Macrophages (MFs) are heterogeneous and metabolically flexible, with metabolism strongly affecting immune activation. A classic response to proinflammatory activation is increased flux through glycolysis with a downregulation of oxidative metabolism, whereas alternative activation is primarily oxidative, which begs the question of whether targeting glucose metabolism is a viable approach to control MF activation. We created a murine model of myeloid-specific glucose transporter GLUT1 (Slc2a1) deletion. Bone marrow-derived MFs (BMDM) from Slc2a1 M2/2 mice failed to uptake glucose and demonstrated reduced glycolysis and pentose phosphate pathway activity. Activated BMDMs displayed elevated metabolism of oleate and glutamine, yet maximal respiratory capacity was blunted in MF lacking GLUT1, demonstrating an incomplete metabolic reprogramming. Slc2a1 M2/2 BMDMs displayed a mixed inflammatory phenotype with reductions of the classically activated pro-and anti-inflammatory markers, yet less oxidative stress. Slc2a1 M2/2 BMDMs had reduced proinflammatory metabolites, whereas metabolites indicative of alternative activation-such as ornithine and polyamines-were greatly elevated in the absence of GLUT1. Adipose tissue MFs of lean Slc2a1 M2/2 mice had increased alternative M2-like activation marker mannose receptor CD206, yet lack of GLUT1 was not a critical mediator in the development of obesity-associated metabolic dysregulation. However, Ldlr 2/2 mice lacking myeloid GLUT1 developed unstable atherosclerotic lesions. Defective phagocytic capacity in Slc2a1 M2/2 BMDMs may have contributed to unstable atheroma formation. Together, our findings suggest that although lack of GLUT1 blunted glycolysis and the pentose phosphate pathway, MF were metabolically flexible enough that inflammatory cytokine release was not dramatically regulated, yet phagocytic defects hindered MF function in chronic diseases.

The pulmonary microvasculature plays a critical role in endotoxin-induced acute lung injury. However, the relevant signaling remain unclear. Specifically the role of endothelial Ca2+ in the induction of endotoxin-mediated responses in lung microvessels remains undefined. Toward elucidating this, we used the isolated blood-perfused rat lung preparation. We loaded microvessels with the Ca2+ indicator, Fura 2 AM and then determined Ca2+ responses to infusions of lipopolysaccharide (LPS) into the microvessels. LPS induced a more than two-fold increase in the amplitude of cytosolic Ca2+ oscillations. Inhibiting inositol 1,4,5 trisphosphate receptors on endoplasmic reticulum (ER) Ca2+ stores with Xestospongin C (XeC), blocked the LPS-induced increase in the Ca2+ oscillation amplitude. However, XeC did not affect entry of external Ca2+ via plasma membrane Ca2+ channels in lung microvascular endothelial cells. This suggested that LPS augmented the oscillations via release of Ca2+ from ER stores. In addition, XeC also blocked LPS-mediated activation and nuclear translocation of nuclear factor-kappa B in lung microvessels. Further, inhibiting ER Ca2+ release blunted increases in intercellular adhesion molecule-1 expression and retention of naïve leukocytes in LPS-treated microvessels. Taken together, the data suggest that LPS-mediated Ca2+ release from ER stores underlies nuclear factor-kappa B activation and downstream inflammatory signaling in lung microvessels. Thus, we show for the first time a role for inositol 1,4,5 trisphosphate-mediated ER Ca2+ release in the induction of LPS responses in pulmonary microvascular endothelium. Mechanisms that blunt this signaling may mitigate endotoxin-induced morbidity.

The metabolic impact of influenza A virus (IAV) infections on immune cells remains largely uncharacterized. However, much is known about the metabolism of IAV infection and immunometabolism. Thus, we will consider four main factors: the metabolic requirements of influenza virus, metabolic reprogramming of immune cells that are mobilized to fight IAV infection, the impact of systemic or local metabolism on the infection and immune response, and vice versa. We will also address the interplay of metabolism and cytokines with a focus on those relevant to IAV infections. Finally, we will limit information on immunometabolism to key cell types critical to fighting IAV infection. We will relate this information to the unique metabolic demands on immune cells and discuss how their translocation through nutrient diverse and changing environments may affect their functions in IAV infection. | ME TABOLIS M AND THE LUNG MICROENVIRONMENT | Steady-state metabolismUnder aerobic conditions, cells sustain themselves catabolically using glycolytic carbons to fuel the tricarboxylic acid (TCA) cycle. AbstractRecent studies support the notion that glycolysis and oxidative phosphorylation are rheostats in immune cells whose bioenergetics have functional outputs in terms of their biology. Specific intrinsic and extrinsic molecular factors function as molecular potentiometers to adjust and control glycolytic to respiratory power output. In many cases, these potentiometers are used by influenza viruses and immune cells to support pathogenesis and the host immune response, respectively. Influenza virus infects the respiratory tract, providing a specific environmental niche, while immune cells encounter variable nutrient concentrations as they migrate in response to infection. Immune cell subsets have distinct metabolic programs that adjust to meet energetic and biosynthetic requirements to support effector functions, differentiation, and longevity in their ever-changing microenvironments. This review details how influenza coopts the host cell for metabolic reprogramming and describes the overlap of these regulatory controls in immune cells whose function and fate are dictated by metabolism. These details are contextualized with emerging evidence of the consequences of influenzainduced changes in metabolic homeostasis on disease progression. K E Y W O R D Shost pathogen, immune response, Immunometabolism, Influenza, metabolism, viral infection, virus | 141 BAHADORAN et Al.

32Infection with the influenza virus triggers an innate immune response aimed at initiating the 33 adaptive response to halt viral replication and spread. However, the metabolic response fueling 34 the molecular mechanisms underlying changes in innate immune cell homeostasis remain 35undefined. Thus, we compared the metabolic response of dendritic cells to that of those infected 36 with active and inactive influenza A virus or treated with toll like receptor agonists. While influenza 37 infects dendritic cells, it does not productively replicate in these cells, and therefore metabolic 38 changes upon infection may represent an adaptive response on the part of the host cells. 39Using quantitative mass spectrometry along with pulse chase substrate utilization assays and 40 metabolic flux measurements, we found global metabolic changes 17 hours post infection, 41including significant changes in carbon commitment via glycolysis and glutaminolysis, as well as 42 ATP production via TCA cycle and oxidative phosphorylation. Influenza infection of dendritic cells 43 led to a metabolic phenotype, distinct from that induced by TLR agonists, with significant 44 resilience in terms of metabolic plasticity. We identified Myc as one transcription factor modulating 45 this response. Restriction of either Myc activity or mitochondrial substrates resulted in significant 46 changes in the innate immune functions of dendritic cells, including reduced motility and T cell 47 activation. Transcriptome analysis of inflammatory dendritic cells isolated following influenza 48 infection showed similar metabolic reprogramming occurs in vivo. Thus, early in the infection 49 process dendritic cells respond with global metabolic restructuring that is present in lung DC 9 50 days following infection and impacts their effector function, suggesting that metabolic switching in 51 dendritic cells plays a vital role in initiating the immune response to influenza infection. 52 53 54 3 Author Summary 55In response to influenza infection we found that dendritic cells, cells that are critical in mounting 56 an effective immune response, undergo a profound metabolic shift. They alter the concentration 57 and location of hundreds of proteins, including c-MYC, mediating a shift to a highly glycolytic 58 phenotype that is also flexible in terms of fueling respiration. Dendritic cells initiate the immune 59 response to influenza and activate the adaptive response allowing viral clearance and manifesting 60 immune memory for protection against subsequent infections. We found that limiting access to 61 specific metabolic pathways or substrates diminished key immune functions. Previously we 62 described an immediate, fixed, hypermetabolic state in infected respiratory epithelial cells. We 63 now show the metabolic responses of epithelial and dendritic cells are distinct. Here, we also 64 demonstrate that dendritic cells tailor their metabolic response to the pathogen or TLR stimulus. 65This metabolic reprogramming occurs rapidly in vitro and it is sustained in ...

Infection with the influenza virus triggers an innate immune response that initiates the adaptive response to halt viral replication and spread. However, the metabolic response fueling the molecular mechanisms underlying changes in innate immune cell homeostasis remain undefined. Although influenza increases parasitized cell metabolism, it does not productively replicate in dendritic cells. To dissect these mechanisms, we compared the metabolism of dendritic cells to that of those infected with active and inactive influenza A virus and those treated with toll-like receptor agonists. Using quantitative mass spectrometry, pulse chase substrate utilization assays and metabolic flux measurements, we found global metabolic changes in dendritic cells 17 hours post infection, including significant changes in carbon commitment via glycolysis and glutaminolysis, as well as mitochondrial respiration. Influenza infection of dendritic cells led to a metabolic phenotype distinct from that induced by TLR agonists, with significant resilience in terms of metabolic plasticity. We identified c-Myc as one transcription factor modulating this response. Restriction of c-Myc activity or mitochondrial substrates significantly changed the immune functions of dendritic cells, such as reducing motility and T cell activation. Transcriptome analysis of inflammatory dendritic cells isolated following influenza infection showed similar metabolic reprogramming occurs in vivo. Thus, early in the infection process, dendritic cells respond with global metabolic restructuring, that is present in inflammatory lung dendritic cells after infection, and this is important for effector function. These findings suggest metabolic switching in dendritic cells plays a vital role in initiating the immune response to influenza infection.