Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

Increased amounts of PGE2 have been detected in the inflamed mucosa of patients with inflammatory bowel disease (IBD). This increase has been attributed to enhanced synthesis rather than reduced catabolism of PGE2. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) plays a major role in the catabolism of PGE2. In this study, we investigated whether amounts of 15-PGDH were altered in inflamed mucosa from patients with IBD. Amounts of 15-PGDH protein and mRNA were markedly reduced in inflamed mucosa from patients with Crohn's disease and ulcerative colitis. In situ hybridization demonstrated that 15-PGDH was expressed in normal colonic epithelium but was virtually absent in inflamed colonic mucosa from IBD patients. Because of the importance of TNF-α in IBD, we also determined the effects of TNF-α on the expression of 15-PGDH in vitro. Treatment with TNF-α suppressed the transcription of 15-PGDH in human colonocytes, resulting in reduced amounts of 15-PGDH mRNA and protein and enzyme activity. In contrast, TNF-α induced two enzymes (cyclooxygenase-2 and microsomal prostaglandin E synthase-1) that contribute to increased synthesis of PGE2. Overexpressing 15-PGDH blocked the increase in PGE2 production mediated by TNF-α. Taken together, these results suggest that reduced expression of 15-PGDH contributes to the elevated levels of PGE2 found in inflamed mucosa of IBD patients. The decrease in amounts of 15-PGDH in inflamed mucosa can be explained at least, in part, by TNF-α-mediated suppression of 15-PGDH transcription.

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An Abundance of Escherichia coli Is Harbored by the Mucosa- Associated Bacterial Flora of Interleukin-2-Deficient Mice

Infection and Immunity ◽

10.1128/iai.72.4.1983-1990.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 1983-1990 ◽

Cited By ~ 34

Author(s):

M. Schuppler ◽

K. Lötzsch ◽

M. Waidmann ◽

I. B. Autenrieth

Keyword(s):

Inflammatory Bowel Disease ◽

16S Rdna ◽

Bowel Disease ◽

Bacterial Flora ◽

Interleukin 2 ◽

Healthy Controls ◽

E Coli ◽

Inflammatory Bowel ◽

Deficient Mice

ABSTRACT Mice deficient in interleukin-2 are well suited for use as an animal model for inflammatory bowel disease. Raised under specific-pathogen-free conditions, interleukin-2-deficient mice develop an inflammatory bowel disease resembling ulcerative colitis in humans. The finding that colitis was attenuated when the mice were kept under germfree conditions implies that the resident intestinal flora is involved in the pathogenesis of colitis. The present study addresses the composition of the mucosa-associated bacterial flora in colon samples from interleukin-2-deficient mice that developed colitis. This was investigated by comparative 16S ribosomal DNA (rDNA) sequence analysis and fluorescence in situ hybridization using rRNA-targeted fluorescent probes to quantify the bacterial populations of the mucosa-associated flora. The investigations revealed distinct differences in the bacterial composition of the mucosa-associated flora between interleukin-2-deficient mice and healthy controls. Fluorescence in situ hybridization identified up to 10% of the mucosa-associated flora in interleukin-2-deficient mice as Escherichia coli, whereas no E. coli was detected in the mucosa from healthy wild-type mice. This finding was consistent with the results from comparative 16S rDNA analysis. About one-third of the clones analyzed from 16S rDNA libraries of interleukin-2-deficient mice represented Enterobacteriaceae, whereas none of the clones analyzed from the healthy controls harbored 16S rDNA from Enterobacteriaceae. The abundance of E. coli in the colonic mucosa of interleukin-2-deficient mice strongly suggests a participation in the pathogenesis of colitis in the interleukin-2-deficient mouse model for inflammatory bowel disease.

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IL-1-driven stromal–neutrophil interactions define a subset of patients with inflammatory bowel disease that does not respond to therapies

Nature Medicine ◽

10.1038/s41591-021-01520-5 ◽

2021 ◽

Author(s):

Matthias Friedrich ◽

Mathilde Pohin ◽

Matthew A. Jackson ◽

Ilya Korsunsky ◽

Samuel J. Bullers ◽

...

Keyword(s):

Inflammatory Bowel Disease ◽

Inflammatory Response ◽

Bowel Disease ◽

Vascular Remodeling ◽

Neutrophil Infiltration ◽

Deep Ulceration ◽

Inflammatory Bowel ◽

Gene Modules ◽

Activated Fibroblasts

AbstractCurrent inflammatory bowel disease (IBD) therapies are ineffective in a high proportion of patients. Combining bulk and single-cell transcriptomics, quantitative histopathology and in situ localization across three cohorts of patients with IBD (total n = 376), we identify coexpressed gene modules within the heterogeneous tissular inflammatory response in IBD that map to distinct histopathological and cellular features (pathotypes). One of these pathotypes is defined by high neutrophil infiltration, activation of fibroblasts and vascular remodeling at sites of deep ulceration. Activated fibroblasts in the ulcer bed display neutrophil-chemoattractant properties that are IL-1R, but not TNF, dependent. Pathotype-associated neutrophil and fibroblast signatures are increased in nonresponders to several therapies across four independent cohorts (total n = 343). The identification of distinct, localized, tissular pathotypes will aid precision targeting of current therapeutics and provides a biological rationale for IL-1 signaling blockade in ulcerating disease.

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