Phenotype remodeling in neutrophilic granulocyte subsets CD64^-CD32⁺CD16⁺CD11B⁺NG, CD64⁺CD32⁺CD16⁺CD11B⁺NG in de novo experimental model of viral-bacterial infection in vitro

A search for new targeted therapeutic strategies based on examining immunopathogenetic mechanisms for emerging co-infections is relevant and may further contribute not only to optimizing choice of immunotropic drugs, but also to achieving positive clinical and immunological remission for abnormal infectious processes. Previously, our studies found that recurrent viral-bacterial respiratory infections are associated with dysfunction of neutrophilic granulocytes (NG) with varying degree of intensity in altered effector properties. NG dysfunctions are often associated with diverse phenotypic profiles characterized by varying density for expression level of functionally significant trigger receptors. The aim of the study was to pinpoint phenotype transformation in CD64^-CD32⁺CD16⁺CD11b⁺, CD64⁺CD32⁺CD16⁺CD11b⁺ neutrophilic granulocytes in experimental model of viral-bacterial infection in vitro. We examined 52 peripheral blood samples collected from 13 healthy adult volunteers. Viral, bacterial and virus-bacterial infection was modelled in vitro by incubating blood-derived cell samples with formyl-methionyl-leucyl-phenylalanine (fMLP), double-stranded RNA (dsRNA) or in combination followed by assessing changes in immunophenotyping of CD64^-CD32⁺CD16⁺CD11b⁺NG, CD64⁺CD32⁺CD16⁺CD11b⁺NG by using using MAbs CD16-ECD, CD64-FITC, CD32-PE, CD11b-PC5 conjugates (Beckman Coulter International SA, France). It was demonstrated that NGs from healthy adult volunteers were dominated by CD64^-CD32⁺CD16⁺CD11b⁺NG as well as minor subset СD64⁺CD32⁺CD16⁺CD11b⁺ NG varying in expression density of membrane molecules. Percentage of the minor subset СD64⁺CD16+CD32⁺CD11b⁺ NG was significantly increased after exposure with dsRNA, fMLP and dsRNA+fMLP compared to untreated samples. Comparative analysis revealed that various immunotropic agents differed in affecting expression of surface receptor molecules CD16, CD32 and unidirectional effects, but of varying magnitude altering CD11b marker both in major and minor subsets. Preincubation with dsRNA followed by adding fMLP allowed to find that they co-stimulated expression of surface receptors in both NG subsets. We generated an experimental model of viral-bacterial co-infection in vitro by using fMLP and dsRNA and observed types of phenotype transformation in CD64^-CD32⁺CD16⁺CD11b⁺ NG and CD64⁺CD32⁺CD16⁺CD11b⁺ NG subsets. This model can be used to evaluate transformation of other NG subset phenotypes, NG functional activity, features of NET formation as well as impact of various immunotropic agents on NG.