Transmigrating neutrophils shape the mucosal microenvironment through localized oxygen depletion to influence resolution of inflammation.

Acute intestinal inflammation involves early accumulation of neutrophils (PMNs) followed by either resolution or progression to chronic inflammation. Based on recent evidence that mucosal metabolism influences disease outcomes, we hypothesized that transmigrating PMNs influence the transcriptional profile of the surrounding mucosa. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Transmigrating PMNs rapidly depleted microenvironmental O2 sufficiently to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). By utilizing HIF reporter mice in an acute colitis model, we investigated the relative contribution of PMNs and the respiratory burst to "inflammatory hypoxia" in vivo. CGD mice, lacking a respiratory burst, developed accentuated colitis compared to control, with exaggerated PMN infiltration and diminished inflammatory hypoxia. Finally, pharmacological HIF stabilization within the mucosa protected CGD mice from severe colitis. In conclusion, transcriptional imprinting by infiltrating neutrophils modulates the host response to inflammation, via localized O2 depletion, resulting in microenvironmental hypoxia and effective inflammatory resolution.

G2A Signaling Dampens Colitic Inflammation via Production of IFN-γ.

Proinflammatory consequences have been described for lysophosphatidylcholine, a lipid product of cellular injury, signaling via the G protein-coupled receptor G2A on myeloid and lymphoid inflammatory cells. This prompted the hypothesis that genetic deletion of G2A would limit intestinal inflammation in a mouse model of colitis induced by dextran sodium sulfate. Surprisingly, G2A(-/-) mice exhibited significantly worsened colitis compared with wild-type mice, as demonstrated by disease activity, colon shortening, histology, and elevated IL-6 and IL-5 in colon tissues. Investigation of inflammatory cells recruited to inflamed G2A(-/-) colons showed significantly more TNF-α(+) and Ly6C(hi)MHCII(-) proinflammatory monocytes and eosinophils than in wild-type colons. Both monocytes and eosinophils were pathogenic as their depletion abolished the excess inflammation in G2A(-/-) mice. G2A(-/-) mice also had less IFN-γ in inflamed colon tissues than wild-type mice. Fewer CD4(+) lymphocytes were recruited to inflamed G2A(-/-) colons, and fewer colonic lymphocytes produced IFN-γ upon ex vivo stimulation. Administration of IFN-γ to G2A(-/-) mice during dextran sodium sulfate exposure abolished the excess colitic inflammation and reduced colonic IL-5 and eosinophil numbers to levels seen in wild-type mice. Furthermore, IFN-γ reduced the numbers of TNF-α(+) monocyte and enhanced their maturation from Ly6C(hi)MHCII(-) to Ly6C(int)MHCII(+) Taken together, the data suggest that G2A signaling serves to dampen intestinal inflammation via the production of IFN-γ, which, in turn, enhances monocyte maturation to a less inflammatory program and ultimately reduces eosinophil-induced injury of colonic tissues.

Control of creatine metabolism by HIF is an endogenous mechanism of barrier regulation in colitis.

Mucosal surfaces of the lower gastrointestinal tract are subject to frequent, pronounced fluctuations in oxygen tension, particularly during inflammation. Adaptive responses to hypoxia are orchestrated largely by the hypoxia-inducible transcription factors (HIFs). As HIF-1α and HIF-2α are coexpressed in mucosal epithelia that constitute the barrier between the lumen and the underlying immune milieu, we sought to define the discrete contribution of HIF-1 and HIF-2 transactivation pathways to intestinal epithelial cell homeostasis. The present study identifies creatine kinases (CKs), key metabolic enzymes for rapid ATP generation via the phosphocreatine-creatine kinase (PCr/CK) system, as a unique gene family that is coordinately regulated by HIF. Cytosolic CKs are expressed in a HIF-2-dependent manner in vitro and localize to apical intestinal epithelial cell adherens junctions, where they are critical for junction assembly and epithelial integrity. Supplementation with dietary creatine markedly ameliorated both disease severity and inflammatory responses in colitis models. Further, enzymes of the PCr/CK metabolic shuttle demonstrate dysregulated mucosal expression in a subset of ulcerative colitis and Crohn disease patients. These findings establish a role for HIF-regulated CK in epithelial homeostasis and reveal a fundamental link between cellular bioenergetics and mucosal barrier.

Eosinophil-mediated signalling attenuates inflammatory responses in experimental colitis.

OBJECTIVE: Eosinophils reside in the colonic mucosa and increase significantly during disease. Although a number of studies have suggested that eosinophils contribute to the pathogenesis of GI inflammation, the expanding scope of eosinophil-mediated activities indicate that they also regulate local immune responses and modulate tissue inflammation. We sought to define the impact of eosinophils that respond to acute phases of colitis in mice. DESIGN: Acute colitis was induced in mice by administration of dextran sulfate sodium, 2,4,6-trinitrobenzenesulfonic acid or oxazolone to C57BL/6J (control) or eosinophil deficient (PHIL) mice. Eosinophils were also depleted from mice using antibodies against interleukin (IL)-5 or by grafting bone marrow from PHIL mice into control mice. Colon tissues were collected and analysed by immunohistochemistry, flow cytometry and reverse transcription PCR; lipids were analysed by mass spectroscopy. RESULTS: Eosinophil-deficient mice developed significantly more severe colitis, and their colon tissues contained a greater number of neutrophils, than controls. This compensatory increase in neutrophils was accompanied by increased levels of the chemokines CXCL1 and CXCL2, which attract neutrophils. Lipidomic analyses of colonic tissue from eosinophil-deficient mice identified a deficiency in the docosahexaenoic acid-derived anti-inflammatory mediator 10, 17- dihydroxydocosahexaenoic acid (diHDoHE), namely protectin D1 (PD1). Administration of an exogenous PD1-isomer (10S, 17S-DiHDoHE) reduced the severity of colitis in eosinophil-deficient mice. The PD1-isomer also attenuated neutrophil infiltration and reduced levels of tumour necrosis factor-α, IL-1ß, IL-6 and inducible NO-synthase in colons of mice. Finally, in vitro assays identified a direct inhibitory effect of PD1-isomer on neutrophil transepithelial migration. CONCLUSIONS: Eosinophils exert a protective effect in acute mouse colitis, via production of anti-inflammatory lipid mediators.

Hypoxia-inducible factor-1 alpha-dependent induction of FoxP3 drives regulatory T-cell abundance and function during inflammatory hypoxia of the mucosa.

Recent studies have demonstrated dramatic shifts in metabolic supply-and-demand ratios during inflammation, a process resulting in localized tissue hypoxia within inflammatory lesions ("inflammatory hypoxia"). As part of the adaptive immune response, T cells are recruited to sites of inflammatory hypoxia. Given the profound effects of hypoxia on gene regulation, we hypothesized that T-cell differentiation is controlled by hypoxia. To pursue this hypothesis, we analyzed the transcriptional consequences of ambient hypoxia (1% oxygen) on a broad panel of T-cell differentiation factors. Surprisingly, these studies revealed selective, robust induction of FoxP3, a key transcriptional regulator for regulatory T cells (Tregs). Studies of promoter binding or loss- and gain-of-function implicated hypoxia-inducible factor (HIF)-1α in inducing FoxP3. Similarly, hypoxia enhanced Treg abundance in vitro and in vivo. Finally, Treg-intrinsic HIF-1α was required for optimal Treg function and Hif1a-deficient Tregs failed to control T-cell-mediated colitis. These studies demonstrate that hypoxia is an intrinsic molecular cue that promotes FoxP3 expression, in turn eliciting potent anti-inflammatory mechanisms to limit tissue damage in conditions of reduced oxygen availability.

Vascular endothelial growth factor C promotes breast cancer progression via a novel antioxidant mechanism that involves regulation of superoxide dismutase 3.

INTRODUCTION: Triple-negative breast cancers, particularly the claudin-low subtype, are highly aggressive and exhibit increased tumor-initiating cell (TIC) characteristics. In this study, we demonstrate that vascular endothelial growth factor C (VEGF-C) is highly expressed in the claudin-low breast cancer subtype and also that it mediates tumor progression, not only through its role in lymphangiogenesis but also through regulating TIC characteristics and the response to reactive oxygen species (ROS). METHODS: VEGF C expression was examined in breast cancer subtypes, and a VEGF C expression signature was derived. VEGF C expression and/or its associated signature was correlated with TIC and chemoresistance signatures. In vitro and in vivo assays were performed to determine whether VEGF-C expression alters TIC characteristics and the response of breast cancer cells to chemotherapy and oxidative stress. Array analysis was used to identify a downstream effector of VEGF-C, superoxide dismutase 3 (Sod3), which was tested for its involvement in VEGF-C-mediated resistance to oxidative stress and enhancement of in vivo metastasis. The VEGF-C-associated receptor neuropilin 2 (Nrp2) was knocked down to determine whether it is required for the observed effects of VEGF-C. Expression of VEGF C and Sod3 was assessed in human breast cancers. RESULTS: VEGF C is highly expressed in claudin-low breast cancers, and VEGF C and the VEGF C signature are associated with TIC-related gene signatures. VEGF-C-knockdown in mammary carcinoma cells decreases TIC properties in vitro and in vivo, sensitizing cells to oxidative stress and chemotherapy. We identified Sod3 as a target of VEGF-C in breast cancer cells by demonstrating that it is required for VEGF-C-mediated cell survival in response to oxidative stress and for VEGF-C-mediated metastasis. We demonstrate that Nrp2 is the VEGF-C-associated receptor that mediates alterations in Sod3 expression and the response of tumor cells to oxidative stress. We show that VEGF C and Sod3 are positively associated in human breast cancer. CONCLUSIONS: We describe a novel mechanism by which VEGF-C contributes to metastasis via its ability to enhance TIC-associated characteristics, particularly the response to ROS. We identified Sod3 as a critical mediator of VEGF-C-induced metastasis, and we provide evidence that the VEGF-C-Sod3 axis plays a role in human breast cancers.

Modulation of tumor fatty acids, through overexpression or loss of thyroid hormone responsive protein spot 14 is associated with altered growth and metastasis.

INTRODUCTION: Spot14 (S14), encoded by the THRSP gene, regulates de novo fatty acid synthesis in the liver, adipose, and lactating mammary gland. We recently showed that S14 stimulated fatty acid synthase (FASN) activity in vitro, and increased the synthesis of fatty acids in mammary epithelial cells in vivo. Elevated de novo fatty acid synthesis is a distinguishing feature of many solid tumors compared with adjacent normal tissue. This characteristic is thought to be acquired during tumor progression, as rapidly proliferating cells have a heightened requirement for membrane phospholipids. Further, overexpression of FASN is sufficient to stimulate cell proliferation. While many studies have focused on the FASN enzyme in cancer biology, few studies have addressed the roles of proteins that modify FASN activity, such as S14. METHODS: Tumor fatty acids were modulated using two mouse models, mouse mammary tumor virus (MMTV)-neu mice overexpressing S14 and MMTV-polyomavirus middle T antigen (PyMT) mice lacking S14, and associations between elevated or impaired fatty acid synthesis on tumor latency, growth, metastasis, and signaling pathways were investigated. We evaluated S14-dependent gene expression profiles in mouse tumors by microarray and used publicly available microarray datasets of human breast tumors. RESULTS: S14 overexpression in the MMTV-Neu transgenic model is associated with elevated medium-chain fatty acids, increased proliferation and a shorter tumor latency, but reduced tumor metastasis compared to controls. Loss of S14 in the MMTV-PyMT model decreased FASN activity and the synthesis of medium-chain fatty acids but did not alter tumor latency. Impaired fatty acid synthesis was associated with reduced solid tumor cell proliferation, the formation of cystic lesions in some animals, and decreased phosphorylation of Src and protein kinase B (Akt). Analysis of gene expression in these mouse and human tumors revealed a relationship between S14 status and the expression of genes associated with luminal epithelial differentiation. CONCLUSIONS: This study demonstrates a potential role for S14 in regulating mammary tumor growth and fatty acid synthesis in vivo. Furthermore, these results suggest that modulating the amount of medium chain fatty acids, by changing the levels of S14, has the potential to impact malignant mammary tumor phenotypes.

Luminal breast cancer metastases and tumor arousal from dormancy are promoted by direct actions of estradiol and progesterone on the malignant cells.

INTRODUCTION: Luminal, estrogen receptor-positive (ER(+)) breast cancers can metastasize but lie dormant for years before recurrences prove lethal. Understanding the roles of estrogen (E) or progestin (P) in development of luminal metastases or in arousal from dormancy is hindered by few preclinical models. We have developed such models. METHODS: Immunocompromised, ovariectomized (ovx'd) mice were intracardiac-injected with luminal or basal human breast cancer cells. Four lines were tested: luminal ER(+)PR(+) cytokeratin 5-negative (CK5(-)) E3 and MCF-7 cells, basal ER(-)PR(-)CK5(+) estrogen withdrawn-line 8 (EWD8) cells, and basal ER(-)PR(-)CK5(-) MDA-MB-231 cells. Development of micrometastases or macrometastases was quantified in ovx'd mice and in mice supplemented with E or P or both. Metastatic deposits were analyzed by immunohistochemistry for luminal, basal, and proliferation markers. RESULTS: ER(-)PR(-) cells generated macrometastases in multiple organs in the absence or presence of hormones. By contrast, ovx'd mice injected with ER(+)PR(+) cells appeared to be metastases-free until they were supplemented with E or E+P. Furthermore, unlike parental ER(+)PR(+)CK5(-) cells, luminal metastases were heterogeneous, containing a significant (6% to 30%) proportion of non-proliferative ER(-)PR(-)CK5(+) cells that would be chemotherapy-resistant. Additionally, because these cells lack receptors, they would also be endocrine therapy-resistant. With regard to ovx'd control mice injected with ER(+)PR(+) cells that appeared to be metastases-free, systematic pathologic analysis of organs showed that some harbor a reservoir of dormant micrometastases that are ER(+) but PR(-). Such cells may also be endocrine therapy- and chemotherapy-resistant. Their emergence as macrometastases can be triggered by E or E+P restoration. CONCLUSIONS: We conclude that hormones promote development of multi-organ macrometastases in luminal disease. The metastases display a disturbing heterogeneity, containing newly emergent ER(-)PR(-) subpopulations that would be resistant to endocrine therapy and chemotherapy. Similar cells are found in luminal metastases of patients. Furthermore, lack of hormones is not protective. While no overt metastases form in ovx'd mice, luminal tumor cells can seed distant organs, where they remain dormant as micrometastases and sheltered from therapies but arousable by hormone repletion. This has implications for breast cancer survivors or women with occult disease who are prescribed hormones for contraception or replacement purposes.

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide.

INTRODUCTION: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer. METHODS: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR. RESULTS: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis. CONCLUSIONS: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

Interleukin 37 expression protects mice from colitis.

IL-37, a newly described member of the IL-1 family, functions as a fundamental inhibitor of innate inflammation and immunity. In the present study, we examined a role for IL-37 during experimental colitis. A transgenic mouse strain was generated to express human IL-37 (hIL-37tg), and these mice were subjected to dextran sulfate sodium (DSS)-induced colitis. Despite the presence of a CMV promoter to drive expression of IL-37, mRNA transcripts were not present in colons at the resting state. Expression was observed only upon disruption of the epithelial barrier, with a six- to sevenfold increase (P = 0.02) on days 3 and 5 after continuous exposure to DSS. During the development of colitis, clinical disease scores were reduced by 50% (P < 0.001), and histological indices of colitis were one-third less in hIL-37tg mice compared with WT counterparts (P < 0.001). Reduced inflammation was associated with decreased leukocyte recruitment into the colonic lamina propria. In addition, release of IL-1ß and TNFα from ex vivo colonic explant tissue was decreased 5- and 13-fold, respectively, compared with WT (P ≤ 0.005), whereas IL-10 was increased sixfold (P < 0.001). However, IL-10 was not required for the anti-inflammatory effects of IL-37 because IL-10-receptor antibody blockade did not reverse IL-37-mediated protection. Mechanistically, IL-37 originating from hematopoietic cells was sufficient to exert anti-inflammatory effects because WT mice reconstituted with hIL-37tg bone marrow were protected from colitis. Thus, IL-37 emerges as key modulator of intestinal inflammation.

Ectopic lymphoid tissue alters the chemokine gradient, increases lymphocyte retention and exacerbates murine ileitis.

BACKGROUND: The earliest endoscopically-evident lesion in Crohn's disease is the aphthous ulcer, which develops over ectopic lymphoid tissues (ie, inducible lymphoid follicles (ILF), tertiary lymphoid tissue (TLT)) in the chronically inflamed intestine. ILF/TLT are induced within effector sites by homeostatic lymphoid chemokines, but their role in the development of intestinal ILF/TLT and in the pathogenesis of Crohn's disease is poorly understood. DESIGN: Using a mouse model of Crohn's-like ileitis (TNFARE) which develops florid induction of ILF/TLT within its terminal ileum, the contribution of the CCR7/CCL19/CCL21 chemokine axis during the development of TLT and its role in disease pathogenesis were assessed. RESULTS: Both CCL19 and CCL21 were increased within the inflamed ileum of TNFARE mice, which resulted in CCR7 internalisation and impaired T cell chemotaxis. ILF/TLT were a major source of CCL19 and CCL21 and increased local synthesis, augmented recruitment/retention of effector, naïve and central memory T cell subsets within the inflamed ileum. Immunoblockade of CCR7 resulted in further effector T cell retention and exacerbation of ileitis. CONCLUSIONS: Induction of ILF/TLT in the chronically inflamed intestine alters the homeostatic CCL19-CCL21 lymphoid-chemokine gradient and increases recruitment/retention of effector CCR7+ T cell subsets within the terminal ileum, contributing to the perpetuation of chronic inflammation. Thus, blockade of CCR7 or its ligands might result in deleterious consequences for subjects with chronic inflammatory diseases.

Heat shock factor 1 drives regulatory T-cell induction to limit murine intestinal inflammation.

The heat shock response is a critical component of the inflammatory cascade that prevents misfolding of new proteins and regulates immune responses. Activation of clusters of differentiation (CD)4+ T cells causes an upregulation of heat shock transcription factor, heat shock factor 1 (HSF1). We hypothesized that HSF1 promotes a pro-regulatory phenotype during inflammation. To validate this hypothesis, we interrogated cell-specific HSF1 knockout mice and HSF1 transgenic mice using in vitro and in vivo techniques. We determined that while HSF1 expression was induced by anti-CD3 stimulation alone, the combination of anti-CD3 and transforming growth factor ß, a vital cytokine for regulatory T cell (Treg) development, resulted in increased activating phosphorylation of HSF1, leading to increased nuclear translocation and binding to heat shock response elements. Using chromatin immunoprecipitation (ChIP), we demonstrate the direct binding of HSF1 to foxp3 in isolated murine CD4+ T cells, which in turn coincided with induction of FoxP3 expression. We defined that conditional knockout of HSF1 decreased development and function of Tregs and overexpression of HSF1 led to increased expression of FoxP3 along with enhanced Treg suppressive function. Adoptive transfer of CD45RBHigh CD4 colitogenic T cells along with HSF1 transgenic CD25+ Tregs prevented intestinal inflammation when wild-type Tregs did not. Finally, overexpression of HSF1 provided enhanced barrier function and protection from murine ileitis. This study demonstrates that HSF1 promotes Treg development and function and may represent both a crucial step in the development of induced regulatory T cells and an exciting target for the treatment of inflammatory diseases with a regulatory T-cell component. SIGNIFICANCE STATEMENT: The heat shock response (HSR) is a canonical stress response triggered by a multitude of stressors, including inflammation. Evidence supports the role of the HSR in regulating inflammation, yet there is a paucity of data on its influence in T cells specifically. Gut homeostasis reflects a balance between regulatory clusters of differentiation (CD)4+ T cells and pro-inflammatory T-helper (Th)17 cells. We show that upon activation within T cells, heat shock factor 1 (HSF1) translocates to the nucleus, and stimulates Treg-specific gene expression. HSF1 deficiency hinders Treg development and function and conversely, HSF1 overexpression enhances Treg development and function. While this work, focuses on HSF1 as a novel therapeutic target for intestinal inflammation, the findings have significance for a broad range of inflammatory conditions.

Dependence of PAX3-FOXO1 chromatin occupancy on ETS1 at important disease-promoting genes exposes new targetable vulnerability in Fusion-Positive Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), a malignancy of impaired myogenic differentiation, is the most common soft tissue pediatric cancer. PAX3-FOXO1 oncofusions drive the majority of the clinically more aggressive fusion-positive rhabdomyosarcoma (FP-RMS). Recent studies have established an epigenetic basis for PAX3-FOXO1-driven oncogenic processes. However, details of PAX3-FOXO1 epigenetic mechanisms, including interactions with, and dependence on, other chromatin and transcription factors, are incompletely understood. We previously identified a novel disease-promoting epigenetic axis in RMS, involving the histone demethylase KDM3A and the ETS1 transcription factor, and demonstrated that this epigenetic axis interfaces with PAX3-FOXO1 both phenotypically and transcriptomically, including co-regulation of biological processes and genes important to FP-RMS progression. In this study, we demonstrate that KDM3A and ETS1 colocalize with PAX3-FOXO1 to enhancers of important disease-promoting genes in FP-RMS, including FGF8, IL4R, and MEST, as well as PODXL, which we define herein as a new FP-RMS-promoting gene. We show that ETS1, which is induced by both PAX3-FOXO1 and KDM3A, exists in complex with PAX3-FOXO1, and augments PAX3-FOXO1 chromatin occupancy. We further show that the PAX3-FOXO1/ETS1 complex can be disrupted by the clinically relevant small molecule inhibitor YK-4-279. YK-4-279 displaces PAX3-FOXO1 from chromatin and interferes with PAX3-FOXO1-dependent gene regulation, resulting in potent inhibition of growth and invasive properties in FP-RMS, along with downregulation of FGF8, IL4R, MEST and PODXL expression. We additionally show that, in some FP-RMS, KDM3A also increases PAX3-FOXO1 levels. Together, our studies illuminate mechanisms of action of the KDM3A/ETS1 regulatory module, and reveal novel targetable mechanisms of PAX3-FOXO1 chromatin complex regulation, in FP-RMS.

Flt3 ligand expands CD103⁺ dendritic cells and FoxP3⁺ T regulatory cells, and attenuates Crohn's-like murine ileitis.

UNLABELLED: BACKGROUND; Imprinting an effector or regulatory phenotype on naïve T cells requires education at induction sites by dendritic cells (DC). Objectives To analyse the effect of inflammation on the frequency of mononuclear phagocytes (MP) and the effect of altering their frequency by administration of Flt3-L in chronic ileitis. METHODS: Using a tumour necrosis factor (TNF) driven model of ileitis (ie, TNFΔARE) that recapitulates many features of Crohn's disease (CD), dynamic changes in the frequency and functional state of MP within the inflamed ileum were assessed by flow cytometry, immunofluorescence and real-time reverse-transcription PCR and by generating CX(3)CR1 GFP-reporter TNFΔARE mice. The effect of Flt3-L supplementation on the severity of ileitis, and the frequency of CD103(+) DC and of FoxP3(+) regulatory T cells was also studied in TNFΔARE mice. RESULTS: CD11c(Hi)/MHCII(+) MP accumulated in inflamed ilea, predominantly mediated by expansion of the CX(3)CR1(+) MP subpopulation. This coincided with a decreased pro-regulatory CD103(+) DC. The phenotype of these MP was that of activated cells, as they expressed increased CD80 and CD86 on their surface. Flt3-ligand administration resulted in a preferential expansion of CD103(+) DC that attenuated the severity of ileitis in 20-week-old TNFΔARE mice, mediated by increased CD4(+)/CD25(+)/FoxP3(+) regulatory T cells. CONCLUSIONS: Results support a role for Flt3-L as a potential therapeutic agent in Crohn's-like ileitis.

SIX1 and EWS/FLI1 co-regulate an anti-metastatic gene network in Ewing Sarcoma.

Ewing sarcoma (ES), which is characterized by the presence of oncogenic fusion proteins such as EWS/FLI1, is an aggressive pediatric malignancy with a high rate of early dissemination and poor outcome after distant spread. Here we demonstrate that the SIX1 homeoprotein, which enhances metastasis in most tumor types, suppresses ES metastasis by co-regulating EWS/FLI1 target genes. Like EWS/FLI1, SIX1 promotes cell growth/transformation, yet dramatically inhibits migration and invasion, as well as metastasis in vivo. We show that EWS/FLI1 promotes SIX1 protein expression, and that the two proteins share genome-wide binding profiles and transcriptional regulatory targets, including many metastasis-associated genes such as integrins, which they co-regulate. We further show that SIX1 downregulation of integrins is critical to its ability to inhibit invasion, a key characteristic of metastatic cells. These data demonstrate an unexpected anti-metastatic function for SIX1, through coordinate gene regulation with the key oncoprotein in ES, EWS/FLI1.

Expression of Six1 in luminal breast cancers predicts poor prognosis and promotes increases in tumor initiating cells by activation of extracellular signal-regulated kinase and transforming growth factor-beta signaling pathways.

INTRODUCTION: Mammary-specific overexpression of Six1 in mice induces tumors that resemble human breast cancer, some having undergone epithelial to mesenchymal transition (EMT) and exhibiting stem/progenitor cell features. Six1 overexpression in human breast cancer cells promotes EMT and metastatic dissemination. We hypothesized that Six1 plays a role in the tumor initiating cell (TIC) population specifically in certain subtypes of breast cancer, and that by understanding its mechanism of action, we could potentially develop new means to target TICs. METHODS: We examined gene expression datasets to determine the breast cancer subtypes with Six1 overexpression, and then examined its expression in the CD24low/CD44+ putative TIC population in human luminal breast cancers xenografted through mice and in luminal breast cancer cell lines. Six1 overexpression, or knockdown, was performed in different systems to examine how Six1 levels affect TIC characteristics, using gene expression and flow cytometric analysis, tumorsphere assays, and in vivo TIC assays in immunocompromised and immune-competent mice. We examined the molecular pathways by which Six1 influences TICs using genetic/inhibitor approaches in vitro and in vivo. Finally, we examined the expression of Six1 and phosphorylated extracellular signal-regulated kinase (p-ERK) in human breast cancers. RESULTS: High levels of Six1 are associated with adverse outcomes in luminal breast cancers, particularly the luminal B subtype. Six1 levels are enriched in the CD24low/CD44+ TIC population in human luminal breast cancers xenografted through mice, and in tumorsphere cultures in MCF7 and T47D luminal breast cancer cells. When overexpressed in MCF7 cells, Six1expands the TIC population through activation of transforming growth factor-beta (TGF-ß) and mitogen activated protein kinase (MEK)/ERK signaling. Inhibition of ERK signaling in MCF7-Six1 cells with MEK1/2 inhibitors, U0126 and AZD6244, restores the TIC population of luminal breast cancer cells back to that observed in control cells. Administration of AZD6244 dramatically inhibits tumor formation efficiency and metastasis in cells that express high levels of Six1 ectopically or endogenously. Finally, we demonstrate that Six1 significantly correlates with phosphorylated ERK in human breast cancers. CONCLUSIONS: Six1 plays an important role in the TIC population in luminal breast cancers and induces a TIC phenotype by enhancing both TGF-ß and ERK signaling. MEK1/2 kinase inhibitors are potential candidates for targeting TICs in breast tumors.

The chemokine receptor CCR9 is required for the T-cell-mediated regulation of chronic ileitis in mice.

BACKGROUND & AIMS: A balance between effector and regulatory T-cell (Treg) responses is required to maintain intestinal homeostasis. To regulate immunity, T cells migrate to the intestine using a combination of adhesion molecules and chemokine receptors. However, it is not known whether the migration pathways of effector cells and Tregs are distinct or shared. We sought to determine whether interaction between the chemokine receptor 9 (CCR9) and its ligand, chemokine ligand 25 (CCL25), allows effectors or Tregs to localize to chronically inflamed small intestine. METHODS: By using a mouse model that develops Crohn's-like ileitis (tumor necrosis factor Δadenosine uracyl-rich element [TNFΔARE] mice) we examined the role of CCL25-CCR9 interactions for effector and Treg traffic using flow cytometry, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, immunoneutralization, and proliferation analyses. RESULTS: In TNFΔARE mice, expression of CCL25 and the frequency of CCR9-expressing lymphocytes increased during late-stage disease. In the absence of CCR9, TNFΔARE mice developed exacerbated disease, compared with their CCR9-sufficient counterparts, which coincided with a deficiency of CD4(+)/CD25(+)/forkhead box P3(+) and CD8(+)/CD103(+) Tregs within the intestinal lamina propria and mesenteric lymph nodes. Furthermore, the CD8(+)/CCR9(+) subset decreased the proliferation of CD4(+) T cells in vitro. Administration of a monoclonal antibody against CCR9 to TNFΔARE mice exacerbated ileitis in vivo, confirming the regulatory role of CD8(+)/CCR9(+) cells. CONCLUSIONS: Signaling of the chemokine CCL25 through its receptor CCR9 induces Tregs to migrate to the intestine. These findings raise concerns about the development of reagents to disrupt this pathway for the treatment of patients with Crohn's disease.

Retinoic acid attenuates ileitis by restoring the balance between T-helper 17 and T regulatory cells.

BACKGROUND & AIMS: Retinoic acid (RA), produced by intestinal epithelial cells (IECs) and dendritic cells (DCs) promotes the induction of regulatory T cells (Tregs) and decreases the induction of T-helper (Th)17 cells. METHODS: We studied the roles of RA in mice that overproduce tumor necrosis factor (TNF) and develop chronic ileitis (TNF_ARE mice). We assessed the frequency and function of CD103+ DCs, Th17 cells, and Tregs by flow cytometry, and we measured expression of cytokines and retinaldehyde dehydrogenase (RALDH) enzymes in ileum samples, DCs, and IECs by real-time polymerase chain reaction. We quantified RA by electrochemical analysis and examined the effect of RA supplementation on TNF-induced ileitis using histologic, coculture, and suppression assays and flow cytometry. RESULTS: Numbers of CD103+ DCs decreased in the inflamed ilea of mice with chronic disease; RA synthetic machinery (RALDH1,2) was down-regulated. Nevertheless, the proportion of CD4+, CD25+, FoxP3+ Tregs increased, indicating an alternate source for RA. IECs responded to reduced levels of RA by up-regulating RALDH3 in vivo and in vitro. Net tissue levels of RA remained lower in TNF+ARE than wild-type mice, indicating that epithelial up-regulation of RALDH3 could not maintain adequate concentrations of RA, probably because of loss of IEC mass. RA supplementation significantly attenuated disease by increasing the number and function of CD103+ DCs and Tregs and reducing Th17 cells. CONCLUSIONS: Reduced levels of RA appear to induce IECs to up-regulate synthesis of RA. RA supplementation attenuates ileitis through its effects on CD103+ DCs, Tregs, and Th17 cells. RA supplementation might offer therapeutic benefit in Crohn's disease.

CCR3 Blockade Attenuates Eosinophilic Ileitis and Associated Remodeling.

Intestinal remodeling and stricture formation is a complication of inflammatory bowel disease (IBD) that often requires surgical intervention. Although eosinophils are associated with mucosal remodeling in other organs and are increased in IBD tissues, their role in IBD-associated remodeling is unclear. Histological and molecular features of ileitis and remodeling were assessed using immunohistochemical, histomorphometric, flow cytometric, and molecular analysis (real-time RT-PCR) techniques in a murine model of chronic eosinophilic ileitis. Collagen protein was assessed by Sircol assay. Using a spontaneous eosinophilic Crohn's-like mouse model SAMP1/SkuSlc, we demonstrate an association between ileitis progression and remodeling over the course of 40 weeks. Mucosal and submucosal eosinophilia increased over the time course and correlated with increased histological inflammatory indices. Ileitis and remodeling increased over the 40 weeks, as did expression of fibronectin. CCR3-specific antibody-mediated reduction of eosinophils resulted in significant decrease in goblet cell hyperplasia, muscularis propria hypertrophy, villus blunting, and expression of inflammatory and remodeling genes, including fibronectin. Cellularity of local mesenteric lymph nodes, including T- and B-lymphocytes, was also significantly reduced. Thus, eosinophils participate in intestinal remodeling, supporting eosinophils as a novel therapeutic target.

Ets2 regulates colonic stem cells and sensitivity to tumorigenesis.

Ets2 has both tumor repressive and supportive functions for different types of cancer. We have investigated the role of Ets2 within intestinal epithelial cells in postnatal mouse colon development and tumorigenesis. Conditional inactivation of Ets2 within intestinal epithelial cells results in over representation of Ets2-deficient colon crypts within young and adult animals. This preferential representation is associated with an increased number of proliferative cells within the stem cell region and an increased rate of crypt fission in young mice that result in larger patches of Ets2-deficient crypts. These effects are consistent with a selective advantage of Ets2-deficient intestinal stem cells in colonizing colonic crypts and driving crypt fission. Ets2-deficient colon crypts have an increased mucosal thickness, an increased number of goblet cells, and an increased density. Mice with Ets2-deficient intestinal cells develop more colon tumors in response to treatment with azoxymethane and dextran sulfate sodium. The selective population of colon crypts, the altered differentiation state and increased sensitivity to carcinogen-induced tumors all indicate that Ets2 deficiency alters colon stem cell number or behavior. Ets2-dependent, epithelial cell-autonomous repression of intestinal tumors may contribute to protection from colon cancer of persons with increased dosage of chromosome 21.