High-affinity antibodies specific to the core region of the tau protein exhibit diagnostic and therapeutic potential for Alzheimer's disease.

BACKGROUND: Recent advances in blood-based biomarker discovery are paving the way for simpler, more accessible diagnostic tools that can detect early signs of Alzheimer's disease (AD). Recent successes in the development of amyloid-targeting immunotherapy approaches mark an important advancement in providing new options for the treatment of AD. We have developed a set of high-affinity monoclonal antibodies (mAbs) to tau protein that have the potential as tools for diagnosis and treatment of AD. METHODS: Sheep were immunised with either full-length tau (1-441) or truncated paired helical filament (PHF)-core tau (297-391). A stringent bio-panning and epitope selection strategy, with a particular focus directed to epitopes within the disease-relevant PHF-core tau, was used to identify single-chain antibodies (scAbs). These scAbs were ranked by affinity for each epitope class, with leads converted to high-affinity mAbs. These antibodies and their potential utility were assessed by their performance in tau immunoassays, as well as their ability to prevent tau aggregation and propagation. Further characterisation of these antibodies was performed by immunohistochemical staining of brain sections and immuno-gold electronmicroscopy of isolated PHFs. RESULTS: Our work resulted in a set of high-affinity antibodies reacting with multiple epitopes spanning the entire tau protein molecule. The tau antibodies directed against the core tau unit of the PHF inhibited pathological aggregation and seeding using several biochemical and cell assay systems. Through staining of brain sections and PHFs, the panel of antibodies revealed which tau epitopes were available, truncated, or occluded. In addition, highly sensitive immunoassays were developed with the ability to distinguish between and quantify various tau fragments. CONCLUSION: This article introduces an alternative immunodiagnostic approach based on the concept of a "tauosome" - the diverse set of tau fragments present within biological fluids. The development of an antibody panel that can distinguish a range of different tau fragments provides the basis for a novel approach to potential diagnosis and monitoring of disease progression. Our results further support the notion that tau immunotherapy targeting the PHF-core needs to combine appropriate selection of both the target epitope and antibody affinity to optimise therapeutic potential.

Single domain antibody-scFv conjugate targeting amyloid ß and TfR penetrates the blood-brain barrier and interacts with amyloid ß.

Neurodegenerative diseases such as Alzheimer's disease (AD) pose substantial challenges to patients and health-care systems, particularly in countries with aging populations. Immunotherapies, including the marketed antibodies lecanemab (Leqembi®) and donanemab (KisunlaTM), offer promise but face hurdles due to limited delivery across the blood-brain barrier (BBB). This limitation necessitates high doses, resulting in increased costs and a higher risk of side effects. This study explores transferrin receptor (TfR)-binding camelid single-domain antibodies (VHHs) for facilitated brain delivery. We developed and evaluated fusion proteins (FPs) combining VHHs with human IgG Fc domains or single-chain variable fragments (scFvs) of the anti-amyloid-beta (Aß) antibody 3D6. In vitro assessments showed varying affinities of the FPs for TfR. In vivo evaluations indicated that specific VHH-Fc and VHH-scFv fusions reached significant brain concentrations, emphasizing the importance of optimal TfR binding affinities. The VHH-scFv fusions were further investigated in mouse models with Aß pathology, showing higher retention compared to wild-type mice without Aß pathology. Our findings suggest that these novel VHH-based FPs hold potential for therapeutic and diagnostic applications in AD, providing a strategy to overcome BBB limitations and enhance brain targeting of antibody-based treatments. Furthermore, our results suggest that a given bispecific TfR-binding fusion format has a window of "optimal" affinity where parenchymal delivery is adequate, while blood pharmacokinetics aligns with the desired application of the fusion protein.

Role and mechanisms of mast cells in brain disorders.

Mast cells serve as crucial effector cells within the innate immune system and are predominantly localized in the skin, airways, gastrointestinal tract, urinary and reproductive tracts, as well as in the brain. Under physiological conditions, brain-resident mast cells secrete a diverse array of neuro-regulatory mediators to actively participate in neuroprotection. Meanwhile, as the primary source of molecules causing brain inflammation, mast cells also function as the "first responders" in brain injury. They interact with neuroglial cells and neurons to facilitate the release of numerous inflammatory mediators, proteases, and reactive oxygen species. This process initiates and amplifies immune-inflammatory responses in the brain, thereby contributing to the regulation of neuroinflammation and blood-brain barrier permeability. This article provides a comprehensive overview of the potential mechanisms through which mast cells in the brain may modulate neuroprotection and their pathological implications in various neurological disorders. It is our contention that the inhibition of mast cell activation in brain disorders could represent a novel avenue for therapeutic breakthroughs.

Microglia bridge brain activity and blood pressure.

Our brain is not an immune-privileged island isolated from peripheries, but how non-neuronal brain cells interact with the peripheral system is not well understood. Wei et al. report that microglia in the hypothalamic paraventricular nucleus (PVN) with unique vasculature can detect ATP derived from hemodynamic disturbance. These microglia in the PVN regulate the response to hypertension via ATP-P2Y12-C/EBPß signaling.

Maternal immune activation upregulates the AU020206-IRFs-STAT1 axis in modulating cytokine production in the brain.

Maternal immune activation (MIA) is reported to increase the risk of psychiatric disorders in the offspring. However, the underlying mechanism remains unclear. Methods: We constructed a MIA mouse model by intraperitoneal injection of LPS into pregnant mice and evaluated the behaviors and gene expression profiles in the brains of the female and male offspring, respectively. Results: We found that the MIA female offspring exhibited increased anxiety and a large number of differentially expressed genes (DEGs) in the brain, which were enriched with candidate gene sets of psychiatric disorders and immune functions. In contrast, the MIA male offspring exhibited no significant abnormal behaviors and only a small number of DEGs that were not enriched with disease genes and immune functions. Therefore, we further pursued the downstream study on the molecular mechanism underlying the increased anxiety in the female offspring. We identified the lncRNA AU020206-IRFs-STAT1-cytokine axis by integrating lncRNA-protein interaction data and TF-promoter interaction data, and verified the axis in vitro and in vivo. Conclusion: This study illustrates that MIA upregulates the AU020206-IRFs-STAT1 axis in controlling the brain immunity linked to abnormal behaviors, providing a basis for understanding the role of MIA in psychiatric disorders.

Endothelial Cell-Derived Soluble CD200 Determines the Ability of Immune Cells to Cross the Blood-Brain Barrier.

The infiltration of immune cells into the central nervous system mediates the development of autoimmune neuroinflammatory diseases. We previously showed that the loss of either Fabp5 or calnexin causes resistance to the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of multiple sclerosis (MS). Here we show that brain endothelial cells lacking either Fabp5 or calnexin have an increased abundance of cell surface CD200 and soluble CD200 (sCD200) as well as decreased T-cell adhesion. In a tissue culture model of the blood-brain barrier, antagonizing the interaction of CD200 and sCD200 with T-cell CD200 receptor (CD200R1) via anti-CD200 blocking antibodies or the RNAi-mediated inhibition of CD200 production by endothelial cells increased T-cell adhesion and transmigration across monolayers of endothelial cells. Our findings demonstrate that sCD200 produced by brain endothelial cells regulates immune cell trafficking through the blood-brain barrier and is primarily responsible for preventing activated T-cells from entering the brain.

Repeated LPS induces training and tolerance of microglial responses across brain regions.

BACKGROUND: Neuroinflammation is involved in the pathogenesis of almost every central nervous system disorder. As the brain's innate immune cells, microglia fine tune their activity to a dynamic brain environment. Previous studies have shown that repeated bouts of peripheral inflammation can trigger long-term changes in microglial gene expression and function, a form of innate immune memory. METHODS AND RESULTS: In this study, we used multiple low-dose lipopolysaccharide (LPS) injections in adult mice to study the acute cytokine, transcriptomic, and microglia morphological changes that contribute to the formation of immune memory in the frontal cortex, hippocampus, and striatum, as well as the long-term effects of these changes on behavior. Training and tolerance of gene expression was shared across regions, and we identified 3 unique clusters of DEGs (2xLPS-sensitive, 4xLPS-sensitive, LPS-decreased) enriched for different biological functions. 2xLPS-sensitive DEG promoters were enriched for binding sites for IRF and NFkB family transcription factors, two key regulators of innate immune memory. We quantified shifts in microglia morphological populations and found that while the proportion of ramified and rod-like microglia mostly remained consistent within brain regions and sexes with LPS treatment, there was a shift from ameboid towards hypertrophic morphological states across immune memory states and a dynamic emergence and resolution of events of microglia aligning end-to-end with repeated LPS. CONCLUSIONS: Together, findings support the dynamic regulation of microglia during the formation of immune memories in the brain and support future work to exploit this model in brain disease contexts.

Microglia and macrophages alterations in the CNS during acute SIV infection: A single-cell analysis in rhesus macaques.

Human Immunodeficiency Virus (HIV) is widely acknowledged for its profound impact on the immune system. Although HIV primarily affects peripheral CD4 T cells, its influence on the central nervous system (CNS) cannot be overlooked. Within the brain, microglia and CNS-associated macrophages (CAMs) serve as the primary targets for HIV and the simian immunodeficiency virus (SIV) in nonhuman primates. This infection can lead to neurological effects and establish a viral reservoir. Given the gaps in our understanding of how these cells respond in vivo to acute CNS infection, we conducted single-cell RNA sequencing (scRNA-seq) on myeloid cells from the brains of three rhesus macaques 12 days after SIV infection, along with three uninfected controls. Our analysis revealed six distinct microglial clusters including homeostatic microglia, preactivated microglia, and activated microglia expressing high levels of inflammatory and disease-related molecules. In response to acute SIV infection, the homeostatic and preactivated microglia population decreased, while the activated and disease-related microglia increased. All microglial clusters exhibited upregulation of MHC class I molecules and interferon-related genes, indicating their crucial roles in defending against SIV during the acute phase. All microglia clusters also upregulated genes linked to cellular senescence. Additionally, we identified two distinct CAM populations: CD14lowCD16hi and CD14hiCD16low CAMs. Interestingly, during acute SIV infection, the dominant CAM population changed to one with an inflammatory phenotype. Specific upregulated genes within one microglia and one macrophage cluster were associated with neurodegenerative pathways, suggesting potential links to neurocognitive disorders. This research sheds light on the intricate interactions between viral infection, innate immune responses, and the CNS, providing valuable insights for future investigations.

Navigating the Neuroimmunomodulation Frontier: Pioneering Approaches and Promising Horizons-A Comprehensive Review.

The research in neuroimmunomodulation aims to shed light on the complex relationships that exist between the immune and neurological systems and how they affect the human body. This multidisciplinary field focuses on the way immune responses are influenced by brain activity and how neural function is impacted by immunological signaling. This provides important insights into a range of medical disorders. Targeting both brain and immunological pathways, neuroimmunomodulatory approaches are used in clinical pain management to address chronic pain. Pharmacological therapies aim to modulate neuroimmune interactions and reduce inflammation. Furthermore, bioelectronic techniques like vagus nerve stimulation offer non-invasive control of these systems, while neuromodulation techniques like transcranial magnetic stimulation modify immunological and neuronal responses to reduce pain. Within the context of aging, neuroimmunomodulation analyzes the ways in which immunological and neurological alterations brought on by aging contribute to cognitive decline and neurodegenerative illnesses. Restoring neuroimmune homeostasis through strategies shows promise in reducing age-related cognitive decline. Research into mood disorders focuses on how immunological dysregulation relates to illnesses including anxiety and depression. Immune system fluctuations are increasingly recognized for their impact on brain function, leading to novel treatments that target these interactions. This review emphasizes how interdisciplinary cooperation and continuous research are necessary to better understand the complex relationship between the neurological and immune systems.

Inflammation and heterogeneity in synucleinopathies.

Neurodegenerative diseases represent a huge healthcare challenge which is predicted to increase with an aging population. Synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), present complex challenges in understanding their onset and progression. They are characterized by the abnormal aggregation of α-synuclein in the brain leading to neurodegeneration. Accumulating evidence supports the existence of distinct subtypes based on the site of α-synuclein aggregation initiation, genetics, and, more recently, neuroinflammation. Mediated by both central nervous system-resident cells, peripheral immune cells, and gut dysbiosis, neuroinflammation appears as a key process in the onset and progression of neuronal loss. Sex-based differences add another layer of complexity to synucleinopathies, influencing disease prevalence - with a known higher incidence of PD in males compared to females - as well as phenotype and immune responses. Biological sex affects neuroinflammatory pathways and the immune response, suggesting the need for sex-specific therapeutic strategies and biomarker identification. Here, we review the heterogeneity of synucleinopathies, describing the etiology, the mechanisms by which the inflammatory processes contribute to the pathology, and the consideration of sex-based differences to highlight the need for personalized therapeutics.

Visualization, Fate Mapping, Ablation, and Mutagenesis of Microglia in the Mouse Brain.

Since the classical studies of Pío del Río-Hortega, microglia research has come a long way. In particular, recent advances in bulk and single-cell (sc) transcriptomics have yielded many fascinating new insights into these intriguing immune cells at the interface with the central nervous system (CNS), both in small animal models and human samples. In parallel, tools developed by advanced mouse genetics have revealed the unique ontogeny of microglia and their striking dynamic interactions with other cells in the brain parenchyma. In this chapter, we will discuss various applications of the Cre/loxP-based approach that have enabled the study of microglia in their physiological context of the mouse brain. We will highlight selected key findings that have shaped our current understanding of these cells and discuss the technical intricacies of the Cre/loxP approach and some remaining challenges.

Specificities of Living Human Microglial Cells.

Microglia are macrophages residing in the central nervous system, where they perform immune surveillance, synaptic remodeling, neurogenesis, and monitor signals arising from brain injuries or potential pathogens.Commonly, rodent models are used for studying microglia because of the available transgenic mouse lines in which specific genetic manipulations are successfully accomplished. However, human and rodents microglia showed significant differences, which are reflected in different morphological and functional properties. These differences are in genetic and transcriptomic, but also in the expression of signaling molecules and age-associated changes.Several strategies are available to study human microglia, as using surgical brain resections from epileptic and tumoral tissues and from post mortem brain samples. In addition, the generation of human-induced pluripotent stem cells (hPSCs) and the possibility to differentiate them in microglia-like cells provide unique opportunities to compare microglia functions between rodents' and human brain.The use of human ex vivo and in vitro brain models allows the study of human microglia, mimicking in vivo conditions. This will be useful for a better understanding of the real live behavior and functions of microglia in the human brain. This chapter aims to highlight significant similarities and differences between human and rodent microglia in order to re-evaluate mouse models of different human brain disorders, proposing the use of in vitro and ex vivo human brain models.Studies on living human microglia in the brain may help to define divergences from animal models and to improve clinical interventions to treat brain pathologies, using alternatives targets.

Comparative insight into the regenerative mechanisms of the adult brain in zebrafish and mouse: highlighting the importance of the immune system and inflammation in successful regeneration.

Regeneration, the complex process of restoring damaged or absent cells, tissues, and organs, varies considerably between species. The zebrafish is a remarkable model organism for its impressive regenerative abilities, particularly in organs such as the heart, fin, retina, spinal cord, and brain. Unlike mammals, zebrafish can regenerate with limited or absent scarring, a phenomenon closely linked to the activation of stem cells and immune cells. This review examines the unique roles played by the immune response and inflammation in zebrafish and mouse during regeneration, highlighting the cellular and molecular mechanisms behind their divergent regenerative capacities. By focusing on zebrafish telencephalic regeneration and comparing it to that of the rodents, this review highlights the importance of a well-controlled, acute, and non-persistent immune response in zebrafish, which promotes an environment conducive to regeneration. The knowledge gained from understanding the mechanisms of zebrafish regeneration holds great promises for the treatment of human neurodegenerative diseases and brain damage (stroke and traumatic brain injuries), as well as for the advancement of regenerative medicine approaches.

Isolating Immune Cells from Mouse Brain and Skull.

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

Preface to the special issue "Neuroimmunology".

This preface introduces the Special Issue on Neuroimmunology in the Journal of Neurochemistry. The basis of neuroimmunology is to understand functional interactions between cells of the immune system and the central nervous system (CNS). These cells communicate across systems because they share signaling molecules and corresponding receptors. Moreover, this cell signaling allows for dynamic bidirectional communication between the immune system and the brain both within the CNS proper as well as across peripheral organs. Because of this, Neuroimmunology intersects with many biological processes including immunity, behavior, endocrinology, metabolism, and pathology. Understanding neuroimmune interactions that influence CNS homeostasis is especially relevant in health and disease. This special issue comprises of 14 articles, representing 9 review articles and 5 original articles, covering the roles of neuroimmunology relevant to CNS injury, CNS & peripheral infections, cancer, Alzheimer's disease, and COVID-19. Thus, these articles highlight different aspects of neuroimmunology and signaling, and represent progress in understanding the consequences of inflammation on key communication pathways between the immune system and the brains.

Fibrin drives thromboinflammation and neuropathology in COVID-19.

Life-threatening thrombotic events and neurological symptoms are prevalent in COVID-19 and are persistent in patients with long COVID experiencing post-acute sequelae of SARS-CoV-2 infection1-4. Despite the clinical evidence1,5-7, the underlying mechanisms of coagulopathy in COVID-19 and its consequences in inflammation and neuropathology remain poorly understood and treatment options are insufficient. Fibrinogen, the central structural component of blood clots, is abundantly deposited in the lungs and brains of patients with COVID-19, correlates with disease severity and is a predictive biomarker for post-COVID-19 cognitive deficits1,5,8-10. Here we show that fibrin binds to the SARS-CoV-2 spike protein, forming proinflammatory blood clots that drive systemic thromboinflammation and neuropathology in COVID-19. Fibrin, acting through its inflammatory domain, is required for oxidative stress and macrophage activation in the lungs, whereas it suppresses natural killer cells, after SARS-CoV-2 infection. Fibrin promotes neuroinflammation and neuronal loss after infection, as well as innate immune activation in the brain and lungs independently of active infection. A monoclonal antibody targeting the inflammatory fibrin domain provides protection from microglial activation and neuronal injury, as well as from thromboinflammation in the lung after infection. Thus, fibrin drives inflammation and neuropathology in SARS-CoV-2 infection, and fibrin-targeting immunotherapy may represent a therapeutic intervention for patients with acute COVID-19 and long COVID.

Cutting Edge: Retinoic Acid Promotes Brain-homing of CD8+ T Cells during Congenital Cytomegalovirus Infection.

The most common congenital viral infection is CMV, which leads to numerous neurologic disabilities. Using a mouse model of congenital CMV, we previously determined that Ag-specific CD8+ T cells traffic to the brain in a CCR9-dependent manner. The mechanism by which these CD8+ T cells acquire a CCR9-dependent "brain-tropic" phenotype remains unclear. In this study, we identify the key factor that imprints brain homing specificity on CD8+ T cells, the source of production, and the location where CCR9 expression is induced. Specifically, we discovered that CCR9 is induced on CD8+ T cells by retinoic acid-producing CD8α+ dendritic cells in the cervical lymph node postinfection. We found that retinoic acid is important for CD8+ T cells to establish tissue residency in the brain. Collectively, our data expand the role of retinoic acid during infection and mechanistically demonstrate how CD8+ T cells are primed to protect the brain during congenital viral infection.

Molecular Mimicry between Toxoplasma gondii B-Cell Epitopes and Neurodevelopmental Proteins: An Immunoinformatic Approach.

Epidemiological studies and meta-analyses have shown a strong association between high seroprevalence of Toxoplasma gondii (T. gondii) and schizophrenia. Schizophrenic patients showed higher levels of anti-Toxoplasma immunoglobulins M and G (IgM and IgG) when compared to healthy controls. Previously, in a rat model, we demonstrated that the progeny of mothers immunized with T. gondii lysates before gestation had behavioral and social impairments during adulthood. Therefore, we suggested that T. gondii infection can trigger autoreactivity by molecularly mimicking host brain proteins. Here, we aimed to identify the occurrence of antigenic mimicry between T. gondii epitopes and host brain proteins. Using a bioinformatic approach, we predicted T. gondii RH-88 B cell epitopes and compared them to human cell-surface proteins involved in brain development and differentiation (BrainS). Five different algorithms for B-cell-epitope prediction were used and compared, resulting in 8584 T. gondii epitopes. We then compared T. gondii predicted epitopes to BrainS proteins by local sequence alignments using BLASTP. T. gondii immunogenic epitopes significantly overlapped with 42 BrainS proteins. Among these overlapping proteins essential for brain development and differentiation, we identified HSP90 and NOTCH receptors as the proteins most likely to be targeted by the maternally generated pathogenic antibodies due to their topological overlap at the extracellular region of their sequence. This analysis highlights the relevance of pregestational clinical surveillance and screening for potential pathogenic anti-T. gondii antibodies. It also identifies potential targets for the design of vaccines that could prevent behavioral and cognitive impairments associated with pre-gestational T. gondii exposure.

Antibiotics Trigger Host Innate Immune Response via Microbiota-Brain Communication in C. elegans.

Besides their direct bactericidal effect, antibiotics have also been suggested to stimulate the host immune response to defend against pathogens. However, it remains unclear whether any antibiotics may stimulate the host immune response by affecting bacterial activity. In this study, reasoning that genetic mutations inhibit bacterial activities and, thereby, may mimic the effects of antibiotics, we performed genome-wide screening and identified 77 E. coli genes whose inactivation induces C. elegans cyp-14A4, representing an innate immune and detoxification response. Further analyses reveal that this host immune response can clearly be induced through either inactivating the E. coli respiratory chain via the bacterial cyoB mutation or using the antibiotic Q203, which is able to enhance host survival when encountering the pathogen Pseudomonas aeruginosa. Mechanistically, the innate immune response triggered by both the cyoB mutation and Q203 is found to depend on the host brain response, as evidenced by their reliance on the host neural gene unc-13, which is required for neurotransmitter release in head neurons. Therefore, our findings elucidate the critical involvement of the microbiota-brain axis in modulating the host immune response, providing mechanistic insights into the role of antibiotics in triggering the host immune response and, thus, facilitating host defense against pathogens.

Activation of neurotoxic A1-reactive astrocytes by SFTS virus infection accelerates fatal brain damage in IFNAR1-/- mice.

Severe fever with thrombocytopenia syndrome (SFTS) has a high mortality rate compared to other infectious diseases. SFTS is particularly associated with a high risk of mortality in immunocompromised individuals, while most patients who die of SFTS exhibit symptoms of severe encephalitis before death. However, the region of brain damage and mechanisms by which the SFTS virus (SFTSV) causes encephalitis remains unknown. Here, we revealed that SFTSV infects the brainstem and spinal cord, which are regions of the brain associated with respiratory function, and motor nerves in IFNAR1-/- mice. Further, we show that A1-reactive astrocytes are activated, causing nerve cell death, in infected mice. Primary astrocytes of SFTSV-infected IFNAR1-/- mice also induced neuronal cell death through the activation of A1-reactive astrocytes. Herein, we showed that SFTSV induces fatal neuroinflammation in the brain regions important for respiratory function and motor nerve, which may underlie mortality in SFTS patients. This study provides new insights for the treatment of SFTS, for which there is currently no therapeutic approach.