Modulation of surface CD11c expression tracks plasticity in murine intestinal tissue eosinophils

Abstract

Intestinal eosinophils are implicated in the inflammatory pathology of eosinophilic gastrointestinal diseases and inflammatory bowel diseases. Eosinophils also contribute to intestinal immunologic and tissue homeostasis and host defense. Recent studies in allergic airway disease suggest functional subphenotypes of eosinophils may underly their pathogenic versus protective roles. However, subphenotypes of intestinal eosinophils have not been defined and are complicated by their constitutive expression of the putative eosinophil inflammatory marker CD11c. Here, we propose a framework for subphenotype characterization of intestinal eosinophils based on relative intensity of surface CD11c expression. Using this flow cytometry framework in parallel with histology and BrdU tracing, we characterize intestinal eosinophil subphenotypes and monitor their plasticity at baseline and within the context of acute allergic and chronic systemic inflammation. Data reveal a conserved continuum of CD11c expression amongst intestinal eosinophils in health and acute disease states that overall tracked with other markers of activation. Oral allergen challenge induced recruitment of eosinophils into small intestinal lamina propria surrounding crypts, followed by in situ induction of CD11c expression in parallel with eosinophil redistribution into intestinal villi. Allergen challenge also elicited eosinophil transepithelial migration and the appearance of CD11c^lo CD11b^hi eosinophils in the intestinal lumen. Chronic inflammation driven by overexpression of TNFα led to a qualitative shift in the relative abundance of CD11c-defined eosinophil subphenotypes favoring CD11c^hi -expressing eosinophils. These findings provide new insights into heterogeneity of intestinal tissue eosinophils and offer a framework for measuring and tracking eosinophil subphenotype versatility in situ in health and disease.

Keywords: allergic inflammation; eosinophil; eosinophil subphenotypes; intestinal innate immune cell; phenotypic plasticity.

FIGURE 1

CD11c expression tracks plasticity along a continuum of eosinophil activation phenotypes. (A) Single-cell suspensions from both LP and IE compartments were assessed by flow cytometry for expression of CD11c. (B) Gating strategy to assign intestinal eosinophils into CD11c^−/lo, CD11c^int, or CD11c^hi subpopulations. (C) Breakdown of LP and IE associated eosinophils based on CD11c-defined subpopulations (N = 5). Expressions of Siglec F (D), CD11b (E), SSC parameter (F), and IL-5Rα (G) were quantified on CD11c-defined subpopulations from LP (open circles) and IE (filled circles) compartments. Each circle represents data from a single mouse (N = 8). (H) CD62L expression assessed on blood (left panel) and total LP (right panel) eosinophils. Shaded histograms, isotype control; open histograms, CD62L. Inset gated on CD11c^−/lo LP eosinophils. Error bars, SD. Statistical analysis, Student’s t-tests. *, p < 0.05

FIGURE 2

Redistribution and CD11c plasticity of intestinal eosinophils following acute food allergen challenge. (A) Experimental timeline for acute food allergy challenge. (B) Eosinophils quantified from jejunum sections stained with anti-MBP are expressed as total eosinophils (eos) per crypt villus unit (CVU) (B) or as a ratio of eosinophils detected within LP surrounding crypts to eosinophils within LP of villi (Bi). Open circles, no OVA; closed circles, +OVA. (C) Breakdown of LP and IE eosinophils based on CD11c subphenotypes in saline- versus OVA-challenged mice at the indicated time points, as determined by flow cytometry of whole small intestine tissue digests (N ≥ 3 mice per time point). (D–F) BrdU⁺ eosinophils detected from jejunum sections (D) or whole small intestine tissue digests (E and F) in saline- or OVA-challenged mice. In (D), jejunum sections from an OVA-challenged mouse on day 27, showing a crypt:villus unit (left panel) or cross sectional villi (right panel) are stained with MBP (eosinophils, red) and BrdU (nuclear, brown). Insets in (D) show BrdU⁻ (Di) and BrdU⁺ (Dii) eosinophils. (Ei and Fi) CD11c expression on BrdU⁺ eosinophils from LP (Di) or IE (Fi) eosinophils of OVA-challenged mice recovered on day 24 (gray histograms) or day 27 (blue histograms). Arrows identify BrdU⁺ eosinophils. Cr, crypt; Vi, villus. Data in (D)–(F) are representative of N = 4 (saline), N = 4 (OVA, day 24) or N = 3 (OVA, day 27) mice per group, from 2 separate experiments. Error bars, SD. Statistical analyses, Student’s t-tests. *, p < 0.05

FIGURE 3

Lumen transmigrated eosinophils are predominantly CD11c^lo CD11b^hi. (A) Frozen sections of jejunum tissue rings from eosinophil reporter mice 4 days after oral allergen challenge. Magnification, 200×. White arrows, noneosinophil (tdTomRed⁺); green arrows, eosinophils (GFP⁺). (B) Frequency of eosinophils (CD45⁺GFP⁺tdTomRed⁻) amongst total CD45⁺ leukocytes recovered from ILF of the small intestine from naïve, saline-, or allergen-challenged C57Bl/6 eosinophil reporter mice. (C) Representative plot from a single mouse (left panel) and compiled data from N = 6 mice (right panel) showing the CD11c subphenotype composition of lumen-migrated eosinophils (d27). (D) Frequency of eosinophils (CD45⁺SSC^hiSiglecF⁺FSC^lo) amongst total CD45⁺ leukocytes recovered from ILF of small intestine from naïve, saline-, or OVA-challenged BALB/c WT mice. (E) Representative plot from a single mouse (left panel) and compiled data from N = 3 mice (right panel) showing the CD11c subphenotype composition of lumen-migrated eosinophils (d24) from BALB/c mice. (F) Comparison of CD11b surface expression on eosinophils from LP, IE, and intestinal lumen (ILF). In (B) and (D) open circles, no OVA; closed circles, +OVA. Statistics in (B) and (D) reflect one-way ANOVA comparing all means back to naïve mice using Dunnett’s multiple comparisons post-hoc test. *, p < 0.05

FIGURE 4

Chronic inflammation shifts the CD11c subphenotype composition of intestinal eosinophils to favor CD11c^int/hi subpopulations. (A) Ileal sections from 12 w/o TNF^ΔARE/+ (right panels) or WT littermate controls (left panels) were stained with anti-MBP to identify eosinophils (DAB-Ni, dark gray cells). (B) CD11c-defined subphenotype composition of LP eosinophils recovered from WT versus age- and gender-matched TNF^ΔARE/+ mice, aged 12–21 w/o. Data combined from N = 4 matched pairs. *, p < 0.05, Student’s t-tests