Leukotrienes and prostaglandins, products of arachidonic acid metabolism, sustain both systemic and lesion-localized inflammation. Tumor-associated Inflammation can also contribute to the pathogenesis of colon cancer. Patients with inflammatory bowel disease (IBD) have increased risk of developing colon cancer. The levels of 5-lipoxygenase (5-LO), the key enzyme for leukotrienes production, are increased in colon cancer specimens and colonic dysplastic lesions. Here we report that Zileuton, a specific 5-LO inhibitor, can prevent polyp formation by efficiently reducing the tumor-associated and systemic inflammation in APCΔ468 mice.

Colorectal cancer (CRC) is associated with the modern lifestyle. Chronic alcohol consumption-a frequent habit of majority of modern societies-increases the risk of CRC. Our group showed that chronic alcohol consumption increases polyposis in a mouse mode of CRC. Here we assess the effect of circadian disruption-another modern life style habit-in promoting alcohol-associated CRC.

Cellular senescence, a stress-induced irreversible growth arrest often characterized by expression of p16(Ink4a) (encoded by the Ink4a/Arf locus, also known as Cdkn2a) and a distinctive secretory phenotype, prevents the proliferation of preneoplastic cells and has beneficial roles in tissue remodelling during embryogenesis and wound healing. Senescent cells accumulate in various tissues and organs over time, and have been speculated to have a role in ageing. To explore the physiological relevance and consequences of naturally occurring senescent cells, here we use a previously established transgene, INK-ATTAC, to induce apoptosis in p16(Ink4a)-expressing cells of wild-type mice by injection of AP20187 twice a week starting at one year of age. We show that compared to vehicle alone, AP20187 treatment extended median lifespan in both male and female mice of two distinct genetic backgrounds. The clearance of p16(Ink4a)-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart and fat, where clearance preserved the functionality of glomeruli, cardio-protective KATP channels and adipocytes, respectively. Thus, p16(Ink4a)-positive cells that accumulate during adulthood negatively influence lifespan and promote age-dependent changes in several organs, and their therapeutic removal may be an attractive approach to extend healthy lifespan.

The targeting of the immune system through immunotherapies to prevent tumor tolerance and immune suppression are at the front lines of breast cancer treatment and research. Human and laboratory studies have attributed breast cancer progression and metastasis to secondary organs such as the bone, to a number of factors, including elevated levels of prostaglandin E2 (PGE2) and the enzyme responsible for its production, cyclooxygenase 2 (COX2). Due to the strong connection of COX2 with immune function, we focused on understanding how variance in COX2 expression manipulates the immune profile in a syngeneic, and immune-competent, mouse model of breast cancer. Though there have been correlative findings linking elevated levels of COX2 and Tregs in other cancer models, we sought to elucidate the mechanisms by which these immuno-suppressive cells are recruited to breast tumor and the means by which they promote tumor tolerance.

Mast cells (MCs) are tissue resident sentinels that mature and orchestrate inflammation in response to infection and allergy. While they are also frequently observed in tumors, the contribution of MCs to carcinogenesis remains unclear. Here, we show that sequential oncogenic events in gut epithelia expand different types of MCs in a temporal-, spatial-, and cytokine-dependent manner. The first wave of MCs expands focally in benign adenomatous polyps, which have elevated levels of IL-10, IL-13, and IL-33, and are rich in type-2 innate lymphoid cells (ILC2s). These vanguard MCs adhere to the transformed epithelial cells and express murine mast cell protease 2 (mMCP2; a typical mucosal MC protease) and, to a lesser extent, the connective tissue mast cell (CTMC) protease mMCP6. Persistence of MCs is strictly dependent on T cell-derived IL-10, and their loss in the absence of IL-10-expressing T cells markedly delays small bowel (SB) polyposis. MCs expand profusely in polyposis-prone mice when T cells overexpress IL-10. The frequency of polyp-associated MCs is unaltered in response to broad-spectrum antibiotics, arguing against a microbial component driving their recruitment. Intriguingly, when polyps become invasive, a second wave of mMCP5+/mMCP6+ CTMCs expands in the tumor stroma and at invasive tumor borders. Ablation of mMCP6 expression attenuates polyposis, but invasive properties of the remaining lesions remain intact. Our findings argue for a multistep process in SB carcinogenesis in which distinct MC subsets, and their elaborated proteases, guide disease progression.

Progressive scarring is ubiquitous postoperatively and in an array of chronic systemic diseases. Recent studies indicate that such scarring has a high female propensity; females are also almost exclusively affected by endometriosis, a common sex steroid-dependent fibrotic disease. Endometriosis-related fibrosis is regulated epigenetically through transcription factor Krüppel-like factor 11 (KLF11). In response to surgical induction of endometriosis, Klf11-/- female mice develop significant fibrosis in contrast to wild-type mice. We therefore hypothesized that female fibrotic predilection was mediated by differential sex steroid regulation of KLF11/collagen 1a1 signaling and investigated the fibrotic response in wild-type and Klf11-/- male and female animals using a sterile peritonitis model. Fibrosis selectively developed in Klf11-/- females. Fibrosis in these animals was almost completely abrogated by ovariectomy. Ovariectomized animals were selectively supplemented with estradiol, medroxyprogesterone acetate (MPA), or dihydrotestosterone; fibrosis was only observed in mice exposed to MPA. Fibrosis therefore selectively developed in Klf11-/- female mice in response to physiological or pharmacological progesterone. The fibrotic response in these animals was also mitigated in response to antiprogestin therapy. Profibrotic gene expression was activated in a primary human peritoneal cell line in response to KLF11 short hairpin RNA and MPA but not estradiol. KLF11/collagen 1a1 signaling previously shown to be linked to fibrosis was thus selectively dysregulated in MPA-treated cells. Our in vivo and in vitro findings in an animal model and human cells, respectively, suggest that progressive fibrotic scarring is a sexually dimorphic response irrespective of etiology; moreover, it is responsive to novel, individualized therapeutic intervention.

Chronic liver scarring from any cause leads to cirrhosis, portal hypertension, and a progressive decline in renal blood flow and renal function. Extreme renal vasoconstriction characterizes hepatorenal syndrome, a functional and potentially reversible form of acute kidney injury in patients with advanced cirrhosis, but current therapy with systemic vasoconstrictors is ineffective in a substantial proportion of patients and is limited by ischemic adverse events. Serelaxin (recombinant human relaxin-2) is a peptide molecule with anti-fibrotic and vasoprotective properties that binds to relaxin family peptide receptor-1 (RXFP1) and has been shown to increase renal perfusion in healthy human volunteers. We hypothesized that serelaxin could ameliorate renal vasoconstriction and renal dysfunction in patients with cirrhosis and portal hypertension.

Mast cells constitutively express ß-catenin and expand in solid tumors such as colon and skin cancer. However, the role of ß-catenin signaling in mast cells and the cause or effect of mast cell expansion and tumor growth has yet to be established. In earlier studies we used mast cell depletion and protease staining approaches, to provide evidence for a causative role of mast cells in small bowel polyposis, and related specific phenotypes and distributions of tumor infiltrating mast cells to stages of tumor growth. Here we report that, stabilization of ß-catenin expands mast cells to promote high incidence of colon polyposis and infrequent small bowel polyps and skin cancer. Expression of a dominant acting ß-catenin in mast cells (5CreCAT) stimulated maturation and expression of granule stored proteases. Both mucosal and connective tissue type mast cells accumulated in colonic small bowel polyps independent of gender, and mice developed chronic systemic inflammation with splenomegaly. Reconstitution of polyposis-prone mice with bone marrow from 5CreCAT mice resulted in focal expansion of connective tissue like mast cells, which are normally rare in benign polyps and characteristically expand during adenoma-to-carcinoma transition. Our findings highlight a hitherto unknown contribution of ß-catenin signaling in mast cells to their maturation and to increased risk of colon cancer.

Many tumors exhibit increased incorporation of sialic acids into cell-surface glycans, which impact the tumor microenvironment. Sialic acid immunoglobulin-like lectins (Siglec) are receptors that recognize sialic acids and modulate immune responses, including responses to tumors. However, the roles of individual sialyltransferases in tumorigenesis and tumor growth are not well understood. Here, we examined the sialyltransferase ST8Sia6, which generated α2,8-linked disialic acids that bind to murine Siglec-E and human Siglec-7 and -9. Increased ST8Sia6 expression was found on many human tumors and associated with decreased survival in several cancers, including colon cancer. Because of this, we engineered MC38 and B16-F10 tumor lines to express ST8Sia6. ST8Sia6-expressing MC38 and B16-F10 tumors exhibited faster growth and led to decreased survival, which required host Siglec-E. ST8Sia6 expression on tumors also altered macrophage polarization toward M2, including upregulation of the immune modulator arginase, which also required Siglec-E. ST8Sia6 also accelerated tumorigenesis in a genetically engineered, spontaneous murine model of colon cancer, decreasing survival from approximately 6 months to 67 days. Thus, ST8Sia6 expression on tumors inhibits antitumor immune responses to accelerate tumor growth.

Recent studies indicate that mural cells of the preglomerular vessels, known as cells of renin lineage (CoRL), contribute to repair and regeneration of injured kidney glomeruli. However, their potential roles in tubulointerstitial disease are less understood. The aim of this study was to better understand CoRL number and distribution following UUO so that future mechanistic studies could be undertaken.

To validate the feasibility of labeling Clostridium novyi-NT (C.novyi-NT) anaerobes with iron-oxide nanoparticles for magnetic resonance imaging (MRI) and demonstrate the potential to use MRI to visualize intra-tumoral delivery of these iron-oxide labeled C.novyi-NT during percutaneous injection procedures.

MicroRNAs, activated by the enzyme Dicer1, control post-transcriptional gene expression. Dicer1 has important roles in the epithelium during nephrogenesis, but its function in stromal cells during kidney development is unknown. To study this, we inactivated Dicer1 in renal stromal cells. This resulted in hypoplastic kidneys, abnormal differentiation of the nephron tubule and vasculature, and perinatal mortality. In mutant kidneys, genes involved in stromal cell migration and activation were suppressed as were those involved in epithelial and endothelial differentiation and maturation. Consistently, polarity of the proximal tubule was incorrect, distal tubule differentiation was diminished, and elongation of Henle's loop attenuated resulting in lack of inner medulla and papilla in stroma-specific Dicer1 mutants. Glomerular maturation and capillary loop formation were abnormal, whereas peritubular capillaries, with enhanced branching and increased diameter, formed later. In Dicer1-null renal stromal cells, expression of factors associated with migration, proliferation, and morphogenic functions including α-smooth muscle actin, integrin-α8, -β1, and the WNT pathway transcriptional regulator LEF1 were reduced. Dicer1 mutation in stroma led to loss of expression of distinct microRNAs. Of these, miR-214, -199a-5p, and -199a-3p regulate stromal cell functions ex vivo, including WNT pathway activation, migration, and proliferation. Thus, Dicer1 activity in the renal stromal compartment regulates critical stromal cell functions that, in turn, regulate differentiation of the nephron and vasculature during nephrogenesis.

Loss of the microvascular (MV) network results in tissue ischemia, loss of tissue function, and is a hallmark of chronic diseases. The incorporation of a functional vascular network with that of the host remains a challenge to utilizing engineered tissues in clinically relevant therapies. We showed that vascular-bed-specific endothelial cells (ECs) exhibit differing angiogenic capacities, with kidney microvascular endothelial cells (MVECs) being the most deficient, and sought to explore the underlying mechanism. Constitutive activation of the phosphatase PTEN in kidney MVECs resulted in impaired PI3K/AKT activity in response to vascular endothelial growth factor (VEGF). Suppression of PTEN in vivo resulted in microvascular regeneration, but was insufficient to improve tissue function. Promoter analysis of the differentially regulated genes in KMVECs suggests that the transcription factor FOXO1 is highly active and RNAseq analysis revealed that hyperactive FOXO1 inhibits VEGF-Notch-dependent tip-cell formation by direct and indirect inhibition of DLL4 expression in response to VEGF. Inhibition of FOXO1 enhanced angiogenesis in human bio-engineered capillaries, and resulted in microvascular regeneration and improved function in mouse models of injury-repair.

Mutations resulting in overexpression of intracellular Notch1 (ICN1) are frequently observed in human T cell acute lymphoblastic leukemia (T-ALL). We have determined the consequences of ICN1 overexpression from retroviral vectors introduced into bone marrow cells. Early consequences are the generation of polyclonal nontumorigenic CD4(+)8(+) T cell receptor (TCR)-alphabeta(+) cells that do not qualify as tumor precursors despite the observation that they overexpress Notch 1 and c-Myc and degrade the tumor suppressor E2A by posttranslational modification. The first tumorigenic cells are detected among more immature CD4(-)8(+)TCR-alphabeta(-) cells that give rise to monoclonal tumors with a single, unique TCR-beta chain and diverse TCR-alpha chains, pinpointing malignant transformation to a stage after pre-TCR signaling and before completion of TCR-alpha rearrangement. In T-ALL, E2A deficiency is accompanied by further transcriptional up-regulation of c-Myc and concomitant dysregulation of the c-Myc-p53 axis at the transcriptional level. Even though the tumors consist of phenotypically heterogeneous cells, no evidence for tumor stem cells was found. As judged by array-based comparative genomic hybridization (array CGH) and spectral karyotype (SKY) analysis, none of the tumors arise because of genomic instability.

Mechanisms of dominant tolerance have evolved within the mammalian immune system to prevent inappropriate immune responses. CD4(+)CD25(+) regulatory T (T(reg)) cells have emerged as central constituents of this suppressive activity. By using multiphoton intravital microscopy in lymph nodes (LNs) of anesthetized mice, we have analyzed how cytotoxic T lymphocytes (CTLs) interact with antigen-presenting target cells in the presence or absence of activated T(reg) cells. Nonregulated CTLs killed their targets at a 6.6-fold faster rate than regulated CTLs. In spite of this compromised effector activity, regulated CTLs exhibited no defect in proliferation, induction of cytotoxic effector molecules and secretory granules, in situ motility, or ability to form antigen-dependent conjugates with target cells. Only granule exocytosis by CTLs was markedly impaired in the presence of T(reg) cells. This selective form of regulation did not require prolonged contact between CTLs and T(reg) cells but depended on CTL responsiveness to transforming growth factor-beta. CTLs quickly regained full killing capacity in LNs upon selective removal of T(reg) cells. Thus, T(reg) cells reversibly suppress CTL-mediated immunity by allowing acquisition of full effector potential but withholding the license to kill.

With increasing age, the kidney undergoes characteristic changes in the glomerular and tubulo-interstitial compartments, which are ultimately accompanied by reduced kidney function. Studies have shown age-related loss of peritubular vessels. Normal peritubular vessel tone, function and survival depend on neighboring pericytes. Pericyte detachment leads to vascular damage, which can be accompanied by their differentiation to fibroblasts and myofibroblasts, a state that favors matrix production. To better understand the fate of pericytes in the aged kidney, 27 month-old mice were studied. Compared to 3 month-old young adult mice, aged kidneys showed a substantial decrease in capillaries, identified by CD31 staining, in both cortex and medulla. This was accompanied by a marked decrease in surrounding NG2+ / PDGFRβ+ pericytes. This decrease was more pronounced in the medulla. Capillaries devoid of pericytes were typically dilated in aged mice. Aged kidneys were also characterized by interstitial fibrosis due to increased collagen-I and -III staining. This was accompanied by an increase in the number of pericytes that acquired a pro-fibrotic phenotype, identified by increased PDGFRβ+ / αSMA+ staining. These findings are consistent with the decline in kidney interstitial pericytes as a critical step in the development of changes to the peritubular vasculature with aging, and accompanying fibrosis.

Endometriosis is a highly prevalent, chronic, heterogeneous, fibro-inflammatory disease that remains recalcitrant to conventional therapy. We previously showed that loss of KLF11, a transcription factor implicated in uterine disease, results in progression of endometriosis. Despite extensive homology, co-expression, and human disease association, loss of the paralog Klf10 causes a unique inflammatory, cystic endometriosis phenotype in contrast to fibrotic progression seen with loss of Klf11. We identify here for the first time a novel role for KLF10 in endometriosis. In an animal endometriosis model, unlike wild-type controls, Klf10(-/-) animals developed cystic lesions with massive immune infiltrate and minimal peri-lesional fibrosis. The Klf10(-/-) disease progression phenotype also contrasted with prolific fibrosis and minimal immune cell infiltration seen in Klf11(-/-) animals. We further found that lesion genotype rather than that of the host determined each unique disease progression phenotype. Mechanistically, KLF10 regulated CD40/CD154-mediated immune pathways. Both inflammatory as well as fibrotic phenotypes are the commonest clinical manifestations in chronic fibro-inflammatory diseases such as endometriosis. The complementary, paralogous Klf10 and Klf11 models therefore offer novel insights into the mechanisms of inflammation and fibrosis in a disease-relevant context. Our data suggests that divergence in underlying gene dysregulation critically determines disease-phenotype predominance rather than the conventional paradigm of inflammation being precedent to fibrotic scarring. Heterogeneity in clinical progression and treatment response are thus likely from disparate gene regulation profiles. Characterization of disease phenotype-associated gene dysregulation offers novel approaches for developing targeted, individualized therapy for recurrent and recalcitrant chronic disease.

TGFβ has both tumor suppressive and tumor promoting effects in colon cancer. Also, TGFβ can affect the extent and composition of inflammatory cells present in tumors, contextually promoting and inhibiting inflammation. While colon tumors display intratumoral inflammation, the contributions of TGFβ to this process are poorly understood. In human patients, we found that epithelial loss of TGFβ signaling was associated with increased inflammatory burden; yet overexpression of TGFβ was also associated with increased inflammation. These findings were recapitulated in mutant APC models of murine tumorigenesis, where epithelial truncation of TGFBR2 led to lethal inflammatory disease and invasive colon cancer, mediated by IL8 and TGFβ1. Interestingly, mutant APC mice with global suppression of TGFβ signals displayed an intermediate phenotype, presenting with an overall increase in IL8-mediated inflammation and accelerated tumor formation, yet with a longer latency to the onset of disease observed in mice with epithelial TGFBR-deficiency. These results suggest that the loss of TGFβ signaling, particularly in colon epithelial cells, elicits a strong inflammatory response and promotes tumor progression. This implies that treating colon cancer patients with TGFβ inhibitors may result in a worse outcome by enhancing inflammatory responses.

Increased toxicant exposure and resultant environmentally induced diseases are a tradeoff of industrial productivity. Dioxin [2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD)], a ubiquitous byproduct, is associated with a spectrum of diseases including endometriosis, a common, chronic disease in women. TCDD activates cytochrome (CYP) p450 metabolic enzymes that alter organ function to cause disease. In contrast, the transcription factor, Krüppel-like factor (KLF) 11, represses these enzymes via epigenetic mechanisms. In this study, we characterized these opposing mechanisms in vitro and in vivo as well as determining potential translational implications of epigenetic inhibitor therapy. KLF11 antagonized TCDD-mediated activation of CYP3A4 gene expression and function in endometrial cells. The repression was pharmacologically replicated by selective use of an epigenetic histone acetyltransferase inhibitor (HATI). We further showed phenotypic relevance of this mechanism using an animal model for endometriosis. Fibrotic extent in TCDD-exposed wild-type animals was similar to that previously observed in Klf11-/- animals. When TCDD-exposed animals were treated with a HATI, Cyp3 messenger RNA levels and protein expression decreased along with disease progression. Fibrotic progression is ubiquitous in environmentally induced chronic, untreatable diseases; this report shows that relentless disease progression can be arrested through targeted epigenetic modulation of protective mechanisms.

Disruption of central circadian rhythms likely mediated by changes in microbiota and a decrease in gut-derived metabolites like short chain fatty acids (SCFAs) negatively impacts colonic barrier homeostasis. We aimed to explore the effects of isolated peripheral colonic circadian disruption on the colonic barrier in a mouse model of colitis and explore the mechanisms, including intestinal microbiota community structure and function.