Publisher Correction: The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

Phospholipid membranes drive abdominal aortic aneurysm development through stimulating coagulation factor activity.

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high mortality and limited treatment options. How blood lipids regulate AAA development is unknown. Here lipidomics and genetic models demonstrate a central role for procoagulant enzymatically oxidized phospholipids (eoxPL) in regulating AAA. Specifically, through activating coagulation, eoxPL either promoted or inhibited AAA depending on tissue localization. Ang II administration to ApoE-/- mice increased intravascular coagulation during AAA development. Lipidomics revealed large numbers of eoxPL formed within mouse and human AAA lesions. Deletion of eoxPL-generating enzymes (Alox12 or Alox15) or administration of the factor Xa inhibitor rivaroxaban significantly reduced AAA. Alox-deficient mice displayed constitutively dysregulated hemostasis, including a consumptive coagulopathy, characterized by compensatory increase in prothrombotic aminophospholipids (aPL) in circulating cell membranes. Intravenously administered procoagulant PL caused clotting factor activation and depletion, induced a bleeding defect, and significantly reduced AAA development. These data suggest that Alox deletion reduces AAA through diverting coagulation away from the vessel wall due to eoxPL deficiency, instead activating clotting factor consumption and depletion in the circulation. In mouse whole blood, ∼44 eoxPL molecular species formed within minutes of clot initiation. These were significantly elevated with ApoE-/- deletion, and many were absent in Alox-/- mice, identifying specific eoxPL that modulate AAA. Correlation networks demonstrated eoxPL belonged to subfamilies defined by oxylipin composition. Thus, procoagulant PL regulate AAA development through complex interactions with clotting factors. Modulation of the delicate balance between bleeding and thrombosis within either the vessel wall or circulation was revealed that can either drive or prevent disease development.

Isolation and culture of murine bone marrow-derived macrophages for nitric oxide and redox biology.

Macrophages are mononuclear phagocytes derived from haematopoietic progenitors that are widely distributed throughout the body. These cells participate in both innate and adaptive immune responses and lie central to the processes of inflammation, development, and homeostasis. Macrophage physiology varies depending on the environment in which they reside and they exhibit rapid functional adaption in response to external stimuli. To study macrophages in vitro, cells are typically cultured ex vivo from the peritoneum or alveoli, or differentiated from myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). BMDMs represent an efficient and cost-effective means of studying macrophage biology. However, the inherent sensitivity of macrophages to biochemical stimuli (such as cytokines, metabolic intermediates, and RNS/ROS) makes it imperative to control experimental conditions rigorously. Therefore, the aim of this study was to establish an optimised and standardised method for the isolation and culture of BMDMs. We used classically activated macrophages isolated from WT and nitric oxide (NO)-deficient mice to develop a standardised culture method, whereby the constituents of the culture media are defined. We then methodically compared our standardised protocol to the most commonly used method of BMDM culture to establish an optimal protocol for the study of nitric oxide (NO)-redox biology and immunometabolism in vitro.

Improved cellular uptake of perfluorocarbon nanoparticles for in vivo murine cardiac 19F MRS/MRI and temporal tracking of progenitor cells.

Herein, we maximize the labeling efficiency of cardiac progenitor cells (CPCs) using perfluorocarbon nanoparticles (PFCE-NP) and 19F MRI detectability, determine the temporal dynamics of single-cell label uptake, quantify the temporal viability/fluorescence persistence of labeled CPCs in vitro, and implement in vivo, murine cardiac CPC MRI/tracking that could be translatable to humans. FuGENEHD-mediated CPC PFCE-NP uptake is confirmed with flow cytometry/confocal microscopy. Epifluorescence imaging assessed temporal viability/fluorescence (up to 7 days [D]). Nonlocalized murine 19F MRS and cardiac MRI studied label localization in terminal/longitudinal tracking studies at 9.4 T (D1-D8). A 4-8 fold 19F concentration increase is evidenced in CPCs for FuGENE vs. directly labeled cells. Cardiac 19F signals post-CPC injections diminished in vivo to ~31% of their values on D1 by D7/D8. Histology confirmed CPC retention, dispersion, and macrophage-induced infiltration. Intra-cardiac injections of PFCE-NP-labeled CPCs with FuGENE can be visualized/tracked in vivo for the first time with 19F MRI.

Tracking Monocyte Recruitment and Macrophage Accumulation in Atherosclerotic Plaque Progression Using a Novel hCD68GFP/ApoE-/- Reporter Mouse-Brief Report.

OBJECTIVE: To create a model of atherosclerosis using green fluorescent protein (GFP)-targeted monocytes/macrophages, allowing analysis of both endogenous GFP+ and adoptively transferred GFP+ myeloid cells in arterial inflammation. APPROACH AND RESULTS: hCD68GFP reporter mice were crossed with ApoE-/- mice. Expression of GFP was localized to macrophages in atherosclerotic plaques and in angiotensin II-induced aortic aneurysms and correlated with galectin 3 and mCD68 expression. Flow cytometry confirmed GFP+ expression in CD11b+/CD64+, CD11c+/MHC-IIHI, and CD11b+/F4/80+ myeloid cells. Adoptive transfer of GFP+ monocytes demonstrated monocyte recruitment to both adventitia and atherosclerotic plaque, throughout the aortic root, within 72 hours. We demonstrated the biological utility of hCD68GFP monocytes by comparing the recruitment of wild-type and CCR2-/- monocytes to sites of inflammation. CONCLUSIONS: hCD68GFP/ApoE-/- mice provide a new approach to study macrophage accumulation in atherosclerotic plaque progression and to identify cells recruited from adoptively transferred monocytes.

Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome.

Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrow-derived monocytes and "alternatively activated" M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide-induced expression of inducible nitric oxide synthase, interleukin-1ß, and interleukin 1 receptor antagonist but not transforming growth factor ß1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids.

Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation in vivo.

The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo, we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood, spleen, and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes, which downregulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages, allowing continued cell tracking during resolution of inflammation. In summary, this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation.

Endothelial cell-specific reactive oxygen species production increases susceptibility to aortic dissection.

BACKGROUND: Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. METHODS AND RESULTS: A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II-mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45(+) inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II-induced vascular smooth muscle cell ROS production. CONCLUSIONS: These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II-mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

A new protective role for S100A9 in regulation of neutrophil recruitment during invasive pneumococcal pneumonia.

The S100A8/A9 heterodimer is abundantly expressed by myeloid cells, especially neutrophils, but its mechanism of action is only partially determined. In this study we investigated S100A8/A9 involvement in the host response to Streptococcus pneumoniae infection making use of S100a9(-/-) mice that lack heterodimer expression in myeloid cells. S100a9(-/-) mice that were infected intranasally with pneumococci rapidly succumbed, with 80% mortality after 48 h, whereas the majority of wild-type mice recovered. Over this time period, S100a9(-/-) mice displayed an average 6-fold reduction in circulating and lung-recruited neutrophils. Taqman analysis of S100a9(-/-) lungs revealed decreased production of a dominant subset of 5 cytokines and chemokines associated with neutrophil recruitment. The greatest differential was with the cytokine granulocyte colony-stimulating factor (G-CSF) that causes bone marrow release of neutrophils into the circulation (1900-fold difference at 48 h). Treating S100a9(-/-) mice with G-CSF reversed their increased susceptibility to infection by enhancing both circulating neutrophils and neutrophil recruitment into infected lungs, by reducing pneumococcal colony forming units, and by elevation of chemokine CXCL1, cytokine IL-6, and endogenous G-CSF proteins. Thus S100A9, potentially with its partner S100A8, makes a major contribution in the host response to pneumococcal infection by increasing circulating neutrophils principally regulation of G-CSF production.

Fractalkine promotes human monocyte survival via a reduction in oxidative stress.

OBJECTIVE: The CX3C chemokine fractalkine (CX3CL1) has a critical role in the development of atherogenesis because apolipoprotein-E-deficient mice lacking CX3CL1 or its receptor CX3CR1 develop smaller plaques and polymorphisms in CX3CR1 are associated with altered risk of cardiovascular disease. CX3CR1 is found on numerous cell types involved in atherogenesis but seems to have a key role in monocyte function. We aimed to elucidate the role of CX3CL1 in human monocyte survival and determine the mechanism by which CX3CL1 spares monocytes from apoptosis. APPROACH AND RESULTS: Primary human monocytes were prepared from healthy donors and subjected to serum-starvation to induce spontaneous apoptosis. The addition of CX3CL1, but not other chemokines tested, promoted monocyte survival in a dose-dependent manner with full-length CX3CL1 (including the mucin stalk) having a more potent antiapoptotic effect than chemokine-domain CX3CL1. The prosurvival effect of CX3CL1 was evident in both monocyte subsets although nonclassical monocytes were more prone to spontaneous apoptosis. In addition, we found that the effect of CX3CL1 was independent of CX3CR1 genotype. Serum-starvation increased the level of intracellular reactive oxygen species, and this was reduced by the addition of CX3CL1. Inhibition of oxidative stress with an antioxidant prevented monocyte apoptosis, indicating that this is the dominant mechanism of cell death targeted by CX3CL1. CONCLUSIONS: CX3CL1 has a substantial and highly reproducible antiapoptotic effect on human monocytes, via a mechanism involving a reduction in oxidative stress. This suggests that CX3CL1 is likely to play a key role in human atherogenesis and may provide a novel therapeutic target in cardiovascular disease.

Tetrahydrobiopterin determines vascular remodeling through enhanced endothelial cell survival and regeneration.

BACKGROUND: Endothelial cell (EC) survival and regeneration are important determinants of the response to vascular injury that leads to neointimal hyperplasia and accelerated atherosclerosis. Nitric oxide (NO) is a key regulator of EC and endothelial progenitor cell function, but the pathophysiological mechanisms that regulate endothelial NO synthase in endothelial regeneration remain unclear. METHODS AND RESULTS: Endothelium-targeted overexpression of GTP cyclohydrolase (GCH) I increased levels of the endothelial NO synthase cofactor, tetrahydrobiopterin, in an EC-specific manner and reduced neointimal hyperplasia in experimental vein grafts in GCH/apolipoprotein E-knockout mice. These effects were mediated through enhanced donor-derived survival and recipient-derived repopulation of GCH transgenic ECs, revealed by tracking studies in Tie2-LacZ/GCH-Tg/apolipoprotein E-knockout recipient mice or donor grafts, respectively. Endothelial GCH overexpression increased endothelial NO synthase coupling and enhanced the proliferative capacity of ECs and circulating endothelial progenitor cell numbers after vascular injury. CONCLUSIONS: These observations indicate that endothelial tetrahydrobiopterin availability modulates neointimal hyperplasia after vascular injury via accelerated EC repopulation and growth. Targeting tetrahydrobiopterin-dependent endothelial NO synthase regulation in the endothelium is a rational therapeutic target to enhance endothelial regeneration and reduce neointimal hyperplasia in vascular injury states.

S100A9 has a protective role in inflammation-induced skin carcinogenesis.

The S100A8/A9 heterodimer is expressed by myeloid cells where its function has been extensively investigated. Immune cell S100A8/A9 promotes proinflammatory effects, and its absence is often associated with lack of leukocyte recruitment resulting in protection in terms of disease progression. S100A8/A9 is also expressed by certain epithelia, either constitutively as in mucosal epithelia or following stimulation as in skin keratinocytes. The role of the heterodimer in this context has not been as frequently explored. In this study, the incidence of skin papillomas induced by 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) in S100a9(-/-) mice has been investigated. Unlike the immune disorders and certain models of cancer, absence of S100A8/A9 caused an increased incidence in skin of papillomas and, subsequently, squamous cell carcinomas. Although associated in S100a9(-/-) mice with increased recruitment of neutrophils and T cells, a bone marrow chimera experiment revealed the major defect to be primarily due to the absence of S100A8/A9 in the skin keratinocytes. S100a9(-/-) skin displayed enhanced Ki-67 expression over the time period of appearance of the papillomas suggesting an effect of S100A8/A9 in regulating proliferation in the epidermal layer. Thus, despite immune cell recruitment in S100a9(-/-) mouse skin that might have been predicted to promote tumor growth, it was the absence of S100A8/A9 in skin keratinocytes that dominated in terms of papilloma formation. The study highlights the importance of the S100A8/A9-expressing skin epidermal layer in controlling skin tumor formation and suggests that the influence of the heterodimer is dependent on the tissue context in which it is expressed.

Endothelial cell repopulation after stenting determines in-stent neointima formation: effects of bare-metal vs. drug-eluting stents and genetic endothelial cell modification.

AIMS: Understanding endothelial cell repopulation post-stenting and how this modulates in-stent restenosis is critical to improving arterial healing post-stenting. We used a novel murine stent model to investigate endothelial cell repopulation post-stenting, comparing the response of drug-eluting stents with a primary genetic modification to improve endothelial cell function. METHODS AND RESULTS: Endothelial cell repopulation was assessed en face in stented arteries in ApoE(-/-) mice with endothelial-specific LacZ expression. Stent deployment resulted in near-complete denudation of endothelium, but was followed by endothelial cell repopulation, by cells originating from both bone marrow-derived endothelial progenitor cells and from the adjacent vasculature. Paclitaxel-eluting stents reduced neointima formation (0.423 ± 0.065 vs. 0.240 ± 0.040 mm(2), P = 0.038), but decreased endothelial cell repopulation (238 ± 17 vs. 154 ± 22 nuclei/mm(2), P = 0.018), despite complete strut coverage. To test the effects of selectively improving endothelial cell function, we used transgenic mice with endothelial-specific overexpression of GTP-cyclohydrolase 1 (GCH-Tg) as a model of enhanced endothelial cell function and increased NO production. GCH-Tg ApoE(-/-) mice had less neointima formation compared with ApoE(-/-) littermates (0.52 ± 0.08 vs. 0.26 ± 0.09 mm(2), P = 0.039). In contrast to paclitaxel-eluting stents, reduced neointima formation in GCH-Tg mice was accompanied by increased endothelial cell coverage (156 ± 17 vs. 209 ± 23 nuclei/mm(2), P = 0.043). CONCLUSION: Drug-eluting stents reduce not only neointima formation but also endothelial cell repopulation, independent of strut coverage. In contrast, selective targeting of endothelial cell function is sufficient to improve endothelial cell repopulation and reduce neointima formation. Targeting endothelial cell function is a rational therapeutic strategy to improve vascular healing and decrease neointima formation after stenting.

Ly6Chi Monocytes Are Metabolically Reprogrammed in the Blood during Inflammatory Stimulation and Require Intact OxPhos for Chemotaxis and Monocyte to Macrophage Differentiation.

Acute inflammation is a rapid and dynamic process involving the recruitment and activation of multiple cell types in a coordinated and precise manner. Here, we investigate the origin and transcriptional reprogramming of monocytes using a model of acute inflammation, zymosan-induced peritonitis. Monocyte trafficking and adoptive transfer experiments confirmed that monocytes undergo rapid phenotypic change as they exit the blood and give rise to monocyte-derived macrophages that persist during the resolution of inflammation. Single-cell transcriptomics revealed significant heterogeneity within the surface marker-defined CD11b+Ly6G-Ly6Chi monocyte populations within the blood and at the site of inflammation. We show that two major transcriptional reprogramming events occur during the initial six hours of Ly6Chi monocyte mobilisation, one in the blood priming monocytes for migration and a second at the site of inflammation. Pathway analysis revealed an important role for oxidative phosphorylation (OxPhos) during both these reprogramming events. Experimentally, we demonstrate that OxPhos via the intact mitochondrial electron transport chain is essential for murine and human monocyte chemotaxis. Moreover, OxPhos is needed for monocyte-to-macrophage differentiation and macrophage M(IL-4) polarisation. These new findings from transcriptional profiling open up the possibility that shifting monocyte metabolic capacity towards OxPhos could facilitate enhanced macrophage M2-like polarisation to aid inflammation resolution and tissue repair.

The downstream regulation of chemokine receptor signalling: implications for atherosclerosis.

Heterotrimeric G-protein-coupled receptors (GPCRs) are key mediators of intracellular signalling, control numerous physiological processes, and are one of the largest class of proteins to be pharmacologically targeted. Chemokine-induced macrophage recruitment into the vascular wall is an early pathological event in the progression of atherosclerosis. Leukocyte activation and chemotaxis during cell recruitment are mediated by chemokine ligation of multiple GPCRs. Regulation of GPCR signalling is critical in limiting vascular inflammation and involves interaction with downstream proteins such as GPCR kinases (GRKs), arrestin proteins and regulator of G-protein signalling (RGS) proteins. These have emerged as new mediators of atherogenesis by functioning in internalisation, desensitisation, and signal termination of chemokine receptors. Targeting chemokine signalling through these proteins may provide new strategies to alter atherosclerotic plaque formation and plaque biology.

Endothelial cell vasodilator dysfunction mediates progressive pregnancy-induced hypertension in endothelial cell tetrahydrobiopterin deficient mice.

BACKGROUND AND PURPOSE: Pregnancy-associated vascular remodelling is essential for both maternal and fetal health. We have previously shown that maternal endothelial cell tetrahydrobiopterin (BH4) deficiency causes poor pregnancy outcomes. Here, we investigated the role and mechanisms of endothelial cell-mediated vasorelaxation function in these outcomes. EXPERIMENTAL APPROACH: The vascular reactivity of mouse aortas and uterine arteries from non-pregnant and pregnant endothelial cell-specific BH4 deficient mice (Gch1fl/flTie2cre mice) was assessed by wire myography. Systolic blood pressure was assessed by tail cuff plethysmography. KEY RESULTS: In late pregnancy, systolic blood pressure was significantly higher (∼24 mmHg) in Gch1fl/flTie2cre mice compared with wild-type littermates. This was accompanied by enhanced vasoconstriction and reduced endothelial-dependent vasodilation in both aorta and uterine arteries from pregnant Gch1fl/flTie2cre mice. In uterine arteries loss of eNOS-derived vasodilators was partially compensated by upregulation of intermediate and large-conductance Ca2+-activated K+ channels. In rescue experiments, oral BH4 supplementation alone did not rescue vascular dysfunction and pregnancy-induced hypertension in Gch1fl/flTie2cre mice. However, combination with the fully reduced folate, 5-methyltetrahydrofolate (5-MTHF), restored endothelial cell vasodilator function and blood pressure. CONCLUSIONS AND IMPLICATIONS: We identify a critical requirement for maternal endothelial cell Gch1/BH4 biosynthesis in endothelial cell vasodilator function in pregnancy. Targeting vascular Gch1 and BH4 biosynthesis with reduced folates may provide a novel therapeutic target for the prevention and treatment of pregnancy-related hypertension.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology.

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Site-directed mutagenesis of the CC chemokine binding protein 35K-Fc reveals residues essential for activity and mutations that increase the potency of CC chemokine blockade.

Chemokines of the CC class are key mediators of monocyte recruitment and macrophage differentiation and have a well documented role in many inflammatory diseases. Blockade of chemokine activity is therefore an attractive target for anti-inflammatory therapy. 35K (vCCI) is a high-affinity chemokine binding protein expressed by poxviruses, which binds all human and murine CC chemokines, preventing their interaction with chemokine receptors. We developed an Fc-fusion protein of 35K with a modified human IgG1 Fc domain and expressed this construct in human embryonic kidney 293T cells. Purified 35K-Fc is capable of inhibiting CC chemokine-induced calcium flux, chemotaxis, and ß-arrestin recruitment in primary macrophages and transfected cells. To elucidate the residues involved in chemokine neutralization, we performed site-directed mutagenesis of six key amino acids in 35K and expressed the mutant Fc-fusion proteins in vitro. We screened the mutants for their ability to block chemokine-induced ß-arrestin recruitment in transfected cells and to inhibit primary macrophage signaling in an electric cell substrate impedance sensing assay. Using a sterile model of acute inflammation, zymosan-induced peritonitis, we confirmed that wild-type 35K-Fc can reduce monocyte recruitment, whereas one mutant (R89A) showed a more pronounced blockade of monocyte influx and another mutant (E143K) showed total loss of function. We believe that 35K-Fc will be a useful tool for exploring the role of CC chemokines in chronic inflammatory pathologies, and we have identified a higher potency form of the molecule that may have potential therapeutic applications in chronic inflammatory disease.

Endothelial GTPCH (GTP Cyclohydrolase 1) and Tetrahydrobiopterin Regulate Gestational Blood Pressure, Uteroplacental Remodeling, and Fetal Growth.

[Figure: see text].