Low dose dietary contamination with deoxynivalenol mycotoxin exacerbates enteritis and colorectal cancer in mice.

BACKGROUND: The mycotoxin deoxynivalenol (DON) is a frequent contaminant of grain and cereal products worldwide. Exposure to DON can cause gastrointestinal inflammation, disturb gut barrier function, and induce gut dysbiosis in vivo under basal conditions, but little is known about the effects of DON ingestion in individuals with pre-existing gastrointestinal disease. OBJECTIVES: Mice were orally exposed to 10 and 100 µg/kg bw/day of DON, corresponding to 10 to 100-fold human tolerable daily intake concentrations, and to the translation in mice of current human daily intake. The effects of DON exposure were explored under steady-state conditions, and in murine models of enteritis and colorectal cancer (CRC). RESULTS: After 8 days of DON exposure, an increase of histomorphological and molecular parameters of epithelial proliferation were observed in normal mice, from the duodenum to the colon. The same exposure in a murine model of indomethacin-induced enteritis led to exacerbation of lesion development and induction of ileal cytokines. DON exposure also worsened the development of colitis-associated CRC in mice as shown by increases in endoscopic and histological colitis scores, tumor grades, and histological hyperplasia. In colon of DON-exposed mice, upstream and downstream ERK signaling genes were upregulated including Mapk1, Mapk3, Map 2k1, Map2k2 core ERK pathway effectors, and Bcl2 and Bcl2l1 antiapoptotic genes. The effects observed in the CRC model were associated with alterations in cecal microbiota taxonomic composition and metabolism of bacterial fucose and rhamnose. Strong Spearman's correlations were revealed between the relative abundance of the changed bacterial genera and CRC-related variables. DISCUSSION: Ingestion of DON mycotoxin at concentrations representative of human real-world exposure worsened the development of indomethacin-induced enteritis and colitis-associated CRC in mice. Our results suggest that even at low doses, which are currently tolerated in the human diet, DON could promote the development of intestinal inflammatory diseases and CRC.

Fungi as Part of the Microbiota and Interactions with Intestinal Bacteria.

The human microbiota consists of bacteria, archaea, viruses, and fungi that build a highly complex network of interactions between each other and the host. While there are many examples for commensal bacterial influence on host health and immune modulation, little is known about the role of commensal fungi inside the gut community. Up until now, fungal research was concentrating on opportunistic diseases caused by fungal species, leaving the possible role of fungi as part of the microbiota largely unclear. Interestingly, fungal and bacterial abundance in the gut appear to be negatively correlated and disruption of the bacterial microbiota is a prerequisite for fungal overgrowth. The mechanisms behind bacterial colonization resistance are likely diverse, including direct antagonism as well as bacterial stimulation of host defense mechanisms. In this work, we will review the current knowledge of the development of the intestinal bacterial and fungal community, the influence of the microbiota on human health and disease, and the role of the opportunistic yeast C. albicans. We will furthermore discuss the possible benefits of commensal fungal colonization. Finally, we will summarize the recent findings on bacterial-fungal interactions.

Galleria mellonella: A Novel Invertebrate Model to Distinguish Intestinal Symbionts From Pathobionts.

Insects and mammals share evolutionary conserved innate immune responses to maintain intestinal homeostasis. We investigated whether the larvae of the greater wax moth Galleria mellonella may be used as an experimental organism to distinguish between symbiotic Bacteroides vulgatus and pathobiotic Escherichia coli, which are mammalian intestinal commensals. Oral application of the symbiont or pathobiont to G. mellonella resulted in clearly distinguishable innate immune responses that could be verified by analyzing similar innate immune components in mice in vivo and in vitro. The differential innate immune responses were initiated by the recognition of bacterial components via pattern recognition receptors. The pathobiont detection resulted in increased expression of reactive oxygen and nitrogen species related genes as well as antimicrobial peptide gene expression. In contrast, the treatment/application with symbiotic bacteria led to weakened immune responses in both mammalian and insect models. As symbionts and pathobionts play a crucial role in development of inflammatory bowel diseases, we hence suggest G. mellonella as a future replacement organism in inflammatory bowel disease research.

Proteome and phosphoproteome analysis of commensally induced dendritic cell maturation states.

Dendritic cells (DCs) can shape the immune system towards an inflammatory or tolerant state depending on the bacterial antigens and the environment they encounter. In this study we provide a proteomic catalogue of differentially expressed proteins between distinct DC maturation states, brought about by bacteria that differ in their endotoxicity. To achieve this, we have performed proteomics and phosphoproteomics on murine DC cultures. Symbiont and pathobiont bacteria were used to direct dendritic cells into a semi-mature and fully-mature state, respectively. The comparison of semi-mature and fully-mature DCs revealed differential expression in 103 proteins and differential phosphorylation in 118 phosphosites, including major regulatory factors of central immune processes. Our analyses predict that these differences are mediated by upstream elements such as SOCS1, IRF3, ABCA1, TLR4, and PTGER4. Our analyses indicate that the symbiont bacterial strain affects DC proteome in a distinct way, by downregulating inflammatory proteins and activating anti-inflammatory upstream regulators. Biological significance In this study we have investigated the responses of immune cells to distinct bacterial stimuli. We have used the symbiont bacterial strain B. vulgatus and the pathobiont E. coli strain to stimulate cultured primary dendritic cells and performed a shotgun proteome analysis to investigate the protein expression and phosphorylation level differences on a genome level. We have observed expression and phosphorylation level differences in key immune regulators, transcription factors and signal transducers. Moreover, our subsequent bioinformatics analysis indicated regulation at several signaling pathways such as PPAR signaling, LXR/RXR activation and glucocorticoid signaling pathways, which are not studied in detail in an inflammation and DC maturation context. Our phosphoproteome analysis showed differential phosphorylation in 118 phosphosites including those belonging to epigenetic regulators, transcription factors and major cell cycle regulators. We anticipate that our study will facilitate further investigation of immune cell proteomes under different inflammatory and non-inflammatory conditions.

Role of CD40 ligation in dendritic cell semimaturation.

BACKGROUND: DC are among the first antigen presenting cells encountering bacteria at mucosal surfaces, and play an important role in maintenance of regular homeostasis in the intestine. Upon stimulation DC undergo activation and maturation and as initiators of T cell responses they have the capacity to stimulate naïve T cells. However, stimulation of naïve murine DC with B. vulgatus or LPS at low concentration drives DC to a semimature (sm) state with low surface expression of activation-markers and a reduced capacity to activate T-cells. Additionally, semimature DC are nonresponsive to subsequent TLR stimulation in terms of maturation, TNF-α but not IL-6 production. Ligation of CD40 is an important mechanism in enhancing DC maturation, function and capacity to activate T-cells. We investigated whether the DC semimaturation can be overcome by CD40 ligation. RESULTS: Upon CD40 ligation smDC secreted IL-12p40 but not the bioactive heterodimer IL-12p70. Additionally, CD40 ligation of smDC resulted in an increased production of IL-6 but not in an increased expression of CD40. Analysis of the phosphorylation pattern of MAP kinases showed that in smDC the p38 phosphorylation induced by CD40 ligation is inhibited. In contrast, phosphorylation of ERK upon CD40 ligation was independent of the DC maturation state. CONCLUSION: Our data show that the semimature differentiation state of DC can not be overcome by CD40 ligation. We suggest that the inability of CD40 ligation in overcoming DC semimaturation might contribute to the tolerogenic phenotype of semimature DC and at least partially account for maintenance of intestinal immune homeostasis.