Gliadin Stimulation of Murine Macrophage Inflammatory Gene Expression and Intestinal Permeability Are MyD88-Dependent: Role of the Innate Immune Response in Celiac Disease

Karen E. Thomas, Anna Sapone, Alessio Fasano and Stefanie N. Vogel
J Immunol February 15, 2006, 176 (4) 2512-2521; DOI: https://doi.org/10.4049/jimmunol.176.4.2512

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    PT-gliadin and its peptides, 33-mer and p31-43, induce intestinal permeability. Intestines from individual mice were treated with medium only, PT-gliadin (1000 μg/ml), or its peptide subunits p31-43 (100 μg/ml) and 33-mer (3.3 μg/ml) in the microsnapwell assay as described in Materials and Methods. Resistance (TEER) was measured over the indicated times. As an additional control, LPS (100 ng/ml) was tested and induced no significant TEER changes. Results represent the mean ± SEM from four mice per treatment.

  • FIGURE 2.
    FIGURE 2.

    PT-gliadin and its peptide derivatives, p31-43 and 33-mer, induce proinflammatory gene expression in murine macrophages. A, Primary macrophage cultures from C57BL/6 mice were treated for 3 h with medium only, LPS (100 ng/ml), PT-gliadin (1000 μg/ml), p31-43 (100 μg/ml), or 33-mer (3.3 μg/ml). Total RNA was extracted and analyzed by RT-PCR with Southern blotting as described in Materials and Methods. B, The same RNA samples in A were also subjected to quantitative real-time PCR as described in Materials and Methods. Induction of all genes by LPS and PT-gliadin were found to be statistically greater than that of medium-treated macrophages. ∗, p < 0.05, as compared with LPS treatment. These data are representative of one of three independent experiments.

  • FIGURE 3.
    FIGURE 3.

    Gliadin, but not LPS, induces zonulin release in murine macrophages. C57BL/6 macrophage cultures were treated for 24 h with medium only, PT-gliadin (10–1000 μg/ml), or LPS (100 ng/ml) as described in the footnotes of Table I. Results represent the mean ± SEM of a single representative experiment. ∗, p < 0.01.

  • FIGURE 4.
    FIGURE 4.

    Gliadin-induced zonulin release in macrophages is MyD88-dependent. Primary macrophages from C57BL/6 (WT) and MyD88−/− (KO, knockout) mice were cultured for 24 h in the presence of medium only, LPS (50 ng/ml), or PT-gliadin (1000 μg/ml). Culture supernatants were collected and assayed for zonulin by ELISA. Results represent mean ± SEM from two separate experiments. ∗, p < 0.01.

  • FIGURE 5.
    FIGURE 5.

    Gliadin-induction of intestinal permeability and zonulin release is MyD88 dependent. Intestinal permeability was measured in the microsnapwell assay in medium- or PT-gliadin (1000 μg/ml)-stimulated intestinal fragments from WT (MyD88+/+) (A) or knockout (MyD88−/−) (B) mice. Culture supernatants were collected from the mucosal side of these same microsnapwell intestinal cultures form WT (MyD88+/+) (C) and MyD88−/− (D) mice and assayed for zonulin release by ELISA. Also shown is the effect of the ZOT ΔG peptide (10 μg/ml) on the intestinal permeability of WT (MyD88+/+) (E) and MyD88−/− (F) mice. The results represent the mean ± SEM of five mice for A–D and three mice for E and F. ∗, p < 0.01.

  • FIGURE 6.
    FIGURE 6.

    PT-gliadin fails to activate signaling through TLR2 or TLR4. CHO cells that express a functional TLR4 (3E10) or both TLR4 and TLR2 (3E10-huTLR2) were stimulated with medium only, LPS (a TLR4 agonist), Pam3Cys (a TLR2 agonist), or PT-gliadin. Signal transduction was measured by an increase in the expression of CD25 detected by FACS analysis as described in Materials and Methods. These data correspond to those tabulated in Table III.

  • FIGURE 7.
    FIGURE 7.

    Gliadin-induction of intestinal permeability (A) and zonulin release (B) are TLR4-independent. Intestinal permeability (A) and zonulin release at 60 min were measured in medium- or PT-gliadin (1000 μg/ml)-stimulated microsnapwell cultures derived from TLR4-defective C3H/HeJ mice (B) and TLR4-responsive C3H/OuJ mice (C) as described in the Fig. 5 legend. The results represent the mean ± SEM from five mice per strain. ∗, p < 0.01.

Tables

  • Table I.

    Gliadin-induced cytokine production in primary murine macrophagesa

    MediumGliadin (0.01 mg/ml)Gliadin (0.1 mg/ml)Gliadin (1.0 mg/ml)LPS (100 ng/ml)
    TNF-αb (X ± SD)≤46.9c≤46.9721.0 ± 2107,845.0 ± 1,94422,755.0 ± 800*
    IL-12 p40d (X ± SD)≤23.4≤23.4122.5 ± 6295.0 ± 3*2,700.0 ± 70*

    • a C57BL/6 macrophages were cultured with medium, gliadin, or LPS for 24 h. Supernatants were assayed by ELISA for the presence of TNF-α and IL-12 p40 as described in Materials and Methods. *, p ≤ 0.01.

    • b The lowest detectable level for the TNF-α ELISA was 46.9 pg/ml.

    • c All samples at the lower limits of detection were assayed at 1/2 dilutions.

    • d The lowest detectable level for the IL-12 p40 ELISA was 23.4 pg/ml.

  • Table II.

    Gliadin-induced TNF-α and IL-12 p40 production in primary murine macrophages is MyD88 dependenta

    WTMyD88−/−
    MediumGliadin (1.0 mg/ml)LPS (50 ng/ml)MediumGliadin (1.0 mg/ml)LPS (50 ng/ml)
    TNF-αb (X ± SD)≤15.6c2,439.2 ± 4276,168.3 ± 460≤15.6263.1 ± 14*345.5 ± 4*
    IL-12 p40d (X ± SD)≤7.848.2 ± 5132.2 ± 26≤7.8≤7.8≤7.8

    • a C57BL/6 (WT) or MyD88−/− macrophages were cultured with medium, gliadin, or LPS for 24 h. Supernatants were assayed by ELISA for the presence of TNF-α and IL-12 p40 as described in Materials and Methods. *, p ≤ 0.01.

    • b The lowest detectable amount for the TNF-α ELISA was 15.6 pg/ml.

    • c All samples at the lower limits of detection were assayed in undiluted supernatants.

    • d The lowest detectable amount for the IL-12 p40 ELISA was 7.8 pg/ml.

  • Table III.

    Gliadin fails to activate CHO cell reporter lines that express functional TLR4 with or without TLR2a

    3E10 (endogenous TLR4 only)3E10-huTLR2 (endogenous TLR4 + huTLR2)
    MediumLPSPam3CysGliadinMediumLPSPam3CysGliadin
    Isotype control0.2b0.20.10.21.21.11.61.1
    Anti-CD254.557.72.11.67.348.371.15.3

    • a Cells were stimulated for 18 h prior to FACS analysis. These data correspond to those presented in Fig. 6⇑.

    • b Data shown as percentage of positively stained cells.

  • Table IV.

    TLR agonist- and IL-1β-induced TNF-α and zonulin production in primary murine macrophagesa

    MediumGliadin (1.0 mg/mlx)LPS (100 ng/ml)Pam3Cys (150 μg/ml)Poly(I:C) (100 μg/ml)IL-1β (25 ng/ml)
    TNF-αb (X ± SD)≤23.4c9,051 ± 91*14,466 ± 3000*1,658 ± 465*2,085 ± 1417*≤23.4
    Zonulin (X ± SD)0.2 ± 0.013.7 ± 0.8*0.18 ± 0.05.1 ± 1.7*0.18 ± 0.12.8 ± 0.1*

    • a C57BL/6 macrophages were cultured with medium, gliadin, LPS, Pam3Cys, poly(I:C), or IL-1β for 24 h. Supernatants were assayed by ELISA for the presence of TNF-α (pg/ml) and zonulin (ng/mg) as described in Materials and Methods. Data are presented as mean ± SD of duplicate cultures from a representative experiment (n = 2). *, p < 0.05.

    • b The lowest detectable level for the TNF-α ELISA was 23.4 pg/ml.

    • c All samples at the lower limits of detection were assayed in undiluted samples.

Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section