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 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

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

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

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

Image_2_Single cell analysis revealed that two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice.jpeg

Recent advances in research suggest that aging has a controllable chronic inflammatory disease aspect. Aging systemic T cells, which secrete pro-inflammatory factors, affect surrounding somatic cells, and accelerate the aging process through chronic inflammation, have attracted attention as potential therapeutic targets in aging. On the other hand, there are few reports on the aging of the intestinal immune system, which differs from the systemic immune system in many ways. In the current study, we investigated the age-related changes in the intestinal immune system, particularly in T cells. The most significant changes were observed in the CD4+ T cells in the small intestinal IEL, with a marked increase in this fraction in old mice and reduced expression of CD27 and CD28, which are characteristic of aging systemic T cells. The proliferative capacity of aging IEL CD4+ T cells was significantly more reduced than that of aging systemic T cells. Transcriptome analysis showed that the expression of inflammatory cytokines was not upregulated, whereas Cd8α, NK receptors, and Granzymes were upregulated in aging IEL CD4+ T cells. Functional analysis showed that aging IEL T cells had a higher cytotoxic function against intestinal tumor organoids in vitro than young IEL T cells. scRNAseq revealed that splenic T cells show a transition from naïve to memory T cells, whereas intestinal T cells show the emergence of a CD8αα+CD4+ T cell fraction in aged mice, which is rarely seen in young cells. Further analysis of the aging IEL CD4+ T cells showed that two unique subsets are increased that are distinct from the systemic CD4+ T cells. Subset 1 has a pro-inflammatory component, with expression of IFNγ and upregulation of NFkB signaling pathways. Subset 2 does not express IFNγ, but upregulates inhibitory molecules and nIEL markers. Expression of granzymes and Cd8a was common to both. These fractions were in opposite positions in the clustering by UMAP and had different TCR repertoires. They may be involved in the suppression of intestinal aging and longevity through anti-tumor immunity, elimination of senescent cells and stressed cells in the aging environment. This finding could be a breakthrough in aging research.</p

Image_4_Single cell analysis revealed that two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice.jpeg

Recent advances in research suggest that aging has a controllable chronic inflammatory disease aspect. Aging systemic T cells, which secrete pro-inflammatory factors, affect surrounding somatic cells, and accelerate the aging process through chronic inflammation, have attracted attention as potential therapeutic targets in aging. On the other hand, there are few reports on the aging of the intestinal immune system, which differs from the systemic immune system in many ways. In the current study, we investigated the age-related changes in the intestinal immune system, particularly in T cells. The most significant changes were observed in the CD4+ T cells in the small intestinal IEL, with a marked increase in this fraction in old mice and reduced expression of CD27 and CD28, which are characteristic of aging systemic T cells. The proliferative capacity of aging IEL CD4+ T cells was significantly more reduced than that of aging systemic T cells. Transcriptome analysis showed that the expression of inflammatory cytokines was not upregulated, whereas Cd8α, NK receptors, and Granzymes were upregulated in aging IEL CD4+ T cells. Functional analysis showed that aging IEL T cells had a higher cytotoxic function against intestinal tumor organoids in vitro than young IEL T cells. scRNAseq revealed that splenic T cells show a transition from naïve to memory T cells, whereas intestinal T cells show the emergence of a CD8αα+CD4+ T cell fraction in aged mice, which is rarely seen in young cells. Further analysis of the aging IEL CD4+ T cells showed that two unique subsets are increased that are distinct from the systemic CD4+ T cells. Subset 1 has a pro-inflammatory component, with expression of IFNγ and upregulation of NFkB signaling pathways. Subset 2 does not express IFNγ, but upregulates inhibitory molecules and nIEL markers. Expression of granzymes and Cd8a was common to both. These fractions were in opposite positions in the clustering by UMAP and had different TCR repertoires. They may be involved in the suppression of intestinal aging and longevity through anti-tumor immunity, elimination of senescent cells and stressed cells in the aging environment. This finding could be a breakthrough in aging research.</p

Image_7_Single cell analysis revealed that two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice.jpeg

Recent advances in research suggest that aging has a controllable chronic inflammatory disease aspect. Aging systemic T cells, which secrete pro-inflammatory factors, affect surrounding somatic cells, and accelerate the aging process through chronic inflammation, have attracted attention as potential therapeutic targets in aging. On the other hand, there are few reports on the aging of the intestinal immune system, which differs from the systemic immune system in many ways. In the current study, we investigated the age-related changes in the intestinal immune system, particularly in T cells. The most significant changes were observed in the CD4+ T cells in the small intestinal IEL, with a marked increase in this fraction in old mice and reduced expression of CD27 and CD28, which are characteristic of aging systemic T cells. The proliferative capacity of aging IEL CD4+ T cells was significantly more reduced than that of aging systemic T cells. Transcriptome analysis showed that the expression of inflammatory cytokines was not upregulated, whereas Cd8α, NK receptors, and Granzymes were upregulated in aging IEL CD4+ T cells. Functional analysis showed that aging IEL T cells had a higher cytotoxic function against intestinal tumor organoids in vitro than young IEL T cells. scRNAseq revealed that splenic T cells show a transition from naïve to memory T cells, whereas intestinal T cells show the emergence of a CD8αα+CD4+ T cell fraction in aged mice, which is rarely seen in young cells. Further analysis of the aging IEL CD4+ T cells showed that two unique subsets are increased that are distinct from the systemic CD4+ T cells. Subset 1 has a pro-inflammatory component, with expression of IFNγ and upregulation of NFkB signaling pathways. Subset 2 does not express IFNγ, but upregulates inhibitory molecules and nIEL markers. Expression of granzymes and Cd8a was common to both. These fractions were in opposite positions in the clustering by UMAP and had different TCR repertoires. They may be involved in the suppression of intestinal aging and longevity through anti-tumor immunity, elimination of senescent cells and stressed cells in the aging environment. This finding could be a breakthrough in aging research.</p

Image_1_Single cell analysis revealed that two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice.jpeg

Recent advances in research suggest that aging has a controllable chronic inflammatory disease aspect. Aging systemic T cells, which secrete pro-inflammatory factors, affect surrounding somatic cells, and accelerate the aging process through chronic inflammation, have attracted attention as potential therapeutic targets in aging. On the other hand, there are few reports on the aging of the intestinal immune system, which differs from the systemic immune system in many ways. In the current study, we investigated the age-related changes in the intestinal immune system, particularly in T cells. The most significant changes were observed in the CD4+ T cells in the small intestinal IEL, with a marked increase in this fraction in old mice and reduced expression of CD27 and CD28, which are characteristic of aging systemic T cells. The proliferative capacity of aging IEL CD4+ T cells was significantly more reduced than that of aging systemic T cells. Transcriptome analysis showed that the expression of inflammatory cytokines was not upregulated, whereas Cd8α, NK receptors, and Granzymes were upregulated in aging IEL CD4+ T cells. Functional analysis showed that aging IEL T cells had a higher cytotoxic function against intestinal tumor organoids in vitro than young IEL T cells. scRNAseq revealed that splenic T cells show a transition from naïve to memory T cells, whereas intestinal T cells show the emergence of a CD8αα+CD4+ T cell fraction in aged mice, which is rarely seen in young cells. Further analysis of the aging IEL CD4+ T cells showed that two unique subsets are increased that are distinct from the systemic CD4+ T cells. Subset 1 has a pro-inflammatory component, with expression of IFNγ and upregulation of NFkB signaling pathways. Subset 2 does not express IFNγ, but upregulates inhibitory molecules and nIEL markers. Expression of granzymes and Cd8a was common to both. These fractions were in opposite positions in the clustering by UMAP and had different TCR repertoires. They may be involved in the suppression of intestinal aging and longevity through anti-tumor immunity, elimination of senescent cells and stressed cells in the aging environment. This finding could be a breakthrough in aging research.</p

Image_3_Single cell analysis revealed that two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice.jpeg

Recent advances in research suggest that aging has a controllable chronic inflammatory disease aspect. Aging systemic T cells, which secrete pro-inflammatory factors, affect surrounding somatic cells, and accelerate the aging process through chronic inflammation, have attracted attention as potential therapeutic targets in aging. On the other hand, there are few reports on the aging of the intestinal immune system, which differs from the systemic immune system in many ways. In the current study, we investigated the age-related changes in the intestinal immune system, particularly in T cells. The most significant changes were observed in the CD4+ T cells in the small intestinal IEL, with a marked increase in this fraction in old mice and reduced expression of CD27 and CD28, which are characteristic of aging systemic T cells. The proliferative capacity of aging IEL CD4+ T cells was significantly more reduced than that of aging systemic T cells. Transcriptome analysis showed that the expression of inflammatory cytokines was not upregulated, whereas Cd8α, NK receptors, and Granzymes were upregulated in aging IEL CD4+ T cells. Functional analysis showed that aging IEL T cells had a higher cytotoxic function against intestinal tumor organoids in vitro than young IEL T cells. scRNAseq revealed that splenic T cells show a transition from naïve to memory T cells, whereas intestinal T cells show the emergence of a CD8αα+CD4+ T cell fraction in aged mice, which is rarely seen in young cells. Further analysis of the aging IEL CD4+ T cells showed that two unique subsets are increased that are distinct from the systemic CD4+ T cells. Subset 1 has a pro-inflammatory component, with expression of IFNγ and upregulation of NFkB signaling pathways. Subset 2 does not express IFNγ, but upregulates inhibitory molecules and nIEL markers. Expression of granzymes and Cd8a was common to both. These fractions were in opposite positions in the clustering by UMAP and had different TCR repertoires. They may be involved in the suppression of intestinal aging and longevity through anti-tumor immunity, elimination of senescent cells and stressed cells in the aging environment. This finding could be a breakthrough in aging research.</p