Publications

Natural killer (NK) cells are a vital part of the innate immune system capable of rapidly clearing mutated or infected cells from the body and promoting an immune response. Here, we find that NK cells activated by viral infection or tumor challenge increase uptake of fatty acids and their expression of carnitine palmitoyltransferase I (CPT1A), a critical enzyme for long-chain fatty acid oxidation. Using a mouse model with an NK cell–specific deletion of CPT1A, combined with stable 13 C isotope tracing, we observe reduced mitochondrial function and fatty acid–derived aspartate production in CPT1A-deficient NK cells. Furthermore, CPT1A-deficient NK cells show reduced proliferation after viral infection and diminished protection against cancer due to impaired actin cytoskeleton rearrangement. Together, our findings highlight that fatty acid oxidation promotes NK cell metabolic resilience, processes that can be optimized in NK cell–based immunotherapies.

Influx of Ca 2+ ions through α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors contributes to neuronal damage in stroke, epilepsy, and neurodegenerative disorders such as ALS. The Ca 2+ permeability of AMPA receptors is largely determined by the glutamate receptor 2 (GluR2) subunit, receptors lacking GluR2 being permeable to Ca 2+ ions. We identified a difference in GluR2 expression in motor neurons from two rat strains, resulting in a difference in vulnerability to AMPA receptor-mediated excitotoxicity both in vitro and in vivo . Astrocytes from the ventral spinal cord were found to mediate this difference in GluR2 expression in motor neurons. The presence of ALS-causing mutant superoxide dismutase 1 in astrocytes abolished their GluR2-regulating capacity and thus affected motor neuron vulnerability to AMPA receptor-mediated excitotoxicity. These results reveal a mechanism through which astrocytes influence neuronal functioning in health and disease.

During vessel sprouting, a migratory endothelial tip cell guides the sprout, while proliferating stalk cells elongate the branch. Tip and stalk cell phenotypes are not genetically predetermined fates, but are dynamically interchangeable to ensure that the fittest endothelial cell (EC) leads the vessel sprout. ECs increase glycolysis when forming new blood vessels. Genetic deficiency of the glycolytic activator PFKFB3 in ECs reduces vascular sprouting by impairing migration of tip cells and proliferation of stalk cells. PFKFB3-driven glycolysis promotes the tip cell phenotype during vessel sprouting, since PFKFB3 overexpression overrules the pro-stalk activity of Notch signaling. Furthermore, PFKFB3-deficient ECs cannot compete with wild-type neighbors to form new blood vessels in chimeric mosaic mice. In addition, pharmacological PFKFB3 blockade reduces pathological angiogenesis with modest systemic effects, likely because it decreases glycolysis only partially and transiently.

Formation and homeostasis of the vascular system requires several coordinated cellular functions, but their precise interplay during development and their relative importance for vascular pathologies remain poorly understood. Here, we investigate the endothelial functions regulated by Cdc42 and their in vivo relevance during angiogenic sprouting and vascular morphogenesis in the postnatal mouse retina. We find that Cdc42 is required for endothelial tip cell selection, directed cell migration and filopodia formation, but dispensable for cell proliferation or apoptosis. While the loss of Cdc42 seem generally compatible with apical-basal polarization and lumen formation in retinal blood vessels, it leads to defective endothelial axial polarization and to the formation of severe vascular malformations in capillaries and veins. Tracking of Cdc42 depleted endothelial cells in mosaic retinas suggests that these capillary-venous malformations arise as a consequence of defective cell migration, when endothelial cells that proliferate at normal rates are unable to re-distribute within the vascular network.

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.

<ns4:p>Background Neutrophils are essential in the early innate immune response to pathogens. Harnessing their antimicrobial powers, without driving excessive and damaging inflammatory responses, represents an attractive therapeutic possibility. The neutrophil population is increasingly recognised to be more diverse and malleable than was previously appreciated. Hypoxic signalling pathways are known to regulate important neutrophil behaviours and, as such, are potential therapeutic targets for regulating neutrophil antimicrobial and inflammatory responses. Methods We used a combination of <ns4:italic>in vivo</ns4:italic> and <ns4:italic>ex vivo</ns4:italic> models, utilising neutrophil and myeloid specific PHD1 or PHD3 deficient mouse lines to investigate the roles of oxygen sensing prolyl hydroxylase enzymes in the regulation of neutrophilic inflammation and immunity. Mass spectrometry and Seahorse metabolic flux assays were used to analyse the role of metabolic shifts in driving the downstream phenotypes. Results We found that PHD1 deficiency drives alterations in neutrophil metabolism and recruitment, in an oxygen dependent fashion. Despite this, PHD1 deficiency did not significantly alter <ns4:italic>ex vivo</ns4:italic> neutrophil phenotypes or <ns4:italic>in vivo</ns4:italic> outcomes in mouse models of inflammation. Conversely, PHD3 deficiency was found to enhance neutrophil antibacterial properties without excessive inflammatory responses. This was not linked to changes in the abundance of core metabolites but was associated with increased oxygen consumption and increased mitochondrial reactive oxygen species (mROS) production. Conclusions PHD3 deficiency drives a favourable neutrophil phenotype in infection and, as such, is an important potential therapeutic target.</ns4:p>

Abstract Hematopoietic stem/progenitor cells (HSC/HPCs) are retained in the bone marrow (BM) niche via receptor-ligand interactions and mobilized from the BM after proteolytic degradation of these retention complexes. Yet, the proteinases and retention signals involved remain incompletely identified. Here, we studied the role of the plasminogen proteinase system with its plasminogen activators PA) tPA and uPA, and active plasmin (Pli) in chemo- and G-CSF-induced mobilization of HPCs and HSCs. Therefore, 5-fluorouracil (5-FU) or G-CSF were administered to mice lacking plasminogen (Plg−/−), tPA (tPA−/−), uPA (uPA−/−), both activators (tPA−/−uPA−/−), uPAR (uPAR−/−), PAI-1 (PAI-1−/−), or α2-antiplasmin (α2-AP−/−). 5-FU treatment in WT mice elevated Pli activity in BM plasma 5-fold, killed 10% of WT mice, and increased number/proliferation of HSC/HPCs in the surviving mice, with full hematopoietic recovery after 3 weeks. In contrast, up to 75% of 5-FU-treated Plg−/−and tPA−/−uPA−/− demised in the early phase of recovery, with reduced number/proliferation of HSC/HPCs and delayed hematopoietic recovery. Following G-CSF, deficiency of Plg or inhibition of Pli by tranexamic acid reduced HPC expansion and HSC translocation in the BM, resulting in impaired HPC/HSC mobilization, by up to 55% and 75%, respectively. Further analysis using uPA−/− and tPA−/− mice revealed that uPA was critical for 5-FU-induced mobilization, whereas tPA was crucial for G-CSF-induced mobilization. In addition, analysis of mice lacking MMP-2, -3, -9, and -12 revealed that 5-FU- and G-CSF-induced mobilization required predominantly MMP-9 and -3, respectively. MMP-3 and MMP-9 activities upon mobilization were reduced in Plg−/− mice, suggesting that Pli activates these MMPs. In the absence of Plg, degradation of fibronectin in the BM and production of soluble Kit ligand (but not SDF1 α) were impaired, indicating additional downstream targets of Pli. uPAR is a membrane-anchored receptor for uPA, which is cleaved into a soluble form (suPAR) by Pli and other proteinases. Interestingly, uPAR was expressed on BM-derived HPC/HSCs, and uPAR deficiency reduced their retention within the BM niche in vitro and in vivo. Furthermore, uPAR−/− mice showed poor HSC/HPC mobilization in response to 5-FU and G-CSF, while suPAR administration in WT mice amplified G-CSF-induced mobilization. Moreover, suPAR levels in the BM were increased in WT but not in Plg−/− mice during mobilization, indicating, all together, that uPAR might be a novel retention signal for HSC/HPCs in the BM niche. Finally, increased Pli activity in PAI-1−/− and α2-AP−/− mice, and WT mice treated with tenecteplase (i.e. recombinant tPA variant used for clinical thrombolysis) enhanced G-CSF-induced mobilization. Importantly, initial results suggest that thrombolytic treatment of individuals after acute myocardial infarction also seemed to stimulate HPC mobilization, extrapolating our findings to man. In conclusion, these genetic and pharmacological data reveal, for the first time, a novel role for uPAR as a retention signal for HSC/HPCs in the BM and suggest that strategies to increase PA or Pli activity might offer novel therapeutic opportunities for HSC/HPC mobilization.

Abstract —The matrix metalloproteinase (MMP) system, which may be activated via the plasminogen (Plg)/plasmin system, is claimed to play a role in matrix degradation and smooth muscle cell migration. To test the role of both systems, expression of fibrinolytic and gelatinolytic activity was quantified after vascular injury in mice with targeted inactivation of tissue-type Plg activator (tPA −/− ), urokinase-type Plg activator (uPA −/− ), or Plg (Plg −/− ). Neointima formation 1 week after vascular injury was impaired in uPA −/− and Plg −/− mice compared with wild-type (WT) mice or tPA −/− mice (reduction of neointimal area to 30% and 10% of WT, respectively). Cell accumulation at the borders of the injury was significantly ( P &lt;0.01) impaired compared with that in WT mice. One week after injury of the femoral artery, tPA-mediated fibrinolytic activity in arterial sections or extracts of WT, uPA −/− , or Plg −/− mice was not altered, whereas uPA activity levels in tPA −/− and Plg −/− mice were 2- to 3-fold higher than in uninjured controls. Total levels (latent plus active) of MMP-2 (gelatinase A) were increased by 2- to 4-fold, whereas the contribution of active MMP-2 represented 38% to 63% of the total in the different genotypes. MMP-9 (gelatinase B) was not detectable in the majority of control arteries, whereas total MMP-9 levels after injury were dramatically increased (up to 50-fold above the detection limit). Active MMP-9 represented 20% to 46% of total MMP-9 in WT, tPA −/− , and uPA −/− mice but was not consistently detectable in Plg −/− mice. Similar results were obtained in carotid arteries. Thus, the unaltered ratios of active and latent MMP-2 suggest that proMMP-2 activation may occur in the absence of tPA, uPA, or Plg, whereas no active MMP-9 was detected in the absence of Plg. The data of this study confirm a role for uPA and Plg but not for tPA in smooth muscle cell migration and neointima formation after vascular injury and indicate that impairment of these phenomena may occur despite the observed increases in MMP-2 or MMP-9 levels after vascular injury.