Hyperglycemia Induces Trained Immunity in Macrophages and Their Precursors and Promotes Atherosclerosis.

BACKGROUND: Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. METHODS: Bone marrow-derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr-/- mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. RESULTS: In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow-derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr-/- mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow-derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. CONCLUSIONS: Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

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.

Enabling public, patient and practitioner involvement in co-designing frailty pathways in the acute care setting.

BACKGROUND: Although not an inevitable part of ageing, frailty is an increasingly common condition in older people. Frail older patients are particularly vulnerable to the adverse effects of hospitalisation, including deconditioning, immobility and loss of independence (Chong et al, J Am Med Dir Assoc 18:638.e7-638.e11, 2017). The 'Systematic Approach to improving care for Frail older patients' (SAFE) study co-designed, with public and patient representatives, quality improvement initiatives aimed at enhancing the delivery of care to frail older patients within an acute hospital setting. This paper describes quality improvement initiatives which resulted from a co-design process aiming to improve service delivery in the acute setting for frail older people. These improvement initiatives were aligned to five priority areas identified by patients and public representatives. METHODS: The co-design work was supported by four pillars of effective and meaningful public and patient representative (PPR) involvement in health research (Bombard et al, Implement Sci 13:98, 2018; Black et al, J Health Serv Res Policy 23:158-67, 2018). These pillars were: research environment and receptive contexts; expectations and role clarity; support for participation and inclusive representation and; commitment to the value of co-learning involving institutional leadership. RESULTS: Five priority areas were identified by the co-design team for targeted quality improvement initiatives: Collaboration along the integrated care continuum; continence care; improved mobility; access to food and hydration and improved patient information. These priority areas and the responding quality improvement initiatives are discussed in relation to patient-centred outcomes for enhanced care delivery for frail older people in an acute hospital setting. CONCLUSIONS: The co-design approach to quality improvement places patient-centred outcomes such as dignity, identity, respectful communication as well as independence as key drivers for implementation. Enhanced inter-personal communication was consistently emphasised by the co-design team and much of the quality improvement initiatives target more effective, respectful and clear communication between healthcare personnel and patients. Measurement and evaluation of these patient-centred outcomes, while challenging, should be prioritised in the implementation of quality improvement initiatives. Adequate resourcing and administrative commitment pose the greatest challenges to the sustainability of the interventions developed along the SAFE pathways. The inclusion of organisational leadership in the co-design and implementation teams is a critical factor in the success of interventions targeting service delivery and quality improvement.

Using geographic information systems to map older people's emergency department attendance for future health planning.

OBJECTIVES: This study aimed to assess the pattern of use of EDs, factors contributing to the visits, geographical distribution and outcomes in people aged 65 years or older to a large hospital in Dublin. METHODS: A retrospective analysis of 2 years of data from an urban university teaching hospital ED in the southern part of Dublin was reviewed for the period 2014-2015 (n=103 022) to capture the records of attenders. All ED presentations by individuals 65 years and older were extracted for analysis. Address-matched records were analysed using QGIS, a geographic information systems (GIS) analysis and visualisation tool to determine straight-line distances travelled to the ED by age. RESULTS: Of the 49 538 non-duplicate presentations in the main database, 49.9% of the total are women and 49.1% are men. A subset comprised of 40 801 had address-matched records. When mapped, the data showed a distinct clustering of addresses around the hospital site but this clustering shows different patterns based on age cohort. Average distances travelled to ED are shorter for people 65 and older compared with younger patients. Average distances travelled for those aged 65-74 was 21 km (n=4177 presentations); for the age group 75-84, 18 km (n=2518 presentations) and 13 km for those aged 85 and older (n=2104 presentations). This is validated by statistical tests on the clustered data. Self-referral rates of about 60% were recorded for each age group, although this varied slightly, not significantly, with age. CONCLUSIONS: Health planning at a regional level should account for the significant number of older patients attending EDs. The use of GIS for health planning in particular can assist hospitals to improve their understanding of the origin of the cohort of older ED patients.

A pivotal role for tryptophan 447 in enzymatic coupling of human endothelial nitric oxide synthase (eNOS): effects on tetrahydrobiopterin-dependent catalysis and eNOS dimerization.

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by NOS. Bioavailability of BH4 is a critical factor in regulating the balance between NO and superoxide production by endothelial NOS (eNOS coupling). Crystal structures of the mouse inducible NOS oxygenase domain reveal a homologous BH4-binding site located in the dimer interface and a conserved tryptophan residue that engages in hydrogen bonding or aromatic stacking interactions with the BH4 ring. The role of this residue in eNOS coupling remains unexplored. We overexpressed human eNOS W447A and W447F mutants in novel cell lines with tetracycline-regulated expression of human GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, to determine the importance of BH4 and Trp-447 in eNOS uncoupling. NO production was abolished in eNOS-W447A cells and diminished in cells expressing W447F, despite high BH4 levels. eNOS-derived superoxide production was significantly elevated in W447A and W447F versus wild-type eNOS, and this was sufficient to oxidize BH4 to 7,8-dihydrobiopterin. In uncoupled, BH4-deficient cells, the deleterious effects of W447A mutation were greatly exacerbated, resulting in further attenuation of NO and greatly increased superoxide production. eNOS dimerization was attenuated in W447A eNOS cells and further reduced in BH4-deficient cells, as demonstrated using a novel split Renilla luciferase biosensor. Reduction of cellular BH4 levels resulted in a switch from an eNOS dimer to an eNOS monomer. These data reveal a key role for Trp-447 in determining NO versus superoxide production by eNOS, by effects on BH4-dependent catalysis, and by modulating eNOS dimer formation.

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.

Temporal trends of gestational malaria in the United States.

BACKGROUND: Although regarded as rare in the United States (US), increased global traffic and importation of malaria from endemic countries may lead to a rise in gestational malaria in the US. METHODS: This multi-year retrospective study analyzed trends in diagnosed cases of gestational malaria from 2002 to 2017 using joinpoint regression models. We also assessed the association between gestational malaria and selected maternal-fetal adverse outcomes. RESULTS: Mothers diagnosed with gestational malaria tended to be older, and the majority of diagnosed cases (52.9%) were among Non-Hispanic (NH) Blacks. Diagnosed cases of gestational malaria are on the rise in the US. Mothers diagnosed with gestational malaria were 5 times as likely (OR = 5.05, 95% CI: 4.05-6.29) to be anemic as compared to those without malaria. Compared to NH-Whites, NH-Black mothers were twice as likely to experience stillbirth, had nearly 50% greater adjusted odds of severe preeclampsia, and had about 30% elevated likelihood for preterm labor. CONCLUSIONS: There is a need to dedicate appropriate resources to identify women that are at risk for gestational malaria in order to prevent related pregnancy complications.

Primary care interventions to address physical frailty among community-dwelling adults aged 60 years or older: A meta-analysis.

INTRODUCTION: The best interventions to address frailty among older adults have not yet been fully defined, and the diversity of interventions and outcome measures makes this process challenging. Consequently, there is a lack of guidance for clinicians and researchers regarding which interventions are most likely to help older persons remain robust and independent. This paper uses meta-analysis to assess effectiveness of primary care interventions for physical frailty among community-dwelling adults aged 60+ and provides an up-to-date synthesis of literature in this area. METHODS: PubMed, CINAHL, Cochrane Register of Controlled Trials, and PEDro databases were searched, and RCTs, controlled pilot studies, or trials with similar study designs addressing frailty in the primary care setting among persons aged 60+ were chosen. Study data was abstracted following PRISMA guidelines, then meta-analysis was performed using the random effects model. RESULTS: 31 studies with a total of 4794 participants were analysed. Interventions using predominantly resistance-based exercise and nutrition supplementation seemed to improve frailty status versus control (RR = 0.62 (CI 0.48-0.79), I2 = 0%). Exercise plus nutrition education also reduced frailty (RR = 0.69 (CI 0.58-0.82), I2 = 0%). Exercise alone seemed effective in reducing frailty (RR = 0.63 (CI 0.47-0.84), I2 = 0%) and improving physical performance (RR = 0.43 (CI 0.18-0.67), I2 = 0%). Exercise alone also appeared superior to control in improving gait speed (SMD = 0.36 (CI 0.10-0.61, I2 = 74%), leg strength (SMD = 0.61 (CI 0.09-1.13), I2 = 87%), and grip strength (Mean Difference = 1.08 (CI 0.02-2.15), I2 = 71%) though a high degree of heterogeneity was observed. Comprehensive geriatric assessment (RR = 0.77 (CI 0.64-0.93), I2 = 0%) also seemed superior to control in reducing frailty. CONCLUSION: Exercise alone or with nutrition supplementation or education, and comprehensive geriatric assessment, may reduce physical frailty. Individual-level factors and health systems resource availability will likely determine configuration of future interventions.

Delaying and reversing frailty: a systematic review of primary care interventions.

BACKGROUND: Recommendations for routine frailty screening in general practice are increasing as frailty prevalence grows. In England, frailty identification became a contractual requirement in 2017. However, there is little guidance on the most effective and practical interventions once frailty has been identified. AIM: To assess the comparative effectiveness and ease of implementation of frailty interventions in primary care. DESIGN AND SETTING: A systematic review of frailty interventions in primary care. METHOD: Scientific databases were searched from inception to May 2017 for randomised controlled trials or cohort studies with control groups on primary care frailty interventions. Screening methods, interventions, and outcomes were analysed in included studies. Effectiveness was scored in terms of change of frailty status or frailty indicators and ease of implementation in terms of human resources, marginal costs, and time requirements. RESULTS: A total of 925 studies satisfied search criteria and 46 were included. There were 15 690 participants (median study size was 160 participants). Studies reflected a broad heterogeneity. There were 17 different frailty screening methods. Of the frailty interventions, 23 involved physical activity and other interventions involved health education, nutrition supplementation, home visits, hormone supplementation, and counselling. A significant improvement of frailty status was demonstrated in 71% (n = 10) of studies and of frailty indicators in 69% (n=22) of studies where measured. Interventions with both muscle strength training and protein supplementation were consistently placed highest for effectiveness and ease of implementation. CONCLUSION: A combination of muscle strength training and protein supplementation was the most effective intervention to delay or reverse frailty and the easiest to implement in primary care. A map of interventions was created that can be used to inform choices for managing frailty.

Nitric Oxide Modulates Metabolic Remodeling in Inflammatory Macrophages through TCA Cycle Regulation and Itaconate Accumulation.

Classical activation of macrophages (M(LPS+IFNγ)) elicits the expression of inducible nitric oxide synthase (iNOS), generating large amounts of NO and inhibiting mitochondrial respiration. Upregulation of glycolysis and a disrupted tricarboxylic acid (TCA) cycle underpin this switch to a pro-inflammatory phenotype. We show that the NOS cofactor tetrahydrobiopterin (BH4) modulates IL-1ß production and key aspects of metabolic remodeling in activated murine macrophages via NO production. Using two complementary genetic models, we reveal that NO modulates levels of the essential TCA cycle metabolites citrate and succinate, as well as the inflammatory mediator itaconate. Furthermore, NO regulates macrophage respiratory function via changes in the abundance of critical N-module subunits in Complex I. However, NO-deficient cells can still upregulate glycolysis despite changes in the abundance of glycolytic intermediates and proteins involved in glucose metabolism. Our findings reveal a fundamental role for iNOS-derived NO in regulating metabolic remodeling and cytokine production in the pro-inflammatory macrophage.

Tetrahydrobiopterin modulates ubiquitin conjugation to UBC13/UBE2N and proteasome activity by S-nitrosation.

Nitric Oxide (NO) is an intracellular signalling mediator, which affects many biological processes via the posttranslational modification of proteins through S-nitrosation. The availability of NO and NOS-derived reactive oxygen species (ROS) from enzymatic uncoupling are determined by the NO synthase cofactor Tetrahydrobiopterin (BH4). Here, using a global proteomics "biotin-switch" approach, we identified components of the ubiquitin-proteasome system to be altered via BH4-dependent NO signalling by protein S-nitrosation. We show S-nitrosation of ubiquitin conjugating E2 enzymes, in particular the catalytic residue C87 of UBC13/UBE2N, leading to impaired polyubiquitylation by interfering with the formation of UBC13~Ub thioester intermediates. In addition, proteasome cleavage activity in cells also seems to be altered by S-nitrosation, correlating with the modification of cysteine residues within the 19S regulatory particle and catalytic subunits of the 20S complex. Our results highlight the widespread impact of BH4 on downstream cellular signalling as evidenced by the effect of a perturbed BH4-dependent NO-Redox balance on critical processes within the ubiquitin-proteasome system (UPS). These studies thereby uncover a novel aspect of NO associated modulation of cellular homeostasis.

A novel role for endothelial tetrahydrobiopterin in mitochondrial redox balance.

The redox co-factor tetrahydrobiopterin (BH4) regulates nitric oxide (NO) and reactive oxygen species (ROS) production by endothelial NOS (eNOS) and is an important redox-dependent signalling molecule in the endothelium. Loss of endothelial BH4 is observed in cardiovascular disease (CVD) states and results in decreased NO and increased superoxide (O2-) generation via eNOS uncoupling. Genetic mouse models of augmented endothelial BH4 synthesis have shown proof of concept that endothelial BH4 can alter CVD pathogenesis. However, clinical trials of BH4 therapy in vascular disease have been limited by systemic oxidation, highlighting the need to explore the wider roles of BH4 to find novel therapeutic targets. In this study, we aimed to elucidate the effects of BH4 deficiency on mitochondrial function and bioenergetics using targeted knockdown of the BH4 synthetic enzyme, GTP Cyclohydrolase I (GTPCH). Knockdown of GTPCH by >90% led to marked loss of cellular BH4 and a striking induction of O2- generation in the mitochondria of murine endothelial cells. This effect was likewise observed in BH4-depleted fibroblasts devoid of NOS, indicating a novel NOS-independent role for BH4 in mitochondrial redox signalling. Moreover, this BH4-dependent, mitochondria-derived ROS further oxidised mitochondrial BH4, concomitant with changes in the thioredoxin and glutathione antioxidant pathways. These changes were accompanied by a modest increase in mitochondrial size, mildly attenuated basal respiratory function, and marked changes in the mitochondrial proteome and cellular metabolome, including the accumulation of the TCA intermediate succinate. Taken together, these data reveal a novel NOS-independent role for BH4 in the regulation of mitochondrial redox signalling and bioenergetic metabolism.