Neutrophil Extracellular Traps Induce Tissue-Invasive Monocytes in Granulomatosis With Polyangiitis

Abstract

Objective: Granulomatosis with polyangiitis (GPA) is a multi-organ vasculitic syndrome typically associated with neutrophil extracellular trap (NET) formation and aggressive tissue inflammation. Manifestations in head and neck (H&N) GPA include septal perforations, saddle-nose deformities, bony erosions of the orbital and sinus walls, middle ear damage and epiglottitis, indicative of bone, cartilage, and connective tissue destruction. Whether H&N-centric lesions engage disease pathways distinctive from the ischemic tissue damage in the lungs, kidneys, skin, and peripheral nerves is unknown. We have compared inflammatory responses triggered by neutrophilic NETs in patients with H&N GPA and systemic GPA (sGPA). Methods: Neutrophils and monocytes were isolated from the peripheral blood of patients with H&N GPA, sGPA, and age/gender matched healthy individuals. Neutrophil NETosis was induced. NETs were isolated and cocultured with monocytes. Gene induction was quantified by RT-PCR, protein upregulation by flow cytometry. Tissue invasiveness of monocytes was measured in a 3D collagen matrix system. Expression of MMP-9 in tissue-residing macrophages was assessed by immunohistochemistry in tissue biopsies. Results: Neutrophils from H&N GPA patients showed more intense NETosis with higher frequencies of netting neutrophils (P < 0.001) and release of higher amounts of NETs (P < 0.001). Isolated NETs from H&N GPA functioned as an inducer of danger-associated molecular patterns in monocytes; specifically, alarmin S100A9. NET-induced upregulation of monocyte S100A9 required recognition of DNA. S100A9 release resulted in the induction of metalloproteinases, including MMP-9, and enabled monocytes to invade into extracellular matrix. Anti-MMP-9 treatment attenuated the tissue invasiveness of monocytes primed with NETs from H&N GPA patients. MMP-9-producing macrophages dominated the tissue infiltrates in naso-sinal biopsies from H&N GPA patients. Conclusion: Distinct disease patterns in GPA are associated with differences in NET formation and NET content. H&N GPA patients with midline cartilaginous and bony lesions are highly efficient in generating NETs. H&N GPA neutrophils trigger the induction of the alarmin S100A9, followed by production of MMP-9, endowing monocytes with tissue-invasive capabilities.

Keywords: NETosis; S100A9; bone destruction; cartilage destruction; granulomatosis with polyangiitis; matrix metalloproteinases; monocytes.

Figure 1

Neutrophil NET formation in patients with GPA correlates with clinical disease pattern. Blood was collected from patients with H&N GPA, systemic GPA, and age-matched healthy individuals. NET formation in isolated neutrophils was assessed by three methods. (A,B) Neutrophils were cultured for 1.5 h and spontaneous netting neutrophils were assessed as SYTOX® Green-positive cells by flow cytometry. Representative results (A) and data from four experiments (B). (C,D) Neutrophils were treated with phorbol myristate acetate (PMA; 20 ng/ml) for 1.5 h. Netting neutrophils were detected as SYTOX® Green-positive cells by flow cytometry. Representative results (C) and data from five experiments (D). (E,F) Neutrophils were activated with PMA (20 ng/ml) for 4 h and stained with SYTOX® Green. Netting neutrophils were identified by confocal microscopy. Representative images (scale bar = 100 μm) (E) and results from five experiments (F). (G,H) Netting was induced with PMA treatment (20 ng/ml; 4 h) and extracellular DNA fibers were stained by propidium iodide (PI). Representative images (scale bar = 50 μm) (G) and results from six experiments (H). (I) Measurement of DNA content in isolated NETs derived from 6 million PMA-treated neutrophils (20 ng/ml; 4 h), quantified spectrophotometrically from nine patients with H&N GPA, nine patients with systemic GPA, and nine healthy individuals. All data are mean ± SEM. (B,D,F,H,I) One-way ANOVA with post-hoc Tukey's multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Neutrophilic NETs induce the alarmin S100A9. Neutrophilic NETs were generated from H&N GPA, systemic GPA, and age-matched healthy individuals and added to CD14⁺ monocytes (5,000 ng/ml) for 24 h. Transcript expression was quantified by RT-PCR. (A) Transcripts for the alarmins S100A8 and S100A9 (n = 5, each group). (B,C) Intracellular expression of S100A9 protein measured by flow cytometry. Representative contour plots (B) and summary results from five experiments (C). (D–G) Transcript expression for IFNα, IFNβ, IFNγ, and IL-1β (n = 5, each group). All data are mean ± SEM. (A,C) One-way ANOVA with post-hoc Tukey's multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns P > 0.05.

Figure 3

Induction of S100A9 requires recognition of NET DNA. Neutrophilic NETs isolated from patients with H&N GPA were treated with DNase for 15 min. Monocytes were stimulated with treated or untreated NETs for 24 h. S100A9 protein expression in monocytes was analyzed by flow cytometry. Representative contour plots (A) and data from seven experiments (B). Paired t-test. **P < 0.01.

Figure 4

S100A9 upregulates monocyte MMP production. Purified monocytes were treated with recombinant S100A9 (100 ng/ml) or vehicle for 24 h. Transcripts for MMP-2, MMP-3, and MMP-9 were quantified by RT-PCR (n = 6) (A). Intracellular MMP-9 protein expression was analyzed by flow cytometry (B,C). Representative histograms (B) and data from seven experiments (C). Paired t-test. *P < 0.05, **P < 0.01.

Figure 5

NETs-induced S100A9 promotes MMP-9 production. NETs were induced from neutrophils of GPA patients and matched controls. Monocytes were treated with isolated NETs for 24 h. (A) Transcripts for MMPs were assessed by RT-PCR. (B,C) Flow cytometrical quantification of intracellular MMP-9 protein. Representative histograms (B) and MFIs from five experiments (C). (D,E) S100A9 receptor binding was blocked with paquinimod in NET-monocyte cocultures. MMP-9 protein was quantified by flow cytometry as in (B). Representative histograms (D) and results from six experiments (E). NETs from H&N GPA patients were treated with DNase for 15 min and added to monocytes for 24 h. Intracellular MMP-9 expression was examined using flow cytometry. Representative histogram (F) and MFIs from six experiments (G). All data are mean ± SEM. (A,C,E) One-way ANOVA with post-hoc Tukey's multiple comparisons test. (G) Paired t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

NET-stimulated monocytes are tissue invasive. Basement membrane matrix was built by coating collagen IV and collagen I layers on top of porous membranes. 100,000 monocytes which were untreated or pre-treated with purified NETs were layered on top of the collagen matrix (A). Invasive capacity of monocytes was quantified by enumerating the cells in the lower chamber after 24 h. Mean ± SEM from 12 experiments (B). NET-stimulated monocytes were incubated with anti–MMP-9 antibody (10 μg/mL) or isotype control IgG. Cells that had penetrated through the matrix layers were counted after 24 h. Mean ± SEM from six experiments (C). One-way ANOVA with post-hoc Tukey's multiple comparisons test (B). Paired t-test (C). **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 7

MMP-9-producing macrophages in GPA tissue lesions. Tissue biopsies were collected from sinal and nasal lesions of patients with H&N GPA. (A) H&E (hematoxylin and eosin)-stained sections. (B,C) Dual-color immunostaining for the macrophage marker PU.1 (nuclei dark gray) and pro-MMP-9 (red). (scale bar = 100 μm).

Figure 8

NET-induced pathology in H&N GPA. In patients with GPA dominated by head and neck manifestations (H&N GPA), neutrophils are highly susceptible to NETosis. NET-DNA is recognized by monocytes and macrophages and induces the alarmin S100A9. In a feed-forward loop, S100A9 binds to TLR4 and triggers the induction of the protease MMP-9. MMP-9-producing monocytes/macrophages have tissue-invasive and destructive capabilities. The pathway correlates with distinct clinical manifestations: invasive sinusitis, bony erosions of the sinal walls, septal perforation, saddle nose deformity, otitis media with bony invasion, invasive growth of orbital pseudotumor.