Metabolic and Proteomic Divergence Is Present in Circulating Monocytes and Tissue-Resident Macrophages from Berkeley Sickle Cell Anemia and ß-Thalassemia Mice.

Sickle cell disease and ß-thalassemia represent hemoglobinopathies arising from dysfunctional or underproduced ß-globin chains, respectively. In both diseases, red blood cell injury and anemia are the impetus for end organ injury. Because persistent erythrophagocytosis is a hallmark of these genetic maladies, it is critical to understand how macrophage phenotype polarizations in tissue compartments can inform on disease progression. Murine models of sickle cell disease and ß-thalassemia allow for a basic understanding of the mechanisms and provide for translation to human disease. A multi-omics approach to understanding the macrophage metabolism and protein changes in two murine models of ß-globinopathy was performed on peripheral blood mononuclear cells as well as spleen and liver macrophages isolated from Berkley sickle cell disease (Berk-ss) and heterozygous B1/B2 globin gene deletion (Hbbth3/+) mice. The results from these experiments revealed that the metabolome and proteome of macrophages are polarized to a distinct phenotype in Berk-ss and Hbbth3/+ compared with each other and their common-background mice (C57BL6/J). Further, spleen and liver macrophages revealed distinct disease-specific phenotypes, suggesting that macrophages become differentially polarized and reprogrammed within tissue compartments. We conclude that tissue recruitment, polarization, and metabolic and proteomic reprogramming of macrophages in Berk-ss and Hbbth3/+ mice may be relevant to disease progression in other tissue.

High-Fat Diet Causes Subfertility and Compromised Ovarian Function Independent of Obesity in Mice.

Excess calorie consumption, particularly of a diet high in fat, is a risk factor for both obesity and reproductive disorders. Animal model studies indicate that elevated dietary fat can influence some reproductive functions independent of obesity. In the current study we sought to determine whether a high-fat diet (HFD) impacts ovarian function, long-term fertility, and local and systemic markers of inflammation independent of obesity. Five-week-old mice were fed either low-fat diet (control group-LF-Ln) or HFD for 10 wk and were divided based on body weight into high-fat obese (HF-Ob: >25 g) and high-fat lean (HF-Ln: <22 g). Ovaries were collected to assess ovarian follicles and to determine the degree of local inflammation. Serum proinflammatory cytokines were also measured. A group of animals was followed for breeding trials for 5 mo while being exposed to LFD or HFD. We found that both 10-wk and 32-wk exposure to HFD resulted in depleted primordial follicles regardless of obesity phenotype. Macrophage counts revealed increased tissue inflammation in the ovary independent of obesity. In addition, serum proinflammatory cytokines were increased in HF-Ln and HF-Ob in comparison to LF-Ln mice. Moreover, HFD had a sustained effect on litter production rate and number of pups per litter regardless of obese phenotype. This study describes for the first time that exposure to HFD causes significant reduction in primordial follicles, compromised fertility, produced higher proinflammatory cytokine levels, and increased ovarian macrophage infiltration, independent of obesity. The negative effects of HFD on primordial follicles may be mediated by increased tissue inflammation.

Moderate hypoxia induces metabolic divergence in circulating monocytes and tissue resident macrophages from Berkeley sickle cell anemia mice.

Introduction: Human and murine sickle cell disease (SCD) associated pulmonary hypertension (PH) is defined by hemolysis, nitric oxide depletion, inflammation, and thrombosis. Further, hemoglobin (Hb), heme, and iron accumulation are consistently observed in pulmonary adventitial macrophages at autopsy and in hypoxia driven rodent models of SCD, which show distribution of ferric and ferrous Hb as well as HO-1 and ferritin heavy chain. The anatomic localization of these macrophages is consistent with areas of significant vascular remodeling. However, their contributions toward progressive disease may include unique, but also common mechanisms, that overlap with idiopathic and other forms of pulmonary hypertension. These processes likely extend to the vasculature of other organs that are consistently impaired in advanced SCD. Methods: To date, limited information is available on the metabolism of macrophages or monocytes isolated from lung, spleen, and peripheral blood in humans or murine models of SCD. Results: Here we hypothesize that metabolism of macrophages and monocytes isolated from this triad of tissue differs between Berkley SCD mice exposed for ten weeks to moderate hypobaric hypoxia (simulated 8,000 ft, 15.4% O2) or normoxia (Denver altitude, 5000 ft) with normoxia exposed wild type mice evaluated as controls. Discussion: This study represents an initial set of data that describes the metabolism in monocytes and macrophages isolated from moderately hypoxic SCD mice peripheral lung, spleen, and blood mononuclear cells.

Extracellular Vesicle Size Reveals Cargo Specific to Coagulation and Inflammation in Pediatric and Adult Sickle Cell Disease.

Aberrant coagulation in sickle cell disease (SCD) is linked to extracellular vesicle (EV) exposure. However, there is no consensus on the contributions of small EVs (SEVs) and large EVs (LEVs) toward underlying coagulopathy or on their molecular cargo. The present observational study compared the thrombin potential of SEVs and LEVs isolated from the plasma of stable pediatric and adult SCD patients. Further, EV lipid and protein contents were analyzed to define markers consistent with activation of thrombin and markers of underlying coagulopathy. Results suggested that LEVs-but not SEVs-from pediatrics and adults similarly enhanced phosphatidylserine (PS)-dependent thrombin generation, and cell membrane procoagulant PS (18:0;20:4 and 18:0;18:1) were the most abundant lipids found in LEVs. Further, LEVs showed activated coagulation in protein pathway analyses, while SEVs demonstrated high levels of cholesterol esters and a protein pathway analysis that identified complement factors and inflammation. We suggest that thrombin potential of EVs from both stable pediatric and adult SCD patients is similarly dependent on size and show lipid and protein contents that identify underlying markers of coagulation and inflammation.

Murine models of sickle cell disease and beta-thalassemia demonstrate pulmonary hypertension with distinctive features.

Sickle cell anemia and ß-thalassemia intermedia are very different genetically determined hemoglobinopathies predisposing to pulmonary hypertension. The etiologies responsible for the associated development of pulmonary hypertension in both diseases are multi-factorial with extensive mechanistic contributors described. Both sickle cell anemia and ß-thalassemia intermedia present with intra and extravascular hemolysis. And because sickle cell anemia and ß-thalassemia intermedia share features of extravascular hemolysis, macrophage iron excess and anemia we sought to characterize the common features of the pulmonary hypertension phenotype, cardiac mechanics, and function as well as lung and right ventricular metabolism. Within the concept of iron, we have defined a unique pulmonary vascular iron accumulation in lungs of sickle cell anemia pulmonary hypertension patients at autopsy. This observation is unlike findings in idiopathic or other forms of pulmonary arterial hypertension. In this study, we hypothesized that a common pathophysiology would characterize the pulmonary hypertension phenotype in sickle cell anemia and ß-thalassemia intermedia murine models. However, unlike sickle cell anemia, ß-thalassemia is also a disease of dyserythropoiesis, with increased iron absorption and cellular iron extrusion. This process is mediated by high erythroferrone and low hepcidin levels as well as dysregulated iron transport due transferrin saturation, so there may be differences as well. Herein we describe common and divergent features of pulmonary hypertension in aged Berk-ss (sickle cell anemia) and Hbbth/3+ (intermediate ß-thalassemia) mice and suggest translational utility as proof-of-concept models to study pulmonary hypertension therapeutics specific to genetic anemias.

Evidence supporting a role for circulating macrophages in the regression of vascular remodeling following sub-chronic exposure to hemoglobin plus hypoxia.

Macrophages are a heterogeneous population with both pro- and anti-inflammatory functions play an essential role in maintaining tissue homeostasis, promoting inflammation under pathological conditions, and tissue repair after injury. In pulmonary hypertension, the M1 phenotype is more pro-inflammatory compared to the M2 phenotype, which is involved in tissue repair. The role of macrophages in the initiation and progression of pulmonary hypertension is well studied. However, their role in the regression of established pulmonary hypertension is not well known. Rats chronically exposed to hemoglobin (Hb) plus hypoxia (HX) share similarities to humans with pulmonary hypertension associated with hemolytic disease, including the presence of a unique macrophage phenotype surrounding distal vessels that are associated with vascular remodeling. These lung macrophages are characterized by high iron content, HO-1, ET-1, and IL-6, and are recruited from the circulation. Depletion of macrophages in this model prevents the development of pulmonary hypertension and vascular remodeling. In this study, we specifically investigate the regression of pulmonary hypertension over a four-week duration after rats were removed from Hb + HX exposure with and without gadolinium chloride administration. Withdrawal of Hb + HX reversed systolic pressures and right ventricular function after Hb + Hx exposure in four weeks. Our data show that depleting circulating monocytes/macrophages during reversal prevents complete recovery of right ventricular systolic pressure and vascular remodeling in this rat model of pulmonary hypertension at four weeks post exposure. The data presented offer a novel insight into the role of macrophages in the processes of pulmonary hypertension regression in a rodent model of Hb + Hx-driven disease.

Agonistic Anti-CD40 Antibody Triggers an Acute Liver Crisis With Systemic Inflammation in Humanized Sickle Cell Disease Mice.

Sickle cell disease (SCD) is an inherited hemolytic disorder, defined by a point mutation in the ß-globin gene. Stress conditions such as infection, inflammation, dehydration, and hypoxia trigger erythrocyte sickling. Sickled red blood cells (RBCs) hemolyze more rapidly, show impaired deformability, and increased adhesive properties to the endothelium. In a proinflammatory, pro-coagulative environment with preexisting endothelial dysfunction, sickled RBCs promote vascular occlusion. Hepatobiliary involvement related to the sickling process, such as an acute sickle hepatic crisis, is observed in about 10% of acute sickle cell crisis incidents. In mice, ligation of CD40 with an agonistic antibody leads to a macrophage activation in the liver, triggering a sequence of systemic inflammation, endothelial cell activation, thrombosis, and focal ischemia. We found that anti-CD40 antibody injection in sickle cell mice induces a systemic inflammatory and hemodynamic response with accelerated hemolysis, extensive vaso-occlusion, and large ischemic infarctions in the liver mimicking an acute hepatic crisis. Administration of the tumor necrosis factor-α (TNF-α) blocker, etanercept, and the heme scavenger protein, hemopexin attenuated end-organ damage. These data collectively suggest that anti-CD40 administration offers a novel acute liver crisis model in humanized sickle mice, allowing for evaluation of therapeutic proof-of-concept.

Hemopexin dosing improves cardiopulmonary dysfunction in murine sickle cell disease.

Hemopexin (Hpx) is a crucial defense protein against heme liberated from degraded hemoglobin during hemolysis. High heme stress creates an imbalance in Hpx bioavailability, favoring heme accumulation and downstream pathophysiological responses leading to cardiopulmonary disease progression in sickle cell disease (SCD) patients. Here, we evaluated a model of murine SCD, which was designed to accelerate red blood cell sickling, pulmonary hypertension, right ventricular dysfunction, and exercise intolerance by exposure of the mice to moderate hypobaric hypoxia. The sequence of pathophysiology in this model tracks with circulatory heme accumulation, lipid oxidation, extensive remodeling of the pulmonary vasculature, and fibrosis. We hypothesized that Hpx replacement for an extended period would improve exercise tolerance measured by critical speed as a clinically meaningful therapeutic endpoint. Further, we sought to define the effects of Hpx on upstream cardiopulmonary function, histopathology, and tissue oxidation. Our data shows that tri-weekly administrations of Hpx for three months dose-dependently reduced heme exposure and pulmonary hypertension while improving cardiac pressure-volume relationships and exercise tolerance. Furthermore, Hpx administration dose-dependently attenuated pulmonary fibrosis and oxidative modifications in the lung and myocardium of the right ventricle. Observations in our SCD murine model are consistent with pulmonary vascular and right ventricular pathology at autopsy in SCD patients having suffered from severe pulmonary hypertension, right ventricular dysfunction, and sudden cardiac death. This study provides a translational evaluation supported by a rigorous outcome analysis demonstrating therapeutic proof-of-concept for Hpx replacement in SCD.

The effect of dietary nitrate supplementation on the speed-duration relationship in mice with sickle cell disease.

Sickle cell disease (SCD) causes exercise intolerance likely due to impaired skeletal muscle function and low nitric oxide (NO) bioavailability. Dietary nitrate improves hemodynamic and metabolic control during exercise in humans and animals. The purpose of this investigation was to assess the impact of nitrate supplementation on exercise capacity as measured by the running speed to exercise duration relationship [critical speed (CS)]in mice with SCD. We tested the hypothesis that nitrate supplementation via beetroot juice (BR) would attenuate the exercise intolerance observed in mice with SCD. Ten wild-type (WT) and 18 Berkley sickle-cell mice (BERK) received water (WT: n = 10, BERK: n = 10) or nitrate-rich BR (BERK+BR: n = 8, nitrate dose 1 mmol/kg/day) for 5 days. Following the supplementation period, all mice performed 3-5 constant-speed treadmill tests that resulted in exhaustion within 1.5 to 20 min. Time to exhaustion vs. treadmill speed was fit to a hyperbolic model to determine CS. CS was significantly lower in BERK vs. WT and BERK+BR with no significant difference between WT and BERK+BR (WT: 36.6 ± 1.6, BERK: 23.8 ± 1.5, BERK+BR: 31.1 ± 2.1 m/min, P < 0.05). Exercise tolerance, measured via CS, was significantly lower in BERK mice relative to WT. However, BERK mice receiving 5 days of nitrate supplementation exhibited no difference in exercise tolerance when compared with WT. These results support the potential utility of a dietary nitrate intervention to improve functionality in SCD patients.NEW & NOTEWORTHY Sickle cell disease compromises muscle O2 delivery resulting in exercise intolerance. Dietary nitrate supplementation increases skeletal muscle blood flow during exercise and may improve exercise capacity in a mouse model of sickle cell disease. We investigated the effects of dietary nitrate supplementation on exercise tolerance in a mouse model of sickle cell disease using the treadmill speed-duration relationship (critical speed). Mice with sickle cell disease provided with a dietary nitrate supplement had a critical speed not significantly different from healthy wild-type mice.

An Hb-mediated circulating macrophage contributing to pulmonary vascular remodeling in sickle cell disease.

Circulating macrophages recruited to the lung contribute to pulmonary vascular remodeling in various forms of pulmonary hypertension (PH). In this study we investigated a macrophage phenotype characterized by intracellular iron accumulation and expression of antioxidant (HO-1), vasoactive (ET-1), and proinflammatory (IL-6) mediators observed in the lung tissue of deceased sickle cell disease (SCD) patients with diagnosed PH. To this end, we evaluated an established rat model of group 5 PH that is simultaneously exposed to free hemoglobin (Hb) and hypobaric hypoxia (HX). Here, we tested the hypothesis that pulmonary vascular remodeling observed in human SCD with concomitant PH could be replicated and mechanistically driven in our rat model by a similar macrophage phenotype with iron accumulation and expression of a similar mixture of antioxidant (HO-1), vasoactive (ET-1), and inflammatory (IL-6) proteins. Our data suggest phenotypic similarities between pulmonary perivascular macrophages in our rat model and human SCD with PH, indicating a potentially novel maladaptive immune response to concomitant bouts of Hb and HX exposure. Moreover, by knocking out circulating macrophages with gadolinium trichloride (GdCl3), the response to combined Hb and hypobaric HX was significantly attenuated in rats, suggesting a critical role for macrophages in the exacerbation of SCD PH.

High-fat diet exposure, regardless of induction of obesity, is associated with altered expression of genes critical to normal ovulatory function.

We evaluated the impact of high-fat diet (HFD) on ovarian gene expression. Female 5-week-old C57BL/6J mice were fed a 60% HFD or standard chow for 10 weeks. HFD-fed mice were then separated into obese (HF-Ob) and lean (HF-Ln) based on body weight. HFD exposure led to impairment of the estrous cycle, changes in hormones affecting reproduction, and decreased primordial follicles regardless of the development of obesity. RNA-sequencing of whole ovaries identified multiple genes with altered expression after HFD, with 25 genes displaying decreased expression in both HF-Ln and HF-Ob mice compared to the chow-fed controls (q < 0.05). Several of these 25 genes are involved in normal ovarian functions, including ovulation (Edn2, Tnfaip6, Errfi1, Prkg2, and Nfil3), luteinization (Edn2), and luteolysis (Nr4a1). Taken together, elevated dietary fat intake, regardless of obesity, is associated with impaired estrous cycle, depletion of the ovarian reserve, and altered expression of genes critical to normal ovulatory function.

Fat-1 Transgene Is Associated With Improved Reproductive Outcomes.

High intake of ω-3 polyunsaturated fatty acids (PUFAs) has been associated with a variety of health benefits. However, the role of ω-3 PUFAs in female reproductive function is unclear, with studies showing both positive and negative effects. The type of diet that ω-3 fatty acids are consumed with, for example, a balanced diet vs a high-fat diet (HFD), may influence how ω-3 fatty acids affect female reproductive function. To address the role of ω-3 PUFAs in female reproduction, we used the fat-1 mouse both with and without HFD exposure. Fat-1 mice constitutively express the fat-1 transgene, allowing the conversion of ω-6 to ω-3 fatty acids to yield an optimal tissue ratio of ω-6 to ω-3 fatty acids (∼1:1). In our study, at 15 weeks of age, fat-1 mice had elevated primordial follicles compared with wild-type controls with both standard chow and HFD feeding. Higher serum levels of the ω-3 docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA) were positively associated with primordial follicle numbers, whereas the ratio of the ω-6 arachidonic acid to EPA + DPA + DHA had the opposite effect. Furthermore, fat-1 mice had increased pregnancy rates and shorter time to pregnancy when fed an HFD compared with wild-type mice. In conclusion, our novel preclinical model suggests that high tissue levels of long-chain ω-3 PUFAs are associated with an improved ovarian reserve and improved reproductive outcomes. Further studies are needed to evaluate ω-3 PUFAs as a potential intervention strategy in women with diminished ovarian reserve.