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During an immune response, macrophages systematically rewire their metabolism in specific ways to support their diversve functions. However, current knowledge of macrophage metabolism is largely concentrated on central carbon metabolism. Using multi-omics analysis, we identified nucleotide metabolism as one of the most significantly rewired pathways upon classical activation. Further isotopic tracing studies revealed several major changes underlying the substantial metabolomic alterations: 1) de novo synthesis of both purines and pyrimidines is shut down at several specific steps; 2) nucleotide degradation activity to nitrogenous bases is increased but complete oxidation of bases is reduced, causing a great accumulation of nucleosides and bases; and 3) cells gradually switch to primarily relying on salvaging the nucleosides and bases for maintaining most nucleotide pools. Mechanistically, the inhibition of purine nucleotide de novo synthesis is mainly caused by nitric oxide (NO)-driven inhibition of the IMP synthesis enzyme ATIC, with NO-independent transcriptional downregulation of purine synthesis genes augmenting the effect. The inhibition of pyrimidine nucleotide de novo synthesis is driven by NO-driven inhibition of CTP synthetase (CTPS) and transcriptional downregulation of thymidylate synthase (TYMS). For the rewiring of degradation, purine nucleoside phosphorylase (PNP) and uridine phosphorylase (UPP) are transcriptionally upregulated, increasing nucleoside degradation activity. However, complete degradation of purine bases by xanthine oxidoreductase (XOR) is inhibited by NO, diverting flux into nucleotide salvage. Inhibiting the activation-induced switch from nucleotide de novo synthesis to salvage by knocking out the purine salvage enzyme hypoxanthine-guanine phosporibosyl transferase ( Hprt ) significantly alters the expression of genes important for activated macrophage functions, suppresses macrophage migration, and increases pyroptosis. Furthermore, knocking out Hprt or Xor increases proliferation of the intracellular parasite Toxoplasma gondii in macrophages. Together, these studies comprehensively reveal the characteristics, the key regulatory mechanisms, and the functional importance of the dynamic rewiring of nucleotide metabolism in classically activated macrophages., Competing Interests: Conflict of Interest The authors declare no competing interest.

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., Competing Interests: Conflict of Interest Statement The authors declare no conflict of interest.

Branched chain α-ketoacid dehydrogenase complex (BCKDC) is the rate limiting enzyme in branched chain amino acid (BCAA) catabolism, a metabolic pathway with great importance for human health. BCKDC belongs to the mitochondrial α-ketoacid dehydrogenase complex family, which also includes pyruvate dehydrogenase complex (PDHC) and oxoglutarate dehydrogenase complex (OGDC). Here we revealed that BCKDC can be substantially inhibited by reactive nitrogen species (RNS) via a mechanism similar to what we recently discovered with PDHC and OGDC - modifying the lipoic arm on its E2 subunit. In addition, we showed that such reaction between RNS and the lipoic arm of the E2 subunit can further promote inhibition of the E3 subunits of α-ketoacid dehydrogenase complexes. We examined the impacts of this RNS-mediated BCKDC inhibition in muscle cells, an important site of BCAA metabolism, and demonstrated that the nitric oxide production induced by cytokine stimulation leads to a strong inhibition of BCKDC activity and BCAA oxidation in myotubes and myoblasts. More broadly, nitric oxide production reduced the level of functional lipoic arms across the multiple α-ketoacid dehydrogenases and led to intracellular accumulation of their substrates (α-ketoacids), reduction of their products (acyl-CoAs), and a lower cellular energy charge. This work revealed a new mechanism for BCKDC regulation, demonstrated its biological significance, and elucidated the mechanistic connection between RNS-driven inhibitory modifications on the E2 and E3 subunits of α-ketoacid dehydrogenases. Together with previous work, we revealed a general mechanism for RNS to inhibit all α-ketoacid dehydrogenases, which has numerous physiological implications across multiple cell types., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this article.

Public health agencies have recommended that the public wear face coverings, including face masks, to mitigate COVID-19 transmission. However, the extent to which the public has adopted this recommendation is unknown. An observational study of 3,271 members of the public in May and June 2020 examined face covering use at grocery stores across Wisconsin. We found that only 41.2% used face coverings. Individuals who appeared to be female or older adults had higher odds of using face coverings. Additionally, location-specific variables such as expensiveness of store, county-level population and county-level COVID-19 case prevalence were associated with increased odds of using face coverings. To our knowledge, this is the first direct observational study examining face covering behavior by the public in the U.S., and our findings have implications for public health agencies during the COVID-19 pandemic., Competing Interests: No author has a conflict of interest.