An Imaging and Computational Algorithm for Efficient Identification and Quantification of Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) are associated with multiple disease pathologies including sepsis, asthma, rheumatoid arthritis, cancer, systemic lupus erythematosus, acute respiratory distress syndrome, and COVID-19. NETs, being a disintegrated death form, suffered inconsistency in their identification, nomenclature, and quantifications that hindered therapeutic approaches using NETs as a target. Multiple strategies including microscopy, ELISA, immunoblotting, flow cytometry, and image-stream-based methods have exhibited drawbacks such as being subjective, non-specific, error-prone, and not being high throughput, and thus demand the development of innovative and efficient approaches for their analyses. Here, we established an imaging and computational algorithm using high content screening (HCS)—cellomics platform that aid in easy, rapid, and specific detection as well as analyses of NETs. This method employed membrane-permeable and impermeable DNA dyes in situ to identify NET-forming cells. Automated algorithm-driven single-cell analysis of change in nuclear morphology, increase in nuclear area, and change in intensities provided precise detection of NET-forming cells and eliminated user bias with other cell death modalities. Further combination with Annexin V staining in situ detected specific death pathway, e.g., apoptosis, and thus, discriminated between NETs, apoptosis, and necrosis. Our approach does not utilize fixation and permeabilization steps that disturb NETs, and thus, allows the time-dependent monitoring of NETs. Together, this specific imaging-based high throughput method for NETs analyses may provide a good platform for the discovery of potential inhibitors of NET formation and/or agents to modulate neutrophil death, e.g., NETosis-apoptosis switch, as an alternative strategy to enhance the resolution of inflammation

Bile Acid Receptor Agonist GW4064 Regulates PPAR� Coactivator-1� Expression Through Estrogen Receptor-Related Receptor

Peroxisome proliferator-activated receptor� coactivator-1� (PGC-1�) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1� an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1� expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1� promoter reporter activity, mRNA, and protein expression. This induction in PGC-1� concomitantly enhances mitochondrial mass and expression of several PGC-1� target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor � (ERR�). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1� mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1� expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERR�-FXRPGC- 1� and small heterodimer partner offers new insights into the biological functions of ERR� and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiolog