Supplementary Table 1 from Dietary Induction of Colonic Tumors in a Mouse Model of Sporadic Colon Cancer

Supplementary Table 2 from Interaction of Muc2 and Apc on Wnt Signaling and in Intestinal Tumorigenesis: Potential Role of Chronic Inflammation

Supplementary Figures 1-8 from Interaction of Muc2 and Apc on Wnt Signaling and in Intestinal Tumorigenesis: Potential Role of Chronic Inflammation

Supplementary Materials and Figure Legends 1-8 from Interaction of Muc2 and Apc on Wnt Signaling and in Intestinal Tumorigenesis: Potential Role of Chronic Inflammation

Supplementary Figures 1-8 from Interaction of Muc2 and Apc on Wnt Signaling and in Intestinal Tumorigenesis: Potential Role of Chronic Inflammation

Supplementary Table 2 from Dietary Induction of Colonic Tumors in a Mouse Model of Sporadic Colon Cancer

Cerebral cavernous malformation (CCM) is a genetic, cerebrovascular disease. Familial CCM is caused by genetic mutations in KRIT1, CCM2, or PDCD10. Disease onset is earlier and more severe in individuals with PDCD10 mutations. Recent studies have shown that lesions arise from excess mitogen-activated protein kinase kinase kinase 3 (MEKK3) signaling downstream of Toll-like receptor 4 (TLR4) stimulation by lipopolysaccharide derived from the gut microbiome. These findings suggest a gut-brain CCM disease axis but fail to define it or explain the poor prognosis of patients with PDCD10 mutations. Here, we demonstrate that the gut barrier is a primary determinant of CCM disease course, independent of microbiome configuration, that explains the increased severity of CCM disease associated with PDCD10 deficiency. Chemical disruption of the gut barrier with dextran sulfate sodium augments CCM formation in a mouse model, as does genetic loss of Pdcd10, but not Krit1, in gut epithelial cells. Loss of gut epithelial Pdcd10 results in disruption of the colonic mucosal barrier. Accordingly, loss of Mucin-2 or exposure to dietary emulsifiers that reduce the mucus barrier increases CCM burden analogous to loss of Pdcd10 in the gut epithelium. Last, we show that treatment with dexamethasone potently inhibits CCM formation in mice because of the combined effect of action at both brain endothelial cells and gut epithelial cells. These studies define a gut-brain disease axis in an experimental model of CCM in which a single gene is required for two critical components: gut epithelial function and brain endothelial signaling.

The mucus layer in the intestine affects several aspects of intestinal biology, encompassing physical, chemical protection, immunomodulation and growth, thus contributing to homeostasis. Mice with genetic inactivation of the Muc2 gene, encoding the MUC2 mucin, the major protein component of mucus, exhibit altered intestinal homeostasis, which is strictly dependent on the habitat, likely due to differing complements of intestinal microbes. Our previous work established that Muc2 deficiency was linked to low chronic inflammation resulting in tumor development in the small, large intestine including the rectum. Here, we report that inactivation of Muc2 alters metabolic pathways in the normal appearing mucosa of Muc2-/- mice. Comparative analysis of gene expression profiling of isolated intestinal epithelial cells (IECs) and the entire intestinal mucosa, encompassing IECs, immune and stromal cells underscored that more than 50% of the changes were common to both sets of data, suggesting that most alterations were IEC-specific. IEC-specific expression data highlighted perturbation of lipid absorption, processing and catabolism linked to altered Pparα signaling in IECs. Concomitantly, alterations of glucose metabolism induced expression of genes linked to de novo lipogenesis, a characteristic of tumor cells. Importantly, gene expression alterations characterizing Muc2-/- IECs are similar to those observed when analyzing the gene expression signature of IECs along the crypt-villus axis in WT B6 mice, suggesting that Muc2-/- IECs display a crypt-like gene expression signature. Thus, our data strongly suggest that decreased lipid metabolism, and alterations in glucose utilization characterize the crypt proliferative compartment, and may represent a molecular signature of pre-neoplastic lesions.

Background and Purpose: Currently, no readily available mitigators exist for acute abdominal radiation injury. Here, we present an animal model for precise and homogenous limb-sparing abdominal irradiation (LSAIR) to study the radiation-induced gastrointestinal syndrome (RIGS). Materials and Methods: The LSAIR technique was developed using the small animal radiation research platform (SARRP) with image guidance capabilities. We delivered LSAIR at doses between 14 and 18 Gy on 8- to 10-week-old male C57BL/6 mice. Histological analysis was performed to confirm that the observed mortality was due to acute abdominal radiation injury. Results: A steep dose–response relationship was found for survival, with no deaths seen at doses below 16 Gy and 100% mortality at above 17 Gy. All deaths occurred between 6 and 10 days after irradiation, consistent with the onset of RIGS. This was further confirmed by histological analysis showing clear differences in the number of regenerative intestinal crypts between animals receiving sublethal (14 Gy) and 100% lethal (18 Gy) radiation. Conclusion: The developed LSAIR technique provides uniform dose delivery with a clear dose response, consistent with acute abdominal radiation injury on histological examination. This model can provide a useful tool for researchers investigating the development of mitigators for accidental or clinical high-dose abdominal irradiation.

Expression of a germline VH3609/D/JH2 IgH in mice results in the generation of B1 B cells with anti-thymocyte/Thy-1 glycoprotein autoreactivity by coexpression of Vk21-5/Jk2 L chain leading to production of serum IgM natural autoantibody. In these same mice, the marginal zone (MZ) B cell subset in spleen shows biased usage of a set of Ig L chains different from B1 B cells, with 30% having an identical Vk19-17/Jk1 L chain rearrangement. This VH3609/Vk19-17 IgM is reactive with intestinal goblet cell granules, binding to the intact large polymatrix form of mucin 2 glycoprotein secreted by goblet cells. Analysis of a μκ B cell AgR (BCR) transgenic (Tg) mouse with this anti–goblet cell/mucin2 autoreactive (AGcA) specificity demonstrates that immature B cells expressing the Tg BCR become MZ B cells in spleen by T cell–independent BCR signaling. These Tg B cells produce AGcA as the predominant serum IgM, but without enteropathy. Without the transgene, AGcA autoreactivity is low but detectable in the serum of BALB/c and C.B17 mice, and this autoantibody is specifically produced by the MZ B cell subset. Thus, our findings reveal that AGcA is a natural autoantibody associated with MZ B cells.

We sought to determine if single-dose external beam radiation therapy (EBRT) could modulate the expression signature of T-cell costimulatory and coinhibitory molecules in human prostate cancer (PCa) cell lines in vitro. We investigated the functional impact of irradiated PCa cells with a modulated costimulatory profile on responder T-cell activity. We used three PCa cell lines (DU145, PC3, and LNCaP) and two epithelial cell lines from noncancerous prostate and lung tissue. After 72 hours of EBRT, surface expression of four immunostimulatory molecules (CD70, CD275/ICOSL, CD134L/OX40L, and CD137L/41BBL) and two immunosuppressive markers (CTLA-4/CD152 and PD-L1/CD274) were evaluated by flow cytometry. We evaluated the impact of several radiation doses and the longevity of modulated expression. We examined the functional impact of radiation-induced modulation of cancer cells by cytotoxic T cells (CTL) cytotoxicity and ELISPOT assay for interferon-gamma (IFN-γ) production. Last, we evaluated whether IFN-γ-induced PD-L1 expression could be reversed by EBRT. After 10 Gy EBRT, expression of OX40L and 41BBL increased in all three PCa cell lines; expression of CD70 and ICOSL increased in PC3 cells. Conversely, a decrease in PD-L1 expression in DU145 and PC3 cells was detectable up to 144 hours after EBRT. No PD-L1 was detected in LNCaP. Epithelial cells from normal prostate were not modulated by radiation. CTL cytolytic activity and IFN-γ production were enhanced by interaction with irradiated PCa cells. Finally, EBRT failed to prevent IFN-γ-induced upregulation of PD-L1. We demonstrate that a single dose of EBRT increased surface expression of costimulatory molecules and decreased the expression of coinhibitory molecules in human PCa cell lines. Changes in irradiated tumor cells led to functional enhancement of T-cell activity, despite EBRT failing to reduce IFN-γ-induced expression of PD-L1. These data suggest that combining radiotherapy with T-cell stimulating immunotherapy may be an attractive strategy for cancer treatment.

Background: The colonic mucus layer plays a critical role in intestinal homeostasis by limiting contact between luminal bacteria and the mucosal immune system. A defective mucus barrier in animal models allows bacterial contact with the intestinal epithelium and results in spontaneous colitis. A defective mucus barrier is also a key feature of active ulcerative colitis (UC). Alterations in the immune compartment due to intestinal bacterial breach in mice lacking the colon mucus barrier have not been characterized and correlated to active UC.Aims: To characterize alterations in the immune compartment due to intestinal bacterial breach in Muc2(-/-) mice, which lack the colon mucus barrier, and correlate the findings to active UC.Methods: Bacterial contact with colon epithelium and penetration into colon tissue was examined in Muc2(-/-) mice and colon biopsies from patients with active UC using fluorescence microscopy and qPCR. Neutrophils, lymphocytes, CD103(+) dendritic cell subsets and macrophages in colon from Muc2(-/-) mice and biopsies from UC patients were quantitated by flow cytometry.Results: Inflamed UC patients and Muc2(-/-) mice had bacteria in contact with the colon epithelium. Bacterial rRNA was present in colonic mucosa in humans and Muc2(-/-) mice and in the draining lymph nodes of mice. Inflamed Muc2(-/-) mice and UC patients had elevated colon neutrophils, T cells and macrophages while a reduced frequency of CD103(+) DCs was present in the inflamed colon of both mice and humans.Conclusions: The parallel features of the colon immune cell compartment in Muc2(-/-) mice and UC patients supports the usefulness of this model to understand the early phase of spontaneous colitis and will provide insight into novel strategies to treat UC.

Purpose/Objective(s)Radiation-induced gastrointestinal syndrome (RIGS) results in crypt cell, enterocyte damage and death, followed by villi shortening, malabsorption, sepsis and death. Agents that accelerate repair and regeneration of irradiated intestinal stem cells are essential for mitigation of RIGS. In order to develop a human intestinal model to screen for radiomitigating agents, we optimized and characterized the in vitro differentiation of skin iPSCs into iHIOs as a robust human intestinal organoid model to screen for radiomitigators.Materials/MethodsHuman iPSCs derived from skin fibroblasts and dental pulp cells were reprogrammed using retroviral vectors expressing the Yamanaka factors (Sox2, Oct4, KLF4 and c-Myc). Directed differentiation of iPSCs to definitive endoderm was induced with Activin A, followed by intestinal specification with FGF4 and Wnt3a exposure. The floating spheroids formed on day 6 were embedded in matrigel promoting intestinal growth and differentiation in the presence of R-spondin 1, noggin and EGF.ResultsRT-PCR analysis with an extensive panel of intestinal stage-specific markers demonstrated clear progression from activin-induced definitive endoderm formation to FGF and Wnt-induced posterior endoderm patterning, hindgut specification, and finally to a 3D culture system to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting iHIOs consisted of a columnar epithelium that was patterned into villus like structure and crypt-like proliferative cells that expressed intestinal stem cell markers (Sox9, CDX2, Lgr5, Bmx1, and Klf4). Further characterization was performed by immunofluorescence for intestinal differentiation markers (Muc2, Lysozyme, Ki67, and E-Cadherin) to identify intestinal cell lineages - enterocytes, goblet, paneth and endocrine cells. The effect of potential radiomitigators is being investigated in this model.ConclusionsWe have developed a protocol to culture iHIOs from human iPSCs. This will provide us a platform to investigate intestinal regeneration post-irradiation and to identify potential radiomitigators to treat RIGS. Purpose/Objective(s)Radiation-induced gastrointestinal syndrome (RIGS) results in crypt cell, enterocyte damage and death, followed by villi shortening, malabsorption, sepsis and death. Agents that accelerate repair and regeneration of irradiated intestinal stem cells are essential for mitigation of RIGS. In order to develop a human intestinal model to screen for radiomitigating agents, we optimized and characterized the in vitro differentiation of skin iPSCs into iHIOs as a robust human intestinal organoid model to screen for radiomitigators. Radiation-induced gastrointestinal syndrome (RIGS) results in crypt cell, enterocyte damage and death, followed by villi shortening, malabsorption, sepsis and death. Agents that accelerate repair and regeneration of irradiated intestinal stem cells are essential for mitigation of RIGS. In order to develop a human intestinal model to screen for radiomitigating agents, we optimized and characterized the in vitro differentiation of skin iPSCs into iHIOs as a robust human intestinal organoid model to screen for radiomitigators. Materials/MethodsHuman iPSCs derived from skin fibroblasts and dental pulp cells were reprogrammed using retroviral vectors expressing the Yamanaka factors (Sox2, Oct4, KLF4 and c-Myc). Directed differentiation of iPSCs to definitive endoderm was induced with Activin A, followed by intestinal specification with FGF4 and Wnt3a exposure. The floating spheroids formed on day 6 were embedded in matrigel promoting intestinal growth and differentiation in the presence of R-spondin 1, noggin and EGF. Human iPSCs derived from skin fibroblasts and dental pulp cells were reprogrammed using retroviral vectors expressing the Yamanaka factors (Sox2, Oct4, KLF4 and c-Myc). Directed differentiation of iPSCs to definitive endoderm was induced with Activin A, followed by intestinal specification with FGF4 and Wnt3a exposure. The floating spheroids formed on day 6 were embedded in matrigel promoting intestinal growth and differentiation in the presence of R-spondin 1, noggin and EGF. ResultsRT-PCR analysis with an extensive panel of intestinal stage-specific markers demonstrated clear progression from activin-induced definitive endoderm formation to FGF and Wnt-induced posterior endoderm patterning, hindgut specification, and finally to a 3D culture system to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting iHIOs consisted of a columnar epithelium that was patterned into villus like structure and crypt-like proliferative cells that expressed intestinal stem cell markers (Sox9, CDX2, Lgr5, Bmx1, and Klf4). Further characterization was performed by immunofluorescence for intestinal differentiation markers (Muc2, Lysozyme, Ki67, and E-Cadherin) to identify intestinal cell lineages - enterocytes, goblet, paneth and endocrine cells. The effect of potential radiomitigators is being investigated in this model. RT-PCR analysis with an extensive panel of intestinal stage-specific markers demonstrated clear progression from activin-induced definitive endoderm formation to FGF and Wnt-induced posterior endoderm patterning, hindgut specification, and finally to a 3D culture system to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting iHIOs consisted of a columnar epithelium that was patterned into villus like structure and crypt-like proliferative cells that expressed intestinal stem cell markers (Sox9, CDX2, Lgr5, Bmx1, and Klf4). Further characterization was performed by immunofluorescence for intestinal differentiation markers (Muc2, Lysozyme, Ki67, and E-Cadherin) to identify intestinal cell lineages - enterocytes, goblet, paneth and endocrine cells. The effect of potential radiomitigators is being investigated in this model. ConclusionsWe have developed a protocol to culture iHIOs from human iPSCs. This will provide us a platform to investigate intestinal regeneration post-irradiation and to identify potential radiomitigators to treat RIGS. We have developed a protocol to culture iHIOs from human iPSCs. This will provide us a platform to investigate intestinal regeneration post-irradiation and to identify potential radiomitigators to treat RIGS.

Guardian of the Gut The intestine is able to tolerate continual exposure to large amounts of commensal bacteria and foreign food antigens without triggering an inappropriate inflammatory immune response. In the large intestine, this immunological tolerance is thought to occur via a physical separation between environment and host imposed by a continuous mucous layer built up from the secreted mucin protein, MUC2. However, in the small intestine, this mucous layer is porous, necessitating an additional layer of immune control. Shan et al. (p. 447 , published online 26 September; see the Perspective by Belkaid and Grainger ) now report that in the small intestine, MUC2 plays an active role in immunological tolerance by activating a transcription factor in resident dendritic cells, thereby selectively blocking their ability to launch an inflammatory response. This work identifies MUC2 as a central mediator of immune tolerance to maintain homeostasis in the gut and possibly at other mucosal surfaces in the body.

ABSTRACT Salmonella enterica serovar Typhimurium is a model organism used to explore the virulence strategies underlying Salmonella pathogenesis. Although intestinal mucus is the first line of defense in the intestine, its role in protection against Salmonella is still unclear. The intestinal mucus layer is composed primarily of the Muc2 mucin, a heavily O-glycosylated glycoprotein. The core 3-derived O-glycans of Muc2 are synthesized by core 3 β1,3- N -acetylglucosaminyltransferase (C3GnT). Mice lacking these glycans still produce Muc2 but display a thinner intestinal mucus barrier. We began our investigations by comparing Salmonella -induced colitis and mucus dynamics in Muc2 -deficient ( Muc2 −/− ) mice, C3GnT −/− mice, and wild-type C57BL/6 (WT) mice. Salmonella infection led to increases in luminal Muc2 secretion in WT and C3GnT −/− mice. When Muc2 −/− mice were infected with Salmonella , they showed dramatic susceptibility to infection, carrying significantly higher cecal and liver pathogen burdens, and developing significantly higher barrier disruption and higher mortality rates, than WT mice. We found that the exaggerated barrier disruption in infected Muc2 −/− mice was invA dependent. We also tested the susceptibility of C3GnT −/− mice and found that they carried pathogen burdens similar to those of WT mice but developed exaggerated barrier disruption. Moreover, we found that Muc2 −/− mice were impaired in intestinal alkaline phosphatase (IAP) expression and lipopolysaccharide (LPS) detoxification activity in their ceca, potentially explaining their high mortality rates during infection. Our data suggest that the intestinal mucus layer (Muc2) and core 3 O-glycosylation play critical roles in controlling Salmonella intestinal burdens and intestinal epithelial barrier function, respectively.

We review the profound effects that components of diets commonly consumed in western societies and linked through population studies to risk for colon cancer have on the development of intestinal cancer in humans and in mouse models. Focus is particularly on levels of vitamin D, interactive with calcium and fat, in establishing probability of tumor development even in mouse genetic models in which there is high penetrance of the disease. These dietary factors have also been used to develop a mouse model of dietary-induced sporadic colon cancer which exhibits similar lag, incidence, and frequency of tumor development, and relative incidence of carcinomas and adenomas, as seen for >90% of colon tumors that arise in the general population later in life. Potential mechanisms influenced by diet that alter probability of tumor development are outlined, including altered patterns of intermediary metabolism, differentiation, and inflammation in the intestinal mucosa, all apparent in the histopathologically normal intestinal mucosa well before neoplastic changes become detectable. This includes pathways by which macrophages signal to intestinal epithelial cells, revealing a new paradigm for how vitamin D may influence tumor development.

Commensal as well as pathogenic bacteria have been implicated as possible triggers of Inflammatory Bowel Disease (IBD). The ability of pathogens to cause episodes of infectious gastroenteritis could play a role in the initiation, and/or exacerbation of IBD. In fact, an increased risk of IBD has been reported in individuals who suffer an acute episode of Salmonella gastroenteritis. For Salmonella to cause gastroenteritis, it has to directly infect the epithelial cells lining the mammalian intestine. Despite many studies exploring Salmonella interactions with epithelial cells, it is unclear how this pathogen evades and survives the array of host defenses found in the mammalian intestine. In particular it is unclear how Salmonella interacts with as well as crosses the mucus layer that protects the underlying intestinal epithelial cells from such noxious agents. Salmonella enterica serovar Typhimurium is a model organism used to test the virulence factors involved in Salmonella pathogenesis. To study Salmonella’s ability to cause intestinal inflammation, most groups use a colitis model relying on streptomycin to displace intestinal commensal microbes, resulting in heavy pathogen colonization of cecal tissues and severe inflammation. Although intestinal mucus is the first line of defense in the mouse GI tract, its role in providing host defense against Salmonella is still unclear. The mucus barrier is made up of the highly glycosylated mucin Muc2, which is secreted by goblet cells. Muc2 glycosylation occurs within the goblet cell and likely has significant implications for the function and effectiveness of the mucus barrier. Glycosylation involves the actions of several enzymes, for example, Core 3- O derived glycans are synthesized by Core 3 β1,3-N-acetylglucosaminyltransferase (C3GnT). Mice lacking these glycans still produce the Muc2 protein, but display a thinner mucus barrier, and show increased susceptibility to chemical induced colitis. We began our investigations by comparing Salmonella induced colitis and mucus dynamics in Muc2 deficient (-/-) mice, C3GnT-/- mice and wildtype C57BL/6 mice. We observed that mucus secretion increased in response to Salmonella infection in C3GnT-/- and C57BL/6, with Salmonella found within the mucus layer. In contrast, Muc2-/- mice showed dramatic susceptibility to Salmonella infection, carrying 100 fold heavier cecal pathogen burdens and developing significantly increased barrier disruption compared with C57BL/6 mice. As a result, Muc2 -/- mice displayed high rates of morbidity and mortality. We also tested the susceptibility of C3GnT -/- mice, finding they carry WT pathogen burdens but developed exaggerated barrier disruption like Muc2 -/- mice. These data suggest that the intestinal mucus layer plays a critical role in controlling Salmonella intestinal burdens, whereas core-3 glycosylation plays an important role in controlling intestinal epithelial barrier function.

The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2(-/-), Muc1(-/-), wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2(-/-) mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1(-/-) mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1(-/-) mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2(-/-)) were hypersensitive to DSS-induced colitis, whereas Muc1(-/-) were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.