IL-33/regulatory T cell axis triggers the development ofa tumor-promoting immune environment inchronic inflammationAmir H. Ameria, Sara Moradi Tuchayia, Anniek Zaalberga, Jong Ho Parka, Kenneth H. Ngoa, Tiancheng Lia, Elena Lopeza,Marco Colonnab, Richard T. Leec, Mari Mino-Kenudsond, and Shadmehr Demehria,1

aCenter for Cancer Immunology and Cutaneous Biology Research Center, Department of Dermatology, Center for Cancer Research, Massachusetts GeneralHospital and Harvard Medical School, Boston, MA 02114; bDepartment of Pathology and Immunology, Washington University School of Medicine, SaintLouis, MO 63110; cDepartment of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138; and dDepartment of Pathology,Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114

Edited by Alexander Y. Rudensky, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved December 26, 2018 (received for review September11, 2018)

Chronic inflammation’s tumor-promoting potential is well-recognized;however, the mechanism underlying the development of this immuneenvironment is unknown. Studying the transition from acute,tumor-suppressive to chronic, tumor-promoting allergic contactdermatitis (ACD) revealed how tumor-promoting chronic inflamma-tion develops. Epidermis-derived interleukin (IL)-33 up-regulationand its induction of regulatory T cell (Treg) accumulation in the skinpreceded the transition from acute to chronic ACD and triggered thetumor-promoting immune environment in chronic ACD. Mice lackingIL-33 were protected from chronic ACD and its skin cancer sequelacompared with wild-type controls (P = 0.0002). IL-33’s direct signal-ing onto Tregs was required for the development of the tumor-promoting immune environment in the skin. IL-33–Treg signalingwas also required for chronic colitis and its associated colorectalcancer development in a colitis model (P < 0.0001). Significantly in-creased IL-33 and Tregs marked the perilesional skin and colon inpatients with cancer-prone chronic inflammatory diseases. Our find-ings elucidate the role of the IL-33/Treg axis in creating a tumor-promoting immune environment in chronic inflammatory diseasesand suggest therapeutic targets for cancer prevention and treat-ment in high-risk patients.

interleukin (IL)-33 | regulatory T cells | chronic inflammation |cancer promotion | allergic contact dermatitis

The association between chronic inflammation and cancer riskhas been appreciated for over a century (1). Chronic in-

flammatory conditions contribute to 15–20% of all cancer-relateddeaths worldwide (2). Besides chronic inflammation-inducedcancers, tumor-promoting immune environments are an essen-tial component of a wide array of sporadic cancers (2). In the skin,chronic inflammation is associated with an aggressive form ofsquamous cell carcinoma (SCC), Marjolin’s ulcer (3). Previously,we described the development of Marjolin’s ulcer in chronicallergic contact dermatitis (ACD) secondary to an allergenic or-thopedic metal implant (4). The extent of chronic inflammation-associated cancers highlights the need to elucidate the pathogenesisof cancer-prone, chronic inflammatory conditions.Cancer-prone, chronic inflammation is commonly described as

a type 2 immune environment containing multiple protumori-genic immune cells and factors, including T helper 2 (Th2) cells,regulatory T cells (Tregs), M2 macrophages, mast cells, eosino-phils, myeloid-derived suppressor cells, interleukin (IL)-6, tumornecrosis factor (TNF)-α, transforming growth factor (TGF)-β,chemokines, and other growth factors (5). Although targetingthese tumor-promoting factors in chronic inflammation may re-duce the rate of cancer development, the complete reversal oftumor promotion by blocking all these effector pathways is im-practical (5). Therefore, it is necessary to explore the upstreamfactors creating a tumor-promoting immune environment in the

first place to identify targets for cancer prevention in chronicinflammation.Chronic ACD is marked by type 2, tumor-promoting immune

responses (4). In stark contrast, acute ACD (i.e., contact hy-persensitivity) is well-known to be tumor-suppressive and ischaracterized by type 1 immunity involving Th1, cytotoxic T, andnatural killer cells (6). The opposite immune environments ofchronic versus acute ACD provide a unique opportunity to de-termine the mechanism of transition from a type 1, tumor-suppressing immunity to a type 2, tumor-promoting immuneenvironment. Th2 cells, Tregs, group 2 innate lymphoid cells(ILC2s), and mast cells are potential candidates for initiating thedevelopment of chronic inflammation (2). The epithelium-derived alarmins, IL-25, IL-33, and thymic stromal lympho-poietin (TSLP), may contribute to the formation of the type 2immune environment of chronic inflammation (7). These alar-mins initiate type 2 allergic inflammation at barrier sites (7).They can activate Tregs, ILC2s, and mast cells upon epithelialdamage in barrier organs (8, 9). It is unknown if alarmins play a

Significance

Chronic inflammatory diseases are well-recognized causes ofcancer and account for up to 20% of all cancer deaths world-wide. However, the mechanism that initiates the developmentof a tumor-promoting immune environment in chronic in-flammation is not known. Using mouse models of chronic skinand colon inflammation and human samples, we show IL-33triggers the transition from tumor-suppressive immunity tochronic, tumor-promoting inflammation through a regulatoryT cell-dependent mechanism. Our findings demonstrate ageneralized dependency of tumor-promoting immune envi-ronments on the IL-33/Treg axis both in the skin and colon.Based on these findings, IL-33/Treg axis blockade may be anattractive therapeutic strategy for the treatment and pre-vention of cancers associated with chronic inflammatory dis-eases and potentiating the antitumor immunity induced bycancer immunotherapeutics.

Author contributions: A.H.A., S.M.T., and S.D. designed research; A.H.A., S.M.T., A.Z.,J.H.P., K.H.N., T.L., and E.L. performed research; M.C., R.T.L., and M.M.-K. contributednew reagents/analytic tools; A.H.A., S.M.T., A.Z., J.H.P., K.H.N., T.L., E.L., M.M.-K., andS.D. analyzed data; and A.H.A. and S.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: sdemehri1@mgh.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1815016116/-/DCSupplemental.

Published online January 29, 2019.

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determining role in the transition from acute to chronicinflammation.Herein, we demonstrate IL-33 up-regulation precedes the

development of a tumor-promoting immune environment inchronic inflammation. We determined that IL-33/Treg axis wasrequired for the transition to a type 2 immune environment andtumor development in chronic ACD and colitis-induced co-lorectal cancer. Finally, we demonstrated increases in IL-33 andTregs are associated with human cancer development in chronicinflammatory diseases. We conclude the IL-33/Treg axis is es-sential for the initiation of a tumor-promoting immune envi-ronment in chronic inflammation.

ResultsIL-33 Overexpression Precedes the Development of a Tumor-Promoting Immune Environment in Chronic ACD. To study thetransition from an acute, tumor-suppressing immunity to achronic, tumor-promoting inflammation in the skin, we searchedfor the timepoint at which the switch from acute to chronic ACDoccurs. One week after abdominal sensitization with 0.5%1-fluoro-2,4-dinitrobenzene (DNFB), mice were challengedwith 0.25% DNFB on their ear three times per week, and earthickness was measured over time. Increased ear thickness,marking acute inflammation and dermal edema during the acutephase of ACD [i.e., contact hypersensitivity (6)], peaked at day14–18 after first challenge in wild-type (Wt) mice, followed by adecrease, reaching a plateau at approximately day 22 (SI Ap-pendix, Fig. S1A). Therefore, we identified day 22 as the transi-tion timepoint from acute to chronic ACD (SI Appendix, Fig.S1A, arrow). Given the role for the cardinal epithelium-derivedalarmins, TSLP, IL-25, and IL-33, in the initiation of a type 2immune response at barrier sites (7), we examined the expressionlevels of TSLP, IL-25, and IL-33 in the back skin of Wt animalsat day 22 post DNFB versus acetone (carrier alone) challenge (SIAppendix, Fig. S1B). Tslp expression was down-regulated (P <0.05), and Il25 expression was not changed in DNFB- comparedwith acetone-treated skin at the transition timepoint (SI Ap-pendix, Fig. S1C). In contrast, Il33 expression was significantlyelevated in DNFB-treated skin at the transition timepoint (SIAppendix, Fig. S1C) and, to a lesser degree, at 24 h after the firstchallenge with DNFB (acute phase; Il33 expression at transitionto acute ratio: 2.32; SI Appendix, Fig. S1D). IL-33 protein levelswere also significantly elevated in DNFB-treated skin during thetransition to chronic ACD (SI Appendix, Fig. S1E). IL-33 was de-tectable in keratinocytes at the transition timepoint, but absent at 24h post first DNFB challenge or in acetone-treated skin (Fig. 1A andSI Appendix, Figs. S1F and S2). IL-33 up-regulation was also de-tectable in the skin of MRL/lpr mice that develop cancer-pronecutaneous lupus inflammation [SI Appendix, Fig. S3 (10)]. IL-33up-regulation at the transition timepoint and its initiating role inthe development of type 2 inflammation (7) strongly suggest its rolein driving the transition from acute to chronic inflammation.

IL-33 Is Required for the Development of a Tumor-Promoting ImmuneEnvironment in ACD. We investigated whether IL-33 is necessaryfor the transition to a tumor-promoting immune environment inchronic ACD. IL-33 deletion had no impact on baseline T cellinfiltrates in the skin (SI Appendix, Fig. S4 A and B) or the acutephase of ACD as measured by ear thickness in DNFB-treatedIL-33 knockout mice (IL-33tm1b/tm1b or IL-33KO) compared withWt mice (SI Appendix, Fig. S5). However, epidermal hyperplasiaand dermal inflammation were significantly blunted in DNFB-treated IL-33KO compared with Wt mice during the transitionto chronic ACD (Fig. 1 B and C). To determine the role of IL-33in regulating tumor promotion in chronic ACD, we sensitized IL-33KO and Wt mice to DNFB, treated their back skin with thecarcinogen, 7,12-dimethylbenz(a)anthracene (DMBA), followedby treatment with DNFB or acetone three times per week on

their back skin for 30 wk to measure their tumorigenesis po-tential (Fig. 1D). IL-33 loss blunted the epidermal hyperplasia inchronic ACD (P = 0.0021; Fig. 1E and SI Appendix, Fig. S6).DNFB-treated IL-33KO mice were protected from chronicACD-induced skin tumors compared with DNFB-treated Wtmice (P = 0.0002, Fig. 1F and SI Appendix, Fig. S6). Further, IL-33KO

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Fig. 1. IL-33 is required for tumor promotion in chronic ACD. (A) Repre-sentative images of IL-33 immunostaining in the Wt skin at the transitiontimepoint relative to acute timepoint and acetone control. (B) Epidermalthickness measured across 10 random high power fields (hpf) per each backskin of IL-33KO and Wt mice treated with DNFB compared with acetonecontrol at the transition timepoint. (C) Representative H&E images of backskin of IL-33KO, Wt, and acetone-treated mice at the transition timepoint.Note the epidermal thickness (brackets) and dermal inflammatory infiltratesin each group. (D) Experimental design for chronic ACD-induced skin carci-nogenesis consisting of initial sensitization with DNFB allergen, treatmentwith DMBA carcinogen, and challenge with DNFB three times a week for 29wk. (E) Epidermal thickness measured across 10 random hpf per back skin ofeach IL-33KO and Wt mice treated with DNFB compared with acetonecontrol at the end of 30-wk carcinogenesis protocol. (F) Skin tumor onset inIL-33KO, Wt, and acetone-treated mice (log-rank test). (G) Final tumorcounts per IL-33KO mice compared with Wt and acetone-treated controls.n = 10 per group; error bars represent the mean ± SD; *P < 0.005, **P <0.0001 by two-tailed Mann–Whitney U test; epidermal thickness measure-ments were performed blindly. (Scale bars, 100 μm.)

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mice developed significantly fewer tumors compared with Wt ani-mals in response to chronic DNFB exposure (Fig. 1G). IL-33 lossdid not impact tumor development in mice subjected to the stan-dard DMBA/TPA skin carcinogenesis protocol (SI Appendix, Fig.S7), suggesting that IL-33’s protumorigenic effect is specific toinflammation-associated, but not sporadic forms of skin cancer.These findings highlight distinct pathways of skin carcinogenesis andIL-33’s essential role in the formation of a tumor-promoting chronicinflammatory state.To determine the cellular target(s) of IL-33, we examined the

immune environment of IL-33KO relative to that of Wt skinat day 22 post DNFB challenge (i.e., transition timepoint, SIAppendix, Fig. S1B). CD4+ and CD8+ T cells were significantlyreduced in DNFB-treated IL-33KO compared with Wt skin, withCD4+ T cell numbers in IL-33KO skin approximating those inacetone-treated controls (SI Appendix, Fig. S8 A and B). We didnot detect any differences between DNFB-treated IL-33KO andWt mice in mast cell, basophil, or ILC2 accumulation in the skin,which are the other direct targets of IL-33 and can drive thedevelopment of a type 2 immune environment (SI Appendix,Figs. S8 C–E and S9). Among CD4+ T cell subsets, Foxp3+ Tregsshowed a marked reduction in DNFB-treated IL-33KO com-pared with Wt skin at the transition timepoint (P = 0.0068; SIAppendix, Fig. S8 F and G). To determine the impact of IL-33loss on the transition from type 1 immunity to type 2 in-flammation in chronic ACD, we analyzed the expression of cy-tokines associated with Th cells (Ifng, Il4, and Il17) and Tregs(Il10) in acute (D1) versus transition (D22) timepoints in IL-33KO and Wt skin-draining lymph nodes. IFN gamma expres-sion, a marker of type 1, acute ACD, was significantly decreasedin Wt mice at transition relative to the acute timepoint; however,IL-33KO mice showed a reversed trend (SI Appendix, Fig. S8H).There was a significant reduction in percent T-bet+ Th1 cells inWt skin at the transition timepoint, which was reversed in IL-33KO skin (SI Appendix, Figs. S8F and S10A). Il4 and Il17expression levels did not change significantly from acute totransition phase (SI Appendix, Fig. S8H). GATA3+ Th2 cells

were not increased in Wt or IL-33KO skin at the transitiontimepoint, suggesting Th2 cells may not play a role in the ini-tiation of a type 2 inflammatory state in chronic ACD (SI

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Fig. 2. ST2 deletion in Tregs blocks chronic ACD-induced skin tumorigenesis. (A) Representative im-ages of CD4+ T cells in DNFB-treated TregST2CKO,IL-33KO, Wt, and acetone-treated Wt skin at theconclusion of 30-wk DMBA/DNFB carcinogenesisprotocol (CK: cytokeratin). (B and C) CD4+ T cells (B)and Tregs (C) quantified in 10 random hpf per skin ofDMBA/DNFB-treated TregST2CKO and IL-33KO com-pared with Wt mice. (D) CD8/Treg ratio in DMBA/DNFB-treated TregST2CKO and IL-33KO skin com-pared with Wt skin. Cell counts were computed usingimmunofluorescence image-based quantificationand flow cytometry. (E) Epidermal thickness mea-sured across 10 random hpf per back skin of eachDMBA/DNFB-treated TregST2CKO mice comparedwith IL-33KO, Wt and DMBA/acetone controls at theend of 30-wk carcinogenesis protocol. (F) Skin tumoronset in DMBA/DNFB-treated TregST2CKO, IL-33KO,Wt, and DMBA/acetone-treated groups (log-ranktest). (G) Tumor counts per mouse in TregST2CKOmice compared with IL-33KO, Wt, and acetone-treated groups. n = 10 TregST2CKO, 13 Wt (in-cluding Foxp3Cre, ST2+/flox), 4 IL-33KO, and 4 acetonecontrol mice; error bars represent the mean ± SD;*P < 0.05, **P < 0.01; ns, not significant by two-tailedMann–Whitney U test; cell counts and epidermalthickness measurements were preformed blindly.(Scale bar, 100 μm.)

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Fig. 3. IL-33 overexpression and Treg accumulation are associated withcancer-prone chronic ACD in human skin. (A) Representative images of IL-33immunostained squamous cell carcinoma (SCC) and perilesional skin of apatient with chronic ACD due to allergenic metal implant compared withage- and gender-matched normal skin (brown nuclear stains). (B) Repre-sentative images of Foxp3 (brown) and CD3 (red) highlighting Treg infil-trates in skin cancer and perilesional skin relative to normal skin. Arrowspoint to Foxp3+ Tregs. (Scale bars, 100 μm.)

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Appendix, Fig. S10B). Il10 expression was significantly elevated atthe transition timepoint inWt mice and blunted in IL-33KOmice (SIAppendix, Fig. S8H). We determined that a significantly higher per-centage of ST2+ Tregs express IL-10 compared with ST2− Tregs inthe skin during transition from acute to chronic ACD (P < 0.0001, SIAppendix, Fig. S11A). Accordingly, there were significantly fewer IL-10+ Tregs, ST2+ Tregs, and IL-10+ ST2+ Tregs in IL-33KO skincompared with Wt controls at the transition timepoint (SI Appendix,Fig. S11 B andC). In contrast, IFNγ+CD4+ T cells and IFNγ+CD8+

T cells were markedly increased in the skin of IL-33KO mice com-pared with Wt controls at the transition timepoint (SI Appendix, Fig.S11 B and D). These findings suggest an essential role for Treg andits associated cytokine, IL-10, downstream of IL-33 in mediating thetransition from acute to chronic ACD.

Loss of ST2 Expression on Tregs Blocks Chronic ACD-Induced SkinCarcinogenesis. Having shown IL-33’s role in the transition totumor-promoting chronic inflammation in association with Tregaccumulation in the skin, we investigated whether the IL-33/Tregaxis is necessary for the transition to a chronic tumor-promotingimmune environment.We utilized Foxp3Cre, ST2flox/flox (TregST2CKO)mouse model in which IL-33 receptor [interleukin 1 receptor like

1 (IL1RL1) or ST2] was deleted specifically on Tregs. As observedin the IL-33KOmice, knocking out ST2 specifically on Tregs had noimpact on baseline T cell infiltrates in the skin (SI Appendix, Fig. S4A and B) or acute ACD inflammation (SI Appendix, Fig. S12A).However, ST2 deletion on Tregs blunted the epidermal hyperplasiain response to repeated DNFB challenge at the transition timepoint(SI Appendix, Fig. S12B). DNFB-treated TregST2CKO skinexhibited decreased CD4+ T cell accumulation, decreased Tregaccumulation, and increased CD8/Treg ratio compared withDNFB-treatedWt skin at the transition timepoint (SI Appendix, Fig.S12 C–G). To test whether IL-33 signaling onto Tregs was requiredfor the transition to a tumor-promoting immune environment inchronic ACD, we subjected TregST2CKO, IL-33KO, and Wt miceto a chronic ACD-associated skin carcinogenesis protocol (Fig. 1D).Compared with DNFB-treated Wt mice, TregST2CKO and IL-33KO mice had significantly reduced CD4+ T cell and Treg accu-mulation in the skin during chronic ACD (Fig. 2A–C). TregST2CKOmaintained a high number of CD8+ T cells in their skin leading tosignificantly elevated CD8/Treg ratio, a marker of antitumor immu-nity, in these animals compared with DNFB-treated Wt mice (Fig.2D and SI Appendix, Fig. S13). Accordingly, epidermal hyper-plasia was markedly blunted in DNFB-treated TregST2CKO

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Fig. 4. ST2 loss on Tregs suppresses colitis-inducedcolorectal cancer in mice. (A) Schematic diagram forcolitis-induced colorectal tumorigenesis in mice,consisting of four cycles of AOM/DSS administrationover 15 wk. (B) Weight loss pattern in TregST2CKOandWt mice during the final DSS treatment cycle. (C)Colon length measurement, from cecum to rectum,in TregST2CKO and Wt mice at the completion ofAOM/DSS carcinogenesis protocol. (D) Representa-tive images of TregST2CKO and Wt colon at thecompletion of AOM/DSS carcinogenesis protocol. (E)Colorectal tumors quantified per TregST2CKO andWt mice at the completion of AOM/DSS carcinogen-esis protocol. (F) Representative macroscopic images ofexposed TregST2CKO and Wt colonic lumen. (G)Representative H&E image of distal colon ofTregST2CKO and Wt mice. (Scale bar, 1 cm.) (H) Flowcytometric analysis of T cells isolated from colon show-ing CD8+ T and CD4+ T cell frequencies in TregST2CKOand Wt colon. Percent cells in each gate are shown onthe flow dot plots. (I) CD4+ and CD8+ T cell infiltrates inTregST2CKO and Wt colons quantified in 10 randomhpf per colon section and averaged across the animalsin each group. CD8+ T cell abundance was determinedusing CD3-stained images and flow cytometry data (SIAppendix, Fig. S20). Stained cells were counted blindly.(J) Representative images of CD4+ T cell in TregST2CKOand Wt colons. (CK, cytokeratin; Scale bars, 100 μm.) (K)Treg frequencies of total CD4+ T cells in TregST2CKOand Wt colon. Treg frequency was determined by flowcytometry. n = 16 TregST2CKO and 14 Wt (includingFoxp3Cre, ST2+/flox) mice; error bars represent themean ± SD; *P < 0.05, **P < 0.01, ***P < 0.0001; ns, notsignificant by two-tailed Mann–Whitney U test.

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mice compared with Wt controls (Fig. 2E and SI Appendix,Fig. S14). Significantly fewer TregST2CKO mice developedskin tumors in response to DMBA/DNFB treatment, and thenumber of tumors per mouse was markedly less amongTregST2CKO mice compared with DMBA/DNFB-treated Wtmice (Fig. 2 F and G). Our findings demonstrate that blockingIL-33 signaling to Tregs recapitulates the tumor-protectiveeffect of global IL-33 loss in chronic inflammation.

IL-33 and Tregs Are Increased in Cancer-Prone Chronic InflammatoryDiseases of Human Skin. We next investigated whether the IL-33/Treg axis was associated with skin cancer development in aclinical case of chronic ACD-associated skin cancer (4). IL-33immunostaining of the patient’s skin cancer and perilesionalskin, distant from the cancer site, revealed IL-33 overexpressionin the keratinocytes compared with normal gender- and age-matched control skin from the same anatomical site (Fig. 3A).We also identified increased numbers of CD3+ Foxp3+ Tregs inthe SCC and the perilesional skin of our patient compared withthe normal skin (Fig. 3B). Importantly, we observed a similarpattern of IL-33 up-regulation in SCC and perilesional skin oftwo skin cancer-prone discoid lupus patients, which was absent incontrol discoid lupus lesions with no skin cancer history andnormal skin [SI Appendix, Fig. S15 (10)]. Therefore, the IL-33/Treg axis may play an important role in skin cancer developmentin cancer-prone inflammatory skin diseases.

ST2 Expression by Tregs Is Critical for Colitis-Induced ColorectalCarcinogenesis in Mice. We extended our studies to chronic in-flammatory bowel disease (IBD)-associated colorectal cancer. Atbaseline, TregST2CKO mice had more CD4+ T cells in theircolon compared with IL-33KO and Wt mice (SI Appendix, Fig.S4 A and C). However, there were no differences in Treg andCD8+ T cell numbers in the colon between the three groups (SIAppendix, Fig. S4 A and C). An AOM/DSS model was used as anestablished model for colitis-induced colorectal cancer in whichmice received four treatment cycles of a carcinogen, azoxy-methane (AOM), injected intraperitoneally followed by a colitis-causing agent, dextran sodium sulfate (DSS), added to thedrinking water over 5 d (Fig. 4A). This carcinogenesis protocolled to a massive IL-33 induction in Wt colon (SI Appendix, Fig.S16). IL-33KO mice did not experience weight loss and de-veloped significantly fewer colorectal tumors compared with Wtmice (SI Appendix, Fig. S17). TregST2CKO mice subjected tothis protocol were also resistant to weight loss, a marker of dis-ease severity, compared with Wt controls (Fig. 4B). Likewise,colon length was significantly greater in the TregST2CKO micecompared with Wt mice (Fig. 4 C and D). TregST2CKO micedeveloped significantly fewer colorectal tumors compared withWt controls (P < 0.0001; Fig. 4 E and F). Tumors were smaller inTregST2CKO compared with Wt colon, and we observed betterpreserved crypt architecture in TregST2CKO relative to Wt colon(Fig. 4 F and G). Flow cytometry demonstrated increased CD8+

T cell frequency in TregST2CKO relative to Wt colon and in-creased PD-1+ CD8+ T cells in the mesenteric lymph nodes ofTregST2CKO compared with Wt mice (Fig. 4H and SI Appendix,Fig. S18). We identified a significant increase in CD8+ T cell countsin TregST2CKO relative to Wt colon, while CD4+ T cell countsremained the same (Fig. 4 I and J). Among CD4+ T cells, Tregpercentage was significantly reduced in TregST2CKO comparedwith Wt colon (P < 0.0001; Fig. 4K). Finally, the CD8/Treg ratio inTregST2CKO colon was significantly higher compared with Wtcontrols (SI Appendix, Fig. S19). These findings support the gen-eralized role of the IL-33/Treg axis as a driver of the tumor-promoting immune environment in chronic inflammation.

IL-33 and Treg Are Increased in Cancer-Prone IBD in Humans.We nextinvestigated whether the IL-33/Treg axis plays a role in humancolons affected by cancer-prone IBD preceding the developmentof colorectal cancer. We collected 19 colorectal cancer speci-mens (12 sporadic and 7 IBD-associated), 13 colitis specimens (7ulcerative colitis and 6 Crohn’s disease) from patients withconcurrent diagnosis of colorectal cancers, and 20 normal colonspecimens. We stained for IL-33 and CD4/Foxp3 using adjacentsections and quantified IL-33+ and Foxp3+ CD4+ T cells within10 randomly selected high-power fields from each specimen in ablinded manner. We observed a significant increase in IL-33+

cells in colorectal cancers and colon tissues with colitis, awayfrom any cancer, compared with the normal colon (P < 0.0005;Fig. 5A). In addition, the number of Tregs in colitis tissues washigher than normal colon (P < 0.001) and comparable to Tregnumbers in colon cancers (Fig. 5B). Correlation analysis betweenIL-33+ cell counts and Treg counts across all specimens revealeda positive correlation (r = 0.321) between IL-33+ cell numberand Treg accumulation in the colon (P = 0.0202; Fig. 5C). Theseoutcomes demonstrate IL-33 up-regulation and its associatedTreg accumulation precede the development of colorectal cancerin cancer-prone IBD.

DiscussionOur clinical and experimental findings demonstrate that IL-33–driven stimulation of Tregs is an essential trigger for the devel-opment of a tumor-promoting immune environment in chronicinflammation. Genetic studies demonstrated that IL-33 leads tothe expansion of IL-10+ Tregs in the skin during the transition

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Fig. 5. IL-33 induction and Treg accumulation in the colon mark high-riskhuman IBD. (A) Representative IL-33 immunostained (brown) tissue sectionsfrom human colorectal cancer, colitis, and normal colon and quantificationof IL-33+ cells in 10 random hpf per specimen (***P < 0.0005; ns, not sig-nificant by two-tailed Mann–Whitney U test). (B) Representative Foxp3(brown) and CD4 (red) immunostained tissue sections from human colorectalcancer, colitis, and normal colon and quantification of Foxp3+ Tregs in 10random hpf per specimen. (Insets, Right) show higher magnification of theboxed areas of the images. Arrowheads point to CD4+ T cells with positiveFoxp3 nuclear stain (i.e., Treg; ***P < 0.001; ns, not significant by two-tailedMann–Whitney U test). (C) Correlation between IL-33+ cell and Treg count incolon cancer, colitis, and normal colon (Student’s t test for the Pearsoncorrelation coefficient). n = 19 colorectal cancer specimens (12 sporadic and7 IBD-associated), 13 cancer-prone colitis specimens (7 ulcerative colitis and 6Crohn’s disease), and 20 normal colons; cells were counted blindly; error barsrepresent the mean ± SD. (Scale bars, 100 μm.)

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from acute to chronic inflammation, which can suppress type 1immunity and promote the development of a tumor-promotingtype 2 inflammation in the skin (11, 12). Deletion of ST2 spe-cifically on Tregs blocked tumor development in cancer-prone,chronic inflammation of the skin and colon. IL-33–expressingcells and Tregs were significantly and specifically increased incancer-prone, chronic inflammatory diseases of skin and colon inhumans. Together, our findings have major implications forcancer prevention in chronic inflammatory diseases, cancertreatment in a large array of malignancies in which IL-33 andTregs are increased, and enhancement of antitumor immunityand the efficacy of immunotherapeutics by blocking the det-rimental effects of the IL-33/Treg axis.Study of ACD and its distinct acute, chronic, and transition

phases enabled us to identify the IL-33/Treg axis as a funda-mental node in regulating the development of a complex tumor-promoting immune environment in the skin and colon. Whilecarcinogenesis in chronic ACD is rare, the rising prevalence ofcontact allergies to nickel (13) and nickel-containing implantssuggests that chronic ACD-induced carcinogenesis is likelyunderreported (4). As we showed in discoid lupus, the IL-33/Treg axis may also play a critical role in skin cancer develop-ment in other cancer-prone inflammatory diseases of the skinincluding hypertrophic lichen planus, chronic wound, andchronic scar (3, 14–16). In colon, IBD is a major risk factor ofcolorectal cancer with 23-fold increased risk among patients withpancolitis (17). In addition, IL-33 up-regulation has been de-tected in established invasive cancers, which may suggest a rolefor IL-33/Treg axis in tumor progression in response to damagedtissue homeostasis (9, 18–20). These observations suggest IL-33blocking antibodies developed for atopic disorders may showefficacy in cancer treatment. However, it is critical to emphasizethe complexity of IL-33 function and its potentially distinct ef-fects on carcinogenesis. While our data demonstrate that IL-33/Treg axis has protumorigenic effects, IL-33 signaling onto otherimmune cell types, such as CD8+ T cells, can be tumor-protective (21). Importantly, while the IL-33/Treg axisserves as a promising therapeutic target in cancer, more work

needs to be done to understand the IL-33 signaling pathwayand determine the potentially distinct impact of IL-33 onTregs versus other immune cell types in these contexts (22),which could dictate the outcome of IL-33 blockade forcancer therapy.While the mechanism underlying the transition from acute to

chronic immune activation has not been previously elucidated,the existence of such a transition has been long reported (23).Recently, it is appreciated that the transition from acute, anti-tumor immunity to chronic inflammation is a common detrimentto the efficacy of cancer immunotherapeutics (5). This issue isevident in the ACD paradigm itself: the tumor-suppressive effectof acute ACD has been used for decades to treat skin cancers(24). However, the repeated application of contact allergens maycreate a tumor-promoting immune environment (4). Therefore,blocking the IL-33/Treg axis may provide an approach to maxi-mize the efficacy of cancer immunotherapeutics by sustaininglong-term antitumor immunity. Further studies are required todetermine whether IL-33/Treg axis blockade would be effectivein reverting to a tumor-suppressing immunity after a chronicinflammatory environment has been established.

Materials and MethodsAnonymized human tissue samples were used in the study. MassachusettsGeneral Hospital Institutional Review Board approved the anonymized tissuestudy. Formalin-fixed paraffin-embedded tissue sections (4 μm) were used formanual (IL-33) or automated immunostaining (CD3/Foxp3 and CD4/Foxp3)using a Ventana Ultra automated staining system (Ventana Medical Sys-tems). Stained cells were counted blindly. Refer to SI Appendix, Materialsand Methods for detailed description of the methods.

ACKNOWLEDGMENTS. We thank Dr. David Fisher, Dr. Ethan Lerner, andDr. Nir Hacohen for critically reading the manuscript. We thank Dr. DianeMathis for providing TregST2CKO mice. IL-33KO mice were generated withsupport from Mucosal Immunology Studies Team (MIST) (Grant U01; RFA-AI-15-023). S.D. holds a Career Award for Medical Scientists from the BurroughsWellcome Fund. A.H.A. was supported by the Howard Hughes Medical Insti-tute. A.H.A., S.M.T., A.Z., J.H.P., K.H.N., T.L., and S.D. were supported bygrants from the Burroughs Wellcome Fund, Sidney Kimmel Foundation,Cancer Research Institute, and NIH (K08AR068619 and DP5OD021353).

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