Pattern of cytokine responses to gram-positive and gram-negative commensal bacteria is profoundly changed when monocytes differentiate into dendritic cells

Abstract

The normal gastrointestinal bacterial flora is crucial for the maturation of acquired immunity via effects on antigen-presenting cells (APCs). Here we investigated how two types of APCs, monocytes and dendritic cells (DCs), react to different bacterial strains typical of the commensal intestinal microflora. Purified human monocytes and monocyte-derived DCs were stimulated with UV-inactivated gram-positive (Lactobacillus plantarum and Bifidobacterium adolescentis) and gram-negative (Escherichia coli and Veillonella parvula) bacterial strains. Monocytes produced higher levels of interleukin 12p70 (IL-12p70) and tumor necrosis factor (TNF), as detected by an enzyme-linked immunosorbent assay, in response to L. plantarum than in response to E. coli and V. parvula. In contrast, DCs secreted large amounts of IL-12p70, TNF, IL-6, and IL-10 in response to E. coli and V. parvula but were practically unresponsive to L. plantarum and B. adolescentis. The lack of a response to the gram-positive strains correlated with lower surface expression of Toll-like receptor 2 (TLR2) on DCs than on monocytes. The surface expression of TLR4 on DCs was undetectable when it was analyzed by flow cytometry, but blocking this receptor decreased the TNF production in response to V. parvula, indicating that TLR4 is expressed at a low density on DCs. Gamma interferon increased the expression of TLR4 on DCs and also potentiated the cytokine response to the gram-negative strains. Our results indicate that when monocytes differentiate into DCs, their ability to respond to different commensal bacteria dramatically changes, and they become unresponsive to probiotic gram-positive bacteria. These results may have important implications for the abilities of different groups of commensal bacteria to regulate mucosal and systemic immunity.

FIG. 1.

IL-12p70 production from monocytes (A) and monocyte-derived DCs (B) (10⁶ cells) after stimulation with the gram-positive organisms L. plantarum and B. adolescentis or the gram-negative organisms E. coli and V. parvula (5 × 10⁷ bacteria) for 24 h in the absence or in the presence of 10 ng of rIFN-γ per ml. The bars indicate the mean cytokine production from six donors, and the error bars indicate the standard error of the mean. Statistical significance refers to the difference between the bacterium and the two gram-positive or two gram-negative organisms, respectively (one asterisk, P < 0.05; two asterisks, P < 0.01). Unstim, unstimulated.

FIG. 2.

TNF, IL-6, and IL-10 production by monocytes and monocyte-derived DCs (10⁶ cells) after stimulation with the gram-positive organisms L. plantarum and B. adolescentis or the gram-negative organisms E. coli and V. parvula (5 × 10⁷ bacteria) for 24 h in the absence or in the presence of 10 ng of rIFN-γ per ml. The bars indicate the mean cytokine production from six donors, and the error bars indicate the standard error of the mean. Statistical significance refers to the difference between the bacterium and the two gram-positive or two gram-negative organisms, respectively (one asterisk, P < 0.05; two asterisks, P < 0.01). Unstim, unstimulated.

FIG. 3.

(A) Phagocytosis of FITC-labeled bacteria by monocytes and DCs. The bars indicate the mean percentage of FITC-positive cells from three donors, and the error bars indicate the standard error of the mean. (B) Photomicrographs of a monocyte and a DC that have phagocytosed FITC-labeled bacteria. Original magnification, ×400.

FIG. 4.

Expression of CD14, TLR2, and TLR4 on monocytes (A) and DCs and in the absence (B) or in the presence (E) of 10 ng of IFN-γ per ml after stimulation for 48 h with L. plantarum or E. coli. The percentages of CD14-positive cells and the geometric mean fluorescence intensity for TLR expression, respectively, are indicated in the upper right corners of the panels. The lines show the data for the isotype control antibodies, and the solid histograms show the expression of TLRs. The results shown are the results of one representative experiment of three experiments performed. (C) Effect on V. pavula-induced TNF production from DCs after blocking of CD14, TLR2, or TLR4. DCs (5 × 10⁵ cells per ml) were incubated with 10 μg of blocking antibody per ml or 10 μg of isotype control antibody per ml for 1 h prior to stimulation with bacteria (5 × 10⁶ cells per ml) for 7 h. The results after blocking were expressed as the percentage of TNF production relative to control production for each individual. The bars indicate the mean percentage of positive cells from three donors, and the error bars indicate the standard error of the mean. (D) Positive control for the effectiveness and specificity of the blocking antibodies used. The experiments were performed as described above, and the cells were stimulated with 0.1 μg of LPS per ml or 1 μg of peptidoglycan per ml. a-CD14, anti-CD14; a-TLR2, anti-TLR2; a-TLR4, anti-TLR4.