Eosinophil-mediated signalling attenuates inflammatory responses in experimental colitis

Abstract

Objective: Eosinophils reside in the colonic mucosa and increase significantly during disease. Although a number of studies have suggested that eosinophils contribute to the pathogenesis of GI inflammation, the expanding scope of eosinophil-mediated activities indicate that they also regulate local immune responses and modulate tissue inflammation. We sought to define the impact of eosinophils that respond to acute phases of colitis in mice.

Design: Acute colitis was induced in mice by administration of dextran sulfate sodium, 2,4,6-trinitrobenzenesulfonic acid or oxazolone to C57BL/6J (control) or eosinophil deficient (PHIL) mice. Eosinophils were also depleted from mice using antibodies against interleukin (IL)-5 or by grafting bone marrow from PHIL mice into control mice. Colon tissues were collected and analysed by immunohistochemistry, flow cytometry and reverse transcription PCR; lipids were analysed by mass spectroscopy.

Results: Eosinophil-deficient mice developed significantly more severe colitis, and their colon tissues contained a greater number of neutrophils, than controls. This compensatory increase in neutrophils was accompanied by increased levels of the chemokines CXCL1 and CXCL2, which attract neutrophils. Lipidomic analyses of colonic tissue from eosinophil-deficient mice identified a deficiency in the docosahexaenoic acid-derived anti-inflammatory mediator 10, 17- dihydroxydocosahexaenoic acid (diHDoHE), namely protectin D1 (PD1). Administration of an exogenous PD1-isomer (10S, 17S-DiHDoHE) reduced the severity of colitis in eosinophil-deficient mice. The PD1-isomer also attenuated neutrophil infiltration and reduced levels of tumour necrosis factor-α, IL-1β, IL-6 and inducible NO-synthase in colons of mice. Finally, in vitro assays identified a direct inhibitory effect of PD1-isomer on neutrophil transepithelial migration.

Conclusions: Eosinophils exert a protective effect in acute mouse colitis, via production of anti-inflammatory lipid mediators.

Keywords: IBD BASIC RESEARCH; IMMUNOREGULATION; INFLAMMATION; LIPID MEDIATORS; MUCOSAL IMMUNOLOGY.

Figure 1

Eosinophilic inflammation in acute colitis. (A) H&E, and major basic protein immunohistochemistry on mouse colons following dextran sodium sulfate (DSS)-colitis. (B) Eosinophil peroxidase immunohistochemistry on human colons from normal and ulcerative colitis subjects. (C) Wild type whole mouse colon analysis by flow cytometry for infiltrating myeloid leucocytes. (D) Colon mRNA analysis for eosinophil-associated chemokines CCL11, CCL24 and CCL5. Scale bars represent 100 and 50 μM, respectively. Data are expressed as means±SEM of 3–7 individual mice per group and represent >3-independent experimental repeats following 6 days of colitis. *p≤0.05.

Figure 2

Protective role for eosinophils in acute colitis. Dextran sodium sulfate (DSS) or water-vehicle was administered to wild type (WT)-mice and PHIL-mice. (A) Representative H&E-stained sections of colons from WT-DSS and PHIL-DSS mice (100× magnification), (B) Colon lengths were assessed upon sacrifice and (C) A histological scoring tool combining injury and inflammation was applied to H&E-stained sections following 6 days of colitis. (D) Kaplan–meier survival curve comparing PHIL-DSS and WT-DSS littermates. (E) Colitis-induced weight loss as a percentage of day zero and (F) overall disease activity index calculated in PHIL compared with WT controls following 8 days of colitis. Data are expressed as means±SEM of 15 individual control mice and seven individual mice per DSS group and represent >3-independent experimental repeats. *p≤0.05, **p≤0.01, ***p≤0.001.

Figure 3

Antibody depletion or myeloablation of eosinophils exacerbates acute colitis. Mice were depleted of eosinophils by antibody mediated interleukin-5 diminution (anti-TRFK-5), induced with dextran sodium sulfate (DSS)-colitis and (A) disease activity, (B) colonic length and (C) histology scores of H&E-stained colon tissues were reported. Bone marrow chimera was performed between PHIL-donors and wild type (WT)-recipients (PHIL->WT) and between WT-donors and WT-recipients (WT->WT) as controls. Age and sex matched PHIL-mice and WT-mice were maintained as controls. Mice were induced with colitis and (D) disease activity, (E) colonic length and (F) histology scores of H&E-stained colon tissues were reported. Data are expressed as means±SEM of 3-7 individual mice per group and represent 2-independent experimental repeats following 6 days of colitis. *†p≤0.05, ††p≤0.01, †††p≤0.001 PHIL->WT-DSS-versus-WT->WT-DSS.

Figure 4

Inflammatory infiltrate in eosinophil null mucosa is predominated by neutrophils. Whole colon flow cytometry analysis for infiltrating myeloid leucocytes in wild type (WT)-colons and PHIL-colons in dextran sodium sulfate (DSS)-colitis. Derivation and quantification of relative proportions of (A and B) neutrophils and eosinophils, (C and D) macrophage and (E and F) dendritic cell infiltrates comparing WT-DSS with PHIL-DSS-colitis. Data are expressed as means±SEM of seven individual mice per group and represent >3-independent experimental repeats following 6 days of colitis. **p≤0.01, Wild Type versus PHIL.

Figure 5

Proinflammatory and type-2 cytokine expression profile in PHIL-mice during acute colitis. mRNA was isolated from whole colons and assessed by Taqman technologies. (A–C) Major acute proinflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 as well as (D–F) Type-2 cytokines IL-4, IL-5 and IL-13 were measured. Data are expressed as means±SEM of 7–10 individual mice per group and represent 2-independent experimental repeats following 6 days of colitis. *p≤0.05, **p≤0.01, ***p≤0.001 versus genotype matched water controls. †p≤0.05, ††p≤0.01, †††p≤0.001 versus wild type-dextran sodium sulfate (DSS).

Figure 6

Chemokine, iNOS and lipid oxygenation enzyme expression profile in PHIL-mice during acute colitis. mRNA was isolated from whole colons and assessed by Taqman technologies. (A–C) Neutrophil-associated transcripts CXCL1/KC, CXCL2 and NOS2 were assessed. (D–H) Lipid oxygenation enzymes critical for lipid mediator biosynthesis PTGS1, PTGS2, ALOX5, ALOX12 and ALOX15 were also measured. Data are expressed as means±SEM of 7–10 individual mice per group and represent 2-independent experimental repeats following 6 days of colitis. *p≤0.05, **p≤0.01, ***p≤0.001 versus genotype-matched water controls. †p≤0.05, ††p≤0.01, †††p≤0.001 versus wild type-dextran sodium sulfate (DSS).

Figure 7

12/15-lipoxygenase (12/15-LOX) lipid derivative deficiency in colitis in PHIL-mice. Whole mouse colons from dextran sodium sulfate (DSS)-colitis and water control wild type (WT)-mice and PHIL-mice were assessed by LC-MS/MS-based mediator lipidomics following 6 days of colitis. Arachidonic acid (AA)-derived, eicosapentaenoic acid-derived and docosahexaenoic acid (DHA)-derived products were quantified by LC-MS/MS, and multiple reaction monitoring. Levels of 12/15-LOX products are boxed in black or blue and 12/15-LOX biosynthetic end products with statistically significant differences are in blue shaded boxes. Data are expressed as means±SEM of 3-H₂O and 6-DSS individual mice. *p≤0.05, **p≤0.01, ***p≤0.001 DSS versus genotype-matched water controls. ††p≤0.01, †††p≤0.001 PHIL-DSS versus WT-DSS.

Figure 8

Protectin D1 (PD1)-isomer (PD1-i) treatment attenuates colitis in PHIL-mice via attenuation of neutrophil migration. PHIL- dextran sodium sulfate (DSS)-colitis mice were treated with PD1-isomer or vehicle via intraperitoneal injection 0.05 mg/kg daily throughout the course of colitis which lasted 6 days. Upon sacrifice, colons were excised and (A) colon length measured. (B) Injury, inflammatory and total histology scores were assessed and (C) representative H&E-stained sections from PHIL-DSS-mice treated with PD1-isomer or vehicle (100× magnification). (D) Proinflammatory cytokine and neutrophil-associated chemokine mRNA production was examined in PD1-isomer versus vehicle-treated PHIL-DSS-mice. (E and F) Flow cytometric analysis of lamina propria infiltrates for neutrophil percent and absolute number comparing PD1-isomer with vehicle-treated PHIL-DSS-controls. Data are expressed as means±SEM of 3–7 individual mice per group, and represent 2 independent experimental repeats. (G) In vitro examination of the effects of PD1-isomer on Formyl–Methionyl–Leucyl–Phenylalanine-induced neutrophil-colon epithelial monolayer (Caco-2) transmigration; dose-dependence and time-dependence. Data are expressed as means±SEM of three individual donors and three independent experimental repeats. *p≤0.05, **p≤0.01 0.2 nM PD1 versus controls. ††p≤0.01, †††p≤0.001 20 nM PD1-isomer versus controls.