Eosinophils suppress Th1 responses and restrict bacterially induced gastrointestinal inflammation

Abstract

Eosinophils are predominantly known for their contribution to allergy. Here, we have examined the function and regulation of gastrointestinal eosinophils in the steady-state and during infection with Helicobacter pylori or Citrobacter rodentium We find that eosinophils are recruited to sites of infection, directly encounter live bacteria, and activate a signature transcriptional program; this applies also to human gastrointestinal eosinophils in humanized mice. The genetic or anti-IL-5-mediated depletion of eosinophils results in improved control of the infection, increased inflammation, and more pronounced Th1 responses. Eosinophils control Th1 responses via the IFN-γ-dependent up-regulation of PD-L1. Furthermore, we find that the conditional loss of IFN-γR in eosinophils phenocopies the effects of eosinophil depletion. Eosinophils further possess bactericidal properties that require their degranulation and the deployment of extracellular traps. Our results highlight two novel functions of this elusive cell type and link it to gastrointestinal homeostasis and anti-bacterial defense.

Figure 1.

Comparative analysis of the evolution and composition of the murine GI LP eosinophil population at steady-state and during H. pylori infection. (A) Frequencies (in percent of live CD45⁺ leukocytes) and absolute counts per organ of CD11b⁺MHCII⁻Ly6G⁻Siglec-F⁺ eosinophils in the three indicated compartments in 8-wk-old mice. n = 6–8; STO, stomach; SI, small intestine; CO, colon. (B) Absolute eosinophil counts per organ at the indicated age (in weeks). n = 4–5 per time point, shown as mean + SEM. (C–L) Eosinophil recruitment to, and activation in, the H. pylori–infected gastric LP at 6 and 12 wk post infection (p.i.), as assessed by flow cytometry and RNA sequencing. (C) Absolute numbers of CD45⁺ leukocytes in the gastric LP; n = 10–15, pooled from two independent studies. (D) Absolute numbers of the indicated gastric LP leukocyte populations of the mice shown in C. (E) Gastric LP eosinophil numbers and frequencies of the mice shown in C. (F) Frequencies of eosinophils in the BM of the mice shown in C, shown with representative FACS plots of eosinophils as identified by their Siglec F staining and granularity and back-gated into FSC/SSC plots of all CD45⁺ live cells. Lineage markers include CD3, CD4, B220, TCRα/β, NK 1.1, Ter119, and GR1. (G) Frequencies of eosinophils in the MLNs of the mice shown in C; representative FACS plots show the same gates as in F. In C–G, four and eight control mice were age-matched to the 6- and 12-wk infection, respectively. (H) Activation state of the gastric LP eosinophils shown in E, as assessed by CD11b and Siglec-F expression and side scatter. MFI, mean fluorescence intensity. (I) Frequencies of degranulated eosinophils as identified by their CD63 expression, of all gastric LP eosinophils at 12 wk p.i. (J) Frequencies of apoptotic eosinophils as identified by Annexin V binding, of all gastric LP eosinophils at 12 wk p.i. relative to controls, shown with representative FACS plots. (K) Heat map of the top 100 most differentially regulated transcripts in FACS-sorted gastric LP eosinophil samples from nine infected relative to six naive mice. (L) Selected log₂ expression ratios of transcripts potentially associated with either regulatory or pro-inflammatory functions of eosinophils. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as calculated by Mann-Whitney test. Horizontal lines in scatter plots indicate medians throughout. Each symbol represents one mouse.

Figure 2.

The antibody-mediated or genetic depletion of eosinophils enhances Th1 and Th17, as well as innate immune responses to H. pylori and improves clearance of the infection. (A–C) Mice were infected with H. pylori and treated with either anti–IL-5 or isotype control antibody for 8 wk. (A) The eosinophil depletion efficiency was ∼80%, as shown in representative FACS plots of gastric LP leukocyte preparations. (B) Gastric mucosal expression of Ifng and Il17 as determined by qRT-PCR and normalized to Hprt. Data are pooled from two independent experiments. (C) H. pylori colonization of the mice shown in B. (D–I) Eosinophil-deficient PHIL mice were infected with H. pylori for 12 wk. (D) The gastric eosinophil depletion efficiency in PHIL mice was >95%, as shown in representative FACS plots (left) and for all mice of the study relative to uninfected WT controls. (E) Neutrophil and CD4⁺ T cell numbers in the gastric LP of infected WT and PHIL mice, relative to uninfected WT controls. (F) Th1 and Th17 cell frequencies in the gastric LP of infected WT and PHIL mice. (G) Gastric mucosal expression of Ifng and Il17 as determined by qRT-PCR and normalized to Hprt. (H) H. pylori colonization of the mice shown in D–G. Data in D–H are representative of two independently conducted experiments. (I) Gastric mucosal expression of the indicated transcripts, as determined by qRT-PCR and normalized to Hprt. Note that PHIL mice were infected for 6 or 12 wk, whereas the anti–IL-5 treatment could only be continued for up to 8 wk. (J–L) Neonatal WT or PHIL mice were intrahepatically reconstituted at birth with BM from WT or PHIL donors, infected at 4 wk of age with H. pylori and sacrificed 6 wk later. (J) Eosinophil numbers in the gastric LP of reconstituted, infected mice, relative to nonreconstituted, infected PHIL mice. (K and L) Th1 and Th17 cell frequencies and H. pylori colonization of the mice shown in J. (M) Representative immunofluorescence microscopy images of FFPE sections of a control (left) and a 12 wk-infected (H. pylori PMSS1, right) WT stomach, stained with antibodies specific for EPX (for eosinophils) and CD3 (for T cells), along with the quantification of eosinophil and T cell counts per 0–225 mm². Bars, 10 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as calculated by Mann-Whitney test.

Figure 3.

Eosinophil depletion enhances colonic microbiota-dependent Th1 responses. (A) GI tissues from six WT and five PHIL mice were analyzed by qRT-PCR for the expression of Ifng, Il17, and Il4, normalized to Hprt. STO, stomach; SI, small intestine; CO, colon. (B and C) Th1, Th17 and Th2 frequencies in the colonic LP of 6-wk-old WT and PHIL mice at steady-state, as assessed by intracellular cytokine staining (B) and staining for the lineage-specific transcription factors T-bet, RORγt, and GATA-3, shown with representative FACS plots (C). (D) Th1 and Th17 cell frequencies in the colonic LP of WT mice treated with anti–IL-5 or isotype control antibody for 10 d and of WT and Eo-Cre^Dtr mice treated with diphtheria toxin (DT) for four days. Data are representative of two to three independent experiments. (E) Colonic mucosal expression of the indicated cytokines, of mice that were subjected to a 7-wk regimen of bacterial eradication by quadruple antibiotic therapy relative to untreated controls. (F) Eosinophil frequencies in the colons of the mice shown in E. (G) Activation state of the colonic LP eosinophils shown in F. (H) Eosinophil frequencies in the MLNs of the mice shown in E–G. Data are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as calculated by Mann-Whitney test.

Figure 4.

Eosinophils encounter RFP⁺H. pylori in the gastric LP and suppress T cell proliferation in a PD-1/PD-L1–dependent manner. (A–C) Mice were infected at 1 wk of age with RFP⁺ or WT (RFP⁻) H. pylori for 3 mo. (A) Frequency of RFP⁺ eosinophils among all gastric LP eosinophils. Representative FACS plots are shown along with the quantification of two pooled experiments. (B) Representative ImageStream-derived image of a SiglecF⁺ eosinophil that colocalizes with multiple RFP⁺ bacteria. Bar, 7 µm. (C) Activation state of RFP⁺ relative to RFP⁻ eosinophils of the same stomach of the mice shown in A. (D) Mice were reconstituted at birth with human cord blood HSCs and infected at 4 wk of age with RFP⁺ H. pylori for 6 wk; frequencies of human eosinophils among all human CD45⁺ leukocytes, and of RFP⁺ human eosinophils among all human gastric LP eosinophils are shown in the top panel. The MFI of the RFP channel is also shown. Bottom panel: activation state of RFP⁺ relative to RFP⁻ human eosinophils of the same stomach, as judged by CD66b and Siglec-8 expression and granularity. (E–H) Naive or H. pylori–infected, in vitro differentiated eosinophils were co-cultured at 1:1 or the indicated ratios with CSFE-labeled immunomagnetically isolated splenic CD4⁺ T cells in the presence of anti-CD3/CD28-coated beads, IL-2, and IL-5. Two different strains of H. pylori (PMSS1 and G27) were used in parallel to infect eosinophil cultures for 18 h. (E) Representative FACS plots of the CFSE dilution of anti-CD3/CD28 bead-activated T cells relative to T cells co-cultured with naive or H. pylori–educated eosinophils. (F) Quantification of the CFSE dilution of T cells co-cultured with the indicated eosinophil:T cell ratios. (G) Quantification of the CFSE dilution of T cells co-cultured with eosinophils that had previously been exposed to the indicated WT and mutant H. pylori strains, with and without separation by a trans-well filter. (H) Quantification of the CFSE dilution of T cells in eosinophil co-cultures, in the presence or absence of the indicated concentrations of anti–PD-L1 blocking antibody. Means +SEM of three technical replicates of one representative experiment of two or three are shown in F–H. (I) PD-L1 expression of eosinophils infected with the indicated H. pylori strains for 18 h. Statistics were calculated by Mann-Whitney test in A–D and by one-way ANOVA with Bonferroni’s correction in F–I and are relative to the control condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

IFN-γ signaling conditions eosinophils to suppress Th1 responses, but is not required for the pathogenic functions of eosinophils in allergic asthma. (A) In vitro differentiated WT or Ifngr^−/− eosinophils were co-cultured for 18 h with H. pylori PMSS1 (multiplicity of infection of 10) with or without 20 ng/ml rIFN-γ and subjected to qRT-PCR of the indicated transcripts. Means of three technical replicates + SEM are shown for one representative of two experiments. (B and C) Gastric LP eosinophils were FACS-sorted from naive and H. pylori–infected (6 wk) mice and subjected to qRT-PCR of the indicated transcripts. n = 6–8. (D–G) Eo-Cre⁺ × Ifngr^fl/fl mice and their Cre-negative littermates (WT) were infected with H. pylori for 6 or 12 wk. (D) Th1 and Th17 cell frequencies in the gastric LP of infected WT and EoCre⁺ × Ifngr^fl/fl mice. (E) Gastric mucosal expression of Ifng and Il17 as determined by qRT-PCR. (F) H. pylori colonization of the mice shown in D and E. (G) PD-L1 expression on gastric LP eosinophils at 12 wk p.i., as assessed by FACS of leukocyte preparations. 6-wk time point data are pooled from two independent studies; 12-wk time point data are representative of two experiments. (H) In vitro differentiated eosinophils were infected with the indicated strains of H. pylori and/or cultured in the presence of 20 ng/ml rIFN-γ for 18 h. PD-L1 expression as assessed by FACS is shown. (I–L) Eo-Cre⁺ × Ifngr^fl/fl mice, their Cre-negative littermates, and PHIL mice were sensitized and challenged with house dust mite allergen. (I) Total leukocytes in 1 ml of bronchoalveolar lavage fluid (BALF). (J) Total eosinophils in 1 ml of BALF. (K and L) Pulmonary inflammation and goblet cell metaplasia, as assessed on stained lung sections. BM, basal membrane. Results in I–L are pooled from two experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as calculated by Mann-Whitney test (B–K) or by one way ANOVA with Bonferroni’s correction (A).

Figure 6.

Eosinophils suppress C. rodentium–specific Th1 and Th17 responses and colitis. (A–H) PHIL mice and their WT littermates were infected with C. rodentium for 12 d. (A) Colonic LP eosinophil numbers of infected PHIL and WT relative to naive WT mice and their activation state. (B) Colonic neutrophil and CD4⁺ T cell numbers of the mice shown in A. (C) Th1 and Th17 cell frequencies in the colonic LP of C. rodentium–infected mice. (D) Cytokine and inflammatory gene expression in the colonic mucosa of C. rodentium–infected mice as determined by qRT-PCR. (E and F) C. rodentium–induced colitis as assessed by histopathological examination of H&E-stained sections. Representative sections of C. rodentium–infected WT and PHIL mice are shown in E, along with colitis scores in F. (G) C. rodentium colonization of the colons and cecums of the mice shown in A–E. (H) EPX expression in the colonic mucosa of the mice shown in A–G, as assessed by ELISA. Data in A–H are representative of two independent experiments. (I) CD63 expression as a marker of eosinophil degranulation, as assessed by FACS of colonic leukocyte preparations from the naive and C. rodentium–infected mice shown in A–H. (J and K) Eo-Cre⁺ × Ifngr^fl/fl mice and their Cre-negative (WT) littermates were infected with C. rodentium for 12 d and assessed with respect to their colonization levels (J) and EPX expression in the colon as assessed by ELISA (K). Bar, 100 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as calculated by Mann-Whitney test.

Figure 7.

Eosinophils contribute to C. rodentium, but not H. pylori control through degranulation and extracellular trap formation. (A) In vitro differentiated eosinophils were co-cultured with either C. rodentium or H. pylori for 6 or 18 h; bacterial viability was determined by live/dead staining and FACS. (B) CD63 expression as a marker of eosinophil degranulation, as assessed by FACS of in vitro infected and naive eosinophil cultures. (C) EPX expression, as assessed by ELISA of the supernatants of eosinophil cultures. (D) Eosinophil extracellular trap (EET) formation by eosinophils infected with C. rodentium, but not H. pylori; white arrowheads point to bacteria caught in EETs. DNase I treatment dissolves EETs. (E and F) Representative confocal immunofluorescence microscopy images of FFPE sections of a C. rodentium–infected colon (12 d, E) and an H. pylori–infected stomach (12 wk, F), along with uninfected control sections of both tissues (EPX, green; propidium iodide, PI, red). Arrows point to regions of high eosinophil density and of colocalized extracellular EPX and EETs. The quantification of eosinophils in C. rodentium–infected colons is shown in E. Bars, 50 µm in low magnification (orange), 10 µm in high magnification images (white). (G) Quantification of eosinophils with EETs in stomachs and colons of four H. pylori–infected and four C. rodentium–infected mice relative to their uninfected controls. Statistics were calculated by Mann-Whitney test in A–E and by one-way ANOVA with Bonferroni’s correction in G. *, P < 0.05; **, P < 0.01; ***, P < 0.001.