Bacillus velezensis inhibits azoxymethane/dextran sulphate sodium induced colitis associated colorectal cancer via the small molecule HeLM

Abstract

Background: Ulcerative colitis (UC) is a chronic inflammatory condition of the colon. There is a direct correlation between the severity and chronicity of colitis and subsequent risk of colitis associated colorectal cancer (CAC). We have recently shown that a strain of Bacillus velezensis (EHv5) can ameliorate colonic inflammation in the acute setting. This was found to be mediated via the secondary metabolite HeLM and its multifactorial interactions on Toll-like receptors. It is yet to be seen if sustained administration will alleviate colitis over a prolonged period and reduce the risk of CAC. This study will examine the therapeutic potential of EHv5 in ameliorating UC in the chronic setting and assess its ability to prevent CAC.

Methods: CAC was induced in mice (BALB/c) by the administration of intraperitoneal azoxymethane (AOM) and chronic colitis by multiple cycles of dextran sulphate sodium (DSS). Mice were divided into four groups: (I) a negative control; (II) a positive control; (III) the treatment arm receiving EHv5; and (IV) an isogenic mutant of EHv5 that does not produce HeLM, but is otherwise identical. Colitis was assessed throughout the experiment using clinical, biochemical, and endoscopic assessments. A novel scoring system was devised, the chronic colitis associated cancer activity index (CACI) and is compared to the established Disease Activity Index (DAI). Faecal blood and serum haematinics were assessed for iron deficiency anaemia. Tumorigenesis was measured at multiple time points endoscopically and histologically at the end of the experiment.

Results: Compared to the positive control, there was a significant reduction in both the severity and chronicity of colitis in the group receiving EHv5 as measured clinically, endoscopically and with faecal calprotectin. This translated to a significant reduction in both the number and grade of tumours-20% of the group receiving treatment developed tumours of any grade, compared to 80% in the positive control. The CACI score was more reflective to the severity of colitis as the experiment progressed than DAI.

Conclusions: EHv5 provides sustained amelioration of chronic inflammation in a murine model of UC. Strikingly, the effect is such that it significantly reduces the risk of CAC tumour development. This protective effect was not seen in the isogenic mutant, confirming HeLM to be the mechanistic mediator of this beneficial effect.

Keywords: Colitis associated colorectal cancer (CAC); Toll-like receptors (TLRs); azoxymethane/dextran sulphate sodium (AOM/DSS); bacillus; inflammatory bowel disease (IBD).

Figure 1

Schematic of the experiment. On the Y axis, a breakdown of each group, number of mice per group, and sex ratio. On the X axis is the timeline of the experiment with the timepoints for each investigation and cycle of colitis. AOM, azoxymethane; CACI, chronic colitis associated with cancer activity index; DAI, Disease Activity Index; DSS, dextran sulphate sodium; IP, intraperitoneal; PBS, phosphatebuffered-solution.

Figure 2

Clinical scores throughout the experiment. For both graphs, α indicates the initiation of DSS (Days 10 and 48) whilst Ω shows the cessation. (A) Graph showing CACI scores over time. On Day-80 (end of experiment) the mean score for EHv5^WT was 1.2 which was significantly different (P=0.047) from the positive control which scored 4.6. This protection from chronic colitis was not seen in the mutant (EHv5^HeLM−), which scored 4.3. (B) The DAI score over time. At the end of the experiment, EHv5^WT was significantly different from the positive control (P=0.02) with a score of 0.6 compared to 3.1. There was no protective effect seen in the mutant group. CACI, chronic colitis associated with cancer activity index; DAI, Disease Activity Index; DSS, dextran sodium sulphate.

Figure 3

Endoscopic and biochemical evaluation of colitis. (A) MCEI scores throughout the experiment. A score of 0 indicates no colitis, 9 is the maximal severity score. EHv5^WT was significantly protected compared to the negative control. (B) Faecal calprotectin levels taken after the cycle of 2.5% DSS. After both cycles, EHv5^WT, whilst elevated, was significantly lower than the positive control. *, P≤0.05; ****, P≤0.0001; ns, non-significant. DSS, dextran sodium sulphate; MCEI, mouse colitis endoscopy index.

Figure 4

Haematological evaluation. (A) Hb (g/dL) levels taken at the end of the experiment. The red line at 12 g/dL indicates the lower boundary for “normal”—i.e., below 12 indicates anaemia. There was no significant difference in Hb between the groups. (B) The percentage of mice in each group with anaemia, analysing the results this way shows that the positive control and the mutant had significant rates of anaemia, a finding not seen in the EHv5^WT group. (C) MCV (fL) values. The red line at 44 fL is the boundary, below which signifies microcytosis, no significant difference between the groups were found. (D) The percentage of mice in each group with microcytosis, with the EHv5^WT group being non-significantly different to the negative control. *, P≤0.05; **, P≤0.01; ns, non-significant. DSS, dextran sulfate sodium; Hb, haemoglobin; MCV, mean corpuscular volume.

Figure 5

Colorectal cancer burden. (A) Kaplan-Meier plot showing the probability of tumour detection during endoscopy throughout the experiment. The EHv5^WT curve was significantly different to the positive control (P<0.001). (B) Captured images during endoscopy, the top row shows normal mucosa, with all subsequent images demonstrating polyps (white star). (C) The mean total size of tumour(s) (mm) per group, with the EHv5^WT demonstrating a significantly reduced mean size of tumour compared to the positive control. (D) The total number of polyps counted at the end of the experiment, grouped into categories. There was no significant difference from “1–2” to “7–8”, there was, however, significant difference in the category “9+”. (E) Representative photos of the opened colorectum. The top left photo shows normal mucosa, as the photos progress there is an increasing tumour burden. Tumour growth identified with a white star. *, P≤0.05; ****, P≤0.0001; ns, non-significant. AOM, azoxymethane; DSS, dextran sodium sulphate.

Figure 6

Histological analysis. (A) Histology grouped into either “no abnormality”, “low grade dysplasia”, or “high grade/adenocarcinoma”. This was determined as per the Royal College of Pathologists standards for colorectal cancer. EHv5^WT provided significant protection from developing high grade dysplasia/adenocarcinoma compared to the positive control. (B) Representative histology slides, stained with hematoxylin and eosin, taken at 10× magnification. For the negative control (a), EHv5^WT group (b), DSS-only (c), and EHv5^HeLM− (d), tumour growth is highlighted with a white star. *, P≤0.05; ***, P≤0.001; ****, P≤0.0001; ns, non-significant. DSS, dextran sodium sulphate; HGD, high grade dysplasia.