Multiparametric Profiling of Neutrophil Function via a High-Throughput Flow Cytometry-Based Assay

Abstract

Neutrophils are a vital component of the innate immune system and play an essential function in the recognition and clearance of bacterial and fungal pathogens. There is great interest in understanding mechanisms of neutrophil dysfunction in the setting of disease and deciphering potential side effects of immunomodulatory drugs on neutrophil function. We developed a high throughput flow cytometry-based assay for detecting changes to four canonical neutrophil functions following biological or chemical triggers. Our assay detects neutrophil phagocytosis, reactive oxygen species (ROS) generation, ectodomain shedding, and secondary granule release in a single reaction mixture. By selecting fluorescent markers with minimal spectral overlap, we merge four detection assays into one microtiter plate-based assay. We demonstrate the response to the fungal pathogen, Candida albicans and validate the assay’s dynamic range using the inflammatory cytokines G-CSF, GM-CSF, TNFα, and IFNγ. All four cytokines increased ectodomain shedding and phagocytosis to a similar degree while GM-CSF and TNFα were more active in degranulation when compared to IFNγ and G-CSF. We further demonstrated the impact of small molecule inhibitors such as kinase inhibition downstream of Dectin-1, a critical lectin receptor responsible for fungal cell wall recognition. Bruton’s tyrosine kinase (Btk), Spleen tyrosine kinase (Syk), and Src kinase inhibition suppressed all four measured neutrophil functions but all functions were restored with lipopolysaccharide co-stimulation. This new assay allows for multiple comparisons of effector functions and permits identification of distinct subpopulations of neutrophils with a spectrum of activity. Our assay also offers the potential for studying the intended and off-target effects of immunomodulatory drugs on neutrophil responses.

Evaluation of outbreak persistence caused by multidrug-resistant and echinocandin-resistant Candida parapsilosis using multidimensional experimental and epidemiological approaches

“…Primary human neutrophils were isolated using the EasySep™ Human Neutrophil Isolation Kit (STEMCELL Technologies), and purity and viability were assessed according to the manufacturer’s instructions. A previously published neutrophil multiparametric assay was utilized [ 30 ], which is able to determine the most prominent neutrophil effector functions, i.e. reactive oxygen species (ROS) production, degranulation (CD66b) staining, and phagocytosis.…”

Characterizing the Inflammatory Profile of Neutrophil-Rich Triple-Negative Breast Cancer

“…CD62L (L-selectin) is an adhesion marker that can identify active neutrophils. Upon neutrophil contact with an inflammatory pathogen, the cells shed CD62L, initiating the inflammatory process and activating neutrophils [35]. In the N1 subpopulation, 58.01%, 62.47%, 68.13%, 53.68%, 42.67%, and 65.39% of cells were activated in DMEM control, RPMI control, N1-polarized, N2-polarized, MCF-7 CM, and MDA-MB-231 CM, respectively; all of which were not statistically different.…”

“…CD11b expression and CD62L cleavage indicate primed neutrophils, and the surface density of CD11b increases during the process. CD66b expression is linked with the activation of secondary granules, and increased expression is associated with increased inflammation [35,36]. Our results showed that the activated N1 population does not undergo high levels of degranulation, where 15.89%, 16.63%, 15.2%, 11.22%, 17.84%, and 13.71% of DMEM control, RPMI control, N1-polarized, N2-polarized, MCF-7 CM, and MDA-MB-231 CM were degranulating, respectively.…”