Julie M. Davies

TLR4 Activates the β-catenin Pathway to Cause Intestinal Neoplasia

Colonic bacteria have been implicated in the development of colon cancer. We have previously demonstrated that toll-like receptor 4 (TLR4), the receptor for bacterial lipopolysaccharide (LPS), is over-expressed in humans with colitis-associated cancer. Genetic epidemiologic data support a role for TLR4 in sporadic colorectal cancer (CRC) as well, with over-expression favoring more aggressive disease. The goal of our study was to determine whether TLR4 played a role as a tumor promoter in sporadic colon cancer. Using immunofluorescence directed to TLR4, we found that a third of sporadic human colorectal cancers over-express this marker. To mechanistically investigate this observation, we used a mouse model that over-expresses TLR4 in the intestinal epithelium (villin-TLR4 mice). We found that these transgenic mice had increased epithelial proliferation as measured by BrdU labeling, longer colonic crypts and an expansion of Lgr5+ crypt cells at baseline. In addition, villin-TLR4 mice developed spontaneous duodenal dysplasia with age, a feature that is not seen in any wild-type (WT) mice. To model human sporadic CRC, we administered the genotoxic agent azoxymethane (AOM) to villin-TLR4 and WT mice. We found that villin-TLR4 mice showed an increased number of colonic tumors compared to WT mice as well as increased β-catenin activation in non-dysplastic areas. Biochemical studies in colonic epithelial cell lines revealed that TLR4 activates β-catenin in a PI3K-dependent manner, increasing phosphorylation of β-cateninSer552, a phenomenon associated with activation of the canonical Wnt pathway. Our results suggest that TLR4 can trigger a neoplastic program through activation of the Wnt/β-catenin pathway. Our studies highlight a previously unexplored link between innate immune signaling and activation of oncogenic pathways, which may be targeted to prevent or treat CRC.

Intestinal Epithelial Toll-Like Receptor 4 Signaling Affects Epithelial Function and Colonic Microbiota and Promotes a Risk for Transmissible Colitis

ABSTRACT Evidence obtained from gene knockout studies supports the role of Toll-like receptor 4 (TLR4) in intestinal inflammation and microbiota recognition. Increased epithelial TLR4 expression is observed in patients with inflammatory bowel disease. However, little is known of the effect of increased TLR4 signaling on intestinal homeostasis. Here, we examined the effect of increased TLR4 signaling on epithelial function and microbiota by using transgenic villin-TLR4 mice that overexpress TLR4 in the intestinal epithelium. Our results revealed that villin-TLR4 mice are characterized by increases in the density of mucosa-associated bacteria and bacterial translocation. Furthermore, increased epithelial TLR4 signaling was associated with an impaired epithelial barrier, altered expression of antimicrobial peptide genes, and altered epithelial cell differentiation. The composition of the colonic luminal and mucosa-associated microbiota differed between villin-TLR4 and wild-type (WT) littermates. Interestingly, WT mice cohoused with villin-TLR4 mice displayed greater susceptibility to acute colitis than singly housed WT mice did. The results of this study suggest that epithelial TLR4 expression shapes the microbiota and affects the functional properties of the epithelium. The changes in the microbiota induced by increased epithelial TLR4 signaling are transmissible and exacerbate dextran sodium sulfate-induced colitis. Together, our findings imply that host innate immune signaling can modulate intestinal bacteria and ultimately the hosts susceptibility to colitis.

The innate immune system and inflammatory bowel disease

Abstract The innate immune system is a key factor in understanding the pathogenesis of inflammatory bowel disease (IBD) and in the hopes of improving its treatment. NOD2, a pattern recognition receptor, was one of the first major susceptibility genes identified in Crohn’s disease (CD). This discovery has been followed by genome-wide association studies that have identified other genes involved in innate immune responses. Most notably, polymorphisms in the interleukin (IL)-23 receptor have also been linked to IBD – both CD and ulcerative colitis. At the core of the innate immune defects associated with IBD is a lack of generating a robust response to control invasive commensal or pathogenic bacteria. The defect sometimes lies in a failure of the epithelium to express antimicrobial peptides or in defective control of intracellular bacteria by phagocytic cells such as dendritic cells, macrophages, or neutrophils. The recent identification of innate lymphoid cells that express the IL-23 receptor and generate both proinflammatory and protective or regulatory responses to commensal or pathogenic bacteria provides another layer of complexity to the interplay of host protection and dysregulated inflammation. Although inhibition of tumor necrosis factor has been highly successful as a strategy in treating IBD, we must better understand the nuanced role of other innate cytokines before we may incorporate these in the treatment of IBD.

Differential regulation of Toll-like receptor signalling in spleen and Peyer's patch dendritic cells.

Toll‐like receptor (TLR) signalling shapes dendritic cell (DC) responses by inducing co‐stimulatory molecule up‐regulation and cytokine secretion while TLR regulatory proteins inhibit this process. We aimed to determine if gene expression of TLRs and TLR regulatory proteins underpins the functionally different lipopolysaccharide (LPS) responses of DCs from murine Peyer’s patches (PP) and spleen and of murine bacteria‐conditioned bone‐marrow‐derived cells. Isolated spleen and PP DCs were analysed for basal expression of TLRs by flow cytometry and real time quantitative reverse transcription polymerase chain reaction (qRT‐PCR). The DCs were stimulated with LPS to determine cytokine secretion by enzyme‐linked immunosorbent assay and expression of TLR regulatory proteins by qRT‐PCR. In vitro results were confirmed following in vivo intraperitoneal LPS injection. In addition, changes in gene expression of TLR regulatory proteins were assessed in bacteria‐conditioned bone‐marrow‐derived cells. Results indicated that surface expression of TLR2 and TLR4 on PP DCs was decreased compared with spleen DCs. The PP DCs secreted a limited profile of cytokines compared with spleen DCs following LPS stimulation. In vivo LPS exposure up‐regulated sigirr, tollip and tmed1 messenger RNA in PP DCs, but not spleen DCs. Similar gene expression changes were observed in bacteria‐conditioned bone‐marrow‐derived cells. Therefore, functionally different LPS responses in PP and spleen DCs reflect their characteristic expression of TLRs and TLR regulatory proteins. Differential regulation of TLR signalling was also evident in bacteria‐conditioned bone‐marrow‐derived cells indicating that bacterial signalling may be a mechanism for inducing altered gene regulation in PP DCs.

Bacterial signalling overrides cytokine signalling and modifies dendritic cell differentiation

Heterogeneity of dendritic cells (DC) is evident in the gut‐associated lymphoid tissue and determined, in part, by incompletely understood local environmental factors. Bacterial signalling is likely to be a dominant influence on precursor cells when recruited to the mucosa. We assessed the influence of commensal bacteria on DC differentiation and function. Murine bone marrow progenitors were exposed to Lactobacillus salivarius, Bifidobacterium breve or Bifidobacterium infantis. Differences in cell surface phenotype and function were assessed. Myeloid differentiation factor 88−/− (MyD88) cells were used to determine the influence of Toll‐like receptor signalling. While bacterial strains varied in impact, there was a consistent dose‐dependent inhibition of DC differentiation with a shift toward a Gr‐1+ CD11b+ monocyte‐like phenotype. A single bacterium on a per cell basis (1 : 1) was sufficient to alter cell phenotype. The effect was only evident in early precursors. Enhanced interleukin‐10 production correlated with increased Forkhead box P3 expression and reduced T‐cell proliferation. The bacterial effect on DC differentiation was found to be MyD88‐dependent. Signalling by enteric commensals through pattern recognition receptors on precursor cells alters DC differentiation and results in cells that are phenotypically monocyte‐like and functionally suppressive. This may account for some of the features of mucosal immune tolerance to the microbiota.

Host-microbe interactions in the small bowel.

Purpose of review The intestine – home to a vast microbiome – balances its immune reactivity on a knifes edge. This review will summarize recent studies examining innate immune signals that shape the microbiota, and how pathogens can usurp protective responses to their advantage. Recent findings Innate signaling uses several pathways to maintain epithelial defense. Toll-like receptor signaling through myeloid differentiation factor 88 maintains segregation between bacteria and the epithelium through production of antimicrobial proteins, and inflammasome signaling mediates efficient goblet cell release of mucus containing granules. Conversely, negative regulators of Toll-like receptor signaling help maintain a healthy microbiota resistant to pathogen infection. Methods to evade immune elimination by pathogens associated with human infections and inflammatory bowel disease are described. Emerging evidence that pattern recognition receptors can differentiate between commensals and pathogens will be examined. Summary The balance of innate signaling in the intestine is crucial to homeostasis: too little and bacteria can directly contact the epithelium, too much depletes the protective microbiota, creating a niche for pathogens. Understanding the dynamic interaction between the immune system and the microbiota in a variety of infection and inflammation models will hopefully translate to new therapies.

The viral mimetic polyinosinic: Polycytidylic acid alters the growth characteristics of small intestinal and colonic crypt cultures

Background & Aims The intestinal epithelium is the first line of defense against enteric pathogens. We investigated the response of small intestinal and colonic crypt cultures to a panel of toll-like receptor ligands to assess the impact of microbial pattern recognition on epithelial growth. Methods Primary murine jejunal enteroids and colonoids were cultured with lipopeptide Pam3CSK4, lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (Poly I:C) for 4 to 6 days. Surface area, budding and survival were assessed. Proliferation and numbers of lysozyme positive cells were quantified by flow cytometry. Gene expression was assessed by Nanostring and qRT-PCR. Results Exposure to Pam3CSK4 and LPS had minimal impact on either enteroids or colonoids. In contrast, Poly I:C increased the surface area of enteroids, while colonoids demonstrated decreased budding. Survival was decreased by Poly I:C in enteroids but not in colonoids. Both enteroids and colonoids exhibited upregulated gene expression of chemokines, but these were increased in magnitude in enteroids. Decreases in gene expression associated with epithelial differentiation and lysozyme positive cells were more apparent in enteroids than in colonoids. Baseline gene expression between enteroids and colonoids differed markedly in levels of stem cell and inflammatory markers. The changes in morphology induced by Poly I:C were mediated by the toll-like receptor adaptor molecule 1 (Ticam1) in enteroids but not in colonoids. Conclusions Poly I:C alters the molecular program of epithelial cells and shifts from absorption and digestion towards defense and inflammation. Diversity of responses to microbial patterns in enteroids and colonoids may underlie differences in susceptibility to infection along the intestinal tract.

Stool Phospholipid Signature is Altered by Diet and Tumors

Intake of saturated fat is a risk factor for ulcerative colitis (UC) and colon cancer. Changes in the microbiota have been implicated in the development of UC and colon cancer. The host and the microbiota generate metabolites that may contribute to or reflect disease pathogenesis. We used lipid class specific quantitative mass spectrometry to assess the phospholipid (PL) profile (phosphatidylcholine [PC], phosphatidylethanolamine [PE], phosphatidylinositol [PI], phosphatidylserine [PS]) of stool from mice fed a high fat (HFD) or control diet with or without induction of colitis-associated tumors using azoxymethane and dextran sodium sulfate. The microbiota was assessed using qPCR for several bacterial groups. Colitis-associated tumors were associated with reduced bulk PI and PE levels in control diet fed mice compared to untreated mice. Significant decreases in the relative quantities of several PC species were found in colitis-associated tumor bearing mice fed either diet. Statistical analysis of the PL profile revealed distinct clustering by treatment group. Partial least squares regression analysis found that the relative quantities of the PS class profile best predicted bacterial abundance of Clostridium leptum and Prevotella groups. Abundance of selected PL species correlated with bacterial group quantities. Thus, we have described that a HFD and colitis-associated tumors are associated with changes in phospholipids and may reflect host-microbial interactions and disease states.

Use of cancer stem cells to investigate the pathogenesis of colitis-associated cancer

Abstract:Colitis-associated cancer (CAC) can develop in patients with inflammatory bowel disease with long-term uncontrolled inflammation. The mutational history and tumor microenvironment observed in CAC patients is distinct from that observed in sporadic colon cancer and suggests a different etiology. Recently, much attention has been focused on understanding the cellular origin of cancer and the cancer stem cells, which is key to growth and progression. Cancer stem cells are often chemo-resistant making them attractive targets for improving patient outcomes. New techniques have rapidly been evolving allowing for a better understanding of the normal intestinal stem cell function and behavior in the niche. Use of these new technologies will be crucial to understanding cancer stem cells in both sporadic and CAC. In this review, we will explore emerging methods related to the study of normal and cancer stem cells in the intestine, and examine potential avenues of investigation and application to understanding the pathogenesis of CAC.

Intestinal Exposure to PCB 153 Induces Inflammation via the ATM/NEMO Pathway

Background: Polychlorinated biphenyls (PCBs) are persistent organic pollutants that adversely affect human health. PCBs bio‐accumulate in organisms important for human consumption. PCBs accumulation in the body leads to activation of the transcription factor NF‐&kgr;B, a major driver of inflammation. Despite dietary exposure being one of the main routes of exposure to PCBs, the gut has been widely ignored when studying the effects of PCBs. Objectives: We investigated the effects of PCB 153 on the intestine and addressed whether PCB 153 affected intestinal permeability or inflammation and the mechanism by which this occurred. Methods: Mice were orally exposed to PCB 153 and gut permeability was assessed. Intestinal epithelial cells (IECs) were collected and evaluated for evidence of genotoxicity and inflammation. A human IEC line (SW480) was used to examine the direct effects of PCB 153 on epithelial function. NF‐&kgr;B activation was measured using a reporter assay, DNA damage was assessed, and cytokine expression was ascertained with real‐time PCR. Results: Mice orally exposed to PCB 153 had an increase in intestinal permeability and inflammatory cytokine expression in their IECs; inhibition of NF‐&kgr;B ameliorated both these effects. This inflammation was associated with genotoxic damage and NF‐&kgr;B activation. Exposure of SW480 cells to PCB 153 led to similar effects as seen in vivo. We found that activation of the ATM/NEMO pathway by genotoxic stress was upstream of NF‐kB activation. Conclusions: These results demonstrate that oral exposure to PCB 153 is genotoxic to IECs and induces downstream inflammation and barrier dysfunction in the intestinal epithelium. HighlightsPCB 153 causes an increase in inflammatory cytokines in intestinal epithelial cells.PCB 153 increases intestinal permeability.PCB 153 activates the transcription factor NF‐&kgr;B to cause these effects.PCB 153 is genotoxic to intestinal epithelial cells.PCB 153 activates NF‐ &kgr;B through the ATM/NEMO pathway.