Luciana R. Muniz

The cytomegalovirus-encoded chemokine receptor US28 promotes intestinal neoplasia in transgenic mice.

US28 is a constitutively active chemokine receptor encoded by CMV (also referred to as human herpesvirus 5), a highly prevalent human virus that infects a broad spectrum of cells, including intestinal epithelial cells (IECs). To study the role of US28 in vivo, we created transgenic mice (VS28 mice) in which US28 expression was targeted to IECs. Expression of US28 was detected in all IECs of the small and large intestine, including in cells expressing leucine rich repeat containing GPCR5 (Lgr5), a marker gene of intestinal epithelial stem cells. US28 expression in IECs inhibited glycogen synthase 3β (GSK-3β) function, promoted accumulation of β-catenin protein, and increased expression of Wnt target genes involved in the control of the cell proliferation. VS28 mice showed a hyperplastic intestinal epithelium and, strikingly, developed adenomas and adenocarcinomas by 40 weeks of age. When exposed to an inflammation-driven tumor model (azoxymethane/dextran sodium sulfate), VS28 mice developed a significantly higher tumor burden than control littermates. Transgenic coexpression of the US28 ligand CCL2 (an inflammatory chemokine) increased IEC proliferation as well as tumor burden, suggesting that the oncogenic activity of US28 can be modulated by inflammatory factors. Together, these results indicate that expression of US28 promotes development of intestinal dysplasia and cancer in transgenic mice and suggest that CMV infection may facilitate development of intestinal neoplasia in humans.

Intestinal antimicrobial peptides during homeostasis, infection, and disease

Antimicrobial peptides (AMPs), including defensins and cathelicidins, constitute an arsenal of innate regulators of paramount importance in the gut. The intestinal epithelium is exposed to myriad of enteric pathogens and these endogenous peptides are essential to fend off microbes and protect against infections. It is becoming increasingly evident that AMPs shape the composition of the commensal microbiota and help maintain intestinal homeostasis. They contribute to innate immunity, hence playing important functions in health and disease. AMP expression is tightly controlled by the engagement of pattern recognition receptors (PRRs) and their impairment is linked to abnormal host responses to infection and inflammatory bowel diseases (IBD). In this review, we provide an overview of the mucosal immune barriers and the intricate crosstalk between the host and the microbiota during homeostasis. We focus on the AMPs and pay particular attention to how PRRs promote their secretion in the intestine. Furthermore, we discuss their production and main functions in three different scenarios, at steady state, throughout infection with enteric pathogens and IBD.

A Critical Role for Dendritic Cells in the Formation of Lymphatic Vessels within Tertiary Lymphoid Structures

Ectopic, or tertiary, lymphoid aggregates often form in chronically inflamed areas. Lymphatic vessels, as well as high endothelial venules, form within these lymphoid aggregates, but the mechanisms underlying their development are poorly understood. Overexpression of the chemokine CCL21 in the thyroid of transgenic mice leads to formation of lymphoid aggregates containing topologically segregated T and B lymphocytes, dendritic cells (DCs), and specialized vasculature, including Lyve-1+/Prox-1+ lymphatic vessels. In this article, we show that adoptive transfer of mature CD4+ T cells into animals expressing CCL21 in a RAG-deficient background promotes the influx of host NK cells and DCs into the thyroid and the formation of new lymphatic vessels within 10 d. This process is dependent on the expression of lymphotoxin ligands by host cells, but not by the transferred CD4+ T cells. Ablation of host DCs, but not NK cells, reduces the formation of new lymphatic vessels in the thyroid. Taken together, these data suggest a critical role for CD11c+ DCs in the induction of lymphangiogenesis in tertiary lymphoid structures.

Different tissue phagocytes sample apoptotic cells to direct distinct homeostasis programs.

Recognition and removal of apoptotic cells by professional phagocytes, including dendritic cells and macrophages, preserves immune self-tolerance and prevents chronic inflammation and autoimmune pathologies. The diverse array of phagocytes that reside within different tissues, combined with the necessarily prompt nature of apoptotic cell clearance, makes it difficult to study this process in situ. The full spectrum of functions executed by tissue-resident phagocytes in response to homeostatic apoptosis, therefore, remains unclear. Here we show that mouse apoptotic intestinal epithelial cells (IECs), which undergo continuous renewal to maintain optimal barrier and absorptive functions, are not merely extruded to maintain homeostatic cell numbers, but are also sampled by a single subset of dendritic cells and two macrophage subsets within a well-characterized network of phagocytes in the small intestinal lamina propria. Characterization of the transcriptome within each subset before and after in situ sampling of apoptotic IECs revealed gene expression signatures unique to each phagocyte, including macrophage-specific lipid metabolism and amino acid catabolism, and a dendritic-cell-specific program of regulatory CD4+ T-cell activation. A common ‘suppression of inflammation’ signature was noted, although the specific genes and pathways involved varied amongst dendritic cells and macrophages, reflecting specialized functions. Apoptotic IECs were trafficked to mesenteric lymph nodes exclusively by the dendritic cell subset and served as critical determinants for the induction of tolerogenic regulatory CD4+ T-cell differentiation. Several of the genes that were differentially expressed by phagocytes bearing apoptotic IECs overlapped with susceptibility genes for inflammatory bowel disease. Collectively, these findings provide new insights into the consequences of apoptotic cell sampling, advance our understanding of how homeostasis is maintained within the mucosa and set the stage for development of novel therapeutics to alleviate chronic inflammatory diseases such as inflammatory bowel disease.

Distinguishing the immunostimulatory properties of noncoding RNAs expressed in cancer cells

Significance Using an approach derived from statistical physics, we quantify transcriptome-wide motif usage in human and murine noncoding RNAs (ncRNAs), determining that most have motif usage consistent with the coding genome. However, an outlier subset of tumor-associated ncRNAs comprises repetitive elements whose motif usage patterns are more typically associated with the genomes of inflammatory pathogens. We demonstrate that a key subset of these elements directly activates the cellular innate immune response. We propose that the innate response in tumors partially originates from direct interaction of immunogenic ncRNAs preferentially expressed in cancer cells with innate pattern recognition receptors. Recent studies have demonstrated abundant transcription of a set of noncoding RNAs (ncRNAs) preferentially within tumors as opposed to normal tissue. Using an approach from statistical physics, we quantify global transcriptome-wide motif use for the first time, to our knowledge, in human and murine ncRNAs, determining that most have motif use consistent with the coding genome. However, an outlier subset of tumor-associated ncRNAs, typically of recent evolutionary origin, has motif use that is often indicative of pathogen-associated RNA. For instance, we show that the tumor-associated human repeat human satellite repeat II (HSATII) is enriched in motifs containing CpG dinucleotides in AU-rich contexts that most of the human genome and human adapted viruses have evolved to avoid. We demonstrate that a key subset of these ncRNAs functions as immunostimulatory “self-agonists” and directly activates cells of the mononuclear phagocytic system to produce proinflammatory cytokines. These ncRNAs arise from endogenous repetitive elements that are normally silenced, yet are often very highly expressed in cancers. We propose that the innate response in tumors may partially originate from direct interaction of immunogenic ncRNAs expressed in cancer cells with innate pattern recognition receptors, and thereby assign a previously unidentified danger-associated function to a set of dark matter repetitive elements. These findings potentially reconcile several observations concerning the role of ncRNA expression in cancers and their relationship to the tumor microenvironment.

A Role for the Epidermal Growth Factor Receptor Signaling in Development of Intestinal Serrated Polyps in Mice and Humans

BACKGROUND & AIMS Epithelial cancers can be initiated by activating mutations in components of the mitogen-activated protein kinase signaling pathway such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), or epidermal growth factor receptor (EGFR). Human intestinal serrated polyps are a heterogeneous group of benign lesions, but some progress to colorectal cancer. Tumors that arise from these polyps frequently contain activating mutations in BRAF or KRAS, but little is known about the role of EGFR activation in their development. METHODS Polyp samples were obtained from adults during screening colonoscopies at Mount Sinai Hospital in New York. We measured levels of EGFR protein and phosphorylation in human serrated polyps by immunohistochemical and immunoblot analyses. We generated transgenic mice that express the ligand for EGFR, Heparin-binding EGF-like growth factor (HB-EGF), in the intestine. RESULTS EGFR and the extracellular-regulated kinases (ERK)1/2 were phosphorylated in serrated areas of human hyperplastic polyps (HPPs), sessile serrated adenomas, and traditional serrated adenomas. EGFR and ERK1/2 were phosphorylated in the absence of KRAS or BRAF activating mutations in a subset of HPP. Transgenic expression of the EGFR ligand HB-EGF in the intestines of mice promoted development of small cecal serrated polyps. Mice that expressed a combination of HB-EGF and US28 (a constitutively active, G-protein-coupled receptor that increases processing of HB-EGF from the membrane) rapidly developed large cecal serrated polyps. These polyps were similar to HPPs and had increased phosphorylation of EGFR and ERK1/2 within the serrated epithelium. Administration of pharmacologic inhibitors of EGFR or MAPK to these transgenic mice significantly reduced polyp development. CONCLUSIONS Activation of EGFR signaling in the intestine of mice promotes development of serrated polyps. EGFR signaling also is activated in human HPPs, sessile serrated adenomas, and traditional serrated adenomas.

Original ResearchBasic and Translational—Alimentary TractA Role for the Epidermal Growth Factor Receptor Signaling in Development of Intestinal Serrated Polyps in Mice and Humans

BACKGROUND & AIMS Epithelial cancers can be initiated by activating mutations in components of the mitogen-activated protein kinase signaling pathway such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), or epidermal growth factor receptor (EGFR). Human intestinal serrated polyps are a heterogeneous group of benign lesions, but some progress to colorectal cancer. Tumors that arise from these polyps frequently contain activating mutations in BRAF or KRAS, but little is known about the role of EGFR activation in their development. METHODS Polyp samples were obtained from adults during screening colonoscopies at Mount Sinai Hospital in New York. We measured levels of EGFR protein and phosphorylation in human serrated polyps by immunohistochemical and immunoblot analyses. We generated transgenic mice that express the ligand for EGFR, Heparin-binding EGF-like growth factor (HB-EGF), in the intestine. RESULTS EGFR and the extracellular-regulated kinases (ERK)1/2 were phosphorylated in serrated areas of human hyperplastic polyps (HPPs), sessile serrated adenomas, and traditional serrated adenomas. EGFR and ERK1/2 were phosphorylated in the absence of KRAS or BRAF activating mutations in a subset of HPP. Transgenic expression of the EGFR ligand HB-EGF in the intestines of mice promoted development of small cecal serrated polyps. Mice that expressed a combination of HB-EGF and US28 (a constitutively active, G-protein-coupled receptor that increases processing of HB-EGF from the membrane) rapidly developed large cecal serrated polyps. These polyps were similar to HPPs and had increased phosphorylation of EGFR and ERK1/2 within the serrated epithelium. Administration of pharmacologic inhibitors of EGFR or MAPK to these transgenic mice significantly reduced polyp development. CONCLUSIONS Activation of EGFR signaling in the intestine of mice promotes development of serrated polyps. EGFR signaling also is activated in human HPPs, sessile serrated adenomas, and traditional serrated adenomas.

Attenuation of TNF-driven murine ileitis by intestinal expression of the viral immunomodulator CrmD

Tumor necrosis factor α (TNFα) is a key pathogenic factor in Crohns disease and rheumatoid arthritis. TNFΔARE mice express high levels of TNFα and present Crohns-like ileitis and arthritis. Alterations in the chemokine network could underline the TNF-driven ileitis. The aim of this study was to evaluate the role of TNF and chemokines in ileitis using ectromelia virus cytokine response modifier D (CrmD), a protein that binds TNFα and a limited number of chemokines. We generated transgenic mice expressing CrmD in intestinal epithelial cells (vCrmD mice) and crossed them with the TNFΔARE mice to test whether CrmD could affect TNF-driven inflammatory processes. During homeostasis, only the number of B cells in the lamina propria was reduced by CrmD expression. Interestingly, CrmD expression in the intestine markedly attenuated the inflammatory infiltrates in the ileum of TNFΔARE mice, but did not affect development of arthritis. Our results suggest that CrmD affects development of ileitis by locally affecting both TNF and chemokine function in the ileum.

Abstract B169: Matrix metalloproteinase-2 and Toll-like receptors modulating immune responses in the tumor microenvironment

Successful anti-tumor immunotherapeutic approaches are improving survival in many malignancies but not all patients respond to these interventions, and several also relapse. This may result from an immunosuppressive tumor microenvironment (TME) that contributes towards tumor growth and metastasis while dampening anti-tumor immune responses. We have recently demonstrated that the metalloproteinases (MMPs) are produced within the TME. This production is associated with several cancers and can compromise tumor infiltrating lymphocyte (TILs) function. In particular, MMP-2 modulates dendritic cell (DC) function to skew T cells towards a deleterious T helper 2 (TH2) phenotype. MMP-2 was shown to inhibit Interleukin-12 (IL-12) and up-regulate OX40L expression thereby promoting TH2 function. Significantly, MMP-2 triggers Toll-like receptor 2 (TLR2) stimulation and NFκB activation, to promote OX40L expression and inflammatory cytokine production. Based on these results, the main purpose of this study is to dissect MMP-2s signaling via TLRs in the TME, which could promote immune suppression and tumor growth. We aim to access the signaling pathways downstream of the MMP-2/TLR2 axis in mouse bone marrow derived cells and immortalized cell lines. We also plan to evaluate MMP-29s role in T cell skewing in vivo and how this could affect tumor growth in the context of mouse B16 F1-induced melanomas. And finally we want to dissect the MMP-2/TLR signaling complex and identify the interactions between MMP-2, TLR2 and other proteins. We stimulated mouse primary cells or cell lines with MMP2, TLR agonists and appropriate controls in vitro and measured inflammatory cytokine secretion by cytometric bead array (CBA). Our preliminary results suggest that similar to our previous findings in humans, mouse bone marrow-derived DCs and Macrophages respond to MMP-2 and secrete pro-inflammatory cytokines such as Tumor Necrosis Factor (TNF) α and IL-6. Furthermore we found that in mouse immortalized bone marrow macrophage cell lines (Im-BMs), MMP-2 signaling is not only TLR2-, but also TLR4- and MyD88-dependent. While TLR2-/- and TLR4-/- Im-BMs do not show impaired TNFα secretion in response to MMP2, TLR2-/-/TLR4-/- double knockout cells demonstrate compromised signaling. Similarly MyD88-/- cells and TRIF-/-/MyD88-/- also showed impaired signaling whereas TRIF-/- cells did not, suggesting that MMP-2 signaling is mainly MyD88 dependent. Altogether, our data suggests an important role for the MMP-2/TLR axis in immune cell signaling that could be of importance in promoting tumor growth and eventually metastasis. Our results revealed that in mouse cells, TLR4 is also needed for MMP-2 signaling, which suggests species-specific requirements of the MMP-2 signaling pathway. Previous work has shown that targeting TLRs and endogenous alarmins are beneficial for control of tumor growth, thus targeting MMP-2 in the TME might be an additional way to control immune suppression in the TME. Citation Format: Luciana Ribeiro Muniz, Mansi Saxena, Nina Bhardwaj. Matrix metalloproteinase-2 and Toll-like receptors modulating immune responses in the tumor microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B169.

Abstract A087: Quantifying the landscape of immunostimulatory tumoral RNA

Recent studies demonstrate an unexpected connection between aberrant transcription of noncoding RNA in tumors and innate immune system activation in the tumor microenvironment. Such RNA is often of unknown function and may consist of typically silenced interspersed elements, satellite repeats, and endogenous retroviruses. For instance, satellite RNA from the pericentromere (particularly HSATII) is abundantly transcribed in several solid tumors - such as pancreatic ductal adenocarcinoma - yet it is virtually silent in normal tissue. The genomic DNA repetitive regions this RNA is transcribed from frequently expand during tumorigenesis. Using novel quantitative methods, we have shown that a set of such repetitive elements, abundantly expressed in tumors, display sequence patterns typically associated with viruses. We therefore predicted they are immunogenic, particularly HSATII. In a novel, theory-experiment collaboration between the laboratories of Professors Benjamin Greenbaum and Nina Bhardwaj, the most significant set of these RNA have been validated as immunostimulatory (HSATII and murine GSAT), capable of activating antigen presenting cells - HSATII stimulated production of IL-6, IL-12 and TNFalpha (Tanne, et al., PNAS, 2015). At the same time a set of recent papers has shown that ERV transcription may be a predictor of immunotherapy response in melanoma. Hence, it is critical to profile key immunostimulatory endogenous RNA in the tumor microenviroment, understand which immune pathways different sets of such RNA activate, and assess the link between the specific pathways activated, prognosis, and immunotherapy. We further profile the landscape of activation and expression for endogenous elements. We have currently demonstrated a key set of noncoding RNAs preferentially expressed in cancer cells have sequence features that are immunostimulatory in humans, and have characterized the range and breadth of the expression of such elements in several solid tumors. We present several new results on the topic and the potential consequences of the aberrant expression of endogenous RNA that mimic pathogen features in cancer. Citation Format: Alexander Solovyov, Antoine Tanne, Luciana Muniz, Simona Cocco, Remi Monasson, Arnold Levine, David T. Ting, Nina Bhardwaj, Benjamin Greenbaum. Quantifying the landscape of immunostimulatory tumoral RNA [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A087.