Review article: the human intestinal virome in health and disease

Background: The human virome consists of animal-cell viruses causing transient infections, bacteriophage (phage) predators of bacteria and archaea, endogenous retroviruses and viruses causing persistent and latent infections. High-throughput, inexpensive, sensitive sequencing methods and metagenomics now make it possible to study the contribution dsDNA, ssDNA and RNA virus-like particles make to the human virome, and in particular the intestinal virome. Aim: To review and evaluate the pioneering studies that have attempted to characterise the human virome and generated an increased interest in understanding how the intestinal virome might contribute to maintaining health, and the pathogenesis of chronic diseases. Methods: Relevant virome-related articles were selected for review following extensive language- and date-unrestricted, electronic searches of the literature. Results: The human intestinal virome is personalised and stable, and dominated by phages. It develops soon after birth in parallel with prokaryotic communities of the microbiota, becoming established during the first few years of life. By infecting specific populations of bacteria, phages can alter microbiota structure by killing host cells or altering their phenotype, enabling phages to contribute to maintaining intestinal homeostasis or microbial imbalance (dysbiosis), and the development of chronic infectious and autoimmune diseases including HIV infection and Crohn's disease, respectively. Conclusions: Our understanding of the intestinal virome is fragmented and requires standardised methods for virus isolation and sequencing to provide a more complete picture of the virome, which is key to explaining the basis of virome-disease associations, and how enteric viruses can contribute to disease aetiologies and be rationalised as targets for interventions

Dysbiosis of the gut microbiota in disease

There is growing evidence that dysbiosis of the gut microbiota is associated with the pathogenesis of both intestinal and extra-intestinal disorders. Intestinal disorders include inflammatory bowel disease, irritable bowel syndrome (IBS), and coeliac disease, while extra-intestinal disorders include allergy, asthma, metabolic syndrome, cardiovascular disease, and obesity. In many of these conditions, the mechanisms leading to disease development involve the pivotal mutualistic relationship between the colonic microbiota, their metabolic products, and the host immune system. The establishment of a ‘healthy’ relationship early in life appears to be critical to maintaining intestinal homeostasis. Whilst we do not yet have a clear understanding of what constitutes a ‘healthy’ colonic microbiota, a picture is emerging from many recent studies identifying particular bacterial species associated with a healthy microbiota. In particular, the bacterial species residing within the mucus layer of the colon, either through direct contact with host cells, or through indirect communication via bacterial metabolites, may influence whether host cellular homeostasis is maintained or whether inflammatory mechanisms are triggered. In addition to inflammation, there is some evidence that perturbations in the gut microbiota is involved with the development of colorectal cancer. In this case, dysbiosis may not be the most important factor, rather the products of interaction between diet and the microbiome. High-protein diets are thought to result in the production of carcinogenic metabolites from the colonic microbiota that may result in the induction of neoplasia in the colonic epithelium. Ever more sensitive metabolomics methodologies reveal a suite of small molecules produced in the microbiome which mimic or act as neurosignallers or neurotransmitters. Coupled with evidence that probiotic interventions may alter psychological endpoints in both humans and in rodent models, these data suggest that CNS-related co-morbidities frequently associated with GI disease may originate in the intestine as a result of microbial dysbiosis. This review outlines the current evidence showing the extent to which the gut microbiota contributes to the development of disease. Based on evidence to date, we can assess the potential to positively modulate the composition of the colonic microbiota and ameliorate disease activity through bacterial intervention

Diet, the intestinal microbiota, and immune health in aging

Many countries are facing aging populations, with those over 65 years of age likely to represent the largest population over the next 10–20 years. Living longer often comes with poor health and, in particular, a decline in the immune function characterized by poor vaccine responses and increased risk of infection and certain cancers. Aging and diet represent major intrinsic and extrinsic factors that influence the makeup and activity of resident intestinal microbes, the microbiota, the efficient functioning of which is essential for sustaining overall health and the effectiveness of the immune system. The provision of elderly specific dietary recommendations appears to be lacking but is necessary since this population has an altered microbiota and immune response and may not respond in the same way as their healthy and younger counterparts. We have reviewed the evidence supporting the role of diet and, in particular, dietary carbohydrate, protein, and fat in influencing the microbiota and its generation of key metabolites that influence the efficient functioning of immune cells during aging, and how dietary intervention might be of benefit in improving the intestinal health and immune status in the elderly

Use of bioengineered human commensal gut bacteria-derived microvesicles for mucosal plague vaccine delivery and immunization

Plague caused by the Gram-negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non-human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V-OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV-based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo-tolerance; needle-free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell-mediated immune responses; and targeting of primary sites of plague infection

Evaluation of an intensive voice treatment to reduce anterior drooling in children with cerebral palsy: Protocol for a concurrent multiple-baseline, single case experimental design study

Anterior drooling is common in children with cerebral palsy (CP) and poses significant risks to the child's health. Causes of drooling include oro-motor dysfunction, inefficient swallowing and reduced sensation in the orofacial musculature. Behavioural interventions are frequently recommended to reduce drooling; however, this is in the absence of high-quality research evidence. This paper describes a protocol for evaluating the effectiveness of the Lee Silverman Voice Treatment LOUD (LSVT LOUD®) in reducing drooling; and optimising speech and swallowing in a group of children with CP. A structured and systematic visual analysis supplemented with statistical analyses will be used to analyse the data. The risk of bias in n-of-1 trials (RoBiNT) Scale [1] guided the design and implementation of the study

Propionate has protective and anti-inflammatory effects on the blood–brain barrier

Production of short-chain fatty acids (SCFAs) from dietary substrates by the gut microbiota is associated with health, with these metabolites influencing the host via the ‘gut–brain axis’. Micromolar quantities of microbially derived SCFAs are taken up from the gut and reach systemic circulation, where they can influence host gene expression through a variety of largely unknown mechanisms. The blood–brain barrier (BBB) is the major interface between the circulation and central nervous system, and is critically involved in the pathogenesis of neuroinflammatory disorders such as stroke and vascular dementia. We hypothesized exposure of the BBB to SCFAs influences barrier integrity and function. To test our hypothesis, we investigated the in vitro effects of a physiologically relevant concentration (1 μM) of propionate upon the human immortalised cerebromicrovascular endothelial cell line hCMEC/D3. Propionate is produced by the microbiota from dietary glucans, and is biologically active via the G protein coupled receptors FFAR2 and FFAR3. It is a highly potent FFAR2 agonist (agonist activity 3.99) and has close to optimal ligand efficiency (-ΔG=1.19 kcal mol-1 atom-1) for this receptor. Notably, FFAR3 is expressed on the vascular endothelium and a likely target for propionate in the BBB. After confirming the presence of FFAR3 on hCMEC/D3 cells, we undertook an unbiased transcriptomic analysis of confluent hCMEC/D3 monolayers treated or not for 24 h with 1 μM propionate, supported by in vitro validation of key findings and assessment of functional endothelial permeability barrier properties. Propionate treatment had a significant (PFDR < 0.1) effect on the expression of 1136 genes: 553 upregulated, 583 downregulated. Propionate inhibited several inflammation-associated pathways: namely, TLR-specific signalling, NFkappaB signalling, and cytosolic DNA-sensing. Functional validation of these findings confirmed the down-regulation of TLR signalling by propionate, achieved primarily through down-regulation of endothelial CD14 expression. Accordingly, propionate prevented LPS-induced increases in paracellular permeability to 70 kDa FITC-dextran and loss of transendothelial electrical resistance. Enrichr analysis indicated the activation by propionate of the NFE2L2 (NRF2)-driven protective response against oxidative stress. Confirming these data, propionate limited free reactive oxygen species induction by the mitochondrial respiratory inhibitor rotenone. Together, these data strongly suggest the SCFA propionate contributes to maintaining BBB integrity and protecting against inflammatory challenge by downregulating BBB responsiveness. In addition to its well-described effects on cholesterol metabolism, maintenance of propionate levels in the circulation may be an additional mechanism whereby a glucan-containing diet protects against neurovascular disease

Apoptosis of Fashigh CD4+ synovial T cells by borrelia-reactive Fas-ligand(high) gamma delta T cells in Lyme arthritis

The function of the minor subset of T lymphocytes bearing the gamma delta T cell antigen receptor is uncertain. Although some gamma delta T cells react to microbial products, responsiveness has only rarely been demonstrated toward a bacterial antigen from a naturally occurring human infection. Synovial fluid lymphocytes from patients with Lyme arthritis contain a large proportion of gamma delta cells that proliferate in response to the causative spirochete, Borrelia burgdorferi. Furthermore, synovial gamma delta T cell clones express elevated and sustained levels of the ligand for Fas (APO-1, CD95) compared to alpha beta T cells, and induce apoptosis of Fashigh CD4+ synovial lymphocytes. The findings suggest that gamma delta T cells contribute to defense in human infections, as well as manifest an immunoregulatory function at inflammatory sites by a Fas-dependent process

Extracellular vesicles produced by the human commensal gut bacterium Bacteroides thetaiotaomicron affect host immune pathways in a cell-type specific manner that are altered in inflammatory bowel disease

The gastrointestinal (GI) tract harbours a complex microbial community, which contributes to its homeostasis. A disrupted microbiome can cause GI-related diseases, including inflammatory bowel disease (IBD), therefore identifying host-microbe interactions is crucial for better understanding gut health. Bacterial extracellular vesicles (BEVs), released into the gut lumen, can cross the mucus layer and access underlying immune cells. To study BEV-host interactions, we examined the influence of BEVs generated by the gut commensal bacterium, Bacteroides thetaiotaomicron, on host immune cells. Single-cell RNA sequencing data and host-microbe protein-protein interaction networks were used to predict the effect of BEVs on dendritic cells, macrophages and monocytes focusing on the Toll-like receptor (TLR) pathway. We identified biological processes affected in each immune cell type and cell-type specific processes including myeloid cell differentiation. TLR pathway analysis highlighted that BEV targets differ among cells and between the same cells in healthy versus disease (ulcerative colitis) conditions. The in silico findings were validated in BEV-monocyte co-cultures demonstrating the requirement for TLR4 and Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) in BEV-elicited NF-kB activation. This study demonstrates that both cell-type and health status influence BEV-host communication. The results and the pipeline could facilitate BEV-based therapies for the treatment of IBD

Regulation of blood-brain barrier integrity by microbiome-associated methylamines and cognition by trimethylamine N-oxide.

BACKGROUND: Communication between the gut microbiota and the brain is primarily mediated via soluble microbe-derived metabolites, but the details of this pathway remain poorly defined. Methylamines produced by microbial metabolism of dietary choline and L-carnitine have received attention due to their proposed association with vascular disease, but their effects upon the cerebrovascular circulation have hitherto not been studied. RESULTS: Here, we use an integrated in vitro/in vivo approach to show that physiologically relevant concentrations of the dietary methylamine trimethylamine N-oxide (TMAO) enhanced blood-brain barrier (BBB) integrity and protected it from inflammatory insult, acting through the tight junction regulator annexin A1. In contrast, the TMAO precursor trimethylamine (TMA) impaired BBB function and disrupted tight junction integrity. Moreover, we show that long-term exposure to TMAO protects murine cognitive function from inflammatory challenge, acting to limit astrocyte and microglial reactivity in a brain region-specific manner. CONCLUSION: Our findings demonstrate the mechanisms through which microbiome-associated methylamines directly interact with the mammalian BBB, with consequences for cerebrovascular and cognitive function. Video abstract