Human cytokine responses induced by gram-positive cell walls of normal intestinal microbiota

Abstract

The normal microbiota plays an important role in the health of the host, but little is known of how the human immune system recognizes and responds to Gram-positive indigenous bacteria. We have investigated cytokine responses of peripheral blood mononuclear cells (PBMC) to Gram-positive cell walls (CW) derived from four common intestinal indigenous bacteria, Eubacterium aerofaciens (Eu.a. ), Eubacterium limosum (Eu.l.), Lactobacillus casei (L.c.), and Lactobacillus fermentum (L.f.). Our results indicate that Gram-positive CW of the normal intestinal microbiota can induce cytokine responses of the human PBMC. The profile, level and kinetics of these responses are similar to those induced by lipopolysaccharide (LPS) or CW derived from a pathogen, Streptococcus pyogenes (S.p.). Bacterial CW are capable of inducing production of a proinflammatory cytokine, tumour necrosis factor-alpha (TNF-alpha), and an anti-inflammatory cytokine, IL-10, but not that of IL-4 or interferon-gamma (IFN-gamma). Monocytes are the main cell population in PBMC to produce TNF-alpha and IL-10. Induction of cytokine secretion is serum-dependent; both CD14-dependent and -independent pathways are involved. These findings suggest that the human cytokine responses induced by Gram-positive CW of the normal intestinal microbiota are similar to those induced by LPS or Gram-positive CW of the pathogens.

Figures

Fig. 1

Production of IL-10 and tumour necrosis factor-alpha (TNF-α) but not of IL-4 and IFN-γ by human peripheral blood mononuclear cells (PBMC) in response to Gram-positive cell walls (CW) derived from members of intestinal normal microbiota or from Streptococcus pyogenes(S.p.). Lipopolysaccharide (LPS) was used as a control. Cells (2 × 106/ml) were incubated with CW (10 μg/ml) or LPS (1 μg/ml) at 37°C for 24 h, thereafter the supernatants were assessed for cytokines by ELISA. Each column represents a mean ± s.d. of eight separate experiments.

Fig. 2

Kinetics and dose effects of IL-10 and tumour necrosis factor-alpha (TNF-α) release triggered by cell walls (CW) from Eubacterium aerofaciens(Eu.a.) or Streptococcus pyogenes(S.p.). Lipopolysaccharide (LPS) was used for comparison. Upper panels: peripheral blood mononuclear cells (PBMC; 2 × 106/ml) were stimulated with CW (10 μg/ml) or LPS (1 μg/ml) for 6 h, 24 h and 48 h at 37°C. Lower panels: cells were stimulated with various concentrations of bacterial CW or LPS (1 μg/ml) at 37°C for 24 h. Each dot represents a mean ± s.d. from two (upper panels) or three (lower panels) separate experiments. •, LPS; ▪, Eu.a. CW; ▴, S.p. CW.

Fig. 3

Influence of polymyxin B on cytokine induction by cell walls (CW) or lipopolysaccharide (LPS). Peripheral blood mononuclear cells (PBMC; 2 × 106/ml) were incubated with CW (10 μg/ml) or different concentrations of LPS. CW or LPS were first mixed with polymyxin B (10 μg/ml) 30 min before incubation with PBMC. Each column represents a mean ± s.d. of three separate experiments. The results for the CW are pooled data obtained with CW of Eubacterium aerofaciens(Eu.a.), Eubacterium limosum(Eu.l.), Lactobacillus casei(L.c.), Lactobacillus fermentum(L.f.) and Streptococcus pyogenes(S.p.). *P < 0.05 compared with the control with no polymyxin B.

Fig. 4

The serum dependence of IL-10 and tumour necrosis factor-alpha (TNF-α) induction by Eubacterium aerofaciens(Eu.a.) or Streptococcus pyogenes(S.p.) cell walls (CW) or by lipopolysaccharide (LPS). Peripheral blood mononuclear cells (PBMC; 2 × 106/ml) were stimulated with CW or LPS at 37°C for 24 h with or without 10% human AB serum. Each column represents a mean ± s.d. of three separate experiments. *P < 0.05 compared with the experiments with serum.

Fig. 5

Comparison of cytokine production from adherent and non-adherent peripheral blood mononuclear cells (PBMC) stimulated with cell walls (CW) or lipopolysaccharide (LPS). Upper panels: the cells (106/ml) were incubated with CW (10 μg/ml) or LPS (0.1 μg/ml) at 37°C for 24 h. *P < 0.05 compared with that of adherent PBMC. Lower panels: non-adherent cells (106/ml) were incubated with LPS in the presence or absence of anti-CD14 MoAb (10 μg/ml). *P < 0.05 compared with the experiments with no antibody. Each column represents a mean ± s.d. of three separate experiments.

Fig. 6

The effect of anti-CD14 MoAb on cytokine production induced by cell walls (CW) or lipopolysaccharide (LPS). Upper panels: adherent peripheral blood mononuclear cells (PBMC; 106/ml) were incubated for 2 h with anti-CD14 MoAb, or with immunoglobulin isotype control, then cultured with CW (10 μg/ml) or LPS (10 ng/ml) at 37°C for 24 h. *P < 0.05 compared with the experiments with no antibody. Lower panels: adherent cells (106/ml) were stimulated with different concentrations of CW of Lactobacillus casei(L.c.) or Streptococcus pyogenes(S.p.) or with LPS in the presence or absence of anti-CD14 MoAb. Results represent means of four separate experiments. •, LPS + anti-CD14; ▪, L.c.+ anti-CD14; Δ, S.p.+ anti-CD14.