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. Keywords: NRF2; erythrophagocytosis; heme; hemolysis; macrophage; tumor-associated macrophage

Hemophagocytic macrophages constitute a major compartment of heme oxygenase expression in sepsis

Schaer DJ, Schaer CA, Schoedon G, Imhof A, Kurrer MO. Hemophagocytic macrophages constitute a major compartment of heme oxygenase expression in sepsis. Objectives: Uncontrolled macrophage activation with hemophagocytosis is a distinctive feature of hemophagocytic syndromes (HPS). We examined whether lympho-histiocytic infiltration of the bone marrow and liver, as well as hemo-/erythrophagocytosis also occurs during sepsis and whether this process could account for the increased production of anti-inflammatory heme-oxygenase (HO-1) products observed during sepsis. Methods: Hemophagocytosis and expression of CD163, HO-1, ferritin as well as CD8 and granzyme-B were examined in post-mortem bone marrow samples from 28 patients with sepsis and from eight control patients. Results: Comparison of samples from non-septic patients with samples from patients with fatal sepsis revealed that the latter group displayed dense lympho-histiocytic bone marrow infiltration with CD163(+)/HO-1(+)/ferritin(+) macrophages as well as with CD8(+) and granzyme-B(+) T-cells. Hemophagocytosis with prominent phagocytosis of erythroid cells was readily apparent in septic patients, implying that this process is a likely stimulus for the up-regulation of macrophage HO-1 expression. Conclusions: Lympho-histiocytic activation with hemophagocytosis is a shared pathophysiologic mechanism in HPS and sepsis. Furthermore, the association of hemophagocytosis with an increase in HO-1 expression may indicate a novel role for this apparently futile process as a negative regulator of inflammation

Targeting Metabolic Vulnerabilities to Overcome Prostate Cancer Resistance: Dual Therapy with Apalutamide and Complex I Inhibition

Prostate cancer (PCa) often becomes drug-treatment-resistant, posing a significant challenge to effective management. Although initial treatment with androgen deprivation therapy can control advanced PCa, subsequent resistance mechanisms allow tumor cells to continue growing, necessitating alternative approaches. This study delves into the specific metabolic dependencies of different PCa subtypes and explores the potential synergistic effects of combining androgen receptor (AR) inhibition (ARN with mitochondrial complex I inhibition (IACS)). We examined the metabolic behaviors of normal prostate epithelial cells (PNT1A), androgen-sensitive cells (LNCaP and C4-2), and androgen-independent cells (PC-3) when treated with ARN, IACS, or a combination. The results uncovered distinct mitochondrial activities across PCa subtypes, with androgen-dependent cells exhibiting heightened oxidative phosphorylation (OXPHOS). The combination of ARN and IACS significantly curbed cell proliferation in multiple PCa cell lines. Cellular bioenergetics analysis revealed that IACS reduced OXPHOS, while ARN hindered glycolysis in certain PCa cells. Additionally, galactose supplementation disrupted compensatory glycolytic mechanisms induced by metabolic reprogramming. Notably, glucose-deprived conditions heightened the sensitivity of PCa cells to mitochondrial inhibition, especially in the resistant PC-3 cells. Overall, this study illuminates the intricate interplay between AR signaling, metabolic adaptations, and treatment resistance in PCa. The findings offer valuable insights into subtype-specific metabolic profiles and propose a promising strategy to target PCa cells by exploiting their metabolic vulnerabilities

Extensive chronic xanthogranulomatous intra-abdominal inflammation due to Mycoplasma hominis mimicking a malignancy: a case report

Introduction: While infectious peritonitis is a common occurrence in patients with liver cirrhosis, Mycoplasma is rarely identified as a causative agent. Case presentation: We report the case of a 43-year-old Caucasian woman presenting with an extensive abdominal conglomerate tumor mimicking malignancy. A histologic specimen showed a xanthogranulomatous inflammation. Subsequently, Mycoplasma hominis was identified as the specific causative infectious agent using a broad-range (eubacterial) polymerase chain reaction. To the best of our knowledge, this is the first reported case of an intra-abdominal Mycoplasma infection presenting as a conglomerate tumor. Conclusion: An unusual presentation of an inflammatory process in the abdomen or an insufficient response to conventional therapy should prompt clinicians to consider atypical infectious agents in the differential diagnosis. This case illustrates the potential of newer diagnostic methods, since certain fastidious microorganisms may not be diagnosed and treated appropriately using conventional means

Down Selection of Polymerized Bovine Hemoglobins for Use as Oxygen Releasing Therapeutics in a Guinea Pig Model

Editor's Highlight: The development of hemoglobin-based oxygen carriers (HBOCs) as a replacement for whole-blood transfusions has been impeded by their systemic toxicity. This paper presents data from a series of HBOCs, demonstrating one candidate that meets predetermined safety criteria. This approach may allow the development of an acceptable blood substitute for human us

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 peri-necrotic hemorrhagic tumor regions. These cells resembled anti-inflammatory 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 IFNγ 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

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

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

BackgroundAgonistic 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.MethodsWe 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.ResultsWe 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 Hmox$^{high}$/Marco$^{high}$/MHCII$^{low}$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.ConclusionsOur study offers a targeted approach to uncouple CD40-augmented antitumor immunity in peripheral tissues from harmful inflammatoxicity in the liver