Differential inflammatory mediator response in vitro from murine macrophages to lactobacilli and pathogenic intestinal bacteria

Abstract

Chronic active colitis (including inflammatory bowel disease - IBD) is maintained by a variety of pro-inflammatory mediators. Certain intestinal bacterial strains may induce colitis, whereas some strains (e.g. Lactobacillus spp.) show a protective effect in colitis owing to their anti-inflammatory activity. In this study, we have examined the production of selected inflammatory cytokines, reactive oxygen species (ROS), nitric oxide (NO) and the expression of haeme oxygenase-1 (HO-1) by murine peritoneal macrophages stimulated in vitro by the intestinal bacterial strains, isolated from mice with colitis. Lactobacillus strains (Lactobacillus reuteri, L. johnsonii, L. animalis/murinus) and two potentially pathogenic bacteria (Escherichia coli and Enterococcus faecalis) induced the production of substantial amounts of cytokines with a strain specific profile. Despite some interstrain differences, all lactobacilli induced production of anti-inflammatory cytokines (IL-10(high), IL-6(low), IL-12p70(low)). Conversely, E. faecalis and E. coli induced the production of proinflammatory cytokines (TNF-alpha, IL-12p70), the cytokines essential for chronic IBD. Macrophages released comparably substantial amounts of ROS in response to all Lactobacillus strains tested, while E. coli and E. faecalis ability to induce generation of ROS was negligible. In contrast to ROS, the production of NO/NO(2) (-) by macrophages activated with all bacterial strains tested was similar. Moreover, for the first time, it has been shown that intestinal bacteria differed in their ability to induce expression of HO-1, a stress-inducible enzyme with antioxidant and anti-inflammatory properties. The beneficial immunoregulatory properties of candidate probiotic bacteria for the treatment of IBD are discussed.

Figure 1

Induction of luminol-dependent chemiluminescence (LCL) by killed intestinal bacteria. Peritoneal exudates cells (5 × 10⁵/well) were stimulated in vitro with bacteria at a ratio 30 : 1 (bacteria/macrophages). LCL signal was monitored as described in Materials and methods (▿, control, non-stimulated macrophages; ▾, opsonized zymosan; □, Lactobacillus reuteri 115; ▪, L. jonhsonii 142; ♦, L. animalis/murnus 148; •, Escherichia coli 877; ○, E. faecalis 107). The figure shows the results of five separate experiments.

Figure 2

Nitrite secretion from macrophages induced by killed intestinal bacteria. Macrophages (5 × 10⁵/well) were cultured with killed bacteria (10⁷/well) for 24 h. After incubation supernatants were collected and ${\text{No}}_2^{-}$ concentration was measured using Griess reagents as described in Materials and methods. Results are the mean +/- SEM of nine separate experiments.

Figure 3

Cytokine secretion from macrophages induced by killed intestinal bacteria. Cytokines TNF-α (A), IL-6 (B), IL-12p40 (C), IL-12p70 (D) and IL-10 (E) were analysed by ELISA in supernatants collected from 24 h cultures of peritoneal macrophages (5 × 10⁵/well) incubated with different number of bacteria (Escherichia coli 877, •; Lactobacillus reuteri 115, □; L. johnsonii 142, ▪; L. animalis/murinus 148, ♦ and E. faecalis 107, ○). Bacterial concentrations ranged between 10⁶ and 10⁸ cfu/well. Data are mean +/- SEM values derived from six independent experiments, each based on macrophages isolated from four mice and tested in duplicate. ND, not detectable; ▵, control; non-stimulated macrophages. Significant differences between bacterial strains, within each concentration level were tested by one-way anova. *P < 0.05 lactobacilli vs E. coli 877, ^†P < 0.05 L.115 vs L.148.

Figure 4

Western blot analysis of haeme oxygenase-1 (HO-1) protein expression in macrophages stimulated with killed intestinal bacteria. (A) Representative Western blot of HO-1 protein expression levels in peritoneal macrophages stimulated for 24 h with Escherichia coli 877 (E.c.), Lactobacillus reuteri 115 (L.115), L. johnsonii 142 (L.142), L. animalis/murinus 148 (L.148) and E. faecalis 107 (E.f.). β-actin, a constitutively expressed protein was used as control. (B) Densitometric analysis of bands from n = 5 experiments. Data are normalized to β-actin levels.