Possible involvement of NETosis in inflammatory processes in the eye: Evidence from a small cohort of patients

Abstract

Purpose: To evaluate whether NETosis is involved in cytokine-induced ocular inflammation and to track neutrophil extracellular traps (NET) complexes in patients with proliferative diabetic retinopathy (PDR).

Methods: For the animal model, the eyes of C57BL/6J mice were intravitreally injected with interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), or saline. Histology and immunofluorescence staining for CD11b, neutrophil elastase (NE), myeloperoxidase (MPO), citrullinated histone 3 (H3Cit), and net-like structure were performed. Vitreous samples were collected from patients with PDR; the PDR1 group had no need for repeated surgical intervention, and the PDR2 group had repeated vitreous bleeding or other complication and controls. Levels of MPO, H3Cit-MPO, and NE-MPO complex were measured with enzyme-linked immunosorbent assay (ELISA).

Results: Massive influx of CD11+ inflammatory cells, involving the anterior and posterior chambers, was observed in the murine eyes 24 h after the IL-8 or TNF-α injections. Cells excreted to their surroundings an extracellular net-like structure positive for NE, MPO, and H3Cit. H3Cit staining was abolished with the DNase I treatment, indicating the presence of extracellular DNA in the net-like structures. The vitreous samples of the patients with PDR2 contained statistically significantly higher levels of MPO (173±230) compared to those of the patients with PDR1 (12.0±33.0, p<0.05) or the controls (0.00, p<0.01). The levels of H3Cit-MPO and NE-MPO complexes were also statistically significantly higher in the patients with PDR2 (776.0±1274, 573.0±911.0, respectively) compared to those in the patients with PDR1 (0, p<0.05) and the controls (0, p<0.05).

Conclusions: This study showed the existence of NETosis in cytokine-induced ocular inflammation in a mouse model and human samples. Furthermore, the extent of NET complex formation was higher in a subset of patients who exhibited more complicated PDR.

Figure 1

Histological evaluation of cytokine-induced ocular inflammation in the anterior and posterior chambers, respectively. A, D: Sham injected. B, E: Interleukin (IL)-8. C, F: Tumor necrosis factor alpha (TNF-α) 24 h post-injection. C = cornea; AC = anterior chamber; V = vitreous. Magnification = 10X. Scale bar = 100 μm.

Figure 2

Neutrophils infiltration into the anterior and posterior chambers of the eye. Sham-injected (A, D), interleukin (IL)-8 (B, E) and tumor necrosis factor alpha (TNF-α) (C, F), 24 h post-injection. No cellular infiltration was observed in the sham-injected eyes (A, E) while abundant CD11b+ cells were seen in the eyes injected with IL-8 (B, E), and TNF-α (C, F). C= cornea; AC = anterior chamber; V = vitreous; GCL = ganglion cell layer. The results show representative images observed in five eyes (n=5/group). Magnification = 63X. Scale bar = 20 μm.

Figure 3

NET analysis in the anterior chamber upon IL-8 or TNF-α injections. The sham-injected eyes were negative for neutrophil extracellular traps (NETs; A, D, G, J, M). Netting neutrophils and NETs were observed in the eyes injected with interleukin (IL)-8 and tumor necrosis factor alpha (TNF-α) and were based on colocalization of neutrophil elastase (NE, red; E, F, arrows), myeloperoxidase (MPO, green; H, I, arrows), and H3Cit (purple; K, L, arrows). 4',6-diamidino-2-phenylindole (DAPI, blue; N, O, arrows) shows the nuclear morphology. Magnification = 63X. Scale bar = 50 μm.

Figure 4

NET analysis in the posterior chamber upon IL-8 or TNF-α injections. The sham-injected eyes were negative for neutrophil extracellular traps (NETs; A, D, G, J, M). Netting neutrophils and NETs were observed in the eyes injected with interleukin (IL)-8 and tumor necrosis factor alpha (TNF-α) and were based on colocalization of neutrophil elastase (NE, red; E, F, arrows), myeloperoxidase (MPO, green; H, I, arrows), and citrullinated histone 3 (H3Cit, purple; K, L, arrows). 4',6-diamidino-2-phenylindole (DAPI, blue; N, O, arrows) shows the nuclear morphology. Magnification = 63X. Scale bar = 50 μm.

Figure 5

The effect of DNase I treatment on extracellular citrullinated DNA. Number of citrullinated histone 3 (H3Cit) loci with and without DNase I treatment in the anterior and posterior chambers of interleukin (IL)-8 (A) and tumor necrosis factor alpha (TNF-α) (B). *Statistically significant; p<0.05, ** highly significant, p<0.001.

Figure 6

Vitreous levels of NET complexes found in patients with PDR. Myeloperoxidase (MPO) (A), citrullinated histone 3-myeloperoxidase (H3Cit-MPO) complex (B), and neutrophil elastase-myeloperoxidase (NE-MPO) complex (C). PDR = proliferative diabetic retinopathy; PDR1 = had no need for repeated surgical intervention; PDR2 = had repeated vitreous bleeding or other complication. *Statistically significant; p<0.05, **, highly statistically significant, p<0.01.