Science - USA (2021-12-24)

RESEARCH ARTICLE

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IMMUNOLOGY

Fibrin is a critical regulator of neutrophil effector

function at the oral mucosal barrier

Lakmali M. Silva1,2, Andrew D. Doyle^3 , Teresa Greenwell-Wild^2 , Nicolas Dutzan2,4, Collin L. Tran^1 ,
Loreto Abusleme2,5, Lih Jiin Juang^6 , Jerry Leung^6 , Elizabeth M. Chun^1 , Andrew G. Lum^2 ,
Cary S. Agler^7 , Carlos E. Zuazo^2 , Megan Sibree^1 , Priyam Jani^8 , Vardit Kram^8 , Daniel Martin^9 ,
Kevin Moss^7 , Michail S. Lionakis^10 , Francis J. Castellino^11 , Christian J. Kastrup6,12,13,
Matthew J. Flick^14 , Kimon Divaris15,16, Thomas H. Bugge^1 , Niki M. Moutsopoulos^2

Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we report that the clotting factor
fibrin is a critical regulator of neutrophil function at the oral mucosal barrier. We demonstrate that
commensal microbiota trigger extravascular fibrin deposition in the oral mucosa. Fibrin engages neutrophils
through theaMb 2 integrin receptor and activates effector functions, including the production of reactive
oxygen species and neutrophil extracellular trap formation. These immune-protective neutrophil functions
become tissue damaging in the context of impaired plasmin-mediated fibrinolysis in mice and humans.
Concordantly, genetic polymorphisms inPLG, encoding plasminogen, are associated with common forms of
periodontal disease. Thus, fibrin is a critical regulator of neutrophil effector function, and fibrin-neutrophil
engagement may be a pathogenic instigator for a prevalent mucosal disease.

T

he study of Mendelian diseases is instru-
mental in uncovering genes and pathways
that are implicated in human immunity
and mucosal homeostasis. One Mende-
lian disease clearly linked to mucosal
immunopathology is plasminogen (PLG) defi-
ciency. PLG is synthesized by the liver and is
present at high concentrations in the circu-
lation and in interstitial fluids. PLG is a
zymogen, which is converted to the active
serine protease plasmin by urokinase or tis-
sue plasminogen activator. Homozygous or
compound heterozygous mutations in the
humanPLGgene typically lead to severe mu-
cosal disease, suggesting a critical role for
this gene in mucosal immunity. Such patients
present with deposition of fibrin at various
mucosal sites leading to oral disease (ligneous
periodontitis); ocular disease (conjunctivitis);
and lung, vaginal, and gastrointestinal disease
( 1 – 3 ). Fibrin, a physiological target of plasmin,
is by itself a proinflammatory molecule, and
many of the abnormal inflammatory responses
associated with PLG deficiency have been linked
to persistent extravascular fibrin deposition.
In the oral mucosa, local deposition of fibrin

is hypothesized to lead to severe soft tissue

and bone destruction around teeth and often

loss of the entire dentition in adolescence ( 4 – 7 ).

Mucosal inflammation in areas surrounding

the dentition and destruction of underlying bone

are also the hallmarks of the common human

oral mucosal disease, periodontitis ( 8 , 9 ).

We hypothesized that insufficient plasmin-

mediated fibrinolysis is a key contributor to

mucosal immunopathology in humans and in

animal models. To understand the mechanistic

links between mucosal fibrin deposition and

immunopathology, we employed an array of

genetically engineered mouse models, comple-

mented by histological and genetic studies in

humans. We chose to study the mechanisms

involved in PLG deficiency–mediated immuno-

pathology at the site of the oral mucosa because

periodontitis is among the most common man-

ifestations in humans with PLG deficiency. This

suggests that this mucosal site is particularly

susceptible to PLG deficiency–related immuno-

pathology. Our work highlights fibrin as a critical

immune regulator of mucosal barrier homeosta-

sis and provides insights into the role of fibrin in

neutrophil activation and tissue immunity.

Results

Plgdeficiency instigates oral mucosal

immunopathology in mice

Plg−/−mice phenocopy PLG-deficient humans

and present with mucosal immunopathology,

including periodontitis, conjunctivitis, and gas-

trointestinal immunopathology ( 10 – 13 ) (Fig. 1A

and fig. S1). The hallmark of periodontitis is

mucosal inflammation and destruction of bone

around the teeth.Plg−/−mice developed peri-

odontal bone loss by 12 weeks of age compared

with their wild-type and heterozygous litter-

mates, and this loss became increasingly severe

at24weeksofage(Fig.1,AandB,andfig.S2A).

Consistent with accelerated periodontal bone

loss inPlg−/−mice, mice double-deficient for

the two physiological PLG activators, tissue

plasminogen activator and urokinase plas-

minogen activator (Plat−/−;Plau−/−), also dis-

played severe periodontal bone loss (fig. S2B).

To further confirm that spontaneous peri-

odontitis inPlg−/−mice was associated with

the loss of enzymatic activity of plasmin, we

used a knock-in mouse model that harbors a

mutation in the catalytic serine residue (S743A),

which renders plasmin catalytically inactive

(PlgS743A/S743A). At the ages of 12 and 24 weeks,

PlgS743A/S743Amice showed significant increases

in periodontal bone loss compared with their

wild-type and heterozygous littermates, con-

firming that plasmin insufficiency drives

periodontal bone loss (Fig. 1C). Consistent

with mucosal lesions in patients with PLG

deficiency, the oral mucosal tissues ofPlg−/−

mice displayed a disruption of tissue architec-

ture associated with the deposition of fibrin

(Fig. 1, D and E). Beyond fibrin deposition, the

oral mucosal immunopathology inPlg−/−mice

was characterized by mucosal edema and loss

of connective tissue attachment with disrup-

tion of the junctional epithelium (fig. S3A).

Thus, we confirm thatPlgdeficiency in mice,

like human disease, leads to severe oral mu-

cosal immunopathology associated with fibrin

deposition.

Elimination of fibrinogen rescues oral mucosal

immunopathology inPlg-deficient mice

To determine whether fibrin accumulation,

secondary to impaired plasmin-mediated fi-

brinolysis, was the trigger for mucosal immuno-

pathology, we generatedPlgand fibrinogen

RESEARCH

Silvaet al.,Science 374 , eabl5450 (2021) 24 December 2021 1 of 11

(^1) Proteases and Tissue Remodeling Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA. (^2) Oral Immunity and Inflammation Section,
National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA.^3 NIDCR Imaging Core, National Institute of Dental and Craniofacial Research, National
Institutes of Health, Bethesda, MD, USA.^4 Department of Conservative Dentistry, Faculty of Dentistry, University of Chile, Santiago, Chile.^5 Department of Pathology and Oral Medicine, Faculty of
Dentistry, University of Chile, Santiago, Chile.^6 Michael Smith Laboratories and Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, BC, Canada.
(^7) Division of Oral and Craniofacial Health Sciences, Adams School of Dentistry, University of North Carolina, Chapel Hill, NC, USA. (^8) Molecular Biology of Bones and Teeth Section, National
Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA.^9 NIDCR Genomics and Computational Biology Core, National Institute of Dental and Craniofacial
Research, National Institutes of Health, Bethesda, MD, USA.^10 Fungal Pathogenesis Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD, USA.^11 WM Keck Center for Transgene Research, University of Notre Dame, Notre Dame, IN, USA.^12 Blood Research Institute, Versiti,
Milwaukee, WI, USA.^13 Departments of Surgery, Biochemistry, Biomedical Engineering, and Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA.^14 Department of
Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC, USA.^15 Division of Pediatric and Public Health, Adams School of Dentistry, University of North Carolina, Chapel
Hill, NC, USA.^16 Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, NC, USA.
*Corresponding author. Email: [email protected] (T.H.B.); [email protected] (N.M.M.)
Present address: Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA.