In vitro Assessment of Efferocytic Capacity of Human Macrophages Using Flow Cytometry.

Clearance of dying cells, named efferocytosis, is a pivotal function of professional phagocytes that impedes the accumulation of cell debris. Efferocytosis can be experimentally assessed by differentially tagging the target cells and professional phagocytes and analyzing by cell imaging or flow cytometry. Here, we describe an assay to evaluate the uptake of apoptotic cells (ACs) by human macrophages in vitro by labeling the different cells with commercially available dyes and analysis by flow cytometry. We detail the methods to prepare and label human macrophages and apoptotic lymphocytes and the in vitro approach to determine AC uptake. This protocol is based on previously published literature and allows for in vitro modeling of the efficiency of AC engulfment during continual efferocytosis process. Also, it can be modified to evaluate the clearance of different cell types by diverse professional phagocytes.

Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory functions and clearance of apoptotic cells.

COVID-19 is a disease of dysfunctional immune responses, but the mechanisms triggering immunopathogenesis are not established. The functional plasticity of macrophages allows this cell type to promote pathogen elimination and inflammation or suppress inflammation and promote tissue remodeling and injury repair. During an infection, the clearance of dead and dying cells, a process named efferocytosis, can modulate the interplay between these contrasting functions. Here, we show that engulfment of SARS-CoV-2-infected apoptotic cells exacerbates inflammatory cytokine production, inhibits the expression of efferocytic receptors, and impairs continual efferocytosis by macrophages. We also provide evidence supporting that lung monocytes and macrophages from severe COVID-19 patients have compromised efferocytic capacity. Our findings reveal that dysfunctional efferocytosis of SARS-CoV-2-infected cell corpses suppresses macrophage anti-inflammation and efficient tissue repair programs and provides mechanistic insights for the excessive production of pro-inflammatory cytokines and accumulation of tissue damage associated with COVID-19 immunopathogenesis.

Near-infrared photodynamic inactivation of S. pneumoniae and its interaction with RAW 264.7 macrophages.

Pneumonia is the main cause of children mortality worldwide, and its major treatment obstacle stems from the microorganisms increasing development of resistance to several antibiotics. Photodynamic therapy has been presenting, for the last decades, promising results for some subtypes of cancer and infections. In this work we aimed to develop a safe and efficient in vitro protocol for photodynamic inactivation of Streptococcus pneumoniae, one of the most commonly found bacteria in pneumonia cases, using two near-infrared light sources and indocyanine green, a FDA approved dye. Photodynamic inactivation experiments with bacteria alone allowed to determine the best parameters for microbial inactivation. Cytotoxicity assays with RAW 264.7 macrophages evaluated the safety of the PDI. To determine if the photodynamic inactivation had a positive or negative effect on the natural killing action of macrophages, we selected and tested fewer indocyanine green concentrations and 10 J/cm2 on macrophage-S. pneumoniae co-cultures. We concluded that ICG has potential as a photosensitizer for near-infrared photodynamic inactivation of S. pneumoniae, producing minimum negative impact on RAW 264.7 macrophages and having a positive interaction with the immune cell's microbicidal action.