Science - USA (2021-12-24)

double-deficient (Plg−/−;Fga−/−) mice. As
expected,Plg−/−;Fga−/−mice did not exhibit
fibrin deposition in mucosal tissues, in con-
trast to theirPlg−/−;Fga+/−littermates (Fig. 1E).
Notably, fibrinogen deficiency completely pre-
vented the periodontal bone loss imposed
byPlgdeficiency (Fig. 1F). Thus, oral mucosal

immunopathology inPlgdeficiency is driven

by fibrin accumulation.

The oral microbiome instigates fibrin-mediated

immunopathology inPlg-deficient mice

To assess the contribution of the microbiome

in fibrin-mediated mucosal immunopathology,

we rederivedPlg−/−mice in a germ-free (GF)

setting. GFPlg−/−mice displayed significantly

reduced mucosal fibrin deposition and asso-

ciated mucosal immunopathology and peri-

odontal bone loss compared with their specific

pathogenÐfree (SPF) counterparts (Fig. 1, G to

I,andfig.S3),whichsuggestsacriticalrolefor

Silvaet al.,Science 374 , eabl5450 (2021) 24 December 2021 2 of 11

A C

DEF

G

B

P< 0.0001

P< 0.0001

P< 0.0001

P= 0.0002

P= 0.0001

Bone loss (mm)

0.4

0.3

0.2

0.1

0.0

Plg: +/+ +/

S743A

S743A/

S743A

+/+

12 weeks 24 weeks

0.5

+/

S743A

S743A/

S743A

Bone loss (mm)

Bone loss (mm)

0.4

0.3

0.2

0.1

0.0

0.5

-0.1

Plg: +/+ +/- -/- +/+ +/- -/-

Bone loss (mm)

P < 0.0001

12 weeks 24 weeks

P= 0.0001

P < 0.0001

P < 0.0001

P < 0.0001

0.4

0.3

0.2

0.1

0.0

P < 0.0001

P= 0.0007P = 0.0002

24 weeks

Plg:

0.4

+/-

+/- +/-

-/-

-/-

-/- +/-

-/-

0.3

0.2

0.1

0.0

Fga:

Plg+/-Fga+/- Plg-/-Fga+/-

Plg+/-Fga-/- Plg-/-Fga-/-

Fibrin(ogen)

SPF GF

P < 0.0001

H

Plg-/-

Plg+/+

Plg-/-

Plg+/+

Plg-/-

Plg+/+

Plg-/-

Tooth

Fibrin(ogen)

Fibrin(ogen)

I

Mucosal edema (

m)

400

300

200

100

0

Attachment loss (

m)

50

0

100

150

P < 0.0001

P < 0.0001

P = 0.0010

P = 0.0004

Plg:+/+ -/-

SPF GF

+/+ -/- Plg: +/+ -/-

SPF GF

+/+ -/-

24 weeks

Fig. 1. Defective fibrinolysis triggers oral mucosal immunopathology.
(A)Plg-deficient mice (Plg−/−) display periodontal bone loss at the ages of 12
and 24 weeks compared withPlg+/+andPlg+/−littermates. (B) Micro-CT
visualization of periodontal bone loss in 24-week-oldPlg+/+andPlg−/−maxillae
(black arrows depict the distance between alveolar bone crest and cementoenamel
junction). (C) Mice with catalytically inactive plasmin (PlgS743A/S743A) develop
significantly increased spontaneous periodontal bone loss. (D) Fraser-Lendrum
staining ofPlg+/+andPlg−/−oral mucosal tissue sections (green indicates
collagen, magenta indicates keratin and fibrin, orange indicates erythrocytes, and

the black arrowhead depicts fibrin deposition). Scale bars, 100mm (left) and 50mm

(right, insets). (E) Immunofluorescence (IF) staining for fibrin(ogen) (gray) in

Plg+/−;Fga+/−,Plg−/−;Fga+/−,Plg+/−;Fga−/−, andPlg−/−;Fga−/−oral tissue sections.

Scale bars, 50mm. (F) Bone loss measurements inPlg+/−;Fga+/−,Plg−/−;Fga+/−,

Plg+/−;Fga−/−, andPlg−/−;Fga−/−mice maxillae. (G) Difference (D) in bone loss

betweenPlg−/−andPlg+/+mice under SPF and GF conditions. (H) Fraser-Lendrum

staining ofPlg+/+andPlg−/−GF mice oral mucosal tissue sections. Scale bars,

100 mm (left) and 50mm (right, insets). (I) Measurements of mucosal edema and

attachment loss inPlg+/+andPlg−/−under SPF and GF conditions.

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