Astrocyte-derived MFG-E8 facilitates microglial synapse elimination in Alzheimer's disease mouse models.

Region-specific synapse loss is an early pathological hallmark in Alzheimer's disease (AD). Emerging data in mice and humans highlight microglia, the brain-resident macrophages, as cellular mediators of synapse loss; however, the upstream modulators of microglia-synapse engulfment remain elusive. Here, we report a distinct subset of astrocytes, which are glial cells essential for maintaining synapse homeostasis, appearing in a region-specific manner with age and amyloidosis at onset of synapse loss. These astrocytes are distinguished by their peri-synaptic processes which are 'bulbous' in morphology, contain accumulated p62-immunoreactive bodies, and have reduced territorial domains, resulting in a decrease of astrocyte-synapse coverage. Using integrated in vitro and in vivo approaches, we show that astrocytes upregulate and secrete phagocytic modulator, milk fat globule-EGF factor 8 (MFG-E8), which is sufficient and necessary for promoting microglia-synapse engulfment in their local milieu. Finally, we show that knocking down Mfge8 specifically from astrocytes using a viral CRISPR-saCas9 system prevents microglia-synapse engulfment and ameliorates synapse loss in two independent amyloidosis mouse models of AD. Altogether, our findings highlight astrocyte-microglia crosstalk in determining synapse fate in amyloid models and nominate astrocytic MFGE8 as a potential target to ameliorate synapse loss during the earliest stages of AD.

Dedicated macrophages organize and maintain the enteric nervous system.

Correct development and maturation of the enteric nervous system (ENS) is critical for survival1. At birth, the ENS is immature and requires considerable refinement to exert its functions in adulthood2. Here we demonstrate that resident macrophages of the muscularis externa (MMϕ) refine the ENS early in life by pruning synapses and phagocytosing enteric neurons. Depletion of MMϕ before weaning disrupts this process and results in abnormal intestinal transit. After weaning, MMϕ continue to interact closely with the ENS and acquire a neurosupportive phenotype. The latter is instructed by transforming growth factor-ß produced by the ENS; depletion of the ENS and disruption of transforming growth factor-ß signalling result in a decrease in neuron-associated MMϕ associated with loss of enteric neurons and altered intestinal transit. These findings introduce a new reciprocal cell-cell communication responsible for maintenance of the ENS and indicate that the ENS, similarly to the brain, is shaped and maintained by a dedicated population of resident macrophages that adapts its phenotype and transcriptome to the timely needs of the ENS niche.

Perivascular cells induce microglial phagocytic states and synaptic engulfment via SPP1 in mouse models of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-ß oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell-cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD.

Microglia modulate neurodegeneration in Alzheimer's and Parkinson's diseases.

Dementia is a rapidly rising global health crisis that silently disables families and ends lives and livelihoods around the world. To date, however, no early biomarkers or effective therapies exist. It is now clear that brain microglia are more than mere bystanders or amyloid phagocytes; they can act as governors of neuronal function and homeostasis in the adult brain. Here, we highlight the fundamental role of microglia as tissue-resident macrophages in neuronal health. Then, we suggest how chronic impairment in microglia-neuron cross-talk may secure the permanence of the failure of synaptic and neuronal function and health in Alzheimer's and Parkinson's diseases. Understanding how to assess and modulate microglia-neuron interactions critical for brain health will be key to developing effective therapies for dementia.

A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment.

While the roles of parenchymal microglia in brain homeostasis and disease are fairly clear, other brain-resident myeloid cells remain less well understood. By dissecting border regions and combining single-cell RNA-sequencing with high-dimensional cytometry, bulk RNA-sequencing, fate-mapping and microscopy, we reveal the diversity of non-parenchymal brain macrophages. Border-associated macrophages (BAMs) residing in the dura mater, subdural meninges and choroid plexus consisted of distinct subsets with tissue-specific transcriptional signatures, and their cellular composition changed during postnatal development. BAMs exhibited a mixed ontogeny, and subsets displayed distinct self-renewal capacity following depletion and repopulation. Single-cell and fate-mapping analysis both suggested that there is a unique microglial subset residing on the apical surface of the choroid plexus epithelium. Finally, gene network analysis and conditional deletion revealed IRF8 as a master regulator that drives the maturation and diversity of brain macrophages. Our results provide a framework for understanding host-macrophage interactions in both the healthy and diseased brain.

Preoperative administration of the 5-HT4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons.

OBJECTIVES: Vagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human. DESIGN: Using Ca2+ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1-5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI. RESULTS: EFS reduced the ATP-induced Ca2+ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery. CONCLUSION: Enteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI. TRIAL REGISTRATION NUMBER: NCT02425774.

Self-Maintaining Gut Macrophages Are Essential for Intestinal Homeostasis.

Macrophages are highly heterogeneous tissue-resident immune cells that perform a variety of tissue-supportive functions. The current paradigm dictates that intestinal macrophages are continuously replaced by incoming monocytes that acquire a pro-inflammatory or tissue-protective signature. Here, we identify a self-maintaining population of macrophages that arise from both embryonic precursors and adult bone marrow-derived monocytes and persists throughout adulthood. Gene expression and imaging studies of self-maintaining macrophages revealed distinct transcriptional profiles that reflect their unique localization (i.e., closely positioned to blood vessels, submucosal and myenteric plexus, Paneth cells, and Peyer's patches). Depletion of self-maintaining macrophages resulted in morphological abnormalities in the submucosal vasculature and loss of enteric neurons, leading to vascular leakage, impaired secretion, and reduced intestinal motility. These results provide critical insights in intestinal macrophage heterogeneity and demonstrate the strategic role of self-maintaining macrophages in gut homeostasis and intestinal physiology.

Muscularis macrophages: Key players in intestinal homeostasis and disease.

Macrophages residing in the muscularis externa of the gastrointestinal tract are highly specialized cells that are essential for tissue homeostasis during steady-state conditions as well as during disease. They are characterized by their unique protective functional phenotype that is undoubtedly a consequence of the reciprocal interaction with their environment, including the enteric nervous system. This muscularis macrophage-neuron interaction dictates intestinal motility and promotes tissue-protection during injury and infection, but can also contribute to tissue damage in gastrointestinal disorders such as post-operative ileus and gastroparesis. Although the importance of muscularis macrophages is clearly recognized, different aspects of these cells remain largely unexplored such their origin, longevity and instructive signals that determine their function and phenotype. In this review, we will discuss the phenotype, functions and origin of muscularis macrophages during steady-state and disease conditions. We will highlight the bidirectional crosstalk with neurons and potential therapeutic strategies that target and manipulate muscularis macrophages to restore their protective signature as a treatment for disease.

Biopsy-derived Intestinal Epithelial Cell Cultures for Pathway-based Stratification of Patients With Inflammatory Bowel Disease.

BACKGROUND: Endoplasmic reticulum [ER] stress was shown to be pivotal in the pathogenesis of inflammatory bowel disease. Despite progress in inflammatory bowel disease [IBD] drug development, not more than one-third of patients achieve steroid-free remission and mucosal healing with current therapies. Furthermore, patient stratification tools for therapy selection are lacking. We aimed to identify and quantify epithelial ER stress in a patient-specific manner in an attempt towards personalised therapy. METHODS: A biopsy-derived intestinal epithelial cell culture system was developed and characterised. ER stress was induced by thapsigargin and quantified with a BiP enzyme-linked immunosorbent assay [ELISA] of cell lysates from 35 patients with known genotypes, who were grouped based on the number of IBD-associated ER stress and autophagy risk alleles. RESULTS: The epithelial character of the cells was confirmed by E-cadherin, ZO-1, and MUC2 staining and CK-18, CK-20, and LGR5 gene expression. Patients with three risk alleles had higher median epithelial BiP-induction [vs untreated] levels compared with patients with one or two risk alleles [p = 0.026 and 0.043, respectively]. When autophagy risk alleles were included and patients were stratified in genetic risk quartiles, patients in Q2, Q3, and Q4 had significantly higher ER stress [BiP] when compared with Q1 [p = 0.034, 0.040, and 0.034, respectively]. CONCLUSIONS: We developed and validated an ex vivo intestinal epithelial cell culture system and showed that patients with more ER stress and autophagy risk alleles have augmented epithelial ER stress responses. We thus presented a personalised approach whereby patient-specific defects can be identified, which in turn could help in selecting tailored therapies.

Strong Upregulation of AIM2 and IFI16 Inflammasomes in the Mucosa of Patients with Active Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by a chronic inflammation of the gut, partly driven by defects in the innate immune system. Considering the central role of inflammasome signaling in innate immunity, we studied inflammasome components in IBD mucosa. METHODS: Expression of genes encoding inflammasome sensor subunits was investigated in colonic mucosal biopsies from 2 cohorts of patients with IBD and controls. RESULTS: A significant upregulation (>2-fold change in expression, false discovery rate <0.05) of the PYHIN inflammasomes AIM2 and IFI16 in active IBD versus controls was found. Also IFI16 was significantly increased in inactive IBD versus controls. Moreover, responders to anti-tumor necrosis factor therapy showed decreased expression of these inflammasomes although IFI16 remained significantly increased in responders showing endoscopic healing versus controls. AIM2 was mainly expressed in epithelial cells, whereas IFI16 was expressed in both lymphocytes and epithelial cells. Functional activation of predominant AIM2/IFI16-mediated inflammasomes in active IBD colon was shown by the presence of the downstream effectors CASP1 and HMGB-1 in inflamed mucosa. CONCLUSIONS: Our results highlight the importance of PYHIN inflammasome signaling in IBD and also link anti-tumor necrosis factor responsiveness to inflammasome signaling. Together, this points to the potential value of the inflammasome pathway as a new therapeutic target for IBD treatment.