The enteric nervous system relays psychological stress to intestinal inflammation.

Mental health profoundly impacts inflammatory responses in the body. This is particularly apparent in inflammatory bowel disease (IBD), in which psychological stress is associated with exacerbated disease flares. Here, we discover a critical role for the enteric nervous system (ENS) in mediating the aggravating effect of chronic stress on intestinal inflammation. We find that chronically elevated levels of glucocorticoids drive the generation of an inflammatory subset of enteric glia that promotes monocyte- and TNF-mediated inflammation via CSF1. Additionally, glucocorticoids cause transcriptional immaturity in enteric neurons, acetylcholine deficiency, and dysmotility via TGF-ß2. We verify the connection between the psychological state, intestinal inflammation, and dysmotility in three cohorts of IBD patients. Together, these findings offer a mechanistic explanation for the impact of the brain on peripheral inflammation, define the ENS as a relay between psychological stress and gut inflammation, and suggest that stress management could serve as a valuable component of IBD care.

How Support of Early Career Researchers Can Reset Science in the Post-COVID19 World.

The COVID19 crisis has magnified the issues plaguing academic science, but it has also provided the scientific establishment with an unprecedented opportunity to reset. Shoring up the foundation of academic science will require a concerted effort between funding agencies, universities, and the public to rethink how we support scientists, with a special emphasis on early career researchers.

Senescent glia link mitochondrial dysfunction and lipid accumulation.

Senescence is a cellular state linked to ageing and age-onset disease across many mammalian species1,2. Acutely, senescent cells promote wound healing3,4 and prevent tumour formation5; but they are also pro-inflammatory, thus chronically exacerbate tissue decline. Whereas senescent cells are active targets for anti-ageing therapy6-11, why these cells form in vivo, how they affect tissue ageing and the effect of their elimination remain unclear12,13. Here we identify naturally occurring senescent glia in ageing Drosophila brains and decipher their origin and influence. Using Activator protein 1 (AP1) activity to screen for senescence14,15, we determine that senescent glia can appear in response to neuronal mitochondrial dysfunction. In turn, senescent glia promote lipid accumulation in non-senescent glia; similar effects are seen in senescent human fibroblasts in culture. Targeting AP1 activity in senescent glia mitigates senescence biomarkers, extends fly lifespan and health span, and prevents lipid accumulation. However, these benefits come at the cost of increased oxidative damage in the brain, and neuronal mitochondrial function remains poor. Altogether, our results map the trajectory of naturally occurring senescent glia in vivo and indicate that these cells link key ageing phenomena: mitochondrial dysfunction and lipid accumulation.

Microglia in antiviral immunity of the brain and spinal cord.

Viral infections of the central nervous system (CNS) are a significant cause of neurological impairment and mortality worldwide. As tissue resident macrophages, microglia are critical initial responders to CNS viral infection. Microglia seem to coordinate brain-wide antiviral responses of both brain resident cells and infiltrating immune cells. This review discusses how microglia may promote this antiviral response at a molecular level, from potential mechanisms of virus recognition to downstream cytokine responses and interaction with antiviral T cells. Recent advancements in genetic tools to specifically target microglia in vivo promise to further our understanding about the precise mechanistic role of microglia in CNS infection.

Immune cell regulation of glia during CNS injury and disease.

Glial cells are abundant in the CNS and are essential for brain development and homeostasis. These cells also regulate tissue recovery after injury and their dysfunction is a possible contributing factor to neurodegenerative and psychiatric disease. Recent evidence suggests that microglia, which are also the brain's major resident immune cells, provide disease-modifying regulation of the other major glial populations, namely astrocytes and oligodendrocytes. In addition, peripheral immune cells entering the CNS after injury and in disease may directly affect microglial, astrocyte and oligodendrocyte function, suggesting an integrated network of immune cell-glial cell communication.

Please eat (only part) of me: synaptic phosphatidylserine cues microglia to feast: Two new studies identify how a common apoptotic cell flag is used to sculpt neural circuits.

Microglia, the brain's tissue-resident macrophages, contribute to the developmental elimination of extranumerary synapses and to pathologic synapse loss in mouse models of neurodegeneration. Two papers published in The EMBO Journal reveal that phosphatidylserine (PS) is a neuronal cue for microglial synapse elimination.

The immune system and psychiatric disease: a basic science perspective.

Mental illness exerts a major burden on human health, yet evidence-based treatments are rudimentary due to a limited understanding of the underlying pathologies. Clinical studies point to roles for the immune system in psychiatric diseases, while basic science has revealed that the brain has an active and multi-cellular resident immune system that interacts with peripheral immunity and impacts behavior. In this perspective, we highlight evidence of immune involvement in human psychiatric disease and review data from animal models that link immune signaling to neuronal function and behavior. We propose a conceptual framework for linking advances in basic neuroimmunology to their potential relevance for psychiatric diseases, based on the subtypes of immune responses defined in peripheral tissues. Our goal is to identify novel areas of focus for future basic and translational studies that may reveal the potential of the immune system for diagnosing and treating mental illnesses.

New tools for studying microglia in the mouse and human CNS.

The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

Neurodevelopmental and Cognitive Outcomes in Children With Intestinal Failure.

OBJECTIVES: Recent advances in medical and surgical management have led to improved long-term survival in children with intestinal failure. Yet, limited data exist on their neurodevelopmental and cognitive outcomes. The aim of the present study was to measure neurodevelopmental outcomes in children with intestinal failure. METHODS: Children enrolled in a regional intestinal failure program underwent prospective neurodevelopmental and psychometric evaluation using a validated scoring tool. Cognitive impairment was defined as a mental developmental index <70. Neurodevelopmental impairment was defined as cerebral palsy, visual or hearing impairment, or cognitive impairment. Univariate analyses were performed using the Wilcoxon rank-sum test. Data are presented as median (range). RESULTS: Fifteen children with a remnant bowel length of 18 (5-85) cm were studied at age 17 (12-67) months. Thirteen patients remained dependent on parenteral nutrition. Twelve (80%) subjects scored within the normal range on cognitive testing. Each child with cognitive impairment was noted to have additional risk factors independent of intestinal failure including cardiac arrest and extreme prematurity. On univariate analysis, cognitive impairment was associated with longer inpatient hospital stays, increased number of surgical procedures, and prematurity (P < 0.02). In total, 4 (27%) children demonstrated findings consistent with neurodevelopmental impairment. CONCLUSIONS: A majority of children with intestinal failure demonstrated normal neurodevelopmental and cognitive outcomes on psychometric testing. These data suggest that children with intestinal failure without significant comorbidity may be at low risk for long-term neurodevelopmental impairment.

Microglia replacement by ER-Hoxb8 conditionally immortalized macrophages provides insight into Aicardi-Goutières Syndrome neuropathology.

Microglia, the brain's resident macrophages, can be reconstituted by surrogate cells - a process termed "microglia replacement." To expand the microglia replacement toolkit, we here introduce estrogen-regulated (ER) homeobox B8 (Hoxb8) conditionally immortalized macrophages, a cell model for generation of immune cells from murine bone marrow, as a versatile model for microglia replacement. We find that ER-Hoxb8 macrophages are highly comparable to primary bone marrow-derived (BMD) macrophages in vitro, and, when transplanted into a microglia-free brain, engraft the parenchyma and differentiate into microglia-like cells. Furthermore, ER-Hoxb8 progenitors are readily transducible by virus and easily stored as stable, genetically manipulated cell lines. As a demonstration of this system's power for studying the effects of disease mutations on microglia in vivo, we created stable, Adar1-mutated ER-Hoxb8 lines using CRISPR-Cas9 to study the intrinsic contribution of macrophages to Aicardi-Goutières Syndrome (AGS), an inherited interferonopathy that primarily affects the brain and immune system. We find that Adar1 knockout elicited interferon secretion and impaired macrophage production in vitro, while preventing brain macrophage engraftment in vivo - phenotypes that can be rescued with concurrent mutation of Ifih1 (MDA5) in vitro, but not in vivo. Lastly, we extended these findings by generating ER-Hoxb8 progenitors from mice harboring a patient-specific Adar1 mutation (D1113H). We demonstrated the ability of microglia-specific D1113H mutation to drive interferon production in vivo, suggesting microglia drive AGS neuropathology. In sum, we introduce the ER-Hoxb8 approach to model microglia replacement and use it to clarify macrophage contributions to AGS.

Microglia are not necessary for maintenance of blood-brain barrier properties in health, but PLX5622 alters brain endothelial cholesterol metabolism.

Microglia, the resident immune cells of the central nervous system, are intimately involved in the brain's most basic processes, from pruning neural synapses during development to preventing excessive neuronal activity throughout life. Studies have reported both helpful and harmful roles for microglia at the blood-brain barrier (BBB) in the context of disease. However, less is known about microglia-endothelial cell interactions in the healthy brain. To investigate the role of microglia at a healthy BBB, we used the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia and analyzed the BBB ultrastructure, permeability, and transcriptome. Interestingly, we found that, despite their direct contact with endothelial cells, microglia are not necessary for the maintenance of BBB structure, function, or gene expression in the healthy brain. However, we found that PLX5622 treatment alters brain endothelial cholesterol metabolism. This effect was independent from microglial depletion, suggesting that PLX5622 has off-target effects on brain vasculature.

Bone mineral density in Jamaican black women after hysterectomy and bilateral oophorectomy: an observational study.

OBJECTIVE: To see if black Jamaican postmenopausal women who had hysterectomy were at increased risk of osteoporosis. To assess the risk of osteoporosis in hysterectomized Jamaican postmenopausal patients. METHOD: We reviewed 809 women (403 hysterectomized and 406 controls) for cardiovascular disease risk. We did a demographic history and examination looking at blood pressure, waist hip ratio and body mass index and investigations done included fasting blood glucose and total and high density lipoprotein (HDL) cholesterol. We also measured bone density at the heel in all women using the Achilles ultrasound bone densitometer looking at T-score and Z-score. RESULTS: There was a significant association of hysterectomy status and bone mineral density (BMD) status with a smaller than expected proportion of women with osteoporosis in the hysterectomy group (χ2 = 18.4; p = 0.001). The mean T-score was significantly higher in the hysterectomized women, adjusting for age, waist circumference and sociodemographic factors. The relationship between the various predictors and BMD was explored by stepwise regression modelling. The factors that were significantly related to low BMD were hysterectomy status, age, waist circumference and being employed. CONCLUSION: Hysterectomy was not found to be a significant risk factor for osteoporosis. The osteoporosis risk among menopausal women in Jamaica appears to be due to other risk factors which probably existed prior to the operation.

Molecular and metabolic heterogeneity of astrocytes and microglia.

Astrocytes and microglia are central players in a myriad of processes in the healthy and diseased brain, ranging from metabolism to immunity. The crosstalk between these two cell types contributes to pathology in many if not all neuroinflammatory and neurodegenerative diseases. Recent advancements in integrative multimodal sequencing techniques have begun to highlight how heterogeneous both cell types are and the importance of metabolism to their regulation. We discuss here the transcriptomic, metabolic, and functional heterogeneity of astrocytes and microglia and highlight their interaction in health and disease.

CRISPR generation of CSF1R-G795A human microglia for robust microglia replacement in a chimeric mouse model.

Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been challenging. Here, we present a protocol to introduce the G795A point mutation using CRISPR-Cas9 into the CSF1R locus of human pluripotent stem cells. We also describe an optimized microglial differentiation technique for transplantation into newborn or adult recipients. We then detail pharmacological paradigms to achieve widespread and near-complete engraftment of human microglia. For complete details on the use and execution of this protocol, please refer to Chadarevian et al. (2023).1.

Engineering an inhibitor-resistant human CSF1R variant for microglia replacement.

Hematopoietic stem cell transplantation (HSCT) can replace endogenous microglia with circulation-derived macrophages but has high mortality. To mitigate the risks of HSCT and expand the potential for microglia replacement, we engineered an inhibitor-resistant CSF1R that enables robust microglia replacement. A glycine to alanine substitution at position 795 of human CSF1R (G795A) confers resistance to multiple CSF1R inhibitors, including PLX3397 and PLX5622. Biochemical and cell-based assays show no discernable gain or loss of function. G795A- but not wildtype-CSF1R expressing macrophages efficiently engraft the brain of PLX3397-treated mice and persist after cessation of inhibitor treatment. To gauge translational potential, we CRISPR engineered human-induced pluripotent stem cell-derived microglia (iMG) to express G795A. Xenotransplantation studies demonstrate that G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand in the presence of PLX3397, replacing endogenous microglia to fully occupy the brain. In sum, we engineered a human CSF1R variant that enables nontoxic, cell type, and tissue-specific replacement of microglia.

Human OPRM1 and murine Oprm1 promoter driven viral constructs for genetic access to µ-opioidergic cell types.

With concurrent global epidemics of chronic pain and opioid use disorders, there is a critical need to identify, target and manipulate specific cell populations expressing the mu-opioid receptor (MOR). However, available tools and transgenic models for gaining long-term genetic access to MOR+ neural cell types and circuits involved in modulating pain, analgesia and addiction across species are limited. To address this, we developed a catalog of MOR promoter (MORp) based constructs packaged into adeno-associated viral vectors that drive transgene expression in MOR+ cells. MORp constructs designed from promoter regions upstream of the mouse Oprm1 gene (mMORp) were validated for transduction efficiency and selectivity in endogenous MOR+ neurons in the brain, spinal cord, and periphery of mice, with additional studies revealing robust expression in rats, shrews, and human induced pluripotent stem cell (iPSC)-derived nociceptors. The use of mMORp for in vivo fiber photometry, behavioral chemogenetics, and intersectional genetic strategies is also demonstrated. Lastly, a human designed MORp (hMORp) efficiently transduced macaque cortical OPRM1+ cells. Together, our MORp toolkit provides researchers cell type specific genetic access to target and functionally manipulate mu-opioidergic neurons across a range of vertebrate species and translational models for pain, addiction, and neuropsychiatric disorders.

Treatment of a genetic brain disease by CNS-wide microglia replacement.

Hematopoietic cell transplantation after myeloablative conditioning has been used to treat various genetic metabolic syndromes but is largely ineffective in diseases affecting the brain presumably due to poor and variable myeloid cell incorporation into the central nervous system. Here, we developed and characterized a near-complete and homogeneous replacement of microglia with bone marrow cells in mice without the need for genetic manipulation of donor or host. The high chimerism resulted from a competitive advantage of scarce donor cells during microglia repopulation rather than enhanced recruitment from the periphery. Hematopoietic stem cells, but not immediate myeloid or monocyte progenitor cells, contained full microglia replacement potency equivalent to whole bone marrow. To explore its therapeutic potential, we applied microglia replacement to a mouse model for Prosaposin deficiency, which is characterized by a progressive neurodegeneration phenotype. We found a reduction of cerebellar neurodegeneration and gliosis in treated brains, improvement of motor and balance impairment, and life span extension even with treatment started in young adulthood. This proof-of-concept study suggests that efficient microglia replacement may have therapeutic efficacy for a variety of neurological diseases.

Myosin-IIA and ICAM-1 regulate the interchange between two distinct modes of T cell migration.

How T cells achieve rapid chemotactic motility under certain circumstances and efficient cell surface surveillance in others is not fully understood. We show that T lymphocytes are motile in two distinct modes: a fast "amoeboid-like" mode, which uses sequential discontinuous contacts to the substrate; and a slower mode using a single continuously translating adhesion, similar to mesenchymal motility. Myosin-IIA is necessary for fast amoeboid motility, and our data suggests that this occurs via cyclical rear-mediated compressions that eliminate existing adhesions while licensing subsequent ones at the front of the cell. Regulation of Myosin-IIA function in T cells is thus a key mechanism to regulate surface contact area and crawling velocity within different environments. This can provide T lymphocytes with motile and adhesive properties that are uniquely suited toward alternative requirements for immune surveillance and response.