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.

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.

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.

Acute Hemolysis and Heme Suppress Anti-CD40 Antibody-Induced Necro-Inflammatory Liver Disease.

Clearance of red blood cells and hemoproteins is a key metabolic function of macrophages during hemolytic disorders and following tissue injury. Through this archetypical phagocytic function, heme is detoxified and iron is recycled to support erythropoiesis. Reciprocal interaction of heme metabolism and inflammatory macrophage functions may modify disease outcomes in a broad range of clinical conditions. We hypothesized that acute hemolysis and heme induce acute anti-inflammatory signals in liver macrophages. Using a macrophage-driven model of sterile liver inflammation, we showed that phenylhydrazine (PHZ)-mediated acute erythrophagocytosis blocked the anti-CD40 antibody-induced pathway of macrophage activation. This process attenuated the inflammatory cytokine release syndrome and necrotizing hepatitis induced by anti-CD40 antibody treatment of mice. We further established that administration of heme-albumin complexes specifically delivered heme to liver macrophages and replicated the anti-inflammatory effect of hemolysis. The anti-inflammatory heme-signal was induced in macrophages by an increased intracellular concentration of the porphyrin independently of iron. Overall, our work suggests that induction of heme-signaling strongly suppresses inflammatory macrophage function, providing protection against sterile liver inflammation.

Hemolysis transforms liver macrophages into antiinflammatory erythrophagocytes.

During hemolysis, macrophages in the liver phagocytose damaged erythrocytes to prevent the toxic effects of cell-free hemoglobin and heme. It remains unclear how this homeostatic process modulates phagocyte functions in inflammatory diseases. Using a genetic mouse model of spherocytosis and single-cell RNA sequencing, we found that erythrophagocytosis skewed liver macrophages into an antiinflammatory phenotype that we defined as MarcohiHmoxhiMHC class IIlo erythrophagocytes. This phenotype transformation profoundly mitigated disease expression in a model of an anti-CD40-induced hyperinflammatory syndrome with necrotic hepatitis and in a nonalcoholic steatohepatitis model, representing 2 macrophage-driven sterile inflammatory diseases. We reproduced the antiinflammatory erythrophagocyte transformation in vitro by heme exposure of mouse and human macrophages, yielding a distinctive transcriptional signature that segregated heme-polarized from M1- and M2-polarized cells. Mapping transposase-accessible chromatin in single cells by sequencing defined the transcription factor NFE2L2/NRF2 as a critical driver of erythrophagocytes, and Nfe2l2/Nrf2 deficiency restored heme-suppressed inflammation. Our findings point to a pathway that regulates macrophage functions to link erythrocyte homeostasis with innate immunity.

Line-selective macrophage activation with an anti-CD40 antibody drives a hemophagocytic syndrome in mice.

Hemophagocytic syndromes comprise a cluster of hyperinflammatory disorders, including hemophagocytic lymphohistiocytosis and macrophage activation syndrome. Overwhelming macrophage activation has long been considered a final common pathway in the pathophysiology of hemophagocytic syndromes leading to the characteristic cytokine storm, laboratory abnormalities, and organ injuries that define the clinical spectrum of the disease. So far, it is unknown whether primary macrophage activation alone can induce the disease phenotype. In this study, we established a novel mouse model of a hemophagocytic syndrome by treating mice with an agonistic anti-CD40 antibody (Ab). The response in wild-type mice is characterized by a cytokine storm, associated with hyperferritinemia, high soluble CD25, erythrophagocytosis, secondary endothelial activation with multiple organ vaso-occlusion, necrotizing hepatitis, and variable cytopenias. The disease is dependent on a tumor necrosis factor-α-interferon-γ-driven amplification loop. After macrophage depletion with clodronate liposomes or in mice with a macrophage-selective deletion of the CD40 gene (CD40flox/flox/LysMCre), the disease was abolished. These data provide a new preclinical model of a hemophagocytic syndrome and reinforce the key pathophysiological role of macrophages.

From an Isolable Acyclic Phosphinosilylene Adduct to Donor-Stabilized Si=E Compounds (E=O, S, Se).

Reaction of the arylchlorosilylene-NHC adduct ArSi(NHC)Cl [Ar=2,6-Trip2C6H3; NHC=(MeC)2(NMe)2C:] 1 with one molar equiv of lithium diphenylphosphanide affords the first stable NHC-stabilized acyclic phosphinosilylene adduct 2 (ArSi(NHC)PPh2), which could be structurally characterized. Compound 2, when reacted with one molar equiv selenium and sulfur, affords the silanechalcogenones 4 a and 4 b (ArSi(NHC)(=E)PPh2, 4 a: E=Se, 4 b: E=S), respectively. Conversion of 2 with an excess of Se and S, through additional insertion of one chalcogen atom into the Si=P bond, leads to 3 a and 3 b (ArSi(NHC)(=E)-E-P(=E)Ph2, 3 a: E=Se, 3 b: E=S), respectively. Additionally, the exposure of 2 to N2O or CO2 yielded the isolable NHC-stabilized silanone 4 c, Ar(NHC)(Ph2P)Si=O.

A Persistent 1,2-Dihydrophosphasilene Adduct.

The reaction of the arylchlorosilylene-NHC adduct ArSi(NHC)Cl [Ar=2,6-Trip2 -C6 H3 ; NHC=(MeC)2 (NMe)2 C] 1 with one molar equiv of LiPH2 (.) dme (dme=1,2-dimethoxyethane) affords the first 1,2-dihydrophosphasilene adduct 2 (ArSi(NHC)(H)PH). The latter is labile in solution and can undergo head-to-tail dimerization to give [ArSi(H)PH]2 3 and "free" NHC. Further stabilization of 2 by complexation with {W(CO)5 } affords the isolable 1,2-dihydrophosphasilene-tungsten complex 4 [ArSi(NHC)(H)P(H)W(CO)5 ]. Additionally, the new 1-silyl-2-hydrophosphasilene ArSi(NHC)(H)PSiMe3 5 could be synthesized and structurally characterized. DFT studies confirmed that the SiP bond in 2 and 4 is mostly zwitterionic with drastically decreased double-bond character.

Adherence to Disease Modifying Drugs among Patients with Multiple Sclerosis in Germany: A Retrospective Cohort Study.

BACKGROUND: Long-term therapies such as disease modifying therapy for Multiple Sclerosis (MS) demand high levels of medication adherence in order to reach acceptable outcomes. The objective of this study was to describe adherence to four disease modifying drugs (DMDs) among statutorily insured patients within two years following treatment initiation. These drugs were interferon beta-1a i.m. (Avonex), interferon beta-1a s.c. (Rebif), interferon beta-1b s.c. (Betaferon) and glatiramer acetate s.c. (Copaxone). METHODS: This retrospective cohort study used pharmacy claims data from the data warehouse of the German Institute for Drug Use Evaluation (DAPI) from 2001 through 2009. New or renewed DMD prescriptions in the years 2002 to 2006 were identified and adherence was estimated during 730 days of follow-up by analyzing the medication possession ratio (MPR) as proxy for compliance and persistence defined as number of days from initiation of DMD therapy until discontinuation or interruption. FINDINGS: A total of 52,516 medication profiles or therapy cycles (11,891 Avonex, 14,060 Betaferon, 12,353 Copaxone and 14,212 Rebif) from 50,057 patients were included into the analysis. Among the 4 cohorts, no clinically relevant differences were found in available covariates. The Medication Possession Ratio (MPR) measured overall compliance, which was 39.9% with a threshold MPR≥0.8. There were small differences in the proportion of therapy cycles during which a patient was compliant for the following medications: Avonex (42.8%), Betaferon (40.6%), Rebif (39.2%), and Copaxone (37%). Overall persistence was 32.3% at the end of the 24 months observation period, i.e. during only one third of all included therapy cycles patients did not discontinue or interrupt DMD therapy. There were also small differences in the proportion of therapy cycles during which a patient was persistent as follows: Avonex (34.2%), Betaferon (33.4%), Rebif (31.7%) and Copaxone (29.8%). CONCLUSIONS: Two years after initiating MS-modifying therapy, only 30-40% of patients were adherent to DMDs.

An amplified Ylidic "half-parent" iminosilane LSi═NH.

The reaction of LSiBr(NH2) (4) (L = CH[(C═CH2)CMe(NAr)2]; Ar = 2,6-iPr2C6H3) with lithium bis(trimethylsilyl)amide in the presence of pyridine or 4-dimethylaminopyridine (DMAP) resulted in the activation of the α C-H bond of pyridine or DMAP, affording the products LSi(dmap)NH2 (6) and LSi(pyridine)NH2 (7a), respectively. Remarkably, this metal-free aromatic C-H activation occurs at room temperature. The emerging aminosilanes were isolated and fully characterized. Isotope labeling experiments and detailed DFT calculations, elucidating the reaction mechanism, were performed and provide compelling evidence of the formation of the "half-parent" iminosilane 1, LSi═NH, which facilitates this transformation due to its amplified ylidic character by the chelate ligand L. Furthermore, the elusive iminosilane 1 could be trapped by benzophenone and trimethylsilylazide affording the corresponding products, 8 and 9, respectively, thereby confirming its formation as a key intermediate.

A donor-stabilized zwitterionic "half-parent" phosphasilene and its unusual reactivity towards small molecules.

The stabilization of the labile, zwitterionic "half-parent" phosphasilene 4 L'Si=PH (L'=CH[(C=CH2)CMe(NAr)2]; Ar=2,6-iPr2C6H3) could now be accomplished by coordination with two different donor ligands (4-dimethylaminopyridine (DMAP) and 1,3,4,5-tetramethylimidazol-2-ylidene), affording the adducts 8 and 9, respectively. The DMAP-stabilized zwitterionic "half-parent" phosphasilene 8 is capable of transferring the elusive parent phosphinidene moiety (:PH) to an unsaturated organic substrate, in analogy to the "free" phosphasilene 4. Furthermore, compounds 4 and 8 show an unusual reactivity of the Si=P moiety towards small molecules. They are capable of adding dimethylzinc and of activating the S-H bonds in H2S and the N-H bonds in ammonia and several organoamines. Interestingly, the DMAP donor ligand of 8 has the propensity to act as a leaving group at the phosphasilene during the reaction. Accordingly, treatment of 8 with H2S affords, under liberation of DMAP, the unprecedented thiosilanoic phosphane LSi=S(PH2) 16 (L=HC(CMe[2,6-iPr2C6H3N])2). Compounds 4 and 8 react with ammonia both affording L'Si(NH2)PH2 17, respectively. In addition, the reaction of 8 with isoproylamine, p-toluidine, and pentafluorophenylhydrazine lead to the corresponding phosphanylsilanes L'Si(PH2)NHR (R=iPr 18 a; R=C6H5-CH3 18 b, R=NH(C6F5) 18 c), respectively.

A fragile zwitterionic phosphasilene as a transfer agent of the elusive parent phosphinidene (:PH).

The simplest parent phosphinidene, :PH (1), has been observed only in the gas phase or low temperature matrices and has escaped rigorous characterization because of its high reactivity. Its liberation and transfer to an unsaturated organic molecule in solution has now been accomplished by taking advantage of the facile homolytic bond cleavage of the fragile Si═P bond of the first zwitterionic phosphasilene LSi=PH (8) (L = CH[(C═CH2)CMe(NAr)2]; Ar = 2,6-(i)Pr2C6H3). The latter bears two highly localized lone pairs on the phosphorus atom due to the LSi═PH ↔ LSi(+)-PH(-) resonance structures. Strikingly, the dissociation of 8 in hydrocarbon solutions occurs even at room temperature, affording the N-heterocyclic silylene LSi: (9) and 1, which leads to oligomeric [PH]n clusters in the absence of a trapping agent. However, in the presence of an N-heterocyclic carbene as an unsaturated organic substrate, the fragile phosphasilene 8 acts as a :PH transfer reagent, resulting in the formation of silylene 9 and phosphaalkene 11 bearing a terminal PH moiety.

The age of the beholder: ERP evidence of an own-age bias in face memory.

Unfamiliar faces from the viewer's own ethnic group are more accurately recognized than other-race faces. The present study examined whether similar effects occur for own-age versus other-age faces, analyzing both behavioural and event-related potential (ERP) measures. Young and elderly participants were to recognize previously studied young and old faces. Whereas young participants demonstrated enhanced recognition memory for own-age faces, no corresponding effect was observed in elderly participants. During recognition tests, enhanced N170 and decreased P2 amplitudes were observed for old faces. Of particular importance, increased N250 amplitudes at right occipito-temporal electrodes as well as enhanced centro-parietal old/new recognition memory effects (more positive ERPs to hits than correct rejections) were observed to own-age compared to other-age faces in the young but not in the elderly participants' ERPs. In young participants, the right occipito-temporal N250 suggests easier access to temporary structural representations for young as compared to old faces, whereas the centro-parietal old/new recognition effect (400-600 ms) suggests an advantage in retrieving episodic information for young faces. The early (<300 ms) neuro-cognitive correlates of the own-age bias in young participants were similar to those of an own-race bias studied previously, suggesting that similar mechanisms underlie these face memory biases. The results are discussed with respect to a perceptual learning account, in which asymmetrical perceptual experience of young and elderly people with faces from different age groups may underlie the differential pattern of own-age effects.