TLR signaling is required for pro-IL-1β synthesis in response to PDWGF.

<p>WT BMDC and MyD88−/−, TRIF−/−, and IL-1R−/− KO BMDC were treated with PDWGF alone or in combination with ATP and (<b>A</b>) IL-1β production was evaluated after 24 h. (<b>B</b>) Cell lysates were evaluated for <i>de novo</i> synthesis of pro-IL-1β (<b>C</b>) WT BMDC and TLR2−/−, TLR4−/−, and TLR2/4−/− KO BMDC were treated with PDWGF alone or in combination with ATP, and IL-1β production was evaluated after 24 h. (<b>D</b>) Cell lysates were evaluated for <i>de novo</i> synthesis of pro-IL-1β. Data in (A) and (C) are expressed as mean ± SD from 5 independent experiments. *P<0.05, **P<0.01 vs. WT BMDC. Blots in (B) and (D) are representative from 3 independent experiments. β-actin was used as a loading control. (<b>E</b>) Celiac PBMC were treated with PDWGF alone or in combination with anti-TLR4 or anti-TLR2 Ab. IL-1β secretion was evaluated after 24 h. LPS was used as a positive control. Mean ± SD, 8 independent experiments. **P<0.01, ***P<0.001 vs. cells without anti-TLR Ab.</p

Genotype frequencies for <i>NALP1</i> (rs12150220) and <i>NALP3</i> (rs10754558).

<p>OR- odds ratio, CI- confidence interval.</p

The role of caspase-1 in PDWGF-treated PBMC.

<p>(<b>A</b>) Caspase-1 inhibitor Z-YVAD-fmk reduced PDWGF-induced IL-1β, but not IL-1α or TNF-α production. Mean ± SD, n = 10 independent experiments, ***P<0.001 compared to PDWGF-treated cells. (<b>B</b>) Western blot analysis for the expression of pro-IL-1β and mature IL-1β and (<b>C</b>) for caspase-1 and caspase-1 p10 in cell lysates (CL) and cell culture supernatants (CS) from PBMC. Representative blots from 5 independent experiments are shown. β-actin was used as a loading control. (<b>D</b>) The fold increase (FI) (densitometry analysis) of the quantity of caspase-1 p10 normalized to non-activated cells. Mean ± SD, n = 5 independent experiments, *P<0.05 compared to non-activated cells. (<b>E, F</b>) Direct activation of caspase-1 in PDWGF-treated PBMC, assessed by flow cytometry using a cell-permeable fluorescent probe. Results are shown as (<b>E</b>) a representative histogram from 2 CD patients and 1 HD; and (<b>F</b>) as the percentage of the MFI from 12 CD patients and 10 HD. The data were normalized to the result from untreated cells, which was set as 100%. Mean ± SD, *P<0.05 compared to untreated cells. CD, celiac disease patients; HD, healthy donors.</p

PDWGF stimulates BMDC to IL-1β production through NLRP3 and ASC.

<p>(<b>A</b>) BMDC from WT, NLRP3−/− and ASC−/− mice were exposed to PDWGF (100 µg/ml) alone for 24 h; or first PDWGF was added for 21.5 h, the subsequently ATP (2 mM) was added for additional 2.5 h. IL-1β was measured in culture supernatants. (<b>B</b>) Flow-cytometric evaluation of PDWGF-induced maturation assessed by CD40, CD80, and CD86 expression on BMDC from WT and NLRP3−/− mice. WT and NLRP3−/− BMDC were cultured with 100 µg/ml of PDWGF (green), as well as 0.1 µg/ml of LPS (red) or 100 µg/ml of OVA (grey-filled) as positive and negative controls, respectively. Isotype controls are represented in black overlays. (<b>C</b>) Cells were preincubated with caspase-1 inhibitor Z-YVAD-fmk for 30 min, and then exposed to PDWGF in combination with ATP. Production of IL-1β was measured in culture supernatants. Results are expressed as mean ± SD from 4 independent experiments. The levels of significance for KO BMDC vs. WT BMDC are indicated as follows: *P<0.05, **P<0.01, and ***P<0.001.</p

PDWGF-induced IL-1β production from celiac patient PBMC is modulated by K+ efflux, but is independent of the P2X7 receptor; as shown by (A) ELISA, mean ± SD, n = 10, ***P<0.001 vs. PDWGF-treated cells; and by (B) Western blot.

<p>Representative blots from 5 independent experiments are shown. (<b>C</b>) Inhibition of ROS modulate PDWGF-induced IL-1β secretion, mean ± SD, n = 10; as well as (D) pro-IL-1β production from PBMC of CD patients. Representative blots from 3 independent experiments are shown. β-actin was used as a loading control. ***P<0.001 vs. PDWGF-treated cells.</p

Reduction and alkylation (R/A) of PDWGF led to abrogated IL-1β production.

<p>PDWGF as well as α-amylase inhibitor (non-treated or R/A treated) were added to celiac PBMC. IL-1β secretion was evaluated after 24 h. Results are shown as the percentage of the cytokine production from 5 CD patients. The data were normalized to the result from PDWGF-treated cells which was set as 100%. Mean ± SD, 5 independent experiments. ***P<0.001 vs. non-treated PDWGF.</p

PDWGF digest induces IL-1β, IL-18, and IL-1α release in monocytes and PBMC from CD patients.

<p>IL-1β (A), IL-18 (B) and IL-1α (C) levels were quantified in cell supernatants by ELISA. Data are given as mean ± SD from 39 patients and 15 healthy donors (HD). *P<0.05, **P<0.01 (CD vs. HD). (<b>D</b>). PDWGF-induced activation of PBMC is not due to LPS contamination. Results are shown as the percentage of the cytokine production from 4 CD patients. The data were normalized to the result from untreated cells which was set as 100%. Mean ± SD, n = 4 independent experiments, ***P<0.001 compared to untreated cells.</p

Quantification of immune cells in the colon of DSS-treated mice.

<p>Numbers of T-cells, regulatory T-cells, and neutrophils in the colon of DSS-treated wt mice and mice lacking TLR2 and/ or TLR4. The average number of cells positive for CD3, FOXP3, or myeloperoxidase from at least five high power fields (HPF, 400×magnification) per animal were determined microscopically in immunostained colon sections of wt mice (WT), and animals lacking TLR2 (TLR2^-/-), TLR4^-/- (TLR4^-/-), or both (TLR2/4^-/-) at day eight after DSS-treatment for seven days (C, grey circles). Healthy animals served as controls (H, white circles). The numbers of analyzed animals are given in parenthesis. Mean values (black bars), and significance levels (as compared to wt animals with colitis) determined by Student's t-test are indicated (^*, <i>P</i><0.05;^**, <i>P</i><0.01; ^***, <i>P</i><0.001).</p

Colitis severity and inflammatory responses in TLR2^-/-, TLR4^-/-, and TLR2/4^-/- mice, as compared to wt controls.

<div><p>(A) Severity of colitis in DSS-treated animals. Clinical outcome of colitis was recorded in wt mice (WT, n = 10) and in mice lacking TLR2 (TLR2^-/-, n = 10), TLR4 (TLR4^-/-, n = 12), or both (TLR2/4^-/-, n = 11). Clinical parameters of colitis were monitored daily (see methods). The data sets from two independent experiments were pooled. Total colitis scores were analyzed at day eight after DSS-treatment for seven days.</p><p>(<b>B</b>) IFN-gamma concentrations in supernatants of colonic MLN cultures from wt and TLR-deficient animals. Supernatants from MLN cultures of wt mice (WT, black bars, n = 3), and animals lacking TLR2 (TLR2^-/-, n = 4), TLR4 (TLR4^-/-, n = 5), or both (TLR2/4^-/-, n = 4) with colitis were analyzed at day eight after DSS-treatment for seven days.</p><p>(<b>A/B</b>) Mean values, standard deviations and significance levels (as compared to the wt animals) determined by Student's t-test are indicated (^***, <i>P</i><0.001).</p></div

Molecular gut flora analysis in mice lacking TLR2 and/or TLR4.

<div><p>Detailed molecular investigations of the colon microbiota in three months old C57BL/10 mice lacking TLR2, TLR4, or both were performed by analysis of 16S rRNA gene libraries (A) or high-resolution DGGE (B) with primers HDA-1-GC and HDA-2 (see methods).</p><p>(<b>A</b>) Results from gene library analysis. Complete 16S rRNA genes were amplified from total luminal colon content DNA isolated from wt mice (WT, n = 3), and from mice (n = 3 each group) lacking TLR2 (TLR2^-/-), TLR4 (TLR4^-/-), or both (TLR2/4^-/-). The amplicons were cloned and sequenced as described in the Methods section. Bars represent percentage amounts of gut bacterial groups indicated on the Y-axis.</p><p>(<b>B</b>) Results from DGGE analysis of PCR-amplified total bacterial 16S rRNA gene fragments. Each lane shows DGGE profiles of the bacterial flora from the colon of healthy wt mice, and animals lacking TLR2 (TLR2^-/-), TLR4 (TLR4^-/-), or both (TLR2/4^-/-). Mice of identical age (three months) originated from two different animal facilities (as indicated above the lanes). DNA bands, which were absent or weak in DGGE profiles from TLR4^-/- and TLR2/4^-/- animals are marked by grey arrows. Bacterial species identified by sequence analysis of 16S rRNA gene fragments in the corresponding bands are indicated on the left. Total numbers of DGGE bands in the profile from wt animals were counted and set to 100% in the reference pattern (RP). Presence and absence of bands in profiles from the respective TLR-deficient animals was recorded and similarity values were calculated and expressed as % similarity to the wt RP. The DGGE profiles are representative for at least three mice per group and experiment. Results were reproduced in two independent experiments.</p></div