Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites

Abstract

Antigen-mediated cross-linking of Immunoglobulin E (IgE) bound to mast cells/basophils via FcɛRI, the high affinity IgE Fc-receptor, is a well-known trigger of allergy. In humans, but not mice, dendritic cells (DCs) also express FcɛRI that is constitutively occupied with IgE. In contrast to mast cells/basophils, the consequences of IgE/FcɛRI signals for DC function remain poorly understood. We show that humanized mice that express FcɛRI on DCs carry IgE like non-allergic humans and do not develop spontaneous allergies. Antigen-specific IgE/FcɛRI cross-linking fails to induce maturation or production of inflammatory mediators in human DCs and FcɛRI-humanized DCs. Furthermore, conferring expression of FcɛRI to DCs decreases the severity of food allergy and asthma in disease-relevant models suggesting anti-inflammatory IgE/FcɛRI signals. Consistent with the improved clinical parameters in vivo, antigen-specific IgE/FcɛRI cross-linking on papain or lipopolysaccharide-stimulated DCs inhibits the production of pro-inflammatory cytokines and chemokines. Migration assays confirm that the IgE-dependent decrease in cytokine production results in diminished recruitment of mast cell progenitors; providing a mechanistic explanation for the reduced mast cell-dependent allergic phenotype observed in FcɛRI-humanized mice. Our study demonstrates a novel immune regulatory function of IgE and proposes that DC-intrinsic IgE signals serve as a feedback mechanism to restrain allergic tissue inflammation.

Figure 1

Expression of FcεRI on DCs results in a DC-specific IgE pool as seen in non-allergic humans. (a) Human peripheral blood DCs of a non-allergic individual carry surface IgE. DCs (in blue) were identified by gating on CD1c⁺ CD19⁻ cells to distinguish them from CD1c⁺CD19⁺ B cells (in green) and analyzed for cell surface-bound IgE. DCs carry significant amounts of IgE but less than CD203⁺ basophils and mast cells (in purple). (b) CD11c⁺ DCs of IgE_R-TG mice carry IgE at steady state. Representative FACS plots of WT- and IgE_R-TG DCs isolated from spleens and quantification of the DC-bound IgE pool using mean fluorescence intensities (MFI) determined by flow cytometric analysis. (c) Baseline serum IgE levels are lower in non-sensitized IgE_R-TG animals. Symbols in are representative of individual mice of ≥ 2 independent experiments (*p < 0.05; ***p< 0.001).

Figure 2

IgE/FcεRI-crosslinking does not induce phenotypic maturation or production of inflammatory cytokines in DCs. (a) Antigen-mediated IgE/FcεRI activation induces phosphorylation of Syk and Erk1/2 in DCs. Splenic DCs were loaded with NP-specific IgE prior to incubation with NP-OVA (see schematic). IgE_R-TG DCs were also stimulated with CpG DNA, or antigen-crosslinking was omitted (NT = not treated). As a control, WT DCs were treated identically. Immunoblots for phospho-Syk, total Syk, phospho-Erk1/2 or total Erk1/2 are shown. (b) IgE/FcεRI-crosslinking fails to upregulate expression of maturation marker molecules in DCs from IgE_R-TG mice and (c) human monocyte-derived DCs. (d) Absence of cytokine secretion by splenic DCs upon antigen-specific IgE/FcεRI-crosslinking. Mean of triplicates +/− SEM, representative experiment (n=2); below detection level (bd) (e) TNF-α secretion from bone-marrow derived mast cells upon antigen-specific IgE/FcεRI-crosslinking. (f) Absence of transcriptional responses in murine DCs after antigen-specific IgE/FcεRI-crosslinking. mRNA expression was determined after 8 h. OVA uptake in the presence of CpG-DNA or papain was compared to IgE/FcεRI-mediated OVA uptake. Fold change compared to DCs that received OVA was calculated, and the mean of triplicates +/− SEM is shown, representative experiment (n=2).

Figure 3

Intestinal DCs express FcεRI and allergic sensitization increases the DC-bound IgE pool of IgE_R-TG animals. (a) FcεRI expression (in red) in the small intestine of IgE_R-TG mice is found on CD11c⁺ DCs (in blue). CD11c⁺ DCs from IgE_R-TG are GFP positive because the transgenic construct contains an IRES-GPF reporter element. (b) FcεRI expression on CD11c⁺ DCs in the human small intestine. Human FcεRIα (green, first panel), CD11c (red, second panel). Mast cells as depicted by c-kit are not found in non-inflamed human tissue using immunofluorescence (row two, red). DAPI-stain for nuclei (blue). (c) and (d) Analysis of OVA-specific IgE and IgG1 levels in serum of sensitized mice before antigen challenge. (e) and (f) Sensitization and antigen challenge increases the amount of surface-bound IgE on DCs of IgE_R-TG mice but not WT animals. DCs were isolated from the spleens and mesenteric lymph nodes (MLNs) of OVA-challenged WT and IgE_R-TG mice, respectively. Sensitized mice are indicated by (+), non-sensitized mice by (−). Symbols are representative of individual mice (n=2).

Figure 4

Food allergic responses are significantly reduced in IgE_R-TG animals. (a) Decreased mast cell-specific transcripts (i.e. mast cell proteases MCPT1, MCPT2, and carboxypeptidase A3 (CPA3)) and lower IL-4, IL-13, CCL-2, and IL-6 expression were detected in IgE_R-TGs. Sensitized mice are indicated by (+), non-sensitized mice by (−). Normalized mRNA counts or fold change in mRNA levels +/− SEM of small intestinal tissue of 4 independent experiments (per experiment tissue of ≥2 mice was pooled) are shown. (b) and (c) Small intestinal tissue sections were stained, and infiltrating mast cells were counted. (d) Assessment of mast cell protease MCPT1 in serum as a systemic readout for IgE-mediated mast cell activation. Serum was collected 1h after oral challenge. Symbols are representative of individual mice (n=2).

Figure 5

Chronic allergic asthma presents with a less severe clinical phenotype in IgE_R-TG animals. (a) Airway hyper-responsiveness (airway resistance, Rn) without and with methacholine challenge. (b) Infiltration of mast cell/basophils in lung tissue quantified by using mRNA levels of the cell-type specific marker FcεRI β-chain. Symbols are representative of individual mice ≥ 2 independent experiments. (c) mRNA profiling of lung tissue after induction of allergic asthma. (d) Counts of neutrophils and eosinophils in the bronchoalveolar lavage fluid of sensitized (+) WT and IgE_R-TG mice. Symbols represent individual mice of 2 independent experiments. Due to the high biological variation of the tissue response no statistical significance was reached.

Figure 6

Reduction of allergic inflammation is regulated by the DC-bound IgE pool. (a) Expression patterns of murine and/or human FcεRIα-chains in WT mice, IgE_R-TG mice, and IgE_R-TG mice backcrossed onto the murine α-chain knock strain (muα KO). Backcrossed animals are referred to as IgE_R-TG mice x muα KO (b) DC-bound IgE is found exclusively on strains that express FcεRI on this cell type after OVA/alum sensitization. (c) Analysis of OVA-specific serum IgE after sensitization. (d) Comparison of intestinal mast cell-specific transcripts of MCPT1 and MCPT2 as readout for the severity of food allergy. Representative experiment (n=3); All animals used for experiments (a-d) were on the C57BL/6 background.

Figure 7

IgE/FcεRI-mediated antigen presentation by DCs does not result in efficient generation of Th2-type effector T cells. (a) Induction of in vivo T cell proliferation. Splenic DCs were pulsed with NP-OVA in vitro (0.5 μg/ml antigen) in the presence (plus) or absence (no) of NP-specific IgE. Representative experiment (n=3). (b) Induction of in vitro T cell proliferation by DCs via IgE-mediated antigen uptake. Soluble OVA was present continuously; OVA-peptide^323-339 was used as control. Mean of triplicates +/− SEM, representative experiment (n≥5). (c) In vitro DC/T cells proliferation assay in the presence of increasing antigen concentrations. Triplicates of representative experiment (n=2). (d) IL-4 and IL13 in DC/T cell co-culture supernatant at day 3. (e) IL-4 and IFN-γ increase in culture supernatants when DC-bound IgE is used for antigen uptake. Expression levels of IL-4 mRNA are compared in T cells that were induced IgE-independently (no IgE) or via the IgE/ FcεRI-mediated uptake (plus IgE). (f) Intracellular cytokine staining for IL-4 and IFN-γ in T cells. Recombinant IL-4 or LPS was added to the antigen presentation assays as indicated. Bar diagram shows percentages of IL-4⁺ and IFN-γ CD4⁺ T cells in DC/T cell co-cultures. Data represent triplicates of biological replicates +/− SEM of a representative experiment (n=3). Representative FACS blots depict gated CD4⁺ T cells at day 7. (g) Recombinant IL-12 was added to the co-cultures and priming of Th1 cells was determined by intracellular staining for IFN-γ at day 7. Data represent triplicates of biological replicates +/− SEM of a representative experiment (n=2).

Figure 8

Antigen-specific IgE/FcεRI-crosslinking on activated DCs inhibits the production of proinflammatory cytokines and chemokines resulting in impaired migration of inflammatory cells. (a) IgE-crosslinking inhibits the papain-induced production of CCL-2, IL-6, and TNF-α by murine DCs. (b) Inhibition of CCL-2 production in human DCs by IgE/FcεRI-crosslinking. (c) The chemokine CCL-2 shows strong chemotactic activity for c-Kit⁺ mast cell progenitors and CD11c⁺ DC. Indicated concentrations of recombinant CCL-2 were added to the medium in the lower wells of transwell plates and the migration of cells from mast cell progenitor cultures towards the chemokine was determined. (d) Antigen-specific IgE/FcεRI-mediated DC activation (OVA plus IgE) inhibits migration of bone-marrow derived mast cell progenitors and DC. DCs were activated as indicated and DC-conditioned supernatants were used for chemotaxis assays. Migratory response of cKit⁺ cells and CD11c⁺ cells towards DC-conditioned supernatants was calculated from 3 independent experiments. Migratory response of DC stimulated with LPS or papain in the absence of IgE/FcεRI-mediated DC activation (OVA alone) was set at 100 %.