Modulation of pro-inflammatory activation of monocytes and dendritic cells by aza-bis-phosphonate dendrimer as an experimental therapeutic agent

Abstract

Introduction: Our objective was to assess the capacity of dendrimer aza-bis-phosphonate (ABP) to modulate phenotype of monocytes (Mo) and monocytes derived dendritic cells (MoDC) activated in response to toll-like receptor 4 (TLR4) and interferon γ (IFN- γ) stimulation.

Methods: Mo (n = 12) and MoDC (n = 11) from peripheral blood of healthy donors were prepared. Cells were preincubated or not for 1 hour with dendrimer ABP, then incubated with lipopolysaccharide (LPS; as a TLR4 ligand) and (IFN-γ) for 38 hours. Secretion of tumor necrosis factor α (TNFα), interleukin (IL) -1, IL-6, IL-12, IL-10 and IL-23 in the culture medium was measured by enzyme-linked immunosorbent assay (ELISA) and Cytokine Bead Array. Differentiation and subsequent maturation of MoDC from nine donors in the presence of LPS were analyzed by flow cytometry using CD80, CD86, CD83 and CD1a surface expression as markers.

Results: Mo and MoDC were orientated to a pro-inflammatory state. In activated Mo, TNFα, IL-1β and IL-23 levels were significantly lower after prior incubation with dendrimer ABP. In activated MoDC, dendrimer ABP promoted IL-10 secretion while decreasing dramatically the level of IL-12. TNFα and IL-6 secretion were significantly lower in the presence of dendrimer ABP. LPS driven maturation of MoDC was impaired by dendrimer ABP treatment, as attested by the significantly lower expression of CD80 and CD86.

Conclusion: Our data indicate that dendrimer ABP possesses immunomodulatory properties on human Mo and MoDC, in TLR4 + IFN-γ stimulation model, by inducing M2 alternative activation of Mo and promoting tolerogenic MoDC.

Figure 1

Structure of dendrimer azabisphosphonate (mw = 5,820 da).

Figure 2

Actions of dendrimer ABP on cytokine production by monocytes. TNFα, IL-1β, IL-6, IL-23, IL-12 and IL-10 were measured in the culture supernatant of activated Mo from 12 donors. Data are presented as box plots. Activation was performed, as indicated, in the presence or in the absence of dendrimer ABP. *P < 0.05; **P = 0.0005. ABP, aza-bis-phosphonate; IL, interleukin; Mo, monocytes; TNF, tumor necrosis factor.

Figure 3

Actions of dendrimer ABP on cytokine production by MoDC. TNFα, IL-6, IL-23, IL-12 and IL-10 were measured in the culture supernatant of activated MoDC from 11 donors. Data are presented as box plots. Activation was performed, as indicated, in the presence or in the absence of dendrimer ABP. *P <0.05; **P <0.005. ABP, aza-bis-phosphonate; IL, interleukin; MoDC, monocyte derived dendritic cells; TNF, tumor necrosis factor.

Figure 4

Dendrimer ABP favors the IL-10 over IL-12 production in MoDC. The ratio of IL-12 to IL-10 production in the presence or in the absence of ABP and LPS + IFN-γ +/− dendrimer ABP was calculated for Mo (A) and MoDC (B). When IL-10 levels were not detectable, detection limit (5 pg/mL) was chosen as the denominator of the ratio. ABP, aza-bis-phosphonate; IFN-γ, interferon-gamma; IL, interleukin; LPS, lipopolysaccharides; Mo, monocytes; MoDC, monocyte derived dendritic cells.

Figure 5

Modulation of MoDC maturation by dendrimer ABP. A. the phenotype of immature MoDC was determined by flow cytometry. Expression of CD14, CD1a, CD11c, CD83, CD80 and CD86 was evaluated after six days of differentiation in the presence of IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF). These results are representative of nine donors. B. Phenotype of mature MoDC was determined by flow cytometry. Markers of MoDC were evaluated after incubation for 38 hours in the presence or in the absence of dendrimer ABP and LPS + IFN-γ. The phenotype of non-manipulated, immature MoDC is shown as the control. These results are representative of nine donors. Mean Fluorescence Intensity (MFI) differences between staining and isotype control are shown. C. Variation of expression of MoDC maturation markers CD1a, CD83, CD80 and CD86. MFI was used to measure the level of expression of markers. Data are presented as the relative percentage of expression, as compared to controls obtained in the absence of stimulation (100%). Bars correspond to the 75^th percentile (IQR). Data are from nine donors. *P <0.05. ABP, aza-bis-phosphonate; IFN-γ, interferon-gamma; IQR, interquartile range; LPS, lipopolysaccharides; MoDC, monocyte derived dendritic cells.