Shaoping Wu

Vitamin D Receptor Negatively Regulates Bacterial-Stimulated NF-κB Activity in Intestine

Vitamin D receptor (VDR) plays an essential role in gastrointestinal inflammation. Most investigations have focused on the immune response; however, how bacteria regulate VDR and how VDR modulates the nuclear factor (NF)-kappaB pathway in intestinal epithelial cells remain unexplored. This study investigated the effects of VDR ablation on NF-kappaB activation in intestinal epithelia and the role of enteric bacteria on VDR expression. We found that VDR(-/-) mice exhibited a pro-inflammatory bias. After Salmonella infection, VDR(-/-) mice had increased bacterial burden and mortality. Serum interleukin-6 in noninfected VDR(+/+) mice was undetectable, but was easily detectable in VDR(-/-) mice. NF-kappaB p65 formed a complex with VDR in noninfected wild-type mouse intestine. In contrast, deletion of VDR abolished VDR/P65 binding. P65 nuclear translocation occurred in colonic epithelial cells of untreated VDR(-/-) mice. VDR deletion also elevated NF-kappaB activity in intestinal epithelia. VDR was localized to the surface epithelia of germ-free mice, but to crypt epithelial cells in conventionalized mice. VDR expression, distribution, transcriptional activity, and target genes were regulated by Salmonella stimulation, independent of 1,25-dihydroxyvitamin D3. Our study demonstrates that commensal and pathogenic bacteria directly regulate colonic epithelial VDR expression and location in vivo. VDR negatively regulates bacterial-induced intestinal NF-kappaB activation and attenuates response to infection. Therefore, VDR is an important contributor to intestinal homeostasis and host protection from bacterial invasion and infection.

Sox2 cooperates with inflammation-mediated Stat3 activation in the malignant transformation of foregut basal progenitor cells.

Sox2 regulates the self-renewal of multiple types of stem cells. Recent studies suggest it also plays oncogenic roles in the formation of squamous carcinoma in several organs, including the esophagus where Sox2 is predominantly expressed in the basal progenitor cells of the stratified epithelium. Here, we use mouse genetic models to reveal a mechanism by which Sox2 cooperates with microenvironmental signals to malignantly transform epithelial progenitor cells. Conditional overexpression of Sox2 in basal cells expands the progenitor population in both the esophagus and forestomach. Significantly, carcinoma only develops in the forestomach, where pathological progression correlates with inflammation and nuclear localization of Stat3 in progenitor cells. Importantly, co-overexpression of Sox2 and activated Stat3 (Stat3C) also transforms esophageal basal cells but not the differentiated suprabasal cells. These findings indicate that basal stem/progenitor cells are the cells of origin of squamous carcinoma and that cooperation between Sox2 and microenvironment-activated Stat3 is required for Sox2-driven tumorigenesis.

Intestinal epithelial vitamin D receptor deletion leads to defective autophagy in colitis

Objective Vitamin D and the vitamin D receptor (VDR) appear to be important immunological regulators of inflammatory bowel diseases (IBD). Defective autophagy has also been implicated in IBD, where interestingly, polymorphisms of genes such as ATG16L1 have been associated with increased risk. Although vitamin D, the microbiome and autophagy are all involved in pathogenesis of IBD, it remains unclear whether these processes are related or function independently. Design We investigated the effects and mechanisms of intestinal epithelial VDR in healthy and inflamed states using cell culture models, a conditional VDR knockout mouse model (VDRΔIEC), colitis models and human samples. Results Absence of intestinal epithelial VDR affects microbial assemblage and increases susceptibility to dextran sulfate sodium-induced colitis. Intestinal epithelial VDR downregulates expressions of ATG16L1 and lysozyme, and impairs antimicrobial function of Paneth cells. Gain and loss-of-function assays showed that VDR levels regulate ATG16L1 and lysozyme at the transcriptional and translational levels. Moreover, low levels of intestinal epithelial VDR correlated with reduced ATG16L1 and representation by intestinal Bacteroides in patients with IBD. Administration of the butyrate (a fermentation product of gut microbes) increases intestinal VDR expression and suppresses inflammation in a colitis model. Conclusions Our study demonstrates fundamental relationship between VDR, autophagy and gut microbial assemblage that is essential for maintaining intestinal homeostasis, but also in contributing to the pathophysiology of IBD. These insights can be leveraged to define therapeutic targets for restoring VDR expression and function.

Lack of Vitamin D Receptor Causes Dysbiosis and Changes the Functions of the Murine Intestinal Microbiome

PURPOSE The microbiome modulates numerous aspects of human physiology and is a crucial factor in the development of various human diseases. Vitamin D deficiency and downregulation of the vitamin D receptor (VDR) are also associated with the pathogenesis of diseases such as inflammatory bowel disease, cancers, obesity, diabetes, and asthma. VDR is a nuclear receptor that regulates the expression of antimicrobial peptides and autophagy regulator ATG16L1. Vitamin D may promote a balanced intestinal microbiome and improve glucose homeostasis in diabetes. However, how VDR regulates microbiome is not well known. In the current study, we hypothesize that VDR status regulates the composition and functions of the intestinal bacterial community. METHODS Fecal and cecal stool samples were harvested from Vdr knockout (Vdr(-/-)) and wild-type mice for bacterial DNA and then sequenced with 454 pyrosequencing. The sequences were denoised and clustered into operational taxonomic units, then queried against the National Center for Biotechnology Information database. Metagenomics were analyzed, and the abundances of genes involved in metabolic pathways were compared by reference to the Kyoto Encyclopedia of Genes and Genomes and Clusters of Orthologous Groups databases. FINDINGS In the Vdr(-/-) mice, Lactobacillus was depleted in the fecal stool, whereas Clostridium and Bacteroides were enriched. Bacterial taxa along the Sphingobacteria-to-Sphingobacteriaceae lineage were enriched, but no genera reached statistical significance. In the cecal stool, Alistipes and Odoribacter were depleted, and Eggerthella was enriched. Notably, all of the taxa upstream of Eggerthella remained unchanged. A comparison of Vdr(-/-) and wild-type samples revealed 40 (26 enriched, 14 depleted) and 72 (41 enriched, 31 depleted) functional modules that were significantly altered in the cecal and fecal microbiomes, respectively (both, P < 0.05), due to the loss of Vdr. In addition to phylogenetic differences in gut microbiome with different intestinal origins, we identify several important pathways, such as nucleotide-binding oligomerization domain-like receptor, affected by Vdr status, including amino acid, carbohydrate, and fatty acid synthesis and metabolism, detoxification, infections, signal transduction, and cancer and other diseases. IMPLICATIONS Our study fills knowledge gaps by having investigated the microbial profile affected by VDR. Insights from our findings can be exploited to develop novel strategies to treat or prevent various diseases by restoring VDR function and healthy microbe-host interactions.

Deletion of vitamin D receptor leads to premature emphysema/COPD by increased matrix metalloproteinases and lymphoid aggregates formation.

Deficiency of vitamin D is associated with accelerated decline in lung function. Vitamin D is a ligand for nuclear hormone vitamin D receptor (VDR), and upon binding it modulates various cellular functions. The level of VDR is reduced in lungs of patients with chronic obstructive pulmonary disease (COPD) which led us to hypothesize that deficiency of VDR leads to significant alterations in lung phenotype that are characteristics of COPD/emphysema associated with increased inflammatory response. We found that VDR knock-out (VDR(-/-)) mice had increased influx of inflammatory cells, phospho-acetylation of nuclear factor-kappaB (NF-κB) associated with increased proinflammatory mediators, and up-regulation of matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MMP-12 in the lung. This was associated with emphysema and decline in lung function associated with lymphoid aggregates formation compared to WT mice. These findings suggest that deficiency of VDR in mouse lung can lead to an early onset of emphysema/COPD because of chronic inflammation, immune dysregulation, and lung destruction.

Salmonella-infected crypt-derived intestinal organoid culture system for host-bacterial interactions

The in vitro analysis of bacterial–epithelial interactions in the intestine has been hampered by a lack of suitable intestinal epithelium culture systems. Here, we report a new experimental model using an organoid culture system to study pathophysiology of bacterial–epithelial interactions post Salmonella infection. Using crypt‐derived mouse intestinal organoids, we were able to visualize the invasiveness of Salmonella and the morphologic changes of the organoids. Importantly, we reported bacteria‐induced disruption of epithelial tight junctions in the infected organoids. In addition, we showed the inflammatory responses through activation of the NF‐κB pathway in the organoids. Moreover, our western blot, PCR, and immunofluorescence data demonstrated that stem cell markers (Lgr5 and Bmi1) were significantly decreased by Salmonella infection (determined using GFP‐labeled Lgr5 organoids). For the first time, we created a model system that recapitulated a number of observations from in vivo studies of the Salmonella‐infected intestine, including bacterial invasion, altered tight junctions, inflammatory responses, and decreased stem cells. We have demonstrated that the Salmonella‐infected organoid culture system is a new experimental model suitable for studying host–bacterial interactions.

Salmonella regulation of intestinal stem cells through the Wnt/β-catenin pathway

Recent studies have revealed that bacteria target stem cells for long‐term survival in a Drosophila model. However, in mammalian models, little is known about bacterial infection and intestinal stem cells. Our study aims at understanding bacterial regulation of the intestinal stem cell in a Salmonella colitis mouse model. We found that Salmonella activates the Wnt/β‐catenin signaling pathway that is known to regulate stem cells. We identified Salmonella protein AvrA that modulates Wnt signaling including upregulating Wnt expression, modifying β‐catenin, increasing total β‐catenin expression, and activating Wnt/β‐catenin transcriptional activity in the intestinal epithelial cells. The numbers of stem cells and proliferative cells increased in the intestine infected with Salmonella expressing AvrA. Our study provides insights into bacterial infection and stem cell maintenance.

Enteric bacterial protein AvrA promotes colonic tumorigenesis and activates colonic beta-catenin signaling pathway.

Salmonella infections can become chronic and increase the risk of cancer. The mechanisms by which specific Salmonella organisms contribute to cancer, however, are still unknown. Live and attenuated Salmonella are used as vectors to target cancer cells, but there have been no systematic studies of the oncogenic potential of chronic Salmonella infections in cancer models. AvrA, a pathogenic product of Salmonella, is inserted into host cells during infection and influences eukaryotic cell pathways. In the current study, we colonized mice with Salmonella AvrA-sufficient or AvrA-deficient Salmonella typhimirium strains and induced inflammation-associated colon cancer by azoxymethane/dextran sulfate sodium (AOM/DSS). We confirmed Salmonella persisted in the colon for up to 45 weeks. Salmonella was identified not only in epithelial cells on the colonic luminal surface and base of the crypts but also in invading tumors. Tumor incidence in the AvrA+infected group was 100% compared with 51.4% in the AOM/DSS group without bacterial gavage and 56.3% in mice infected with the AvrA- strain. Infection with AvrA+ strain also altered tumor distribution from the distal to proximal colon that might reflect changes in the microbiome. AvrA-expressing bacteria also upregulated beta-catenin signaling as assessed by decreased beta-catenin ubiquitination, increased nuclear beta-catenin and increased phosphorylated-beta-catenin (Ser552), a marker of proliferating stem-progenitor cells. Other β-catenin targets increased by AvrA included Bmi1, a cancer stem cell marker, matrix metalloproteinase-7, and cyclin D1. In summary, AvrA-expressing Salmonella infection activates β-catenin signals and enhances colonic tumorigenesis. Our findings provide important new mechanistic insights into how a bacterial protein targets proliferating stem-progenitor cells and contributes to cancer development. Our observations also raise a note of caution regarding the use of mutant Salmonella organisms as vectors for anti-cancer therapy. Finally, these studies could suggest biomarkers (such as AvrA level in gut) to assess cancer risk in susceptible individuals and infection-related dysregulation of β-catenin signaling in cancer.

Leaky intestine and impaired microbiome in an amyotrophic lateral sclerosis mouse model

Emerging evidence has demonstrated that intestinal homeostasis and the microbiome play essential roles in neurological diseases, such as Parkinsons disease. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons and muscle atrophy. Currently, there is no effective treatment. Most patients die within 3–5 years due to respiratory paralysis. Although the death of motor neurons is a hallmark of ALS, other organs may also contribute to the disease progression. We examined the gut of an ALS mouse model, G93A, which expresses mutant superoxide dismutase (SOD1G93A), and discovered a damaged tight junction structure and increased permeability with a significant reduction in the expression levels of tight junction protein ZO‐1 and the adherens junction protein E‐cadherin. Furthermore, our data demonstrated increased numbers of abnormal Paneth cells in the intestine of G93A mice. Paneth cells are specialized intestinal epithelial cells that can sense microbes and secrete antimicrobial peptides, thus playing key roles in host innate immune responses and shaping the gut microbiome. A decreased level of the antimicrobial peptides defensin 5 alpha was indeed found in the ALS intestine. These changes were associated with a shifted profile of the intestinal microbiome, including reduced levels of Butyrivibrio Fibrisolvens, Escherichia coli, and Fermicus, in G93A mice. The relative abundance of bacteria was shifted in G93A mice compared to wild‐type mice. Principal coordinate analysis indicated a difference in fecal microbial communities between ALS and wild‐type mice. Taken together, our study suggests a potential novel role of the intestinal epithelium and microbiome in the progression of ALS.

Salmonella Infection Upregulates the Leaky Protein Claudin-2 in Intestinal Epithelial Cells

Background Tight junctions seal the space between adjacent epithelial cells. Mounting evidence suggests that tight junction proteins play a key role in the pathogenesis of human disease. Claudin is a member of the tight junction protein family, which has 24 members in humans. To regulate cellular function, claudins interact structurally and functionally with membrane and scaffolding proteins via their cytoplasmic domain. In particular, claudin-2 is known to be a leaky protein that contributes to inflammatory bowel disease and colon cancer. However, the involvement of claudin-2 in bacterial infection in the intestine remains unknown. Methods/Principal Findings We hypothesized that Salmonella elevates the leaky protein claudin-2 for its own benefit to facilitate bacterial invasion in the colon. Using a Salmonella-colitis mouse model and cultured colonic epithelial cells, we found that pathogenic Salmonella colonization significantly increases the levels of claudin-2 protein and mRNA in the intestine, but not that of claudin-3 or claudin-7 in the colon, in a time-dependent manner. Immunostaining studies showed that the claudin-2 expression along the crypt-villous axis postinfection. In vitro, Salmonella stimulated claudin-2 expression in the human intestinal epithelial cell lines SKCO15 and HT29C19A. Further analysis by siRNA knockdown revealed that claudin-2 is associated with the Salmonella-induced elevation of cell permeability. Epithelial cells with claudin-2 knockdown had significantly less internalized Salmonella than control cells with normal claudin-2 expression. Inhibitor assays demonstrated that this regulation is mediated through activation of the EGFR pathway and the downstream protein JNK. Conclusion/Significance We have shown that Salmonella targets the tight junction protein claudin-2 to facilitate bacterial invasion. We speculate that this disruption of barrier function contributes to a new mechanism by which bacteria interact with their host cells and suggests the possibility of blocking claudin-2 as a potential therapeutic strategy to prevent bacterial invasion.