Molecular players involved in the interaction between beneficial bacteria and the immune system

The human gastrointestinal tract is a very complex ecosystem, in which there is a continuous interaction between nutrients, host cells, and microorganisms. The gut microbiota comprises trillions of microbes that have been selected during evolution on the basis of their functionality and capacity to survive in, and adapt to, the intestinal environment. Host bacteria and our immune system constantly sense and react to one another. In this regard, commensal microbes contribute to gut homeostasis, whereas the necessary responses are triggered against enteropathogens. Some representatives of our gut microbiota have beneficial effects on human health. Some of the most important roles of these microbes are to help to maintain the integrity of the mucosal barrier, to provide nutrients such as vitamins, or to protect against pathogens. In addition, the interaction between commensal microbiota and the mucosal immune system is crucial for proper immune function. This process is mainly performed via the pattern recognition receptors of epithelial cells, such as Toll-like or Nod-like receptors, which are able to recognize the molecular effectors that are produced by intestinal microbes. These effectors mediate processes that can ameliorate certain inflammatory gut disorders, discriminate between beneficial and pathogenic bacteria, or increase the number of immune cells or their pattern recognition receptors (PRRs). This review intends to summarize the molecular players produced by probiotic bacteria, notably Lactobacillus and Bifidobacterium strains, but also other very promising potential probiotics, which affect the human immune system. This Document is Protected by copyright and was first published by Frontiers. All rights reserved. it is reproduced with permission.This research was funded by grants AGL2013-44039-R and AGL2013-44761-P from the Spanish “Plan Estatal de I+D+I.” BS and AH were recipients of a Ramón y Cajal postdoctoral contract and a Formación de Personal Investigador—Research Personnel Training (FPI) grant, respectively, from the Spanish Ministry of Economy and Competitiveness.Peer Reviewe

Interaction of intestinal microorganisms with the human host in the framework of autoimmune diseases

Autoimmune diseases, such as systemic lupus erythematosus (SLE), are caused by a complex interaction of environmental-, genetic-, and sex-related factors. Although SLE has traditionally been considered independent from the microbiota, recent work published during the last 5 years suggests a strong connection between SLE and the composition of our gut commensals as one of the main environmental factors linked to this disease. Preliminary data have evidenced that (i) interaction of certain microbial-derived molecules with specific cell receptors and (ii) the influence of certain commensal microorganisms over specific immune cell subsets plays an important role in the pathogenesis of SLE and SLE-like diseases. In addition, epigenetic changes driven by certain microbial groups have been recently proposed as an additional link between gut microbiota and SLE. As immune responses elicited against commensal bacteria are deeply dependent on the composition of the latter, and as microbial populations can be modified by dietary interventions, identifying the precise gut microorganisms responsible for worsening the SLE symptoms is of crucial importance for this and other SLE-related diseases, including antiphospholipid syndrome or lupus nephritis. In this minireview, the current knowledge on the relationships between microbes and SLE and SLE-related diseases is compiled and discussed.This research was funded by Grants AGL2013-44039-R and AGL2010-14952 from the Spanish “Plan Estatal de I + D + I”. BS and AH were recipients of a Ramón y Cajal postdoctoral contract and a FPI grant, respectively, from the Spanish Ministry of Economy and Competitiveness.Peer Reviewe

Bifidobacteria exhibit social behavior through carbohydrate resource sharing in the gut

Bifidobacteria are common and frequently dominant members of the gut microbiota of many animals, including mammals and insects. Carbohydrates are considered key carbon sources for the gut microbiota, imposing strong selective pressure on the complex microbial consortium of the gut. Despite its importance, the genetic traits that facilitate carbohydrate utilization by gut microbiota members are still poorly characterized. Here, genome analyses of 47 representative Bifidobacterium (sub)species revealed the genes predicted to be required for the degradation and internalization of a wide range of carbohydrates, outnumbering those found in many other gut microbiota members. The glycan-degrading abilities of bifidobacteria are believed to reflect available carbon sources in the mammalian gut. Furthermore, transcriptome profiling of bifidobacterial genomes supported the involvement of various chromosomal loci in glycan metabolism. The widespread occurrence of bifidobacterial saccharolytic features is in line with metagenomic and metatranscriptomic datasets obtained from human adult/infant faecal samples, thereby supporting the notion that bifidobacteria expand the human glycobiome. This study also underscores the hypothesis of saccharidic resource sharing among bifidobacteria through species-specific metabolic specialization and cross feeding, thereby forging trophic relationships between members of the gut microbiota.We thank GenProbio srl for financial support of the Laboratory of Probiogenomics. This work was financially supported by a FEMS Jensen Award to FT, and by a Ph.D. fellowship (Spinner 2013, Regione Emilia Romagna) to S.D. DvS and FT are members of The APC Microbiome Institute, while DvS is also a member of the Alimentary Glycoscience Research Cluster, both funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan (Grant numbers SFI/12/RC/2273 and 08/SRC/B1393, respectively). This work was also partially supported by Fondazione Caritro, by the EU FP7 (PCIG13- GA-2013-618833), and by MIUR “Futuro in Ricerca” E68C13000500001 to NS. Furthermore, this project has been funded in part with funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272200900018C.Peer Reviewe

Association of levels of antibodies from patients with inflammatory bowel disease with extracellular proteins of food and probiotic bacteria

Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG) developed against extracellular protein fractions produced by different food bacteria from the genera Bifidobacterium and Lactobacillus. We used a sera collection consisting of healthy individuals (HC, n=50), Crohn's disease patients (CD, n=37), and ulcerative colitis patients (UC, n=15). Levels of IgA antibodies developed against a cell-wall hydrolase from Lactobacillus casei subsp. rhamnosus GG (CWH) were significantly higher in the IBD group (P<0.002; n=52). The specificity of our measurements was confirmed by measuring IgA antibodies developed against the CWH peptide 365-VNTSNQTAAVSAS- 377. IBD patients appeared to have different immune response to food bacteria. This paper sets the basis for developing systems for early detection of IBD, based on the association of high levels of antibodies developed against extracellular proteins from food and probiotic bacteria. © 2014 Arancha Hevia et al.Borja Sánchez and Arancha Hevia were recipients of a Ramón y Cajal postdoctoral contract and a FPI Grant, respectively, from the Spanish Ministry of Economy and Competitiveness. Research in our group is supported by Grants AGL2010-14952 and RM2010-00012-00-00 from the Spanish Plan Nacional de I+D.Peer Reviewe

Human colon-derived soluble factors modulate gut microbiota composition

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).-- et al.The commensal microbiota modulates immunological and metabolic aspects of the intestinal mucosa contributing to development of human gut diseases including inflammatory bowel disease. The host/microbiota interaction often referred to as a crosstalk, mainly focuses on the effect of the microbiota on the host neglecting effects that the host could elicit on the commensals. Colonic microenvironments from three human healthy controls (obtained from the proximal and distal colon, both in resting conditions and after immune - IL-15-and microbiota - LPS-in vitro challenges) were used to condition a stable fecal population. Subsequent 16S rRNA gene-based analyses were performed to study the effect induced by the host on the microbiota composition and function. Non-supervised principal component analysis (PCA) showed that all microbiotas, which had been conditioned with colonic microenvironments clustered together in terms of relative microbial composition, suggesting that soluble factors were modulating a stable fecal population independently from the treatment or the origin. Our findings confirmed that the host intestinal microenvironment has the capacity to modulate the gut microbiota composition via yet unidentified soluble factors. These findings indicate that an appropriate understanding of the factors of the host mucosal microenvironment affecting microbiota composition and function could improve therapeutic manipulation of the microbiota composition.BS and AH were recipients of a Ramón y Cajal postdoctoral contract and a FPI grant, respectively, from the Spanish Ministry of Economy and Competitiveness. The authors gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC). This research was funded by the BBSRC Institute Strategic Programme for Gut Health and Food Safety BB/J004529/1. This research was also funded by Grants AGL2010-14952 and AGL2013-44039-R from the Spanish “Plan Estatal de I + D + i,” and by Grant EM2014/046 from the “Plan galego de investigación, innovación e crecemento 2011-2015.”Peer Reviewe

Allergic patients with long-term asthma display low levels of bifidobacterium adolescentis

Accumulated evidence suggests a relationship between specific allergic processes, such as atopic eczema in children, and an aberrant fecal microbiota. However, little is known about the complete microbiota profile of adult individuals suffering from asthma. We determined the fecal microbiota in 21 adult patients suffering allergic asthma (age 39.43 ± 10.98 years old) and compare it with the fecal microbiota of 22 healthy controls (age 39.29 ± 9.21 years old) using culture independent techniques. An Ion-Torrent 16S rRNA gene-based amplification and sequencing protocol was used to determine the fecal microbiota profile of the individuals. Sequence microbiota analysis showed that the microbial alpha-diversity was not significantly different between healthy and allergic individuals and no clear clustering of the samples was obtained using an unsupervised principal component analysis. However, the analysis of specific bacterial groups allowed us to detect significantly lower levels of bifidobacteria in patients with long-term asthma. Also, in allergic individuals the Bifidobacterium adolescentis species prevailed within the bifidobacterial population. The reduction in the levels on bifidobacteria in patients with long-term asthma suggests a new target in allergy research and opens possibilities for the therapeutic modulation of the gut microbiota in this group of patients

Ranking the impact of human health disorders on gut metabolism: Systemic lupus erythematosus and obesity as study cases

Multiple factors have been shown to alter intestinal microbial diversity. It remains to be seen, however, how multiple collective pressures impact the activity in the gut environment and which, if any, is positioned as a dominant driving factor determining the final metabolic outcomes. Here, we describe the results of a metabolome-wide scan of gut microbiota in 18 subjects with systemic lupus erythematosus (SLE) and 17 healthy control subjects and demonstrate a statistically significant difference (p < 0.05) between the two groups. Healthy controls could be categorized (p < 0.05) based on their body mass index (BMI), whereas individuals with SLE could not. We discuss the prevalence of SLE compared with BMI as the dominant factor that regulates gastrointestinal microbial metabolism and provide plausible explanatory causes. Our results uncover novel perspectives with clinical relevance for human biology. In particular, we rank the importance of various pathophysiologies for gut homeostasis.The present investigation was funded by the Spanish Ministry of Economy and Competitiveness and the Federal Ministry of Education and Research (BMBF) within the ERA NET PathoGenoMics2 program, grant number 0315441A. This work was further funded by grants BFU2008-04501-E, BFU2008-04398-E, SAF2009-13032-C02-01, SAF2012-31187 and CSD2007-00005, BIO2011-25012, AGL2010-14952 and AGL2006-11697/ALI from the Spanish Ministry of Economy and Competitiveness, and Prometeo/2009/092 from Generalitat Valenciana (Spain). The authors gratefully acknowledge the financial support provided by the European Regional Development Fund (ERDF)

Intestinal dysbiosis associated with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is the prototypical systemic autoimmune disease in humans and is characterized by the presence of hyperactive immune cells and aberrant antibody responses to nuclear and cytoplasmic antigens, including characteristic anti-double-stranded DNA antibodies. We performed a cross-sectional study in order to determine if an SLE-associated gut dysbiosis exists in patients without active disease. A group of 20 SLE patients in remission, for which there was strict inclusion and exclusion criteria, was recruited, and we used an optimized Ion Torrent 16S rRNA gene-based analysis protocol to decipher the fecal microbial profiles of these patients and compare them with those of 20 age- and sex-matched healthy control subjects. We found diversity to be comparable based on Shannon's index. However, we saw a significantly lower Firmicutes/Bacteroidetes ratio in SLE individuals (median ratio, 1.97) than in healthy subjects (median ratio, 4.86; P < 0.002). A lower Firmicutes/Bacteroidetes ratio in SLE individuals was corroborated by quantitative PCR analysis. Notably, a decrease of some Firmicutes families was also detected. This dysbiosis is reflected, based on in silico functional inference, in an overrepresentation of oxidative phosphorylation and glycan utilization pathways in SLE patient microbiota. IMPORTANCE: Growing evidence suggests that the gut microbiota might impact symptoms and progression of some autoimmune diseases. However, how and why this microbial community influences SLE remains to be elucidated. This is the first report describing an SLE-associated intestinal dysbiosis, and it contributes to the understanding of the interplay between the intestinal microbiota and the host in autoimmune disorders

Caracterización funcional de la microbiota intestinal en algunos trastornos inmunológicos

Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de Oviedo.--2016-06-03 Trabajo disponible en: http://hdl.handle.net/10651/38993La microbiota intestinal es el conjunto de microorganismos que habita en inmunológicoel intestino humano de manera natural, estableciendo una relación estrecha con el hospedador que la alberga, al que le proporciona una serie de beneficios entre los que cabe destacar la función barrera contra bacterias potencialmente patógenas, el aprovechamiento de energía de alimentos no digeribles, y la maduración y desarrollo del sistema inmunológico. Hasta el momento, tanto estudios en humanos como en animales han demostrado que cambios en las poblaciones microbianas intestinales pueden tener consecuencias para la salud, y existen numerosos trabajos científicos que relacionan a una microbiota intestinal alterada (disbiosis intestinal), con procesos inflamatorios, asma alérgicos, enfermedades autoinmunes e incluso cáncer. El objetivo general de esta Tesis Doctoral es generar nuevo conocimiento sobre la composición de la microbiota intestinal en el marco de dos trastornos inmunológicos, el lupus eritematoso sistémico (LES) y el asma. A través del uso de técnicas independientes de cultivo, se analizaron muestras fecales y se determinó el perfil microbiano de grupos de individuos que padecían estas enfermedades, comparándolo con el de individuos sanos. De esta manera los resultados de esta Tesis Doctoral han contribuido a entender la relación entre la disbiosis intestinal que presenta la microbiota de estos individuos y su estado inmunológico. Para el caso del LES se estudiaron 20 mujeres de entre 34 y 68 años de edad, y se compararon con un grupo control sano. En asma alérgico se analizaron muestras de 21 pacientes de edades entre 24 y 57 años y se compararon con un grupo de 22 controles sanos de mismo sexo y rango de edad. Durante la primera parte de esta Tesis se optimizaron metodologías y protocolos de tratamiento de muestras fecales humanas.Peer reviewe

Caracterización funcional de la microbiota intestinal en algunos trastornos inmunológicos

Tesis con mención internacional. Tesis doctoral por el sistema de compendio de publicacionesLa microbiota intestinal es el conjunto de microorganismos que habita en inmunológicoel intestino humano de manera natural, estableciendo una relación estrecha con el hospedador que la alberga, al que le proporciona una serie de beneficios entre los que cabe destacar la función barrera contra bacterias potencialmente patógenas, el aprovechamiento de energía de alimentos no digeribles, y la maduración y desarrollo del sistema inmunológico. Hasta el momento, tanto estudios en humanos como en animales han demostrado que cambios en las poblaciones microbianas intestinales pueden tener consecuencias para la salud, y existen numerosos trabajos científicos que relacionan a una microbiota intestinal alterada (disbiosis intestinal), con procesos inflamatorios, asma alérgicos, enfermedades autoinmunes e incluso cáncer. El objetivo general de esta Tesis Doctoral es generar nuevo conocimiento sobre la composición de la microbiota intestinal en el marco de dos trastornos inmunológicos, el lupus eritematoso sistémico (LES) y el asma. A través del uso de técnicas independientes de cultivo, se analizaron muestras fecales y se determinó el perfil microbiano de grupos de individuos que padecían estas enfermedades, comparándolo con el de individuos sanos. De esta manera los resultados de esta Tesis Doctoral han contribuido a entender la relación entre la disbiosis intestinal que presenta la microbiota de estos individuos y su estado inmunológico. Para el caso del LES se estudiaron 20 mujeres de entre 34 y 68 años de edad, y se compararon con un grupo control sano. En asma alérgico se analizaron muestras de 21 pacientes de edades entre 24 y 57 años y se compararon con un grupo de 22 controles sanos de mismo sexo y rango de edad. Durante la primera parte de esta Tesis se optimizaron metodologías y protocolos de tratamiento de muestras fecales humanas. Posteriormente se extrajo el ADN de muestras fecales de los distintos individuos para establecer los perfiles de composición microbianos de cada grupo (LES y asma) mediante la amplificación y análisis de la secuencia del gen del ARN ribosomal 16S bacteriano. En el grupo de LES se obtuvieron niveles diferentes de los dos grupos bacterianos mayoritarios que colonizan el intestino, Bacteroidetes y Firmicutes, con un ratio Firmicutes/Bacteroidetes mucho menor que para los controles sanos. Por otro lado se realizó la cuantificación de metabolitos producidos por la microbiota intestinal de individuos con LES, en comparación con el grupo control, mediante metabolómica, observándose un pequeño grupo de compuestos relacionados con estrés oxidativo producidos por la microbiota de LES. También se observaron diferencias entre individuos sanos de alto y bajo índice de masa corporal, factor que no fue determinante en el caso del LES. Por otro lado, en el grupo de asmáticos no hallamos diferencias significativas en la biodiversidad microbiana en comparación con un grupo de controles sanos, ni tampoco en las abundancias relativas de ningún grupo taxonómico salvo para la especie Bifidobacterium adolescentis, para la que se observaron niveles menores en pacientes con respecto a la duración del proceso asmático. Por último, y mediante experimentos in vitro, se observó que la microbiota aislada de pacientes con LES induce una respuesta Th17, y que la suplementación de esta microbiota con Bifidobacterium bifidum o con una mezcla de las especies comensales Ruminococcus obeum y Blautia coccoides, corrigió en cierta medida el desequilibrio inmunológico, bien reduciendo la diferenciación de células T vírgenes hacia Thelper en el caso de la bifidobacteria, o bien a través de una reducción dosis-dependiente del ratio IL-17/IFNɣ en el caso de la mezcla de Firmicutes, lo cual se interpretó como una reducción de la diferenciación de células T vírgenes a células Th1.   SUMMARY Gut microbiota is the global community of microorganisms that naturally inhabit the human gastrointestinal tract, establishing tight interactions with their host and exerting a great number of benefits. For instance, it contributes to the gut barrier function inhibiting the colonization by potential bacterial pathogens, to energy harvesting from non-digestible foods, and to the development and maturation of the immune system. Several human and animal model studies have shown some changes in the gut bacterial populations that can have important consequences in health, and there is a broad range of scientific works relating an imbalanced intestinal microbiota, also known as dysbiosis, with inflammatory processes, allergy, autoimmune diseases or even cancer. The main aim of this Doctoral Thesis was the generation of novel knowledge about the gut microbiota composition in the frame of two immune disorders, systemic lupus erythematosus (SLE), and allergic asthma. By means of culture-independent techniques, fecal samples from the individuals of both groups were analyzed and compared with healthy controls. In this way we have contributed with this Thesis to understand the relationship between gut dysbiosis and the immunological state of the host. In the SLE group we analyzed a group of 20 women aged of 34-68 years, and their gut microbiotas were compared with a control group of 20 healthy women. For the allergic asthma group, the intestinal microbiota of 21 patients, men and women, whose age was comprised between 24 and 57 years old was analyzed and compared with a control group of 22 healthy individuals of the same sex and age ranges. First of all we optimized the methodologies and protocols to analyze the fecal microbiota in SLE and allergy. We set up a protocol to separate the fecal microbiota from its complex matrix without altering its microbial composition significantly, and we also extracted the DNA from the samples to establish the bacterial profiles of each population group, using a 16S rARN gene-based profiling. In the SLE group we obtained different bacterial abundances within the Phyla Bacteroidetes (B) and Firmicutes (F), and a decreased F/B ratio compared to healthy controls. We also quantified metabolites produced by the microbiota of SLE and we observed higher levels of a small group of metabolites that were related to oxidative stress; we also identified some differences in metabolites produced by healthy controls with high and low body mass index. In the allergic asthma group we found no significant differences in the biodiversity of the gut microbiota compared with controls. However, we observed decreased abundancies of Bifidobacterium adolescentis in individuals with long-term asthma. We also found, in co-culture in vitro experiments, that the isolated microbiota of SLE induces a Th17 immune response, and that the supplementation with Bifidobacterium bifidum and with a mix of the commensal bacteria Ruminococcus obeum y Blautia coccoides can revert the immunological state of SLE, by reducing T cell differentiation to Thelper, or inducing a dose-dependent decrease in the IL-17/IFNɣ proinflammatory ratio