Dissertation

The Therapeutic Effects of Cathelicidin-Encoding Lactococcus lactis on Murine Ulcerative Colitis

Wong, Ching Man

2012

Overview

Ulcerative colitis (UC) is an idiopathic inflammatory bowel disease (IBD). The mainstay of drug treatment is to relieve inflammation. However the aetiology of UC is multi-factorial while most of the anti-inflammatory drugs, such as sulfasalazine, aim at single target with severe side effects. Therefore, a multi-targeted drug with low systemic toxicity is warranted. Cathelicidin, a host defense peptide, shows anti-microbial and anti-inflammatory effects. Indeed intra-rectal administration of mouse cathelicidin (mCRAMP) alleviated murine colitis. To improve therapeutic efficacy and reduce inconvenience of administration, Lactococcus lactis (L. lactis ) was constructed to encode cathelicidin. L. lactis is a lactic acid probiotic which could resist gastric acid and be able to produce and deliver cathelicidin to the colon when given orally. Murine colitis was induced by 3% dextran sulphate sodium (DSS) given in drinking water for 7 days. Mice were given intragastrically with the following preparations once daily: (1) water, (2) DSS, (3, 4) DSS + 1010 cfu L. lactis with or without nisin induction, (5-8) DSS + 10 8 or 1010 cfu mCRAMP-encoding L. lactis with (N4I) or without nisin induction, (9) DSS + 0.5% sodium carboxymethylcellulose (CMC-Na) and (10) DSS + 600 mg/kg sulfasalazine suspended in 0.5 % CMC-Na. To study the preventive effects, mice received the above treatments together with DSS administration. N4I but not the other probiotic preparations suppressed inflammation by reducing neutrophil infiltration, lipid peroxidation and inflammatory cytokines expressions. Crypt structure and mucus-secreting layer were conserved together with the reduction of apoptosis and intestinal microbiota. In contrast, sulfasalazine could only suppress inflammation but not the destruction of colonic structure. To further examine the therapeutic effects, mice received treatments for 4 consecutive days after the inflammation formation. Similarly, only N4I promoted colonic mucosal recovery and preserved colon structure and mucus-secreting layer. These actions are likely mediated through cell proliferation stimulation and apoptosis suppression. Again, sulfasalazine had no effects on colon tissue reconstitution. The direct anti-inflammatory action of mCRAMP was also studied. Mouse macrophage RAW 264.7 cells were stimulated by lipoteichoic acid and lipopolysaccharide to mimic bacteria-induced inflammation during UC. mCRAMP prevented tumour necrosis factor-α secretion and IκBα phosphorylation followed by nuclear factor-κB (NF-κB) suppression. The inflammatory enzymes including inducible nitric oxide synthase and cyclooxygenase-2 were also reduced. It was postulated that mCRAMP might directly interact with the bacterial toxins to reduce receptor complex binding and/or reduce NF-κB suppression in macrophages. The repairing action of mCRAMP on mucosal damage was studied in mouse colon cells. mCRAMP incubation reduced the wound size by promoting cell migration through the G-protein coupled receptor and epidermal growth factor receptor transactivation followed by the mitogen-activated protein kinases activation. In conclusion, the present study demonstrates for the first time the protective and therapeutic roles of mCRAMP-encoding L. lactis in UC. It was the mCRAMP secreted from the probiotic to produce both anti-inflammatory and anti-bacterial actions and further promote mucosal repair. mCRAMP-encoding L. lactis is a multi-targeted agent for IBD. It has a great potential to be a new therapeutic agent better than sulfasalazine for the treatment of UC.

• Publisher: ProQuest Dissertations & Theses
• Subject: Pharmacology
• ISBN: 9781267942418, 126794241X