Distinct Microbial Taxa Are Associated with LDL-Cholesterol Reduction after 12 Weeks of Lactobacillus plantarum Intake in Mild Hypercholesterolemia: Results of a Randomized Controlled Study

Probiotic microbes such as Lactobacillus may reduce serum total cholesterol (TC) and low-density lipoprotein (LDL) cholesterol. The objective of this study was to assess the effect of Lactobacillus plantarum strains CECT7527, CECT7528, and CECT7529 (LP) on the serum lipids, cardiovascular parameters, and fecal gut microbiota composition in patients with mild hypercholesterolemia. A randomized, double-blinded, placebo-controlled clinical trial with 86 healthy adult participants with untreated elevated LDL cholesterol ≥ 160 mg/dl was conducted. Participants were randomly allocated to either placebo or LP (1.2 × 109 CFU/d) for 12 weeks. LDL, HDL, TC, and triglycerides (TG), cardiovascular parameters (blood pressure, arterial stiffness), and fecal gut microbiota composition (16S rRNA gene sequencing) were assessed at baseline and after 12 weeks. Both groups were comparable regarding age, sex, and LDL-C at baseline. LDL-C decreased (mean decrease − 6.6 mg/dl ± − 14.0 mg/dl, P time*group = 0.006) in the LP group but not in the placebo group. No effects were observed on HDL, TG, or cardiovascular parameters or overall gut microbiota composition. Responders to LP intervention (> 5% LDL-C reduction) were characterized by higher BMI, pronounced TC reduction, higher abundance of fecal Roseburia, and lower abundance of Oscillibacter. In conclusion, 12 weeks of L. plantarum intake moderately reduced LDL-C and TC as compared to placebo. LDL-C-lowering efficacy of L. plantarum strains may potentially be dependent on individual difference in the gut microbiota. Trial registration: DRKS00020384, dated 07/01/2020

Characterization of serum biomarkers and antibody responses against Prevotella spp. in preclinical and new-onset phase of rheumatic diseases

IntroductionThe characterization of the influence of the microbiota on the development and drug responses during rheumatic diseases has intensified in recent years. The role of specific bacteria during disease development has become a central research question. Notably, several lines of evidence point to distinct microbes, e.g., Prevotella copri (P. copri) being targeted by antibodies in clinical phases of rheumatic diseases.MethodsIn the present study, we compiled a broad collection of human serum samples from individuals at risk of developing RA, chronic RA patients as well as patients with new-onset of rheumatic diseases. We evaluated the presence of inflammatory biomarkers in our serum collection as well as serum antibody responses against novel, genetically distinct isolates of P. copri and several oral pathobionts.ResultsOur analysis revealed the presence of increased levels of inflammatory markers already in pre-clinical and new onset rheumatoid arthritis. However, antibody reactivity against the microbes did not differ between patient groups. Yet, we observed high variability between the different P. copri strains. We found total serum IgG levels to slightly correlate with IgG antibody responses against P. copri, but no relation between the latter and presence or prevalence of P. copri in the intestine.DiscussionIn conclusion, our work underlined the importance of strain-level characterization and its consideration during further investigations of host-microbiota interactions and the development of microbiome-based therapeutic approaches for treating rheumatic diseases

Extension of the Segatella copri complex to 13 species with distinct large extrachromosomal elements and associations with host conditions

The Segatella copri (formerly Prevotella copri) complex (ScC) comprises taxa that are key members of the human gut microbiome. It was previously described to contain four distinct phylogenetic clades. Combining targeted isolation with large-scale metagenomic analysis, we defined 13 distinct Segatella copri-related species, expanding the ScC complex beyond four clades. Complete genome reconstruction of thirteen strains from seven species unveiled the presence of genetically diverse large circular extrachromosomal elements. These elements are consistently present in most ScC species, contributing to intra- and inter-species diversities. The nine species-level clades present in humans display striking differences in prevalence and intraspecies genetic makeup across human populations. Based on a meta-analysis, we found reproducible associations between members of ScC and the male sex and positive correlations with lower visceral fat and favorable markers of cardiometabolic health. Our work uncovers genomic diversity within ScC, facilitating a better characterization of the human microbiome

Untersuchung des Einflusses der Darmmikrobiota auf Krankheitsentstehung und Behandlungserfolg verschiedener rheumatischer Erkrankungen

Rheumatic diseases encompass multiple distinct disorders and affect millions of people worldwide. Rheumatic diseases represent a significant burden for the society and are expected to increase in the coming years due to demographic aging, increasing obesity in the population and other risk factors. It is therefore crucial to develop effective strategies for managing these diseases. Substantial advances in understanding the pathophysiology of rheumatic diseases have been made during the last years. However, the major challenge in the field of rheumatology remains the identification of disease etiology and risk factors as well as reliable biomarkers, which facilitate disease diagnosis and more effective, personalized treatment strategies. In recent years, there is emerging evidence for a contribution of the intestinal microbiota to the development of rheumatic diseases. Several studies have linked alterations in the microbial composition to specific rheumatic diseases. In particular, the species Prevotella copri has been associated with the occurrence of rheumatoid arthritis (RA) and has been attributed to possess RA-specific immunostimulatory properties. Yet, whether P. copri is actively involved in RA disease development remains unclear. A detailed characterization of the arthritogenic potential of the P. copri species complex has been limited due to the lack of P. copri strain availability in public collections. In the present work, I established a strain collection comprising multiple genetically distinct isolates from the P. copri species complex by performing targeted isolation from human fecal samples. Using several of the novel isolates, I evaluated their ability to provoke immune responses in the serum of patients with new onset of distinct rheumatic diseases as well as in individuals at risk for developing RA. The results revealed antibody responses to be similar between rheumatic patient and control groups, which suggested P. copri to not have a particular role in rheumatic diseases. Nonetheless, the distinct P. copri isolates were identified to have different immunogenic capacities, which highlighted the wide variability in the overall functional abilities of the species complex. Furthermore, the microbial community structure during disease onset of distinct rheumatic disorders was analyzed using a combination of 16S rRNA gene and metagenomic sequencing. In addition, metagenomic sequencing was applied to assess taxonomic and functional variations in the gut microbiota associated with response to the common anti-rheumatic drug methotrexate (MTX). Our findings revealed a reduction of bacteria producing short chain fatty acids (SCFAs) in new-onset axial spondyloarthritis (axSpA) patients as well as in psoriatic arthritis (PsA) patients with insufficient MTX treatment response. This suggested a beneficial impact of SCFAs and SCFA-producing bacteria in rheumatic diseases. In addition to assessing the bacterial community structure in the gut during disease onset, I performed IgA sequencing to identify bacterial communities actively targeted by IgA in the intestine of new onset rheumatic disease patients. This especially demonstrated patients newly diagnosed with axSpA, PsA and reactive arthritis (ReA) to possess similar IgA-coating profiles, which strikingly differed from RA and non-rheumatic disease (NRD) patients. Altogether, the here performed analyses of microbial compositions during disease onset and treatment of distinct rheumatic diseases together with the investigation of interactions between microbial communities and the immune system of rheumatic patients advances our understanding about the role of gut bacteria in the pathogenesis of rheumatic disorders.Rheumatische Erkrankungen umfassen eine Vielzahl unterschiedlicher Krankheiten und betreffen Millionen von Menschen weltweit. Rheumatische Erkrankungen stellen eine große Belastung für die Gesellschaft dar, und es wird erwartet, dass sie in den kommenden Jahren aufgrund von demografischer Alterung, zunehmendem Übergewicht der Bevölkerung und anderen Risikofaktoren zunehmen wird. Es ist daher von entscheidender Bedeutung, wirksame Strategien für die Behandlung dieser Krankheiten zu entwickeln. In den letzten Jahren wurden erhebliche Fortschritte im Verständnis der Krankheitsentstehung rheumatischer Erkrankungen erzielt. Die größte Herausforderung auf dem Gebiet der Rheumatologie ist jedoch nach wie vor die Identifizierung von Krankheitsursachen und Risikofaktoren sowie von zuverlässigen Biomarkern, die eine Krankheitsdiagnose und wirksamere, personalisierte Behandlungsstrategien ermöglichen. Seit einiger Zeit mehren sich die Hinweise, dass die Darmmikrobiota zur Entstehung rheumatischer Erkrankungen beiträgt. Mehrere Studien haben gezeigt, dass Veränderungen in der mikrobiellen Zusammensetzung mit bestimmten rheumatischen Erkrankungen einhergehen. Insbesondere wird die Spezies Prevotella copri mit dem Auftreten von Rheumatoider Arthritis (RA) in Verbindung gebracht und ihr RA-spezifische, immunstimulierende Eigenschaften zugeschrieben. Ob P. copri jedoch aktiv an der Entstehung von RA beteiligt ist, ist bisher unklar. Eine detaillierte Charakterisierung des arthritogenen Potenzials des P. copri-Arten-Komplexes war bisher nur begrenzt möglich, da lediglich wenige P. copri-Stämme in öffentlichen Sammlungen verfügbar waren. In der vorliegenden Arbeit wurde durch gezielte Isolierung aus humanen Stuhlproben eine P. copri-Stammsammlung erstellt, die mehrere, genetisch unterschiedliche Isolate aus dem P. copri-Arten-Komplex umfasst. Durch die Verwendung einiger dieser neuen Isolate wurde deren Fähigkeit zur Stimulierung von Immunreaktionen im Serum von Patienten mit neu diagnostizierten rheumatischen Erkrankungen sowie bei Personen mit einem Risiko für die Entwicklung von RA untersucht. Hierbei wurde festgestellt, dass die Antikörperreaktionen zwischen Patienten- und Kontrollgruppen ähnlich ausfallen, was darauf hindeutet, dass P. copri keine spezifische Rolle in rheumatischen Erkrankungen einnimmt. Allerdings wurde gezeigt, dass die verschiedenen P. copri-Isolate unterschiedliche immunogene Fähigkeiten besitzen, was die große Variabilität im funktionellen Potential des Arten-Komplexes verdeutlicht. Darüber hinaus wurde in dieser Arbeit die Zusammensetzung der mikrobiellen Gemeinschaft im Darm während des Krankheitsausbruchs verschiedener rheumatischer Erkrankungen mittels einer Kombination aus 16S rRNA-Gen- und Metagenom-Sequenzierung analysiert. Metagenomische Sequenzierung wurde zudem verwendet, um taxonomische und funktionelle Variationen in der Darmmikrobiota im Zusammenhang mit dem Ansprechen auf das Antirheumatikum Methotrexat zu bewerten. Wir konnten zeigen, dass neu-diagnostizierte Patienten mit axialer Spondyloarthritis (axSpA) sowie Psoriasis-Arthritis-Patienten (PsA), die nicht auf MTX-Behandlung ansprechen, eine Verringerung von Bakterien, die kurzkettige Fettsäuren (SCFAs) produzieren, vorweisen. Dies lässt auf eine positive Wirkung von SCFAs und SCFA-produzierenden Bakterien bei rheumatischen Erkrankungen schließen. In dieser Arbeit wurde außerdem IgA-Sequenzierung durchgeführt, um Bakteriengemeinschaften zu identifizieren, die im Darm von Patienten mit neu diagnostizierten rheumatischen Erkrankungen aktiv von IgA erkannt und gebunden werden. Hierbei konnte gezeigt werden, dass die Zusammensetzung an IgA-gebundenen Bakterien in Patienten mit neu diagnostizierter axSpA, PsA und reaktiver Arthritis (ReA) ähnlich sind, diese sich aber von RA-Patienten und Patienten mit nicht-rheumatischen Erkrankungen (NRD) unterscheiden. Insgesamt tragen die hier durchgeführten Analysen der mikrobiellen Zusammensetzung während eines rheumatischen Krankheitsausbruches und medikamentöser Behandlung sowie die Untersuchung der Wechselwirkungen zwischen der Mikrobiota und dem Immunsystem von Rheumapatienten zu einem besseren Verständnis der Rolle von Darmbakterien bei der Pathogenese rheumatischer Erkrankungen bei

Assessment of the Gut Microbiota during Juice Fasting with and without Inulin Supplementation: A Feasibility Study in Healthy Volunteers

Prebiotic inulin consumption provides health benefits to the host and has also been associated with a reduction in hunger cravings. We conducted a pilot crossover study to investigate the feasibility of a juice fasting intervention with and without inulin supplementation. We also examined trends of how the microbial community in the human gut adapts to juice fasting as well as to inulin intake during juice fasting. Six healthy volunteers were fasting for three consecutive days consuming a total of 300 kcal daily provided by vegetable juices, framed by two days with a total daily calorie intake of 800 kcal, respectively. During one fasting period, participants consumed additionally 24 g of inulin daily. Stool samples were collected for the analysis of the microbial composition using 16S rRNA gene sequencing. Although no significant uniform changes were observed on the microbiome, quantitative changes in the microbial composition suggest a stronger decrease in alpha-diversity after fasting compared to the fasting intervention with additional inulin intake. The intake of inulin did not affect compliance for the fasting intervention but appeared to increase relative abundance of Bifidobacteria in participants who tolerated it well. Further studies with a larger sample size to overcome inter-individual microbiota differences are warranted to verify our observations

Modulation of inflammatory responses by gastrointestinal Prevotella spp. - From associations to functional studies.

Numerous studies have associated alterations in the gut microbiota composition with almost every known inflammatory disease. However, proving the biological relevance of distinct microbial signatures and linking specific microorganisms to host phenotypes, remains a considerable challenge. Correspondingly, increased abundance of members of Prevotella genus within microbial communities colonizing distinct mucosal surfaces has been found in individuals diagnosed with rheumatoid arthritis, periodontitis, metabolic disorders, and intestinal and vaginal dysbiosis. Still, the role of Prevotella spp. in the incidence of these diseases continues to be debated. For many years, poor understanding of Prevotella biology could be in large part attributed to the lack of experimental tools. However, in the recent years significant advances have been made towards overcoming these limitations, including increased number of isolates and improved understanding of genetic diversity. Besides discussing the most relevant associations between Prevotella spp. and inflammatory disorders, in the present review we examine the recent efforts to expand the Prevotella experimental "toolbox" and we highlight remaining experimental challenges that should advance future research and our understanding of Prevotella-host interplay

Brief report : assessment of mucosal barrier integrity using serological biomarkers in preclinical stages of rheumatoid arthritis

Background The pathogenesis of rheumatoid arthritis (RA) is believed to initiate at mucosal sites. The so-called ‘mucosal origin hypothesis of RA’ postulates an increased intestinal permeability before disease onset. Several biomarkers, including lipopolysaccharide binding protein (LBP) and intestinal fatty acid binding protein (I-FABP), have been proposed to reflect gut mucosa permeability and integrity, while serum calprotectin is a new inflammation marker proposed in RA. Methods We analyzed serum samples of individuals genetically at increased risk of RA in a nested-case-control study. Participants from a longitudinal cohort of first-degree relatives of RA patients (SCREEN-RA cohort) were divided into three pre-clinical stages of RA, based on the presence of risk factors for subsequent RA onset: 1) low-risk healthy asymptomatic controls; 2) intermediate-risk individuals without symptoms, but with RA-associated auto-immunity; 3) high-risk individuals with clinically suspect arthralgias. Five patients with newly diagnosed RA were also sampled. Serum LBP, I-FABP and calprotectin were measured using commercially available ELISA kits. Results We included 180 individuals genetically at increased risk for RA: 84 asymptomatic controls, 53 individuals with RA-associated autoimmunity and 38 high risk individuals. Serum LBP, I-FAPB or calprotectin concentrations did not differ between individuals in different pre-clinical stages of RA. Conclusion Based on the serum biomarkers LBP, I-FABP and calprotectin, we could not detect any evidence for intestinal injury in pre-clinical stages of RA.</p

A versatile genetic toolbox for Prevotella copri enables studying polysaccharide utilization systems.

Prevotella copri is a prevalent inhabitant of the human gut and has been associated with plant-rich diet consumption and diverse health states. The underlying genetic basis of these associations remains enigmatic due to the lack of genetic tools. Here, we developed a novel versatile genetic toolbox for rapid and efficient genetic insertion and allelic exchange applicable to P. copri strains from multiple clades. Enabled by the genetic platform, we systematically investigated the specificity of polysaccharide utilization loci (PULs) and identified four highly conserved PULs for utilizing arabinan, pectic galactan, arabinoxylan, and inulin, respectively. Further genetic and functional analysis of arabinan utilization systems illustrate that P. copri has evolved two distinct types of arabinan-processing PULs (PULAra ) and that the type-II PULAra is significantly enriched in individuals consuming a vegan diet compared to other diets. In summary, this genetic toolbox will enable functional genetic studies for P. copri in future

Distinct Polysaccharide Utilization Determines Interspecies Competition between Intestinal Prevotella spp.

CD81 plays a role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus. Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified an allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant was enhanced. These data are consistent with cholesterol switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in health and disease

Image_2_Characterization of serum biomarkers and antibody responses against Prevotella spp. in preclinical and new-onset phase of rheumatic diseases.jpg

IntroductionThe characterization of the influence of the microbiota on the development and drug responses during rheumatic diseases has intensified in recent years. The role of specific bacteria during disease development has become a central research question. Notably, several lines of evidence point to distinct microbes, e.g., Prevotella copri (P. copri) being targeted by antibodies in clinical phases of rheumatic diseases.MethodsIn the present study, we compiled a broad collection of human serum samples from individuals at risk of developing RA, chronic RA patients as well as patients with new-onset of rheumatic diseases. We evaluated the presence of inflammatory biomarkers in our serum collection as well as serum antibody responses against novel, genetically distinct isolates of P. copri and several oral pathobionts.ResultsOur analysis revealed the presence of increased levels of inflammatory markers already in pre-clinical and new onset rheumatoid arthritis. However, antibody reactivity against the microbes did not differ between patient groups. Yet, we observed high variability between the different P. copri strains. We found total serum IgG levels to slightly correlate with IgG antibody responses against P. copri, but no relation between the latter and presence or prevalence of P. copri in the intestine.DiscussionIn conclusion, our work underlined the importance of strain-level characterization and its consideration during further investigations of host-microbiota interactions and the development of microbiome-based therapeutic approaches for treating rheumatic diseases.</p