Cross-Species Single-Cell Analysis Reveals Divergence of the Primate Microglia Program.

Microglia, the brain-resident immune cells, are critically involved in many physiological and pathological brain processes, including neurodegeneration. Here we characterize microglia morphology and transcriptional programs across ten species spanning more than 450 million years of evolution. We find that microglia express a conserved core gene program of orthologous genes from rodents to humans, including ligands and receptors associated with interactions between glia and neurons. In most species, microglia show a single dominant transcriptional state, whereas human microglia display significant heterogeneity. In addition, we observed notable differences in several gene modules of rodents compared with primate microglia, including complement, phagocytic, and susceptibility genes to neurodegeneration, such as Alzheimer's and Parkinson's disease. Our study provides an essential resource of conserved and divergent microglia pathways across evolution, with important implications for future development of microglia-based therapies in humans.

DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas.

Granulomas are immune cell aggregates formed in response to persistent inflammatory stimuli. Granuloma macrophage subsets are diverse and carry varying copy numbers of their genomic information. The molecular programs that control the differentiation of such macrophage populations in response to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent inflammatory stimuli, pathways previously linked to oncogene-initiated carcinogenesis instruct a long-lived granuloma-resident macrophage differentiation program that regulates granulomatous tissue remodeling.

Ontogeny and homeostasis of CNS myeloid cells.

Myeloid cells in the central nervous system (CNS) represent a heterogeneous class of innate immune cells that contribute to the maintenance of tissue homeostasis differentially during development and adulthood. The subsets of CNS myeloid cells identified so far, including parenchymal microglia and non-parenchymal meningeal, perivascular and choroid-plexus macrophages, as well as disease-associated monocytes, have classically been distinguished on the basis of their surface epitope expression, localization and morphology. However, studies using cell-specific targeting, in vivo imaging, single-cell expression analysis and other sophisticated tools have now increased the depth of knowledge of this immune-cell compartment and call for reevaluation of the traditional views on the origin, fate and function of distinct CNS myeloid subsets. The concepts of CNS macrophage biology that are emerging from these new insights have broad implications for the understanding and treatment of CNS diseases.

Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease.

The immune cells of the central nervous system (CNS) comprise parenchymal microglia and at the CNS border regions meningeal, perivascular, and choroid plexus macrophages (collectively called CNS-associated macrophages, CAMs). While previous work has shown that microglial properties depend on environmental signals from the commensal microbiota, the effects of microbiota on CAMs are unknown. By combining several microbiota manipulation approaches, genetic mouse models, and single-cell RNA-sequencing, we have characterized CNS myeloid cell composition and function. Under steady-state conditions, the transcriptional profiles and numbers of choroid plexus macrophages were found to be tightly regulated by complex microbiota. In contrast, perivascular and meningeal macrophages were affected to a lesser extent. An acute perturbation through viral infection evoked an attenuated immune response of all CAMs in germ-free mice. We further assessed CAMs in a more chronic pathological state in 5xFAD mice, a model for Alzheimer's disease, and found enhanced amyloid beta uptake exclusively by perivascular macrophages in germ-free 5xFAD mice. Our results aid the understanding of distinct microbiota-CNS macrophage interactions during homeostasis and disease, which could potentially be targeted therapeutically.

Seed-induced Aß deposition alters neuronal function and impairs olfaction in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß (Aß) which ultimately forms plaques. These Aß deposits can be induced in APP transgenic mouse models by prion-like seeding. It has been widely accepted that anosmia and hyposmia occur during the early stages of AD, even before cognitive deficits are present. In order to determine the impact of seed-induced Aß deposits on olfaction, we performed intracerebral injections of seed-competent brain homogenate into the olfactory bulb of young pre-depositing APP transgenic mice. Remarkably, we observed a dramatic olfactory impairment in those mice. Furthermore, the number of newborn neurons as well as the activity of cells in the mitral cell layer was decreased. Notably, exposure to an enriched environment reduced Aß seeding, vivified neurogenesis and most importantly reversed olfactory deficits. Based on our findings, we conclude that altered neuronal function as a result of induced Aß pathology might contribute to olfactory dysfunction in AD.

Seed-induced Aß deposition is modulated by microglia under environmental enrichment in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by severe neuronal loss as well as the accumulation of amyloid-ß (Aß), which ultimately leads to plaque formation. Although there is now a general agreement that the aggregation of Aß can be initiated by prion-like seeding, the impact and functional consequences of induced Aß deposits (Aß seeding) on neurons still remain open questions. Here, we find that Aß seeding, representing early stages of plaque formation, leads to a dramatic decrease in proliferation and neurogenesis in two APP transgenic mouse models. We further demonstrate that neuronal cell death occurs primarily in the vicinity of induced Aß deposits culminating in electrophysiological abnormalities. Notably, environmental enrichment and voluntary exercise not only revives adult neurogenesis and reverses memory deficits but, most importantly, prevents Aß seeding by activated, phagocytic microglia cells. Our work expands the current knowledge regarding Aß seeding and the consequences thereof and attributes microglia an important role in diminishing Aß seeding by environmental enrichment.

Stimulated Raman histology in the neurosurgical workflow of a major European neurosurgical center - part A.

Histopathological diagnosis is the current standard for the classification of brain and spine tumors. Raman spectroscopy has been reported to allow fast and easy intraoperative tissue analysis. Here, we report data on the intraoperative implementation of a stimulated Raman histology (SRH) as an innovative strategy offering intraoperative near real-time histopathological analysis. A total of 429 SRH images from 108 patients were generated and analyzed by using a Raman imaging system (Invenio Imaging Inc.). We aimed at establishing a dedicated workflow for SRH serving as an intraoperative diagnostic, research, and quality control tool in the neurosurgical operating room (OR). First experiences with this novel imaging modality were reported and analyzed suggesting process optimization regarding tissue collection, preparation, and imaging. The Raman imaging system was rapidly integrated into the surgical workflow of a large neurosurgical center. Within a few minutes of connecting the device, the first high-quality images could be acquired in a "plug-and-play" manner. We did not encounter relevant obstacles and the learning curve was steep. However, certain prerequisites regarding quality and acquisition of tissue samples, data processing and interpretation, and high throughput adaptions must be considered. Intraoperative SRH can easily be integrated into the workflow of neurosurgical tumor resection. Considering few process optimizations that can be implemented rapidly, high-quality images can be obtained near real time. Hence, we propose SRH as a complementary tool for the diagnosis of tumor entity, analysis of tumor infiltration zones, online quality and safety control and as a research tool in the neurosurgical OR.

Neuropathological interpretation of stimulated Raman histology images of brain and spine tumors: part B.

Intraoperative histopathological examinations are routinely performed to provide neurosurgeons with information about the entity of tumor tissue. Here, we quantified the neuropathological interpretability of stimulated Raman histology (SRH) acquired using a Raman laser imaging system in a routine clinical setting without any specialized training or prior experience. Stimulated Raman scattering microscopy was performed on 117 samples of pathological tissue from 73 cases of brain and spine tumor surgeries. A board-certified neuropathologist - novice in the interpretation of SRH - assessed image quality by scoring subjective tumor infiltration and stated a diagnosis based on the SRH images. The diagnostic accuracy was determined by comparison to frozen hematoxylin-eosin (H&E)-stained sections and the ground truth defined as the definitive neuropathological diagnosis. The overall SRH imaging quality was rated high with the detection of tumor cells classified as inconclusive in only 4.2% of all images. The accuracy of neuropathological diagnosis based on SRH images was 87.7% and was non-inferior to the current standard of fast frozen H&E-stained sections (87.3 vs. 88.9%, p = 0.783). We found a substantial diagnostic correlation between SRH-based neuropathological diagnosis and H&E-stained frozen sections (κ = 0.8). The interpretability of intraoperative SRH imaging was demonstrated to be equivalent to the current standard method of H&E-stained frozen sections. Further research using this label-free innovative alternative vs. conventional staining is required to determine to which extent SRH-based intraoperative decision-making can be streamlined in order to facilitate the advancement of surgical neurooncology.

Transcriptome of microglia reveals a species-specific expression profile in bovines with conserved and new signature genes.

Evidence is growing that microglia adopt different roles than monocyte-derived macrophages (MDM) during CNS injury. However, knowledge about their function in the pathogenesis of neuroinfections is only rudimentary. Cattle are frequently affected by neuroinfections that are either zoonotic or related to diseases in humans, and, hence, studies of bovine neuroinfections as a natural disease model may generate fundamental data on their pathogenesis potentially translatable to humans. We investigated the transcriptomic landscape and lineage markers of bovine microglia and MDM. Although bovine microglia expressed most microglial signature genes known from humans and mice, they exhibited a species-specific transcriptomic profile, including strikingly low expression of TMEM119 and enrichment of the two scavenger receptors MEGF10 and LY75. P2RY12 was amongst the most enriched genes in bovine microglia, and antibodies against P2RY12 labeled specifically resting microglia, but also reactive microglia within neuroinfection foci in-situ. On the other hand, F13A1 was amongst the most enriched genes in bovine monocytes and MDM and, additionally, the encoded protein was expressed in-situ in monocytes and MDM in the inflamed brain but not in microglia, making it a promising marker for infiltrating MDM in the brain. In culture, primary bovine microglia downregulated signature genes, expressed markers of activation, and converged their transcriptome to MDM. However, they retained several microglia signature genes that clearly distinguished them from bovine MDM, making them a promising in-vitro tool to study mechanisms of microglia-pathogen interactions.

How microbiota shape microglial phenotypes and epigenetics.

Among the myeloid cells in the central nervous system (CNS) microglia are the main representatives of the innate immune system. Microglial fulfil tasks beyond phagocytosing debris and host defense against invading microorganism. During brain development microglia guide for example neurons for proper CNS formation, in adulthood they maintain tissue homeostasis and in aging microglia may become pro-inflammatory and finally exhausted. Recently, several endogenous and exogenous factors were identified that essentially shape the microglial phenotype during both steady-state and pathological conditions. On the one hand, microglia receive inputs from CNS-endogenous sources for example, via crosstalk with other glial cells and neurons but on the other hand microglia are also highly modulated by external signals. Among them, host microbiota-the host's resident bacteria-are vital regulators of the CNS innate immune system. This review summarizes key extrinsic and intrinsic factors, with special focus on the host microbiota, that essentially influence microglia functions and states during development, homeostasis, and disease.

Microglia Plasticity During Health and Disease: An Immunological Perspective.

Microglia are macrophages of the central nervous system (CNS) that continuously scrutinize their environment for damage. They colonize the cephalic mesenchyme during embryogenesis and actively shape the developing neuronal network by immune-mediated mechanisms. Upon CNS maturation, microglia drastically change phenotype and function. During health, adult microglia contribute to homeostasis, but also the establishment and resolution of inflammatory conditions. Fulfillment of these distinct tasks requires these long-lived cells to accurately adjust to their changing environment. Deciphering microglia responsiveness to divergent stimuli is central to understanding this cell type and for eventual microglia manipulation to potentially reduce disease burden. Here we discuss new aspects of myeloid cell biology in general with special emphasis on the shifting role of microglia during establishment and protection of CNS integrity.

Communicating systems in the body: how microbiota and microglia cooperate.

Microglia are tissue macrophages of the central nervous system (CNS). Their key tasks are immune surveillance as well as responding to infections or other pathological states such as neurological diseases or injury. In recent years it has been discovered that microglia are additionally crucial for the maintenance of brain homeostasis during development and adulthood by adjusting the neuronal network and phagocytosing neuronal debris. Microglia persist in the CNS throughout the life of the organism and self-renew without engraftment of bone-marrow-derived cells. Until recently it remained unknown what controls their maturation and activation under homeostatic conditions. In this review we discuss new aspects of the interaction between host microbiota and brain function with special focus on the brain-resident innate immune cells, the microglia.

Distinct Pattern of Membrane Formation With Spinal Cerebrospinal Fluid Leaks in Spontaneous Intracranial Hypotension.

BACKGROUND AND OBJECTIVES: To systematically describe pertinent, intraoperative anatomic findings encountered when approaching spinal cerebrospinal fluid (CSF) leaks and CSF-venous fistulas in spontaneous intracranial hypotension (SIH). METHODS: In a retrospective study, we included surgically treated patients suffering from SIH at our institution from April 2018 to March 2022. Anatomic, intraoperative data were extracted from operative notes and supplemented with data from surgical videos and images. Prominent anatomic features were compared among different types of CSF leaks. RESULTS: The study cohort consists of 120 patients with a mean age of 45.2 years. We found four distinct patterns of spinal membranes specifically associated with different types of CSF leaks: (i) thick, dorsal membranes, which were hypervascular and may mimic the dura (pseudodura); (ii) thin, lateral membranes encapsulating a ventral epidural CSF compartment (confining the spinal longitudinal extradural CSF collection); (iii) ventral membranes constituting a transdural funnel-like CSF channel; and (iv) lateral membranes forming spinal cysts/meningeal diverticulae associated with lateral CSF leaks. The latter three types resemble a layer of arachnoid herniated through the dural defect. CONCLUSION: We describe four distinct spinal (neo-)membranes in association with spinal CSF leaks. Formation of these membranes, or emergence by herniation of arachnoid through a dural defect, constitutes a specific pathoanatomic feature of patients with SIH and CSF leaks. Recognition of these membranes is of paramount importance for diagnosis and treatment of patients with spinal CSF leaks.

Advanced diffusion imaging reveals microstructural characteristics of primary CNS lymphoma, allowing differentiation from glioblastoma.

Background: Primary CNS lymphoma (PCNSL) and glioblastoma (GBM) both represent frequent intracranial malignancies with differing clinical management. However, distinguishing PCNSL from GBM with conventional MRI can be challenging when atypical imaging features are present. We employed advanced dMRI for noninvasive characterization of the microstructure of PCNSL and differentiation from GBM as the most frequent primary brain malignancy. Methods: Multiple dMRI metrics including Diffusion Tensor Imaging, Neurite Orientation Dispersion and Density Imaging, and Diffusion Microstructure Imaging were extracted from the contrast-enhancing tumor component in 10 PCNSL and 10 age-matched GBM on 3T MRI. Imaging findings were correlated with cell density and axonal markers obtained from histopathology. Results: We found significantly increased intra-axonal volume fractions (V-intra and intracellular volume fraction) and microFA in PCNSL compared to GBM (all P < .001). In contrast, mean diffusivity (MD), axial diffusivity (aD), and microADC (all P < .001), and also free water fractions (V-CSF and V-ISO) were significantly lower in PCNSL (all P < .01). Receiver-operating characteristic analysis revealed high predictive values regarding the presence of a PCNSL for MD, aD, microADC, V-intra, ICVF, microFA, V-CSF, and V-ISO (area under the curve [AUC] in all >0.840, highest for MD and ICVF with an AUC of 0.960). Comparative histopathology between PCNSL and GBM revealed a significantly increased cell density in PCNSL and the presence of axonal remnants in a higher proportion of samples. Conclusions: Advanced diffusion imaging enables the characterization of the microstructure of PCNSL and reliably distinguishes PCNSL from GBM. Both imaging and histopathology revealed a relatively increased cell density and a preserved axonal microstructure in PCNSL.

Anti-PD-1 cancer immunotherapy induces central nervous system immune-related adverse events by microglia activation.

Cancer treatment with anti-PD-1 immunotherapy can cause central nervous system immune-related adverse events (CNS-irAEs). The role of microglia in anti-PD-1 immunotherapy-induced CNS-irAEs is unclear. We found that anti-PD-1 treatment of mice caused morphological signs of activation and major histocompatibility complex (MHC) class II up-regulation on microglia. Functionally, anti-PD-1 treatment induced neurocognitive deficits in mice, independent of T cells, B cells, and natural killer cells. Instead, we found that microglia mediated these CNS-irAEs. Single-cell RNA sequencing revealed major transcriptional changes in microglia upon anti-PD-1 treatment. The anti-PD-1 effects were mediated by anti-PD-1 antibodies interacting directly with microglia and were not secondary to peripheral T cell activation. Using a proteomics approach, we identified spleen tyrosine kinase (Syk) as a potential target in activated microglia upon anti-PD-1 treatment. Syk inhibition reduced microglia activation and improved neurocognitive function without impairing anti-melanoma effects. Moreover, we analyzed CNS tissue from a patient cohort that had received anti-PD-1 treatment. Imaging mass cytometry revealed that anti-PD-1 treatment of patients was associated with increased surface marker expression indicative of microglia activation. In summary, we identified a disease-promoting role for microglia in CNS-irAEs driven by Syk and provide an inhibitor-based approach to interfere with this complication after anti-PD-1 immunotherapy.