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

Platzer B, Baker K, Vera MP, Singer K, Panduro M, Lexmond WS, Turner D, Vargas SO, Kinet JP, Maurer D, Baron RM, Blumberg RS, Fiebiger E - Mucosal Immunol (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology and Nutrition, Boston Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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-peptide323-339 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).
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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-peptide323-339 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).

Mentions: The inhibitory activity of IgE/FcεRI-bearing DCs described above is in potential disagreement with the Th2-promoting function of IgE/FcεRI-mediated antigen presentation noted previously.31, 46 We therefore revisited the role of the IgE presentation pathway in T cell proliferation and Th2-type T cell priming. First, we confirmed the extraordinary sensitivity of this antigen presentation pathway31, 47 with DCs from IgER-TG animals. We isolated splenic DCs, pre-loaded them with NP-IgE and pulsed with NP-OVA for 1 h. We then injected these DCs into WT recipient mice to assess induction of T cell proliferation in vivo. Analysis of the draining lymph nodes revealed that only IgE-loaded DCs induced a strong proliferative response of adoptively transferred CFSE-labeled OVA-specific T cells in the low antigen concentration range (Figure 7a, second antigen concentration: Supplementary Figure S8a). In contrast to the Th2-promoting stimulus papain,14, 48 IgE/FcεRI mediated-antigen uptake by DCs failed to induce an inflammatory Th2 profile in CD4+T cells in vivo (Supplementary Figure S8b). To rule out that the Th2 response was simply under the detection limit in this assay, we confirmed these data with in vitro antigen presentation assays. Here, we altered the antigen loading conditions to allow for a more physiological setting. In contrast to previous studies where DCs were pulsed with antigen in the cold, our loading conditions allowed for IgE-dependent and IgE-independent antigen uptake in parallel over prolonged periods of time at 37°C. As noted previously, when IgE-loaded DCs from IgER-TG animals were used, T cell proliferation was induced in a low antigen concentration range that otherwise failed to initiate T cell responses. Titration experiments over a broader antigen concentration range demonstrated that IgE/FcεRI-mediated antigen uptake by DCs increased T cell proliferation most effectively at low concentrations (≤0.5 μg/ml), whereas no significant impact on the proliferative responses was observed at higher concentrations (Figure 7c). In line with previously published data,31 day 3 supernatants of the DC/T cell co-cultures contained more IL-4 and IL-13 when IgE/FcεRI-mediated uptake was used for antigen sampling (Figure 7d). However, antigen titration experiments showed that this early IL-4 production by T cells was a response to increased antigen uptake by the DCs at a low antigen concentration rather than a specific consequence of IgE/FcεRI signaling (Figure 7e). At the higher antigen concentration, we actually observed less IL-4 after IgE/FcεRI-mediated uptake when compared to fluid phase uptake (Figure 7e). Since priming of naïve T cells into fully differentiated T effector cells requires more than 3-4 days, we next analyzed the effector T cell phenotype at day 7 by staining for intracellular cytokines. Notably, T cells that were induced after IgE-mediated antigen presentation failed to differentiate into IL-4+ Th2 cells (Figure 7f). Since in vivo basophils and innate lymphoid cells have been demonstrated to provide additional IL-4, which is essential for Th2 cell priming via DCs 48, we added recombinant IL-4 to the DC/T cell co-cultures. Importantly, even in the presence of exogenous IL-4, IgE/FcεRI-mediated antigen presentation failed to generate more efficient Th2 effector responses than seen in the controls (Figure 7e and Supplementary Figure S8c). Addition of LPS to the DC/T cell co-cultures results in a Th1-type response as evident by the presence of a high percentage of IFN-γ+ T cells (Figure 7f). IgE/FcεRI-mediated antigen presentation did not diminish the induction of Th1 cells by LPS and rather reduced the numbers of IL-4+ T cells in LPS cultures (Figure 7f and Supplementary Figure S8c). To test if increased antigen uptake through IgE/FcεRI can promote Th1 responses, we added IL-12 or CpG DNA to the DC/T cell co-cultures. We detected more IFN-γ+ T cells in the presence of IL-12 and IgE (Figure 8g). Similar to IL-12, we found more IFN-γ and less IL-13 production in the presence of CpG DNA and IgE (Supplementary Figure S8d). This data suggest that IgE/FcεRI-mediated antigen uptake can, in fact, increase Th1 immune responses when the DCs receive Th1-supporting stimuli during antigen presentation. We also tested whether IgE/FcεRI-mediated antigen uptake could promote the priming of inducible regulatory T cells (iTreg). Addition of TGF-β1 to DC/T cell co-cultures resulted in the de novo generation of CD25+Foxp3+ iTregs and IL-10 expressing CD4+ T cells from purified naïve CD25− OT-II T cells. No significant difference in iTreg priming or IL-10+ CD4+ T cells was detectable in the presence or absence of IgE-mediated antigen uptake through DCs (Supplementary Figure S8e and S8f).


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

Platzer B, Baker K, Vera MP, Singer K, Panduro M, Lexmond WS, Turner D, Vargas SO, Kinet JP, Maurer D, Baron RM, Blumberg RS, Fiebiger E - Mucosal Immunol (2014)

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-peptide323-339 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).
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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-peptide323-339 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).
Mentions: The inhibitory activity of IgE/FcεRI-bearing DCs described above is in potential disagreement with the Th2-promoting function of IgE/FcεRI-mediated antigen presentation noted previously.31, 46 We therefore revisited the role of the IgE presentation pathway in T cell proliferation and Th2-type T cell priming. First, we confirmed the extraordinary sensitivity of this antigen presentation pathway31, 47 with DCs from IgER-TG animals. We isolated splenic DCs, pre-loaded them with NP-IgE and pulsed with NP-OVA for 1 h. We then injected these DCs into WT recipient mice to assess induction of T cell proliferation in vivo. Analysis of the draining lymph nodes revealed that only IgE-loaded DCs induced a strong proliferative response of adoptively transferred CFSE-labeled OVA-specific T cells in the low antigen concentration range (Figure 7a, second antigen concentration: Supplementary Figure S8a). In contrast to the Th2-promoting stimulus papain,14, 48 IgE/FcεRI mediated-antigen uptake by DCs failed to induce an inflammatory Th2 profile in CD4+T cells in vivo (Supplementary Figure S8b). To rule out that the Th2 response was simply under the detection limit in this assay, we confirmed these data with in vitro antigen presentation assays. Here, we altered the antigen loading conditions to allow for a more physiological setting. In contrast to previous studies where DCs were pulsed with antigen in the cold, our loading conditions allowed for IgE-dependent and IgE-independent antigen uptake in parallel over prolonged periods of time at 37°C. As noted previously, when IgE-loaded DCs from IgER-TG animals were used, T cell proliferation was induced in a low antigen concentration range that otherwise failed to initiate T cell responses. Titration experiments over a broader antigen concentration range demonstrated that IgE/FcεRI-mediated antigen uptake by DCs increased T cell proliferation most effectively at low concentrations (≤0.5 μg/ml), whereas no significant impact on the proliferative responses was observed at higher concentrations (Figure 7c). In line with previously published data,31 day 3 supernatants of the DC/T cell co-cultures contained more IL-4 and IL-13 when IgE/FcεRI-mediated uptake was used for antigen sampling (Figure 7d). However, antigen titration experiments showed that this early IL-4 production by T cells was a response to increased antigen uptake by the DCs at a low antigen concentration rather than a specific consequence of IgE/FcεRI signaling (Figure 7e). At the higher antigen concentration, we actually observed less IL-4 after IgE/FcεRI-mediated uptake when compared to fluid phase uptake (Figure 7e). Since priming of naïve T cells into fully differentiated T effector cells requires more than 3-4 days, we next analyzed the effector T cell phenotype at day 7 by staining for intracellular cytokines. Notably, T cells that were induced after IgE-mediated antigen presentation failed to differentiate into IL-4+ Th2 cells (Figure 7f). Since in vivo basophils and innate lymphoid cells have been demonstrated to provide additional IL-4, which is essential for Th2 cell priming via DCs 48, we added recombinant IL-4 to the DC/T cell co-cultures. Importantly, even in the presence of exogenous IL-4, IgE/FcεRI-mediated antigen presentation failed to generate more efficient Th2 effector responses than seen in the controls (Figure 7e and Supplementary Figure S8c). Addition of LPS to the DC/T cell co-cultures results in a Th1-type response as evident by the presence of a high percentage of IFN-γ+ T cells (Figure 7f). IgE/FcεRI-mediated antigen presentation did not diminish the induction of Th1 cells by LPS and rather reduced the numbers of IL-4+ T cells in LPS cultures (Figure 7f and Supplementary Figure S8c). To test if increased antigen uptake through IgE/FcεRI can promote Th1 responses, we added IL-12 or CpG DNA to the DC/T cell co-cultures. We detected more IFN-γ+ T cells in the presence of IL-12 and IgE (Figure 8g). Similar to IL-12, we found more IFN-γ and less IL-13 production in the presence of CpG DNA and IgE (Supplementary Figure S8d). This data suggest that IgE/FcεRI-mediated antigen uptake can, in fact, increase Th1 immune responses when the DCs receive Th1-supporting stimuli during antigen presentation. We also tested whether IgE/FcεRI-mediated antigen uptake could promote the priming of inducible regulatory T cells (iTreg). Addition of TGF-β1 to DC/T cell co-cultures resulted in the de novo generation of CD25+Foxp3+ iTregs and IL-10 expressing CD4+ T cells from purified naïve CD25− OT-II T cells. No significant difference in iTreg priming or IL-10+ CD4+ T cells was detectable in the presence or absence of IgE-mediated antigen uptake through DCs (Supplementary Figure S8e and S8f).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology and Nutrition, Boston Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

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.

No MeSH data available.


Related in: MedlinePlus