the commensal microbiome. Consistent with
these findings, antibiotic treatment of mice
with defects in fibrinolysis (Plg−/−,Plat−/−;
Plau−/−, andPlgS743A/S743A) also significantly
ameliorated disease (fig. S4, A to C).Plg−/−
mice did not exhibit any significant shifts in
oral microbiome community structure and
composition, nor any specific microbial con-
stituents associated with triggering immu-
nopathology (at 12 and 16 weeks of age; fig. S4,
D to J), which is consistent with a lack of mu-
cosal bacterial infection. Thus, the commensal
microbiome is a trigger for oral mucosal im-
munopathology in the setting of defective
fibrinolysis.

Oral mucosal lesions inPlg-deficient mice
are neutrophil dominated

We next aimed to dissect the mechanisms of
fibrin-mediated mucosal immunopathology.
To this end, we took an unbiased approach
and evaluated global transcriptomic changes
by RNA sequencing (RNA-seq) of oral mu-
cosal (gingival) tissues ofPlg−/−mice compared
with wild-type littermates. Principal compo-
nents analysis of RNA-seq data revealed a
distinct transcriptome in the mucosal tis-
sues ofPlg−/−mice (Fig. 2A, fig. S5, and table
S1). Gene ontology (GO) analysis of the most
significantly up-regulated pathways inPlgde-
ficiency revealed neutrophil migration as the
top differentially expressed pathway, followed
by inflammation, extracellular matrix disas-
sembly, cytokine signaling, leukocyte migra-
tion, and response to bacterial molecules (Fig.
2B and table S2). Examination of the most
significantly differentially expressed genes
betweenPlg−/−and wild-type mice revealed
up-regulation of genes associated with neutro-
phil recruitment and granulopoiesis, includ-
ing chemokine (C-X-C motif) ligand (Cxcl) 1 to
3 and 5 , colony-stimulating factor 3 (Csf3),
and Csf3 receptor (Csf3r). Genes involved in
neutrophil and monocyte activation were also
up-regulated, including pro-platelet basic
protein (Ppbp), ADAM metallopeptidase do-
main 8 (Adam8), triggering receptor expressed
on myeloid cells 1 (Trem1), C-type lectin do-
main family 4 member E (Clec4e), and CD300
molecule like family member B (Cd300lb) (Fig.
2C). Finally, several matrix metalloproteases
(Mmp8to 10 and 12 ), interleukins (Il1aand
Il1b), and prostaglandin-endoperoxide syn-
thase 2 (Ptgs2) that are involved in extracel-
lular matrix remodeling and inflammation
were also up-regulated inPlg−/−mice.
We next evaluated the gingival inflamma-
tory infiltrate by flow cytometry (fig. S6A).
Consistent with the transcriptomic findings,
the oral mucosal inflammatory infiltrate in
Plg−/−mice was dominated by neutrophils
(live CD45+CD11b+Cd11cneg-midLy6G+)atboth
early (12 weeks) and late (24 weeks) time
points (Fig. 2, D and E). The absolute number

of neutrophils was significantly increased in

Plg−/−mice at all time points, whereas their

proportion increased significantly in late stages

of immunopathology. Notably, neutrophils

were the only cell subset that significantly

increased at early time points of immuno-

pathology (12 weeks).

There was no increase in monocyte or macro-

phage cells (live SSCintCD45+CD11b+Cd11cneg-mid

Ly6G−) at 12 weeks, although absolute counts

increased at late stages of immunopath-

ology (24 weeks) (Fig. 2, D and F). B cell (live

CD45+B220+), T cell (live CD45+TCRb+), and

dendritic cell (live CD45+CD11Chi) numbers

and proportions did not change at any stage

of disease (fig. S6, B to D). Immunostain-

ing for fibrin(ogen) and Ly6G—amarkerfor

neutrophils—in gingival tissues confirmed that

neutrophils accumulated in increased numbers

in gingival tissues ofPlg−/−mice and were often

found in proximity to fibrin(ogen) deposits (Fig.

2G). Thus, oral mucosal lesions inPlg−/−mice

are dominated by neutrophils and likely reflect

a neutrophil-mediated immunopathology.

Silvaet al.,Science 374 , eabl5450 (2021) 24 December 2021 3 of 11

A B

Positive regulation of neutrophil migration

Inflammatory response

Extracellular matrix dissassembly

Cytokine-mediated signaling pathway

Positive regulation of leukocyte migration

Response to molecule of bacterial origin

-log 10 (P value)

C

D

−20

−10

0

10

20

−40 −20 0 20

Plg-/-

Plg+/+

1

2

3

1

2

3

PC1: 44.78% variance

PC2: 25.57% variance

E

32.21±9.83 42.58±6.83

14.14±2.83 37.18±4.41

49.38±9.32 37.70±5.96

67.03±8.57 51.73±6.73

Plg+/+ Plg-/-

CD11b

Ly6G

12 Weeks

24

Weeks

F

0

300

400

500

Neutrophils/1

(^50)
μm
2
+/+ -/-
-/-
Plg:
Fga:+/+ +/+
+/+
P< 0.0001
P< 0.0001
100
200
Ly6GFibrinogen
0
2000
4000
6000

Cell

s
Plg
P = 0.0425
0
500
1000
1500
P = 0.0080
0
2000
4000
3000
n.s.

Cells

P = 0.0190
(^0204060) %
n.s.
P = 0.0014
Plg+/+
Plg-/-
Plg+/+
Plg-/-
12
24
%
Plg+/+ n.s.
Plg-/-
Plg+/+
Plg-/-
12
24
0 20 40 6080
024 6 8 10
Neutrophil recruitment
Cxcl5
Cxcl1
Cxcl3
Cxcl2
Csf3
Csf3r
Plg+/+ Plg-/-
Ppbp
Adam8
Trem1
Clec4e
Cd300lb
Mmp12
Mmp10
Mmp9
Il1a
Il1b
Mmp8
Ptgs2
Myeloid cell activation
ECM
dissassembly
1 - -0.5 0 0 5. 1
+/+ -/- +/+ -/-
G
1000
Plg
12 weeks 24 weeks
+/+ -/- +/+ -/-
n.s.
2000
1000
0
500
1500
-/-
Fibrin(ogen) Ly 6 G -/-
Fig. 2. Lesions of mucosal immunopathology inPlgdeficiency are dominated by neutrophils.
(A) Principal coordinates analysis (PCoA) plot showing the distribution of differentially expressed genes
in oral mucosa of 12-week-oldPlg+/+andPlg−/−mice. (B) GO biological processes up-regulated inPlg−/−
mice in ascending order ofPvalue. (C) Heatmaps showing significantly differentially expressed genes
involved in granulopoiesis or neutrophil recruitment, myeloid cell activation, and inflammation or extracellular
matrix (ECM) disassembly inPlg−/−compared withPlg+/+gingival tissues. (D) Flow cytometry analysis of
12- and 24-week-old mouse oral mucosal tissues. Contour plots show changes in neutrophil and monocyte or
macrophage populations in percentages. (EandF) Absolute count and percentages of neutrophils (E)
and monocytes or macrophages (F) in 12- and 24-week-oldPlg+/+andPlg−/−mouse gingiva. (G) Fibrin(ogen)
(green) and Ly6G (neutrophils; magenta) IF staining of 24-week-oldPlg−/−mouse oral mucosal sections
showing extravascular localization of fibrin deposits and neutrophils. Yellow arrowheads indicate blood
vessels. Scale bars, 50mm. On the right, there is a quantification of the number of neutrophils per 10^5 mm^2
of stained tissue sections.
RESEARCH | RESEARCH ARTICLE