Siglec-F inhibition reduces esophageal eosinophilia and angiogenesis in a mouse model of eosinophilic esophagitis

Abstract

Objectives: Eosinophilic esophagitis (EoE) is a disorder characterized histologically by tissue eosinophilia. Sialic acid-binding immunoglobulin-like lectin (Siglec-F) is a receptor highly expressed on mouse eosinophils and mediates eosinophilic apoptosis. We investigated whether administration of an anti-Siglec-F Ab would reduce esophageal eosinophilic inflammation and remodeling in a mouse model of egg ovalbumin (OVA)-induced EoE.

Subjects and methods: Three groups of mice were studied (no OVA, OVA + anti-Siglec-F Ab, and OVA + isotype control Ab). Mice were sensitized intraperitoneally and then challenged chronically with intraesophageal OVA. Levels of esophageal eosinophils and features of remodeling (angiogenesis, vascular endothelial growth factor expression, deposition of fibronectin, basal zone hyperplasia, and fibrosis) were quantitated by immunohistochemistry and image analysis.

Results: Administration of an anti-Siglec-F Ab to OVA-challenged mice significantly reduced levels of esophageal eosinophils, down to levels noted in non-OVA-challenged mice. The anti-Siglec-F Ab also reduced features of OVA-induced remodeling, including angiogenesis, basal zone hyperplasia, and fibronectin deposition. The reduced angiogenesis in anti-Siglec-F Ab-treated mice was associated with reduced numbers of vascular endothelial growth factor-positive cells in the esophagus. The anti-Siglec-F antibody did not significantly reduce esophageal fibrosis as assessed by trichrome staining.

Conclusions: Administration of an anti-Siglec-F antibody significantly decreased the number of eosinophils in the esophagus in a mouse model of OVA-induced EoE. The reduction in eosinophilic inflammation was associated with a significant decrease in levels of angiogenesis, deposition of fibronectin, and basal zone hyperplasia. Studies in this pre-clinical model of EoE suggest that Siglec-F (and its human paralog Siglec-8) may be novel therapeutic targets to reduce eosinophilic inflammation in EoE.

FIGURE 1

Experimental egg OVA EoE protocol. Mice were sensitized intraperitoneally (ip) on day 0 and day 14 and challenged intraesophageally (IE) on days 28, 30, 32, 35, 37, 39, 42, 44, 46, 49, 51, and 53. One hour before each OVA challenge, an anti-Siglec-F or isotype control antibody was administered ip. Mice were sacrificed 24 hours after last administration of intraesophageal OVA (day 54) and their esophagi were analyzed.

FIGURE 2

Eosinophils in the esophagus. Hematoxylin and anti-mouse major basic protein immunostain of esophagus. A, No OVA. B, OVA + control Ab. C, OVA + control Ab (40× magnification of panel B). D, OVA + anti-Siglec-F Ab.

FIGURE 3

Eosinophil quantitation in esophagus, peripheral blood, and bone marrow. A, Esophagus. The number of eosinophils per square millimeter of esophageal lamina propria was quantitated. Intraesophageal OVA challenge induced a significant accumulation of eosinophils (OVA vs no OVA, P<0.0001). Administration of an anti-Siglec-F antibody to OVA-challenged mice significantly reduced the number of esophageal eosinophils (P= 0.003) to levels similar to those in the no-OVA group (n = 8 mice/group). B, Peripheral blood. The number of eosinophils was quantitated in Wright-Giemsa-stained peripheral blood. A significant increase in the percentage of eosinophils was noted in mice challenged with OVA compared to the non-OVA–exposed group (P= 0.0006). The administration of an anti-Siglec-F Ab to OVA-challenged mice significantly reduced the degree of peripheral blood eosinophilia compared to the group challenged with OVA and administered a control Ab (P= 0.04) (n = 16 mice/group). C, Bone marrow. The number of eosinophils was quantitated in Wright-Giemsa-stained bone marrow. A significant increase in the percentage of eosinophils was noted in OVA-challenged mice compared to the non-OVA-exposed group (P= 0.0002). The administration of an anti-Siglec-F Ab to OVA-challenged mice significantly reduced the degree of bone marrow eosinophilia compared to the group challenged with OVA and administered a control Ab (P= 0.0002) (n = 8 mice/group).

FIGURE 4

Effect of anti-Siglec-F antibody (Ab) on apoptosis. Bone marrow from mice chronically challenged with oral ovalbumin (OVA) and treated with either an anti-Siglec-F or control Ab was processed for TUNEL staining and the number of TUNEL-positive cells quantitated by immunohistology. The number of TUNEL-positive cells in mice chronically challenged with oral OVA was significantly increased in the bone marrow of anti-Siglec-F Ab compared to control Ab-treated mice (P= 0.02).

FIGURE 5

Angiogenesis in the esophagus. Hematoxylin and PECAM immunostain of esophagus. A, No OVA. B, OVA + control Ab. C, OVA + control Ab (40× magnification of panel B). D, OVA + anti-Siglec-F Ab.

FIGURE 6

OVA-induced angiogenesis and vascular endothelial growth factor (VEGF) expression in the esophagus. A–C, Detection of cells expressing PECAM and major basic protein (MBP). Esophageal sections from wild-type mice that had been subjected to chronic oral OVA challenge were immunostained with both an anti-PECAM Ab (immunofluoresce green) and an anti-MBP Ab (immunofluoresce red). L indicates esophageal lumen. Arrow in A points to PECAM-positive blood vessel and * indicates PECAM-positive cells, which do not colocalize with MBP (B and C). D, Angiogenesis. A significant increase in the number of small blood vessels was noted in the lamina propria of mice challenged with OVA compared to the non-OVA-exposed group (P<0.001). The administration of an anti-Siglec-F Ab to OVA-challenged mice significantly reduced the number of small vessels compared with mice challenged with OVA and administered a control Ab (P<0.001). E, VEGF-positive cells. A significant increase in the number of VEGF-positive cells was noted in OVA-challenged mice compared to the non-OVA-exposed group (P<0.004). The administration of an anti-Siglec-F Ab to OVA-challenged mice significantly reduced the number of VEGF-positive cells compared with the group challenged with OVA and administered a control Ab (P<0.02) (n = 8 mice/group).

FIGURE 7

Esophageal fibronectin deposition and TGF-β1-positive cells. A, Fibronectin. A significant increase in lamina propria fibronectin deposition was noted in OVA-challenged mice compared to the non-OVA-exposed group (P= 0.05). The administration of an anti-Siglec-F Ab to OVA-challenged mice significantly reduced the amount of fibronectin deposition compared with the group challenged with OVA and administered a control Ab (P= 0.0005). B, TGF-β1-positive cells. A significant increase in the number of TGF-β1-positive cells was noted in OVA-challenged mice compared with the non-OVA-exposed group (P= 0.0001). The administration of an anti-Siglec-F Ab to OVA-challenged mice resulted in a nonsignificant trend to reduced numbers of TGF-β1-positive cells (P= 0.18) (n = 8 mice/group).