In vitro effects of recombinant IFNα2B on the content of antigen-presenting CD66b⁺CD16⁺CD33⁺HLA^-DR⁺ subset of neutrophils in children with acute osteomyelitis

Negative impact of S. aureus, seems to be a sufficient condition for the spread of the infectious process in the bone in acute osteomyelitis (AOM) due to its altered elimination caused by dysfunction of the immune system (IS), in particular, of neutrophilic granulocytes (NG). Correction of NG dysfunction in AOM under the influence of immunotropic substances and cytokines via modulation of the NG phenotypic subsets is of sufficient interest. Our aim was to evaluate the in vitro effects of recombinant IFNá2b on the number and phenotype of CD66b⁺CD16⁺CD33⁺HLA-DR^-, CD66b⁺CD16⁺CD33⁺HLA-DR⁺ subsets and on phagocytic function of neutrophilic granulocytes in acute osteomyelitis in children.

The study of peripheral blood (PB) samples from children aged 8-15 years was carried out as follows: patients with АOM (n = 24) comprised study group 1 (SG1), healthy children (n = 13) were included into comparison group (CG). PB samples of children with AOM were incubated with recIFNá2b (50 IU/µL, 60 min, 37 °C.) in the study group 1a (SG1a). Before and after incubation with recIFNá2b, the number of NG subsets CD66b⁺CD16⁺CD33⁺HLA-DR^-, CD66b⁺CD16⁺CD33⁺HLA-DR⁺ and the density values of receptor expression by fluorescence intensity (MFI) were also determined (FC 500, Beckman Coulter, США). Phagocytic activity of NCs was evaluated as the contents of actively phagocytic NCs (%PhAN), volume of the engulfed S. aureus (strain 209) by assessing their phagocytic number (PhN), phagocytic index (PhI). Bacterial killing was determined as the percentages of microbe digestion (%D), digestion index (DI).

The cells from AOM patients revealed a subset expressing the HLA-DR receptor – СD66b⁺CD16⁺CD33⁺HLA-DR⁺NG, which is absent in the PB of CG children. The cells with primed phenotype exhibited an increased expression density of activation receptors CD16 and CD66b. Incubation of PB in AOM with recIFNá2b led to an increased proportion of CD66b⁺CD16⁺CD33⁺HLA-DR⁺ NG subset which showed active phagocytosis and improved digestion processes. The present study shows the emergence of activated subset of “long-lived” CD66b⁺CD16⁺CD33⁺HLA-DR⁺ NCs in children with AOM. This subpopulation has APC features, by presenting AG to T lymphocytes, with preserved effector properties. In an in vitro experimental system, a positive effect of recIFNá2b was demonstrated, leading to an increased number of NGs of the CD66b⁺CD16⁺CD33⁺HLA-DR⁺ subset and recovery of S. aureus phagocytosis by NGs, thus being promising in the future for development of new approaches to optimization of complex therapy in the postoperative period of AOM treatment, prevention of complications and the opportunity to alleviate the disorders in the immune system.