Histological analysis of colon sections from wild-type and DCIR^−/− mice.

<p>Paraffin sections of the colon from untreated or 3% DSS-treated wild-type and DCIR^−/− mice were prepared at day seven and were stained with hematoxylin and eosin (H&E) for histological evaluation in a blinded manner. (<b>A</b>) Representative images of paraffin-embedded sections of the rectal part of the colon are shown (40x magnification). Arrows indicate a severe ulcer in the colon from DCIR^−/− mice. Each colon was divided into three segments of identical length (oral, middle, rectal) which were separately analyzed. The degree of leukocyte infiltration (<b>B</b>) and mucosal erosion/ulceration (<b>C</b>) was graded from none (score 0) to mild (score 1), moderate (score 3), or severe (score 4). The scores for both, cell infiltration as well as mucosal ulceration in the rectal part of the colon from DCIR^−/− mice were significantly increased compared to wild-type mice. Data are expressed as mean + SEM (n = 5). The <i>p</i>-values were determined using Mann-Whitney’s U test (*<i>p</i><0.05, **<i>p</i><0.01). Significance is indicated by asterisks (*), ns = no significance.</p

MCL and DCIR recognize commensal intestinal microbiota and modulate APC and T cell cytokine production.

<p>Binding of MCL- and DCIR-hFc fusion proteins to stained gut microbes was analyzed by flow cytometry. (<b>A</b>) Representative dot plots of one binding experiment with MCL- and DCIR-hFc, with hFc as negative control, and with MGL1-hFc as positive control. Gating and frequencies indicate binding events of CLR-hFc fusion proteins to commensal microbiota. For analysis, it was first gated on Syto 61 positive events ( = stained microbiota) followed by gating on PE positive events ( = CLR-Fc fusion proteins). Data are representative of three independent experiments (triplicates each). (<b>B</b>) MCL^−/− and wild-type BMMs or (<b>C</b>) DCIR^−/− and wild-type BMDCs were stimulated with various concentrations of heat-killed gut microbiota, LPS or coated zymosan for 18 h (triplicates each). TNF-α levels in the culture supernatants were determined by ELISA. TNF-α production was significantly increased for MCL^−/− BMMs and DCIR^−/− BMDCs compared to wild-type APCs. Data are representative of three independent experiments. For analysis of T cell activation, purified OT-II transgenic T cells were co-cultivated with BMMs or BMDCs in the presence of heat-killed gut microbiota and 30 µg/mL OVA for 72 h. (<b>D</b>) IL-2 and (<b>E</b>) IFN-γ levels were determined in the culture supernatants of stimulated MCL^−/− and wild-type BMMs. Similarly, (<b>F</b>) IL-2 and (<b>G</b>) IFN-γ levels were analyzed in the culture supernatants of stimulated DCIR^−/− and wild-type BMDCs. Data are representative of three independent experiments (triplicates each) and are expressed as mean + SEM. The <i>p</i>-values were determined with unpaired Student’s t-test (*<i>p</i><0.05, **<i>p</i><0.01). Significance is indicated by asterisks (*), ns = no significance.</p

Scoring for assessment of the disease activity index.

<p>Scoring for assessment of the disease activity index.</p

Scoring for the histological evaluation of intestinal lesions.

<p>Scoring for the histological evaluation of intestinal lesions.</p

Local cytokine concentrations in the colon of wild-type, MCL^−/−, and DCIR^−/− mice.

<p>Colons from untreated wild-type mice or from wild-type and MCL^−/− mice (n = 6) (<b>A</b>), or wild-type and DCIR^−/− mice (n = 7 for wild-type and n = 8 for DCIR^−/− mice) (<b>B</b>) treated with 3% DSS for seven consecutive days were homogenized and used for cytokine determination by cytometric bead array. Data are expressed as mean + SEM. Significance is indicated by asterisks (*), ns = no significance.</p

Histological analysis of colon sections from wild-type and MCL^−/− mice.

<p>Paraffin sections of the colon from untreated or 3% DSS-treated wild-type and MCL^−/− mice were prepared at day seven and were stained with hematoxylin and eosin (H&E) for histological evaluation in a blinded manner. Each colon was divided into three segments of identical length (oral, middle, rectal) which were separately analyzed. (<b>A</b>) Representative images of paraffin-embedded sections of the rectal part of the colon are shown (40x magnification). The degree of leukocyte infiltration (<b>B</b>) and mucosal erosion/ulceration (<b>C</b>) was graded from none (score 0) to mild (score 1), moderate (score 3), or severe (score 4). Data are expressed as mean + SEM (n = 8 for wild-type and n = 5 for MCL^−/− mice). The <i>p</i>-values were determined using Mann-Whitney’s U test. Significance is indicated by asterisks (*), ns = no significance.</p

Clinical symptoms of DSS-induced colitis in MCL^−/− and DCIR^−/− mice.

<p>Colitis was induced in wild-type, MCL^−/− and DCIR^−/− mice by adding 3% DSS into the drinking water for seven consecutive days. (<b>A</b>) MCL and DCIR mRNA levels were analyzed by semi-quantitative RT-PCR in colon of untreated and DSS-treated wild-type mice (6 mice per group). As a control, RNA was also isolated from colon of untreated MCL^−/− or DCIR^−/− mice. β-actin expression levels were determined in all samples to ensure the same quantity and quality of cDNA. MCL mRNA expression in colon of DSS-treated mice was increased compared to the colon of untreated mice while DCIR mRNA levels did not differ between untreated and DSS-treated mice. Disease severity was determined by the daily body weight loss for MCL^−/− mice (n = 11) (<b>B</b>), and for DCIR^−/− mice (n = 18) (<b>E</b>), the colon length for MCL^−/− mice (<b>C</b>), and for DCIR^−/− mice (<b>F</b>), and a disease activity index (DAI) based on blood occurrence, stool consistency and body weight loss for MCL^−/− mice (n = 11 for wild-type and n = 10 for MCL^−/− mice) (<b>D</b>), and for DCIR^−/− mice (n = 13 for wild-type and n = 12 for DCIR^−/− mice) (<b>G</b>). Data shown are the combined results of two (MCL) or three (DCIR) independent experiments. On day six, one MCL^−/− mouse had to be euthanized due to the defined humane endpoints. MCL^−/− mice displayed an increased body weight loss compared to wild-type mice, with a significant difference on day 5 and 6. The colon length and DAI did not vary between MCL^−/− and wild-type mice. No significant difference in body weight, colon length, and DAI between DCIR^−/− and wild-type mice was observed. Data are expressed as mean + SEM. The <i>p</i>-values were determined with Mann-Whitney’s U test (*<i>p</i><0.05). Significance is indicated by asterisks (*), ns = no significance.</p