Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance

Abstract

Background: Prophylactic efficacy against colitis following lactobacillus consumption in interleukin 10 (IL-10) knockout (KO) mice has been reported. Whether this applies equally to other probiotic strains is unknown, and the mechanism is unclear.

Aims: (1) To compare the effect of feeding Lactobacillus salivarius subspecies salivarius 433118 and Bifidobacterium infantis 35624 against placebo on enterocolitis, the intestinal microflora, and (2) to compare the systemic immunological response to in vitro microbial challenge in probiotic fed and control IL-10 KO mice.

Methods: Three groups of 10 IL-10 KO mice were fed fermented milk products containing Lb salivarius 433118 at 10(9) CFU/ml, B infantis 35624 at 10(8) CFU/ml, and unmodified milk, respectively, for 19 weeks. Faecal samples were taken at regular intervals to confirm gut transit, recovery of fed probiotics, and to assess the impact on the microflora. At sacrifice, the bowels were histologically scored. Cytokine production from Peyers' patches and splenocytes was measured in vitro by ELISA.

Results: Faecal recovery of probiotics was confirmed in all probiotic fed mice but not in controls. Colonic and caecal inflammatory scores were significantly decreased in both groups of probiotic fed mice (p<0.05). Proinflammatory cytokine production by Peyers' patches and splenocytes was significantly reduced in probiotic fed animals whereas transforming growth factor beta (TGF-beta) levels were maintained.

Conclusion: Both Lactobacillus salivarius 433118 and Bifidobacterium infantis 35624 significantly attenuate colitis in this murine model. Attenuation of colitis is associated with a reduced ability to produce Th1-type cytokines systemically and mucosally, while levels of TGF-beta are maintained.

Figures

Figure 1

Gastrointestinal inflammatory score, including the ileum, caecum, and colon, in probiotic and control fed groups (*p

Figure 2

Recovery of Bifidobacteria infantis 35624 (A) and Lactobacillus salivarius 118 (B) in stool from probiotic fed mice. Results are expressed as mean (SEM) colony forming units/g (CFU/g). No probiotics were isolated from mice in the placebo group.

Figure 3

Culture independent molecular profile (16s rDNA DGGE) of faecal flora from control fed mice and probiotic fed mice (A). Lane M, molecular marker; lanes 5.1–5.6, control fed mice; lanes 6.1–6.6, Lactobacillus salivarius subspecies salivarius UCC118 fed mice; Lane Lb, pure culture of Lb salivarius UCC118. The accompanying dendrogram (B) shows similarity indices for the dominant flora, as assessed by image analysis of the gel. The banding profiles for the two groups of mice are distinct and cluster separately.

Figure 4

Cytokine production by stimulated splenocytes in placebo fed and probiotic fed mice. Results are expressed as mean (SEM) cytokine levels per group. There was a significant reduction in the proinflammatory cytokines (interferon γ (IFN) (A), tumour necrosis factor α (TNF) (C), and interleukin 12 (IL-12) (B)) with maintenance of the anti- inflammatory transforming growth factor β (TGF) (D) (*p

Figure 5

Resting in vitro production of tumour necrosis factor α (TNF) and interferon γ (IFN) by intestinal mononuclear cells from Peyers’ patches in placebo fed and probiotic fed mice (Bifidobacterium infantis 35624). Results are expressed as mean (SEM) cytokine levels per group (*p<0.05; n=8).