CD47 blockade ameliorates autoimmune vasculitis via efferocytosis of neutrophil extracellular traps

Abstract

Neutrophil extracellular trap (NET) formation contributes to immune defense and is a distinct form of cell death. Excessive NET formation is found in patients with anti-neutrophil cytoplasmic antibody-associated (ANCA-associated) vasculitis (AAV), contributing to disease progression. The clearance of dead cells by macrophages, a process known as efferocytosis, is regulated by the CD47-mediated "don't eat me" signal. Hence, we hypothesized that pathogenic NETs in AAV escape from efferocytosis via the CD47 signaling pathway, resulting in the development of necrotizing vasculitis. Immunostaining for CD47 in human renal tissues revealed high CD47 expression in crescentic glomerular lesions of patients with AAV. In ex vivo studies, ANCA-induced netting neutrophils increased the expression of CD47 with the reduction of efferocytosis. After efferocytosis, macrophages displayed proinflammatory phenotypes. The blockade of CD47 in spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mice ameliorated renal disease and reduced myeloperoxidase-ANCA (MPO-ANCA) titers with a reduction in NET formation. Thus, CD47 blockade would protect against developing glomerulonephritis in AAV via restored efferocytosis of ANCA-induced NETs.

Keywords: Autoimmunity; Nephrology; Vasculitis.

Figure 1. CD47 expression on human renal tissue.

(A) Representative IHC images for CD47 in MGA, AAV, LN class IV, and LN class V patients. Scale bars: 50 μm. (B) Quantification of CD47⁺ area of renal biopsy sections from patients with MGA (n = 8), AAV (n = 7), LN class IV (n = 4), and LN class V (n = 4) as a percentage of glomerular area. Data are shown as mean ± SD. *P <0.05, **P < 0.01 (1-way ANOVA with post hoc Dunnett’s multiple-comparison test).

Figure 2. CD47 expression on human neutrophils.

Nonstimulated, TNF-α–primed neutrophils incubated with healthy-IgGs or ANCA-IgGs and PMA-induced netting neutrophils were examined. (A) Representative images of CD47 and DAPI staining of neutrophils. Red indicates CD47; blue indicates DAPI staining. Scale bars: 10 μm. (B) Representative FCM plots of the gating strategy and histogram of CD47 expression on neutrophils. (C) Mean fluorescence intensity of CD47 in B. Data are shown as mean ± SEM. ***P < 0.001 (1-way ANOVA with post hoc Dunnett’s multiple-comparison test).

Figure 3. Efferocytosis of human neutrophils via CD47 signaling.

(A) Representative images of macrophages after 3 hours of incubation with CMFDA-labeled nonstimulated, apoptotic neutrophils, neutrophils incubated with TNF-α + healthy-IgGs, ANCA-induced NETs, and NETs treated with CT-Ab, anti-CD47 mAb, anti-CD47 mAb + FcR blocker, or anti-CD47 F(ab’)2 fragments. Cocultures were rinsed before imaging. Green indicates neutrophils. Scale bars: 50 μm. (B) Efferocytosis rate (the percentage of CMFDA⁺ macrophages) in A. (C) Representative time-lapse images of efferocytosis assay of neutrophils. Green indicates neutrophils, red indicates macrophages. Scale bars: 50 μm. Arrowheads indicate the engulfed neutrophils by macrophages. (D) Quantification of the number of engulfed nonstimulated (n =3), apoptotic neutrophils (n =3), NETs (n =2), and NETs treated with CT-Ab (n =3) or anti-CD47 mAb (n =3) every 7 minutes. Statistical analysis was performed using 1-way ANOVA, followed by Dunnett’s multiple-comparison test compared with NETs treated with CT-Ab. (E) mRNA expressions of IL1B, IL8, MCP1, and TNFA by efferocytosis. Total RNA was extracted from macrophages after 2 hours of their exposure to nonstimulated neutrophils, NETs treated with CT-Ab or anti-CD47 mAb, and monocultured macrophages (n =4 for each). Data are shown as mean ± SEM. *P <0.05, ** P <0.01, ***P <0.001 (1-way ANOVA with post hoc Dunnett’s multiple-comparison test).

Figure 4. Blockade of CD47 protected mice from spontaneous development of vasculitis.

Eight-week-old SCG/Kj mice were i.p. injected with CT-Ab or anti-CD47 mAb every 5 days for 2 weeks (n =6 for each). (A) Results of serological tests (as assessed by Mann-Whitney U test) and histopathology. The glomerular activity score was assessed. Scale bars: 100 μm (low magnification) and 50 μm (high magnification). (B) Representative images of citH3 staining and quantitative analysis of glomeruli for MPO⁺/citH3⁺ double-positive area. White dotted lines and yellow arrowheads indicated glomeruli and MPO⁺/citH3⁺ double-positive cells, respectively. Green indicates MPO; red indicates citH3; blue indicates DAPI staining. Scale bars: 50 μm. (C) mRNA expression of Ifna, Ifng, Mcp1, Prf1, and Il1b in the whole kidney. Data are shown as mean ± SD. *P <0.05, **P <0.01 (Student’s unpaired t test).

Figure 5. The immune cell profiles of the kidneys and systemic immune responses in SCG/Kj mice treated with CD47 blockade.

(A) Representative images and quantitative analysis of glomeruli by immunostaining for macrophages. White dotted lines and yellow arrowheads indicate glomeruli and CD68⁺ cells, respectively. Scale bars: 50 μm. (B) mRNA expressions of Nos2 and Cd206 in whole kidney. (C) Representative images and quantitative analysis of glomeruli by immunostaining for neutrophils. Scale bars: 50 μm. (D) The results of serum IgG. (E) The results of serum MPO-ANCA titer. Data are shown as mean ± SD. *P <0.05 (Student’s unpaired t test).