ROLE OF RGDSK HELICAL ROSETTE NANOTUBES (RGDSK-HRNs) IN INTESTINAL MUCOSAL IMMUNE RESPONSE TO E. COLI INFECTION
Date
2020-01-21
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Degree Level
Doctoral
Abstract
Integrin αvβ3 is a transmembrane receptor recognizing arginine-glycine-aspartic acid (RGD) tripeptide. However, the expression and functions of integrin αvβ3 in intestinal epithelium in diarrhea, particularly due to E. coli infection, have been poorly understood. Therefore, we performed a series of experiments to fill the gap information.
We found that integrin αvβ3 is localized on the plasma membrane, the cytoplasm, and the nucleus of IPEC1 cells. The expression of integrin αvβ3 on IPEC1 decreased at 15 minutes but returned to normal after 90 minutes of infection with E. coli F4 (P<0.05). The light, fluorescent and electron microscopy, western blots, and 96-well plate binding assays showed the presence of integrin αvβ3-like protein domain in E. coli F4. The data from binding assays on 96-well plates also suggests that E. coli F4 may have an RGD-like sequence binding to host integrin αvβ3.
Immunohistochemistry showed normal porcine jejuna strongly expressed integrin αvβ3 in the nucleus and the apical surface of epithelia as well as crypts. Integrin αvβ3 expression was decreased in the epithelium but increased in the vascular endothelium of the jejunum infected with E. coli or E. coli associated with Salmonella (P<0.05). Immunogold electron microscope confirmed the presence of integrin αvβ3 in the porcine jejunal epithelium and E. coli.
Flow cytometry showed that RGDSK-HRNs did not significantly increase dead IPEC1. RGDSK-HRNs improved IPEC1 cell survival upon E. coli infection compared with E. coli infection alone group (P<0.05). Western blot showed that in E. coli infection, RGDSK-HRNs-FITC significantly decreased the level of p-p53 compared with monoclonal anti-integrin αvβ3 antibody treatment, and the level of p-p38MAPK compared with RGDSK-FITC group (P<0.05).
The data from ex vivo villus adhesion assays showed that RGDSK-HRNs-FITC significantly reduced the number of E. coli adhering to villi for up to 12 hours compared with the E. coli-only challenged group (P<0.05). Both RGDSK-FITC peptide and monoclonal anti-integrin αvβ3 antibody were effective in inhibiting the E. coli binding to villi for up to 24 hours compared with the E. coli-only challenged group (P<0.05). Consistently, in the in vivo porcine gut loop model, RGDSK-HRNs-FITC significantly decreased the number of E. coli binding to villi compared with E. coli treatment group (P<0.05). RGDSK-HRNs-FITC did not significantly decrease the number of E. coli colonies in the supernatant.
In conclusion, our study highlighted that integrin αvβ3 was involved in E. coli colonization in the porcine intestine. Also, the novel RGDSK-HRNs-FITC could inhibit the attachment of E. coli to the epithelium, suggesting a potential intervention in combination with anti-microbial and other treatments for E. coli infection in the future.
Description
Keywords
integrin αvβ3, E. coli, nanotubes, RGDSK, IPEC1, porcine intestinal epithelium, porcine jejunal villi, porcine gut loop model, equine jejunum, equine lung
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Veterinary Biomedical Sciences
Program
Veterinary Biomedical Sciences