Activation induces shift in nutrient utilization that differentially impacts cell functions in human neutrophils.

Neutrophils - the first responders in innate immunity - perform a variety of effector functions associated with specific metabolic demand. To maintain fitness and support functions, neutrophils have been found to utilize extracellular glucose, intracellular glycogen, and other alternative substrates. However, the quantitative contribution of these nutrients under specific conditions and the relative dependence of various cell functions on specific nutrients remain unclear. Here, using ex vivo and in vivo isotopic tracing, we reveal that under resting condition, human peripheral blood neutrophils, in contrast to in vitro cultured human neutrophil-like cell lines, rely on glycogen as a major direct source of glycolysis and pentose phosphate pathway. Upon activation with a diversity of stimuli, neutrophils undergo a significant and often rapid nutrient preference shift, with glucose becoming the dominant metabolic source thanks to a multi-fold increase in glucose uptake mechanistically mediated by the phosphorylation and translocation of GLUT1. At the same time, cycling between gross glycogenesis and glycogenolysis is also substantially increased, while the net flux favors sustained or increased glycogen storage. The shift in nutrient utilization impacts neutrophil functions in a function-specific manner. The activation of oxidative burst specifically depends on the utilization of extracellular glucose rather than glycogen. In contrast, the release of neutrophil traps can be flexibly supported by either glucose or glycogen. Neutrophil migration and fungal control is promoted by the shift away from glycogen utilization. Together, these results quantitatively characterize fundamental features of neutrophil metabolism and elucidate how metabolic remodeling shapes neutrophil functions upon activation.

Switching to the cyclic pentose phosphate pathway powers the oxidative burst in activated neutrophils.

Neutrophils are cells at the frontline of innate immunity that can quickly activate effector functions to eliminate pathogens upon stimulation. However, little is known about the metabolic adaptations that power these functions. Here we show rapid metabolic alterations in neutrophils upon activation, particularly drastic reconfiguration around the pentose phosphate pathway, which is specifically and quantitatively coupled to an oxidative burst. During this oxidative burst, neutrophils switch from glycolysis-dominant metabolism to a unique metabolic mode termed 'pentose cycle', where all glucose-6-phosphate is diverted into oxidative pentose phosphate pathway and net flux through upper glycolysis is reversed to allow substantial recycling of pentose phosphates. This reconfiguration maximizes NADPH yield to fuel superoxide production via NADPH oxidase. Disruptions of pentose cycle greatly suppress oxidative burst, the release of neutrophil extracellular traps and pathogen killing by neutrophils. Together, these results demonstrate the remarkable metabolic flexibility of neutrophils, which is essential for their functions as the first responders in innate immunity.

Effects of blunt force injuries in third-instar Drosophila larvae persist through metamorphosis and reduce adult lifespan.

Blunt force injuries are a significant cause of disability and death worldwide. Here, we describe a Drosophila melanogaster model of blunt force injury that can be used to investigate cellular and molecular mechanisms that underlie the short-term and long-term effects of injuries sustained at a juvenile stage of development. Injuries inflicted in late third-instar larvae using the spring-based High-Impact Trauma (HIT) device robustly activated the humoral defense response process of melanization and caused larval and pupal lethality. Additionally, adults that developed from injured larvae had reduced lifespans, indicating that cellular and molecular mechanisms activated by blunt force injuries in larvae persist through metamorphosis and adult development. Previously, the HIT device has been used to investigate genetic and environmental factors underlying mechanisms that contribute to consequences of blunt force injuries incurred in adult flies. This work expands use of the HIT device to a juvenile stage of development, offering the opportunity to investigate whether the consequences of blunt force injuries involve different factors and mechanisms at different stages of development.

A methionine-Mettl3-N6-methyladenosine axis promotes polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic disorder marked by numerous progressively enlarging kidney cysts. Mettl3, a methyltransferase that catalyzes the abundant N6-methyladenosine (m6A) RNA modification, is implicated in development, but its role in most diseases is unknown. Here, we show that Mettl3 and m6A levels are increased in mouse and human ADPKD samples and that kidney-specific transgenic Mettl3 expression produces tubular cysts. Conversely, Mettl3 deletion in three orthologous ADPKD mouse models slows cyst growth. Interestingly, methionine and S-adenosylmethionine (SAM) levels are also elevated in ADPKD models. Moreover, methionine and SAM induce Mettl3 expression and aggravate ex vivo cyst growth, whereas dietary methionine restriction attenuates mouse ADPKD. Finally, Mettl3 activates the cyst-promoting c-Myc and cAMP pathways through enhanced c-Myc and Avpr2 mRNA m6A modification and translation. Thus, Mettl3 promotes ADPKD and links methionine utilization to epitranscriptomic activation of proliferation and cyst growth.

Metalloproteinase PAPP-A regulation of IGF-1 contributes to polycystic kidney disease pathogenesis.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end-stage renal disease (ESRD). The treatment options for ADPKD are limited. We observed an upregulation in several IGF-1 pathway genes in the kidney of Pkd1RC/RC mice, a model of ADPKD. Pregnancy-associated plasma protein A (PAPP-A), a metalloproteinase that cleaves inhibitory IGF binding proteins (IGFBPs), increasing the local bioactivity of IGF-1, was highly induced in the kidney of ADPKD mice. PAPP-A levels were high in cystic fluid and kidneys of humans with ADPKD. Our studies further showed that PAPP-A transcription in ADPKD was mainly regulated through the cAMP/CREB/CBP/p300 pathway. Pappa deficiency effectively inhibited the development of cysts in the Pkd1RC/RC mice. The role of PAPP-A in cystic disease appears to be regulation of the IGF-1 pathway and cellular proliferation in the kidney. Finally, preclinical studies demonstrated that treatment with a monoclonal antibody that blocks the proteolytic activity of PAPP-A against IGFBP4 ameliorated ADPKD cystic disease in vivo in Pkd1RC/RC mice and ex vivo in embryonic kidneys. These data indicated that the PAPP-A/IGF-1 pathway plays an important role in the growth and expansion of cysts in ADPKD. Our findings introduce a therapeutic strategy for ADPKD that involves the inhibition of PAPP-A.