Dataset Information

Dysbiosis of intestinal microbiota and decrease in paneth cell antimicrobial peptide level during acute necrotizing pancreatitis in rats.

ABSTRACT:

Objectives

Intestinal barrier dysfunction plays an important role in acute necrotizing pancreatitis (ANP) and intestinal microbiota dysbiosis was involved in intestinal barrier failure. Paneth cells protect intestinal barrier and are associated with intestinal microbiota. Here, we investigated changes in intestinal microbiota and antimicrobial peptides of Paneth cells in ileum during ANP.

Methods

Rats with ANP were established by retrograde injection of 3.5% sodium taurocholate into biliopancreatic duct and sacrificed at 24h and 48h, respectively. Injuries of pancreas and distal ileum were evaluated by histopathological score. Intestinal barrier function was assessed by plasma diamine oxidase activity (DAO) and D-lactate. Systemic and intestinal inflammation was evaluated by TNF?, IL-1? and IL-17A concentration by ELISA, respectively. 16S rRNA high throughput sequencing on fecal samples was used to investigate the changes in intestinal microbiota in the ANP group at 48h. Lysozyme and ?-defensin5 were measured by real-time PCR, western blot and immunofluoresence.

Results

ANP rats had more severe histopathological injuries in pancreas and distal ileum, injured intestinal barrier and increased expression of TNF?, IL-1? and IL-17A in plasma and distal ileum compared with those of the sham-operated (SO) group. Principal component analysis (PCA) showed structural segregation between the SO and ANP groups. Operational taxonomic unit (OTU) number and ACE index revealed decreased microbiota diversity in the ANP group. Taxonomic analysis showed dysbiosis of intestinal microbiota structure. At phyla level, Saccharibacteria and Tenericutes decreased significantly. At genus level, Escherichia-Shigella and Phascolarctobacterium increased significantly, while Candidatus_Saccharimonas, Prevotellaceae_UCG-001, Lachnospiraceae_UCG-001, Ruminiclostridium_5 and Ruminococcaceae_UCG-008 decreased significantly. Lysozyme and ?-defensin5 mRNA expression levels decreased significantly in ANP group at 48h. Protein expression of lysozyme decreased in ANP groups at 24h and 48h. Meanwhile, the relative abundance of Escherichia-Shigella correlated inversely with the decrease in lysozyme.

Conclusion

The disorder in intestinal microbiota and decreases of Paneth cell antimicrobial peptides might participate in the pathogenesis of intestinal barrier dysfunction during ANP.

SUBMITTER: Chen J

PROVIDER: S-EPMC5404871 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Dysbiosis of intestinal microbiota and decrease in paneth cell antimicrobial peptide level during acute necrotizing pancreatitis in rats.

Chen Jing J Huang Chunlan C Wang Jingjing J Zhou Hui H Lu Yingying Y Lou Lihong L Zheng Junyuan J Tian Ling L Wang Xingpeng X Cao Zhongwei Z Zeng Yue Y

PloS one 20170425 4

<h4>Objectives</h4>Intestinal barrier dysfunction plays an important role in acute necrotizing pancreatitis (ANP) and intestinal microbiota dysbiosis was involved in intestinal barrier failure. Paneth cells protect intestinal barrier and are associated with intestinal microbiota. Here, we investigated changes in intestinal microbiota and antimicrobial peptides of Paneth cells in ileum during ANP.<h4>Methods</h4>Rats with ANP were established by retrograde injection of 3.5% sodium taurocholate in ...[more]

PMID: 28441432

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Project description:Hypertriglyceridemia (HTG) aggravates the course of acute pancreatitis (AP). Intestinal barrier dysfunction is implicated in the pathogenesis of AP during which dysbiosis of intestinal microbiota contributes to the dysfunction in intestinal barrier. However, few studies focus on the changes in intestine during HTG-related acute necrotizing pancreatitis (ANP). Here, we investigated the changes in intestinal microbiota and Paneth cell antimicrobial peptides (AMPs) in HTG-related ANP (HANP) in rats. Rats fed a high-fat diet to induce HTG and ANP was induced by retrograde injection of 3.5% sodium taurocholate into biliopancreatic duct. Rats were sacrificed at 24 and 48 h, respectively. Pancreatic and ileal injuries were evaluated by histological scores. Intestinal barrier function was assessed by plasma diamine oxidase activity and D-lactate level. Systemic and intestinal inflammation was evaluated by tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, and IL-17A expression. 16S rRNA high throughput sequencing was used to investigate changes in intestinal microbiota diversity and structure. AMPs (α-defensin5 and lysozyme) expression was measured by real-time polymerase chain reaction (PCR) and immunofluorescence. The results showed that compared with those of normal-lipid ANP (NANP) groups, the HANP groups had more severe histopathological injuries in pancreas and distal ileum, aggravated intestinal barrier dysfunction and increased TNFα, IL-1β, and IL-17A expression in plasma and distal ileum. Principal component analysis showed structural segregation between the HANP and NANP group. α-Diversity estimators in the HANP group revealed decreased microbiota diversity compared with that in NANP group. Taxonomic analysis showed dysbiosis of intestinal microbiota structure. In the HANP group, at phyla level, Candidatus_Saccharibacteria and Tenericutes decreased significantly, whereas Actinobacteria increased. At genus level, Allobaculum, Bifidobacterium, and Parasutterella increased significantly, while Alloprevotella, Anaerotruncus, Candidatus_Saccharimonas, Christensenellaceae_R-7_group, Rikenellaceae_RC9_gut_group, Ruminiclostridium_5, Ruminococcaceae_UCG-005, and Ruminococcaceae_UCG-014 decreased. Compared with those in the NANP rats, mRNA expression of lysozyme and α-defensin5 and protein expression of lysozyme decreased significantly in the HANP rats. Moreover, in the NANP rats and the HANP rats, Allobaculum abundance was inversely correlated with lysozyme expression, while Anaerotruncus abundance was positively correlated with it by Spearman test. In conclusion, intestinal microbiota dysbiosis and decreased AMPs of Paneth cells might participate in the pathogenesis of intestinal barrier dysfunction in HANP.

Project description:Enteric antimicrobial peptides secreted from Paneth cells, including alpha-defensins (in mice named cryptdins), are key effector molecules of innate immunity in the small intestine. The importance of Paneth cells alpha-defensins emerged from studies of enteric bacterial infection in genetically modified mice, as well as from recent studies linking reduced levels of these alpha-defensins to Crohn's disease localized to the ileum. However, analysis of expression of Paneth cell alpha-defensins is incomplete. We therefore performed a comprehensive evaluation of the distribution of antimicrobial molecules along the mouse small intestinal tract to identify potential variations in regional expression.In conventionally reared mice, the repertoire of Paneth cell antimicrobials differs between duodenum and ileum. In contrast to the uniform expression of most Paneth cell antimicrobials, both cryptdin 4 and cryptdin-related sequences (CRS) 4C peptides were expressed at progressively increasing amounts (101- and 104-fold, respectively) comparing duodenum and ileum. In tissues other than the small intestine, expression of CRS peptides was noted in thymus and caecum. Most Paneth cell products were also produced in the small intestine of germ-free mice at levels similar to those in controls, however CRS4C and RegIIIgamma had reduced levels in the former (3- and 8-fold, respectively). No significant changes in expression levels of Paneth cell antimicrobial peptides was observed after oral challenge with either Salmonella enterica serovar typhimurium or Listeria monocytogenes, supporting current notions on the constitutive nature of this defensive system.The repertoire of antimicrobial peptides changes along the small intestinal tract, and a subset of these molecules are up-regulated upon colonization, but not in response to enteric bacterial pathogens. The changes detected upon colonization suggest that Paneth cell antimicrobial peptides may play an important role in commensal microbial homeostasis, in addition to their proposed role in protection against infection. In addition, the differential expression of CRS4C along the small intestine suggests mechanisms of regulation that are distinct from other Paneth cell derived antimicrobial peptides.

Project description:BackgroundAcute necrotizing pancreatitis (NP), a severe form of acute pancreatitis (AP), has higher mortality and worse outcome than non-necrotizing pancreatitis (non-NP). Infected NP is a devastating subgroup of NP. To date neither NP nor infected NP has robust prediction strategies, which may delay early recognition and timely intervention. Recent studies revealed correlations between disturbed gut microbiota and AP severity. Some features of intestinal microbiota have the potential to become biomarkers for NP prediction.MethodsWe performed 16S rRNA sequencing to analyze gut microbiota features in 20 healthy controls (HC), and 58 AP patients on hospital admission. The AP patients were later classified into NP and non-NP groups based on subsequent diagnostic imaging features. Random forest regression model and ROC curve were applied for NP and infected NP prediction. PIRCUSt2 was used for bacterial functional pathway prediction analysis.ResultsWe found that the three groups (HC, NP, and non-NP) had distinct microorganism composition. NP patients had reduced microbial diversity, higher abundance of Enterobacteriales, but lower abundance of Clostridiales and Bacteroidales compared with the non-NP group. Correlation analyses displayed that intestine bacterial taxonomic alterations were related to severity, ICU admission, and prognosis. By pathway prediction, species more abundant in NP patients had positive correlation with synthesis and degradation of ketone bodies, and benzoate degradation. Enterococcus faecium (ASV2) performed best in discriminating NP and non-NP patients. Finegoldia magna (ASV3) showed the maximal prediction capacity among all ASVs and had comparable accuracy with Balthazar CT to detect patients with infected NP.ConclusionsOur study suggests that NP patients have distinct intestinal microbiota on admission compared to non-NP patients. Dysbiosis of intestinal microbiota might influence NP progression through ketone body or benzoate metabolism. Enterococcus faecium and Finegoldia magna are potential predictors for NP and infected NP. Our findings explore biomarkers which may inform clinical decision-making in AP and shed light on further studies on NP pathophysiology and management.

Dataset Information

Dysbiosis of intestinal microbiota and decrease in paneth cell antimicrobial peptide level during acute necrotizing pancreatitis in rats.

Objectives

Methods

Results

Conclusion

Publications

Dysbiosis of intestinal microbiota and decrease in paneth cell antimicrobial peptide level during acute necrotizing pancreatitis in rats.

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