In vitro dendritic cell maturation isolated from healthy people and patients with Staphylococcus aureuscaused chronic osteomyelitis

Abstract

Here we present the data comparing maturation of peripheral blood mononuclear cell-derived dendritic cells (DCs) isolated from healthy volunteers and Staphylococcus aureus-positive patients with chronic osteomyelitis. Dendritic cells were cultured in a standard maturation cell medium (RPMI-1640, supplemented with antibiotics, L-glutamine, 15% calf embryonic serum) added with interleukin-4 and granulocyte-macrophage colony-stimulating factor, followed by adding a stimulating factor cocktail containing interleukin-1β, tumor necrosis factor-α, interleukin-6, and prostaglandin E2. Dendritic cell maturation was analyzed by estimating visual characteristics under Zeiss ODSERVER.Z1 inverted microscope using Axiovision Rel.4.8 imaging software as well as light and phase-contrast microscopy at magnification of ×40, ×100, ×200, ×400, ×630. Dendritic cell immunophenotyping was carried out by using a panel of anti-human monoclonal antibodies: anti-CD80 FITC-conjugated, anti-CD86 (B7–2) PE-conjugated, anti-HLA-DR PC7-conjugated (all from Beckman Coulter, USA), anti-CD14 PerCP-Cy5.5-conjugated, anti-CD83 APC-conjugated, anti-CD40 PE-Cy7-conjugated (Becton Dickinson, USA) as well as isotype-matched control antibodies on the FACS Canto II f low cytometer (Becton Dickinson, USA). It was shown that while maturation dendritic cells derived both from patients or volunteers increased in size and underwent dendrite formation. Moreover, expression of CD86, CD83, CD80, and CD40 markers on dendritic cells derived from patients vs. volunteers was lowered. However, DC stimulation resulted in significantly increased percentage of DCs positive for CD83 DCs co-stimulation molecules CD86, CD80, CD40 chronic osteomyelitis. However, such differences found in immature DCs in both groups disappeared upon maturation, so that expression of the key markers on day 10 was maintained at close level. In particular, expression of CD83 differentiation marker and the CD80 co-stimulation molecule on DCs from patients vs. volunteers was increased stronger. Thus, a maturation potential in DCs isolated from patients with Staphylococcus aureus-caused chronic osteomyelitis was not impaired in vitro. The data obtained open up an opportunity to use dendritic cells as a natural adjuvant-substituting component for development of individualized vaccines in treatment and prevention of recurrent chronic osteomyelitis.

Keywords

• chronic osteomyelitis
• staphylococcus aureus
• dendritic cells
• maturation
• phenotyping
• cd-receptors