MO244CD47 BLOCKADE AMELIORATES AUTOIMMUNE VASCULITIS VIA THE EFFEROCYTOSIS OF NEUTROPHIL EXTRACELLULAR TRAPS*

Background and Aims Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterized by systemic necrotizing vasculitis in small vessels. The necrotic lesions consist of ANCA-mediated neutrophil extracellular traps (NETs) which represent a form of lytic cell death. The persistent NETs serve as autoantigens against ANCAs and cause organ damage in a vicious cycle. Considering dead cells are essentially cleared by phagocytic cells as a process of efferocytosis, why the NETs persist in tissue remains unclear. During efferocytosis, macrophages engulf apoptotic cells to prevent the leakage of intracellular components including toxic enzyme into the surrounding cells and these processes are regulated by the expression of CD47 as a “don’t eat me” signal. In this study, we hypothesized that ANCA-mediated NETs in AAV escape from efferocytosis via the up-regulation of CD47 and the persistent NETs amplify the disease. Method Human data: Human kidney biopsy specimens from patients with AAV and minor glomerular abnormality (MGA, as a case control) were subjected to immunohistochemistry (IHC) staining for CD47. In vitro: The expression of CD47 on neutrophils was evaluated by flow cytometry (FCM). Human neutrophils from healthy donor were treated with ANCA-IgGs from MPO-AAV patients or control IgGs. For the efferocytosis assay, macrophages were co-incubated with unstimulated, apoptotic, and ANCA-IgGs treated neutrophils in the presence of anti-CD47 monoclonal antibody (mAb) or a control antibody. The neutrophils were labeled with CFMDA cell tracker (fluorescent probe) and the efferocytosis was evaluated as neutrophil engulfed (CFMDA positive) macrophages using fluorescent microscopy. In vivo: Spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mice (8-week-old age) were treated with intraperitoneal injection of anti-CD47 mAb or a control antibody every 5 days for two weeks. The severity of glomerulonephritis was assessed by the levels of serum creatinine, haematuria, mRNA expression of pro-inflammatory genes, and histopathological findings. To address the immune response against the CD47 blockade, the titre of MPO-ANCA and the number of splenic cell subset was assessed by ELISA and FCM analysis, respectively. Results Human data: The IHC analysis of human renal specimens revealed that the positive area of CD47 of AAV was greater than that of MGA. In particular, the CD47-overexpressed cells were seen in glomeruli with necrotic crescent formation. In vitro: Mean fluorescence intensity (MFI) of CD47 in ANCA-IgGs treated neutrophils (NETs) was significantly higher than that in control IgGs treated neutrophils (ANCA-IgG; 442±21.4 a.u. vs control IgG; 402±10 a.u., p<0.05). In efferocytosis assay, apoptotic neutrophils were engulfed by macrophages (efferocytosis rate/ apoptotic neutrophil; 20.5±3.8%, live neutrophils; 0.9±0.5%). The efferocytosis rate of ANCA-induced NETs significantly decreased compared to apoptotic neutrophil, but anti-CD47 mAb improved the efferocytosis of ANCA-NETs (efferocytosis rate/ anti-CD47 mAb; 19.1±4.2%, control antibody; 7.7±2.2%, p<0.05). In vivo: the renal histopathological severity score, serum creatinine level of AAV mice treated with anti-CD47 mAb decreased compared to that of AAV mice treated with a control antibody (anti-CD47 mAb; 0.96±0.30 vs control antibody; 0.61±0.32 mg/dL). Although there was no significant difference in the number of splenic cells between anti-CD47 and control antibody treated mice, CD47 blockade therapy significantly reduced serum MPO-ANCA titre (28.5±10.4 vs 45.2±14.5 μg/mL) and renal mRNA expression (IFNα, IFNγ, MCP-1 and perforin) of AAV mice. Conclusion ANCA-mediated NETs might escape from efferocytosis through up-regulation of CD47 and provoke necrotizing vasculitis. CD47 blockade could be a potential novel therapeutic strategy for AAV.

Abstract 17501: Splenic CD169 + Tim4 + Marginal Zone Metallophilic Macrophages Are Essential for Wound Healing and Resolution of Inflammation After Myocardial Infarction

Fidelity of wound healing after myocardial infarction (MI) is an important determinant of subsequent adverse cardiac remodeling and failure. Infiltrating Ly6C hi blood monocytes are a key component of this healing response; however, the importance of other monocyte/macrophage populations is unclear. Here, we demonstrate in mice an essential role for splenic CD169 + Tim4 + marginal zone metallophilic macrophages (MMMs) in post-MI wound healing. CD169 + Tim4 + MMMs circulate as Ly6C low monocytes, and dynamically populate the naïve heart. After acute MI, mobilization robustly increases to the heart, where these macrophages are obligatory for apoptotic neutrophil clearance and the induction of pro-resolving and reparative tissue macrophages. Splenic MMMs are both necessary and sufficient forpost-MI wound healing, and limit late pathological remodeling.Pharmacological expansion of the splenic marginal zone during acute MI alleviates inflammation and cardiac remodeling. Finally, humans with acute MI also exhibit expansion of blood CD169 + monocytes. Hence, splenic CD169 + Tim4 + MMMs are required for pro-resolving and reparative responses after MI, and can be manipulated for therapeutic benefit to limit long-term heart failure.

Defective intestinal repair after short-term high fat diet due to loss of efferocytosis

Defective tissue repair is a hallmark of many inflammatory disorders including, inflammatory bowel disease (IBD). While it is clear that high fat diets (HFD) can exacerbate inflammatory disease by increasing inflammation, the direct effect of lipids on tissue homeostasis and repair remains undefined. We show here that short term exposure to HFD directly impairs barrier repair after intestinal epithelial damage by interfering with recognition and uptake of apoptotic neutrophils by intestinal macrophages. Apoptotic neutrophil uptake induces macrophage IL-10 production, which is lacking after intestinal damage in the context of HFD. Overexpression of IL-10 rescues repair defects after HFD treatment, but not if epithelial cells lack the IL-10 receptor, highlighting the key role of IL-10 in barrier repair. These findings demonstrate a previously unidentified mechanism by which dietary lipids directly interfere with homeostatic processes required to maintain tissue integrity.

NADPH oxidase activation regulates apoptotic neutrophil clearance by murine macrophages

Key Points Efferocytosis of ACs by inflammatory macrophages activates NADPH oxidase in a CD11b-TLR2/4-MyD88–dependent manner. ROS generated positively regulate acidification and proteolysis in efferosomes, and limit cross-presentation of AC-associated antigens.

Neutrophils induce proangiogenic T cells with a regulatory phenotype in pregnancy

Although neutrophils are known to be fundamental in controlling innate immune responses, their role in regulating adaptive immunity is just starting to be appreciated. We report that human neutrophils exposed to pregnancy hormones progesterone and estriol promote the establishment of maternal tolerance through the induction of a population of CD4+ T cells displaying a GARP+CD127loFOXP3+ phenotype following antigen activation. Neutrophil-induced T (niT) cells produce IL-10, IL-17, and VEGF and promote vessel growth in vitro. Neutrophil depletion during murine pregnancy leads to abnormal development of the fetal-maternal unit and reduced empbryo development, with placental architecture displaying poor trophoblast invasion and spiral artery development in the maternal decidua, accompanied by significantly attenuated niT cell numbers in draining lymph nodes. Using CD45 congenic cells, we show that induction of niT cells and their regulatory function occurs via transfer of apoptotic neutrophil-derived proteins, including forkhead box protein 1 (FOXO1), to T cells. Unlike in women with healthy pregnancies, neutrophils from blood and placental samples of preeclamptic women fail to induce niT cells as a direct consequence of their inability to transfer FOXO1 to T cells. Finally, neutrophil-selective FOXO1 knockdown leads to defective placentation and compromised embryo development, similar to that resulting from neutrophil depletion. These data define a nonredundant function of neutrophil–T cell interactions in the regulation of vascularization at the maternal–fetal interface.

Apoptosis and Bcl-2 Expression in Irradiated Lungs and the Effect of Pentoxifylline

We measured number of bcl-2, apoptotic, neutrophil, and surfactant apoprotein D (SP-D) positive cells in irradiated rat lungs during different time points after the sublethal whole-thorax irradiation of rats. We also investigated the influence of pentoxifylline (PTX) therapy on these markers. Wistar rats were given 15 Gy thoracic irradiation and PTX (35 mg/kg) twice a week. Animals were examined histologically and imunohistochemically at intervals from 1-12 weeks. In non-treated rats compared with treated rats, bcl-2 expression was significantly inhibited from 4 weeks after irradiation. A higher apoptosis presence in non-treated rats from 4 weeks was found and apoptosis development in PTX-treated animals was delayed and started 8 weeks after irradiation. Similar differences were measured during neutrophil granulocytes examination. Neutrophil penetration in non-treated rats was found 5 weeks after irradiation in contrast to the RP onset of PTX-treated animals 8 weeks after irradiation. The number of SP-D positive cells in non-treated rats observed until 5 weeks after irradiation was higher than in the control group. PTX-treated animals expressed higher number of SP-D positive cells during the whole experiment than the control group. We suggest that apoptosis is linked to neutrophil granulocyte actions during the RP onset and that PTX-therapy causes diminished inflammation development.