Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens

Journal name:
Nature
Volume:
471,
Pages:
220–224
Date published:
DOI:
doi:10.1038/nature09849
Received
Accepted
Published online

Under physiological conditions the gut-associated lymphoid tissues not only prevent the induction of a local inflammatory immune response, but also induce systemic tolerance to fed antigens1, 2. A notable exception is coeliac disease, where genetically susceptible individuals expressing human leukocyte antigen (HLA) HLA-DQ2 or HLA-DQ8 molecules develop inflammatory T-cell and antibody responses against dietary gluten, a protein present in wheat3. The mechanisms underlying this dysregulated mucosal immune response to a soluble antigen have not been identified. Retinoic acid, a metabolite of vitamin A, has been shown to have a critical role in the induction of intestinal regulatory responses4, 5, 6. Here we find in mice that in conjunction with IL-15, a cytokine greatly upregulated in the gut of coeliac disease patients3, 7, retinoic acid rapidly activates dendritic cells to induce JNK (also known as MAPK8) phosphorylation and release the proinflammatory cytokines IL-12p70 and IL-23. As a result, in a stressed intestinal environment, retinoic acid acted as an adjuvant that promoted rather than prevented inflammatory cellular and humoral responses to fed antigen. Altogether, these findings reveal an unexpected role for retinoic acid and IL-15 in the abrogation of tolerance to dietary antigens.

At a glance

Figures

  1. IL-15-activated dendritic cells in the presence of retinoic acid prevent induction of Foxp3+ regulatory T cells.
    Figure 1: IL-15-activated dendritic cells in the presence of retinoic acid prevent induction of Foxp3+ regulatory T cells.

    a, 105 CD4+ Foxp3 T cells were cultured with 4×104 MLN dendritic cells (DCs) isolated from wild-type (WT) or Dd-IL-15 transgenic (Dd-IL-15tg) mice with anti-CD3 alone or combined with IL-15, TGF-β and RA. The percentages of Foxp3+ cells are shown. Graph depicts pooled data±s.e.m. (n = 3). b, RAG1−/−OT-IICD45 congenic CD25CD4+ T cells were transferred into wild-type and Dd-IL-15 transgenic mice that were fed OVA in drinking water for five days (black dots) or by gavage every other day for 10 days (grey dots). Treg-cell conversion was assessed in the MLN by intracellular staining for Foxp3 and detected by flow cytometry. The absolute numbers of converted CD4+ Foxp3+ T cells are shown. Data are representative of two experiments performed independently. The decrease in the number of converted Treg cells was associated with a significant decrease in the number of detectable transferred T cells in Dd-IL-15 transgenic mice (data not shown). This is probably due to the inability to detect inflammatory T cells that are more susceptible to cell death than Foxp3+ Treg cells, which express anti-apoptotic factors. c, Ly5.2+OT-II T cells were transferred into Ly5.1+ and Dd-IL-15tg-Ly5.1+ recipient mice that were fed OVA or OVA and RA five times during ten days. The absolute number of CD4+Foxp3+Ly5.2+ converted T cells in the MLN is shown as in a. d, As in a, CD4+Foxp3 T cells were cultured with splenic (SPL) dendritic cells isolated from wild-type and IL-12p40−/− mice. The percentages of Foxp3+ cells are indicated. Graph depicts three pooled experiments±s.e.m. *P<0.05, **P<0.01, ***P<0.001 (unpaired Student’s t-test).

  2. Retinoic acid exerts an adjuvant effect on IL-15-mediated inflammatory T-cell responses.
    Figure 2: Retinoic acid exerts an adjuvant effect on IL-15-mediated inflammatory T-cell responses.

    a, CD4+ T cells were cultured with wild-type splenic dendritic cells with the indicated cytokines. Representative histograms gated on CD4+ T cells show IFN-γ expression (n = 5). b, Dd-IL-15 transgenic and wild-type mice were fed PBS (sham), OVA, RA, or a mixture of OVA and RA. IFN-γ secretion by lamina propria (LP) cells re-stimulated for 24h with OVA. The results are the means of triplicate samples obtained from two independent experiments. c, CD4+ T cells were cultured with splenic dendritic cells isolated from wild-type or IL-12p40−/− mice as described in a. Intracellular staining for IFN-γ of gated CD4 T cells is shown. Results are representative of two experiments. d, Levels of IL-12p40 in the MLN of wild-type and Dd-IL-15 transgenic mice fed OVA, or a mixture of OVA and the RAR antagonist LE135. The results are the means of triplicate samples obtained from two independent experiments. Similar results were obtained for IL-12p70 and IL-23 (data not shown). e, IFN-γ secretion by lamina propria cells isolated from Dd-IL-15 transgenic mice fed PBS (sham), OVA and LE135. The results are the means of triplicate samples obtained from two independent experiments. f, g, Dd-IL-15 transgenic mice were fed OVA and treated with blocking anti-IL-12p40, anti-IL-15 and TMβ-1 (anti-IL-2Rβ) or isotype control monoclonal antibodies. The levels of IL-12p40 in the MLN (f) and IFN-γ in lamina propria cells re-stimulated overnight with OVA (g) were quantified. When anti-IL-15 and anti-IL-12 treatment experiments were performed in parallel, control mice received a mixture of corresponding isotype controls. Data represent two pooled experiments (n = 6 mice per group) except for the anti-IL-12 treatment (n = 3 individual mice). *P<0.05, **P<0.01, ***P<0.001 (unpaired Student’s t-test).

  3. Retinoic acid and IL-15 act in synergy to induce dendritic cells with proinflammatory properties in a JNK-dependent manner.
    Figure 3: Retinoic acid and IL-15 act in synergy to induce dendritic cells with proinflammatory properties in a JNK-dependent manner.

    a, Concentration-dependent JNK phosphorylation in wild-type or IL-2Rβ−/− bone-marrow-derived dendritic cells upon IL-15 stimulation analysed by western blot (left panel) and quantified (right panel). b, IL-12p70 secretion after overnight culture of wild-type splenic dendritic cells with increasing doses of RA, with and without IL-15. Results are mean values±s.e.m. (n = 3). c, CD4+Foxp3 T cells were cultured with wild-type splenic dendritic cells with anti-CD3 alone or combined with IL-15, TGF-β and increasing doses of RA. The percentages of Foxp3+ cells are shown. Graph depicts pooled data±s.e.m. (n = 3). d, Concentration-dependent JNK phosphorylation in wild-type bone-marrow-derived dendritic cells upon IL-15 (0.01ngml−1) and increasing doses of RA stimulation by western blot (left panel) and quantified (right panel). e, Concentration-dependent JNK phosphorylation in wild-type bone-marrow-derived dendritic cells upon stimulation with a RARα agonist (AM580). f, IL-12p70 secretion after overnight culture of wild-type and RARα−/− bone-marrow-derived dendritic cells with IL-15 alone or combined with RA. Data are shown as means and s.e.m. (n = 2). g, CD4+Foxp3 T cells were cultured with splenic dendritic cells isolated from wild-type or JNK2−/− mice with the indicated cytokines. The percentages of Foxp3+ and IFN-γ+ among CD4+ T cells are indicated. h, JNK phosphorylation in bone-marrow-derived dendritic cells pretreated with cyclohexamide (CHX) or actinomycin D (AD) before stimulation with 0.1nM RA. Results are representative of two independent experiments. *P<0.05, **P<0.01 (unpaired Student’s t-test).

  4. DQ8-Dd-IL-15 transgenic mice fed gliadin mimic early stages of coeliac disease reflecting dysregulation in the adaptive immune response to gluten.
    Figure 4: DQ8-Dd-IL-15 transgenic mice fed gliadin mimic early stages of coeliac disease reflecting dysregulation in the adaptive immune response to gluten.

    ad, DQ8 and DQ8-Dd-IL-15 transgenic mice were fed gliadin every other day for ten days. a, IFN-γ secretion by lamina propria cells after overnight culture with gliadin. b, c, Anti-gliadin IgG, anti-gliadin IgA, anti-TG2 IgG and anti-TG2 IgA titres from serum collected fifteen days after feeding. Antibody titres were detected by ELISA and calculated according to the formula: (OD450nm of sample – OD450nm of blank) × serum dilution, where OD is optical density d, Quantification of IELs among IECs in small intestines fifteen days after the last gliadin feeding. Two independent counts of IELs among 200 IECs were performed for each mouse. Data are shown as means and s.e.m. (n = 3 individual mice). Similar results were obtained in another set of experiments where mice were fed with α-gliadin. e, IL-15 and IL-12 expression in the lamina propria of coeliac disease patients. Immunohistochemical stainings for IL-15 in gut tissue from an active coeliac disease (CD) patient (left panel). Lamina propria cells were harvested from biopsies obtained from control (n = 14) or active coeliac disease patients (n = 7) and assayed for levels of IL-15 and IL-12p70 by ELISA (middle and right panels). Equal concentration of total proteins was analysed for each sample. **P<0.01, ***P<0.001 (unpaired Student’s t-test). f, Proposed model for the co-adjuvant effects of RA and IL-15 in the intestinal mucosa. Under inflammatory conditions, the expression of the proinflammatory cytokine IL-15 is upregulated in the lamina propria of the small intestine. Through the synergistic action of IL-15 and RA, dendritic cells acquire the ability to release inflammatory cytokines, particularly IL-12 and IL-23. These inflammatory mediators then act in concert with RA to prevent the induction of Foxp3+ Treg cells and drive TH1 and potentially TH17 polarization. In turn, inflammatory T cells may provide help to B cells to produce specific IgG and IgA antibodies.

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Author information

  1. These authors contributed equally to this work.

    • R. W. DePaolo &
    • V. Abadie

Affiliations

  1. Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA

    • R. W. DePaolo,
    • V. Abadie,
    • F. Tang,
    • H. Fehlner-Peach,
    • W. Wang,
    • C. Semrad,
    • S. S. Kupfer &
    • B. Jabri
  2. Mucosal Immunology Unit, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

    • J. A. Hall &
    • Y. Belkaid
  3. Immunology Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    • J. A. Hall
  4. Department of Dermatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA

    • E. V. Marietta
  5. Department of Immunology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA

    • E. V. Marietta
  6. US Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 800 Buchanan Street, Albany, California 94710, USA

    • D. D. Kasarda
  7. Metabolism Branch, National Cancer Institute, Bethesda, Maryland 20892-1374, USA

    • T. A. Waldmann
  8. Department of Medicine, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA

    • J. A. Murray
  9. Department of Pediatrics, University of Chicago, Chicago, Illinois 60637, USA

    • S. Guandalini &
    • B. Jabri
  10. Department of Pathology, University of Chicago, Chicago, Illinois 60637, USA

    • B. Jabri

Contributions

R.W.D. and V.A. provided input into the conceptual development and execution of the studies, as well as preparation of the manuscript. F.T., H.F.-P., J.A.H. and W.W. provided technical assistance and input into data analyses. J.A.M. and E.V.M. helped with the analysis of the humanized HLA-DQ8 transgenic mice. D.D.K. provided preparations of α-gliadin used in the feeding experiments, T.A.W. provided TMβ-1 antibody, and Y.B. helped with the realization of T-cell transfer experiments and provided us with RARα-deficient bone-marrow. C.S., S.K. and S.G. followed patients with coeliac disease and provided intestinal biopsies for cytokines analysis. Y.B., J.A.M., D.D.K. and T.A.W. participated in discussion and review of the manuscript. B.J. conceived the idea, wrote the manuscript and supervised all investigations.

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The authors declare no competing financial interests.

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