The Short Chain Fatty Acid Butyrate Imprints an Antimicrobial Program in Macrophages.

Host microbial cross-talk is essential to maintain intestinal homeostasis. However, maladaptation of this response through microbial dysbiosis or defective host defense toward invasive intestinal bacteria can result in chronic inflammation. We have shown that macrophages differentiated in the presence of the bacterial metabolite butyrate display enhanced antimicrobial activity. Butyrate-induced antimicrobial activity was associated with a shift in macrophage metabolism, a reduction in mTOR kinase activity, increased LC3-associated host defense and anti-microbial peptide production in the absence of an increased inflammatory cytokine response. Butyrate drove this monocyte to macrophage differentiation program through histone deacetylase 3 (HDAC3) inhibition. Administration of butyrate induced antimicrobial activity in intestinal macrophages in vivo and increased resistance to enteropathogens. Our data suggest that (1) increased intestinal butyrate might represent a strategy to bolster host defense without tissue damaging inflammation and (2) that pharmacological HDAC3 inhibition might drive selective macrophage functions toward antimicrobial host defense.

Pharmacologically induced weight loss is associated with distinct gut microbiome changes in obese rats.

BACKGROUND: Obesity, metabolic disease and some psychiatric conditions are associated with changes to relative abundance of bacterial species and specific genes in the faecal microbiome. Little is known about the impact of pharmacologically induced weight loss on distinct microbiome species and their respective gene programs in obese individuals. METHODOLOGY: Using shotgun metagenomics, the composition of the microbiome was obtained for two cohorts of obese female Wistar rats (n = 10-12, total of 82) maintained on a high fat diet before and after a 42-day treatment with a panel of four investigatory or approved anti-obesity drugs (tacrolimus/FK506, bupropion, naltrexone and sibutramine), alone or in combination. RESULTS: Only sibutramine treatment induced consistent weight loss and improved glycaemic control in the obese rats. Weight loss was associated with reduced food intake and changes to the faecal microbiome in multiple microbial taxa, genes, and pathways. These include increased ß-diversity, increased relative abundance of multiple Bacteroides species, increased Bacteroides/Firmicutes ratio and changes to abundance of genes and species associated with obesity-induced inflammation, particularly those encoding components of the flagellum and its assembly. CONCLUSIONS: Sibutramine-induced weight loss in obese rats is associated with improved metabolic health, and changes to the faecal microbiome consistent with a reduction in obesity-induced bacterially-driven inflammation.

Sequencing depth and coverage: key considerations in genomic analyses.

Sequencing technologies have placed a wide range of genomic analyses within the capabilities of many laboratories. However, sequencing costs often set limits to the amount of sequences that can be generated and, consequently, the biological outcomes that can be achieved from an experimental design. In this Review, we discuss the issue of sequencing depth in the design of next-generation sequencing experiments. We review current guidelines and precedents on the issue of coverage, as well as their underlying considerations, for four major study designs, which include de novo genome sequencing, genome resequencing, transcriptome sequencing and genomic location analyses (for example, chromatin immunoprecipitation followed by sequencing (ChIP-seq) and chromosome conformation capture (3C)).

Tuning the Transcriptional Response to Hypoxia by Inhibiting Hypoxia-inducible Factor (HIF) Prolyl and Asparaginyl Hydroxylases.

The hypoxia-inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/ß-heterodimeric transcription factor that regulates the expression of hundreds of genes in a tissue context-dependent manner. The major hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1-3) and an asparaginyl hydroxylase (factor-inhibiting HIF (FIH)). PHD catalysis regulates HIFα levels, and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of specific HIF target gene transcription is unknown. We report studies using small molecule HIF hydroxylase inhibitors that investigate the extent to which HIF target gene expression is induced by PHD or FIH inhibition. The results reveal substantial differences in the role of prolyl and asparaginyl hydroxylation in regulating hypoxia-responsive genes in cells. PHD inhibitors with different structural scaffolds behave similarly. Under the tested conditions, a broad-spectrum 2-oxoglutarate dioxygenase inhibitor is a better mimic of the overall transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in the transcriptional induction of a subset of genes not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.

CGAT: computational genomics analysis toolkit.

Computational genomics seeks to draw biological inferences from genomic datasets, often by integrating and contextualizing next-generation sequencing data. CGAT provides an extensive suite of tools designed to assist in the analysis of genome scale data from a range of standard file formats. The toolkit enables filtering, comparison, conversion, summarization and annotation of genomic intervals, gene sets and sequences. The tools can both be run from the Unix command line and installed into visual workflow builders, such as Galaxy.

Predicting long non-coding RNAs using RNA sequencing.

The advent of next-generation sequencing, and in particular RNA-sequencing (RNA-seq), technologies has expanded our knowledge of the transcriptional capacity of human and other animal, genomes. In particular, recent RNA-seq studies have revealed that transcription is widespread across the mammalian genome, resulting in a large increase in the number of putative transcripts from both within, and intervening between, known protein-coding genes. Long transcripts that appear to lack protein-coding potential (long non-coding RNAs, lncRNAs) have been the focus of much recent research, in part owing to observations of their cell-type and developmental time-point restricted expression patterns. A variety of sequencing protocols are currently available for identifying lncRNAs including RNA polymerase II occupancy, chromatin state maps and - the focus of this review - deep RNA sequencing. In addition, there are numerous analytical methods available for mapping reads and assembling transcript models that predict the presence and structure of lncRNAs from RNA-seq data. Here we review current methods for identifying lncRNAs using large-scale sequencing data from RNA-seq experiments and highlight analytical considerations that are required when undertaking such projects.

Alterations in the gut microbiome implicate key taxa and metabolic pathways across inflammatory arthritis phenotypes.

Musculoskeletal diseases affect up to 20% of adults worldwide. The gut microbiome has been implicated in inflammatory conditions, but large-scale metagenomic evaluations have not yet traced the routes by which immunity in the gut affects inflammatory arthritis. To characterize the community structure and associated functional processes driving gut microbial involvement in arthritis, the Inflammatory Arthritis Microbiome Consortium investigated 440 stool shotgun metagenomes comprising 221 adults diagnosed with rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis and 219 healthy controls and individuals with joint pain without an underlying inflammatory cause. Diagnosis explained about 2% of gut taxonomic variability, which is comparable in magnitude to inflammatory bowel disease. We identified several candidate microbes with differential carriage patterns in patients with elevated blood markers for inflammation. Our results confirm and extend previous findings of increased carriage of typically oral and inflammatory taxa and decreased abundance and prevalence of typical gut clades, indicating that distal inflammatory conditions, as well as local conditions, correspond to alterations to the gut microbial composition. We identified several differentially encoded pathways in the gut microbiome of patients with inflammatory arthritis, including changes in vitamin B salvage and biosynthesis and enrichment of iron sequestration. Although several of these changes characteristic of inflammation could have causal roles, we hypothesize that they are mainly positive feedback responses to changes in host physiology and immune homeostasis. By connecting taxonomic alternations to functional alterations, this work expands our understanding of the shifts in the gut ecosystem that occur in response to systemic inflammation during arthritis.

Genetic and environmental factors shape the host response to Helicobacter hepaticus: insights into IBD pathogenesis.

Pathobionts are members of the gut microbiota with the capacity to cause disease when there is malfunctioning intestinal homeostasis. These organisms are thought to be major contributors to the pathogenesis of inflammatory bowel disease (IBD), a group of chronic inflammatory disorders driven by dysregulated responses towards the microbiota. Over two decades have passed since the discovery of Helicobacter hepaticus, a mouse pathobiont which causes colitis in the context of immune deficiency. During this time, we have developed a detailed understanding of the cellular players and cytokine networks which drive H. hepaticus immunopathology. However, we are just beginning to understand the microbial factors that enable H. hepaticus to interact with the host and influence colonic health and disease. Here we review key H. hepaticus-host interactions, their relevance to other exemplar pathobionts and how when maladapted they drive colitis. Further understanding of these pathways may offer new therapeutic approaches for IBD.

Tissue-dependent transcriptional and bacterial associations in primary sclerosing cholangitis-associated inflammatory bowel disease.

Background: Primary sclerosing cholangitis (PSC) is a disease of the bile duct and liver. However, patients frequently have co-morbidities including inflammatory bowel disease (IBD) and colorectal cancer. Colorectal cancer risk in patients with PSC-associated ulcerative colitis (PSC/UC) is elevated relative to patients with ulcerative colitis (UC) alone, reasons for which remain obscure. Further, clinical and immunological features, and involved intestinal sites differ between PSC/UC and UC. Understanding the molecular and microbial basis for differences in cancer risk between these two patient groups and how these differ across intestinal sites is important for the development of therapies to prevent colorectal cancer development in at-risk individuals.   Methods: We employed ribonucleic acid sequencing (RNA-seq) analysis of biopsy samples across three intestinal tissue locations (ileum, caecum and rectum) in patients with PSC/UC (n = 8), UC (n = 10) and healthy controls (n = 12) to determine tissue-dependent transcriptional alterations in PSC/UC. We also performed 16S ribosomal RNA (rRNA) amplicon sequencing to determine bacterial associations with PSC/UC and host-microbiome associations. Results: Tissue-defining transcriptional signatures revealed that the ileum was enriched for genes involved in lipid and drug metabolism, the caecum for activated immune cells and the rectum for enteric neurogenesis. Transcriptional alterations relative to healthy control samples were largely shared between patients with PSC/UC or UC although were distinct across tissue locations. Nevertheless, we observed reduced expression of gamma-glutamyl transferase 1 ( GGT1) specifically in the ileum and caecum of patients with PSC/UC. Analysis of the bacterial component of the microbiome revealed high inter-individual variability of microbiome composition and little evidence for tissue-dependency. We observed a reduction in Parabacteroides relative abundance in the rectum of patients with PSC/UC. Conclusions: The role of gamma-glutamyl transferase in maintaining the redox environment through the glutathione salvage pathway makes our observed alterations a potential pathway to PSC-associated colorectal cancer.

Accurate identification and quantification of commensal microbiota bound by host immunoglobulins.

BACKGROUND: Identifying which taxa are targeted by immunoglobulins can uncover important host-microbe interactions. Immunoglobulin binding of commensal taxa can be assayed by sorting bound bacteria from samples and using amplicon sequencing to determine their taxonomy, a technique most widely applied to study Immunoglobulin A (IgA-Seq). Previous experiments have scored taxon binding in IgA-Seq datasets by comparing abundances in the IgA bound and unbound sorted fractions. However, as these are relative abundances, such scores are influenced by the levels of the other taxa present and represent an abstract combination of these effects. Diversity in the practical approaches of prior studies also warrants benchmarking of the individual stages involved. Here, we provide a detailed description of the design strategy for an optimised IgA-Seq protocol. Combined with a novel scoring method for IgA-Seq datasets that accounts for the aforementioned effects, this platform enables accurate identification and quantification of commensal gut microbiota targeted by host immunoglobulins. RESULTS: Using germ-free and Rag1-/- mice as negative controls, and a strain-specific IgA antibody as a positive control, we determine optimal reagents and fluorescence-activated cell sorting (FACS) parameters for IgA-Seq. Using simulated IgA-Seq data, we show that existing IgA-Seq scoring methods are influenced by pre-sort relative abundances. This has consequences for the interpretation of case-control studies where there are inherent differences in microbiota composition between groups. We show that these effects can be addressed using a novel scoring approach based on posterior probabilities. Finally, we demonstrate the utility of both the IgA-Seq protocol and probability-based scores by examining both novel and published data from in vivo disease models. CONCLUSIONS: We provide a detailed IgA-Seq protocol to accurately isolate IgA-bound taxa from intestinal samples. Using simulated and experimental data, we demonstrate novel probability-based scores that adjust for the compositional nature of relative abundance data to accurately quantify taxon-level IgA binding. All scoring approaches are made available in the IgAScores R package. These methods should improve the generation and interpretation of IgA-Seq datasets and could be applied to study other immunoglobulins and sample types. Video abstract.

Genetic influences on attention deficit hyperactivity disorder symptoms from age 2 to 3: a quantitative and molecular genetic investigation.

BACKGROUND: A twin study design was used to assess the degree to which additive genetic variance influences ADHD symptom scores across two ages during infancy. A further objective in the study was to observe whether genetic association with a number of candidate markers reflects results from the quantitative genetic analysis. METHOD: We have studied 312 twin pairs at two time-points, age 2 and age 3. A composite measure of ADHD symptoms from two parent-rating scales: The Child Behavior Checklist/1.5 - 5 years (CBCL) hyperactivity scale and the Revised Rutter Parent Scale for Preschool Children (RRPSPC) was used for both quantitative and molecular genetic analyses. RESULTS: At ages 2 and 3 ADHD symptoms are highly heritable (h2 = 0.79 and 0.78, respectively) with a high level of genetic stability across these ages. However, we also observe a significant level of genetic change from age 2 to age 3. There are modest influences of non-shared environment at each age independently (e2 = 0.22 and 0.21, respectively), with these influences being largely age-specific. In addition, we find modest association signals in DAT1 and NET1 at both ages, along with suggestive specific effects of 5-HTT and DRD4 at age 3. CONCLUSIONS: ADHD symptoms are heritable at ages 2 and 3. Additive genetic variance is largely shared across these ages, although there are significant new effects emerging at age 3. Results from our genetic association analysis reflect these levels of stability and change and, more generally, suggest a requirement for consideration of age-specific genotypic effects in future molecular studies.

Foxp3+ T reg cells control psoriasiform inflammation by restraining an IFN-I-driven CD8+ T cell response.

Psoriasis is a complex inflammatory skin disease affecting ∼3% of the population worldwide. Although type I interferons (IFN-I) are thought to be involved in its pathogenesis, the details of this relationship remain elusive. Here we show that in a murine model of imiquimod-driven psoriatic skin inflammation, Foxp3+ regulatory T cells (T reg cells) control inflammation severity by restraining IFN-I. Depletion of T reg cells induces IFN-I and IFN-stimulated gene expression, and leads to accumulation of CD8+ T cells in lesional skin. Mononuclear phagocytes (MNPs) were the source of IFN-I, and their depletion reversed the effect of T reg cell depletion. Blockade of IFN-I signaling abolished CD8+ T cell infiltration and excess inflammation in the skin of T reg cell-depleted mice. Depletion of CD8+ T cells attenuated pathology, confirming their role as critical effector cells downstream of IFN-I. Our results describe an unexpected role for T reg cells in restraint of an MNP-IFN-I-driven CD8+ T cell response during psoriasiform skin inflammation. These findings highlight a pathway with potential relevance for the treatment of early-stage disease.

Alpha kinase 1 controls intestinal inflammation by suppressing the IL-12/Th1 axis.

Inflammatory bowel disease (IBD) are heterogenous disorders of the gastrointestinal tract caused by a spectrum of genetic and environmental factors. In mice, overlapping regions of chromosome 3 have been associated with susceptibility to IBD-like pathology, including a locus called Hiccs. However, the specific gene that controls disease susceptibility remains unknown. Here we identify a Hiccs locus gene, Alpk1 (encoding alpha kinase 1), as a potent regulator of intestinal inflammation. In response to infection with the commensal pathobiont Helicobacter hepaticus (Hh), Alpk1-deficient mice display exacerbated interleukin (IL)-12/IL-23 dependent colitis characterized by an enhanced Th1/interferon(IFN)-γ response. Alpk1 controls intestinal immunity via the hematopoietic system and is highly expressed by mononuclear phagocytes. In response to Hh, Alpk1-/- macrophages produce abnormally high amounts of IL-12, but not IL-23. This study demonstrates that Alpk1 promotes intestinal homoeostasis by regulating the balance of type 1/type 17 immunity following microbial challenge.

Long-term effects of gestational nicotine exposure and food-restriction on gene expression in the striatum of adolescent rats.

Gestational exposure to environmental toxins such as nicotine may result in detectable gene expression changes in later life. To investigate the direct toxic effects of prenatal nicotine exposure on later brain development, we have used transcriptomic analysis of striatal samples to identify gene expression differences between adolescent Lister Hooded rats exposed to nicotine in utero and controls. Using an additional group of animals matched for the reduced food intake experienced in the nicotine group, we were also able to assess the impact of imposed food-restriction on gene expression profiles. We found little evidence for a role of gestational nicotine exposure on altered gene expression in the striatum of adolescent offspring at a significance level of p<0.01 and |log2 fold change >0.5|, although we cannot exclude the possibility of nicotine-induced changes in other brain regions, or at other time points. We did, however, find marked gene expression differences in response to imposed food-restriction. Food-restriction resulted in significant group differences for a number of immediate early genes (IEGs) including Fos, Fosb, Fosl2, Arc, Junb, Nr4a1 and Nr4a3. These genes are associated with stress response pathways and therefore may reflect long-term effects of nutritional deprivation on the development of the stress system.