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.

Inflammation Drives MicroRNAs to Limit Hepatocyte Bile Acid Transport in Murine Biliary Atresia.

BACKGROUND: Biliary atresia (BA) is an inflammatory pediatric cholangiopathy with only surgical means for treatment. Many contributors to bile acid synthesis and transport have previously been reported to be downregulated in patients with BA; yet, the driving factors of the abnormal bile acid synthesis and transport in regard to BA have not been previously studied. MATERIALS AND METHODS: Wild type or Ig-α-/- mice were injected with salt solution (control) or rotavirus on day of life 0, and analyses were performed on day of life 14. The mRNA levels of bile acid transporters/nuclear receptors and liver microRNAs (miRNAs) were compared between groups. A mouse hepatocyte cell line was used to examine the effects of innate cytokines on miRNA levels and bile acid transporter/nuclear receptor expression and miRNAs on bile acid transporter/nuclear receptor expression. RESULTS: BA mice had significantly increased mRNA expression of innate cytokines and miRNAs known to bind bile acid transporters/nuclear receptors (miRNAs -22-5p, -34a-5p, and -222-3p) and decreased mRNA expression of bile acid transporters and nuclear receptors. In vitro, TNF-α and IL-1ß decreased BSEP and CYP7A1 while increasing miRNA-34a-5p and miRNA 222-3p. LXR, SHP, CYP7A1, NTCP, and MRP2 were decreased by miRNA-34a-5p, whereas miRNA-222-3p decreased NTCP and MRP4. TNF-α and IL-1ß increased expression of miRNAs 34a-5p and 222-3p and these miRNAs then decrease expression of multiple bile acid transporters and nuclear receptors. CONCLUSIONS: Loss of bile acid transporters increases hepatotoxicity via bile acid retention. Therapeutic agents that increase bile acid transport or nuclear receptor functioning should be investigated in BA.

Fragile X related protein 1 (FXR1P) regulates proliferation of adult neural stem cells.

Fragile X related protein 1 (FXR1P) is a member of the fragile X family of RNA-binding proteins, which includes FMRP and FXR2P. Both FMRP and FXR2P regulate neurogenesis, a process affected in a number of neurological and neuropsychiatric disorders, including fragile X syndrome. Although FXR1P has been implicated in various developmental processes and neuropsychiatric diseases, its role in neurodevelopment is not well understood. The goal of the present study was to elucidate the function of FXR1P in adult neurogenesis. We used an inducible mouse model that allows us to investigate how FXR1P deficiency in adult neural stem cells (aNSCs) affects proliferation and neuronal differentiation. Deletion of FXR1 in aNSCs resulted in fewer adult-born cells in the dentate gyrus (DG) overall, reducing populations across different stages of neurogenesis, including radial glia-like cells, intermediate progenitors, neuroblasts, immature neurons and neurons. We hypothesized that this reduction in new cell numbers resulted from impaired proliferation, which we confirmed both in vivo and in vitro. We discovered that FXR1P-deficient aNSCs have altered expression of a select number of cell-cycle genes, and we identified the mRNA of cyclin-dependent kinase inhibitor 1A (Cdkn1a, p21) as a direct target of FXR1P. Restoration of p21 mRNA to wild-type levels rescued the proliferation deficit in cells lacking FXR1P, demonstrating that p21 is a mediator of FXR1P in aNSCs. These results indicate that FXR1P plays an important role in regulating aNSC self-renewal and maintenance in the adult brain, which may have implications for a number of neurodevelopmental and psychiatric disorders.

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.

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.