Defining blood-induced microglia functions in neurodegeneration through multiomic profiling.

Blood protein extravasation through a disrupted blood-brain barrier and innate immune activation are hallmarks of neurological diseases and emerging therapeutic targets. However, how blood proteins polarize innate immune cells remains largely unknown. Here, we established an unbiased blood-innate immunity multiomic and genetic loss-of-function pipeline to define the transcriptome and global phosphoproteome of blood-induced innate immune polarization and its role in microglia neurotoxicity. Blood induced widespread microglial transcriptional changes, including changes involving oxidative stress and neurodegenerative genes. Comparative functional multiomics showed that blood proteins induce distinct receptor-mediated transcriptional programs in microglia and macrophages, such as redox, type I interferon and lymphocyte recruitment. Deletion of the blood coagulation factor fibrinogen largely reversed blood-induced microglia neurodegenerative signatures. Genetic elimination of the fibrinogen-binding motif to CD11b in Alzheimer's disease mice reduced microglial lipid metabolism and neurodegenerative signatures that were shared with autoimmune-driven neuroinflammation in multiple sclerosis mice. Our data provide an interactive resource for investigation of the immunology of blood proteins that could support therapeutic targeting of microglia activation by immune and vascular signals.

Author Correction: The immunology of blood: connecting the dots at the neurovascular interface.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation.

Oxidative stress is a central part of innate immune-induced neurodegeneration. However, the transcriptomic landscape of central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available. Here, we provide the oxidative stress innate immune cell atlas in neuroinflammatory disease and report the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells identified a core oxidative stress gene signature coupled to coagulation and glutathione-pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen and oxidative stress gene network analysis identified the glutathione-regulating compound acivicin, with potent therapeutic effects that decrease oxidative stress and axonal damage in chronic and relapsing multiple sclerosis models. Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and may enable discovery of selective neuroprotective strategies.

Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.

Coagulation and immunity: Caught in the fibrin web.

In this issue of Immunity, Vega-Pérez et al. (2021) reveal the formation of a dynamic multicellular aggregate within a fibrin scaffold consisting of large peritoneal macrophages, B1 cells, neutrophils, and monocytes during antibacterial immunity in the peritoneum. Anticoagulants targeting thrombin or peritoneal macrophage depletion by clodronate impaired efficient control of E. coli infection.

Fibrinogen inhibits sonic hedgehog signaling and impairs neonatal cerebellar development after blood-brain barrier disruption.

Cerebellar injury in preterm infants with central nervous system (CNS) hemorrhage results in lasting neurological deficits and an increased risk of autism. The impact of blood-induced pathways on cerebellar development remains largely unknown, so no specific treatments have been developed to counteract the harmful effects of blood after neurovascular damage in preterm infants. Here, we show that fibrinogen, a blood-clotting protein, plays a central role in impairing neonatal cerebellar development. Longitudinal MRI of preterm infants revealed that cerebellar bleeds were the most critical factor associated with poor cerebellar growth. Using inflammatory and hemorrhagic mouse models of neonatal cerebellar injury, we found that fibrinogen increased innate immune activation and impeded neurogenesis in the developing cerebellum. Fibrinogen inhibited sonic hedgehog (SHH) signaling, the main mitogenic pathway in cerebellar granule neuron progenitors (CGNPs), and was sufficient to disrupt cerebellar growth. Genetic fibrinogen depletion attenuated neuroinflammation, promoted CGNP proliferation, and preserved normal cerebellar development after neurovascular damage. Our findings suggest that fibrinogen alters the balance of SHH signaling in the neurovascular niche and may serve as a therapeutic target to mitigate developmental brain injury after CNS hemorrhage.

Fibrin promotes oxidative stress and neuronal loss in traumatic brain injury via innate immune activation.

BACKGROUND: Traumatic brain injury (TBI) causes significant blood-brain barrier (BBB) breakdown, resulting in the extravasation of blood proteins into the brain. The impact of blood proteins, especially fibrinogen, on inflammation and neurodegeneration post-TBI is not fully understood, highlighting a critical gap in our comprehension of TBI pathology and its connection to innate immune activation. METHODS: We combined vascular casting with 3D imaging of solvent-cleared organs (uDISCO) to study the spatial distribution of the blood coagulation protein fibrinogen in large, intact brain volumes and assessed the temporal regulation of the fibrin(ogen) deposition by immunohistochemistry in a murine model of TBI. Fibrin(ogen) deposition and innate immune cell markers were co-localized by immunohistochemistry in mouse and human brains after TBI. We assessed the role of fibrinogen in TBI using unbiased transcriptomics, flow cytometry and immunohistochemistry for innate immune and neuronal markers in Fggγ390-396A knock-in mice, which express a mutant fibrinogen that retains normal clotting function, but lacks the γ390-396 binding motif to CD11b/CD18 integrin receptor. RESULTS: We show that cerebral fibrinogen deposits were associated with activated innate immune cells in both human and murine TBI. Genetic elimination of fibrin-CD11b interaction reduced peripheral monocyte recruitment and the activation of inflammatory and reactive oxygen species (ROS) gene pathways in microglia and macrophages after TBI. Blockade of the fibrin-CD11b interaction was also protective from oxidative stress damage and cortical loss after TBI. CONCLUSIONS: These data suggest that fibrinogen is a regulator of innate immune activation and neurodegeneration in TBI. Abrogating post-injury neuroinflammation by selective blockade of fibrin's inflammatory functions may have implications for long-term neurologic recovery following brain trauma.

Fibrinogen in neurological diseases: mechanisms, imaging and therapeutics.

The blood coagulation protein fibrinogen is deposited in the brain in a wide range of neurological diseases and traumatic injuries with blood-brain barrier (BBB) disruption. Recent research has uncovered pleiotropic roles for fibrinogen in the activation of CNS inflammation, induction of scar formation in the brain, promotion of cognitive decline and inhibition of repair. Such diverse roles are possible in part because of the unique structure of fibrinogen, which contains multiple binding sites for cellular receptors and proteins expressed in the nervous system. The cellular and molecular mechanisms underlying the actions of fibrinogen are beginning to be elucidated, providing insight into its involvement in neurological diseases, such as multiple sclerosis, Alzheimer disease and traumatic CNS injury. Selective drug targeting to suppress the damaging functions of fibrinogen in the nervous system without affecting its beneficial effects in haemostasis opens a new fibrinogen therapeutics pipeline for neurological disease.

Metabolic control of TH17 and induced Treg cell balance by an epigenetic mechanism.

Metabolism has been shown to integrate with epigenetics and transcription to modulate cell fate and function. Beyond meeting the bioenergetic and biosynthetic demands of T-cell differentiation, whether metabolism might control T-cell fate by an epigenetic mechanism is unclear. Here, through the discovery and mechanistic characterization of a small molecule, (aminooxy)acetic acid, that reprograms the differentiation of T helper 17 (TH17) cells towards induced regulatory T (iTreg) cells, we show that increased transamination, mainly catalysed by GOT1, leads to increased levels of 2-hydroxyglutarate in differentiating TH17 cells. The accumulation of 2-hydroxyglutarate resulted in hypermethylation of the Foxp3 gene locus and inhibited Foxp3 transcription, which is essential for fate determination towards TH17 cells. Inhibition of the conversion of glutamate to α-ketoglutaric acid prevented the production of 2-hydroxyglutarate, reduced methylation of the Foxp3 gene locus, and increased Foxp3 expression. This consequently blocked the differentiation of TH17 cells by antagonizing the function of transcription factor RORγt and promoted polarization into iTreg cells. Selective inhibition of GOT1 with (aminooxy)acetic acid ameliorated experimental autoimmune encephalomyelitis in a therapeutic mouse model by regulating the balance between TH17 and iTreg cells. Targeting a glutamate-dependent metabolic pathway thus represents a new strategy for developing therapeutic agents against TH17-mediated autoimmune diseases.

Blood Coagulation Factor Fibrinogen in Tumor Pathogenesis of Central Nervous System B-Cell Lymphoma.

Central nervous system (CNS) lymphoma is an extranodal non-Hodgkin B-cell lymphoma characterized by malignant lymph tissue arising in the brain or spinal cord, associated with inflammation and blood-brain barrier (BBB) disruption. Although BBB disruption is known to occur in patients with CNS lymphoma, a direct link between these two has not been shown. Herein, abundant deposition of the blood coagulation protein fibrinogen around B-cell lymphoma was detected in CNS lymphoma patients and in the CNS parenchyma in an orthotopic mouse model. Functional enrichment analysis of unbiased cerebrospinal fluid proteomics of CNS B-cell lymphoma patients showed that coagulation protein networks were highly connected with tumor-associated biological signaling pathways. In vivo two-photon imaging demonstrated that lymphoma growth was associated with BBB disruption, and in vitro experiments identified a role for fibrinogen in promoting lymphoma cell adhesion. Overall, these results identify perivascular lymphoma clustering at sites of fibrinogen deposition, and suggest that fibrinogen may be a target for pharmacologic intervention in metastatic B-cell lymphoma associated with BBB disruption.

BMP receptor blockade overcomes extrinsic inhibition of remyelination and restores neurovascular homeostasis.

Extrinsic inhibitors at sites of blood-brain barrier disruption and neurovascular damage contribute to remyelination failure in neurological diseases. However, therapies to overcome the extrinsic inhibition of remyelination are not widely available and the dynamics of glial progenitor niche remodelling at sites of neurovascular dysfunction are largely unknown. By integrating in vivo two-photon imaging co-registered with electron microscopy and transcriptomics in chronic neuroinflammatory lesions, we found that oligodendrocyte precursor cells clustered perivascularly at sites of limited remyelination with deposition of fibrinogen, a blood coagulation factor abundantly deposited in multiple sclerosis lesions. By developing a screen (OPC-X-screen) to identify compounds that promote remyelination in the presence of extrinsic inhibitors, we showed that known promyelinating drugs did not rescue the extrinsic inhibition of remyelination by fibrinogen. In contrast, bone morphogenetic protein type I receptor blockade rescued the inhibitory fibrinogen effects and restored a promyelinating progenitor niche by promoting myelinating oligodendrocytes, while suppressing astrocyte cell fate, with potent therapeutic effects in chronic models of multiple sclerosis. Thus, abortive oligodendrocyte precursor cell differentiation by fibrinogen is refractory to known promyelinating compounds, suggesting that blockade of the bone morphogenetic protein signalling pathway may enhance remyelinating efficacy by overcoming extrinsic inhibition in neuroinflammatory lesions with vascular damage.

Breaking barriers in postoperative delirium.

Systemic perturbations such as peripheral surgical trauma induce neurovascular, inflammatory, and cognitive changes. The blood-brain barrier is a key interface between the periphery and the central nervous system, and is critically involved in regulating neuroimmune interactions to maintain overall homeostasis. Mounting evidence suggests that blood-brain barrier dysfunction is a hallmark of ageing and multiple neurological conditions including Alzheimer's disease. We discuss a recent study published in the British Journal of Anaesthesia that describes blood-brain barrier changes and neuroinflammation in patients with postoperative delirium after non-intracranial surgery.

Extracellular vesicle fibrinogen induces encephalitogenic CD8+ T cells in a mouse model of multiple sclerosis.

Extracellular vesicles (EVs) are emerging as potent mediators of intercellular communication with roles in inflammation and disease. In this study, we examined the role of EVs from blood plasma (pEVs) in an experimental autoimmune encephalomyelitis mouse model of central nervous system demyelination. We determined that pEVs induced a spontaneous relapsing-remitting disease phenotype in MOG35-55-immunized C57BL/6 mice. This modified disease phenotype was found to be driven by CD8+ T cells and required fibrinogen in pEVs. Analysis of pEVs from relapsing-remitting multiple sclerosis patients also identified fibrinogen as a significant portion of pEV cargo. Together, these data suggest that fibrinogen in pEVs contributes to the perpetuation of neuroinflammation and relapses in disease.

Differential effects of partial and complete loss of TREM2 on microglial injury response and tauopathy.

Alzheimer's disease (AD), the most common form of dementia, is characterized by the abnormal accumulation of amyloid plaques and hyperphosphorylated tau aggregates, as well as microgliosis. Hemizygous missense variants in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) are associated with elevated risk for developing late-onset AD. These variants are hypothesized to result in loss of function, mimicking TREM2 haploinsufficiency. However, the consequences of TREM2 haploinsufficiency on tau pathology and microglial function remain unknown. We report the effects of partial and complete loss of TREM2 on microglial function and tau-associated deficits. In vivo imaging revealed that microglia from aged TREM2-haploinsufficient mice show a greater impairment in their injury response compared with microglia from aged TREM2-KO mice. In transgenic mice expressing mutant human tau, TREM2 haploinsufficiency, but not complete loss of TREM2, increased tau pathology. In addition, whereas complete TREM2 deficiency protected against tau-mediated microglial activation and atrophy, TREM2 haploinsufficiency elevated expression of proinflammatory markers and exacerbated atrophy at a late stage of disease. The differential effects of partial and complete loss of TREM2 on microglial function and tau pathology provide important insights into the critical role of TREM2 in AD pathogenesis.

Microglial NFκB-TNFα hyperactivation induces obsessive-compulsive behavior in mouse models of progranulin-deficient frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most common dementia before 65 years of age. Haploinsufficiency in the progranulin (GRN) gene accounts for 10% of all cases of familial FTD. GRN mutation carriers have an increased risk of autoimmune disorders, accompanied by elevated levels of tissue necrosis factor (TNF) α. We examined behavioral alterations related to obsessive-compulsive disorder (OCD) and the role of TNFα and related signaling pathways in FTD patients with GRN mutations and in mice lacking progranulin (PGRN). We found that patients and mice with GRN mutations displayed OCD and self-grooming (an OCD-like behavior in mice), respectively. Furthermore, medium spiny neurons in the nucleus accumbens, an area implicated in development of OCD, display hyperexcitability in PGRN knockout mice. Reducing levels of TNFα in PGRN knockout mice abolished excessive self-grooming and the associated hyperexcitability of medium spiny neurons of the nucleus accumbens. In the brain, PGRN is highly expressed in microglia, which are a major source of TNFα. We therefore deleted PGRN specifically in microglia and found that it was sufficient to induce excessive grooming. Importantly, excessive grooming in these mice was prevented by inactivating nuclear factor κB (NF-κB) in microglia/myeloid cells. Our findings suggest that PGRN deficiency leads to excessive NF-κB activation in microglia and elevated TNFα signaling, which in turn lead to hyperexcitability of medium spiny neurons and OCD-like behavior.

Spatiotemporal distribution of fibrinogen in marmoset and human inflammatory demyelination.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system. Although it has been extensively studied, the proximate trigger of the immune response remains uncertain. Experimental autoimmune encephalomyelitis in the common marmoset recapitulates many radiological and pathological features of focal multiple sclerosis lesions in the cerebral white matter, unlike traditional experimental autoimmune encephalomyelitis in rodents. This provides an opportunity to investigate how lesions form as well as the relative timing of factors involved in lesion pathogenesis, especially during early stages of the disease. We used MRI to track experimental autoimmune encephalomyelitis lesions in vivo to determine their age, stage of development, and location, and we assessed the corresponding histopathology post-mortem. We focused on the plasma protein fibrinogen-a marker for blood-brain barrier leakage that has also been linked to a pathogenic role in inflammatory demyelinating lesion development. We show that fibrinogen has a specific spatiotemporal deposition pattern, apparently deriving from the central vein in early experimental autoimmune encephalomyelitis lesions <6 weeks old, and preceding both demyelination and visible gadolinium enhancement on MRI. Thus, fibrinogen leakage is one of the earliest detectable events in lesion pathogenesis. In slightly older lesions, fibrinogen is found inside microglia/macrophages, suggesting rapid phagocytosis. Quantification demonstrates positive correlation of fibrinogen deposition with accumulation of inflammatory cells, including microglia/macrophages and T cells. The peak of fibrinogen deposition coincides with the onset of demyelination and axonal loss. In samples from chronic multiple sclerosis cases, fibrinogen was found at the edge of chronic active lesions, which have ongoing demyelination and inflammation, but not in inactive lesions, suggesting that fibrinogen may play a role in sustained inflammation even in the chronic setting. In summary, our data support the notion that fibrinogen is a key player in the early pathogenesis, as well as sustained inflammation, of inflammatory demyelinating lesions.

Oxygen-dependent cleavage of the p75 neurotrophin receptor triggers stabilization of HIF-1α.

Homeostatic control of oxygen availability allows cells to survive oxygen deprivation. Although the transcription factor hypoxia-inducible factor 1α (HIF-1α) is the main regulator of the hypoxic response, the upstream mechanisms required for its stabilization remain elusive. Here, we show that p75 neurotrophin receptor (p75(NTR)) undergoes hypoxia-induced γ-secretase-dependent cleavage to provide a positive feed-forward mechanism required for oxygen-dependent HIF-1α stabilization. The intracellular domain of p75(NTR) directly interacts with the evolutionarily conserved zinc finger domains of the E3 RING ubiquitin ligase Siah2 (seven in absentia homolog 2), which regulates HIF-1α degradation. p75(NTR) stabilizes Siah2 by decreasing its auto-ubiquitination. Genetic loss of p75(NTR) dramatically decreases Siah2 abundance, HIF-1α stabilization, and induction of HIF-1α target genes in hypoxia. p75(NTR-/-) mice show reduced HIF-1α stabilization, vascular endothelial growth factor (VEGF) expression, and neoangiogenesis after retinal hypoxia. Thus, hypoxia-induced intramembrane proteolysis of p75(NTR) constitutes an apical oxygen-dependent mechanism to control the magnitude of the hypoxic response.