Published OnlineFirst September 16, 2016; DOI: 10.1158/2326-6066.CIR-15-0038 Research Article Cancer Immunology Research Restoring Retinoic Acid Attenuates Intestinal Inflammation and Tumorigenesis in APCMin/þ Mice Hweixian Leong Penny1, Tyler R. Prestwood1, Nupur Bhattacharya1, Fionna Sun1, Justin A. Kenkel1, Matthew G. Davidson1, Lei Shen1, Luis A. Zuniga2, E. Scott Seeley1, Reetesh Pai3, Okmi Choi1, Lorna Tolentino1, Jinshan Wang4, Joseph L. Napoli4, and Edgar G. Engleman1 Abstract Chronic intestinal inflammation accompanies familial adeno- exhibited further reductions in intestinal RA with concomitant matous polyposis (FAP) and is a major risk factor for colorectal increases in inflammation and tumor burden. Conversely, resto- cancer in patients with this disease, but the cause of such inflam- ration of RA by pharmacologic blockade of the RA-catabolizing mation is unknown. Because retinoic acid (RA) plays a critical role enzyme CYP26A1 attenuated inflammation and diminished in maintaining immune homeostasis in the intestine, we hypoth- tumor burden. To investigate the effect of RA deficiency on the esized that altered RA metabolism contributes to inflammation gut immune system, we studied lamina propria dendritic cells and tumorigenesis in FAP. To assess this hypothesis, we analyzed (LPDC) because these cells play a central role in promoting þ RA metabolism in the intestines of patients with FAP as well as tolerance. APCMin/ LPDCs preferentially induced Th17 cells, but þ APCMin/ mice, a model that recapitulates FAP in most respects. reverted to inducing Tregs following restoration of intestinal RA We also investigated the impact of intestinal RA repletion and in vivo or direct treatment of LPDCs with RA in vitro. These findings þ depletion on tumorigenesis and inflammation in APCMin/ mice. demonstrate the importance of intestinal RA deficiency in tumor- þ Tumors from both FAP patients and APCMin/ mice displayed igenesis and suggest that pharmacologic repletion of RA could striking alterations in RA metabolism that resulted in reduced reduce tumorigenesis in FAP patients. Cancer Immunol Res; 4(11); þ intestinal RA. APCMin/ mice placed on a vitamin A–deficient diet 917–26. Ó2016 AACR. Introduction stabilization and nuclear accumulation of b-catenin leads to dysregulated signaling that initiates the multi-hit progression Several lines of epidemiologic evidence show that chronic to carcinoma (2–4). Although most patients with this disorder intestinal inflammation drives the formation and growth of eventually require colectomy, chronic therapy with nonsteroi- intestinal adenomas and carcinomas (1). Intestinal inflammation dal anti-inflammatory drugs can slow adenoma growth and accompanies familial adenomatous polyposis (FAP), an inherited þ forestall malignant transformation (6). In the APCMin/ mouse disorder in which numerous adenomas develop in the intestine model, which recapitulates FAP in most respects, chronic intes- and eventually undergo malignant transformation. In FAP, as tinal inflammation mediated by a loss of IL10-secreting regu- in the majority of sporadic colorectal cancers, tumorigenesis is latory T cells (Treg) and an increase in IL17A-producing Th17 initiated by mutations in the tumor suppressor gene, APC (2– cells promotes tumor growth that is dependent upon IL17A 4), which is part of the destruction complex that phosphor- (7–9). The genetic events involved in the progression of disease ylates b-catenin, targeting it for ubiquitination and subsequent in this model, such as FAP, are well delineated. Nonetheless, proteolytic degradation (5). In cells lacking functional APC,the the molecular and cellular mechanisms that induce inflamma- tion have not been elucidated. The vitamin A metabolite retinoic acid (RA) plays critical roles 1Department of Pathology, Stanford University School of Medicine (Blood Center), Palo Alto, California. 2Department of Immunology, in embryonic development, fertility, and vision (10). In addi- Veterans Administration Hospital, Palo Alto, California. 3Department tion, it is a key regulator of the immune system (11). For example, of Pathology, Stanford University, Stanford, California. 4Department RA is required for the generation of inducible Tregs, which help of Nutritional Science and Toxicology, University of California, Berke- ley, Berkeley, California. to maintain immune homeostasis in sites such as the intestine (12–15). Bioactive RA is derived from vitamin A (retinol) Note: Supplementary data for this article are available at Cancer Immunology through a multistep process that involves several enzymes, Research Online (http://cancerimmunolres.aacrjournals.org/). including alcohol dehydrogenases (ADH), short-chain dehydro- H.L. Penny, T.R. Prestwood, and N. Bhattacharya contributed equally to this genase reductases (RDH), and retinaldehyde dehydrogenases article. (RALDH; refs. 10, 16). RA is then catabolized to inactive meta- Corresponding Author: Edgar G. Engleman, Stanford University School of bolites by cytochrome p450 (CYP) family members, mainly Medicine, 3373 Hillview Avenue, Palo Alto, CA 94304-1204. Phone: 650-723- CYP26A1. 7960; Fax: 650-725-0592; E-mail: [email protected] In light of the importance of RA in regulating immunity and its doi: 10.1158/2326-6066.CIR-15-0038 potential role in modulating intestinal inflammation, we inves- Ó2016 American Association for Cancer Research. tigated the impact of this metabolite on tumorigenesis and www.aacrjournals.org 917 Downloaded from cancerimmunolres.aacrjournals.org on September 25, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst September 16, 2016; DOI: 10.1158/2326-6066.CIR-15-0038 Penny et al. þ inflammation in human FAP and APCMin/ mice. The experi- Flow-cytometric analysis ments described in this report reveal that RA metabolism is Isolated LP cells from the small intestine and mesenteric lymph þ abnormal in both APCMin/ mice and FAP, resulting in reduced node (MLN) cells were resuspended in 1% BSA in PBS (FACS RA concentrations. We find that restoration of normal intestinal buffer). After Fc blockade with anti-FcgRIII/II (BD Biosciences), þ RA concentrations ameliorates tumorigenesis in APCMin/ mice. cells were stained with Live/Dead Blue (Invitrogen) or PI, and Our data show that lamina propria dendritic cells (LPDC) isolated mAbs against Thy1.2 and CD4 (Biolegend). Intracellular Foxp3 from the small intestine, which normally contribute to the main- and cytokines were stained using mAbs against Foxp3, IL10, and tenance of intestinal tolerance by inducing Tregs, are sensitive to IL17A (eBioscience) per manufacturer's instructions. IL17 intra- changes in RA concentration. Whereas LPDCs in healthy control cellular cytokine staining in freshly isolated LP was performed þ mice induce the formation of Tregs, APCMin/ LPDCs induce after stimulating LP cells for 5 hours in PMA, ionomycin, and proinflammatory Th17 cells, but revert to a tolerogenic phenotype brefeldin A. Flow-cytometric analysis was performed on a LSRII when RA is reconstituted, in vivo. flow cytometer (BD Biosciences). Materials and Methods Immunoblotting Tissue lysates were prepared using RIPA lysis buffer. The fol- Mouse breeding and care lowing primary antibodies were used for immunoblotting: anti- All animal experiments described herein were approved by the ALDH1A1 (Protein tech; 15910-1-AP), anti-ALDH1A2 (Abcam; Stanford University Institutional Animal Care and Use Commit- ab75674), and anti–b-actin (Cell Signaling Technology; 8457S). Min/þ tee. Breeding pairs of APC male and WT C57BL/6 female Primary and secondary HRP-labeled antibodies were used at mice were purchased from The Jackson Laboratory and bred on- 1:500 and 1:2,000 dilutions, respectively. Detection was per- –/– site. OT-II TCR transgenic Rag mice were purchased from formed with SuperSignal West Femto Maximum Sensitivity Sub- Taconic. All mice were housed in an American Association for strate (Thermo Scientific). the Accreditation of Laboratory Animal Care accredited animal facility and maintained in pathogen-free conditions on standard T-cell differentiation assay rodent chow ad libitum unless otherwise stated. þ Sorted CD11chi MHCII DCs (2 Â 104) were cocultured with þ þ À 1 Â 105 MACS-enriched CD4 CD62L Foxp3 na€ve T cells from Isolation of DCs the spleen and lymph nodes of OT-II TCR-transgenic mice, along Mice were euthanized and small intestines were excised while with OVA – peptide (New England Peptide) and 1 ng/mL removing fat and mesentery. Peyer's patches were excised and 323 339 recombinant human transforming growth factor (TGF)-b1 intestines were filleted. Luminal contents and epithelial cells were (Peprotech). Unless otherwise stated, a 200 nmol/L to 1 mmol/L removed by vigorous stirring in HBSS in petri dishes. The intes- peptide range was used for the standard Treg induction assay, tinal tissue was then cut into 5-mm sections that were trans- while a 200 mmol/L peptide concentration was used for Th17 ferred into a glass scintillation vial (Fisher) containing 10% FCS, induction. After 96 hours, cells were harvested and analyzed for 2.5 mmol/L EDTA, and 2 mmol/L HEPES in HBSS containing a intracellular Foxp3 or intracellular cytokines. For the latter, cells magnetic stir bar. Vials were placed into a 37C incubator on a 9- were restimulated at the 90-hour coculture time point with plate- position magnetic stir plate at 400 rpm for 20 minutes. Tissue bound anti-CD3 (145-2C11) and anti-CD28 (37.51; Biolegend), pieces were then filtered through a sieve to remove residual