Resident intestinal eosinophils constitutively express antigen presentation markers and include two phenotypically distinct subsets of eosinophils

Abstract

Intestinal eosinophils are implicated in homeostatic and disease-associated processes, yet the phenotype of intestinal tissue-dwelling eosinophils is poorly defined and their roles in intestinal health or disease remain enigmatic. Here we probed the phenotype and localization of eosinophils constitutively homed to the small intestine of naive mice at baseline, and of antigen-sensitized mice following intestinal challenge. Eosinophils homed to the intestinal lamina propria of naive mice were phenotypically distinguished from autologous blood eosinophils, and constitutively expressed antigen-presenting cell markers, suggesting that intestinal eosinophils, unlike blood eosinophils, may be primed for antigen presentation. We further identified a previously unrecognized resident population of CD11c^hi eosinophils that are recovered with intraepithelial leucocytes, and that are phenotypically distinct from both lamina propria and blood eosinophils. To better visualize intestinal eosinophils in situ, we generated eosinophil reporter mice wherein green fluorescent protein expression is targeted to both granule-delimiting and plasma membranes. Analyses of deconvolved fluorescent z-section image stacks of intestinal tissue sections from eosinophil reporter mice revealed eosinophils within intestinal villi exhibited dendritic morphologies with cellular extensions that often contacted the basement membrane. Using an in vivo model of antigen acquisition in antigen-sensitized mice, we demonstrate that both lamina propria-associated and intraepithelium-associated eosinophils encounter, and are competent to acquire, lumen-derived antigen. Taken together these data provide new foundational insights into the organization and functional potential of intestinal tissue-dwelling eosinophils, including the recognition of different subsets of resident intestinal eosinophils, and constitutive expression of antigen-presenting cell markers.

Keywords: eosinophil; eosinophilic gastrointestinal diseases; intraepithelial; mucosal immunity; non-classical antigen presentation.

Figure 1

Resident intestinal eosinophils are phenotypically distinct from autologous blood eosinophils. (a) Paraffin sections of small intestinal tissue from BALB/c wild‐type mice stained with fast green and neutral red. Eosinophils (arrows) are identified within lamina propria associated with crypts and villi. (b,c) SSC^hi SiglecF^hi eosinophils are identified by flow cytometry of lamina propria (LP) cells recovered from digested small intestinal tissues from wild‐type (b) or eosinophil‐deficient (c) mice. Plots are gated on live, CD45⁺ leucocytes (see Supplementary material, Fig. S1). (d–f) Surface expression of Siglec F (d), CD11b (e) or CD11c (f) on eosinophils from autologous blood (empty histogram) or intestinal lamina propria (shaded histogram). Dashed line, isotype control staining.

Figure 2

Resident intestinal lamina propria (LP) eosinophils constitutively express an antigen‐presenting cell phenotype. (a) MHC II expression on eosinophils gated from autologous whole blood (top), LP eosinophils (middle) and MHC II⁺ non‐eosinophils from the LP (bottom). (b) MHC II expression on LP eosinophils compared with isotype control staining. (c) CD80 expression on LP eosinophils.

Figure 3

A subset of eosinophils that is phenotypically distinct from both blood and lamina propria (LP) eosinophils is recovered with intestinal intraepithelial (IE) leucocytes. (a,b) Flow cytometry of IE cells isolated from BALB/c wild‐type (a) or C57BL/6 EoCre^+/− mTmG^fl/fl (b) mice. Plots shown are gated on total live, CD45⁺ cells. (b, i) Morphology and HEMA 3 staining of GFP⁺ cells sorted from IE preparations confirm the identity of IE eosinophils. (c) IE leucocytes were isolated from human resected tissue recovered from a patient following ileocaecectomy. Total IE cells recovered were spun onto a cytospin and stained using the HEMA 3 staining kit. (d–h) Surface expression of Siglec F (d), CD11b (e), CD11c (f), MHC II (g) and CD80 (h) on blood, LP and IE eosinophils recovered from BALB/c wild‐type mice is expressed as delta mean fluorescence intensity (ΔMFI). *P < 0·05. Data are pooled from n = 12 (LP, IE) mice. Pooled data expressing ΔMFI of autologous blood from three of the mice is provided for reference (open bars). (b, i; c) Scale bars, 10 µm.

Figure 4

Visualization of eosinophils in situ in EoCre^+/− mTmG^fl/fl eosinophil reporter mice. Frozen sections of small intestinal tissues recovered from naive EoCre^+/− mTmG^fl/fl mice were mounted with Hoechst‐containing media and analysed by fluorescence microscopy. Membrane‐targeted tdTomato expression (left column) demarcates cell membranes of non‐eosinophils. GFP⁺ eosinophils (middle column, and arrows in merged images) are visualized within the lamina propria (LP) proximate to crypts (Cr, a) and throughout villi (b,c), often in apparent direct contact with the basement membrane (c, ci). Original magnification, 600×. Scale bars 20 µm (a‐c) and 10 µm (ci).

Figure 5

Villous eosinophils extend dendritic processes that contact the basement membrane. (a) A deconvolved image stack (0·4‐µm z‐step) reveals eosinophil dendritic processes that traverse multiple focal planes. Even numbered slices of the image stack between 8 and 30 are shown. Note that slices 14 through 20 confirm continuity between the cell body visible in slice 10 and the dendritic processes in contact with the basement membrane (demarcated by arrows) in slices 20–24. Original magnification, 600×. Scale bars, 20 µm (top image) and 10 µm (slices). (b) Three‐dimensional rendering of panel (a). Scale bars, 10 µm.

Figure 6

Both lamina propria (LP) and intraepithelially (IE) associated eosinophils access and acquire lumen‐derived antigen in vivo. Fluorescently tagged ovalbumin (OVA) (or unlabelled OVA) was injected into intestinal loops of anaesthetized mice previously sensitized with OVA in alum adjuvant. After 45 min intestinal loops were removed and rinsed, and LP and IE cells isolated and analysed by flow cytometry for acquisition of the fluorescent signal. Percentages of OVA⁺ eosinophils (gated on live, CD45⁺ SSC^hi SiglecF⁺ cells) isolated with LP (a) or IE (b) cells are shown. In (c,d) surface expression of CD11c is assessed on total (c) and OVA⁺ (d) eosinophil populations.