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IntroductionNeutrophils are highly abundant innate immune cells that are constantly produced from myeloid progenitors in the bone marrow. Differentiated neutrophils can perform an arsenal of effector functions critical for host defense. This study aims to quantitatively understand neutrophil mitochondrial metabolism throughout differentiation and activation, and to elucidate the impact of mitochondrial metabolism on neutrophil functions.MethodsTo study metabolic remodeling throughout neutrophil differentiation, murine ER-Hoxb8 myeloid progenitor-derived neutrophils and human induced pluripotent stem cell-derived neutrophils were assessed as models. To study the metabolic remodeling upon neutrophil activation, differentiated ER-Hoxb8 neutrophils and primary human neutrophils were activated with various stimuli, including ionomycin, monosodium urate crystals, and phorbol 12-myristate 13-acetate. Characterization of cellular metabolism by isotopic tracing, extracellular flux analysis, metabolomics, and fluorescence-lifetime imaging microscopy revealed dynamic changes in mitochondrial metabolism.ResultsAs neutrophils mature, mitochondrial metabolism decreases drastically, energy production is offloaded from oxidative phosphorylation, and glucose oxidation through the TCA cycle is substantially reduced. Nonetheless, mature neutrophils retain the capacity for mitochondrial metabolism. Upon stimulation with certain stimuli, TCA cycle is rapidly activated. Mitochondrial pyruvate carrier inhibitors reduce this re-activation of the TCA cycle and inhibit the release of neutrophil extracellular traps. Treatment with these inhibitors also impacts neutrophil redox status, migration, and apoptosis without significantly changing overall bioenergetics.ConclusionsTogether, these results demonstrate that mitochondrial metabolism is dynamically remodeled and plays a significant role in neutrophils. Furthermore, these findings point to the therapeutic potential of mitochondrial pyruvate carrier inhibitors in a range of conditions where dysregulated neutrophil response drives inflammation and contributes to pathology.

Neutrophils are critical for host defense against fungi. However, the short life span and lack of genetic tractability of primary human neutrophils has limited in vitro analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils. Here, we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against Aspergillus fumigatus. GATA1 knockout (KO) iNeutrophils have increased maturation, antifungal pattern recognition receptor expression and more readily execute neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal ?-glucan, including an upregulation of the pentose phosphate pathway (PPP), similar to primary human neutrophils in vitro. Furthermore, we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill A. fumigatus but is not necessary for the upregulation of PPP. Collectively, these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response. Author SummaryNeutrophils are important first responders to fungal infections, and understanding their antifungal functions is essential to better elucidating disease dynamics. Primary human neutrophils are short lived and do not permit genetic manipulation, limiting their use to study neutrophil-fungal interactions in vitro. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils for in vitro analyses. In this report we show that GATA1-deficient iPSCs generate neutrophils (iNeutrophils) that are more mature than wild-type iNeutrophils and display increased antifungal activity against the human fungal pathogen Aspergillus fumigatus. We also show that GATA1-deficient iNeutrophils have increased expression of antifungal receptors than wild-type cells and shift their metabolism and execute neutrophil antifungal functions at levels comparable to primary human neutrophils. Deletion of the integrin CD18 blocks the ability of GATA1-deficient iNeutrophils to kill and control the growth of A. fumigatus, demonstrating an important role for this integrin in iNeutrophil antifungal activity. Collectively, these findings support the use of iNeutrophils as a model to study neutrophil antifungal immunity.

The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM), an embryonic germ layer that gives rise to kidneys. In order to achieve this goal, establishing an efficient, stable and low-cost method for differentiating IM cells using small molecules is required. In this study, we identified two retinoids, AM580 and TTNPB, as potent IM inducers by high-throughput chemical screening, and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator, CHIR99021, combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step, directly inducing IM cells by activating Wnt, retinoic acid (RA), and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy.