Anti-inflammatory effects of Lactococcus lactis NCDO 2118 during the remission period of chemically induced colitis

Three L. lactis strains were used in this study: L. lactis subsp. lactis NCDO 2118 [50], L. lactis subsp. lactis IL1403 [51], and L. lactis subsp. cremoris MG1363 [52]. They were grown at 30°C in M17 medium (Difco) containing 0.5% glucose (GM17) without agitation or in the same medium solidified with 1.5% agar for 18 hours.

Caco-2 cells (ATCC HTB-37), a human colon adenocarcinoma cell line, were cultured in RPMI medium (Sigma) supplemented with 10% (v/v) of fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 1 mM sodium pyruvate solution in an atmosphere containing 5% CO₂ at 37°C.

Caco-2 cells were seeded at 3×10⁵ cells/well in 24-well plates and incubated at 37°C with 5% CO₂ for 24 hours before treatment. Secretion of the pro-inflammatory cytokine IL-8 by the cells was induced by the addition of human recombinant IL-1β (BD Biosciences) to a final concentration of 10 ng/mL. L. lactis cultures in the stationary phase of growth were fractionated by centrifugation into supernatant and cells, and each fraction was co-incubated with Caco-2 cells. Bacterial cells were washed 2 times with PBS (137 mM/L NaCl, 2.7 mM/L KCl, 10 mM/L Na₂HPO4 • 2 H₂O, 2 mM/L KH2PO4) and added at a multiplicity of infection (MOI) of 5. The supernatant was filtered at a final concentration of 10% (v/v). Caco-2 cells that were not treated with IL-1β were used as controls. After 6 hours of co-incubation, the supernatant of cell cultures was collected and stored at -80°C until analysis. IL-8 levels were measured using a Human IL-8 ELISA Kit (BD Biosciences) following the manufacturer's instructions. Data from three independent experiments were analysed.

Chemical colitis was induced by replacing the drinking water of mice with a 2% (w/v) aqueous solution of dextran sodium sulphate (DSS, MP Biomedicals) for 7 consecutive days. Subsequently, mice received either GM17 medium (DSS group) or L. lactis NCDO 2118 (DSS + NCDO2118 group) orally for four consecutive days. Fresh total cultures of NCDO2118 (bacteria plus supernatant at a stationary phase of growth) were prepared daily before being offered to the mice. Because each mouse drank approximately 5 mL of culture per day (data not shown), the total dose of bacteria per mouse was estimated to be 5×10⁹ bacteria/day. Mice were sacrificed either at day 14 (immediately following the oral treatment) or after a second DSS cycle (at day 21). The control groups of mice received M17 or L. lactis alone. Throughout the experimental period, all mice had unlimited access to food. A schematic representation of the experimental procedure is shown in Figure 2A.

DSS-induced colitis was assessed macroscopically by scoring three major clinical signs—weight loss, diarrhea, and rectal bleeding—at day 14 and day 21 as described by Cooper et al.[53]. Body weight loss was calculated as the difference between the initial and actual weight. Diarrhea was determined by assessing mucus/faecal material adhering to anal fur and was confirmed by the presence or absence of faecal pellet formation and continuous fluid faecal material in the colon. Rectal bleeding was defined as diarrhea containing visible blood and gross rectal bleeding. The three major clinical signs (weight loss, diarrhea, and occult/gross bleeding) were scored separately. The macroscopic score was calculated from the score of the clinical signs using the following formula: (weight loss score) + (diarrhea score) + (rectal bleeding score). After the mice were sacrificed, their spleens, mesenteric lymph nodes and colons were excised. Spleens and mesenteric lymph nodes were used for cell population analysis. Colon samples were fixed in formalin and processed for histological analysis. Hematoxylin-eosin-stained sections were blindly scored based on a previously described semi-quantitative scoring system [54]. The following features were graded: extent of destruction of normal mucosal architecture (0: normal; 1, 2 and 3: mild, moderate and extensive damage, respectively), presence and degree of cellular infiltration (0: normal; 1, 2 and 3: mild, moderate and transmural infiltration, respectively), extent of muscle thickening (0: normal; 1, 2 and 3: mild, moderate and extensive thickening, respectively), presence or absence of crypt abscesses (0: absent; 1: present) and the presence or absence of goblet cell depletion (0: absent; 1: present). Scores for each feature were summed up to a maximum possible score of 11.

Colon samples were weighed and homogenised in PBS containing 0.05% (v/v) Tween-20, 0.1 mM phenylmethylsulphonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA and 20 KIU Aprotinin A using a tissue homogeniser (100 mg tissue/ml buffer) [43]. Suspensions were centrifuged at 12,000 g for 20 min at 4°C, and the supernatants were collected for the cytokine assay. The plates were coated with purified monoclonal antibodies reactive for the cytokines IL-6, IL-12, IFN-γ, IL-17, IL-10, TGF-β and TNF-α (BD-Pharmingen) overnight at 4°C. On the following day, the wells were washed, the supernatants were added and the plate was incubated overnight at 4°C. On the third day, biotinylated monoclonal antibodies against the cytokines were added, and the plates were incubated for 2 hours at room temperature. Colour was developed at room temperature with 100 μl/well of orthophenylenediamine (1 mg/ml) and 0.04% (v/v) H₂O₂ substrate in sodium citrate buffer. The reaction was interrupted by the addition of 20 μl/well of 2 N H₂SO₄. The absorbance was measured at 492 nm using a microplate reader (BIO-RAD).

The levels of sIgA were determined by ELISA. Briefly, 96-well plates (NUNC) were coated with Ig goat anti-mouse UNLB antibody in coating buffer (pH 9.6) overnight at 4°C. The wells were washed and blocked with 200 μl of PBS containing 0.25% casein for 1 h at room temperature. Samples were added to the plates and incubated for 1 h at 37°C. The plates were then washed, peroxidase-streptavidin goat anti-mouse IgA-HRP (Southern Biotechnology) diluted 1:10000 was added, and the plates were incubated for 1 h at 37°C. Colour was developed at room temperature with 100 μl/well of orthophenylenediamine (1 mg/ml) (SIGMA) and 0.04% H₂O₂ substrate in sodium citrate buffer. The reaction was interrupted by the addition of 20 μl/well of 2 N H₂SO₄. The absorbance was measured at 492 nm using an ELISA microplate reader (Bio-Rad).

Fluorescein isothiocyanate-conjugated (FITC) mAbs against CD69, CD25 and CD11c; phycoerythrin (PE)-conjugated mAbs against CD45RB and CD11b; and biotinylated antibodies against CD103 were utilised. Streptavidin-Cy5-Chrome was purchased from BD Biosciences. The biotinylated anti-human LAP (TGF-β) antibody was purchased from R&D Systems. Surface staining was performed according to standard procedures at density of 1×10⁶ cells (isolated from the spleen or mesenteric lymph nodes) per well. The samples were analysed in a FACScan instrument (BD), and the results were analysed by FlowJo Software (Tree Star Inc.).

The results were expressed as the mean ± standard error of the mean (SEM). Normal distribution of the samples was confirmed by the Kolmogorov–Smirnov test. The significance of differences among groups was determined by Student’s t-test or analysis of variance (ANOVA) (Tukey’s post test). Means were considered significantly different when P < 0.05.