The human microbiome and juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common rheumatic disease in childhood. The pathogenesis of JIA is thought to be the result of a combination of host genetic and environmental triggers. However, the precise factors that determine one’s susceptibility to JIA remain to be unravelled. The microbiome has received increasing attention as a potential contributing factor to the development of a wide array of immune-mediated diseases, including inflammatory bowel disease, type 1 diabetes and rheumatoid arthritis. Also in JIA, there is accumulating evidence that the composition of the microbiome is different from healthy individuals. A growing body of evidence indeed suggests that, among others, the microbiome may influence the development of the immune system, the integrity of the intestinal mucosal barrier, and the differentiation of T cell subsets. In turn, this might lead to dysregulation of the immune system, thereby possibly playing a role in the development of JIA. The potential to manipulate the microbiome, for example by faecal microbial transplantation, might then offer perspectives for future therapeutic interventions. Before we can think of such interventions, we need to first obtain a deeper understanding of the cause and effect relationship between JIA and the microbiome. In this review, we discuss the existing evidence for the involvement of the microbiome in JIA pathogenesis and explore the potential mechanisms through which the microbiome may influence the development of autoimmunity in general and JIA specifically.

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title: Juvenile idiopathic arthritis.
abstract: This study attempts to determine the clinical manifestations, severity and immunological features of JIA and its influence on growth and associated cardiac involvement in children below 16 years of age. This is a cross sectional study in a tertiary referral center on 50 consecutive children below the age of 16 years. Each patient was thoroughly examined and scored on Juvenile Arthritis  Relevant blood tests, cardiac and ophthalmic evaluation was done. Growth patterns were noted. There was an overall equal sex ratio, though there was a male preponderance in the systemic and oligoarthritis groups. Disturbance of growth frequently occurred in children suffering from JIA. Cardiac involvement should be looked for in cases of JIA. Significant number of cases of PSRA is diagnosed in children presenting with chronic arthritis.

title: Oligoarticular and polyarticular JIA: epidemiology and pathogenesis
abstract: Juvenile idiopathic arthritis (JIA) refers to a group of chronic childhood arthropathies of unknown etiology, currently classified into subtypes primarily on the basis of clinical features. Research has focused on the hypothesis that these subtypes arise through distinct etiologic pathways. In this Review, we discuss four subtypes of JIA: persistent oligoarticular, extended oligoarticular, rheumatoid-factor-positive polyarticular and rheumatoid-factor-negative polyarticular. These subtypes differ in prevalence between ethnic groups and are associated with different HLA alleles. Non-HLA genetic risk factors have also been identified, some of which reveal further molecular differences between these subtypes, while others suggest mechanistic overlap. Investigations of immunophenotypes also provide insights into subtype differences: adaptive immunity seems to have a prominent role in both polyarticular and oligoarticular JIA, and the more-limited arthritis observed in persistent oligoarticular JIA as compared with extended oligoarticular JIA may reflect more-potent immunoregulatory T-cell activity in the former. Tumor necrosis factor seems to be a key mediator of both polyarticular and oligoarticular JIA, especially in the extended oligoarticular subtype, although elevated levels of other cytokines are also observed. Limited data on monocytes, dendritic cells, B cells, natural killer T cells and neutrophils suggest that the contributions of these cells differ across subtypes of JIA. Within each subtype, however, common pathways seem to drive joint damage.

title: Role of the gut microbiota in immunity and inflammatory disease
abstract: The mammalian intestine is colonized by trillions of microorganisms, most of which are bacteria that have co-evolved with the host in a symbiotic relationship. The collection of microbial populations that reside on and in the host is commonly referred to as the microbiota. A principal function of the microbiota is to protect the intestine against colonization by exogenous pathogens and potentially harmful indigenous microorganisms via several mechanisms, which include direct competition for limited nutrients and the modulation of host immune responses. Conversely, pathogens have developed strategies to promote their replication in the presence of competing microbiota. Breakdown of the normal microbial community increases the risk of pathogen infection, the overgrowth of harmful pathobionts and inflammatory disease. Understanding the interaction of the microbiota with pathogens and the host might provide new insights into the pathogenesis of disease, as well as novel avenues for preventing and treating intestinal and systemic disorders.

title: The human microbiome: at the interface of health and disease
abstract: Interest in the role of the microbiome in human health has burgeoned over the past decade with the advent of new technologies for interrogating complex microbial communities. The large-scale dynamics of the microbiome can be described by many of the tools and observations used in the study of population ecology. Deciphering the metagenome and its aggregate genetic information can also be used to understand the functional properties of the microbial community. Both the microbiome and metagenome probably have important functions in health and disease; their exploration is a frontier in human genetics.

title: Perturbation of the Human Microbiome as a Contributor to Inflammatory Bowel Disease
abstract: The human microbiome consist of the composite genome of native flora that have evolved with humanity over millennia and which contains 150-fold more genes than the human genome. A “healthy” microbiome plays an important role in the maintenance of health and prevention of illness, inclusive of autoimmune disease such as inflammatory bowel disease (IBD). IBD is a prevalent spectrum of disorders, most notably defined by Crohn’s disease (CD) and ulcerative colitis (UC), which are associated with considerable suffering, morbidity, and cost. This review presents an outline of the loss of a normal microbiome as an etiology of immune dysregulation and IBD pathogenesis initiation. We, furthermore, summarize the knowledge on the role of a healthy microbiome in terms of its diversity and important functional elements and, lastly, conclude with some of the therapeutic interventions and modalities that are now being explored as potential applications of microbiome-host interactions.

title: Toward defining the autoimmune microbiome for type 1 diabetes
abstract: Several studies have shown that gut bacteria have a role in diabetes in murine models. Specific bacteria have been correlated with the onset of diabetes in a rat model. However, it is unknown whether human intestinal microbes have a role in the development of autoimmunity that often leads to type 1 diabetes (T1D), an autoimmune disorder in which insulin-secreting pancreatic islet cells are destroyed. High-throughput, culture-independent approaches identified bacteria that correlate with the development of T1D-associated autoimmunity in young children who are at high genetic risk for this disorder. The level of bacterial diversity diminishes overtime in these autoimmune subjects relative to that of age-matched, genotype-matched, nonautoimmune individuals. A single species, Bacteroides ovatus, comprised nearly 24% of the total increase in the phylum Bacteroidetes in cases compared with controls. Conversely, another species in controls, represented by the human firmicute strain CO19, represented nearly 20% of the increase in Firmicutes compared with cases overtime. Three lines of evidence are presented that support the notion that, as healthy infants approach the toddler stage, their microbiomes become healthier and more stable, whereas, children who are destined for autoimmunity develop a microbiome that is less diverse and stable. Hence, the autoimmune microbiome for T1D may be distinctly different from that found in healthy children. These data also suggest bacterial markers for the early diagnosis of T1D. In addition, bacteria that negatively correlated with the autoimmune state may prove to be useful in the prevention of autoimmunity development in high-risk children.

title: The microbiome and rheumatoid arthritis
abstract: Humans are not (and have never been) alone. From the moment we are born, millions of micro-organisms populate our bodies and coexist with us rather peacefully for the rest of our lives. This microbiome represents the totality of micro-organisms (and their genomes) that we necessarily acquire from the environment. Micro-organisms living in or on us have evolved to extract the energy they require to survive, and in exchange they support the physiological, metabolic and immune capacities that have contributed to our evolutionary success. Although currently categorized as an autoimmune disorder and regarded as a complex genetic disease, the ultimate cause of rheumatoid arthritis (RA) remains elusive. It seems that interplay between predisposing genetic factors and environmental triggers is required for disease manifestation. New insights from DNA sequence-based analyses of gut microbial communities and a renewed interest in mucosal immunology suggest that the microbiome represents an important environmental factor that can influence autoimmune disease manifestation. This Review summarizes the historical clues that suggest a possible role for the microbiota in the pathogenesis of RA, and will focus on new technologies that might provide scientific evidence to support this hypothesis.

title: Autoimmune host-microbiota interactions at barrier sites and beyond.
abstract: The microbiota is considered to be an important factor influencing the pathogenesis of autoimmunity at both barrier sites and internal organs. Impinging on innate and adaptive immunity, commensals exert protective or detrimental effects on various autoimmune animal models. Human microbiome studies of autoimmunity remain largely descriptive, but suggest a role for dysbiosis in autoimmune disease. Humanized gnotobiotic approaches have advanced our understanding of immune-commensal interactions, but little is known about the mechanisms in autoimmunity. We propose that, similarly to infectious agents, the microbiota mediates autoimmunity via bystander activation, epitope spread, and, particularly under homeostatic conditions, via crossreactivity. This review presents an overview of the current literature concluding with outstanding questions in this field.

title: The Effect of Diet on the Human Gut Microbiome: A Metagenomic Analysis in Humanized Gnotobiotic Mice
abstract: A translational medicine pipeline is described where human gut microbial communities and diets are re-created in gnotobiotic mice and the impact on microbe and host is defined using metagenomics. Are You a Man or a Mouse?  Both Comedian Bill Maher targets obese people with his satire almost as often as he does politicians. But clinical obesity is no joke. The World Health Organization estimates the number of obese people worldwide to be 300 million. Add to that the fact that obesity increases one’s risk for a whole stable of serious illnesses—type II diabetes, stroke, and some cancers—and you have one large global disease burden. Scientists and sociologists cite several hypotheses regarding the causes of the obesity epidemic, such as minimal physical exercise, high-fructose corn syrup, and diets of low-cost, large-portion, fat-filled foods. But pinpointing obesity triggers in humans is hard because of uncontrollable genetic, cultural, and environmental variables. Recently, researchers have thrown another element into the mix: the human gut microbiota. A massive number of microbes make the human gut their home. Highly diverse and numbering in the tens of trillions, our microbial companions help shape our human physiology, including effects on metabolism. The extent of their influence is now the subject of intense study in large part because high-capacity, moderately priced DNA sequencing has allowed our microbial communities and their collections of genes (“the microbiome”) to be characterized without having to culture the component organisms. However, this is a challenging business: Studying the factors that shape the assembly and operations of these communities is difficult to do in humans, given our varied genotypes, our difficult-to-document choices of what we eat, and our different environmental exposures.  To recreate a model human gut ecosystem, they transplanted human fecal matter into germ-free mice. They show that the transplant was remarkably successful: Recipient animals carried a collection of bacteria that mimicked the human donor’s microbiota. Moreover, the transplanted community could be transmitted from generation to generation of gnotobiotic mice. When these humanized animals were switched from a low-fat, plant-rich diet to a high-fat, high-sugar diet, the microbiota was changed after only 1 day on the junk-food binge. The authors were able to measure microbiome gene content and expression to further understand how the community responded to this diet shift. Like their Homo sapiens counterparts, Western diet–fed humanized mice become obese. Remarkably, this increased adiposity phenotype can be transmitted to other mice, at least for a time, by transplanting their gut microbiota to germ-free recipients. Human microbiome investigators are seeking to extend their descriptive studies. The humanized mice described by Turnbaugh et al. now provide a well-controlled system not only for assaying the functional properties of gut communities harvested from humans with different phenotypes, but also for conducting proof-of-principle “clinical” trials to show how a “host” of factors, including our diets, may influence our microbiota and how in turn our microbiota shapes our health and disease predispositions. Diet and nutritional status are among the most important modifiable determinants of human health. The nutritional value of food is influenced in part by a person’s gut microbial community (microbiota) and its component genes (microbiome). Unraveling the interrelations among diet, the structure and operations of the gut microbiota, and nutrient and energy harvest is confounded by variations in human environmental exposures, microbial ecology, and genotype. To help overcome these problems, we created a well-defined, representative animal model of the human gut ecosystem by transplanting fresh or frozen adult human fecal microbial communities into germ-free C57BL/6J mice. Culture-independent metagenomic analysis of the temporal, spatial, and intergenerational patterns of bacterial colonization showed that these humanized mice were stably and heritably colonized and reproduced much of the bacterial diversity of the donor’s microbiota. Switching from a low-fat, plant polysaccharide–rich diet to a high-fat, high-sugar “Western” diet shifted the structure of the microbiota within a single day, changed the representation of metabolic pathways in the microbiome, and altered microbiome gene expression. Reciprocal transplants involving various combinations of donor and recipient diets revealed that colonization history influences the initial structure of the microbial community but that these effects can be rapidly altered by diet. Humanized mice fed the Western diet have increased adiposity; this trait is transmissible via microbiota transplantation. Humanized gnotobiotic mice will be useful for conducting proof-of-principle “clinical trials” that test the effects of environmental and genetic factors on the gut microbiota and host physiology.

title: Long-term impacts of antibiotic exposure on the human intestinal microbiota.
abstract: Although it is known that antibiotics have short-term impacts on the human microbiome, recent evidence demonstrates that the impacts of some antibiotics remain for extended periods of time. In addition, antibiotic-resistant strains can persist in the human host environment in the absence of selective pressure. Both molecular- and cultivation-based approaches have revealed ecological disturbances in the microbiota after antibiotic administration, in particular for specific members of the bacterial community that are susceptible or alternatively resistant to the antibiotic in question. A disturbing consequence of antibiotic treatment has been the long-term persistence of antibiotic resistance genes, for example in the human gut. These data warrant use of prudence in the administration of antibiotics that could aggravate the growing battle with emerging antibiotic-resistant pathogenic strains.

title: Faecal microbiome in new-onset juvenile idiopathic arthritis
abstract: Alterations in the intestinal microbial flora have been linked with autoimmune diseases. Our objective was to analyse the composition of the faecal microbiome of children with new-onset juvenile idiopathic arthritis (JIA) compared to healthy controls, and to identify specific gut bacteria associated with JIA. Stool samples from patients were taken at the time of diagnosis of JIA. The microbiome profiles of samples of 30 children with JIA (mean age 6.2 years, 22 girls) were analysed with 16S region-based sequencing profiling and compared to the stool samples of healthy controls (n = 27, mean age 5.4 years, 18 girls). The proportion of bacteria belonging to the phylum Firmicutes was significantly lower in children with JIA [21 % (95 % confident interval [CI]: 17–25 %)] compared to controls [33 % (95 % CI: 26–41 %), p = 0.009]. Bacteria belonging to Bacteroidetes were significantly more abundant in JIA [78 % (95 % CI: 74–82 %)] than in control samples [65 % (95 % CI: 57–73 %), p = 0.008]. Shared operational taxonomic units (OTUs) between the groups revealed that genera Actinobacteria and Fusobacteria were present only in JIA patients and Lentisphaerae only in controls. In summary, faecal flora in JIA is characterised by a low level of Firmicutes and an abundance of Bacteroidetes, resembling the aberration reported in type 1 diabetes. We suggest that alterations in the intestinal microbial flora may challenge the mucosal immune system of genetically susceptible subjects predisposing to local proinflammatory cascades, thus contributing to the development of JIA.

title: Repeated Exposure to Antibiotics in Infancy: A Predisposing Factor for Juvenile Idiopathic Arthritis or a Sign of This Group’s Greater Susceptibility to Infections?
abstract: Previous exposure to antibiotics has been associated with the pathogenesis of several autoimmune diseases. Our objective was to explore whether childhood exposure to antibiotics would be associated with the risk of developing juvenile idiopathic arthritis (JIA).  The material was collected from national registers containing all children born in 2000–2010 in Finland and diagnosed with JIA by the end of December 2012 (n = 1298) and appropriate controls (n = 5179) matched for age, sex, and place of birth. All purchases of antibiotics were collected from birth until the index date (i.e., the date of special reimbursement for JIA medications). A conditional logistic regression was performed to evaluate the association between the exposure to antibiotics and the risk of JIA.  The risk of JIA increased with the number of antibiotic purchases from birth to the index date: for ≥ 1 purchases versus none, OR 1.6, 95% CI 1.3–1.9 with an upward trend in OR (p < 0.001). Antibiotic groups lincosamides and cephalosporins showed the strongest association with JIA (OR 6.6, 95% CI 3.7–11.7, and OR 1.6, 95% CI 1.4–1.8, respectively). Overall exposure to antibiotics before 2 years of age was associated with an increased risk of JIA (OR 1.4, 95% CI 1.2–1.6), with the trend test of OR (p < 0.001).  Previous early and repeated exposure to antibiotics may predispose individuals to develop JIA. Alternatively, the apparent association may reflect shared susceptibility to infections and JIA.

title: Antibiotic Exposure and Juvenile Idiopathic Arthritis: A Case–Control Study
abstract: Recent evidence has linked childhood antibiotic use and microbiome disturbance to autoimmune conditions. This study tested the hypothesis that antibiotic exposure was associated with newly diagnosed juvenile idiopathic arthritis (JIA).  We performed a nested case–control study in a population-representative medical records database from the United Kingdom. Children with newly diagnosed JIA were compared with age- and gender-matched control subjects randomly selected from general practices containing at least 1 case, excluding those with inflammatory bowel disease, immunodeficiency, or other systemic rheumatic diseases. Conditional logistic regression was used to examine the association between antibacterial antibiotics (including number of antibiotic courses and timing) and JIA after adjusting for significant confounders.  Any antibiotic exposure was associated with an increased rate of developing JIA (adjusted odds ratio: 2.1 [95% confidence interval: 1.2–3.5]). This relationship was dose dependent (adjusted odds ratio over 5 antibiotic courses: 3.0 [95% confidence interval: 1.6–5.6]), strongest for exposures within 1 year of diagnosis, and did not substantively change when adjusting for number or type of infections. In contrast, nonbacterial antimicrobial agents (eg, antifungal, antiviral) were not associated with JIA. In addition, antibiotic-treated upper respiratory tract infections were more strongly associated with JIA than untreated upper respiratory tract infections.  Antibiotics were associated with newly diagnosed JIA in a dose- and time-dependent fashion in a large pediatric population. Antibiotic exposure may play a role in JIA pathogenesis, perhaps mediated through alterations in the microbiome.

title: A core gut microbiome in obese and lean twins
abstract: The human distal gut harbours a vast ensemble of microbes (the microbiota) that provide important metabolic capabilities, including the ability to extract energy from otherwise indigestible dietary polysaccharides. Studies of a few unrelated, healthy adults have revealed substantial diversity in their gut communities, as measured by sequencing 16S rRNA genes, yet how this diversity relates to function and to the rest of the genes in the collective genomes of the microbiota (the gut microbiome) remains obscure. Studies of lean and obese mice suggest that the gut microbiota affects energy balance by influencing the efficiency of calorie harvest from the diet, and how this harvested energy is used and stored. Here we characterize the faecal microbial communities of adult female monozygotic and dizygotic twin pairs concordant for leanness or obesity, and their mothers, to address how host genotype, environmental exposure and host adiposity influence the gut microbiome. Analysis of 154 individuals yielded 9,920 near full-length and 1,937,461 partial bacterial 16S rRNA sequences, plus 2.14 gigabases from their microbiomes. The results reveal that the human gut microbiome is shared among family members, but that each person’s gut microbial community varies in the specific bacterial lineages present, with a comparable degree of co-variation between adult monozygotic and dizygotic twin pairs. However, there was a wide array of shared microbial genes among sampled individuals, comprising an extensive, identifiable ‘core microbiome’ at the gene, rather than at the organismal lineage, level. Obesity is associated with phylum-level changes in the microbiota, reduced bacterial diversity and altered representation of bacterial genes and metabolic pathways. These results demonstrate that a diversity of organismal assemblages can nonetheless yield a core microbiome at a functional level, and that deviations from this core are associated with different physiological states (obese compared with lean).

title: Dense genotyping of immune-related disease regions identifies 14 new susceptibility loci for juvenile idiopathic arthritis
abstract: We used the Immunochip array to analyze 2,816 individuals with juvenile idiopathic arthritis (JIA), comprising the most common subtypes (oligoarticular and rheumatoid factor–negative polyarticular JIA), and 13,056 controls. We confirmed association of 3 known JIA risk loci (the human leukocyte antigen (HLA) region, PTPN22 and PTPN2) and identified 14 loci reaching genome-wide significance (P < 5 × 10−8) for the first time. Eleven additional new regions showed suggestive evidence of association with JIA (P < 1 × 10−6). Dense mapping of loci along with bioinformatics analysis refined the associations to one gene in each of eight regions, highlighting crucial pathways, including the interleukin (IL)-2 pathway, in JIA disease pathogenesis. The entire Immunochip content, the HLA region and the top 27 loci (P < 1 × 10−6) explain an estimated 18, 13 and 6% of the risk of JIA, respectively. In summary, this is the largest collection of JIA cases investigated so far and provides new insight into the genetic basis of this childhood autoimmune disease.

title: The Etiology of Juvenile Idiopathic Arthritis
abstract: Over the years, the commonly used term to describe juvenile idiopathic arthritis (JIA) has changed. By definition, JIA includes all types of arthritis with no apparent cause, lasting more than 6 weeks, in patients aged less than 16 years at onset. JIA pathogenesis is still poorly understood: the interaction between environmental factors and multiple genes has been proposed as the most relevant working mechanism to the development of JIA. The concept that various microbes that colonize or infect not only the mucosal surfaces, like the oral cavity, but also the airways and gut might trigger autoimmune processes, resulting in chronic arthritides, and JIA was first drafted at the outset of last century. JIA development might be initiated and sustained by the exposure to environmental factors, including infectious agents which affect people at a young age, depending on the underlying genetic predisposition to synovial inflammation. Many data from patients with JIA suggest a scenario in which different external antigens incite multiple antigen-specific pathways, cytotoxic T cell responses, activation of classical complement cascade, and production of proinflammatory cytokines. In this review, emphasis is paid not only to the potential role of parvovirus B19 and Epstein-Barr virus in primis but also to the general involvement of different bacteria as Salmonella spp., Shigella spp., Campylobacter spp., Mycoplasma pneumoniae, Chlamydophila pneumoniae, Bartonella henselae, and Streptococcus pyogenes for the development of immune-mediated arthritides during childhood. No unequivocal evidence favoring or refuting these associations has been clearly proved, and today, the strict definition of JIA etiology remains unknown. The infection can represent a random event in a susceptible individual, or it can be a necessary factor in JIA development, always in combination with a peculiar genetic background. Further studies are needed in order to address the unsolved questions concerning this issue.

title: Do infections trigger juvenile idiopathic arthritis?
abstract: Juvenile idiopathic arthritis (JIA) is a disease that was prominent with increased inflammation response in immune system, appeared mostly with peripheral arthritis and endogenous and exogenous antigens play a role in the pathogenesis of disease. Two major reasons were thinking to be considerably important. First of them is immunological predisposition and the second one is environmental factors. Infections are considered to be the most important between environmental factors but also stress and trauma are also important in the etiology of the disease. However, the relation between JIA and infections is not clearly defined but the relation between adult chronic arthritis and infections was well-defined. A total of 70 patients, 26 with primer JIA, 20 with recurrent JIA, 24 healthy control were included in this study. Mycoplasma pneumoniae, Chlamydophila pneumoniae and C. Jejuni were detected in 4, 1 and 1 of 10 (38.46%) patients with primer JIA, respectively. Salmonella enteritidis, EBV, M. pneumoniae, C. jejuni and Borrelia burgdorferi were detected in 1, 2, 2, 2, and 1 of the 8(40%) patients with recurrent JIA, respectively. S. enteritidis were isolated in feces culture and also identified by agglutination method. Infection was detected in total 18 (39.13%) of patient groups. C. pneumoniae and C. jejuni were detected in 1 and 1 of 2(8.33) healthy control groups, respectively. Throat culture positivity was not detected in any of the patient and healthy control groups. In conclusion, etiopathogenesis of JIA is not clearly understood and suggested that various factors can trigger the disease and it is the most common rheumatoid disease of childhood. However, there are some studies focusing especially on one infectious agent but this is the first study including such a big range of infectious agents in the literature for the microorganisms that can be suggested to have a role in the etiopathogenesis of JIA. We have a conclusion in the light of our results and suggest that some microorganisms can trigger and increase the intensity of clinical situation according to the case. When we evaluate the primer and recurrent JIA groups; M. pneumoniae and C. jejuni come forward and seen common in JIA cases. We also suggest that the pre-diagnosis of microorganisms, which can play a role as primarily or by intervening in the etiopathogenesis of JIA and adding specific antimicrobial therapy to the standard JIA therapy, it is possible to perform new, extended, especially molecular based serial case studies.

title: Selective recruitment of polarized T cells expressing CCR5 and CXCR3 to the inflamed joints of children with juvenile idiopathic arthritis.
abstract: To study the expression of chemokine receptors CCR5 and CXCR3 and the Th1/Th2 cytokine balance in children with oligoarticular or polyarticular juvenile idiopathic arthritis (JIA).



Using 3-color immunofluorescence, we studied the expression of CCR5 and CXCR3 on, and T cell cytokine production by, paired samples of synovial fluid (SF) and peripheral blood (PB) T cells from 20 patients with oligoarticular- or polyarticular-onset JIA. Chemokine and cytokine phenotypes were also compared within the CD45RO+,CD3+ subsets. CCR5 genotypes were confirmed by polymerase chain reaction typing and sequencing.



In the majority of samples, the number of T cells that were CCR5+ and CXCR3+ was higher in SF than in PB, and this difference was significant. One child was homozygous for the null A32 CCR5 allele; 4 others had lower expression of CXCR3 in SF than in blood. All samples showed strongly Th1-type cytokine production by synovial T cells compared with that by PB T cells. Both features were also markedly polarized within the synovial CD45RO+ subset compared with PB CD45RO+ T cells.



The high expression of CCR5 and CXCR3 and high interferon-gamma:interleukin-4 ratios suggest a type 1 phenotype of SF T cells in JIA. The difference between CD45RO+ T cells from SF and from PB suggests that specific activation events have occurred in synovial T cells. We suggest that the highly activated, Th1-type phenotype of T cells within the chronically inflamed joints of children with JIA may reflect specific recruitment events that contribute to the polarization of these cells.

title: Interleukin-17–producing T cells are enriched in the joints of children with arthritis, but have a reciprocal relationship to regulatory T cell numbers
abstract: To identify interleukin-17 (IL-17)–producing T cells from patients with juvenile idiopathic arthritis (JIA), and investigate their cytokine production, migratory capacity, and relationship to Treg cells at sites of inflammation, as well as to test the hypothesis that IL-17+ T cell numbers correlate with clinical phenotype in childhood arthritis.  Flow cytometry was used to analyze the phenotype, cytokine production, and chemokine receptor expression of IL-17–producing T cells in peripheral blood and synovial fluid mononuclear cells from 36 children with JIA, in parallel with analysis of forkhead box P3 (FoxP3)–positive Treg cells. Migration of IL-17+ T cells toward CCL20 was assessed by a Transwell assay. Synovial tissue was analyzed by immunohistochemistry for IL-17 and IL-22.  IL-17+ T cells were enriched in the joints of children with JIA as compared with the blood of JIA patients (P = 0.0001) and controls (P = 0.018) and were demonstrated in synovial tissue. IL-17+ T cell numbers were higher in patients with extended oligoarthritis, the more severe subtype of JIA, as compared with patients with persistent oligoarthritis, the milder subtype (P = 0.046). Within the joint, there was an inverse relationship between IL-17+ T cells and FoxP3+ Treg cells (r = 0.61, P = 0.016). IL-17+,CD4+ T cells were uniformly CCR6+ and migrated toward CCL20, but synovial IL-17+ T cells had variable CCR4 expression. A proportion of IL-17+ synovial T cells produced IL-22 and interferon-γ.  This study is the first to define the frequency and characteristics of “Th17” cells in JIA. We suggest that these highly proinflammatory cells contribute to joint pathology, as indicated by relationships with clinical phenotypes, and that the balance between IL-17+ T cells and Treg cells may be critical to outcome.

title: Evidence of the transient nature of the Th17 phenotype of CD4+CD161+ T cells in the synovial fluid of patients with juvenile idiopathic arthritis.
abstract: To investigate the phenotype and function of CD4+ T cells in synovial fluid (SF) from the affected joints of children with oligoarticular-onset juvenile idiopathic arthritis (JIA), and to establish a possible link with disease activity.



CD4+ T cells were obtained from the peripheral blood (PB) and SF of 23 children with oligoarticular-onset JIA, as well as from the PB of 15 healthy children. The cells were analyzed for the expression of CXCR3, CCR6, and CD161 and for the production of interferon-γ and interleukin-17A (IL-17A). Spectratyping and clonotype analyses were performed to assess different T cell subsets.



The numbers of CD4+CD161+ cells showing either the Th1 or the Th17/Th1 phenotype were higher in the SF than in the PB of children with JIA. The few Th17 cells from JIA SF underwent a spontaneous shift to the Th1 phenotype in vitro, whereas Th17 cells from the PB of healthy children shifted only in the presence of JIA SF; this effect was neutralized by antibody blockade of IL-12 activity. Spectratyping and clonotype analyses showed a similar skewing of the T cell receptor V(β) repertoire in both CD161+ Th17 cells and CD161+ Th1 cells derived from the SF of the same JIA patient. The frequencies of CD4+CD161+ cells, particularly the Th17/Th1 cells, in the JIA SF positively correlated with the erythrocyte sedimentation rate and levels of C-reactive protein.



These findings suggest that a shifting of CD4+CD161+ T cells from Th17 to the Th17/Th1 or Th1 phenotype can occur in the SF of children with oligoarticular-onset JIA, and indicate that the accumulation of these cells is correlated with parameters of inflammation. Thus, the results support the hypothesis that these cells may play a role in JIA disease activity.

title: Regulatory T-cells in autoimmune diseases: challenges, controversies and--yet--unanswered questions.
abstract: Regulatory T cells (Tregs) are central to the maintenance of self-tolerance and tissue homeostasis. Markers commonly used to define human Tregs in the research setting include high expression of CD25, FOXP3 positivity and low expression/negativity for CD127. Many other markers have been proposed, but none unequivocally identifies bona fide Tregs. Tregs are equipped with an array of mechanisms of suppression, including the modulation of antigen presenting cell maturation and function, the killing of target cells, the disruption of metabolic pathways and the production of anti-inflammatory cytokines. Treg impairment has been reported in a number of human autoimmune conditions and includes Treg numerical and functional defects and conversion into effector cells in response to inflammation. In addition to intrinsic Treg impairment, resistance of effector T cells to Treg control has been described. Discrepancies in the literature are common, reflecting differences in the choice of study participants and the technical challenges associated with investigating this cell population. Studies differ in terms of the methodology used to define and isolate putative regulatory cells and to assess their suppressive function. In this review we outline studies describing Treg frequency and suppressive function in systemic and organ specific autoimmune diseases, with a specific focus on the challenges faced when investigating Tregs in these conditions.

title: Neutrophils in pediatric autoimmune disease
abstract: Purpose of reviewAs first immune responders, neutrophils are essential mediators of host defense, and also contribute to chronic pathologic inflammation at the crossroads of innate and adaptive immunity. In this review, we will highlight the current understanding of the role of neutrophils in pediatric rheumatology, with a focus on juvenile idiopathic arthritis (JIA) and lupus. Recent findingsIn inflamed tissues, neutrophils extrude neutrophil extracellular traps containing autoantigen that potentially drives lupus and rheumatoid factor-positive JIA. However, the contribution of neutrophil extracellular traps to pathogenesis remains an area of intense investigation. In JIA joints, neutrophils are activated to such an extent that associated circulating levels of S100A proteins may serve as biomarkers, correlating with disease activity, predicting response to treatment and heralding flares. Beyond the effects of ‘normal’ activation, neutrophils in JIA and lupus display dysregulation in gene expression, subset activation, and apoptosis. SummaryThe role of neutrophils in pediatric rheumatology is an understudied area, but garnering increasing attention. Although clearly implicated in JIA and lupus, the specific contributions of neutrophils to pathogenesis and the use of neutrophil activity surrogates as biomarkers require further study. Clarification of these outstanding issues will have implications for diagnosis and treatment of pediatric rheumatologic conditions.

title: T Cell Expression of Granulocyte–Macrophage Colony-Stimulating Factor in Juvenile Arthritis Is Contingent Upon Th17 Plasticity
abstract: Granulocyte–macrophage colony stimulating factor (GM‐CSF) is a potent inflammatory mediator that is responsible for recruitment and activation of innate immune cells. Recent data from murine studies have identified Th17 cells as a key source of GM‐CSF and suggest that T cell–derived GM‐CSF is instrumental in the induction of autoimmune disease. The present study was undertaken to analyze the expression of T cell–derived GM‐CSF in the joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the differentiation of Th17 cells and how this relates to GM‐CSF+ T helper cells.

title: An integrated catalog of reference genes in the human gut microbiome
abstract: Many analyses of the human gut microbiome depend on a catalog of reference genes. Existing catalogs for the human gut microbiome are based on samples from single cohorts or on reference genomes or protein sequences, which limits coverage of global microbiome diversity. Here we combined 249 newly sequenced samples of the Metagenomics of the Human Intestinal Tract (MetaHit) project with 1,018 previously sequenced samples to create a cohort from three continents that is at least threefold larger than cohorts used for previous gene catalogs. From this we established the integrated gene catalog (IGC) comprising 9,879,896 genes. The catalog includes close-to-complete sets of genes for most gut microbes, which are also of considerably higher quality than in previous catalogs. Analyses of a group of samples from Chinese and Danish individuals using the catalog revealed country-specific gut microbial signatures. This expanded catalog should facilitate quantitative characterization of metagenomic, metatranscriptomic and metaproteomic data from the gut microbiome to understand its variation across populations in human health and disease.

title: A human gut microbial gene catalogue established by metagenomic sequencing
abstract: To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, ∼150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.

title: Role of the Microbiota in Immunity and Inflammation
abstract: The microbiota plays a fundamental role on the induction, training, and function of the host immune system. In return, the immune system has largely evolved as a means to maintain the symbiotic relationship of the host with these highly diverse and evolving microbes. When operating optimally, this immune system-microbiota alliance allows the induction of protective responses to pathogens and the maintenance of regulatory pathways involved in the maintenance of tolerance to innocuous antigens. However, in high-income countries, overuse of antibiotics, changes in diet, and elimination of constitutive partners, such as nematodes, may have selected for a microbiota that lack the resilience and diversity required to establish balanced immune responses. This phenomenon is proposed to account for some of the dramatic rise in autoimmune and inflammatory disorders in parts of the world where our symbiotic relationship with the microbiota has been the most affected.

title: Immune-microbiota interactions in health and disease.
abstract: Recent studies have revealed that the intestinal microbiota plays an important role in host physiology and pathophysiology in health and disease. One of the major mechanisms by which the gut microbiota influences the host is through its interactions with and effects on the host immune system. In this review, we discuss the reciprocal interactions between the host immune system and the gut microbiota, with a particular focus on individual microbes that impact the host through dramatic and specific interactions with the adaptive immune system. We highlight the idea that the presence or absence of specific immunologically important members of the microbiota can determine disease susceptibility and propose that the identification and characterization of these bacteria in humans will eventually allow us to elucidate the role of microbiota composition in human disease.

title: Modulation of intestinal barrier by intestinal microbiota: pathological and therapeutic implications.
abstract: Mammals and their intestinal microbiota peacefully coexist in a mutualistic relationship. Commensal bacteria play an active role in shaping and modulating physiological processes in the host, which include, but are not restricted to, the immune system and the intestinal barrier. Both play a crucial role in containing intestinal bacteria and other potentially noxious luminal antigens within the lumen and mucosal compartment. Although mutualism defines the relationship between the host and the intestinal microbiota, disruptions in this equilibrium may promote disease. Thus, alterations in gut microbiota (dysbiosis) have been linked to the recent increased expression of obesity, allergy, autoimmunity, functional and inflammatory disorders such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). In this article, we review the evidence supporting a role of gut microbiota in regulating intestinal barrier function. We discuss the hypothesis that microbial factors can modulate the barrier in ways that can prevent or promote gastrointestinal disease. A better understanding of the role of the intestinal microbiota in maintaining a functional intestinal barrier may help develop targeted strategies to prevent and treat disease.

title: Adaptation of Solitary Intestinal Lymphoid Tissue in Response to Microbiota and Chemokine Receptor CCR7 Signaling1
abstract: Besides Peyer’s patches, solitary intestinal lymphoid tissue (SILT) provides a structural platform to efficiently initiate immune responses in the murine small intestine. SILT consists of dynamic lymphoid aggregates that are heterogeneous in size and composition, ranging from small clusters of mostly lineage-negative cells known as cryptopatches to larger isolated lymphoid follicles rich in B cells. In this study, we report that in chemokine receptor CCR7-deficient mice SILT is enlarged, although unchanged in frequency and cellular composition compared with wild-type mice. This phenotype is conferred by bone marrow-derived cells and is independent of the presence of intestinal bacteria. Remarkably, particularly small-sized SILT predominates in germfree wild-type mice. Colonization of wild-type mice with commensal bacteria provokes an adjustment of the spectrum of SILT to that observed under specific pathogen-free conditions by the conversion of pre-existing lymphoid structures into larger-sized SILT. In conclusion, our findings establish that intestinal microbes influence the manifestation of gut-associated lymphoid tissues and identify CCR7 signaling as an endogeneous factor that controls this process.

title: Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis
abstract: Intestinal homeostasis is critical for efficient energy extraction from food and protection from pathogens. Its disruption can lead to an array of severe illnesses with major impacts on public health, such as inflammatory bowel disease characterized by self-destructive intestinal immunity. However, the mechanisms regulating the equilibrium between the large bacterial flora and the immune system remain unclear. Intestinal lymphoid tissues generate flora-reactive IgA-producing B cells, and include Peyer's patches and mesenteric lymph nodes, as well as numerous isolated lymphoid follicles (ILFs). Here we show that peptidoglycan from Gram-negative bacteria is necessary and sufficient to induce the genesis of ILFs in mice through recognition by the NOD1 (nucleotide-binding oligomerization domain containing 1) innate receptor in epithelial cells, and β-defensin 3- and CCL20-mediated signalling through the chemokine receptor CCR6. Maturation of ILFs into large B-cell clusters requires subsequent detection of bacteria by toll-like receptors. In the absence of ILFs, the composition of the intestinal bacterial community is profoundly altered. Our results demonstrate that intestinal bacterial commensals and the immune system communicate through an innate detection system to generate adaptive lymphoid tissues and maintain intestinal homeostasis.

title: Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine.
abstract: The requirements for in vivo steady state differentiation of IL-17-producing T-helper (Th17) cells, which are potent inflammation effectors, remain obscure. We report that Th17 cell differentiation in the lamina propria (LP) of the small intestine requires specific commensal microbiota and is inhibited by treating mice with selective antibiotics. Mice from different sources had marked differences in their Th17 cell numbers and animals lacking Th17 cells acquired them after introduction of bacteria from Th17 cell-sufficient mice. Differentiation of Th17 cells correlated with the presence of cytophaga-flavobacter-bacteroidetes (CFB) bacteria in the intestine and was independent of toll-like receptor, IL-21 or IL-23 signaling, but required appropriate TGF-beta activation. Absence of Th17 cell-inducing bacteria was accompanied by increase in Foxp3+ regulatory T cells (Treg) in the LP. Our results suggest that composition of intestinal microbiota regulates the Th17:Treg balance in the LP and may thus influence intestinal immunity, tolerance, and susceptibility to inflammatory bowel diseases.

title: Induction of Intestinal Th17 Cells by Segmented Filamentous Bacteria
abstract: The gastrointestinal tract of mammals is inhabited by hundreds of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. How commensal microbiota influence the host immune system is poorly understood. We show here that colonization of the small intestine of mice with a single commensal microbe, segmented filamentous bacterium (SFB), is sufficient to induce the appearance of CD4(+) T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. SFB adhere tightly to the surface of epithelial cells in the terminal ileum of mice with Th17 cells but are absent from mice that have few Th17 cells. Colonization with SFB was correlated with increased expression of genes associated with inflammation and antimicrobial defenses and resulted in enhanced resistance to the intestinal pathogen Citrobacter rodentium. Thus, manipulation of this commensal-regulated pathway may provide new opportunities for enhancing mucosal immunity and treating autoimmune disease.

title: Th17 and Regulatory T Cells in Mediating and Restraining Inflammation
abstract: The vertebrate immune system is poised in a state of equilibrium that permits accurate and rapid protective responses against pathogens but curtails potential for causing harm to the host through targeting of "self" and provoking overexuberant inflammatory processes. In this Review we discuss this balance achieved in large part by interactions of different classes of T lymphocytes that have potent pro- or anti-inflammatory activity in the context of genetic and environmental factors, particularly the commensal microbiota.

title: Induction of Colonic Regulatory T Cells by Indigenous Clostridium Species
abstract: Bacteria of the genus Clostridium promote the induction of suppressor T cells in the colons of mice. CD4+ T regulatory cells (Tregs), which express the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, Tregs were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota, particularly clusters IV and XIVa of the genus Clostridium, promoted Treg cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor–β and affected Foxp3+ Treg number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic immunoglobulin E responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.

title: Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells
abstract: Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4+ CD45RBhi T cells in Rag1−/− mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host–microbe interactions establish immunological homeostasis in the gut.

title: Interleukin-17 and type 17 helper T cells.
abstract: Type 17 helper T cells (Th17) — a third class of T cells — and their major cytokine, interleukin-17, are essential for the defense against certain fungi, extracellular bacteria, and Mycobacterium tuberculosis. Defective control of Th17 cells and production of interleukin-17 are prominent in chronic inflammation and several immunoinflammatory disorders. Monoclonal antibodies against cytokines produced by Th17 cells are being investigated in clinical trials.

title: Molecular Mimicry as a Mechanism of Autoimmune Disease
abstract: A variety of mechanisms have been suggested as the means by which infections can initiate and/or exacerbate autoimmune diseases. One mechanism is molecular mimicry, where a foreign antigen shares sequence or structural similarities with self-antigens. Molecular mimicry has typically been characterized on an antibody or T cell level. However, structural relatedness between pathogen and self does not account for T cell activation in a number of autoimmune diseases. A proposed mechanism that could have been misinterpreted for molecular mimicry is the expression of dual T cell receptors (TCR) on a single T cell. These T cells have dual reactivity to both foreign and self-antigens leaving the host vulnerable to foreign insults capable of triggering an autoimmune response. In this review, we briefly discuss what is known about molecular mimicry followed by a discussion of the current understanding of dual TCRs. Finally, we discuss three mechanisms, including molecular mimicry, dual TCRs, and chimeric TCRs, by which dual reactivity of the T cell may play a role in autoimmune diseases.

title: Isolation from Heart Valves of Glycopeptides which share Immunological Properties with Streptococcus haemolyticus Group A Polysaccharides
abstract: THERE is evidence of immunological cross-reaction between structural glycoproteins extracted from human or bovine heart valves and parietal polysaccharides of haematolytic group A streptococci1,2. Further investigations have led to the isolation of dialysable glycopeptides, which produce the common antigenicity.

title: Molecular mimicry in T cell-mediated autoimmunity: Viral peptides activate human T cell clones specific for myelin basic protein
abstract: Abstract
 
 Structural similarity between viral T cell epitopes and self-peptides could lead to the induction of an autoaggressive T cell response. Based on the structural requirements for both MHC class 11 binding and TCR recognition of an immunodominant myelin basic protein (MBP) peptide, criteria for a data base search were developed in which the degeneracy of amino acid side chains required for MHC class 11 binding and the conservation of those required for T cell activation were considered. A panel of 129 peptides that matched the molecular mimicry motif was tested on seven MBP-specific T cell clones from multiple sclerosis patients. Seven viral and one bacterial peptide efficiently activated three of these clones. Only one peptide could have been identified as a molecular mimic by sequence alignment. The observation that a single T cell receptor can recognize quite distinct but structurally related peptides from multiple pathogens has important implications for understanding the pathogenesis of autoimmunity.

title: Virus-specific CD4(+) memory-phenotype T cells are abundant in unexposed adults.
abstract: Although T cell memory is generally thought to require direct antigen exposure, we found an abundance of memory-phenotype cells (20%-90%, averaging over 50%) of CD4(+) T cells specific to viral antigens in adults who had never been infected. These cells express the appropriate memory markers and genes, rapidly produce cytokines, and have clonally expanded. In contrast, the same T cell receptor (TCR) specificities in newborns are almost entirely naïve, which might explain the vulnerability of young children to infections. One mechanism for this phenomenon is TCR cross-reactivity to environmental antigens, and in support of this, we found extensive cross-recognition by HIV-1 and influenza-reactive T lymphocytes to other microbial peptides and expansion of one of these after influenza vaccination. Thus, the presence of these memory-phenotype T cells has significant implications for immunity to novel pathogens, child and adult health, and the influence of pathogen-rich versus hygienic environments.

title: Acute Gastrointestinal Infection Induces Long-Lived Microbiota-Specific T Cell Responses
abstract: Recognizing Escaped Commensals In order to coexist peacefully, the billions of bacteria in our gut and our immune system have reached a détente. An intestinal mucosal firewall exists, so bacteria remain localized to the gut, where the immune system is tightly regulated so that these bacteria are tolerated. Enteric infections, however, lead to a breach in this mucosal firewall, resulting in exposure of the peripheral immune system to the intestinal bacterial contents. What is the result? Using oral Toxoplasma gondii infection in mice, Hand et al. (p. 1553, published online 23 August) show that, besides the T. gondii–specific T cell response, a commensal bacteria–specific T cell response is elicited. The CD4+ T cell–specific response was tracked to a commensal-derived flagellin, and these T cells expanded after T. gondii infection and formed long-lived memory cells able to respond to subsequent challenges. Thus, enteric infections can lead to the formation of commensal bacteria–specific, long-lived memory T cells that reside throughout the body—which may play a role in intestinal pathologies such as inflammatory bowel disease. Enteric infections induce lasting adaptive immunity against commensal bacteria that may play a role in intestinal problems. The mammalian gastrointestinal tract contains a large and diverse population of commensal bacteria and is also one of the primary sites of exposure to pathogens. How the immune system perceives commensals in the context of mucosal infection is unclear. Here, we show that during a gastrointestinal infection, tolerance to commensals is lost, and microbiota-specific T cells are activated and differentiate to inflammatory effector cells. Furthermore, these T cells go on to form memory cells that are phenotypically and functionally consistent with pathogen-specific T cells. Our results suggest that during a gastrointestinal infection, the immune response to commensals parallels the immune response against pathogenic microbes and that adaptive responses against commensals are an integral component of mucosal immunity.

title: Mucosal immune responses to microbiota in the development of autoimmune disease.
abstract: The etiology of most systemic autoimmune diseases remains unknown. There is often a preclinical period of systemic autoimmunity prior to the onset of clinically classifiable disease; established and emerging data suggest that dysregulated immune interactions with commensal microbiota may play a role in the initial generation of autoimmunity in this preclinical period. This article reviews potential mechanisms by which alterations of healthy microbiota may induce autoimmunity as well as mucosal microbial associations with autoimmune diseases. If mucosal microbiota lead to the development of autoimmunity, these mucosal sites, microorganisms, and immunologic mechanisms can be targeted to prevent the onset of systemic autoimmune disease.

title: Epitope spreading in immune-mediated diseases: implications for immunotherapy
abstract: Evidence continues to accumulate supporting the hypothesis that tissue damage during an immune response can lead to the priming of self-reactive T and/or B lymphocytes, regardless of the specificity of the initial insult. This review will focus primarily on epitope spreading at the T-cell level. Understanding the cellular and molecular basis of epitope spreading in various chronic immune-mediated human diseases and their animal models is crucial to understanding the pathogenesis of these diseases and to the ultimate goal of designing antigen-specific treatments.

title: Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: implications for autoimmunity in rheumatoid arthritis.
abstract: To investigate protein citrullination by the periodontal pathogen Porphyromonas gingivalis as a potential mechanism for breaking tolerance to citrullinated proteins in rheumatoid arthritis (RA).



The expression of endogenous citrullinated proteins was analyzed by immunoblotting of cell extracts from P gingivalis and 10 other oral bacteria. P gingivalis-knockout strains lacking the bacterial peptidylarginine deiminases (PADs) or gingipains were created to assess the role of these enzymes in citrullination. Citrullination of human fibrinogen and α-enolase by P gingivalis was studied by incubating live wild-type and knockout strains with the proteins and analyzing the products by immunoblotting and mass spectrometry.



Endogenous protein citrullination was abundant in P gingivalis but lacking in the other oral bacteria. Deletion of the bacterial PAD gene resulted in complete abrogation of protein citrullination. Inactivation of arginine gingipains, but not lysine gingipains, led to decreased citrullination. Incubation of wild-type P gingivalis with fibrinogen or α-enolase caused degradation of the proteins and citrullination of the resulting peptides at carboxy-terminal arginine residues, which were identified by mass spectrometry.



Our findings demonstrate that among the oral bacterial pathogens tested, P gingivalis is unique in its ability to citrullinate proteins. We further show that P gingivalis rapidly generates citrullinated host peptides by proteolytic cleavage at Arg-X peptide bonds by arginine gingipains, followed by citrullination of carboxy-terminal arginines by bacterial PAD. Our results suggest a novel model where P gingivalis-mediated citrullination of bacterial and host proteins provides a molecular mechanism for generating antigens that drive the autoimmune response in RA.

title: Altered microbiota associated with abnormal humoral immune responses to commensal organisms in enthesitis-related arthritis
abstract: IntroductionPrior studies have established altered microbiota and immunologic reactivity to enteric commensal organisms in inflammatory bowel disease (IBD). Since intestinal inflammation is present in a subset of patients with both pediatric and adult spondyloarthritis (SpA), we hypothesized that SpA patients may also have altered microbiota and immune responsiveness to enteric organisms.MethodsStool and blood specimens were collected from children with enthesitis-related arthritis (ERA) and non-inflammatory controls. DNA purified from stool was subject to PCR amplification and sequencing of the variable IV region from the 16S rDNA gene. IgA and IgG Enzyme-linked Immunosorbent Assays (ELISAs) were performed on select species of bacteria in most subjects.ResultsTwenty-five children with ERA and 13 controls were included. The ERA patients had less Faecalibacterium prausnitzii (3.8% versus 10%, P = 0.008) and lachnospiraceae family (12 versus 7.0%, P = 0.020), a statistically significant increase in bifidobacterium (1.8% versus 0%, P = 0.032) and a non-statistically significant increase in Bacteroides (21% versus 11%, P = 0.150). Akkermansia muciniphila was abundant (>2%) in 7/27 ERA patients but none of the controls (P = 0.072.) Cluster analysis revealed two clusters of ERA patients: Cluster one (n = 8) was characterized by high levels of Bacteroides genus, while a second (n = 15) cluster had similar levels as the controls. Seven of 17 (41%) of the ERA subjects in Cluster 2 compared to 0/8 of the subjects in Cluster 1 had abundant Akkermansia muciniphila (P = 0.057). Serum IgA and IgG antibody levels against F. prausnitzii and B. fragilis were similar between patients and controls, whereas the two groups showed divergent responses when the fecal relative abundances of F. prausnitzii and Bacteroides were compared individually against IgA antibody levels recognizing F. prausnitzii and B. fragilis, respectively.ConclusionThe abundance of F. prausnitzii in the stool among patients with ERA is reduced compared to controls, and Bacteroides and A. muciniphila are identified as associative agents in subsets of ERA patients. Differences in the humoral responses to these bacteria may contribute to disease.

title: The intestinal microbiome in spondyloarthritis
abstract: dysbiosis in the gut is emerging as a common component in various inflammatory disorders including spondyloarthritis (SpA). The depth of this influence has begun to be realized with next-generation sequencing of the gut microbiome providing unbiased assessment of previously uncharted bacterial populations. Recent findingsDecreased numbers of Firmicutes, a major phyla of gut commensals, especially the species Faecalibacterium prausnitzii and Clostridium leptum have been found in various inflammatory disorders including SpA and inflammatory bowel disease (IBD), and could be an important link between SpA and gut inflammation. Multiple studies in ankylosing spondylitis, psoriatic arthritis, juvenile SpA, and animal models of SpA are revealing common bacterial associations among these diseases as well as IBD. SummaryWe are beginning to appreciate the complex relationship between the gut microbiome and host immune regulation and dysregulation in health and disease. Potentially important differences have been revealed in SpA, but cause and effect relationships remain far from established. Many critical questions remain to be answered before we can apply new knowledge to improve therapeutics in SpA.

title: Increased bacterial urease activity in faeces in juvenile chronic arthritis: evidence of altered intestinal microflora?
abstract: The intestinal microflora was indirectly evaluated in juvenile chronic arthritis (JCA) by analysing enzyme activities--urease, beta-glucosidase and beta-glucuronidase--in faeces. In 18 out of 26 JCA patients, the illness had been diagnosed during the past year. The control group was composed of eight age-matched control patients and 18 family members of JCA patients (3-36 yr). The mean [95% confidence interval (CI)] urease activity, but not the activities of beta-glucosidase and beta-glucuronidase, in faeces from the JCA group differed from that in the control group: 32.3 (26.6-38.1) nmol/min/mg protein vs 24.0 (16.8-31.6), P = 0.07. The difference was more marked in a comparison of JCA patients with family members (P = 0.03). In a subgroup of subjects, the effect of 10 days oral bacteriotherapy with Lactobacillus GG on faecal enzyme activities was then investigated (n = 8 JCA patients, n = 8 control patients). This short-term oral bacteriotherapy reduced the increased urease activity in faeces of JCA patients. Keeping in mind the small number of subjects, it may be inferred from the present results that the increased urease activity in JCA is specific for the disease, suggesting altered intestinal microflora in JCA.

title: Dietary therapy with Lactobacillus GG, bovine colostrum or bovine immune colostrum in patients with juvenile chronic arthritis: Evaluation of effect on gut defence mechanisms
abstract: The effect of dietary therapy with a human Lactobacillus strain GG (ATCC 53103), bovine colostrum, or bovine immune colostrum with specific antibodies against anaerobic intestinal bacteria on gut defence mechanisms were studied in juvenile chronic arthritis. Thirty patients with juvenile chronic arthritis were randomly allocated to receive a freeze-dried powder of Lactobacillus GG, or bovine colostrum, or bovine immune colostrum, for a two-week period. Immunologic and non-immunologic gut defence mechanisms were indirectly investigated in blood and faecal samples. In patients receiving Lactobacillus GG, the median (interquartile range) frequency of immunoglobulin-secreting cells, determined by enzyme-linked immunospot assay, increased in the IgA class from 1840 (690–2530) to 3480 (1030–13 170)/106 cells; p=0.02. Likewise the median (interquartile range) frequency of specific antibody-secreting cells against dietary antigens increased during the Lactobacillus GG therapy in the IgM class from 3.8 (1.4–5.0) to 11.2 (5.0–30.0)/106 cells; p=0.02. In addition, Lactobacillus GG therapy decreased the median (interquartile range) activity of faecal urease, which has been associated with mucosal tissue damage, from 40.3 (21.7–54.3) to 28.6 (24.5–49.4) nmol. min−1 (mg protein)−1; p=0.10, while, in patients receiving bovine colostrum, faecal urease activity increased (from 42.2 to 80.6; p=0.04). All findings were transient. We suggest that gut defence mechanisms are disturbed in juvenile chronic arthritis and we further suggest that orally administered Lactobacillus GG has a potential to reinforce the mucosal barrier mechanisms in juvenile chronic arthritis.

title: Increased gut permeability in juvenile chronic arthritides. A multivariate analysis of the diagnostic parameters.
abstract: This study was aimed at evaluating intestinal permeability (IP) in patients with oligoarticular juvenile idiopathic arthritis (o-JIA), spondyloarthropathy (SpA) associated with inflammatory bowel disease (IBD) and other forms of juvenile-onset chronic arthritiis (OIA) using the lactulose/mannitol (L/M) test in comparison with other non-invasive parameters of gut involvement.



A series of 26 children affected with o-JIA and 14 with either SpA/IBD or OIA were assessed for IP. The urinary L/M ratio was measured by gas chromatography. The erythrocyte sedimentation rate (ESR), C reactive protein (CRP), and faecal alpha 1 antitrypsin concentrations were also evaluated. Ten o-JIA patients displayed active arthritis while in 16 the disease was under control. Among the OIA patients, 11 were affected with psoriatic arthritis and the remaining 3 with chronic reactive arthritis. 14 patients with SpA-IBD had active synovitis or spine inflammation. 14 eo-pJCA and 22 OIA and SpA-IBD patients, respectively, were receiving NSAID therapy.



The mean L/M ratios for the Spa-IBD (0.07 +/- 0.02, mean +/- SD), OIA (0.05 +/- 0.02) and o-JIA (0.04 +/- 0.02) patients were significantly higher (p < 0.001, p = 0.022 and p = 0.01, respectively) than those found in controls (0.02 +/- 0.01). Logistic regression analysis disclosed a positive correlation between the L/M ratio and the presence of gastrointestinal manifestations (p = 0.011). The type of disease (p = 0.28), the disease activity in the JCA patient group (p = 0.24) and NSAID administration (p = 0.210) did not seem to significantly influence the L/M ratio.



All of the subtypes of juvenile chronic arthritides that we studied displayed an increased IP. Hence, gut wall inflammation (albeit asymptomatic) may also be present in o-JIA patients. The SpA-IBD patients with gastrointestinal symptoms displayed the highest mean L/M ratio values. The L/M test seemed to correlate with histopathological features of the gut mucosa. The L/M ratio was shown to be a highly sensitive but poorly specific test for predicting gut inflammatory disease compared to other non-invasive screening tests.

title: PReS-FINAL-2160: Intestinal microbiome in polyarticular juvenile idiopathic arthritis: a pilot study
abstract: The intestinal microbiome may play a role in the pathogenesis of Juvenile Idiopathic Arthritis (JIA). In IBD patients an overall decrease in microbial diversity of the intestinal microbiota has been observed. Studies comparing intestinal microbiome in children with JIA and healthy controls have not been conducted to date.

title: Antibiotics and the developing infant gut microbiota and resistome.
abstract: The microbial communities colonizing the human gut are tremendously diverse and highly personal. The composition and function of the microbiota play important roles in human health and disease, and considerable research has focused on understanding the ecological forces shaping these communities. While it is clear that factors such as diet, genotype of the host, and environment influence the adult gut microbiota community composition, recent work has emphasized the importance of early-life assembly dynamics in both the immediate and long-term personalized nature of the gut microbiota. While the mature adult gut microbiota is believed to be relatively stable, the developing infant gut microbiota (IGM) is highly dynamic and prone to disruption by external factors, including antibiotic exposure. Studies have revealed both transient and persistent alterations to the adult gut microbiota community resulting from antibiotic treatment later in life. As antibiotics are routinely prescribed at a greater rate in the first years of life, the impact of these interventions on the developing IGM is emerging as a key research priority. In addition to understanding the impact of these disruptions on the infant gut microbial architecture and related host diseases, we need to understand the contribution of early life antibiotics to the selection of antibiotic resistance gene reservoirs in the microbiota, and their threat to successful treatment of infectious disease. Here we review the current understanding of the developmental progression of the IGM and the impact of antibiotic therapies on its composition and encoded reservoir of antibiotic resistance genes.

title: Effect of antimicrobial agents on the ecological balance of human microflora.
abstract: The normal microflora acts as a barrier against colonisation of potentially pathogenic microorganisms and against overgrowth of already present opportunistic microorganisms. Control of growth of opportunistic microorganisms is termed colonisation resistance. Administration of antimicrobial agents, therapeutically or as prophylaxis, causes disturbances in the ecological balance between the host and the normal microflora. Most studies on the impact of antimicrobial agents on normal microflora have been carried out on the intestinal flora. Less is known on the effects on oropharyngeal, skin, and vaginal microflora. Disturbances in the microflora depend on the properties of the agents as well as of the absorption, route of elimination, and possible enzymatic inactivation and/or binding to faecal material of the agents. The clinically most common disturbances in the intestinal microflora are diarrhoea and fungal infections that usually cease after the end of treatment. A well-balanced microflora prevents establishment of resistant microbial strains. By using antimicrobial agents that do not disturb colonisation resistance, the risk of emergence and spread of resistant strains between patients and dissemination of resistant determinants between microorganisms is reduced. In this article, the potential ecological effects of administration of antimicrobial agents on the intestinal, oropharyngeal, and vaginal microflora are summarised. The review is based on clinical studies published during the past 10 years.

title: Clindamycin-induced enrichment and long-term persistence of resistant Bacteroides spp. and resistance genes.
abstract: The aim was to study the long-term consequences of 1 week clindamycin administration regarding selection and persistence of resistance, resistance determinants and diversity of the Bacteroides spp. in the intestinal microflora.



A total of 1306 Bacteroides isolates were collected from constitutively cultured faecal samples during a 2 year period from eight healthy volunteers. The strains were identified by biochemical and genotyping methods. MIC values were determined by the agar dilution method and presence of resistance genes was screened by real-time PCR.



Ecological changes in the intestinal microflora persisting up to 24 months were recorded after a 7 day clindamycin administration to four healthy volunteers. Compared to a control group, not exposed to clindamycin, an enrichment and stabilization of resistant Bacteroides strains and resistance determinants were discovered up to 2 years after clindamycin exposure.



The results indicate that even a short-term antibiotic administration can cause long-term alterations in the commensal microbiota of individual subjects, detectable 2 years after dosing. The recorded selection and persistence of resistant strains and resistance genes, illustrates the importance of increasing our knowledge of the role of the abundant intestinal microbial community as a reservoir for spread of resistance.

title: The Toll-Like Receptor 2 Pathway Establishes Colonization by a Commensal of the Human Microbiota
abstract: Signaling through innate immune receptors promotes commensal bacteria colonization of the gut. Mucosal surfaces constantly encounter microbes. Toll-like receptors (TLRs) mediate recognition of microbial patterns to eliminate pathogens. By contrast, we demonstrate that the prominent gut commensal Bacteroides fragilis activates the TLR pathway to establish host-microbial symbiosis. TLR2 on CD4+ T cells is required for B. fragilis colonization of a unique mucosal niche in mice during homeostasis. A symbiosis factor (PSA, polysaccharide A) of B. fragilis signals through TLR2 directly on Foxp3+ regulatory T cells to promote immunologic tolerance. B. fragilis lacking PSA is unable to restrain T helper 17 cell responses and is defective in niche-specific mucosal colonization. Therefore, commensal bacteria exploit the TLR pathway to actively suppress immunity. We propose that the immune system can discriminate between pathogens and the microbiota through recognition of symbiotic bacterial molecules in a process that engenders commensal colonization.

title: Differential Effects of Antibiotic Therapy on the Structure and Function of Human Gut Microbiota
abstract: The human intestinal microbiota performs many essential functions for the host. Antimicrobial agents, such as antibiotics (AB), are also known to disturb microbial community equilibrium, thereby having an impact on human physiology. While an increasing number of studies investigate the effects of AB usage on changes in human gut microbiota biodiversity, its functional effects are still poorly understood. We performed a follow-up study to explore the effect of ABs with different modes of action on human gut microbiota composition and function. Four individuals were treated with different antibiotics and samples were taken before, during and after the AB course for all of them. Changes in the total and in the active (growing) microbiota as well as the functional changes were addressed by 16S rRNA gene and metagenomic 454-based pyrosequencing approaches. We have found that the class of antibiotic, particularly its antimicrobial effect and mode of action, played an important role in modulating the gut microbiota composition and function. Furthermore, analysis of the resistome suggested that oscillatory dynamics are not only due to antibiotic-target resistance, but also to fluctuations in the surviving bacterial community. Our results indicated that the effect of AB on the human gut microbiota relates to the interaction of several factors, principally the properties of the antimicrobial agent, and the structure, functions and resistance genes of the microbial community.

title: Paediatric rheumatology--a global perspective.
abstract: This chapter aims to give a global perspective to paediatric rheumatology. The main points covered are the incidence, recognition of paediatric autoimmune diseases, and ethnic/geographic distribution. The most prevalent disease is juvenile idiopathic arthritis; robust data are still required for childhood-onset systemic lupus erythematosus, dermatomyositis, and scleroderma. Mimicking or overlapping infections are a major challenge in developing countries, and immunization policies in our patients in these areas need specific attention. The delivery of paediatric rheumatology care is also overviewed. Discrepancies in health-care resources and priorities are found in developing countries. Although most anti-rheumatic treatments are available worldwide, they are prohibitively expensive in many countries. For more traditional anti-rheumatic drugs there is still an ongoing need for good core outcome data across the world to ensure valid comparisons. Parent/patient education has been implemented worldwide in paediatric rheumatology through the power of the Internet. Physician and undergraduate training goals must be met to facilitate competent musculoskeletal assessment, a proper understanding of age-dependent variations, diagnosis, referral to specialists, and improved standards of care.

title: Human gut microbiome viewed across age and geography
abstract: Gut microbial communities represent one source of human genetic and metabolic diversity. To examine how gut microbiomes differ among human populations, here we characterize bacterial species in fecal samples from 531 individuals, plus the gene content of 110 of them. The cohort encompassed healthy children and adults from the Amazonas of Venezuela, rural Malawi and US metropolitan areas and included mono- and dizygotic twins. Shared features of the functional maturation of the gut microbiome were identified during the first three years of life in all three populations, including age-associated changes in the genes involved in vitamin biosynthesis and metabolism. Pronounced differences in bacterial assemblages and functional gene repertoires were noted between US residents and those in the other two countries. These distinctive features are evident in early infancy as well as adulthood. Our findings underscore the need to consider the microbiome when evaluating human development, nutritional needs, physiological variations and the impact of westernization.

title: Review: Microbiome in Inflammatory Arthritis and Human Rheumatic Diseases
abstract: Ever since its modern conception as a medical discipline, the study of microorganisms has paralleled the many technological advances in microbiology. In the 17th century, the inventor of the microscope, Antony van Leeuwenhoek, was also the first to describe—in the plaque of his own gums—the millions of microorganisms (or “animalcules”) that reside within us. It is that multitude of microbes populating most human body cavities and surfaces, including its genetic and enzymatic composition, that defines our microbiome (1). For centuries, the role of the microbiome as a potential determinant to health and disease has been rather ignored. This has been true in most fields of human research, but particularly so in autoimmune and rheumatic conditions. The reasons are multifactorial. Chief among those was the advent of Koch’s postulates in the late 1800s, which exerted a profound influence on how investigators thought about causality in medicine (2). Unknown at the time, however, were the facts that asymptomatic carriers are a common feature of many infectious diseases and that several microorganisms are fastidious in nature, with complex nutritional requirements for growth. The latter fundamentally prevented the study of bacteria within the context of a dynamic biologic community, the role of commensal taxa, the downstream molecular events, and the resulting immune interactions between microorganisms and their host. Consequently, only the prevalent microbiologic techniques in the past were used to characterize unique agents capable of triggering clinical rheumatic syndromes. Over time, the search led to correlative studies of specific bacteria and viruses in the etiopathogenesis of these disorders, most notably rheumatoid arthritis (RA), psoriasis, inflammatory bowel disease (IBD), and the related spondyloarthritides (SpA), including ankylosing spondylitis (AS) and reactive arthritis (ReA). The revolution of culture-independent, highthroughput microbial DNA sequencing, in parallel with the resurgence of interest in and further understanding of mucosal immunity, has exponentially advanced our knowledge of the interplay between our microbes and ourselves. That profound, bidirectional interaction and its consequences in physiology and disease have led to a whole new field of research. Despite the relative novelty of the human microbiome as a discipline, a substantial body of evidence has accumulated addressing its potential involvement in the pathogenesis of rheumatic disease (3). In this article, we will critically review the available data from animal and human studies, focusing on the role of the intestinal microbiome in RA, psoriatic arthritis (PsA), and SpA. The role of the microbiome in autoimmunity and in other rheumatic diseases has been reviewed elsewhere (4,5) and is beyond the scope of this article. We include a description of the prospects of microbiome manipulation for therapeutic purposes and conclude by delineating challenges and opportunities in the field. Supported by the NIH (National Institute of Arthritis and Musculoskeletal and Skin Diseases grants K23-AR-064318 to Dr. Scher and RC2-AR-058986 through the American Recovery and Reinvestment Act of 2009 to Drs. Littman and Abramson), the Judith and Stewart Colton Center for Autoimmunity, and the Riley Family Foundation.  U. Scher, MD, Steven B. Abramson, MD: New York University School of Medicine and New York University Hospital for Joint Diseases, New York, New York; Dan R. Littman, MD, PhD: Kimmel Center for Biology and Medicine of the Skirball Institute and New York University School of Medicine, New York, New York. Drs. Scher, Littman, and Abramson have a patent application pending on microbiota and byproducts for diagnostics and therapeutics in rheumatic disease. Address correspondence to Jose U. Scher, MD, Division of Rheumatology, New York University Hospital for Joint Diseases, 301 East 17th Street, Room 1608, New York, NY 10003.  2015; accepted in revised form June 23, 2015.

title: Treating Clostridium difficile infection with fecal microbiota transplantation.
abstract: Clostridium difficile infection is increasing in incidence, severity, and mortality. Treatment options are limited and appear to be losing efficacy. Recurrent disease is especially challenging; extended treatment with oral vancomycin is becoming increasingly common but is expensive. Fecal microbiota transplantation is safe, inexpensive, and effective; according to case and small series reports, about 90% of patients are cured. We discuss the rationale, methods, and use of fecal microbiota transplantation.

title: Targeted Restoration of the Intestinal Microbiota with a Simple, Defined Bacteriotherapy Resolves Relapsing Clostridium difficile Disease in Mice
abstract: Relapsing C. difficile disease in humans is linked to a pathological imbalance within the intestinal microbiota, termed dysbiosis, which remains poorly understood. We show that mice infected with epidemic C. difficile (genotype 027/BI) develop highly contagious, chronic intestinal disease and persistent dysbiosis characterized by a distinct, simplified microbiota containing opportunistic pathogens and altered metabolite production. Chronic C. difficile 027/BI infection was refractory to vancomycin treatment leading to relapsing disease. In contrast, treatment of C. difficile 027/BI infected mice with feces from healthy mice rapidly restored a diverse, healthy microbiota and resolved C. difficile disease and contagiousness. We used this model to identify a simple mixture of six phylogenetically diverse intestinal bacteria, including novel species, which can re-establish a health-associated microbiota and clear C. difficile 027/BI infection from mice. Thus, targeting a dysbiotic microbiota with a defined mixture of phylogenetically diverse bacteria can trigger major shifts in the microbial community structure that displaces C. difficile and, as a result, resolves disease and contagiousness. Further, we demonstrate a rational approach to harness the therapeutic potential of health-associated microbial communities to treat C. difficile disease and potentially other forms of intestinal dysbiosis.