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We previously showed that exposure to a high-sugar and moderate-fat diet (i.e., Western diet, WD) in mice induces appreciable skin inflammation and enhances the susceptibility to imiquimod-induced psoriasiform dermatitis (PsD), suggesting that dietary components may render the skin susceptible to psoriatic inflammation. Herein, utilizing an IL-23 minicircle (MC)-based model with features of both PsD and psoriatic arthritis (PsA), we showed that intake of WD for 10 weeks predisposed mice not only to skin but also joint inflammation. Both WD-induced skin and joint injuries were associated with an expansion of IL-17A-producing γδ T cells and increased expression of Th17 cytokines. After IL-23 MC delivery, WD-fed mice had reduced microbial diversity and pronounced dysbiosis. Treatment with broad-spectrum antibiotics suppressed IL-23-mediated skin and joint inflammation in WD-fed mice. Strikingly, reduced skin and joint inflammation with a partial reversion of the gut microbiota were noted when mice switched from a WD to a standard diet after IL-23 MC delivery. These findings reveal that short-term WD intake-induced dysbiosis is accompanied by enhanced psoriasis-like skin and joint inflammation. Modifications toward a healthier dietary pattern should be considered in patients with psoriatic skin and/or joint disease.

In the past three decades, extraordinary advances have been made in the understanding of the pathogenesis of, and treatment options for, inflammatory arthritides, including rheumatoid arthritis and spondyloarthritis. The use of methotrexate and subsequently biologic therapies (such as TNF inhibitors, among others) and oral small molecules have substantially improved clinical outcomes for many patients with inflammatory arthritis; for others, however, these agents do not substantially improve their symptoms. The emerging field of pharmacomicrobiomics, which investigates the effect of variations within the human gut microbiome on drugs, has already provided important insights into these therapeutics. Pharmacomicrobiomic studies have demonstrated that human gut microorganisms and their enzymatic products can affect the bioavailability, clinical efficacy and toxicity of a wide array of drugs through direct and indirect mechanisms. This discipline promises to facilitate the advent of microbiome-based precision medicine approaches in inflammatory arthritis, including strategies for predicting response to treatment and for modulating the microbiome to improve response to therapy or reduce drug toxicity. Pharmacomicrobiomics studies investigating the effect of variations within the human gut microbiome on drugs have provided insights into therapeutics used for inflammatory arthritis, which could facilitate microbiome-based precision medicine approaches in rheumatology.

OBJECTIVE Although oral methotrexate (MTX) remains the anchor drug for rheumatoid arthritis (RA), up to 50% of patients do not achieve a clinically adequate outcome. In addition, there is a lack of prognostic tools for treatment response prior to drug initiation. This study was undertaken to investigate whether interindividual differences in the human gut microbiome can aid in the prediction of MTX efficacy in new-onset RA. METHODS We performed 16S ribosomal RNA gene and shotgun metagenomic sequencing on the baseline gut microbiomes of drug-naive patients with new-onset RA (n = 26). Results were validated in an additional independent cohort (n = 21). To gain insight into potential microbial mechanisms, we conducted ex vivo experiments coupled with metabolomics analysis to evaluate the association between microbiome-driven MTX depletion and clinical response. RESULTS Our analysis revealed significant associations of the abundance of gut bacterial taxa and their genes with future clinical response (q < 0.05), including orthologs related to purine and MTX metabolism. Machine learning techniques were applied to the metagenomic data, resulting in a microbiome-based model that predicted lack of response to MTX in an independent group of patients. Finally, MTX levels remaining after ex vivo incubation with distal gut samples from pretreatment RA patients significantly correlated with the magnitude of future clinical response, suggesting a possible direct effect of the gut microbiome on MTX metabolism and treatment outcomes. CONCLUSION Taken together, these findings are the first step toward predicting lack of response to oral MTX in patients with new-onset RA and support the value of the gut microbiome as a possible prognostic tool and as a potential target in RA therapeutics.

SUMMARY Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enables the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms allow E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof of concept for the extension to other phage-bacterial pairs of interest., Graphical Abstract, In brief Lam et al. show that filamentous bacteriophage can be harnessed as agents of gene delivery to bacteria colonizing the gastrointestinal tract. Using M13 to deliver CRISPR-Cas9, they demonstrate sequence-specific targeting of GFP-marked E. coli in the gut and show that CRISPR-Cas9 can induce genomic deletions at the target site.

In the past three decades, extraordinary advances have been made in the understanding of the pathogenesis of, and treatment options for, inflammatory arthritides, including rheumatoid arthritis and spondyloarthritis. The use of methotrexate and subsequently biologic therapies (such as TNF inhibitors, among others) and oral small molecules have substantially improved clinical outcomes for many patients with inflammatory arthritis; for others, however, these agents do not substantially improve their symptoms. The emerging field of pharmacomicrobiomics, which investigates the effect of variations within the human gut microbiome on drugs, has already provided important insights into these therapeutics. Pharmacomicrobiomic studies have demonstrated that human gut microorganisms and their enzymatic products can affect the bioavailability, clinical efficacy and toxicity of a wide array of drugs through direct and indirect mechanisms. This discipline promises to facilitate the advent of microbiome-based precision medicine approaches in inflammatory arthritis, including strategies for predicting response to treatment and for modulating the microbiome to improve response to therapy or reduce drug toxicity.

Emerging evidence suggests that the use of non-antibiotic drugs can alter the human gut microbiota with unknown consequences for treatment outcomes. Here, we use a combination of in vitro, in vivo, and ex vivo methods to demonstrate that the first-line therapy for rheumatoid arthritis (RA), methotrexate (MTX), has off-target effects on the human gut microbiota, resulting in decreases in Bacteroidetes, which tend to be more sensitive. Longitudinal analyses of the gut microbiotas of RA patients revealed that MTX-induced shifts in bacterial relative abundance are associated with improved drug response and transplant experiments in gnotobiotic mice show that these shifts lead to reduced inflammation. Specific MTX-modulated taxa are associated with immune activation. Together, these results suggest that the mechanism-of-action of non-antibiotic drugs may be due in part to off-target effects on the gut microbiota, while providing a critical first step towards explaining long-standing differences in drug response between patients.

Bacterial activation of T helper 17 (Th17) cells exacerbates mouse models of autoimmune disease, yet the relevant human-associated bacteria for Th17-driven disease remain elusive. Here, we show the prevalent human gut Actinobacterium Eggerthella lenta induces intestinal Th17 activation by upregulating interleukin (IL)-17 expression through a cell- and antigen-independent mechanism. E. lenta is enriched in inflammatory bowel disease patients and worsens colitis severity in a Rorc-dependent manner. We exploited natural E. lenta strain-level variation to identify a bacterial enzyme (Cardiac glycoside reductase 2, Cgr2) necessary and sufficient to induce IL-17a. The cgr2 gene is enriched in the gut microbiomes of autoimmune subjects and correlates with disease severity. Finally, we leveraged the sensitivity of Cgr2 to dietary arginine to prevent E. lenta-induced intestinal inflammation. Together, these results expand the mechanisms through which bacteria shape mucosal immunity and support a role of human gut bacterial metabolism in driving Th17-dependent autoimmune diseases.

Bacterial activation of T helper 17 (Th17) cells exacerbates mouse models of autoimmunity, but how human-associated bacteria impact Th17-driven disease remains elusive. We show that human gut Actinobacterium Eggerthella lenta induces intestinal Th17 activation by lifting inhibition of the Th17 transcription factor Rorγt through cell- and antigen-independent mechanisms. E. lenta is enriched in inflammatory bowel disease (IBD) patients and worsens colitis in a Rorc-dependent manner in mice. Th17 activation varies across E. lenta strains, which is attributable to the cardiac glycoside reductase 2 (Cgr2) enzyme. Cgr2 is sufficient to induce interleukin (IL)-17a, a major Th17 cytokine. cgr2+ E. lenta deplete putative steroidal glycosides in pure culture; related compounds are negatively associated with human IBD severity. Finally, leveraging the sensitivity of Cgr2 to dietary arginine, we prevented E. lenta-induced intestinal inflammation in mice. Together, these results support a role for human gut bacterial metabolism in driving Th17-dependent autoimmunity.

Aberrant activation of Th17 cells by the gut microbiota contributes to autoimmunity; however, the mechanisms responsible and their diet-dependence remain unclear. Here, we show that the autoimmune disease-associated gut Actinobacterium Eggerthella lenta increases intestinal Th17 cells and worsens colitis in a Rorc-dependent and strain-variable manner. A single genomic locus predicted Th17 accumulation. A gene within this locus, encoding the Cgr2 enzyme, was sufficient to increase Th17 cells. Levels of cgr2 were increased in stool from patients with rheumatoid arthritis compared to healthy controls. Dietary arginine blocked E. lenta-induced Th17 cells and colitis. These results expand the mechanisms through which bacteria shape mucosal immunity and demonstrate the feasibility of dissecting the complex interactions between diet, the gut microbiota, and autoimmune disease.One Sentence SummaryAn autoimmune disease-associated bacterium triggers disease due to a diet-dependent enzyme that regulates mucosal immunity.

Background Early treatment initiation in rheumatoid arthritis (RA) is fundamental to avoid chronic joint destruction and disability. Despite remarkable advances in RA therapeutics, oral methotrexate (MTX) remains the anchor drug and mainstay of treatment worldwide (1,2). However, MTX bioavailability has a wide inter-individual variability and >50% of patients with moderate or severe RA show no or suboptimal improvement in their symptoms in response to MTX (1,3). The reasons for these disparities in treatment response remain unclear. Prior studies have shown that the biotransformation of MTX is altered in germ-free and microbiome-depleted mice (4), prompting us to hypothesize that inter-individual differences in the human gut microbiome could impact drug bioavailability and thus clinical efficacy. Objectives To determine differences in the microbiome of drug-naive, new onset RA (NORA) patients that could predict response to MTX therapy. Methods We performed 16S rRNA gene and shotgun metagenomic sequencing on the baseline gut microbiomes of 27 drug-naive, NORA patients. Results Our analysis revealed significant associations between the abundance of gut bacterial taxa and genes including gene families related with purine and methotrexate metabolism. Machine learning techniques were applied to this metagenomic data, resulting in a robust predictive model based on bacterial gene abundance that accurately predicted response to MTX therapy in an independent group of patients. Finally, MTX available levels remaining after ex vivo incubation with distal gut samples from pre-treatment RA patients significantly correlated with the magnitude of future clinical response, suggesting a direct effect of the gut microbiome on MTX bioavailability and response to therapy. Conclusion Together, these results provide the first step towards predicting response to oral MTX in NORA patients and support the utility of the gut microbiome as a prognostic tool and perhaps even as a target for manipulation in the treatment of rheumatic and autoimmune disease. References [1] Detert, J. et al. Ann Rheum Dis72, 844-850, doi:10.1136/annrheumdis-2012-201612 (2013). [2] Favalli, E. G. et al. Autoimmunity reviews13, 1102-1108, doi:10.1016/j.autrev.2014.08.026 (2014). [3] Emery, P. et al. Lancet372, 375-382, doi:10.1016/S0140-6736(08)61000-4 (2008). [4] Valerino, D. M. et al. Biochem Pharmacol21, 821-831 (1972). Disclosure of Interests Sandrine Isaac: None declared, Alejandro Artacho: None declared, Renuka Nayak: None declared, Alejandra Flor: None declared, Steven Abramson: None declared, Pamela Rosenthal: None declared, Leonor Puchades: None declared, Andrew Patterson: None declared, Antonio Pineda-Lucena: None declared, Peter Turnbaugh Consultant for: P.J.T is on the scientific advisory board for Kaleido, Seres, SNIPRbiome, uBiome, and WholeBiome; there is no direct overlap between the current study and these consulting duties., Carles Ubeda: None declared, Jose Scher Grant/research support from: Pfizer, Novartis, Consultant for: Janssen, UCB, Novartis, Amgen

The trillions of microorganisms (microbiota) found within the human gut play a critical role in shaping the immune system, yet these complex microbial communities are also highly sensitive to numerous environmental factors. While much of the focus to date has been on dietary intake, emerging data has begun to suggest that the use of pharmaceutical drugs, even those that are not considered to be antibiotics, can alter the human gut microbiota with unknown consequences for treatment outcomes. Here, we use a combination ofin vitro, in vivo, andex vivomethods to demonstrate that the first-line therapy for rheumatoid arthritis (RA), methotrexate (MTX), has off-target effects on the human gut microbiota, resulting in a significant growth advantage for drug-resistant Firmicutes over the Bacteroidetes, which tend to be more sensitive. Longitudinal analyses of the gut microbiotas of RA patients revealed that MTX-induced shifts in bacterial relative abundance are associated with improved drug response and transplant experiments in gnotobiotic mice show that these shifts lead to reduced inflammation. Together, these results suggest that the mechanism-of-action of non-antibiotic drugs may be due in part to off-target effects on the gut microbiota, while providing a critical first step towards explaining long-standing differences in drug response between patients.

Immunomodulatory drugs can inhibit bacterial growth, yet their mechanism of action, spectrum, and clinical relevance remain unknown. Methotrexate (MTX), a first-line rheumatoid arthritis (RA) treatment, inhibits mammalian dihydrofolate reductase (DHFR), but whether it directly impacts gut bacteria is unclear. We show that MTX broadly alters the human gut microbiota. Drug sensitivity varied across strains, but the mechanism of action against DHFR appears conserved between mammalian and bacterial cells. RA patient microbiotas were sensitive to MTX, and changes in gut bacterial taxa and gene family abundance were distinct between responders and non-responders. Transplantation of post-treatment samples into germ-free mice given an inflammatory trigger led to reduced immune activation relative to pre-treatment controls, enabling identification of MTX-modulated bacterial taxa associated with intestinal and splenic immune cells. Thus, conservation in cellular pathways across domains of life can result in broad off-target drug effects on the human gut microbiota with consequences for immune function.

Cells respond to variable environments by changing gene expression and gene interactions. To study how human cells response to stress, we analyzed the expression of >5000 genes in cultured B cells from nearly 100 normal individuals following endoplasmic reticulum stress and exposure to ionizing radiation. We identified thousands of genes that are induced or repressed. Then, we constructed coexpression networks and inferred interactions among genes. We used coexpression and machine learning analyses to study how genes interact with each other in response to stress. The results showed that for most genes, their interactions with each other are the same at baseline and in response to different stresses; however, a small set of genes acquired new interacting partners to engage in stress-specific responses. These genes with altered interacting partners are associated with diseases in which endoplasmic reticulum stress response or sensitivity to radiation has been implicated. Thus, our findings showed that to understand disease-specific pathways, it is important to identify not only genes that change expression levels but also those that alter interactions with other genes.

Objective Previous studies have shown that differential DNA methylation is associated with SLE susceptibility. How DNA methylation affects the clinical heterogeneity of SLE has not been fully defined. We conducted this study to identify differentially methylated CpG sites associated with nephritis among women with SLE. Methods The methylation status of 428 229 CpG sites across the genome was characterised for peripheral blood cells from 322 women of European descent with SLE, 80 of whom had lupus nephritis, using the Illumina HumanMethylation450 BeadChip. Multivariable linear regression adjusting for population substructure and leucocyte cell proportions was used to identify differentially methylated sites associated with lupus nephritis. The influence of genetic variation on methylation status was investigated using data from a genome-wide association study of SLE. Pathway analyses were used to identify biological processes associated with lupus nephritis. Results We identified differential methylation of 19 sites in 18 genomic regions that was associated with nephritis among patients with SLE (false discovery rate q

Background Clinicians have known for centuries that there is substantial variability between patients in their response to medications—some individuals exhibit a miraculous recovery while others fail to respond at all. Still others experience dangerous side effects. The hunt for the factors responsible for this variation has been aided by the ability to sequence the human genome, but this just provides part of the picture. Here, we discuss the emerging field of study focused on the human microbiome and how it may help to better predict drug response and improve the treatment of human disease. Discussion Various clinical disciplines characterize drug response using either continuous or categorical descriptors that are then correlated to environmental and genetic risk factors. However, these approaches typically ignore the microbiome, which can directly metabolize drugs into downstream metabolites with altered activity, clearance, and/or toxicity. Variations in the ability of each individual’s microbiome to metabolize drugs may be an underappreciated source of differences in clinical response. Complementary studies in humans and animal models are necessary to elucidate the mechanisms responsible and to test the feasibility of identifying microbiome-based biomarkers of treatment outcomes. Summary We propose that the predictive power of genetic testing could be improved by taking a more comprehensive view of human genetics that encompasses our human and microbial genomes. Furthermore, unlike the human genome, the microbiome is rapidly altered by diet, pharmaceuticals, and other interventions, providing the potential to improve patient care by re-shaping our associated microbial communities.

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) results in the condition called “ER stress,” which induces the unfolded protein response (UPR), a complex cellular process that includes changes in expression of many genes. Failure to restore homeostasis in the ER is associated with human diseases. To identify the underlying changes in gene expression in response to ER stress, we induced ER stress in human B cells and then measured gene expression at ten time points. We followed up those results by studying cells from 60 unrelated people. We rediscovered genes that were known to play a role in the ER-stress response and uncovered several thousand genes that are not known to be involved. Two of these are VLDLR and INHBE, which showed significant increase in expression after ER stress in B cells and in primary fibroblasts. To study the links between UPR and disease susceptibility, we identified ER-stress-responsive genes that are associated with human diseases and assessed individual differences in the ER-stress response. Many of the UPR genes are associated with Mendelian disorders, such as Wolfram syndrome, and complex diseases, including amyotrophic lateral sclerosis and diabetes. Data from two independent samples showed extensive individual variability in ER-stress response. Additional analyses with monozygotic twins revealed significant correlations within twin pairs in their responses to ER stress, thus showing evidence for heritable variation among individuals. These results have implications for basic understanding of ER function and its role in disease susceptibility.

Genes interact in networks to orchestrate cellular processes. Analysis of these networks provides insights into gene interactions and functions. Here, we took advantage of normal variation in human gene expression to infer gene networks, which we constructed using correlations in expression levels of more than 8.5 million gene pairs in immortalized B cells from three independent samples. The resulting networks allowed us to identify biological processes and gene functions. Among the biological pathways, we found processes such as translation and glycolysis that co-occur in the same subnetworks. We predicted the functions of poorly characterized genes, including CHCHD2 and TMEM111, and provided experimental evidence that TMEM111 is part of the endoplasmic reticulum-associated secretory pathway. We also found that IFIH1, a susceptibility gene of type 1 diabetes, interacts with YES1, which plays a role in glucose transport. Furthermore, genes that predispose to the same diseases are clustered nonrandomly in the coexpression network, suggesting that networks can provide candidate genes that influence disease susceptibility. Therefore, our analysis of gene coexpression networks offers information on the role of human genes in normal and disease processes.

Summary Drosophila melanogaster has emerged as an important model system for the study of both stem cell biology and aging. Much is known about how molecular signals from the somatic niche regulate adult stem cells in the germline, and a variety of environmental factors as well as single point mutations have been shown to affect lifespan. Relatively little is known, however, about how aging affects specific populations of cells, particularly adult stem cells that may be susceptible to aging-related damage. Here we show that male germline stem cells (GSCs) are lost from the stem cell niche during aging, but are efficiently replaced to maintain overall stem cell number. We also find that the division rate of GSCs slows significantly during aging, and that this slowing correlates with a reduction in the number of somatic hub cells that contribute to the stem cell niche. Interestingly, slowing of stem cell division rate was not observed in long-lived methuselah mutant flies. We finally investigated whether two mechanisms that are thought to be used in other adult stem cell types to minimize the effects of aging were operative in this system. First, in many adult tissues stem cells exhibit markedly fewer cell cycles relative to transit-amplifying cells, presumably protecting the stem cell pool from replication-associated damage. Second, at any given time not all stem cells actively cycle, leading to ‘clonal succession’ from the reserve pool of initially quiescent stem cells. We find that neither of these mechanisms is used in Drosophila male GSCs.

Using genetic and molecular analyses, we identified over 1,000 polymorphic regulators that regulate expression levels of human genes., Expression levels of human genes vary extensively among individuals. This variation facilitates analyses of expression levels as quantitative phenotypes in genetic studies where the entire genome can be scanned for regulators without prior knowledge of the regulatory mechanisms, thus enabling the identification of unknown regulatory relationships. Here, we carried out such genetic analyses with a large sample size and identified cis- and trans-acting polymorphic regulators for about 1,000 human genes. We validated the cis-acting regulators by demonstrating differential allelic expression with sequencing of transcriptomes (RNA-Seq) and the trans-regulators by gene knockdown, metabolic assays, and chromosome conformation capture analysis. The majority of the regulators act in trans to the target (regulated) genes. Most of these trans-regulators were not known to play a role in gene expression regulation. The identification of these regulators enabled the characterization of polymorphic regulation of human gene expression at a resolution that was unattainable in the past., Author Summary Cellular characteristics and functions are determined largely by gene expression and expression levels differ among individuals, however it is not clear how these levels are regulated. While many cis-acting DNA sequence variants in promoters and enhancers that influence gene expression have been identified, only a few polymorphic trans-regulators of human genes are known. Here, we used human B-cells from individuals belonging to large families and identified polymorphic trans-regulators for about 1,000 human genes. We validated these results by gene knockdown, metabolic perturbation studies and chromosome conformation capture assays. Although these regulatory relationships were identified in cultured B-cells, we show that some of the relationships were also found in primary fibroblasts. The large number of regulators allowed us to better understand gene expression regulation, to uncover new gene functions, and to identify their roles in disease processes. This study shows that genetic variation is a powerful tool not only for gene mapping but also to study gene interaction and regulation.

Diet can protect from autoimmune disease; however, whether diet acts via the host and/or microbiome remains unclear. Here, we use a ketogenic diet (KD) as a model to dissect these complex interactions. A KD rescued the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis in a microbiota-dependent fashion. Dietary supplementation with a single KD-dependent host metabolite (β-hydroxybutyrate, βHB) rescued EAE whereas transgenic mice unable to produce βHB in the intestine developed more severe disease. Transplantation of the βHB-shaped gut microbiota was protective. Lactobacillus sequence variants were associated with decreased T helper 17 (Th17) cell activation in vitro . Finally, we isolated a L. murinus strain that protected from EAE, which was phenocopied by the Lactobacillus metabolite indole lactic acid. Thus, diet alters the immunomodulatory potential of the gut microbiota by shifting host metabolism, emphasizing the utility of taking a more integrative approach to study diet-host-microbiome interactions.