Tissue-specific Mechanisms of Bile Acid Homeostasis and Activation of FXR-FGF19 Signaling in Preterm and Term Neonatal Pigs

Background & Aims: The tissue specific molecular mechanisms involved in perinatal liver and intestinal FXR-FGF19 signaling are poorly defined. Our aim was to establish how gestational age and feeding status affect bile acid synthesis pathway, bile acid pool size, ileal response to bile acid stimulation, genes involved in bile acid-FXR-FGF19 signaling and plasma FGF19 in neonatal pigs. Methods Term (n=23) and preterm (n=33) pigs were born via cesarean section at 100% and 90% gestation, respectively. Plasma FGF19, hepatic bile acid and oxysterol profiles, and FXR target gene expression was assessed in pigs at birth and after a bolus feed on day 3 of life. Pig ileal tissue explants were used to measure signaling response to bile acids. Results Preterm pigs had smaller, more hydrophobic bile acid pools, lower plasma FGF19, and blunted FXR-mediated ileal response to bile acid stimulation than term pigs. GATA-4 expression was higher in jejunum than ileum, and was higher in preterm than term pig ileum. Hepatic oxysterol analysis suggested dominance of the alternative pathway of bile acid synthesis in neonates, regardless of gestational age and persists in preterm pigs after feeding on day 3. Conclusion These results highlight the tissue-specific molecular basis for the immature enterohepatic bile acid signaling via FXR-FGF19 in preterm pigs and may have implications for disturbances of bile acid homeostasis and metabolism in preterm infants.

Absorption and Distribution of Toltrazuril and Toltrazuril Sulfone in Plasma, Intestinal Tissues and Content of Piglets after Oral or Intramuscular Administration

Piglet coccidiosis due to Cystoisospora suis is a major cause of diarrhea and poor growth worldwide. It can effectively be controlled by application of toltrazuril (TZ), and oral formulations have been licensed for many years. Recently, the first parenteral formulation containing TZ in combination with iron (gleptoferron) was registered in the EU for the prevention of coccidiosis and iron deficiency anemia, conditions in suckling piglets requiring routine preventive measures. This study evaluated the absorption and distribution of TZ and its main metabolite, toltrazuril sulfone (TZ-SO2), in blood and intestinal tissues after single oral (20 mg/kg) or single intramuscular (45 mg/piglet) application of TZ. Fifty-six piglets were randomly allocated to the two treatment groups. Animals were sacrificed 1-, 5-, 13-, and 24-days post-treatment and TZ and TZ-SO2 levels were determined in blood, jejunal tissue, ileal tissue, and mixed jejunal and ileal content (IC) by high performance liquid chromatography (HPLC). Intramuscular application resulted in significantly higher and more sustained concentrations of both compounds in plasma, intestinal tissue, and IC. Higher concentrations after oral dosing were only observed one day after application of TZ in jejunum and IC. Toltrazuril was quickly metabolized to TZ-SO2 with maximum concentrations on day 13 for both applications. Remarkably, TZ and TZ-SO2 accumulated in the jejunum, the primary predilection site of C. suis, independently of the administration route, which is key to their antiparasitic effect.

Apoptosis levels in bovine Johne’s disease ileal lesions and association with bacterial numbers

Johne’s disease (JD) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). While it is generally accepted that MAP employs immune subversion mechanisms, aspects of the host-pathogen relationship are not fully understood. We sampled 3 ileal tissue sections from 17 naturally infected cattle ( n = 51 sections) to analyze differences in cell types, apoptosis, and phagocytic cells. Diffuse multibacillary (DM) was the most common lesion type ( n = 17) followed by diffuse intermediate (DI; n = 15). DM lesions had significantly greater proportion of Treg cells (CD3+ FoxP3+) relative to all CD3+ T cells as compared to DI forms ( P < .05). CD68+ individual cell size was significantly smaller in DM than in diffuse lymphocytic (DL) forms ( P < .05). Area of caspase-3 positivity (apoptosis) was greater in DM lesions than DL ( P < .05) and DI ( P < .0001), and was linked to higher numbers of MAP within the macrophage.

Transcriptome Analysis Reveals the Genes Involved in Growth and Metabolism in Muscovy Ducks

Muscovy ducks are among the best meat ducks in the world. The objective of this study was to identify genes related to growth metabolism through transcriptome analysis of the ileal tissue of Muscovy ducks. Duck ileum samples with the highest (H group, n = 5 ) and lowest (L group, n = 5 ) body weight were selected from two hundred 70-day-old Muscovy ducks for transcriptome analysis by RNA sequencing. In the screening of differentially expressed genes (DEGs) between the H and L groups, a total of 602 DEGs with a fold change no less than 2 were identified, among which 285 were upregulated and 317 were downregulated. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that glutathione metabolism, pyrimidine metabolism, and protein digestion and absorption processes played a vital role in regulating growth and metabolism. The results showed that 7 genes related to growth and metabolism, namely, ANPEP, ENPEP, UPP1, SLC2A2, SLC6A19, NME4, and LOC106034733, were significantly expressed in group H, which was consistent with the phenotype results. The validation of these 7 genes using real-time quantitative PCR results indicated that the expression level of ENPEP was significantly different between the H and L groups ( P < 0.05 ). This study provides a theoretical basis for exploring the influence of the ileum on growth and metabolism in ducks.

Prediction of Johne’s disease state based on quantification of T cell markers and their interaction with macrophages in the bovine intestine

Cell-mediated immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) are regulated by various types of T lymphocytes. The aim of this study was to quantitate T cell subsets in the mid-ileum of cows naturally infected with MAP to identify differences during different stages of infection, and to determine whether these subsets could be used as predictors of disease state. Immunofluorescent labeling of T cell subsets and macrophages was performed on frozen mid-ileal tissue sections archived from naturally infected dairy cows in either subclinical or clinical disease status, and noninfected control cows. Comprehensive IF staining for CD4, CD8α, TcR1-N24 (gamma delta), FoxP3, CXCR3 and CCR9 served to define T cell subsets and was correlated with macrophages present. Clinically affected cows demonstrated significantly higher numbers of CXCR3+ (Th1-type) and CCR9+ (total small intestinal lymphocytes) cells at the site of infection compared to the subclinical cows and noninfected controls. Further, predictive modeling indicated a significant interaction between CXCR3+ and AM3K+ (macrophages) cells, suggesting that progression to clinical disease state aligns with increased numbers of these cell types at the site of infection. The ability to predict disease state with this model was improved from previous modeling using immunofluorescent macrophage data. Predictive modelling indicated an interaction between CXCR3+ and AM3K+ cells, which could more sensitively detect subclinical cows compared to clinical cows. It may be possible to use this knowledge to improve and develop an assay to detect subclinically infected animals with more confidence during the early stages of the disease.

Whole tissue homogenization preferable to mucosal scraping in determining the temporal profile of segmented filamentous bacteria in the ileum of weanling rats

Segmented filamentous bacteria (SFB) are thought to play a role in small intestine immunological maturation. Studies in weanling mice have shown a positive correlation between ileal SFB abundance and plasma and faecal interleukin 17 (IL-17) and immunoglobulin A (IgA) concentrations. Although the first observation of SFB presence was reported in rats, most studies use mice. The size of the mouse ileum is a limitation whereas the rat could be a suitable alternative for sufficient samples. Changes in SFB abundance over time in rats were hypothesized to follow the pattern reported in mice and infants. We characterized the profile of SFB colonization in the ileum tissue and contents and its correlation with two immune markers of gastrointestinal tract (GIT) maturation. We also compared two published ileum collection techniques to determine which yields data on SFB abundance with least variability. Whole ileal tissue and ileal mucosal scrapings were collected from 20- to 32-day-old Sprague-Dawley rats. SFB abundance was quantified from proximal, middle and distal ileal tissues, contents and faeces by quantitative PCR using SFB-specific primers. Antibody-specific ELISAs were used to determine IL-17 and IgA concentrations. Significant differences in SFB abundance were observed from whole and scraped tissues peaking at day 22. Variability in whole ileum data was less, favouring it as a better collection technique. A similar pattern of SFB abundance was observed in ileum contents and faeces peaking at day 24, suggesting faeces can be a proxy for ileal SFB abundance. SFB abundance at day 26 was higher in females than males across all samples. There were significant differences in IgA concentration between days 20, 30 and 32 and none in IL-17 concentration, which was different from reports in mice and infants.

Diving into inflammation: a pilot study exploring the dynamics of the immune-microbiota axis in ileal tissue layers of patients with Crohn’s disease

Background and aims Crohn’s Disease (CD) pathogenesis is still unclear. Disorders in the mucosal immunoregulation and its crosstalk with the microbiota may represent an important component in tissue injury. We aimed to characterize the molecular immune response distribution within the ileal layers and to evaluate the correlated microbiota in pathological/healthy settings comparing first surgery/relapse clinical conditions. Methods We enrolled 12 CD patients. A comprehensive analysis of ileal mucosa, submucosa and serosa broad-spectrum cytokines’ panel was performed through a multiplex approach. In addition, ileal microbiota composition was assessed through Next Generation Sequencing. Results We observed a distinct profile (of IL1-α, IL-1β, IL-4, IL-8, ICAM-1, E-Selectin, P-Selectin, IP-10, IL 6, and IL 18) across the CD vs healthy ileal layers; and a different distribution of IFN-γ, P-Selectin, IL-27 and IL-21 in first surgery vs relapse patients. In addition, the phylum Tenericutes, the family of Ruminococcaceae, and the genus Mesoplasma and Mycoplasma were significantly enriched in pathological setting. Significant microbiota differences were observed between relapse vs first surgery patients regarding the class Bacteroidia, the genus of Prevotella, Flavobacterium, Tepidimonas and Escherichia/Shigella. Finally, the abundance of the genus Mycoplasma was positively correlated with IL-18. Conclusions We describe a dissimilarity of cytokines’ distribution and microbiota composition within the CD and the adjacent healthy ileal tissue layers and between first operation and surgical relapse. Our results give a potential insight into the dynamics of the gut microbiota-immune axis in CD patients, leading to new biomarkers’ detection.

Ileal transcriptomic analysis in paediatric Crohn’s disease reveals IL17- and NOD-signalling expression signatures in treatment-naïve patients and identifies epithelial cells driving differentially expressed genes

Background/Aims Crohn’s disease (CD) arises through host-environment interaction. Abnormal gene expression results from disturbed pathway activation or response to bacteria. We aimed to determine activated pathways and driving cell types in paediatric CD. Methods - We employed contemporary targeted autoimmune RNA sequencing, in parallel to single-cell sequencing, to ileal tissue derived from paediatric CD and controls. Weighted-gene-co-expression-network-analysis (WGCNA) was performed and differentially expressed genes (DEGs) were determined. We integrated clinical data to determine co-expression modules associated with outcomes. Results - Twenty-seven treatment-naive CD (TN-CD), 26 established-CD patients and 17 controls were included. WGCNA revealed a 31-gene signature characterising TN-CD patients, but not established-CD, or controls. The CSF3R gene is a hub within this module and is key in neutrophil expansion and differentiation. Antimicrobial genes including S100A12 and the calprotectin subunit S100A9 were significantly upregulated in TN CD compared to controls (p=2.61x10 -15 and p=9.13x10 -14, respectively) and established-CD (both p=0.0055). Gene-enrichment analysis confirmed upregulation of the IL17-, NOD- and Oncostatin-M-signalling pathways in TN-CD patients, identified in both WGCNA and DEG analyses. An upregulated gene-signature was enriched for transcripts promoting Th17-cell differentiation and correlated with prolonged time to relapse (correlation-coefficient-0.36, p=0.07).Single-cell sequencing of TN-CD patients identified specialised epithelial cells driving differential expression of S100A9. Cell groups, determined by single-cell gene-expression, demonstrated enrichment of IL17-signalling in monocytes and epithelial cells. Conclusion - Ileal tissue from treatment naïve paediatric patients is significantly upregulated for genes driving IL17-, NOD- and Oncostatin-M-signalling. This signal is driven by a distinct subset of epithelial cells expressing antimicrobial gene transcripts.

Lactobacillus rhamnosus V5 Prevents Salmonella enterica Serovar Typhimurium Invasion in Cell Culture and Mice Infection

Aims: The aim of this study was to evaluate the protective capacity of the exopolysaccharide-producing Lactobacillus rhamnosus V5 against invasion in vitro and in vivo with S. typhimurium. Methodology: We tested the antimicrobial activity of the compound extracted from the lactobacilli against S. typhimurium directly, also we tested the interference of this compound in S. typhimurium adherence and invasion of HeLa and HEp-2 cells (in vitro testings). For in vivo experiments, we used 16 BALB/c female mice. Through gavage method we introduced L. rhamnosus as probiotic and then infected mice with S. enterica serovar typhimurium. After euthanasia, spleen, liver and Peyer's patches removed for microbiological and histopathological analysis. Results: The results showed that lactobacilli were able to produce antimicrobial compounds against S. typhimurium. These lactobacilli inhibited the adhesion and invasion of S. typhimurium in HeLa and HEp-2 cells, respectively. The challenge assay in the murine model demonstrated a decrease in pathogen translocation in the spleen and liver from mice treated with probiotic as well as protection of ileal tissue in lactobacilli-treated mice. The histopathological analysis demonstrated the presence of prominent lymphoid nodules in the ileum from the non-treated lactobacilli mice. Conclusion: Our results suggest that L. rhamnosus improved the effectiveness of the intestinal barrier and, thus, could be a potential probiotic to control salmonellosis.

Reduced expression of COVID-19 host receptor, ACE2 is associated with small bowel inflammation, more severe disease, and response to anti-TNF therapy in Crohn’s disease

Angiotensin-Converting Enzyme 2 (ACE2) has been identified as the host receptor for SARS-coronavirus 2 (SARS-CoV-2) which has infected millions world-wide and likely caused hundreds of thousands of deaths. Utilizing transcriptomic data from four cohorts taken from Crohn’s disease (CD) and non-inflammatory bowel disease (IBD) subjects, we observed evidence of increased ACE2 mRNA in ileum with demographic features that have been associated with poor outcomes in COVID-19 including age and raised BMI. ACE2 was downregulated in CD compared to controls in independent cohorts. Within CD, ACE2 expression was reduced in inflamed ileal tissue and also remarkably, from un-involved tissue in patients with a worse prognosis in both adult and pediatric cohorts. In active CD, small bowel ACE2 expression was restored by anti-TNF therapy particularly in anti-TNF responders. Collectively our data suggest that ACE2 downregulation is associated with inflammation and worse outcomes in CD.