Controlled Access. Restriction lift date: 2026-12-31
Identification of bacteria-regulated mechanisms for the pathogenesis of inflammatory bowel diseases (IBD) and development of upconverting nanoparticle (UCNP) luminescence imaging for the monitoring of gut bacteria
University College Cork
Inflammatory bowel disease (IBD) is a chronic inflammatory state of the gastrointestinal tract, including two inflammatory conditions Crohn’s disease (CD) and ulcerative colitis (UC). The environment, the gut microbiota, the genetic make-up and the immune response are believed to contribute to the aetiology of the disease. Non-steroidal anti-inflammatory drugs (NSAIDs) are believed to exacerbate inflammation in patients with IBD. However, the literature demonstrates no consensus on the association between NSAID use and IBD, with some studies reporting that only high dose NSAID treatment are more likely for IBD exacerbation or relapse. In this study, using the piroxicam-accelerated model of colitis in interlukin-10 deficient (IL-10-/-) mice, we showed that mice fed 100 ppm piroxicam in food for 9 days (high dose) followed by 5 days of regular chow, develop colitis. The colitic phenotype was associated with activation of caspase-8, NLRP3 inflammasome and apoptosis and independent of initial gut microbiota. However, 5 days exposure (low dose) to 100 ppm piroxicam did not lead to colitis development. Adherent-invasive Escherichia coli (AIEC) is widely prevalent and heterogeneously present in the mucosa of IBD patients, particularly in Crohn’s disease. We hypothesise that the presence of AIEC in the gut of IBD patients might explain the NSAID-induced inflammation in IBD under low dose treatment (5 days piroxicam treatment). Indeed, 5 days piroxicam exposure was sufficient to induce colitis in AIEC-precolonised animals, indicating a synergism between AIEC colonisation and piroxicam treatment. Inhibition of NLR family pyrin domain containing 3 (NLRP3) or Caspase-8 activity ameliorated colitis. This synergism was lost under high dose piroxicam (9 days exposure) treatment. Our data indicate that under low dose NSAID treatment, AIEC can potentiate NSAID-induced inflammation in IL-10-/- mice by regulating the intestinal epithelial function and the immune response, highlighting its potential role in NSAID-induced inflammation in IBD patients exposed to low dose NSAIDs. Although, under high dose, NSAID itself is sufficient to induce colitis. This data suggests that the lack of consistency in the association between NSAID use and IBD could be explained by the NSAID dose and the presence of AIEC in the patients with IBD. Future studies should consider both these factors while studying association between NSAID use and symptomatic worsening in IBD. The role of microbes in IBD is supported by various studies performed in animal models where germ free mice are protected from intestinal inflammation. However, the precise interaction(s) between microbes and the host are not well understood. In vivo imaging techniques using custom designed fluorescent probes and bioluminescence have been classically used to track gut microbes. Several disadvantages while using fluorescent probes include autofluorescence, photobleaching and photodamage. To overcome these limitations, we propose to develop upconverting nanoparticles (UCNPs) luminescence imaging for monitoring of gut bacteria. Their unique property of photon upconversion enables them to convert low energy near-infrared (NIR) light into higher energy visible/NIR light offering greater tissue penetrance and signal-to-noise ratio. Here, we investigated the possibility of using UCNP 1) to image a single gut bacterium (e.g., AIEC) using UCNP-conjugated anti-E. coli antibody, and 2) to image the endogenous gut bacteria using metabolic labelling of bacterial peptidoglycan with azido-D-amino acids and strained-cyclooctynes such as Dibenzocyclooctyne (DBCO) -functionalised UCNPs. We showed a proof of principle for both these approaches using a fluorescent dye, but we were not able to replicate it with UCNP. Preliminary data suggests that the size difference between the UCNPs and the fluorescent dyes may be one of the potential reasons for lack of labelling. Future experiments should consider using smaller UCNPs and/or use a polyethylene glycol (PEG) linker between DBCO and UCNPs to increase the length of DBCO-UCNP construct and reduce the steric hinderance for better stability of UCNP-bacteria conjugation.
AIEC , Piroxicam , Inflammatory bowel disease , Cell death , IL10−/− mice , Inflammasome , UCNP
Singh, R. 2022.Identification of bacteria-regulated mechanisms for the pathogenesis of inflammatory bowel diseases (IBD) and development of upconverting nanoparticle (UCNP) luminescence imaging for the monitoring of gut bacteria. PhD Thesis, University College Cork.