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Nowhere in biomedical research does a cell type seem as simultaneously vital and vexing as the neutrophil. Human immunity deteriorates rapidly in the absence of neutrophils whereas their dysfunctional abundance can cause misery and harm, as exemplified by cystic fibrosis (CF), neutrophilic asthma, and chronic obstructive pulmonary disorders (COPDs). The goal of treating the underlying causes of these diseases without impairing immunity has been elusive, leading to calls for improved understanding of neutrophil plasticity and the possibility that it enables generation of pathological neutrophil subsets.1,2 To this end, Forrest, Tirouvanziam, and colleagues report development of a tissue culture method that recreates several of the pathological phenotypes observed in airway neutrophils from CF patients, summarized by the memorable acronym “GRIM” for their granule-releasing, immunoregulatory, and metabolic attributes. Among the most intriguing and important aspects of their study is its demonstration of microenvironment as a determinant of functional responsiveness in human neutrophils, an epigenetic plasticity previously suspected as a contributor to CF2 and other inflammatory diseases.3 New assays involving cultured human cells are a welcome addition to the existing animal models used in preclinical drug testing. This is because most, 80%, of all drugs that show promise in mouse or other animal studies goon to fail in phase I or II human clinical trials.4 Numerous conceptual advances in immunology have been made using mice,5 but it seems our species are not sufficiently alike to consistently predict clinical success. One recent comparison of neuro-inflammatory pathways undertaken to understand the high rate of failure of antiinflammatory treatments for Alzheimer’s disease6 found that whereas up to 60% of inflammatory cell or cytokine interactions were similar in mouse and humans, 10–15% were “reversed.” The remaining interactions that were evaluated were unique to humans, with no known counterparts in themouse. That additional methods of preclinical testing are needed should not be surprising given the estimated 100 million year evolutionary distance between rodents and humans.7 The report from Forrest et al. thus adds a potentially important tool to the drug development toolbox, as well as further evidence of neutrophil plasticity that is reshaping perceptions about their functionally dynamic responsiveness in health and disease. Neutrophil accumulation in airway disease is somewhat unusual in that it involves transepithelial migration into the air-bearing lumen of the lung in addition to transendothelial extravasation from the circulatory system. To replicate the additional transepithelial step, Forrest et al. used a clever twist of standard 3D tissue culture methods in which a human cell line derived from secretory club cells, H441, was initially grown at an air-liquid interface on top of a porous, polystyrene scaffold that had been coated with collagen. After an airway-like epithelial surface had formed, the sealed cell layer and its supporting scaffold were turned upside down into culture medium containing dissociated sputum from CF patients (Fig. 1). Purified blood neutrophils loaded into the top of the newly exposed matrix of the scaffold were observed to migrate from within the matrix, through the sealed H441 cell layer and into the lower culture medium where they expressed phenotypes similar to those found in airway neutrophils collected from CF patients, such as increased intracellular reactive oxygen species (ROS), pinocytosis and cell surface CD63 (a marker of elastase exocytosis) and decreased CD16 (FcγRIIIB, the low affinity receptor for IgG that is shed from activated neutrophils). These “GRIM” neutrophil phenotypes did not appear if transepithelial migration was instigated by a control chemoattractant, the leukotriene LTB4; nor did it appear if neutrophils were cultured directlywith dissociatedCF sputum. Hence, it was only the combination of transepithelial migration and exposure to factor(s) present in patient sputum that elicited GRIM phenotypic changes in human neutrophils. Another phenotypic component of theGRIMneutrophil hypothesis is increased expression of immunoregulatory effectors, arginase-1 and PD-L1, known to downregulate T cell responses. These immunoregulatoryoutcomes arenot necessarily exclusive toCFdiseasebecause they eventually appeared inneutrophils that had transmigrated toward sputum from healthy control participants. Surface display of CD16 and CD63 also shifted after transmigration to control sputum, but again was markedly delayed. At least some phenotypes of GRIM neutrophils may therefore need to be considered as kinetic rather than absolute markers of pathological conditioning. Given this dynamic, and the fact that ex vivo systems often fail to achieve or maintain the same cellular steady states observed in vivo, selection of appropriate time points will be needed when deploying the new culture method for mechanistic studies of neutrophil plasticity or evaluation of drug candidates. As can often be said after a first report, “the proof will be in the tasting of the pudding” with respect to determining the value of the Tirouvanziam transepithelial culture system in basic neutrophil research or drug development. Many interesting parameters beg to be assessed in the course of vetting the GRIM phenotype hypothesis. For example, which factors in CF sputum are responsible for pathological reprogramming of neutrophils? The authors note that CF sputum samples are easily collected because they are (unfortunately) coughed up in abundance and easily processed such that identification and reconstitution of the