Science - USA (2021-12-24)

Fibrin(ogen)-mediated periodontal
immunopathology depends on its myeloid
integrinaMb 2 -binding motif
We next aimed to test whether neutrophil
engagement of fibrin throughaMb 2 in gingi-
val tissues is critical for neutrophil retention
and/or activation and associated immuno-
pathology. Periodontal bone loss was completely
prevented inPlg−/−mice by the elimination
of theaMb 2 -binding motif from fibrin(ogen)
(Plg−/−;Fgg390-396A/390-396A) compared with
Plg−/−andPlg−/−;Fgg+/390-396Alittermates (Fig. 3,
A and B). The prevention of periodontal bone
loss inPlg−/−;Fgg390-396A/390-396Amice was not
a consequence of diminished gingival fibrin
accumulation, as gingival tissues showed in-
creased fibrin(ogen) accumulation inPlg−/−;
Fgg+/390-396AandPlg−/−;Fgg390-396A/390-396A
mice compared withPlg+/−;Fgg+/390-396Alitter-

mates (Fig. 3, C and D). Prevention of alveolar

bone loss inPlg−/−;Fgg+/390-396Amice was also

not a consequence of abolished gingival neu-

trophil accumulation, as tissue immunostaining

(Ly6G) (Fig. 3, C and E) and flow cytometry

(Fig.3,FandG)demonstratedthatlesionsof

Plg−/−;Fgg390-396A/390-396A mice accumulated

neutrophils in a manner comparable to those

ofPlg−/−;Fgg+/390-396Alittermates.

Plg+/−;Fgg390-396A/390-396Amice showed a

reduction of bone loss compared with their

Plg+/−;Fgg+/390-396Alittermates (Fig. 3A). Thus,

we examined whether the fibrin(ogen)ÐaMb 2

integrin interaction contributes to periodontal

bone loss also under normal fibrinolytic condi-

tions. Wild-type mice are known to develop spon-

taneous periodontal bone loss with age, which

becomes significant between 10 and 24 weeks

(Fig. 3H) ( 14 ). However,Fgg390-396A/390-396Amice

showed significantly less periodontal bone

loss thanFgg+/+andFgg+/390-396Alittermate

controls (Fig. 3I). Thus, the local engagement of

fibrin(ogen) by neutrophils throughaMb 2 is

essential for mucosal immunopathology and

associated periodontal bone loss under defec-

tive or normal fibrinolytic conditions.

Engagement of fibrin(ogen) throughaMb 2

integrin regulates neutrophil effector functions

Next, we established an in vitro system to study

the effects of engagement of neutrophils with

fibrin(ogen) purified from either wild-type

orFgg390-396A/390-396Amice. In agreement with

previous studies ( 15 ), human and mouse neu-

trophils showed a reduced adherence to mutant

fibrin compared with wild-type fibrin (Fig. 4A

and fig. S7A). A similar reduction in adher-

ence was noted whenaM-deficient neutrophils

Silvaet al.,Science 374 , eabl5450 (2021) 24 December 2021 4 of 11

0.0

0.1

0.2

0.3

0.4

Bone loss (mm)

A

Plg: +/- -/- -/-

Fgg: +/390-396A

+/-

390-396A/390-396A

P = 0.001

P < 0.0001

P < 0.0001

B

Plg-/-Fgg+/390-396A

Plg-/-Fgg390-396A/390-396A

24 weeks

C

FG

0

300

400

500

Neutrophils/10

5 μm

2

+/+ -/-

-/-

Plg: -/-

Fga: +/++/+

-/-

100

200

P< 0.0001

P< 0.0001

0.0

0.2

0.4

0.6

0.8P= 0.0022

P= 0.0023

MFI (Fibrin/DAPI)

Plg-/-Fga-/-

0

500

1000

1500

2000

Count of Neutrophils

Plg: +/- -/- +/-

Fgg: +/390-396A

-/-

390-396A/390-396A

24 weeks

P= 0.0053

P= 0.0021

P= 0.0271

P= 0.0096

Ly6GHi/CD11b+/CD45+/Live

28.7±1.8 59.5±10.3

76.3±4.8

77.9±9.6

43.5±8.2

74.5±11.8

29.8±5.9

72.8±7.9

CD11b

Ly6G

Plg-/-Fgg390-396A/390-396A

Plg+/-Fgg+/390-396A Plg-/-Fgg+/390-396A

Plg+/-Fgg390-396A/390-396A

Plg-/-Fgg390-396A/390-396A

390-396A/390-396A

Fibrin(ogen)

Ly6G

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

Fgg:

0.2

0.3

0.0

0.1

Bone loss (mm)

10 24

Weeks

Plg+/+

P= 0.0267

H I

P= 0.0036

P= 0.0009

Fgg: 390-396A/

390-396A

24 weeks

+/

390-396A

+/+

Fibrin(ogen)

Ly6G

0.0

0.2

0.3

0.1

+/+ -/-

-/-

Plg: -/-

Fga: +/++/+

-/-

Fgg: 390-396A/390-396A

D E

Fig. 3. Myeloid integrinaMb 2 -binding motif on fibrin(ogen) mediates
plasminogen deficiencyÐinduced oral immunopathology.(A) Bone loss
measurement inPlg+/−;Fgg+/390-396A,Plg−/−;Fgg+/390-396A,Plg+/−;Fgg390-396A/390-396A,
andPlg−/−;Fgg390-396A/390-396Amice maxillae. (B) Micro-CT visualization of
periodontal bone loss of maxilla (black arrows depict the distance between alveolar
bone crest and cementoenamel junction). (C) IF staining of fibrin(ogen) (green) and
Ly6G (neutrophils; magenta) inPlg+/+;Fga+/+,Plg−/−;Fga+/+,Plg−/−;Fgg390-396A/390-396A,
andPlg−/−;Fga−/−mouse gingival sections. Scale bars, 50mm. (DandE) Graphs

showing the mean fluorescence intensity (MFI) of fibrin(ogen) or DAPI staining (D)

and the number of neutrophils per 10^5 mm^2 of stained tissue sections (E). (F) Flow

cytometry analysis of 24-week-old gingiva. Contour plots show neutrophil and

monocyte or macrophage populations in percentages. (G) Counts of neutrophils in

24-week-oldPlg+/−;Fgg+/390-396A,Plg−/−;Fgg+/390-396A,Plg−/−;Fgg390-396A/390-396A,

andPlg+/−;Fgg390-396A/390-396Amouse gingival sections. (H) Bone loss measurement

in wild-type 10- and 24-week-old maxillae. (I) Bone loss measurement inFgg+/+,

Fgg+/390-396AandFgg390-396A/390-396Amice maxillae at 24 weeks of age.

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