ELISA measurement of IL-15Rα (A) and IL-15 (B) proteins and IL-15–IL-15Rα complex (D) expression in lysates of poly I:C–stimulated BMDCs at the indicated time points

(C) ELISA determination of IL-15 protein levels after dissociation of IL-15–IL-15Rα protein complexes with 0.01% SDS and boiling. Lines represent data from WT (▪), (▴), and (▾) BMDCs. All data are representative of at least three separate experiments.<p><b>Copyright information:</b></p><p>Taken from "IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1213-1225.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373851.</p><p></p

ELISA determination of sIL-15Rα, IL-15, and IL-15–sIL-15Rα proteins in sera from intact (A–C, on left) or chimeric (D–F, on right) mice stimulated with 25 μg/g poly I:C or PBS

Sera were collected 24 h after stimulation and IL-15 (B and E), sIL-15Rα (A and D), and IL-15–sIL-15Rα complex (C and F) protein levels were measured by ELISA. Each symbol reflects values obtained from individual mice.<p><b>Copyright information:</b></p><p>Taken from "IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1213-1225.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373851.</p><p></p

(A) ELISA measurement of IL-12 secretion by poly I:C–stimulated BMDCs of the indicated genotypes

(B) Flow cytometric analysis of DC activation after poly I:C stimulation. Histograms of poly I:C–stimulated DCs (gray-filled histograms) of the indicated genotypes indicating surface expression levels of the surface activation markers CD40 and CD86. (C) ELISA determination of IFN-γ secretion by NK cells co-cultured with WT, 15KO, RαKO, or mixed 15KO:RαKO BMDCs. BMDCs were treated with PBS or poly I:C for 18 h, after which NK cells were co-cultured with activated DCs for an additional 6 h. Poly I:C–stimulated cultures are indicated by black bars, and PBS-stimulated control cultures are indicated by white bars. Note that significant poly I:C–induced NK cell activation occurs only in the presence of WT DCs. (D and E) Flow cytometric measurement of NK cell (NK1.1) activation by poly I:C (p(I:C)) –stimulated DC–NK cell co-cultures. Elevated CD69 surface staining reflects initial (TLR-dependent, IL-15–independent) activation of NK cells. IFN-γ and granzyme B expression by NK cells were performed by intracellular staining 6 and 12 h after stimulation, respectively. Numbers indicate the percentage of cells in the indicated gate. Note that NK cells express significant levels of both IFN-γ and granzyme B only when activated by WT DCs. Plots are representative of three separate experiments.<p><b>Copyright information:</b></p><p>Taken from "IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1213-1225.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373851.</p><p></p

Activation of IFN-γ secretion by NK cells co-cultured with various combinations of poly I:C–stimulated BMDCs and supernatants from these BMDCs

BMDCs from various genotypes (indicated along the x axis of graph) were activated with poly I:C and then supplemented with supernatants exchanged from similarly activated BMDCs. Genotypes of DCs from which supernatants were derived are indicated by individual columns (gray, WT; white, 15KO; black, RαKO; checkered, mixture of 15KO and RαKO). ELISA quantitation of NK cell IFN-γ secretion is indicated on the y axis. Note that WT DCs activate NK cells regardless of the type of supernatant added. Note also that WT DC–derived supernatants containing IL-15–sIL-15Rα complexes fail to support or augment NK cell activation.<p><b>Copyright information:</b></p><p>Taken from "IL-15Rα chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1213-1225.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373851.</p><p></p

A20 expression in IECs restricts colon tumorigenesis.

<p>(A) immunoblot analysis of isolated IECs indicating efficient deletion of A20 from the small bowel (SB) and colon (C) of villin-Cre A20^FL/FL APC^min/+ mice (fl/fl) compared to control villin-Cre A20^+/+ APC^min/+ mice (+/+) mice. GAPDH is shown as a loading control. (B) Tumor number (left panel) and aggregate tumor size (right panel) in colons of A20 (fl/fl) and wild-type (+/+) mice harboring APC^min mutation. (C) Tumor numbers in small intestines of A20 (fl/fl) and wild-type (+/+) mice harboring APC^min mutation. (D) Colon and small intestine lengths from (fl/fl) and wild-type (+/+) mice harboring APC^min mutation. Each point represents one mouse. Lines indicate mean values. (f) Hematoxylin and eosin staining (upper panels) and Ki-67 and cleaved caspase-3 immunostaining (lower panels) of colonic sections from villin-Cre A20^FL/FL APC^min/+ mice (fl/fl) and control villin-Cre A20^+/+ APC^min/+ mice. 40X magnification shown.</p

A20 supports β-catenin ubiquitination and degradation through an interaction with the destruction box.

<p>(A) Luciferase assay showing transcriptional activity of a β-catenin dependent TCF/LEF4 reporter in RKO cells. Cells were treated with A20 specific or control siRNA and recombinant human wnt3a (rhwnt3a) as indicated. Relative luciferase units (RLU) are shown. **indicates p<0.01. (B) Co-precipitation of A20 with Axin. RKO cells transfected with the indicated expression plasmids were lysed, immunoprecipiated (IP) for the indicated epitope tag, and immunoblotted (IB) for the indicated proteins. Cells were stimulated with rhwnt3a or control as indicated for four hours. Input levels of MYC, FLAG, and GAPDH are shown as controls below. (C) Co-precipitation of partial A20 proteins with Axin. Co-transfection experiments as in (B). Input levels of MYC, FLAG, and GAPDH shown below. (D) A20 suppresses wnt3a stimulated induction of β-catenin expression. Immunoblot analyses of active and total β-catenin expression in RKO cells treated with A20 specific or control siRNA. A20 and GAPDH levels shown below as loading control. (E) A20 supports wnt3a stimulated β-catenin ubiquitination. RKO cells were treated with A20 specific or control siRNAs and wnt3a for the indicated times. Lysates were immunoprecipitated for β-catenin followed by immunoblotting for ubiquitin. Input amounts of beta-catenin, A20, and GAPDH proteins shown below as controls. All data are representative of three or more independent experiments.</p

A20 Restricts Wnt Signaling in Intestinal Epithelial Cells and Suppresses Colon Carcinogenesis

<div><p>Colon carcinogenesis consists of a multistep process during which a series of genetic and epigenetic adaptations occur that lead to malignant transformation. Here, we have studied the role of A20 (also known as TNFAIP3), a ubiquitin-editing enzyme that restricts NFκB and cell death signaling, in intestinal homeostasis and tumorigenesis. We have found that A20 expression is consistently reduced in human colonic adenomas than in normal colonic tissues. To further investigate A20’s potential roles in regulating colon carcinogenesis, we have generated mice lacking A20 specifically in intestinal epithelial cells and interbred these with mice harboring a mutation in the adenomatous polyposis coli gene (APC^min). While A20^FL/FL villin-Cre mice exhibit uninflamed intestines without polyps, A20^FL/FL villin-Cre APC^min/+ mice contain far greater numbers and larger colonic polyps than control APC^min mice. We find that A20 binds to the β-catenin destruction complex and restricts canonical wnt signaling by supporting ubiquitination and degradation of β-catenin in intestinal epithelial cells. Moreover, acute deletion of A20 from intestinal epithelial cells in vivo leads to enhanced expression of the β-catenin dependent genes cyclinD1 and c-myc, known promoters of colon cancer. Taken together, these findings demonstrate new roles for A20 in restricting β-catenin signaling and preventing colon tumorigenesis.</p> </div

Acute deletion of A20 from IECs leads to increased levels of Cyclin D1 and MYC mRNA in vivo.

<p>Villin-ER/Cre A20^FL/FL (fl/fl) and control Villin-ER/Cre A20^+/+ (+/+) were injected with 1 mg of tamoxifen daily for 5 days. IECs were then isolated and studied for expression of A20 (upper panel), Cyclin D1 (middle panel), and MYC (lower panel) mRNAs by qPCR. Each point represents one mouse. *indicates p<0.05; **indicates p<0.01.</p

Human colonic adenomas express less A20 than normal colonic mucosa.

<p>Expression of A20 (top panel), cyclin D1 (middle panel) and c-myc (bottom panel) mRNAs in normal colonic mucosa and colonic adenomas, as quantitated by the Genome Expression Omnibus (GDS2947). Relative expression levels are shown. **indicates p<0.01.</p