Protists protecting food tolerance.

Celiac disease (CeD) is an immune disorder characterized by gluten intolerance that can be unleashed by enteric viral infections in mice. However, Sanchez-Medina et al. recently identified a murine commensal protist, Tritrichomonas arnold, that protects against reovirus-induced intolerance to dietary protein by counteracting virus-induced epithelial stress and proinflammatory dendritic cell (DC) activation.

Lymph node sharing between pancreas, gut, and liver leads to immune crosstalk and regulation of pancreatic autoimmunity.

Lymph nodes (LNs) are critical sites for shaping tissue-specific adaptive immunity. However, the impact of LN sharing between multiple organs on such tailoring is less understood. Here, we describe the drainage hierarchy of the pancreas, liver, and the upper small intestine (duodenum) into three murine LNs. Migratory dendritic cells (migDCs), key in instructing adaptive immune outcome, exhibited stronger pro-inflammatory signatures when originating from the pancreas or liver than from the duodenum. Qualitatively different migDC mixing in each shared LN influenced pancreatic ß-cell-reactive T cells to acquire gut-homing and tolerogenic phenotypes proportional to duodenal co-drainage. However, duodenal viral infections rendered non-intestinal migDCs and ß-cell-reactive T cells more pro-inflammatory in all shared LNs, resulting in elevated pancreatic islet lymphocyte infiltration. Our study uncovers immune crosstalk through LN co-drainage as a powerful force regulating pancreatic autoimmunity.

RO6807936 as a novel positron emission tomography (PET) radiotracer for in vitro and in vivo visualization and quantification of beta-site amyloid precursor protein cleaving enzyme (BACE1) in the rodent and baboon brain.

The beta-site amyloid precursor protein cleaving enzyme (BACE1) is responsible for initiating the generation of beta-amyloid, the major constituent of amyloid plaques in Alzheimer's disease (AD). The purpose of this study was to develop a specific BACE1 radioligand for visualization of the distribution pattern and quantification of the BACE1 protein in the rodent and monkey brain both in vitro by autoradiography and in vivo by positron emission tomography (PET). The BACE1 inhibitor RO6807936 originating from an in-house chemical drug optimization program was selected based on its PET tracer-like physicochemical properties and a favorable pharmacokinetic profile. Saturation binding analysis of [3 H]RO6807936 revealed specific and high-affinity binding (KD = 2.9 nM) and a low Bmax value (4.3 nM) of the BACE1 protein in native rat brain membranes. [3 H]RO6807936 binding showed a ubiquitous distribution on rat brain slices in vitro with higher levels in the CA3 pyramidal cell layer and the granule cell layer of the hippocampus. In a next step, RO6807936 was successfully radiolabeled with carbon-11 and showed acceptable uptake in the baboon brain as well as a widespread and rather homogeneous distribution consistent with rodent data. In vivo blockade studies with a specific BACE1 inhibitor reduced uptake of the tracer to homogenous levels across brain regions and demonstrated specificity of the signal. Our data warrant further profiling of this PET tracer candidate in humans to investigate BACE1 expression in normal individuals and those with AD and as an imaging biomarker for target occupancy studies in clinical drug trials.

Oral alloantigen exposure promotes donor-specific tolerance in a mouse model of minor-mismatched skin transplantation.

Oral antigen exposure is a powerful, non-invasive route to induce immune tolerance to dietary antigens, and has been modestly successful at prolonging graft survival in rodent models of transplantation. To harness the mechanisms of oral tolerance for promoting long-term graft acceptance, we developed a mouse model where the antigen ovalbumin (OVA) was introduced orally prior to transplantation with skin grafts expressing OVA. Oral OVA treatment pre-transplantation promoted permanent graft acceptance and linked tolerance to skin grafts expressing OVA fused to the additional antigen 2W. Tolerance was donor-specific, as secondary donor-matched, but not third-party allografts were spontaneously accepted. Oral OVA treatment promoted an anergic phenotype in OVA-reactive CD4+ and CD8+ conventional T cells (Tconvs) and expanded OVA-reactive Tregs pre-transplantation. However, skin graft acceptance following oral OVA resisted partial depletion of Tregs and blockade of PD-L1. Mechanistically, we revealed a role for the proximal gut draining lymph nodes (gdLNs) in mediating this effect, as an intestinal infection that drains to the proximal gdLNs prevented tolerance induction. Our study extends previous work applying oral antigen exposure to transplantation and serves as proof of concept that the systemic immune mechanisms supporting oral tolerance are sufficient to promote long-term graft acceptance.

Intestinal immune compartmentalization: implications of tissue specific determinants in health and disease.

The emerging concept of tissue specific immunity has opened the gates to new inquiries into what factors drive immune cell niche adaptation and the implications on immune homeostasis, organ specific immune diseases, and therapeutic efficacy. These issues are particularly complicated at barrier sites, which are directly exposed to an ever-changing environment. In particular, the gastrointestinal (GI) tract faces even further challenges given the profound functional and structural differences along its length, raising the possibility that it may even have to be treated as multiple organs when seeking to answer these questions. In this review, we evaluate what is known about the tissue intrinsic and extrinsic factors shaping immune compartments in the intestine. We then discuss the physiological and pathological consequences of a regionally distinct immune system in a single organ, but also discuss where our insight into the role of the compartment for disease development is still very limited. Finally, we discuss the technological and therapeutic implications this compartmentalization has. While the gut is perhaps one of the most intensely studied systems, many of these aspects apply to understanding tissue specific immunity of other organs, most notably other barrier sites such as skin, lung, and the urogenital tract.

Paying a Price Twice: Dose-Dependent Effects of Treg Cell-Derived TGF- ß on Tolerance.

Although many immune cells can secrete TGF-ß, whether all sources of TGF-ß are functionally equivalent is unknown. In this issue, Turner et al. uncover the importance of T regulatory (Treg) cell-intrinsic Tgfb1 gene dose in the prevention of autoimmunity and allergic disease.

Compartmentalized gut lymph node drainage dictates adaptive immune responses.

The intestinal immune system has the challenging task of tolerating foreign nutrients and the commensal microbiome, while excluding or eliminating ingested pathogens. Failure of this balance leads to conditions such as inflammatory bowel diseases, food allergies and invasive gastrointestinal infections1. Multiple immune mechanisms are therefore in place to maintain tissue integrity, including balanced generation of effector T (TH) cells and FOXP3+ regulatory T (pTreg) cells, which mediate resistance to pathogens and regulate excessive immune activation, respectively1-4. The gut-draining lymph nodes (gLNs) are key sites for orchestrating adaptive immunity to luminal perturbations5-7. However, it is unclear how they simultaneously support tolerogenic and inflammatory reactions. Here we show that gLNs are immunologically specific to the functional gut segment that they drain. Stromal and dendritic cell gene signatures and polarization of T cells against the same luminal antigen differ between gLNs, with the proximal small intestine-draining gLNs preferentially giving rise to tolerogenic responses and the distal gLNs to pro-inflammatory T cell responses. This segregation permitted the targeting of distal gLNs for vaccination and the maintenance of duodenal pTreg cell induction during colonic infection. Conversely, the compartmentalized dichotomy was perturbed by surgical removal of select distal gLNs and duodenal infection, with effects on both lymphoid organ and tissue immune responses. Our findings reveal that the conflict between tolerogenic and inflammatory intestinal responses is in part resolved by discrete gLN drainage, and encourage antigen targeting to specific gut segments for therapeutic immune modulation.

SnapShot: Gut Immune Niches.

The intestinal milieu changes along the proximal to distal axis and across its tissue wall, according to the luminal content and tissue function. Correspondingly, highly specialized immune compartments can be found in each intestinal niche. To view this SnapShot, open or download the PDF.

Corrigendum: Commensal bacteria make GPCR ligands that mimic human signalling molecules.

This corrects the article DOI: 10.1038/nature23874.

Commensal bacteria make GPCR ligands that mimic human signalling molecules.

Commensal bacteria are believed to have important roles in human health. The mechanisms by which they affect mammalian physiology remain poorly understood, but bacterial metabolites are likely to be key components of host interactions. Here we use bioinformatics and synthetic biology to mine the human microbiota for N-acyl amides that interact with G-protein-coupled receptors (GPCRs). We found that N-acyl amide synthase genes are enriched in gastrointestinal bacteria and the lipids that they encode interact with GPCRs that regulate gastrointestinal tract physiology. Mouse and cell-based models demonstrate that commensal GPR119 agonists regulate metabolic hormones and glucose homeostasis as efficiently as human ligands, although future studies are needed to define their potential physiological role in humans. Our results suggest that chemical mimicry of eukaryotic signalling molecules may be common among commensal bacteria and that manipulation of microbiota genes encoding metabolites that elicit host cellular responses represents a possible small-molecule therapeutic modality (microbiome-biosynthetic gene therapy).

Classical dendritic cells are required for dietary antigen-mediated induction of peripheral T(reg) cells and tolerance.

Oral tolerance prevents pathological inflammatory responses to innocuous foreign antigens by peripheral regulatory T cells (pT(reg) cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure for the 'instruction' of naive CD4(+) T cells to differentiate into pT(reg) cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs were dispensable, while classical dendritic cells (cDCs) were critical, for pT(reg) cell induction and oral tolerance. CD11b(-) cDCs from the gut-draining lymph nodes efficiently induced pT(reg) cells and, conversely, loss of transcription factor IRF8-dependent CD11b(-) cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in the induction of pT(reg) cells and their redundancy during the development of oral tolerance.

Absence of MHC class II on cDCs results in microbial-dependent intestinal inflammation.

Conventional dendritic cells (cDCs) play an essential role in host immunity by initiating adaptive T cell responses and by serving as innate immune sensors. Although both innate and adaptive functions of cDCs are well documented, their relative importance in maintaining immune homeostasis is poorly understood. To examine the significance of cDC-initiated adaptive immunity in maintaining homeostasis, independent of their innate activities, we generated a cDC-specific Cre mouse and crossed it to a floxed MHC class II (MHCII) mouse. Absence of MHCII on cDCs resulted in chronic intestinal inflammation that was alleviated by antibiotic treatment and entirely averted under germ-free conditions. Uncoupling innate and adaptive functions of cDCs revealed that innate immune functions of cDCs are insufficient to maintain homeostasis and antigen presentation by cDCs is essential for a mutualistic relationship between the host and intestinal bacteria.

Role of retinoic acid in the stability of the T-helper-type 1 lineage and implications for autoimmunity.

BACKGROUND: CD4 T cells with features of both T-helper-type 1 (Th1) and 17 (Th17) cells have been implicated in several autoimmune diseases suggesting that plasticity among CD4 T-cell lineages is potentially pathogenic. However, the factors that regulate T-cell lineage stability are largely unknown. Retinoic acid (RA) is synthesised at sites of inflammation. We hypothesised that retinoic acid, a profound epigenetic modifier, could regulate T-cell lineage stability. METHODS: We used a mouse model in which retinoic acid signalling is specifically ablated within the T-cell compartment through overexpression of a dominant negative retinoic acid receptor α (RARα) (dnRARα mice) to investigate its role in the regulation of Th1 lineage stability. Genome-wide ChIP-seq analysis was done to identify RARα targets. In parallel, we performed global mapping of regulatory regions, termed enhancers, to gain mechanistic insight into retinoic acid regulation of T-cell fate. The in-vivo relevance of our findings was determined in a model of oral antigen-induced intestinal inflammation. FINDINGS: We found that retinoic acid is crucial for maintenance of the Th1 lineage. Abrogation of retinoic acid signalling in Th1 cells resulted in loss of T-bet expression and STAT4 activity. Th1 cells from dnRARα mice showed enhanced plasticity with the emergence of hybrid Th1-Th17 and Th17 effector cells. Global analysis of RARα binding and enhancer mapping revealed that RA-RARα directly regulated enhancer activity at Th1 lineage defining genes while repressing genes that regulate Th17 cell fate. Retinoic acid inhibition of Th1 plasticity was essential for maintaining appropriate Th cell responses in vivo and preventing autoimmune intestinal inflammation. INTERPRETATION: Our study has identified RA-RARα as a key component of the regulatory network governing maintenance and plasticity of Th1 cells and defines a new pathway for the development of pathogenic Th17 cells. Retinoids might be novel therapeutic agents for Th17-associated autoimmune diseases. FUNDING: Wellcome Trust.

Detection of FGF15 in plasma by stable isotope standards and capture by anti-peptide antibodies and targeted mass spectrometry.

Fibroblast growth factor 15 (FGF15) has been proposed as a postprandial hormone that signals from intestine to liver to regulate bile acid and carbohydrate homeostasis. However, detecting FGF15 in blood using conventional techniques has proven difficult. Here, we describe a stable isotope standards and capture by anti-peptide antibodies (SISCAPA) assay that combines immuno-enrichment with selected reaction monitoring (SRM) mass spectrometry to overcome this issue. Using this assay, we show that FGF15 circulates in plasma in an FXR and circadian rhythm-dependent manner at concentrations that activate its receptor. Consistent with the proposed endocrine role for FGF15 in liver, mice lacking hepatocyte expression of the obligate FGF15 co-receptor, ß-Klotho, have increased bile acid synthesis and reduced glycogen storage despite having supraphysiological plasma FGF15 concentrations. Collectively, these data demonstrate that FGF15 functions as a hormone and highlight the utility of SISCAPA-SRM as a sensitive assay for detecting low-abundance proteins in plasma.

Retinoic acid is essential for Th1 cell lineage stability and prevents transition to a Th17 cell program.

CD4(+) T cells differentiate into phenotypically distinct T helper cells upon antigenic stimulation. Regulation of plasticity between these CD4(+) T-cell lineages is critical for immune homeostasis and prevention of autoimmune disease. However, the factors that regulate lineage stability are largely unknown. Here we investigate a role for retinoic acid (RA) in the regulation of lineage stability using T helper 1 (Th1) cells, traditionally considered the most phenotypically stable Th subset. We found that RA, through its receptor RARα, sustains stable expression of Th1 lineage specifying genes, as well as repressing genes that instruct Th17-cell fate. RA signaling is essential for limiting Th1-cell conversion into Th17 effectors and for preventing pathogenic Th17 responses in vivo. Our study identifies RA-RARα as a key component of the regulatory network governing maintenance and plasticity of Th1-cell fate and defines an additional pathway for the development of Th17 cells.

Serum amyloid A proteins take retinol for a ride.

Vitamin A plays pleiotropic roles in the immune system. A recent eLife paper by Hooper and colleagues shows that hepatic and intestinal serum amyloid A proteins, which are induced in response to infection, can transport vitamin A metabolites to tissues and thus impact immune responses both locally and systemically.

Systematic proteomic analysis identifies ß-site amyloid precursor protein cleaving enzyme 2 and 1 (BACE2 and BACE1) substrates in pancreatic ß-cells.

Expansion of functional islet ß-cell mass is a physiological process to compensate for increased insulin demand. Deficiency or pharmacological inhibition of the plasma membrane protease BACE2 enhances pancreatic ß-cell function and proliferation, and therefore BACE2 is a putative target for the therapeutic intervention under conditions of ß-cell loss and dysfunction. To gain a molecular understanding of BACE2 function, we performed a systematic and quantitative proteomic analysis to map the natural substrate repertoire of BACE2 and its homologue BACE1 in ß-cells. Loss- and gain-of-function studies of in vitro and in vivo models identified specific and functionally heterogeneous targets. Our analysis revealed non-redundant roles of BACE1/2 in ectodomain shedding with BACE1 regulating a broader and BACE2 a more distinct set of ß-cell-enriched substrates including two proteins of the seizure 6 protein family (SEZ6L and SEZ6L2). Lastly, our study provides insights into the global ß-cell sheddome and secretome, an important prerequisite to uncover novel mechanisms contributing to ß-cell homeostasis and a resource for therapeutic target and biomarker discoveries.

Tmem27 dimerization, deglycosylation, plasma membrane depletion, and the extracellular Phe-Phe motif are negative regulators of cleavage by Bace2.

The pancreatic ß-cell surface protein Tmem27 is promotes the preservation of functional ß-cell mass. It is a selective substrate of the protease Bace2, yet the intramolecular features of Tmem27 that regulate its processing by this sheddase have not been characterized. In particular, the importance of homodimerization, glycosylation, trafficking to the plasma membrane (PM), the existence of multiple cleavage sites, and the amino acid residues that govern these features are currently unknown. Using Tmem27 mutational analysis and multiple biochemical approaches, we here show that Tmem27 dimerization is a dynamic process mediated by its intracellular cysteine residue and that prevents Tmem27 cleavage, that extracellular asparagine glycosylation is essential for Tmem27 trafficking to the PM and its processing by Bace2, that the amount of Tmem27 at the PM is proportional to its total cell levels upon glucose stimulation and Bace2 inhibition, and that the double phenylalanine motif in the Tmem27 cleavage site is an intramolecular Bace2 inhibitor. These findings define structural properties of Tmem27 that affect the susceptibility to its protease Bace2 and have implications for the efficiency with which Tmem27 and other Bace2 substrates are cleaved in normal and disease states.

Bace2 is a ß cell-enriched protease that regulates pancreatic ß cell function and mass.

Decreased ß cell mass and function are hallmarks of type 2 diabetes. Here we identified, through a siRNA screen, beta site amyloid precursor protein cleaving enzyme 2 (Bace2) as the sheddase of the proproliferative plasma membrane protein Tmem27 in murine and human ß cells. Mice with functionally inactive Bace2 and insulin-resistant mice treated with a newly identified Bace2 inhibitor both display augmented ß cell mass and improved control of glucose homeostasis due to increased insulin levels. These results implicate Bace2 in the control of ß cell maintenance and provide a rational strategy to inhibit this protease for the expansion of functional pancreatic ß cell mass.