Homeostatic T helper 17 cell responses triggered by complex microbiota are maintained in ex vivo intestinal tissue slices.

Segmented filamentous bacteria (SFB) are members of the commensal intestinal microbiome. They are known to contribute to the postnatal maturation of the gut immune system, but also to augment inflammatory conditions in chronic diseases such as Crohn's disease. Living primary tissue slices are ultrathin multicellular sections of the intestine and provide a unique opportunity to analyze tissue-specific immune responses ex vivo. This study aimed to investigate whether supplementation of the gut flora with SFB promotes T helper 17 (Th17) cell responses in primary intestinal tissue slices ex vivo. Primary tissue slices were prepared from the small intestine of healthy Taconic mice with SFB-positive and SFB-negative microbiomes and stimulated with anti-CD3/CD28 or Concanavalin A. SFB-positive and -negative mice exhibited distinct microbiome compositions and Th17 cell frequencies in the intestine and complex microbiota including SFB induced up to 15-fold increase in Th17 cell-associated mediators, serum amyloid A (SAA), and immunoglobulin A (IgA) responses ex vivo. This phenotype could be transmitted by co-housing of mice. Our findings highlight that changes in the gut microbiome can be observed in primary intestinal tissue slices ex vivo. This makes the system very attractive for disease modeling and assessment of new therapies.

Discrimination of pancreato-biliary cancer and pancreatitis patients by non-invasive liquid biopsy.

BACKGROUND: Current diagnostics for the detection of pancreato-biliary cancers (PBCs) need to be optimized. We therefore propose that methylated cell-free DNA (cfDNA) derived from non-invasive liquid biopsies serves as a novel biomarker with the ability to discriminate pancreato-biliary cancers from non-cancer pancreatitis patients. METHODS: Differentially methylated regions (DMRs) from plasma cfDNA between PBCs, pancreatitis and clinical control samples conditions were identified by next-generation sequencing after enrichment using methyl-binding domains and database searches to generate a discriminatory panel for a hybridization and capture assay with subsequent targeted high throughput sequencing. RESULTS: The hybridization and capture panel, covering around 74 kb in total, was applied to sequence a cohort of 25 PBCs, 25 pancreatitis patients, 25 clinical controls, and seven cases of Intraductal Papillary Mucinous Neoplasia (IPMN). An unbiased machine learning approach identified the 50 most discriminatory methylation markers for the discrimination of PBC from pancreatitis and controls resulting in an AUROC of 0.85 and 0.88 for a training (n = 45) and a validation (n = 37) data set, respectively. The panel was also able to distinguish high grade from low grade IPMN samples. CONCLUSIONS: We present a proof of concept for a methylation biomarker panel with better performance and improved discriminatory power than the current clinical marker CA19-9 for the discrimination of pancreato-biliary cancers from non-cancerous pancreatitis patients and clinical controls. This workflow might be used in future diagnostics for the detection of precancerous lesions, e.g. the identification of high grade IPMNs vs. low grade IPMNs.