Experimental in vitro reprogramming of CD64-CD32+CD16+CD11b+ and CD64+CD32+CD16+CD11b+ neutrophilic granulocytes by arginyl-alpha-aspartyl-lysyl-valyl-tyrosyl-arginine in modelled virus-bacterial coinfection

Abstract

Disruption of neutrophilic granulocyte (NG) functioning underlies one of the key causes for negative polymicrobial synergism during virus-microbial co-infections. In connection with this, it is important to develop experimental models for viral-bacterial co-infections in vitro, which might allow to uncover NG involvement in effector events and assess reorganization of inter-connected functionally relevant NG receptors in response to various immunotropic agents. Understanding molecular mechanisms related to any molecule including drug molecules provides its safe use allowing them to become drug of choice. NG functional activity is associated with surface receptors CD64, CD32, CD16, CD11b, which are assigned to several NG subsets exhibiting distinct phenotypes, and their activation leads to complex processes of pathogen elimination. Study objective: to elucidate types of phenotype transition for NG subsets CD64-CD32+CD16+CD11b+ and СD64+CD32+CD16+CD11b+ and assess an opportunity for phenotype reprogramming exposed to hexapeptide arginyl-alpha-aspartyl-lysyl-valyl-tyrosyl-arginine (НP) in experimental in vitro model of viral-bacterial coinfection. Materials and methods. We examined 39 samples of peripheral blood (PВ) collected from healthy adult volunteers (7 women, 6 men) aged 21 to 32 years, subdivided into 3 groups: comparison group 1 (intact NG); comparison group 2 – model of viral-bacterial infection; Study group – to assess HP effects. Model of viral-bacterial co-infection was created by incubating PВ samples sequentially with dsRNA (10-7M) for 60 min followed by fMLP (10-7M) for 60 min, 37 °С. To assess HP effect, PC samples preincubated with dsRNA and fMLP were next exposed to HP (10-6 g/L) for 60 min at 37 °С. We analyzed percentage of CD64-CD32+CD16+CD11b+ and СD64+CD32+CD16+CD11b+NG subsets as well as receptor expression density (MFI) by flow cytometry (FC 500, Beckman Coulter, USA) using MAbs (Beckman Coulter International SA, France). Results. Transformation of CD64-CD32+CD16+CD11b+ and СD64+CD32+CD16+CD11b+NG subset phenotype was experimentally revealed in virus-bacterial model manifested as upregulated expression of all receptors examined. Our data on HP effects ambiguously demonstrated phenotype transformation in CD64-CD32+CD16+CD11b+NG, СD64+CD32+CD16+CD11b+NG in in vitro viral – bacterial coinfection model contributing to recovery of proper NG response.