were plated on wild-type fibrin and compared
with wild-type neutrophils (Fig. 4B). Thus,aMb 2
is important for neutrophil adhesion to fibrin.
To assess the effect of neutrophil engagement
of fibrin on neutrophil effector functions, we
studied the production of reactive oxygen spe-
cies (ROS) and neutrophil extracellular trap
(NET) formation (NETosis) by human neutro-
phils plated on wild-type or mutant fibrin. We
found that when neutrophils were in associa-
tion with wild-type fibrin, ROS production per
cell (Fig. 4, C and D) was significantly increased
compared with neutrophils plated on fibrin
lacking theaMb 2 -binding motif. The proportion
of NETosis events, as visualized by an increase in
cellular size and subsequent externalization of
DNA (Fig. 4, E to G; fig. S7, B and C; and Movie
1), was also significantly increased when neu-
trophils plated on wild-type fibrin were com-
pared with fibrin lacking theaMb 2 -binding
motif. Fibrin-induced NETosis was further
confirmed by positive staining for extracellular
citrullinated histone (Cit-H3) and myeloperox-
idase (MPO) (Fig. 4H and fig. S7D). Consistent
with fibrin-mediated NETosis in vivo, we ob-
served enhanced diffuse extracellular staining
for Cit-H3 and MPO inPlg−/−mice (Fig. 5A).
Extracellular Cit-H3 and MPO were markedly
reduced when the fibrin-neutrophil interaction
was abrogated inPlg−/−;Fgg390-396A/390-396Amice
(Fig. 5A). Thus, the local engagement of fibrin
(ogen) by neutrophils throughaMb 2 is important
for neutrophil activation.
Fibrin-induced NETosis mediates periodontal
immunopathology inPlg-deficient mice
To understand whether NETosis contributes
to the periodontal disease phenotype in vivo,
we inhibited the accumulation of extracellularDNA and NETosis inPlg−/−mice with pharma-
cologic and genetic tools.Plg−/−mice treated
with deoxyribonuclease (DNase) I intraperito-
neally (i.p.) from weeks 8 to 20 exhibited re-
duced alveolar bone loss compared with theSilvaet al.,Science 374 , eabl5450 (2021) 24 December 2021 5 of 11
AFgg390-396AFgg+/+0200400600Count of cells/field
Fgg: 390-396A/
390-396A+/+P = 0.0008NeutrophilsB051015
P = 0.0256Fgg:C D01020304001020304050Count of cells/fieldNeutrophilsP = 0.0020Wild-type fibrinFgg:P = 0.003402460102030h% NETosingneutrophilsP = 0.0013E F G246-4-202468nmol/million cells hFgg+/+
Fgg390-396A/390-396AP = 0.0176Fgg+/+
+PMAFgg390-396A/390-396A+PMA4 h 5 h
Neutrophil NETs
NucleiNeutrophilsFgg+/+
Fgg390-396A/390-396AHuman NeutrophilsHDAPIDAPI MPOCitH3390-396A/
390-396A+/+390-396A/
390-396A+/+Fig. 4. Fibrin(ogen)-neutrophil interaction throughaMb 2 -binding triggers
key neutrophil effector functions.(A) Human neutrophil binding to
Fgg390-396A/390-396Afibrin compared with wild-type fibrin. Scale bars, 200mm.
(B) Binding of wild-type andaMintegrinÐdeficient (aM−/−) mouse neutrophils
to wild-type fibrin. Scale bars, 200mm. (C to H) Neutrophils were plated
on wild-type or Fgg390-396Afibrin. (C) Representative graph of neutrophil ROS
production over time (15 nM MPO). (D) ROS production at 5-hour time point
(n= 15). (E) NETs (magenta) at 4- and 5-hour time points for neutrophils plated
on wild-type and Fgg390-396Afibrin. Scale bars, 50mm. (F) NETosis progression
over time (1 to 5 hours) (see Movie 1). (G) Quantification of NETosing cells
at 5-hour time point (n= 7). (H) Cit-H3 and MPO staining (gray) of NETs at
5-hour time point. Scale bars, 50mm.Movie 1. NETosis with wild-type or Fgg390-396Afibrin.Random video for a time-lapse series showing live
neutrophils (Hoechst; blue) and those undergoing either NETosis (magenta overlay) or apoptosis (cytox
green; green) after stimulation with 15 nM PMA plated on either wild-type or Fgg390-396Afibrin.RESEARCH | RESEARCH ARTICLE