Microbial and transcriptional differences elucidate atopic dermatitis heterogeneity across skin sites.

It is well established that different sites in healthy human skin are colonized by distinct microbial communities due to different physiological conditions. However, few studies have explored microbial heterogeneity between skin sites in diseased skin, such as atopic dermatitis (AD) lesions. To address this issue, we carried out deep analysis of the microbiome and transcriptome in the skin of a large cohort of AD patients and healthy volunteers, comparing two physiologically different sites: upper back and posterior thigh. Microbiome samples and biopsies were obtained from both lesional and nonlesional skin to identify changes related to the disease process. Transcriptome analysis revealed distinct disease-related gene expression profiles depending on anatomical location, with keratinization dominating the transcriptomic signatures in posterior thigh, and lipid metabolism in the upper back. Moreover, we show that relative abundance of Staphylococcus aureus is associated with disease severity in the posterior thigh, but not in the upper back. Our results suggest that AD may select for similar microbes in different anatomical locations-an "AD-like microbiome," but distinct microbial dynamics can still be observed when comparing posterior thigh to upper back. This study highlights the importance of considering the variability across skin sites when studying the development of skin inflammation.

Immune-microbiota interaction in Finnish and Russian Karelia young people with high and low allergy prevalence.

BACKGROUND: After the Second World War, the population living in the Karelian region was strictly divided by the "iron curtain" between Finland and Russia. This resulted in different lifestyle, standard of living, and exposure to the environment. Allergic manifestations and sensitization to common allergens have been much more common on the Finnish compared to the Russian side. OBJECTIVE: The remarkable allergy disparity in the Finnish and Russian Karelia calls for immunological explanations. METHODS: Young people, aged 15-20 years, in the Finnish (n = 69) and Russian (n = 75) Karelia were studied. The impact of genetic variation on the phenotype was studied by a genome-wide association analysis. Differences in gene expression (transcriptome) were explored from the blood mononuclear cells (PBMC) and related to skin and nasal epithelium microbiota and sensitization. RESULTS: The genotype differences between the Finnish and Russian populations did not explain the allergy gap. The network of gene expression and skin and nasal microbiota was richer and more diverse in the Russian subjects. When the function of 261 differentially expressed genes was explored, innate immunity pathways were suppressed among Russians compared to Finns. Differences in the gene expression paralleled the microbiota disparity. High Acinetobacter abundance in Russians correlated with suppression of innate immune response. High-total IgE was associated with enhanced anti-viral response in the Finnish but not in the Russian subjects. CONCLUSIONS AND CLINICAL RELEVANCE: Young populations living in the Finnish and Russian Karelia show marked differences in genome-wide gene expression and host contrasting skin and nasal epithelium microbiota. The rich gene-microbe network in Russians seems to result in a better-balanced innate immunity and associates with low allergy prevalence.

Soil exposure modifies the gut microbiota and supports immune tolerance in a mouse model.

BACKGROUND: Sufficient exposure to natural environments, in particular soil and its microbes, has been suggested to be protective against allergies. OBJECTIVE: We aim at gaining more direct evidence of the environment-microbiota-health axis by studying the colonization of gut microbiota in mice after exposure to soil and by examining immune status in both a steady-state situation and during allergic inflammation. METHODS: The gastrointestinal microbiota of mice housed on clean bedding or in contact with soil was analyzed by using 16S rRNA gene sequencing, and the data were combined with immune parameters measured in the gut mucosa, lung tissue, and serum samples. RESULTS: We observed marked differences in the small intestinal and fecal microbiota composition between mice housed on clean bedding or in contact with soil, with a higher proportion of Bacteroidetes relative to Firmicutes in the soil group. The housing environment also influenced mouse intestinal gene expression, as shown by upregulated expression of the immunoregulatory markers IL-10, forkhead box P3, and cytotoxic T lymphocyte-associated protein 4 in the soil group. Importantly, using the murine asthma model, we found that exposure to soil polarizes the immune system toward TH1 and a higher level of anti-inflammatory signaling, alleviating TH2-type allergic responses. The inflammatory status of the mice had a marked influence on the composition of the gut microbiota, suggesting bidirectional communication along the gut-lung axis. CONCLUSION: Our results provide evidence of the role of environmentally acquired microbes in alleviating against TH2-driven inflammation, which relates to allergic diseases.

Genome-Scale Model and Omics Analysis of Metabolic Capacities of Akkermansia muciniphila Reveal a Preferential Mucin-Degrading Lifestyle.

The composition and activity of the microbiota in the human gastrointestinal tract are primarily shaped by nutrients derived from either food or the host. Bacteria colonizing the mucus layer have evolved to use mucin as a carbon and energy source. One of the members of the mucosa-associated microbiota is Akkermansia muciniphila, which is capable of producing an extensive repertoire of mucin-degrading enzymes. To further study the substrate utilization abilities of A. muciniphila, we constructed a genome-scale metabolic model to test amino acid auxotrophy, vitamin biosynthesis, and sugar-degrading capacities. The model-supported predictions were validated by in vitro experiments, which showed A. muciniphila to be able to utilize the mucin-derived monosaccharides fucose, galactose, and N-acetylglucosamine. Growth was also observed on N-acetylgalactosamine, even though the metabolic model did not predict this. The uptake of these sugars, as well as the nonmucin sugar glucose, was enhanced in the presence of mucin, indicating that additional mucin-derived components are needed for optimal growth. An analysis of whole-transcriptome sequencing (RNA-Seq) comparing the gene expression of A. muciniphila grown on mucin with that of the same bacterium grown on glucose confirmed the activity of the genes involved in mucin degradation and revealed most of these to be upregulated in the presence of mucin. The transcriptional response was confirmed by a proteome analysis, altogether revealing a hierarchy in the use of sugars and reflecting the adaptation of A. muciniphila to the mucosal environment. In conclusion, these findings provide molecular insights into the lifestyle of A. muciniphila and further confirm its role as a mucin specialist in the gut.IMPORTANCEAkkermansia muciniphila is among the most abundant mucosal bacteria in humans and in a wide range of other animals. Recently, A. muciniphila has attracted considerable attention because of its capacity to protect against diet-induced obesity in mouse models. However, the physiology of A. muciniphila has not been studied in detail. Hence, we constructed a genome-scale model and describe its validation by transcriptomic and proteomic approaches on bacterial cells grown on mucus and glucose, a nonmucus sugar. The results provide detailed molecular insight into the mucus-degrading lifestyle of A. muciniphila and further confirm the role of this mucin specialist in producing propionate and acetate under conditions of the intestinal tract.

Akkermansia muciniphila Adheres to Enterocytes and Strengthens the Integrity of the Epithelial Cell Layer.

Akkermansia muciniphila is a Gram-negative mucin-degrading bacterium that resides in the gastrointestinal tracts of humans and animals. A. muciniphila has been linked with intestinal health and improved metabolic status in obese and type 2 diabetic subjects. Specifically, A. muciniphila has been shown to reduce high-fat-diet-induced endotoxemia, which develops as a result of an impaired gut barrier. Despite the accumulating evidence of the health-promoting effects of A. muciniphila, the mechanisms of interaction of the bacterium with the host have received little attention. In this study, we used several in vitro models to investigate the adhesion of A. muciniphila to the intestinal epithelium and its interaction with the host mucosa. We found that A. muciniphila adheres strongly to the Caco-2 and HT-29 human colonic cell lines but not to human colonic mucus. In addition, A. muciniphila showed binding to the extracellular matrix protein laminin but not to collagen I or IV, fibronectin, or fetuin. Importantly, A. muciniphila improved enterocyte monolayer integrity, as shown by a significant increase in the transepithelial electrical resistance (TER) of cocultures of Caco-2 cells with the bacterium. Further, A. muciniphila induced interleukin 8 (IL-8) production by enterocytes at cell concentrations 100-fold higher than those for Escherichia coli, suggesting a very low level of proinflammatory activity in the epithelium. In conclusion, our results demonstrate that A. muciniphila adheres to the intestinal epithelium and strengthens enterocyte monolayer integrity in vitro, suggesting an ability to fortify an impaired gut barrier. These results support earlier associative in vivo studies and provide insights into the interaction of A. muciniphila with the host.

Akkermansia muciniphila uses human milk oligosaccharides to thrive in the early life conditions in vitro.

Akkermansia muciniphila is a well-studied anaerobic bacterium specialized in mucus degradation and associated with human health. Because of the structural resemblance of mucus glycans and free human milk oligosaccharides (HMOs), we studied the ability of A. muciniphila to utilize human milk oligosaccharides. We found that A. muciniphila was able to grow on human milk and degrade HMOs. Analyses of the proteome of A. muciniphila indicated that key-glycan degrading enzymes were expressed when the bacterium was grown on human milk. Our results display the functionality of the key-glycan degrading enzymes (α-L-fucosidases, ß-galactosidases, exo-α-sialidases and ß-acetylhexosaminidases) to degrade the HMO-structures 2'-FL, LNT, lactose, and LNT2. The hydrolysation of the host-derived glycan structures allows A. muciniphila to promote syntrophy with other beneficial bacteria, contributing in that way to a microbial ecological network in the gut. Thus, the capacity of A. muciniphila to utilize human milk will enable its survival in the early life intestine and colonization of the mucosal layer in early life, warranting later life mucosal and metabolic health.

Action and function of Akkermansia muciniphila in microbiome ecology, health and disease.

The discovery of Akkermansia muciniphila has opened new avenues for the use of this abundant intestinal symbiont in next generation therapeutic products, as well as targeting microbiota dynamics. A. muciniphila is known to colonize the mucosal layer of the human intestine where it triggers both host metabolic and immune responses. A. muciniphila is particularly effective in increasing mucus thickness and increasing gut barrier function. As a result host metabolic markers ameliorate. The mechanism of host regulation is thought to involve the outer membrane composition, including the type IV pili of A. muciniphila, that directly signal to host immune receptors. At the same time the metabolic activity of A. muciniphila leads to the production of short chain fatty acids that are beneficial to the host and microbiota members. This contributes to host-microbiota and microbe-microbe syntrophy The mucolytic activity and metabolite production make A. muciniphila a key species in the mucus layer, stimulating beneficial mucosal microbial networks. This well studied member of the microbiota has been studied in three aspects that will be further described in this review: i) A. muciniphila characteristics and mucin adaptation, ii) its role as key species in the mucosal microbiome, and iii) its role in host health.

Pili-like proteins of Akkermansia muciniphila modulate host immune responses and gut barrier function.

Gut barrier function is key in maintaining a balanced response between the host and its microbiome. The microbiota can modulate changes in gut barrier as well as metabolic and inflammatory responses. This highly complex system involves numerous microbiota-derived factors. The gut symbiont Akkermansia muciniphila is positively correlated with a lean phenotype, reduced body weight gain, amelioration of metabolic responses and restoration of gut barrier function by modulation of mucus layer thickness. However, the molecular mechanisms behind its metabolic and immunological regulatory properties are unexplored. Herein, we identify a highly abundant outer membrane pili-like protein of A. muciniphila MucT that is directly involved in immune regulation and enhancement of trans-epithelial resistance. The purified Amuc_1100 protein and enrichments containing all its associated proteins induced production of specific cytokines through activation of Toll-like receptor (TLR) 2 and TLR4. This mainly leads to high levels of IL-10 similar to those induced by the other beneficial immune suppressive microorganisms such as Faecalibacterium prausnitzii A2-165 and Lactobacillus plantarum WCFS1. Together these results indicate that outer membrane protein composition and particularly the newly identified highly abundant pili-like protein Amuc_1100 of A. muciniphila are involved in host immunological homeostasis at the gut mucosa, and improvement of gut barrier function.

A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice.

Obesity and type 2 diabetes are associated with low-grade inflammation and specific changes in gut microbiota composition. We previously demonstrated that administration of Akkermansia muciniphila to mice prevents the development of obesity and associated complications. However, the underlying mechanisms of this protective effect remain unclear. Moreover, the sensitivity of A. muciniphila to oxygen and the presence of animal-derived compounds in its growth medium currently limit the development of translational approaches for human medicine. We have addressed these issues here by showing that A. muciniphila retains its efficacy when grown on a synthetic medium compatible with human administration. Unexpectedly, we discovered that pasteurization of A. muciniphila enhanced its capacity to reduce fat mass development, insulin resistance and dyslipidemia in mice. These improvements were notably associated with a modulation of the host urinary metabolomics profile and intestinal energy absorption. We demonstrated that Amuc_1100, a specific protein isolated from the outer membrane of A. muciniphila, interacts with Toll-like receptor 2, is stable at temperatures used for pasteurization, improves the gut barrier and partly recapitulates the beneficial effects of the bacterium. Finally, we showed that administration of live or pasteurized A. muciniphila grown on the synthetic medium is safe in humans. These findings provide support for the use of different preparations of A. muciniphila as therapeutic options to target human obesity and associated disorders.

Characterization of Outer Membrane Proteome of Akkermansia muciniphila Reveals Sets of Novel Proteins Exposed to the Human Intestine.

Akkermansia muciniphila is a common member of the human gut microbiota and belongs to the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum. Decreased levels of A. muciniphila have been associated with many diseases, and thus it is considered to be a beneficial resident of the intestinal mucus layer. Surface-exposed molecules produced by this organism likely play important roles in colonization and communication with other microbes and the host, but the protein composition of the outer membrane (OM) has not been characterized thus far. Herein we set out to identify and characterize A. muciniphila proteins using an integrated approach of proteomics and computational analysis. Sarkosyl extraction and sucrose density-gradient centrifugation methods were used to enrich and fractionate the OM proteome of A. muciniphila. Proteins from these fractions were identified by LC-MS/MS and candidates for OM proteins derived from the experimental approach were subjected to computational screening to verify their location in the cell. In total we identified 79 putative OM and membrane-associated extracellular proteins, and 23 of those were found to differ in abundance between cells of A. muciniphila grown on the natural substrate, mucin, and those grown on the non-mucus sugar, glucose. The identified OM proteins included highly abundant proteins involved in secretion and transport, as well as proteins predicted to take part in formation of the pili-like structures observed in A. muciniphila. The most abundant OM protein was a 95-kD protein, termed PilQ, annotated as a type IV pili secretin and predicted to be involved in the production of pili in A. muciniphila. To verify its location we purified the His-Tag labeled N-terminal domain of PilQ and generated rabbit polyclonal antibodies. Immunoelectron microscopy of thin sections immunolabeled with these antibodies demonstrated the OM localization of PilQ, testifying for its predicted function as a type IV pili secretin in A. muciniphila. As pili structures are known to be involved in the modulation of host immune responses, this provides support for the involvement of OM proteins in the host interaction of A. muciniphila. In conclusion, the characterization of A. muciniphila OM proteome provides valuable information that can be used for further functional and immunological studies.

The function of our microbiota: who is out there and what do they do?

Current meta-omics developments provide a portal into the functional potential and activity of the intestinal microbiota. The comparative and functional meta-omics approaches have made it possible to get a molecular snap shot of microbial function at a certain time and place. To this end, metagenomics is a DNA-based approach, metatranscriptomics studies the total transcribed RNA, metaproteomics focuses on protein levels and metabolomics describes metabolic profiles. Notably, the metagenomic toolbox is rapidly expanding and has been instrumental in the generation of draft genome sequences of over 1000 human associated microorganisms as well as an astonishing 3.3 million unique microbial genes derived from the intestinal tract of over 100 European adults. Remarkably, it appeared that there are at least 3 clusters of co-occurring microbial species, termed enterotypes, that characterize the intestinal microbiota throughout various continents. The human intestinal microbial metagenome further revealed unique functions carried out in the intestinal environment and provided the basis for newly discovered mechanisms for signaling, vitamin production and glycan, amino-acid and xenobiotic metabolism. The activity and composition of the microbiota is affected by genetic background, age, diet, and health status of the host. In its turn the microbiota composition and activity influence host metabolism and disease development. Exemplified by the differences in microbiota composition and activity between breast- as compared to formula-fed babies, healthy and malnourished infants, elderly and centenarians as compared to youngsters, humans that are either lean or obese and healthy or suffering of inflammatory bowel diseases (IBD). In this review we will focus on our current understanding of the functionality of the human intestinal microbiota based on all available metagenome, metatranscriptome, and metaproteome results.