. 2021 Dec 24;374(6575):eabl5450.

doi: 10.1126/science.abl5450. Epub 2021 Dec 24.

Fibrin is a critical regulator of neutrophil effector function at the oral mucosal barrier

Lakmali M Silva^{1

2}, Andrew D Doyle³, Teresa Greenwell-Wild², Nicolas Dutzan^{2

4}, Collin L Tran¹, Loreto Abusleme^{2

5}, Lih Jiin Juang⁶, Jerry Leung⁶, Elizabeth M Chun¹, Andrew G Lum², Cary S Agler⁷, Carlos E Zuazo², Megan Sibree¹, Priyam Jani⁸, Vardit Kram⁸, Daniel Martin⁹, Kevin Moss⁷, Michail S Lionakis¹⁰, Francis J Castellino¹¹, Christian J Kastrup^{6

12

13}, Matthew J Flick¹⁴, Kimon Divaris^{15

16}, Thomas H Bugge¹, Niki M Moutsopoulos²

Affiliations

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.

PMID: 34941394
PMCID: PMC11960105
DOI: 10.1126/science.abl5450

Fibrin is a critical regulator of neutrophil effector function at the oral mucosal barrier

Lakmali M Silva et al. Science. 2021.

. 2021 Dec 24;374(6575):eabl5450.

doi: 10.1126/science.abl5450. Epub 2021 Dec 24.

Authors

Affiliations

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.

PMID: 34941394
PMCID: PMC11960105
DOI: 10.1126/science.abl5450

Abstract

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 the α_Mβ₂ 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 in PLG, 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.

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Figures

**Fig. 1.. Defective fibrinolysis triggers oral mucosal immunopathology.**
(A) *Plg*-deficient mice (*Plg*^−/−) display periodontal bone loss at the age of 12 and 24 weeks compared with *Plg*^+/+ and *Plg*^+/− littermates. (B) μCT visualization of periodontal bone loss in 24-weeks-old *Plg*^+/+ and *Plg*^−/− maxillae (black arrows depict the distance between alveolar bone crest and cementoenamel junction). (C) Mice with catalytically inactive plasmin (*Plg*^S743A/S743A) develop significantly increased spontaneous periodontal bone loss. (D) Fraser–Lendrum staining of *Plg*^+/+ and *Plg*^−/− oral mucosal tissue sections (Green: collagen; magenta: keratin, fibrin; and orange: erythrocytes; black arrow depicts fibrin deposition). Scale bar: 100 μm and inset scale bar: 50 μm. (E) Immunofluorescence (IF) staining for fibrin(ogen) (gray) in *Plg*^+/−*;Fga*^+/−, *Plg*^−/−*;Fga*^+/−*, Plg*^+/−*;Fga*^−/− and *Plg*^−/−*;Fga*^−/− oral tissue sections. Scale bar: 50 μm. (F) Bone loss measurements in *Plg*^+/−*;Fga*^+/−*, Plg*^−/−*;Fga*^+/−, *Plg*^+/−*;Fga*^−/− and *Plg*^−/−*;Fga*^−/− mice maxillae. (G) Difference (Δ) in bone loss between *Plg*^−/− and *Plg*^+/+ mice under SPF and GF conditions. (H) Fraser–Lendrum staining of *Plg*^+/+ and *Plg*^−/− GF mice oral mucosal tissue sections. Scale bar: 100 μm and inset scale bar: 50; and (I) Measurements of mucosal edema and attachment loss in *Plg*^+/+ and *Plg*^−/− under SPF and GF conditions.

**Fig. 2.. Lesions of mucosal immunopathology in Plg deficiency are dominated by neutrophils.**
(A) Principal coordinates analysis (PCoA) plot showing the distribution of differentially expressed genes in oral mucosa of 12-week-old *Plg*^+/+ and *Plg*^−/− mice. (B) Gene ontology (GO) biological processes upregulated in *Plg*^−/− mice in ascending order of P-value. (C) Heat maps showing significantly differentially expressed genes involved in granulopoiesis/neutrophil recruitment, myeloid cell activation and inflammation/extracellular matrix disassembly in *Plg*^−/− compared with *Plg*^+/+ gingival tissues. (D) Flow cytometry analysis of 12- and 24-week-old mouse oral mucosal tissues. Contour plots show changes in neutrophil and monocyte/macrophage populations in percentages. Absolute count and percentages of (E) neutrophils and (F) monocytes/macrophages in 12- and 24-week-old *Plg*^+/+ and *Plg*^−/− mouse gingiva. (G) Fibrin(ogen) (green) and Ly6G (neutrophils; magenta) IF staining of 24-week-old *Plg*^−/− mouse oral mucosal sections showing extravascular localization of fibrin deposits and neutrophils. Yellow arrowheads indicate blood vessels. Scale bar: 50 μm and quantification of number of neutrophils per 10⁵ μm² of stained tissue sections.

**Fig. 3.. Myeloid integrin α_Mβ₂-binding motif on fibrin(ogen) mediates plasminogen deficiency-induced oral immunopathology.**
(A) Bone loss measurement in *Plg*^+/−*;Fgg*^+/390-396A, *Plg*^−/−*;Fgg*^+/390-396A, *Plg*^+/−*;Fgg*^{390-396A/390-396A}, *Plg*^−/−*;Fgg*^{390-396A/390-396A} mice maxillae. (B) μCT visualization of periodontal bone loss of maxilla (black arrows depict the distance between alveolar bone crest and cemento–enamel junction). (C) IF staining of fibrin(ogen) (green) and Ly6G (neutrophils; magenta) in *Plg*^+/+*;Fga*^+/+, *Plg*^−/−*;Fga*^+/+, *Plg*^−/−*;Fgg*^{390-396A/390-396A} and *Plg*^−/−*;Fga*^−/− mouse gingival sections. Scale bar: 50 μm. Graphs below show mean fluorescence intensity (MFI) of fibrin(ogen)/DAPI staining, and number of neutrophils per 10⁵ μm² of stained tissue sections. (D) Flow cytometry analysis of 24-week-old gingiva. Contour plots show neutrophil and monocyte/macrophage populations in percentages. (E) Counts of neutrophils in 24-week-old *Plg*^+/−*;Fgg*^+/390-396A, *Plg*^−/−*;Fgg*^+/390-396A, *Plg*^−/−*;Fgg*^{390-396A/390-396A} and *Plg*^+/−*;Fgg*^{390-396A/390-396A} mouse gingival sections. (F) Bone loss measurement in wild-type 10- and 24-week-old maxillae. (G) Bone loss measurement in *Fgg*^+/+, *Fgg*^+/390-396A and *Fgg*^{390-396A/390-396A} mice maxillae at 24 weeks of age.

**Fig. 4.. Fibrin(ogen)-neutrophil interaction through α_Mβ₂-binding triggers key neutrophil effector functions.**
(A) Human neutrophil binding to *Fgg*^{390-396A/390-396A} fibrin compared to wild-type fibrin. Scale bar: 200 μm. (B) Binding of wild-type and α_M integrin-deficient (α_M^−/−) mouse neutrophils to wild-type fibrin. Scale bar: 200 μm. (C to H) Neutrophils were plated on wild-type or Fgg^390-396A fibrin. (C) Representative graph of neutrophil reactive oxygen species (ROS) production over time (15 nM MPO). (D) ROS production at 5-hour time point (n=15). (E) Neutrophil extracellular traps (NETs; magenta) at 4- and 5-hour time points for neutrophils plated on wild-type and Fgg^390-396A fibrin. Scale bars: 50 μm. (F) Cit-H3 and MPO staining (gray) of NETs at 5-hour time point. Scale bar: 50 μm. (G) NETosis progression over time (1 to 5 hours) (see Movie 1). (H) Quantification of NETosing cells at 5-hour time point (n=7).

**Fig. 5.. NETosis mediates periodontal immunopathology in Plg-deficient mice.**
(A) Immunofluorescence staining of Cit-H3 and MPO (yellow) in *Plg*^+/+, *Plg*^−/− and *Plg*^−/−*;Fgg*^{390-396A/390-396A} mouse oral mucosal tissue sections. Scale bars: 50 μm. Quantification of percentage of Cit-H3 and MPO stained area, respectively. (B and C) *Plg*^−/−-control (Ctrl) and *Plg*^−/−-DNase I-treated mice. (B) Bone loss and (C) staining for Cit-H3 and MPO (yellow) in gingiva. Scale bars: 50 μm (D) *Plg*^+/−*;Ela*^+/− and *Plg*^−/−*;Ela*^+/−, *Plg*^−/−*;Ela*^−/− and *Plg*^+/−*;Ela*^−/− mice bone loss measurements. (E and F) siRNA treatment. (E) Plasma fibrinogen levels. (F) Bone-loss measurements of *Plg*^−/−-siLuc- and *Plg*^−/−-siFbg-treated mice.

**Fig. 6.. Fibrin–neutrophil axis as an initiator in common forms of periodontitis.**
(A) Fraser–Lendrum staining for fibrin(ogen) and immunohistochemistry (IHC) staining of MPO in oral mucosal tissues from patients with severe periodontitis and healthy controls. Scale bars: 100 μm. Regional association plots of *PLG* (area ±110-kb flanking *PLG*) with (C) severe disease, and (D) high Aa subgingival colonization in the dental atherosclerosis risk in communities study (D-ARIC) population.

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Comment in

Fibrin sparks inflammation in the oral mucosa.
Vicanolo T, Hidalgo A. Vicanolo T, et al. Science. 2021 Dec 24;374(6575):1559-1560. doi: 10.1126/science.abn0399. Epub 2021 Dec 23. Science. 2021. PMID: 34941413

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Fibrin is a critical regulator of neutrophil effector function at the oral mucosal barrier

Affiliations

Fibrin is a critical regulator of neutrophil effector function at the oral mucosal barrier

Authors

Affiliations

Abstract

Figures

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LinkOut - more resources

Full Text Sources

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