-
ing 153 SNPs. The selection of the specific
boundaries was based upon simulation and
actual data applications of MAGENTA ( 65 )
that were used to obtain gene-centric asso-
ciation estimates. This was done by synthe-
sizing single-marker association signals in
the definedPLGgene region into a single
association estimate forPLG(Z score and cor-
responding gene-centricPvalue), accounting
for the number of SNPs tested and their local
linkage disequilibrium (LD) structure, based
on the human genome build 36. Here, we ob-
tainedPLG-centric estimates of association
with clinical diagnoses and microbiological
periodontal parameters and report geneP
values, as well as information for the lead (i.e.,
lowestPvalue) SNP in the gene region. In this
study, we formally tested for fivePLG-trait
associations (mild periodontitis, severe gingival
inflammation, severe periodontitis, highAa,
and highPgcolonization). Because the P^3 sys-
tem includes six clinical disease groups (includ-
ing localized disease and patterns of tooth loss),
and because four periodontal pathogen coloni-
zation traits have been previously reported ( 63 ),
we applied a conservative Bonferroni multiple
testing correction assuming 10 independent
tests (critical valueP<5×10−^3 ). Finally, to
illustrate and provide more information about
the genomic context of the identifiedPLGlocus
associations, we generated regional association
(Locus Zoom) plots ( 66 ) using single-marker as-
sociation results in areas ±110 kb flankingPLG.Statistical analysis
For in vivo assays, unpaired Student’sttest, or
one-way analysis of variance (ANOVA) (when
>2 groups) was performed. For in vitro assays
with human neutrophils, results were displayed
as means ± SEM, and two-tailed Wilcoxon
signed-rank test was performed. For in vitro
assays with mouse neutrophils, results were
displayed as means ± SEM, and Wilcoxon
signed-rank test was performed for paired
values and Mann-WhitneyUtest for unpaired
samples. For time point analyses (ROS and
NETosis), linear regression analysis was per-
formed to test whether differences in slopes
were significantly different. Confidence in-
terval lines (95%) are displayed on graphs.
All statistical analyses were performed on
Graph Pad Prism (v7).Pvalues for principal
coordinates analysis (PCoA) plot of global mi-
crobial community structure of oral samples
fromPlgmice were determined using analysis
of molecular variance (AMOVA). Microbial
diversity was measured through the nonpa-
rametric version of the Shannon diversity
index of oral samples.Pvalues were deter-
mined using Mann-WhitneyUtest. Statisti-
cal significance of microbiome composition at
the operational taxonomic unit (OTU) level
was determined via LEfSe ( 67 ).REFERENCESANDNOTES- K. Tefset al., Molecular and clinical spectrum of type I
plasminogen deficiency: A series of 50 patients.Blood 108 ,
3021 – 3026 (2006). doi:10.1182/blood-2006-04-017350;
pmid: 16849641 - V. Schuster, B. Hügle, K. Tefs, Plasminogen deficiency.
J. Thromb. Haemost. 5 , 2315–2322 (2007). doi:10.1111/
j.1538-7836.2007.02776.x; pmid: 17900274 - J. Frimodt-Møller, Conjunctivitis ligneosa combined with a dental
affection. Report of a case.Acta Ophthalmol. 51 , 34–38 (1973).
doi:10.1111/j.1755-3768.1973.tb08243.x; pmid: 4739674 - I. Kurtulus Waschulewskiet al., Immunohistochemical analysis
of the gingiva with periodontitis of type I plasminogen
deficiency compared to gingiva with gingivitis and periodontitis
and healthy gingiva.Arch. Oral Biol. 72 , 75–86 (2016).
doi:10.1016/j.archoralbio.2016.07.013; pmid: 27552374 - U.Ertas,N.Saruhan,O.Gunhan,Ligneousperiodontitisinachild
with plasminogen deficiency.Niger. J. Clin. Pract. 20 , 1656– 1658
(2017). doi:10.4103/1119-3077.224133; pmid: 29379003 - S. H. Neeringet al., Periodontitis associated with plasminogen
deficiency: A case report.BMC Oral Health 15 , 59 (2015).
doi:10.1186/s12903-015-0045-3; pmid: 25971786 - M. El-Darouti, A. A. Zayed, G. Y. El-Kamah, M. I. Mostafa,
Ligneous conjunctivitis with oral mucous membrane
involvement and decreased plasminogen level.Pediatr.
Dermatol. 26 , 448–451 (2009). doi:10.1111/j.1525-
1470.2009.00951.x; pmid: 19689523 - R. P. Darveau, Periodontitis: A polymicrobial disruption of host
homeostasis.Nat. Rev. Microbiol. 8 , 481–490 (2010).
doi:10.1038/nrmicro2337; pmid: 20514045 - G. Hajishengallis, Periodontitis: From microbial immune
subversion to systemic inflammation.Nat. Rev. Immunol. 15 ,
30 – 44 (2015). doi:10.1038/nri3785; pmid: 25534621 - T. H. Bugge, M. J. Flick, C. C. Daugherty, J. L. Degen,
Plasminogen deficiency causes severe thrombosis but is
compatible with development and reproduction.Genes Dev. 9 ,
794 – 807 (1995). doi:10.1101/gad.9.7.794; pmid: 7705657 - T. H. Buggeet al., Loss of fibrinogen rescues mice from the
pleiotropic effects of plasminogen deficiency.Cell 87 , 709– 719
(1996). doi:10.1016/S0092-8674(00)81390-2; pmid: 8929539 - R. Sulniute, T. Lindh, M. Wilczynska, J. Li, T. Ny, Plasmin is
essential in preventing periodontitis in mice.Am. J. Pathol. 179 ,
819 – 828 (2011). doi:10.1016/j.ajpath.2011.05.003;
pmid: 21704601
Silvaet al.,Science 374 , eabl5450 (2021) 24 December 2021 10 of 11
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