Induction by a lactic acid bacterium of a population of CD4(+) T cells with low proliferative capacity that produce transforming growth factor beta and interleukin-10

Abstract

We investigated whether certain strains of lactic acid bacteria (LAB) could antagonize specific T-helper functions in vitro and thus have the potential to prevent inflammatory intestinal immunopathologies. All strains tested induced various levels of both interleukin-12 (IL-12) and IL-10 in murine splenocytes. In particular, Lactobacillus paracasei (strain NCC2461) induced the highest levels of these cytokines. Since IL-12 and IL-10 have the potential to induce and suppress Th1 functions, respectively, we addressed the impact of this bacterium on the outcome of CD4(+) T-cell differentiation. For this purpose, bacteria were added to mixed lymphocyte cultures where CD4(+) T-cells from naive BALB/c mice were stimulated weekly in the presence of irradiated allogeneic splenocytes. In these cultures, L. paracasei NCC2461 strongly inhibited the proliferative activity of CD4(+) T cells in a dose-dependent fashion. This was accompanied by a marked decrease of both Th1 and Th2 effector cytokines, including gamma interferon, IL-4, and IL-5. In contrast, IL-10 was maintained and transforming growth factor beta (TGF-beta) was markedly induced in a dose-dependent manner. The bacteria were not cytotoxic, because cell viability was not affected after two rounds of stimulation. Thus, unidentified bacterial components from L. paracasei NCC2461 induced the development of a population of CD4(+) T cells with low proliferative capacity that produced TGF-beta and IL-10, reminiscent of previously described subsets of regulatory cells implicated in oral tolerance and gut homeostasis.

FIG. 1

Induction of IL-12p40 (A) and IL-10 (B) by various strains of lactobacilli. Among the bacterial strains used were L. johnsonii NCC533, L. acidophilus NCC90, L. gasseri NCC2493, L. paracasei NCC2461, and two strains of L. casei, Shirota and GG. Bacteria were added to spleen cultures at concentrations of 10⁷ CFU/ml (black boxes), 10⁶ CFU/ml (grey boxes), or 10⁵ CFU/ml (white boxes). Error bars indicate the SEM of triplicate cultures.

FIG. 2

Inhibition of CD4⁺ T-cell proliferation by L. paracasei NCC2461. CD4⁺ T cells were primed in medium alone (−) or in the presence of L. paracasei NCC2461 (10⁷ or 10⁶ CFU/ml), LPS (1 μg/ml), or a blocking MAb against IL-4 (1D11; 10 μg/ml). Proliferation was measured 48 h after the third stimulation. Error bars indicate the SEM of triplicate cultures.

FIG. 3

Inhibition of Th1 and Th2 effector cytokines by L. paracasei NCC2461 in MLR. CD4⁺ T cells were primed in medium alone (−) or in the presence of L. paracasei NCC2461 (10⁷ or 10⁶ CFU/ml), LPS (1 μg/ml), or a blocking anti-IL-4 MAb (1D11; 10 μg/ml). Cytokine levels were measured 48 h after the third stimulation. Error bars indicate the SEM of triplicate cultures.

FIG. 4

L. paracasei NCC2461 does not suppress IL-10 and induces TGF-β production by CD4⁺ T cells in MLR. CD4⁺ T cells were primed in medium alone (−) or in the presence of L. paracasei NCC2461 (10⁷ or 10⁶ CFU/ml), LPS (1 μg/ml), or a blocking anti-IL-4 MAb (1D11; 10 μg/ml). Cytokine levels were measured 48 h after the third stimulation. Error bars indicate the SEM of triplicate cultures.