Haptoglobin Attenuates Cerebrospinal Fluid Hemoglobin-Induced Neurological Deterioration in Sheep.

Secondary brain injury (SBI) occurs with a lag of several days post-bleeding in patients with aneurysmal subarachnoid hemorrhage (aSAH) and is a strong contributor to mortality and long-term morbidity. aSAH-SBI coincides with cell-free hemoglobin (Hb) release into the cerebrospinal fluid. This temporal association and convincing pathophysiological concepts suggest that CSF-Hb could be a targetable trigger of SBI. However, sparse experimental evidence for Hb's neurotoxicity in vivo defines a significant research gap for clinical translation. We modeled the CSF-Hb exposure observed in aSAH patients in conscious sheep, which allowed us to assess neurological functions in a gyrencephalic species. Twelve animals were randomly assigned for 3-day bi-daily intracerebroventricular (ICV) injections of either Hb or Hb combined with the high-affinity Hb scavenger protein haptoglobin (Hb-Hp, CSL888). Repeated CSF sampling confirmed clinically relevant CSF-Hb concentrations. This prolonged CSF-Hb exposure over 3 days resulted in disturbed movement activity, reduced food intake, and impaired observational neuroscores. The Hb-induced neurotoxic effects were significantly attenuated when Hb was administered with equimolar haptoglobin. Preterminal magnetic resonance imaging (MRI) showed no CSF-Hb-specific structural brain alterations. In both groups, histology demonstrated an inflammatory response and revealed enhanced perivascular histiocytic infiltrates in the Hb-Hp group, indicative of adaptive mechanisms. Heme exposure in CSF and iron deposition in the brain were comparable, suggesting comparable clearance efficiency of Hb and Hb-haptoglobin complexes from the intracranial compartment. We identified a neurological phenotype of CSF-Hb toxicity in conscious sheep, which is rather due to neurovascular dysfunction than structural brain injury. Haptoglobin was effective at attenuating CSF-Hb-induced neurological deterioration, supporting its therapeutic potential.

Hemorrhage-activated NRF2 in tumor-associated macrophages drives cancer growth, invasion, and immunotherapy resistance.

Microscopic hemorrhage is a common aspect of cancers, yet its potential role as an independent factor influencing both cancer progression and therapeutic response is largely ignored. Recognizing the essential function of macrophages in red blood cell disposal, we explored a pathway that connects intratumoral hemorrhage with the formation of cancer-promoting tumor-associated macrophages (TAMs). Using spatial transcriptomics, we found that NRF2-activated myeloid cells possessing characteristics of procancerous TAMs tend to cluster in perinecrotic hemorrhagic tumor regions. These cells resembled antiinflammatory erythrophagocytic macrophages. We identified heme, a red blood cell metabolite, as a pivotal microenvironmental factor steering macrophages toward protumorigenic activities. Single-cell RNA-Seq and functional assays of TAMs in 3D cell culture spheroids revealed how elevated intracellular heme signals via the transcription factor NRF2 to induce cancer-promoting TAMs. These TAMs stabilized epithelial-mesenchymal transition, enhancing cancer invasiveness and metastatic potential. Additionally, NRF2-activated macrophages exhibited resistance to reprogramming by IFN-γ and anti-CD40 antibodies, reducing their tumoricidal capacity. Furthermore, MC38 colon adenocarcinoma-bearing mice with NRF2 constitutively activated in leukocytes were resistant to anti-CD40 immunotherapy. Overall, our findings emphasize hemorrhage-activated NRF2 in TAMs as a driver of cancer progression, suggesting that targeting this pathway could offer new strategies to enhance cancer immunity and overcome therapy resistance.

Clinical potential of automated convolutional neural network-based hematoma volumetry after aneurysmal subarachnoid hemorrhage.

OBJECTIVES: Cerebrospinal fluid hemoglobin has been positioned as a potential biomarker and drug target for aneurysmal subarachnoid hemorrhage-related secondary brain injury (SAH-SBI). The maximum amount of hemoglobin, which may be released into the cerebrospinal fluid, is defined by the initial subarachnoid hematoma volume (ISHV). In patients without external ventricular or lumbar drain, there remains an unmet clinical need to predict the risk for SAH-SBI. The aim of this study was to explore automated segmentation of ISHV as a potential surrogate for cerebrospinal fluid hemoglobin to predict SAH-SBI. METHODS: This study is based on a retrospective analysis of imaging and clinical data from 220 consecutive patients with aneurysmal subarachnoid hemorrhage collected over a five-year period. 127 annotated initial non-contrast CT scans were used to train and test a convolutional neural network to automatically segment the ISHV in the remaining cohort. Performance was reported in terms of Dice score and intraclass correlation. We characterized the associations between ISHV and baseline cohort characteristics, SAH-SBI, ventriculoperitoneal shunt dependence, functional outcome, and survival. Established clinical (World Federation of Neurosurgical Societies, Hunt & Hess) and radiological (modified Fisher, Barrow Neurological Institute) scores served as references. RESULTS: A strong volume agreement (0.73 Dice, range 0.43 - 0.93) and intraclass correlation (0.89, 95% CI, 0.81-0.94) were shown. While ISHV was not associated with the use of antithrombotics or cardiovascular risk factors, there was strong evidence for an association with a lower Glasgow Coma Scale at hospital admission. Aneurysm size and location were not associated with ISHV, but the presence of intracerebral or intraventricular hemorrhage were independently associated with higher ISHV. Despite strong evidence for a positive association between ISHV and SAH-SBI, the discriminatory ability of ISHV for SAH-SBI was insufficient. The discriminatory ability of ISHV was, however, higher regarding ventriculoperitoneal shunt dependence and functional outcome at three-months follow-up. Multivariate survival analysis provided strong evidence for an independent negative association between survival probability and both ISHV and intraventricular hemorrhage. CONCLUSIONS: The proposed algorithm demonstrates strong performance in volumetric segmentation of the ISHV on the admission CT. While the discriminatory ability of ISHV for SAH-SBI was similar to established clinical and radiological scores, it showed a high discriminatory ability for ventriculoperitoneal shunt dependence and functional outcome at three-months follow-up.

Haptoglobin Treatment for Aneurysmal Subarachnoid Hemorrhage: Review and Expert Consensus on Clinical Translation.

Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating form of stroke frequently affecting young to middle-aged adults, with an unmet need to improve outcome. This special report focusses on the development of intrathecal haptoglobin supplementation as a treatment by reviewing current knowledge and progress, arriving at a Delphi-based global consensus regarding the pathophysiological role of extracellular hemoglobin and research priorities for clinical translation of hemoglobin-scavenging therapeutics. After aneurysmal subarachnoid hemorrhage, erythrocyte lysis generates cell-free hemoglobin in the cerebrospinal fluid, which is a strong determinant of secondary brain injury and long-term clinical outcome. Haptoglobin is the body's first-line defense against cell-free hemoglobin by binding it irreversibly, preventing translocation of hemoglobin into the brain parenchyma and nitric oxide-sensitive functional compartments of cerebral arteries. In mouse and sheep models, intraventricular administration of haptoglobin reversed hemoglobin-induced clinical, histological, and biochemical features of human aneurysmal subarachnoid hemorrhage. Clinical translation of this strategy imposes unique challenges set by the novel mode of action and the anticipated need for intrathecal drug administration, necessitating early input from stakeholders. Practising clinicians (n=72) and scientific experts (n=28) from 5 continents participated in the Delphi study. Inflammation, microvascular spasm, initial intracranial pressure increase, and disruption of nitric oxide signaling were deemed the most important pathophysiological pathways determining outcome. Cell-free hemoglobin was thought to play an important role mostly in pathways related to iron toxicity, oxidative stress, nitric oxide, and inflammation. While useful, there was consensus that further preclinical work was not a priority, with most believing the field was ready for an early phase trial. The highest research priorities were related to confirming haptoglobin's anticipated safety, individualized versus standard dosing, timing of treatment, pharmacokinetics, pharmacodynamics, and outcome measure selection. These results highlight the need for early phase trials of intracranial haptoglobin for aneurysmal subarachnoid hemorrhage, and the value of early input from clinical disciplines on a global scale during the early stages of clinical translation.

Association between iron deficiency and hospitalization rate in community-dwelling older adults: A 3-year prospective observational study of DO-HEALTH.

BACKGROUND: Iron deficiency (ID) is associated with negative health outcomes in older adults. However, data on the impact of ID on the number of hospitalizations and length of hospital stay (LOS) is lacking. OBJECTIVE: To explore the associations between baseline ID and the number of hospitalizations and between baseline ID and at least one LOS ≥5 days in community-dwelling older adults. METHODS: This is a secondary observational analysis of a randomized controlled trial including 2157 community-dwelling adults aged ≥70 years without major diseases at baseline. The main exposure was defined as ID (soluble transferrin receptor [sTfR] concentrations >28.1 nmol/L) at baseline. The primary outcome was the number of hospitalizations over a 3-year follow-up. The secondary outcome was having at least one LOS ≥5 days over the study period among individuals with one or more hospitalizations. Interaction between ID and anemia (hemoglobin <130 g/L for men and <120 g/L for women) was also investigated. RESULTS: Baseline sTfR concentration was determined in 2141 participants (median age 74.0 years). At 3 year, 1497 hospitalizations were reported with an incidence rate of hospitalization of 0.26 per person-year (95% CI: 0.24, 0.28). Overall, baseline ID was associated with a 24% increased incidence rate of hospitalization (incidence rate ratio: 1.24; 95% CI: 1.05, 1.45) over 3 years. This association was independent of anemia status at baseline since the interaction between ID and anemia at baseline was not significant. Moreover, ID was not significantly associated with having a LOS ≥5 days (OR: 1.40; 95% CI: 1.00, 1.97) among participants with at least one hospitalization over 3 years. CONCLUSIONS: ID is associated with increased hospitalization rate and not associated with LOS ≥5 days among generally healthy older adults. Efforts to minimize ID in older adults may improve overall health and optimize healthcare costs.

Continuous population-level monitoring of SARS-CoV-2 seroprevalence in a large European metropolitan region.

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

Blood oxygenation-level dependent cerebrovascular reactivity imaging as strategy to monitor CSF-hemoglobin toxicity.

OBJECTIVES: Cell-free hemoglobin in the cerebrospinal fluid (CSF-Hb) may be one of the main drivers of secondary brain injury after aneurysmal subarachnoid hemorrhage (aSAH). Haptoglobin scavenging of CSF-Hb has been shown to mitigate cerebrovascular disruption. Using digital subtraction angiography (DSA) and blood oxygenation-level dependent cerebrovascular reactivity imaging (BOLD-CVR) the aim was to assess the acute toxic effect of CSF-Hb on cerebral blood flow and autoregulation, as well as to test the protective effects of haptoglobin. METHODS: DSA imaging was performed in eight anesthetized and ventilated sheep (mean weight: 80.4 kg) at baseline, 15, 30, 45 and 60 minutes after infusion of hemoglobin (Hb) or co-infusion with haptoglobin (Hb:Haptoglobin) into the left lateral ventricle. Additionally, 10 ventilated sheep (mean weight: 79.8 kg) underwent BOLD-CVR imaging to assess the cerebrovascular reserve capacity. RESULTS: DSA imaging did not show a difference in mean transit time or cerebral blood flow. Whole-brain BOLD-CVR compared to baseline decreased more in the Hb group after 15 minutes (Hb vs Hb:Haptoglobin: -0.03 ± 0.01 vs -0.01 ± 0.02) and remained diminished compared to Hb:Haptoglobin group after 30 minutes (Hb vs Hb:Haptoglobin: -0.03 ± 0.01 vs 0.0 ± 0.01), 45 minutes (Hb vs Hb:Haptoglobin: -0.03 ± 0.01 vs 0.01 ± 0.02) and 60 minutes (Hb vs Hb:Haptoglobin: -0.03 ± 0.02 vs 0.01 ± 0.01). CONCLUSION: It is demonstrated that CSF-Hb toxicity leads to rapid cerebrovascular reactivity impairment, which is blunted by haptoglobin co-infusion. BOLD-CVR may therefore be further evaluated as a monitoring strategy for CSF-Hb toxicity after aSAH.

Antibody-induced erythrophagocyte reprogramming of Kupffer cells prevents anti-CD40 cancer immunotherapy-associated liver toxicity.

BACKGROUND: Agonistic anti-CD40 monoclonal antibodies (mAbs) have emerged as promising immunotherapeutic compounds with impressive antitumor effects in mouse models. However, preclinical and clinical studies faced dose-limiting toxicities mediated by necroinflammatory liver disease. An effective prophylactic treatment for liver immune-related adverse events that does not suppress specific antitumor immunity remains to be found. METHODS: We used different mouse models and time-resolved single-cell RNA-sequencing to characterize the pathogenesis of anti-CD40 mAb induced liver toxicity. Subsequently, we developed an antibody-based treatment protocol to selectively target red blood cells (RBCs) for erythrophagocytosis in the liver, inducing an anti-inflammatory liver macrophage reprogramming. RESULTS: We discovered that CD40 signaling in Clec4f+ Kupffer cells is the non-redundant trigger of anti-CD40 mAb-induced liver toxicity. Taking advantage of the highly specific functionality of liver macrophages to clear antibody-tagged RBCs from the blood, we hypothesized that controlled erythrophagocytosis and the linked anti-inflammatory signaling by the endogenous metabolite heme could be exploited to reprogram liver macrophages selectively. Repeated low-dose administration of a recombinant murine Ter119 antibody directed RBCs for selective phagocytosis in the liver and skewed the phenotype of liver macrophages into a Hmoxhigh/Marcohigh/MHCIIlow anti-inflammatory phenotype. This unique mode of action prevented necroinflammatory liver disease following high-dose administration of anti-CD40 mAbs. In contrast, extrahepatic inflammation, antigen-specific immunity, and antitumor activity remained unaffected in Ter119 treated animals. CONCLUSIONS: Our study offers a targeted approach to uncouple CD40-augmented antitumor immunity in peripheral tissues from harmful inflammatoxicity in the liver.

MyD88-TLR4-dependent choroid plexus activation precedes perilesional inflammation and secondary brain edema in a mouse model of intracerebral hemorrhage.

BACKGROUND: The functional neurological outcome of patients with intracerebral hemorrhage (ICH) strongly relates to the degree of secondary brain injury (ICH-SBI) evolving within days after the initial bleeding. Different mechanisms including the incitement of inflammatory pathways, dysfunction of the blood-brain barrier (BBB), activation of resident microglia, and an influx of blood-borne immune cells, have been hypothesized to contribute to ICH-SBI. Yet, the spatiotemporal interplay of specific inflammatory processes within different brain compartments has not been sufficiently characterized, limiting potential therapeutic interventions to prevent and treat ICH-SBI. METHODS: We used a whole-blood injection model in mice, to systematically characterized the spatial and temporal dynamics of inflammatory processes after ICH using 7-Tesla magnetic resonance imaging (MRI), spatial RNA sequencing (spRNAseq), functional BBB assessment, and immunofluorescence average-intensity-mapping. RESULTS: We identified a pronounced early response of the choroid plexus (CP) peaking at 12-24 h that was characterized by inflammatory cytokine expression, epithelial and endothelial expression of leukocyte adhesion molecules, and the accumulation of leukocytes. In contrast, we observed a delayed secondary reaction pattern at the injection site (striatum) peaking at 96 h, defined by gene expression corresponding to perilesional leukocyte infiltration and correlating to the delayed signal alteration seen on MRI. Pathway analysis revealed a dependence of the early inflammatory reaction in the CP on toll-like receptor 4 (TLR4) signaling via myeloid differentiation factor 88 (MyD88). TLR4 and MyD88 knockout mice corroborated this observation, lacking the early upregulation of adhesion molecules and leukocyte infiltration within the CP 24 h after whole-blood injection. CONCLUSIONS: We report a biphasic brain reaction pattern after ICH with a MyD88-TLR4-dependent early inflammatory response of the CP, preceding inflammation, edema and leukocyte infiltration at the lesion site. Pharmacological targeting of the early CP activation might harbor the potential to modulate the development of ICH-SBI.

Erythrophagocytes in hemolytic anemia, wound healing, and cancer.

Hemolysis is a ubiquitous pathology defined as premature red blood cell destruction within the circulation or local tissues. One of the most archetypal functions of macrophages is phagocytosis of damaged or extravasated red blood cells, preventing the extracellular release of toxic hemoglobin and heme. Upon erythrophagocytosis, spiking intracellular heme concentrations drive macrophage transformation into erythrophagocytes, leveraging antioxidative and iron recycling capacities to defend against hemolytic stress. This unique phenotype transformation is coordinated by a regulatory network comprising the transcription factors BACH1, SPI-C, NRF2, and ATF1. Erythrophagocytes negatively regulate inflammation and immunity and may modulate disease-specific outcomes in hemolytic anemia, wound healing, atherosclerosis, and cancer. In this opinion article, we outline the known and presumed functions of erythrophagocytes and their implications for therapeutic innovation and research.

A model to visualize the fate of iron after intracranial hemorrhage using isotopic tracers and elemental bioimaging.

Hemoglobin-iron is a red blood cell toxin contributing to secondary brain injury after intracranial bleeding. We present a model to visualize an intracerebral hematoma and secondary hemoglobin-iron distribution by detecting 58Fe-labeled hemoglobin (Hb) with laser ablation-inductively coupled plasma-mass spectrometry on mouse brain cryosections after stereotactic whole blood injection for different time periods. The generation of 58Fe-enriched blood and decisive steps in the acute hemorrhage formation and evolution were evaluated. The model allows visualization and quantification of 58Fe with high spatial resolution and striking signal-to-noise ratio. Script-based evaluation of the delocalization depth revealed ongoing 58Fe delocalization in the brain even 6 days after hematoma induction. Collectively, the model can quantify the distribution of Hb-derived iron post-bleeding, providing a methodological framework to study the pathophysiological basis of cell-free Hb toxicity in hemorrhagic stroke.

The HeMoVal study protocol: a prospective international multicenter cohort study to validate cerebrospinal fluid hemoglobin as a monitoring biomarker for aneurysmal subarachnoid hemorrhage related secondary brain injury.

INTRODUCTION: Preclinical studies provided a strong rationale for a pathophysiological link between cell-free hemoglobin in the cerebrospinal fluid (CSF-Hb) and secondary brain injury after subarachnoid hemorrhage (SAH-SBI). In a single-center prospective observational clinical study, external ventricular drain (EVD) based CSF-Hb proved to be a promising biomarker to monitor for SAH-SBI. The primary objective of the HeMoVal study is to prospectively validate the association between EVD based CSF-Hb and SAH-SBI during the first 14 days post-SAH. Secondary objectives include the assessment of the discrimination ability of EVD based CSF-Hb for SAH-SBI and the definition of a clinically relevant range of EVD based CSF-Hb toxicity. In addition, lumbar drain (LD) based CSF-Hb will be assessed for its association with and discrimination ability for SAH-SBI. METHODS: HeMoVal is a prospective international multicenter observational cohort study. Adult patients admitted with aneurysmal subarachnoid hemorrhage (aSAH) are eligible. While all patients with aSAH are included, we target a sample size of 250 patients with EVD within the first 14 day after aSAH. Epidemiologic and disease-specific baseline measures are assessed at the time of study inclusion. In patients with EVD or LD, each day during the first 14 days post-SAH, 2 ml of CSF will be sampled in the morning, followed by assessment of the patients for SAH-SBI, co-interventions, and complications in the afternoon. After 3 months, a clinical follow-up will be performed. For statistical analysis, the cohort will be stratified into an EVD, LD and full cohort. The primary analysis will quantify the strength of association between EVD based CSF-Hb and SAH-SBI in the EVD cohort based on a generalized additive model. Secondary analyses include the strength of association between LD based CSF-Hb and SAH-SBI in the LD cohort based on a generalized additive model, as well as the discrimination ability of CSF-Hb for SAH-SBI based on receiver operating characteristic (ROC) analyses. DISCUSSION: We hypothesize that this study will validate the value of CSF-Hb as a biomarker to monitor for SAH-SBI. In addition, the results of this study will provide the potential base to define an intervention threshold for future studies targeting CSF-Hb toxicity after aSAH. STUDY REGISTRATION: ClinicalTrials.gov Identifier NCT04998370 . Date of registration: August 10, 2021.

Hemolysis, free hemoglobin toxicity, and scavenger protein therapeutics.

During hemolysis, erythrophagocytes dispose damaged red blood cells. This prevents the extracellular release of hemoglobin, detoxifies heme, and recycles iron in a linked metabolic pathway. Complementary to this process, haptoglobin and hemopexin scavenge and shuttle the red blood cell toxins hemoglobin and heme to cellular clearance. Pathological hemolysis outpaces macrophage capacity and scavenger synthesis across a diversity of diseases. This imbalance leads to hemoglobin-driven disease progression. To meet a void in treatment options, scavenger protein-based therapeutics are in clinical development.

The oral ferroportin inhibitor vamifeport improves hemodynamics in a mouse model of sickle cell disease.

Sickle cell disease (SCD) is an inherited hemolytic anemia caused by a single point mutation in the ß-globin gene of hemoglobin that leads to synthesis of sickle hemoglobin (HbS) in red blood cells (RBCs). HbS polymerizes in hypoxic conditions, leading to intravascular hemolysis, release of free hemoglobin and heme, and increased adhesion of blood cells to the endothelial vasculature, which causes painful vaso-occlusion and organ damage. HbS polymerization kinetics are strongly dependent on the intracellular HbS concentration; a relatively small reduction in cellular HbS concentration may prevent HbS polymerization and its sequelae. We hypothesized that iron restriction via blocking ferroportin, the unique iron transporter in mammals, might reduce HbS concentration in RBCs, thereby decreasing hemolysis, improving blood flow, and preventing vaso-occlusive events. Indeed, vamifeport (also known as VIT-2763), a clinical-stage oral ferroportin inhibitor, reduced hemolysis markers in the Townes model of SCD. The RBC indices of vamifeport-treated male and female Townes mice exhibited changes attributable to iron-restricted erythropoiesis: decreased corpuscular hemoglobin concentration mean and mean corpuscular volume, as well as increased hypochromic and microcytic RBC fractions. Furthermore, vamifeport reduced plasma soluble VCAM-1 concentrations, which suggests lowered vascular inflammation. Accordingly, intravital video microscopy of fluorescently labeled blood cells in the microvasculature of Townes mice treated with vamifeport revealed diminished adhesion to the endothelium and improved hemodynamics. These preclinical data provide a strong proof-of-concept for vamifeport in the Townes model of SCD and support further development of this compound as a potential novel therapy in SCD.

Prevalence and incidence of iron deficiency in European community-dwelling older adults: an observational analysis of the DO-HEALTH trial.

BACKGROUND AND AIM: Iron deficiency is associated with increased morbidity and mortality in older adults. However, data on its prevalence and incidence among older adults is limited. The aim of this study was to investigate the prevalence and incidence of iron deficiency in European community-dwelling older adults aged ≥ 70 years. METHODS: Secondary analysis of the DO-HEALTH trial, a 3-year clinical trial including 2157 community-dwelling adults aged ≥ 70 years from Austria, France, Germany, Portugal and Switzerland. Iron deficiency was defined as soluble transferrin receptor (sTfR) > 28.1 nmol/L. Prevalence and incidence rate (IR) of iron deficiency per 100 person-years were examined overall and stratified by sex, age group, and country. Sensitivity analysis for three commonly used definitions of iron deficiency (ferritin < 45 µg/L, ferritin < 30 µg/L, and sTfR-ferritin index > 1.5) were also performed. RESULTS: Out of 2157 participants, 2141 had sTfR measured at baseline (mean age 74.9 years; 61.5% women). The prevalence of iron deficiency at baseline was 26.8%, and did not differ by sex, but by age (35.6% in age group ≥ 80, 29.3% in age group 75-79, 23.2% in age group 70-74); P < 0.0001) and country (P = 0.02), with the highest prevalence in Portugal (34.5%) and the lowest in France (24.4%). As for the other definitions of iron deficiency, the prevalence ranged from 4.2% for ferritin < 30 µg/L to 35.3% for sTfR-ferritin index > 1.5. Occurrences of iron deficiency were observed with IR per 100 person-years of 9.2 (95% CI 8.3-10.1) and did not significantly differ by sex or age group. The highest IR per 100 person-years was observed in Austria (20.8, 95% CI 16.1-26.9), the lowest in Germany (6.1, 95% CI 4.7-8.0). Regarding the other definitions of iron deficiency, the IR per 100 person-years was 4.5 (95% CI 4.0-4.9) for ferritin < 45 µg/L, 2.4 (95% CI 2.2-2.7) for ferritin < 30 µg/L, and 12.2 (95% CI 11.0-13.5) for sTfR-ferritin index > 1.5. CONCLUSIONS: Iron deficiency is frequent among relatively healthy European older adults, with people aged ≥ 80 years and residence in Austria and Portugal associated with the highest risk.

Spatial transcriptome data from coronal mouse brain sections after striatal injection of heme and heme-hemopexin.

Hemorrhagic stroke is a major cause of morbidity and mortality worldwide. Secondary mechanisms of brain injury adversely affect functional outcome in patients after intracranial hemorrhage. Potential drivers of intracranial hemorrhage-related secondary brain injury are hemoglobin and its downstream degradation products released from lysed red blood cells, such as free heme. We established a mouse model with stereotactic striatal injection of heme-albumin to gain insights into the toxicity mechanisms of free heme in the brain and assess the therapeutic potential of heme binding and biochemical neutralization by hemopexin. We defined the dose-dependent transcriptional effect of heme or heme-hemopexin exposure 24 h after injection by spatial transcriptome analysis of lesion-centered coronal cryosections. The spatial transcriptome was interpreted in a multimodal approach along with histology, magnetic resonance imaging, and behavioral data and reported in the associated research article "Spatial transcriptome analysis defines heme as a hemopexin-targetable inflammatoxin in the brain" [1]. The spatially resolved transcriptome dataset made available here is intended for continued analysis of free heme toxicity in the brain, which is of potential pathophysiological and therapeutic significance in the context of a wide range of neurovascular and neurodegenerative diseases.

Heme-stress activated NRF2 skews fate trajectories of bone marrow cells from dendritic cells towards red pulp-like macrophages in hemolytic anemia.

Heme is an erythrocyte-derived toxin that drives disease progression in hemolytic anemias, such as sickle cell disease. During hemolysis, specialized bone marrow-derived macrophages with a high heme-metabolism capacity orchestrate disease adaptation by removing damaged erythrocytes and heme-protein complexes from the blood and supporting iron recycling for erythropoiesis. Since chronic heme-stress is noxious for macrophages, erythrophagocytes in the spleen are continuously replenished from bone marrow-derived progenitors. Here, we hypothesized that adaptation to heme stress progressively shifts differentiation trajectories of bone marrow progenitors to expand the capacity of heme-handling monocyte-derived macrophages at the expense of the homeostatic generation of dendritic cells, which emerge from shared myeloid precursors. This heme-induced redirection of differentiation trajectories may contribute to hemolysis-induced secondary immunodeficiency. We performed single-cell RNA-sequencing with directional RNA velocity analysis of GM-CSF-supplemented mouse bone marrow cultures to assess myeloid differentiation under heme stress. We found that heme-activated NRF2 signaling shifted the differentiation of bone marrow cells towards antioxidant, iron-recycling macrophages, suppressing the generation of dendritic cells in heme-exposed bone marrow cultures. Heme eliminated the capacity of GM-CSF-supplemented bone marrow cultures to activate antigen-specific CD4 T cells. The generation of functionally competent dendritic cells was restored by NRF2 loss. The heme-induced phenotype of macrophage expansion with concurrent dendritic cell depletion was reproduced in hemolytic mice with sickle cell disease and spherocytosis and associated with reduced dendritic cell functions in the spleen. Our data provide a novel mechanistic underpinning of hemolytic stress as a driver of hyposplenism-related secondary immunodeficiency.

Iron deficiency and biomarkers of inflammation: a 3-year prospective analysis of the DO-HEALTH trial.

BACKGROUND: The longitudinal association between iron deficiency and inflammatory biomarkers levels has not been fully explored among relatively healthy older adults. AIMS: To assess whether iron deficiency at baseline and at any yearly follow-up time point, with or without anemia, was associated with changes from baseline in high-sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6) levels over 3 years. METHODS: This is a post-hoc observational analysis of DO-HEALTH, a double-blind, randomized controlled trial including 2157 European community-dwelling adults age 70+. The outcomes were changes from baseline in hs-CRP and IL-6 levels, measured at 12, 24, and 36 months of follow-up. Iron deficiency was defined by soluble transferrin receptor levels > 28.1 nmol/L and baseline anemia by hemoglobin levels < 130 g/L for men and < 120 g/L for women. RESULTS: In total, 2141 participants were included in the analyses (mean age: 74.9 years, 61.5% of women, 26.8% with iron deficiency). Baseline iron deficiency was associated with greater increase in IL-6 levels (mean difference in change: 0.52 ng/L, 95%CI 0.03-1.00, P = .04) over 3 years. Iron deficiency at any yearly time point was associated with higher increases in hs-CRP (mean difference in change: 1.62 mg/L, 95%CI 0.98-2.26, P < .001) and IL-6 levels (mean difference in change: 1.33 ng/L, 95%CI 0.87-1.79, P < .001) over 3 years. No significant interaction between iron deficiency and anemia was found, suggesting that the results are independent of the anemic status. CONCLUSIONS: These findings suggest that iron deficiency may play a role in low-grade chronic inflammation among relatively healthy older adults.

Spatial transcriptome analysis defines heme as a hemopexin-targetable inflammatoxin in the brain.

After intracranial hemorrhage, heme is released from cell-free hemoglobin. This red blood cell component may drive secondary brain injury at the hematoma‒brain interface. This study aimed to generate a spatially resolved map of transcriptome-wide gene expression changes in the heme-exposed brain and to define the potential therapeutic activity of the heme-binding protein, hemopexin. We stereotactically injected saline, heme, or heme‒hemopexin into the striatum of C57BL/6J mice. After 24 h, we elucidated the two-dimensional spatial transcriptome by sequencing 21760 tissue-covered features, at a mean transcript coverage of 3849 genes per feature. In parallel, we studied the extravasation of systemically administered fluorescein isothiocyanate labeled (FITC)-dextran, magnetic resonance imaging features indicative of focal edema and perfusion, and neurological functions as translational correlates of heme toxicity. We defined a cerebral heme-response signature by performing bidimensional differential gene expression analysis, based on unsupervised clustering and manual segmentation of sequenced features. Heme exerted a consistent and dose-dependent proinflammatory activity in the brain, which occurred at minimal exposures, below the toxicity threshold for the induction of vascular leakage. We found dose-dependent regional divergence of proinflammatory heme signaling pathways, consistent with reactive astrocytosis and microglial activation. Co-injection of heme with hemopexin attenuated heme-induced gene expression changes and preserved the homeostatic microglia signature. Hemopexin also prevented heme-induced disruption of the blood‒brain barrier and radiological and functional signals of heme injury in the brain. In conclusion, we defined heme as a potent inflammatoxin that may drive secondary brain injury after intracerebral hemorrhage. Co-administration of hemopexin attenuated the heme-derived toxic effects on a molecular, cellular, and functional level, suggesting a translational therapeutic strategy.

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.