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.

Serotonin reduction in post-acute sequelae of viral infection.

Post-acute sequelae of COVID-19 (PASC, "Long COVID") pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes.

Lipid-Associated Macrophages Control Metabolic Homeostasis in a Trem2-Dependent Manner.

Immune cells residing in white adipose tissue have been highlighted as important factors contributing to the pathogenesis of metabolic diseases, but the molecular regulators that drive adipose tissue immune cell remodeling during obesity remain largely unknown. Using index and transcriptional single-cell sorting, we comprehensively map all adipose tissue immune populations in both mice and humans during obesity. We describe a novel and conserved Trem2+ lipid-associated macrophage (LAM) subset and identify markers, spatial localization, origin, and functional pathways associated with these cells. Genetic ablation of Trem2 in mice globally inhibits the downstream molecular LAM program, leading to adipocyte hypertrophy as well as systemic hypercholesterolemia, body fat accumulation, and glucose intolerance. These findings identify Trem2 signaling as a major pathway by which macrophages respond to loss of tissue-level lipid homeostasis, highlighting Trem2 as a key sensor of metabolic pathologies across multiple tissues and a potential therapeutic target in metabolic diseases.

A microbiome-dependent gut-brain pathway regulates motivation for exercise.

Exercise exerts a wide range of beneficial effects for healthy physiology1. However, the mechanisms regulating an individual's motivation to engage in physical activity remain incompletely understood. An important factor stimulating the engagement in both competitive and recreational exercise is the motivating pleasure derived from prolonged physical activity, which is triggered by exercise-induced neurochemical changes in the brain. Here, we report on the discovery of a gut-brain connection in mice that enhances exercise performance by augmenting dopamine signalling during physical activity. We find that microbiome-dependent production of endocannabinoid metabolites in the gut stimulates the activity of TRPV1-expressing sensory neurons and thereby elevates dopamine levels in the ventral striatum during exercise. Stimulation of this pathway improves running performance, whereas microbiome depletion, peripheral endocannabinoid receptor inhibition, ablation of spinal afferent neurons or dopamine blockade abrogate exercise capacity. These findings indicate that the rewarding properties of exercise are influenced by gut-derived interoceptive circuits and provide a microbiome-dependent explanation for interindividual variability in exercise performance. Our study also suggests that interoceptomimetic molecules that stimulate the transmission of gut-derived signals to the brain may enhance the motivation for exercise.

ß-Hydroxybutyrate suppresses colorectal cancer.

Colorectal cancer (CRC) is among the most frequent forms of cancer, and new strategies for its prevention and therapy are urgently needed1. Here we identify a metabolite signalling pathway that provides actionable insights towards this goal. We perform a dietary screen in autochthonous animal models of CRC and find that ketogenic diets exhibit a strong tumour-inhibitory effect. These properties of ketogenic diets are recapitulated by the ketone body ß-hydroxybutyrate (BHB), which reduces the proliferation of colonic crypt cells and potently suppresses intestinal tumour growth. We find that BHB acts through the surface receptor Hcar2 and induces the transcriptional regulator Hopx, thereby altering gene expression and inhibiting cell proliferation. Cancer organoid assays and single-cell RNA sequencing of biopsies from patients with CRC provide evidence that elevated BHB levels and active HOPX are associated with reduced intestinal epithelial proliferation in humans. This study thus identifies a BHB-triggered pathway regulating intestinal tumorigenesis and indicates that oral or systemic interventions with a single metabolite may complement current prevention and treatment strategies for CRC.

Intestinal Tolerance, with a Little Help from Our Microbial Friends.

The intestinal immune system performs a tightrope walk between anti-pathogen immunity and tolerance to innocuous luminal antigens. In this issue of Immunity, Kim et al. (2018) demonstrate that the microbiota is driving tolerance to food antigens and dampening inflammatory responses through CX3CR1+ mononuclear phagocytes.

Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke.

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.

Astrocyte TrkB promotes brain injury and edema formation in ischemic stroke.

Following ischemic stroke astrocytes undergo rapid molecular and functional changes that may accentuate tissue damage. In this study we identified the neurotrophin receptor TrkB in astrocytes as a key promoter of acute CNS injury in ischemic stroke. In fact, TrkB protein was strongly upregulated in astrocytes after human and experimental stroke, and transgenic mice lacking astrocyte TrkB displayed significantly smaller lesion volume, lower brain atrophy and better motor performance than control animals after transient middle cerebral artery occlusion. Neuropathological studies evidenced that edema directly correlated with astrogliosis and was limited in transgenic mice. Importantly, adaptive levels of the water channel AQP4 was astrocyte TrkB-dependent as AQP4 upregulation after stroke did not occur in mice lacking astrocyte TrkB. In vitro experiments with wild-type and/or TrkB-deficient astrocytes highlighted TrkB-dependent upregulation of AQP4 via activation of HIF1-alpha under hypoxia. Collectively, our observations indicate that TrkB signaling in astrocytes contributes to the development of edema and worsens cerebral ischemia.

Extracellular Vesicles Containing IL-4 Modulate Neuroinflammation in a Mouse Model of Multiple Sclerosis.

Extracellular vesicles (EVs) play a major role in cell-to-cell communication in physiological and pathological conditions, and their manipulation may represent a promising therapeutic strategy. Microglia, the parenchymal mononuclear phagocytes of the brain, modulate neighboring cells also through the release of EVs. The production of custom EVs filled with desired molecules, possibly targeted to make their uptake cell specific, and their administration in biological fluids may represent a valid approach for drug delivery. We engineered a murine microglia cell line, BV-2, to release EVs overexpressing the endogenous "eat me" signal Lactadherin (Mfg-e8) on the surface to target phagocytes and containing the anti-inflammatory cytokine IL-4. A single injection of 107 IL-4+Mfg-e8+ EVs into the cisterna magna modulated established neuroinflammation and significantly reduced clinical signs in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Injected IL-4+Mfg-e8+ EVs target mainly phagocytes (i.e., macrophages and microglia) surrounding liquoral spaces, and their cargo promote the upregulation of anti-inflammatory markers chitinase 3-like 3 (ym1) and arginase-1 (arg1), significantly reducing tissue damage. Engineered EVs may represent a biological drug delivery tool able to deliver multiple functional molecules simultaneously to treat neuroinflammatory diseases.

Modeling type 1 diabetes progression using machine learning and single-cell transcriptomic measurements in human islets.

Type 1 diabetes (T1D) is a chronic condition in which beta cells are destroyed by immune cells. Despite progress in immunotherapies that could delay T1D onset, early detection of autoimmunity remains challenging. Here, we evaluate the utility of machine learning for early prediction of T1D using single-cell analysis of islets. Using gradient-boosting algorithms, we model changes in gene expression of single cells from pancreatic tissues in T1D and non-diabetic organ donors. We assess if mathematical modeling could predict the likelihood of T1D development in non-diabetic autoantibody-positive donors. While most autoantibody-positive donors are predicted to be non-diabetic, select donors with unique gene signatures are classified as T1D. Our strategy also reveals a shared gene signature in distinct T1D-associated models across cell types, suggesting a common effect of the disease on transcriptional outputs of these cells. Our study establishes a precedent for using machine learning in early detection of T1D.

No Evidence for Persistent Enteroviral B Infection of Pancreatic Islets in Patients With Type 1 Diabetes and Prediabetes From RNA Sequencing Data.

Persistent enterovirus B infection has been proposed as an important contributor to the etiology of type 1 diabetes. We leveraged extensive bulk RNA-sequencing (RNA-seq) data from α-, ß-, and exocrine cells, as well as islet single-cell RNA-seq data from the Human Pancreas Analysis Program (HPAP), to evaluate the presence of enterovirus B sequences in the pancreas of patients with type 1 diabetes and prediabetes (no diabetes but positive for autoantibodies). We examined all available HPAP data for either assay type, including donors without diabetes and with type 1 and type 2 diabetes. To assess the presence of viral reads, we analyzed all reads not mapping to the human genome with the taxonomic classification system Kraken2 and its full viral database augmented to encompass representatives for all 28 enterovirus B serotypes for which a complete genome is available. As a secondary approach, we input the same sequence reads into the STAR aligner using these 28 enterovirus B genomes as the reference. No enterovirus B sequences were detected by either approach in any of the 243 bulk RNA libraries or in any of the 79 single-cell RNA libraries. While we cannot rule out the possibility of a very-low-grade persistent enterovirus B infection in the donors analyzed, our data do not support the notion of chronic viral infection by these viruses as a major driver of type 1 diabetes.

Targeting lipid nanoparticles to the blood brain barrier to ameliorate acute ischemic stroke.

After more than 100 failed drug trials for acute ischemic stroke (AIS), one of the most commonly cited reasons for the failure has been that drugs achieve very low concentrations in the at-risk penumbra. To address this problem, here we employ nanotechnology to significantly enhance drug concentration in the penumbra's blood-brain barrier (BBB), whose increased permeability in AIS has long been hypothesized to kill neurons by exposing them to toxic plasma proteins. To devise drug-loaded nanocarriers targeted to the BBB, we conjugated them with antibodies that bind to various cell adhesion molecules on the BBB endothelium. In the transient middle cerebral artery occlusion (tMCAO) mouse model, nanocarriers targeted with VCAM antibodies achieved the highest level of brain delivery, nearly 2 orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles loaded with either a small molecule drug (dexamethasone) or mRNA (encoding IL-10) reduced cerebral infarct volume by 35% or 73%, respectively, and both significantly lowered mortality rates. In contrast, the drugs delivered without the nanocarriers had no effect on AIS outcomes. Thus, VCAM-targeted lipid nanoparticles represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS. Graphical abstract: Acute ischemic stroke induces upregulation of VCAM. We specifically targeted upregulated VCAM in the injured region of the brain with drug- or mRNA-loaded targeted nanocarriers. Nanocarriers targeted with VCAM antibodies achieved the highest brain delivery, nearly orders of magnitude higher than untargeted ones. VCAM-targeted nanocarriers loaded with dexamethasone and mRNA encoding IL-10 reduced infarct volume by 35% and 73%, respectively, and improved survival rates.

A subpopulation of lipogenic brown adipocytes drives thermogenic memory.

Sustained responses to transient environmental stimuli are important for survival. The mechanisms underlying long-term adaptations to temporary shifts in abiotic factors remain incompletely understood. Here, we find that transient cold exposure leads to sustained transcriptional and metabolic adaptations in brown adipose tissue, which improve thermogenic responses to secondary cold encounter. Primary thermogenic challenge triggers the delayed induction of a lipid biosynthesis programme even after cessation of the original stimulus, which protects from subsequent exposures. Single-nucleus RNA sequencing and spatial transcriptomics reveal that this response is driven by a lipogenic subpopulation of brown adipocytes localized along the perimeter of Ucp1hi adipocytes. This lipogenic programme is associated with the production of acylcarnitines, and supplementation of acylcarnitines is sufficient to recapitulate improved secondary cold responses. Overall, our data highlight the importance of heterogenous brown adipocyte populations for 'thermogenic memory', which may have therapeutic implications for leveraging short-term thermogenesis to counteract obesity.

NAD precursors cycle between host tissues and the gut microbiome.

Nicotinamide adenine dinucleotide (NAD) is an essential redox cofactor in mammals and microbes. Here we use isotope tracing to investigate the precursors supporting NAD synthesis in the gut microbiome of mice. We find that dietary NAD precursors are absorbed in the proximal part of the gastrointestinal tract and not available to microbes in the distal gut. Instead, circulating host nicotinamide enters the gut lumen and supports microbial NAD synthesis. The microbiome converts host-derived nicotinamide into nicotinic acid, which is used for NAD synthesis in host tissues and maintains circulating nicotinic acid levels even in the absence of dietary consumption. Moreover, the main route from oral nicotinamide riboside, a widely used nutraceutical, to host NAD is via conversion into nicotinic acid by the gut microbiome. Thus, we establish the capacity for circulating host micronutrients to feed the gut microbiome, and in turn be transformed in a manner that enhances host metabolic flexibility.

Pharmacological activation of STING blocks SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, resulting millions of infections and deaths with few effective interventions available. Here, we demonstrate that SARS-CoV-2 evades interferon (IFN) activation in respiratory epithelial cells, resulting in a delayed response in bystander cells. Since pretreatment with IFNs can block viral infection, we reasoned that pharmacological activation of innate immune pathways could control SARS-CoV-2 infection. To identify potent antiviral innate immune agonists, we screened a panel of 75 microbial ligands that activate diverse signaling pathways and identified cyclic dinucleotides (CDNs), canonical STING agonists, as antiviral. Since CDNs have poor bioavailability, we tested the small molecule STING agonist diABZI, and found that it potently inhibits SARS-CoV-2 infection of diverse strains including variants of concern (B.1.351) by transiently stimulating IFN signaling. Importantly, diABZI restricts viral replication in primary human bronchial epithelial cells and in mice in vivo. Our study provides evidence that activation of STING may represent a promising therapeutic strategy to control SARS-CoV-2.

NAD+ flux is maintained in aged mice despite lower tissue concentrations.

NAD+ is an essential coenzyme for all living cells. NAD+ concentrations decline with age, but whether this reflects impaired production or accelerated consumption remains unclear. We employed isotope tracing and mass spectrometry to probe age-related changes in NAD+ metabolism across tissues. In aged mice, we observed modest tissue NAD+ depletion (median decrease ∼30%). Circulating NAD+ precursors were not significantly changed, and isotope tracing showed the unimpaired synthesis of nicotinamide from tryptophan. In most tissues of aged mice, turnover of the smaller tissue NAD+ pool was modestly faster such that absolute NAD+ biosynthetic flux was maintained, consistent with more active NAD+-consuming enzymes. Calorie restriction partially mitigated age-associated NAD+ decline by decreasing consumption. Acute inflammatory stress induced by LPS decreased NAD+ by impairing synthesis in both young and aged mice. Thus, the decline in NAD+ with normal aging is relatively subtle and occurs despite maintained NAD+ production, likely due to increased consumption.

Interferon-γ/Interleukin-27 Axis Induces Programmed Death Ligand 1 Expression in Monocyte-Derived Dendritic Cells and Restores Immune Tolerance in Central Nervous System Autoimmunity.

Antigen (Ag)-specific tolerance induction by intravenous (i. v.) injection of high-dose auto-Ags has been explored for therapy of autoimmune diseases, including multiple sclerosis (MS). It is thought that the advantage of such Ag-specific therapy over non-specific immunomodulatory treatments would be selective suppression of a pathogenic immune response without impairing systemic immunity, thus avoiding adverse effects of immunosuppression. Auto-Ag i.v. tolerance induction has been extensively studied in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and limited clinical trials demonstrated that it is safe and beneficial to a subset of MS patients. Nonetheless, the mechanisms of i.v. tolerance induction are incompletely understood, hampering the development of better approaches and their clinical application. Here, we describe a pathway whereby auto-Ag i.v. injected into mice with ongoing clinical EAE induces interferon-gamma (IFN-γ) secretion by auto-Ag-specific CD4+ T cells, triggering interleukin (IL)-27 production by conventional dendritic cells type 1 (cDC1). IL-27 then, via signal transducer and activator of transcription 3 activation, induces programmed death ligand 1 (PD-L1) expression by monocyte-derived dendritic cells (moDCs) in the central nervous system of mice with EAE. PD-L1 interaction with programmed cell death protein 1 on pathogenic CD4+ T cells leads to their apoptosis/anergy, resulting in disease amelioration. These findings identify a key role of the IFN-γ/IL-27/PD-L1 axis, involving T cells/cDC1/moDCs in the induction of i.v. tolerance.

The path toward using microbial metabolites as therapies.

Metabolites have emerged as the quintessential effectors mediating the impact of the commensal microbiome on human physiology, both locally at the sites of microbial colonization and systemically. The endocrine activity of the microbiome and its involvement in a multitude of complex diseases has made microbiome-modulated metabolites an attractive target for the development of new therapies. Several properties make metabolites uniquely suited for interventional strategies: natural occurrence in a broad range of concentrations, functional pleiotropy, ease of administration, and tissue bioavailability. Here, we provide an overview of recently discovered physiological effects of microbiome-associated small molecules that may serve as the first examples of metabolite-based therapies. We also highlight challenges and obstacles that the field needs to overcome on the path toward successful clinical trials of microbial metabolites for human disease.

IL-27, but not IL-35, inhibits neuroinflammation through modulating GM-CSF expression.

IL-27 and IL-35 are heterodimeric cytokines, members of the IL-12 family and considered to have immunomodulatory properties. Their role during neuroinflammation had been investigated using mutant mice devoid of either one of their subunits or lacking components of their receptors, yielding conflicting results. We sought to understand the therapeutic potential of IL-27 and IL-35 delivered by gene therapy in neuroinflammation. We constructed lentiviral vectors expressing IL-27 and IL-35 from a single polypeptide chain, and we validated in vitro their biological activity. We injected IL-27 and IL-35-expressing lentiviral vectors into the cerebrospinal fluid (CSF) of mice affected by experimental neuroinflammation (EAE), and performed clinical, neuropathological and immunological analyses. Both cytokines interfere with neuroinflammation, but only IL-27 significantly modulates disease development, both clinically and neuropathologically. IL-27 protects from autoimmune inflammation by inhibiting granulocyte macrophages colony-stimulating factor (GM-CSF) expression in CD4+ T cells and by inducing program death-ligand 1 (PD-L1) expression in both CNS-resident and CNS-infiltrating myeloid cells. We demonstrate here that IL-27 holds therapeutic potential during neuroinflammation and that IL-27 inhibits GM-CSF and induces pd-l1 mRNA in vivo.