L1-RTP levels in AOM+DSS colitis mouse model.

<p>(<b>A</b>) Schematic time schedule of the AOM+DSS colitis mouse model. Arrows indicate the time-points associated with the acute inflammatory and tumor phases for harvesting the colon tissues and/or tumors. (<b>B</b>) L1-RTP levels were analyzed with genomic DNA extracted from whole distal colon tissues and/or tumors. The EGFP copy/genome was calculated by using ß-actin as internal control. Samples labeled “Acute AOM+DSS” were obtained during the acute phase in panel A, and others were collected during the tumor phase. AOM+DSS indicates residual colon tissue after removing visible tumors. Data were shown as mean ± SD. P values were calculated with an unpaired two-tailed t test.</p

Correlation between DNA methylation levels of the human L1 promoter and L1-RTP levels.

<p>DNA methylation levels of the transgenic human L1 promoter were analyzed by bisulfite pyrosequencing with the primers targeting only the human L1 promoter sequence. Correlation between the levels of human L1 promoter DNA methylation and L1-RTP levels were analyzed using whole distal colon tissues with AOM+ acute DSS colitis (no label), isolated colonic ECs (EC) or LP (LP) fractions from AOM+DSS colitis, aberrant crypt foci (ACF) induced by AOM, whole colon of a naïve mouse, or the 2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP)-treated mice (a positive control of human L1-RTP reporter [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116072#pone.0116072.ref008" target="_blank">8</a>]). The P value was determined by a two-tailed Pearson’s calculation.</p

Cell type-specific L1-RTP levels in acute colitis mice.

<p>(A) Representative photograph of a section after microdissection for epithelial cells (EC, top), lamina propria (LP, bottom) and the remaining tissues (including submucosa and muscle layer) of colon section by laser microdissection. (B) L1-RTP levels were analyzed in colonic ECs, the LP, and the submucosa plus muscle layer (SM) isolated by microdissection from mice in the acute colitis phase. The relative L1-RTP value was normalized by using ß-actin as an internal control. Each plot indicates an individual mouse. P values were calculated with an unpaired one-tailed t test. (C) L1-RTP levels were examined in isolated ECs or sorted EpCAM negative-gated non-EC fractions by flow cytometry from mice in the acute colitis phase, as follows: T cells (EpCAM^-CD3⁺), B cells (EpCAM^-B220⁺), macrophage/dendritic cells (MΦ/DCs, EpCAM^-CD11b⁺/CD11c⁺/F4/80⁺), Neutrophils (EpCAM^-Gr^-1⁺) and other cell types, most likely mesenchymal cells (MES, EpCAM^-CD3^-B220^-CD11b^-CD11c^-F4/80^-Gr-1^-). Levels of L1-RTP from pooled cell fractions obtained from four mice are shown.</p

Chemokine Receptor CCR8 Is Required for Lipopolysaccharide-Triggered Cytokine Production in Mouse Peritoneal Macrophages

<div><p>Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (<i>CCR8^-</i>^/-) and wild-type (WT) mice. We found that <i>CCR8^-/-</i> PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca²⁺ flux and CCL1-driven PMφ aggregation. Similar to <i>CCR8^-/-</i> PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. <i>CCR8^-/-</i> PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in <i>CCR8^-/-</i> mice, administration of R243 attenuated peritoneal adhesions <i>in vivo</i>. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of <i>CCR8^-/-</i> mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.</p></div

Scheme of TLR4 and CCR8 signaling cross talk in mouse PMφ.

<p>LPS triggers TLR4 signaling and induces NF-κB and MAPK activation, possibly together with CCR8 activation, which results in the production of TNF-α, IL-6, and IL-10. CCR8 is required for full activation of both signaling pathways. Production of IL-6 and TNF-α involves both the NF-κB and MAPK pathways. IL-10 production is largely dependent on the JNK pathway and is mediated by CCR8 activation. In addition to the antagonistic effect on CCR8, possible sites of action of R243 are indicated.</p

LPS-induced cytokine production by mouse PMφ is dependent on CCR8.

<p>(A) PMφ or (B) BMMφ collected from naïve WT or <i>CCR8^-/-</i> mice were stimulated with LPS (100 ng/mL) Pam3CSK4 (Pam, 100 ng/mL), zymosan (Zym, 1 μg/mL), PolyI:C (Pol, 100 μg/mL), flagellin (Fla, 1 μg/mL), or CpG-ODN (CpG, 1 μg/mL) for 24 h. The levels of cytokines in the culture medium were measured. (C) PMφ or BMMφ prepared from WT or CCR8^-/- mice was stimulated with LPS as above for 3 or 8 hours, and mRNA levels for IL-10 was measured with quantitative RT-PCR. Values were normalized using 18 s rRNA and shown as relative expression levels to WT cells without LPS of each time point. In (A–C), data are shown as mean plus a standard deviation (SD) determined from triplicate assays for each condition. *Statistically significant difference between WT and <i>CCR8^-/-</i> cells (unpaired two-tailed t test). (D) PMφ or BMMφ gated as F4/80⁺ cells were obtained from naïve WT mice and stained with anti-CCR8 antibody (solid line) or without antibody (shaded histogram) analyzed with flow cytometory.</p

<i>CCR8</i> deficiency and R243 attenuated postoperative peritoneal adhesions and acute colitis <i>in vivo</i>.

<p>WT, <i>CCR8^-/-</i>, or WT mice administered with R243 were compared. (A) Mouse peritoneal adhesions were induced through formation of peritoneal ischemic buttons (Exp 1, single R243 dose on day 0), by ablation of the cecum with an electric scalpel (Exp 2, three R243 doses on days 0, 2, and 4), and laparotomy and abrasion of the cecum with gauze (Exp 3, single R243 dose on day 0). Each dot represents an individual mouse. The short bar indicates mean value. *Statistically significant difference compared with the control (WT) according to an unpaired two-tailed t test. (B) Colitis was induced by intrarectal administration of TNBS. R243 was injected intraperitoneally on days 0 and 2. Other mice were given vehicle solution. Each dot represents an individual mouse. The short bar indicates a mean value. *Statistically significant difference from WT control (Mann-Whitney test). (C) Typical histological findings of colitis. Frozen sections were stained with hematoxylin and eosin. Bar = 200 μm. Arrows indicate transmural lesion with peritoneal adhesions.</p

CCR8 ligand secretion was not required for LPS-induced IL-10 secretion.

<p>WT PMφ were stimulated with LPS (100 ng/mL) in the presence of the indicated concentrations of anti-CCL1, anti-CCL8, or control IgG (rat and rabbit, respectively, all F(ab) fragments) for 24 h. IL-10 in the culture supernatant was then measured. Data are shown as the percent of the level in the culture medium of cells stimulated only with LPS. Results are expressed as the mean plus standard deviation (SD) of two or three assays. *Statistically significant difference from LPS only cultures (Mann-Whitney test).</p

Target of CCR8 signaling and R243 in LPS-induced cytokine production by PMφ.

<p>(A) WT PMφ were stimulated with LPS (100 ng/mL) for 24 h in the absence (none) or presence (low, medium, and high indicate 0.5, 5, and 50 μM, respectively) of SN50, U0120 (0.1, 1, 10 μM), SB203580 (0.1, 1, 10 μM), or SP600125 (0.5, 5, 50 μM). The levels of cytokines in the culture medium were measured and are shown as percent relative to the LPS-stimulated cells. *Statistically significant difference between cells treated with LPS plus inhibitors and cells treated with LPS only (P<0.05, Mann-Whitney test). (B) WT (control), <i>CCR8^-/-</i> PMφ or WT PMφ treated with R243 were stimulated with LPS (100 ng/mL) for the indicated times. Some cells were stimulated with 100 ng/ml of CCL1 without LPS. Each phosphoprotein was quantified using a multiplex phosphoprotein detection assay. The intensity relative to unstimulated WT PMφ (0 min) results are expressed as the mean with standard deviation (SD) of three assays. *Statistically significant difference compared with control (two-way ANOVA with Bonferroni post-tests).</p

Localization of TLR4 and LPS in WT, <i>CCR8^-/-</i>, and R243-treated PMφ.

<p>(A) WT PMφ, <i>CCR8^-/-</i> PMφ or WT PMφ in the presence of R243 (5 μM) were stimulated with or without FITC-conjugated LPS (1 mg/mL) for 45 min and stained with biotin-conjugated anti-TLR4/MD2 and TRITC (red)-streptavidin. (B) WT PMφ were stimulated with FITC (green)-conjugated LPS, as in panel A, with or without R243 (5 μM) and stained with anti-CCR8 antibody and anti-sheep IgG-conjugated with Texas Red (red). Fluorescent images were merged and overlaid on the differential interference contrasted image. Bar = 20 μm.</p