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

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

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