IgA Function in Relation to the Intestinal Microbiota.

IgA is the dominant immunoglobulin isotype produced in mammals, largely secreted across the intestinal mucosal surface. Although induction of IgA has been a hallmark feature of microbiota colonization following colonization in germ-free animals, until recently appreciation of the function of IgA in host-microbial mutualism has depended mainly on indirect evidence of alterations in microbiota composition or penetration of microbes in the absence of somatic mutations in IgA (or compensatory IgM). Highly parallel sequencing techniques that enable high-resolution analysis of either microbial consortia or IgA sequence diversity are now giving us new perspectives on selective targeting of microbial taxa and the trajectory of IgA diversification according to induction mechanisms, between different individuals and over time. The prospects are to link the range of diversified IgA clonotypes to specific antigenic functions in modulating the microbiota composition, position and metabolism to ensure host mutualism.

Gut microbiota elicits a protective immune response against malaria transmission.

Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:ß-galactoside-α1-3-galactosyltransferase (α1,3GT) gene, which ablated the expression of the Galα1-3Galß1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against Plasmodium spp. infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both Plasmodium spp. and the human gut pathobiont E. coli O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in α1,3GT-deficient mice, which produce protective anti-α-gal Abs when colonized by E. coli O86:B7. Anti-α-gal Abs target Plasmodium sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by Anopheles mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.

Antibodies Set Boundaries Limiting Microbial Metabolite Penetration and the Resultant Mammalian Host Response.

Although the mammalian microbiota is well contained within the intestine, it profoundly shapes development and metabolism of almost every host organ. We questioned the range and depth of microbial metabolite penetration into the host, and how this is modulated by intestinal immunity. Chemically identical microbial and host metabolites were distinguished by stable isotope tracing from 13C-labeled live non-replicating Escherichia coli, differentiating 12C host isotopes with high-resolution mass spectrometry. Hundreds of endogenous microbial compounds penetrated 23 host tissues and fluids after intestinal exposure: subsequent 12C host metabolome signatures included lipidemia, reduced glycolysis, and inflammation. Penetrant bacterial metabolites from the small intestine were rapidly cleared into the urine, whereas induced antibodies curtailed microbial metabolite exposure by accelerating intestinal bacterial transit into the colon where metabolite transport mechanisms are limiting. Pervasive penetration of microbial molecules can cause extensive host tissue responses: these are limited by immune and non-immune intestinal mucosal adaptations to the microbiota.

Nlrp6- and ASC-Dependent Inflammasomes Do Not Shape the Commensal Gut Microbiota Composition.

The gut microbiota regulate susceptibility to multiple human diseases. The Nlrp6-ASC inflammasome is widely regarded as a hallmark host innate immune axis that shapes the gut microbiota composition. This notion stems from studies reporting dysbiosis in mice lacking these inflammasome components when compared with non-littermate wild-type animals. Here, we describe microbial analyses in inflammasome-deficient mice while minimizing non-genetic confounders using littermate-controlled Nlrp6-deficient mice and ex-germ-free littermate-controlled ASC-deficient mice that were all allowed to shape their gut microbiota naturally after birth. Careful microbial phylogenetic analyses of these cohorts failed to reveal regulation of the gut microbiota composition by the Nlrp6- and ASC-dependent inflammasomes. Our results obtained in two geographically separated animal facilities dismiss a generalizable impact of Nlrp6- and ASC-dependent inflammasomes on the composition of the commensal gut microbiota and highlight the necessity for littermate-controlled experimental design in assessing the influence of host immunity on gut microbial ecology.

Mucosal or systemic microbiota exposures shape the B cell repertoire.

Colonization by the microbiota causes a marked stimulation of B cells and induction of immunoglobulin, but mammals colonized with many taxa have highly complex and individualized immunoglobulin repertoires1,2. Here we use a simplified model of defined transient exposures to different microbial taxa in germ-free mice3 to deconstruct how the microbiota shapes the B cell pool and its functional responsiveness. We followed the development of the immunoglobulin repertoire in B cell populations, as well as single cells by deep sequencing. Microbial exposures at the intestinal mucosa generated oligoclonal responses that differed from those of germ-free mice, and from the diverse repertoire that was generated after intravenous systemic exposure to microbiota. The IgA repertoire-predominantly to cell-surface antigens-did not expand after dose escalation, whereas increased systemic exposure broadened the IgG repertoire to both microbial cytoplasmic and cell-surface antigens. These microbial exposures induced characteristic immunoglobulin heavy-chain repertoires in B cells, mainly at memory and plasma cell stages. Whereas sequential systemic exposure to different microbial taxa diversified the IgG repertoire and facilitated alternative specific responses, sequential mucosal exposure produced limited overlapping repertoires and the attrition of initial IgA binding specificities. This shows a contrast between a flexible response to systemic exposure with the need to avoid fatal sepsis, and a restricted response to mucosal exposure that reflects the generic nature of host-microbial mutualism in the mucosa.

Neuronal programming by microbiota regulates intestinal physiology.

Neural control of the function of visceral organs is essential for homeostasis and health. Intestinal peristalsis is critical for digestive physiology and host defence, and is often dysregulated in gastrointestinal disorders1. Luminal factors, such as diet and microbiota, regulate neurogenic programs of gut motility2-5, but the underlying molecular mechanisms remain unclear. Here we show that the transcription factor aryl hydrocarbon receptor (AHR) functions as a biosensor in intestinal neural circuits, linking their functional output to the microbial environment of the gut lumen. Using nuclear RNA sequencing of mouse enteric neurons that represent distinct intestinal segments and microbiota states, we demonstrate that the intrinsic neural networks of the colon exhibit unique transcriptional profiles that are controlled by the combined effects of host genetic programs and microbial colonization. Microbiota-induced expression of AHR in neurons of the distal gastrointestinal tract enables these neurons to respond to the luminal environment and to induce expression of neuron-specific effector mechanisms. Neuron-specific deletion of Ahr, or constitutive overexpression of its negative feedback regulator CYP1A1, results in reduced peristaltic activity of the colon, similar to that observed in microbiota-depleted mice. Finally, expression of Ahr in the enteric neurons of mice treated with antibiotics partially restores intestinal motility. Together, our experiments identify AHR signalling in enteric neurons as a regulatory node that integrates the luminal environment with the physiological output of intestinal neural circuits to maintain gut homeostasis and health.

Development of non-alcoholic steatohepatitis is associated with gut microbiota but not with oxysterol enzymes CH25H, EBI2, or CYP7B1 in mice.

Liver steatosis is the most frequent liver disorder and its advanced stage, non-alcoholic steatohepatitis (NASH), will soon become the main reason for liver fibrosis and cirrhosis. The "multiple hits hypothesis" suggests that progression from simple steatosis to NASH is triggered by multiple factors including the gut microbiota composition. The Epstein Barr virus induced gene 2 (EBI2) is a receptor for the oxysterol 7a, 25-dihydroxycholesterol synthesized by the enzymes CH25H and CYP7B1. EBI2 and its ligand control activation of immune cells in secondary lymphoid organs and the gut. Here we show a concurrent study of the microbial dysregulation and perturbation of the EBI2 axis in a mice model of NASH.We used mice with wildtype, or littermates with CH25H-/-, EBI2-/-, or CYP7B1-/- genotypes fed with a high-fat diet (HFD) containing high amounts of fat, cholesterol, and fructose for 20 weeks to induce liver steatosis and NASH. Fecal and small intestinal microbiota samples were collected, and microbiota signatures were compared according to genotype and NASH disease state.We found pronounced differences in microbiota composition of mice with HFD developing NASH compared to mice did not developing NASH. In mice with NASH, we identified significantly increased 33 taxa mainly belonging to the Clostridiales order and/ or the family, and significantly decreased 17 taxa. Using an Elastic Net algorithm, we suggest a microbiota signature that predicts NASH in animals with a HFD from the microbiota composition with moderate accuracy (area under the receiver operator characteristics curve = 0.64). In contrast, no microbiota differences regarding the studied genotypes (wildtype vs knock-out CH25H-/-, EBI2-/-, or CYP7B1-/-) were observed.In conclusion, our data confirm previous studies identifying the intestinal microbiota composition as a relevant marker for NASH pathogenesis. Further, no link of the EBI2 - oxysterol axis to the intestinal microbiota was detectable in the current study.

Innate lymphoid cell characterization in the rat and their correlation to gut commensal microbes.

Innate lymphoid cells (ILCs) are important for tissue immune homeostasis, and are thoroughly characterized in mice and humans. Here, we have performed in-depth characterization of rat ILCs. Rat ILCs were identified based on differential expression of transcription factors and lack of lineage markers. ILC3s represented the major ILC population of the small intestine, while ILC2s were infrequent but most prominent in liver and adipose tissue. Two major subsets of group 1 ILCs were defined. Lineage- T-bet+ Eomes+ cells were identified as conventional NK cells, while lineage- T-bet+ Eomes- cells were identified as the probable rat counterpart of ILC1s based on their selective expression of the ILC marker CD200R. Rat ILC1s were particularly abundant in liver and intestinal tissues, and were functionally similar to NK cells. Single-cell transcriptomics of spleen and liver cells confirmed the main division of NK cells and ILC1-like cells, and demonstrated Granzyme A as an additional ILC1 marker. We further report differential distributions of NK cells and ILCs along the small and large intestines, and the association of certain bacterial taxa to frequencies of ILCs. In conclusion, we provide a framework for future studies of ILCs in diverse rat experimental models, and novel data on the potential interplay between commensals and intestinal ILCs.

Antenatal gut microbiome profiles and effect on pregnancy outcome in HIV infected and HIV uninfected women in a resource limited setting.

BACKGROUND: Human immunodeficiency virus (HIV) severely damages the epithelial cells of the gut lining leading to an inflamed leaky gut, translocation of microbial products, and dysbiosis resulting in systemic immune activation. Also, microbiota composition and maternal gut function can be altered in pregnancy through changes in the immune system and intestinal physiology. The aim of this study was to investigate the gut microbiota in HIV-infected and HIV-uninfected pregnant women and to compare and identify the association between gut microbial composition and adverse birth outcomes. RESULTS: A total of 94 pregnant women (35 HIV-infected and 59 HIV-uninfected controls) were recruited in Harare from 4 polyclinics serving populations with relatively poor socioeconomic status. Women were of a median age of 28 years (interquartile range, IQR: 22.3-32.0) and 55% of women were 35 weeks gestational age at enrolment (median 35.0 weeks, IQR: 32.5-37.2). Microbiota profiling in these participants showed that species richness was significantly lower in the HIV-infected pregnant women compared to their HIV-uninfected peers and significant differences in ß-diversity using Bray-Curtis dissimilarity were observed. In contrast, there was no significant difference in α-diversity between immune-compromised (CD4+ < 350 cells/µL) and immune-competent HIV-infected women (CD4+ ≥ 350 cells/µL) even after stratification by viral load suppression. HIV infection was significantly associated with a reduced abundance of Clostridium, Turicibacter, Ruminococcus, Parabacteroides, Bacteroides, Bifidobacterium, Treponema, Oscillospira, and Faecalibacterium and a higher abundance of Actinomyces, and Succinivibrio. Low infant birth weight (< 2500 g) was significantly associated with high abundances of the phylum Spirochaetes, the families Spirochaeteceae, Veillonellaceae, and the genus Treponema. CONCLUSION: The results reported here show that the species richness and taxonomy composition of the gut microbiota is altered in HIV-infected pregnant women, possibly reflecting intestinal dysbiosis. Some of these taxa were also associated with low infant birth weight.

The Bern Birth Cohort (BeBiCo) to study the development of the infant intestinal microbiota in a high-resource setting in Switzerland: rationale, design, and methods.

BACKGROUND: Microbiota composition is fundamental to human health with the intestinal microbiota undergoing critical changes within the first two years of life. The developing intestinal microbiota is shaped by maternal seeding, breast milk and its complex constituents, other nutrients, and the environment. Understanding microbiota-dependent pathologies requires a profound understanding of the early development of the healthy infant microbiota. METHODS: Two hundred and fifty healthy pregnant women (≥20 weeks of gestation) from the greater Bern area will be enrolled at Bern University hospital's maternity department. Participants will be followed as mother-baby pairs at delivery, week(s) 1, 2, 6, 10, 14, 24, 36, 48, 96, and at years 5 and 10 after birth. Clinical parameters describing infant growth and development, morbidity, and allergic conditions as well as socio-economic, nutritional, and epidemiological data will be documented. Neuro-developmental outcomes and behavior will be assessed by child behavior checklists at and beyond 2 years of age. Maternal stool, milk, skin and vaginal swabs, infant stool, and skin swabs will be collected at enrolment and at follow-up visits. For the primary outcome, the trajectory of the infant intestinal microbiota will be characterized by 16S and metagenomic sequencing regarding composition, metabolic potential, and stability during the first 2 years of life. Secondary outcomes will assess the cellular and chemical composition of maternal milk, the impact of nutrition and environment on microbiota development, the maternal microbiome transfer at vaginal or caesarean birth and thereafter on the infant, and correlate parameters of microbiota and maternal milk on infant growth, development, health, and mental well-being. DISCUSSION: The Bern birth cohort study will provide a detailed description and normal ranges of the trajectory of microbiota maturation in a high-resource setting. These data will be compared to data from low-resource settings such as from the Zimbabwe-College of Health-Sciences-Birth-Cohort study. Prospective bio-sampling and data collection will allow studying the association of the microbiota with common childhood conditions concerning allergies, obesity, neuro-developmental outcomes , and behaviour. Trial registration The trial has been registered at www. CLINICALTRIALS: gov , Identifier: NCT04447742.

The Swiss Primary Hypersomnolence and Narcolepsy Cohort study (SPHYNCS): Study protocol for a prospective, multicentre cohort observational study.

Narcolepsy type 1 (NT1) is a disorder with well-established markers and a suspected autoimmune aetiology. Conversely, the narcoleptic borderland (NBL) disorders, including narcolepsy type 2, idiopathic hypersomnia, insufficient sleep syndrome and hypersomnia associated with a psychiatric disorder, lack well-defined markers and remain controversial in terms of aetiology, diagnosis and management. The Swiss Primary Hypersomnolence and Narcolepsy Cohort Study (SPHYNCS) is a comprehensive multicentre cohort study, which will investigate the clinical picture, pathophysiology and long-term course of NT1 and the NBL. The primary aim is to validate new and reappraise well-known markers for the characterization of the NBL, facilitating the diagnostic process. Seven Swiss sleep centres, belonging to the Swiss Narcolepsy Network (SNaNe), joined the study and will prospectively enrol over 500 patients with recent onset of excessive daytime sleepiness (EDS), hypersomnia or a suspected central disorder of hypersomnolence (CDH) during a 3-year recruitment phase. Healthy controls and patients with EDS due to severe sleep-disordered breathing, improving after therapy, will represent two control groups of over 50 patients each. Clinical and electrophysiological (polysomnography, multiple sleep latency test, maintenance of wakefulness test) information, and information on psychomotor vigilance and a sustained attention to response task, actigraphy and wearable devices (long-term monitoring), and responses to questionnaires will be collected at baseline and after 6, 12, 24 and 36 months. Potential disease markers will be searched for in blood, cerebrospinal fluid and stool. Analyses will include quantitative hypocretin measurements, proteomics/peptidomics, and immunological, genetic and microbiota studies. SPHYNCS will increase our understanding of CDH and the relationship between NT1 and the NBL. The identification of new disease markers is expected to lead to better and earlier diagnosis, better prognosis and personalized management of CDH.

Determination of sand fly fauna and molecular detection of Leishmania in sand flies in Antalya Province, Southern Turkey.

Visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are diseases transmitted by infected female sand flies. Since the eradication of malaria in Turkey, CL is the main vector-borne disease in the country, with more than 2000 cases per year, making it a significant public health problem. The aims of this study were to carry out an entomological survey in Antalya Province, an endemic area for CL in the Mediterranean Region of Turkey, to identify sand fly fauna and to screen female specimens for the presence of Leishmania parasites (Leishmania infantum, L. tropica, L. major, and L. donovani) using molecular analysis. Sand flies were collected in 42 localities of seven districts in Antalya Province using CDC miniature light traps in two different periods, June 2012 and September 2013. The specimens were kept in 96% ethanol until the dissection was done. The head and genitalia of the specimens were cut for preparing individual slides for species identification. The rest of the body of female specimens was kept separately. The specimens were identified at the species level, and 27 pools were generated according to the locations and species for screening the presence of Leishmania. A commercial kit was used for DNA extractions. Real-time and conventional polymerase chain reaction (PCR) targeting the internal transcribed spacer region (ITS1) were then performed. In total, 1306 specimens comprising nine species belonging to the Phlebotomus genus were collected in the study region, with Phlebotomus neglectus/syriacus (38.82%) the most abundant, followed by P. alexandri (21.67%) and P. tobbi (20.44%). In the 27 pools, Leishmania infantum DNA was detected in four pools containing P. neglectus/syriacus and one pool containing P. tobbi. In conclusion, the sand fly fauna in the Antalya Province is diverse. The probable vector sand fly species are P. neglectus/syriacus and P. tobbi with high dominance (59.26%), which indicates a high risk of CL transmission. The data presented here may help to shed more light on the transmission cycles of the Leishmania parasite in this CL endemic area.

Association of Alterations in Intestinal Microbiota With Impaired Psychological Function in Patients With Inflammatory Bowel Diseases in Remission.

BACKGROUND & AIMS: Depression and anxiety are frequent comorbidities with inflammatory bowel diseases (IBD). Alterations to the intestinal microbiome promote not only intestinal inflammation but also psychologic function. We studied the interactions between the composition of the intestinal microbiota and psychological outcomes in patients with IBD in Switzerland. METHODS: We performed a prospective study of psychological comorbidities and quality of life (QoL) in 171 participants in the Swiss IBD Cohort Study with IBD in remission. Participants complete the Hospital Anxiety and Depression Scale, Perceived Stress Questionnaire, the 36-Item Short Form Survey, and the IBD QoL Questionnaire. Microbes were collected from intestinal biopsies and analyzed by 16S rRNA high-throughput sequencing. RESULTS: Microbiomes of patients with higher perceived stress had significantly lower alpha diversity. Anxiety and depressive symptoms were significantly associated with beta diversity. We found a negative correlation between psychological distress and abundance of Clostridia, Bacilli, Bacteroidia, and Beta- and Gamma-proteobacteria. Psychological distress was also associated with decreases in operational taxonomic units from the lineages of Lachnospiraceae, Fusobacteriaceae, Ruminococcaceae, Veillonellaceae, Alcaligenaceae, Desulfovibrionaceae, and Bacteroidaceae families. The relative abundance of Bifidobacterium in patients with Crohn's disease and Desulfovibrio in patients with ulcerative colitis correlated with depression, whereas abundance of Sutterella, RF 32, and Lactococcus correlated with quality of life in patients with Crohn's disease. CONCLUSIONS: We identified correlations between the composition of the intestinal microbiota in patients with IBD and remission, psychological well-being, and QoL. Further studies should investigate how intestinal inflammation, the microbiome, and microbial metabolites affect psychological well-being and whether these components are mono- or bi-directionally linked.

FXR modulates the gut-vascular barrier by regulating the entry sites for bacterial translocation in experimental cirrhosis.

BACKGROUND & AIMS: Pathological bacterial translocation (PBT) in cirrhosis is the hallmark of spontaneous bacterial infections, increasing mortality several-fold. Increased intestinal permeability is known to contribute to PBT in cirrhosis, although the role of the mucus layer has not been addressed in detail. A clear route of translocation for luminal intestinal bacteria is yet to be defined, but we hypothesize that the recently described gut-vascular barrier (GVB) is impaired in experimental portal hypertension, leading to increased accessibility of the vascular compartment for translocating bacteria. MATERIALS: Cirrhosis was induced in mouse models using bile-duct ligation (BDL) and CCl4. Pre-hepatic portal-hypertension was induced by partial portal vein ligation (PPVL). Intestinal permeability was compared in these mice after GFP-Escherichia coli or different sized FITC-dextrans were injected into the intestine. RESULTS: Healthy and pre-hepatic portal-hypertensive (PPVL) mice lack translocation of FITC-dextran and GFP-E. coli from the small intestine to the liver, whereas BDL and CCl4-induced cirrhotic mice demonstrate pathological translocation, which is not altered by prior thoracic-duct ligation. The mucus layer is reduced in thickness, with loss of goblet cells and Muc2-staining and expression in cirrhotic but not PPVL mice. These changes are associated with bacterial overgrowth in the inner mucus layer and pathological translocation of GFP-E. coli through the ileal epithelium. GVB is profoundly altered in BDL and CCl4-mice with Ileal extravasation of large-sized 150 kDa-FITC-dextran, but only slightly altered in PPVL mice. This pathological endothelial permeability and accessibility in cirrhotic mice is associated with augmented expression of PV1 in intestinal vessels. OCA but not fexaramine stabilizes the GVB, whereas both FXR-agonists ameliorate gut to liver translocation of GFP-E. coli. CONCLUSIONS: Cirrhosis, but not portal hypertension per se, grossly impairs the endothelial and muco-epithelial barriers, promoting PBT to the portal-venous circulation. Both barriers appear to be FXR-modulated, with FXR-agonists reducing PBT via the portal-venous route. LAY SUMMARY: For intestinal bacteria to enter the systemic circulation, they must cross the mucus and epithelial layer, as well as the gut-vascular barrier. Cirrhosis disrupts all 3 of these barriers, giving bacteria access to the portal-venous circulation and thus, the gut-liver axis. Diminished luminal bile acid availability, cirrhosis and the associated reduction in farnesoid x receptor (FXR) signaling seem, at least partly, to mediate these changes, as FXR-agonists reduce bacterial translocation via the portal-venous route to the liver in cirrhosis.

Associations between gut microbiota characteristics and non-motor symptoms following pharmacological and surgical treatments in Parkinson's disease patients.

BACKGROUND: The gut microbiota has been implicated in Parkinson's disease (PD), with alterations observed in microbial composition and reduced microbial species richness, which may influence gastrointestinal symptoms in PD patients. It remains to be determined whether the severity of gastrointestinal symptoms correlates with microbiota variations in PD patients treated pharmacologically or with subthalamic nucleus deep brain stimulation (STN-DBS) therapy. This study aims to explore how these treatments affect gut microbiota and gastrointestinal symptoms in PD, identifying specific microbial differences associated with each treatment modality. METHODS: A total of 42 individuals diagnosed with PD, along with 38 age-matched household control participants, contributed stool samples for microbiota characterization. Differences in the gut microbiota across various groups of PD patients and their households were identified through comprehensive sequencing of the 16S rRNA gene amplicon sequencing. KEY RESULTS: Differences in microbial communities were observed between PD patients and controls, as well as between PD patients receiving pharmacological treatment and those with STN-DBS. Pharmacologically treated advanced PD patients have higher gastrointestinal dysfunctions. Gut microbiota profile linked to STN-DBS and reduced levodopa consumption, characterized by its anti-inflammatory properties, might play a role in diminishing gastrointestinal dysfunction relative to only pharmacological treatments. CONCLUSIONS & INFERENCES: Advanced PD patients on medication exhibit more gastrointestinal issues, despite relatively stable microbial diversity, indicating a complex interaction between gut microbiota, PD progression, and treatment effects. An imbalanced gut-brain axis, particularly due to reduced butyrate production, may lead to constipation by affecting the enteric nervous system, which emphasizes the need to incorporate gut microbiome insights into treatment strategies.

Neonatal microbiota colonization drives maturation of primary and secondary goblet cell mediated protection in the pre-weaning colon.

In the distal colon, mucus secreting goblet cells primarily confer protection from luminal microorganisms via generation of a sterile inner mucus layer barrier structure. Bacteria-sensing sentinel goblet cells provide a secondary defensive mechanism that orchestrates mucus secretion in response to microbes that breach the mucus barrier. Previous reports have identified mucus barrier deficiencies in adult germ-free mice, thus implicating a fundamental role for the microbiota in programming mucus barrier generation. In this study, we have investigated the natural neonatal development of the mucus barrier and sentinel goblet cell-dependent secretory responses upon postnatal colonization. Combined in vivo and ex vivo analyses of pre- and post-weaning colonic mucus barrier and sentinel goblet cell maturation demonstrated a sequential microbiota-dependent development of these primary and secondary goblet cell-intrinsic protective functions, with dynamic changes in mucus processing dependent on innate immune signalling via MyD88, and development of functional sentinel goblet cells dependent on the NADPH/Dual oxidase family member Duox2. Our findings therefore identify new mechanisms of microbiota-goblet cell regulatory interaction and highlight the critical importance of the pre-weaning period for the normal development of colonic barrier function.

D-Lactate: Implications for Gastrointestinal Diseases.

D-lactate is produced in very low amounts in human tissues. However, certain bacteria in the human intestine produce D-lactate. In some gastrointestinal diseases, increased bacterial D-lactate production and uptake from the gut into the bloodstream take place. In its extreme, excessive accumulation of D-lactate in humans can lead to potentially life-threatening D-lactic acidosis. This metabolic phenomenon is well described in pediatric patients with short bowel syndrome. Less is known about a subclinical rise in D-lactate. We discuss in this review the pathophysiology of D-lactate in the human body. We cover D-lactic acidosis in patients with short bowel syndrome as well as subclinical elevations of D-lactate in other diseases affecting the gastrointestinal tract. Furthermore, we argue for the potential of D-lactate as a marker of intestinal barrier integrity in the context of dysbiosis. Subsequently, we conclude that there is a research need to establish D-lactate as a minimally invasive biomarker in gastrointestinal diseases.

Benchmarking microbial DNA enrichment protocols from human intestinal biopsies.

Shotgun metagenomic sequencing is a powerful tool for studying bacterial communities in their natural habitats or sites of infection, without the need for cultivation. However, low microbial signals in metagenomic sequencing can be overwhelmed by host DNA contamination, resulting in decreased sensitivity for microbial read detection. Several commercial kits and other methods have been developed to enrich bacterial sequences; however, these assays have not been tested extensively for human intestinal tissues yet. Therefore, the objective of this study was to assess the effectiveness of various wet-lab and software-based approaches for depleting host DNA from microbiome samples. Four different microbiome DNA enrichment methods, namely the NEBNext Microbiome DNA Enrichment kit, Molzym Ultra-Deep Microbiome Prep, QIAamp DNA Microbiome kit, and Zymo HostZERO microbial DNA kit, were evaluated, along with a software-controlled adaptive sampling (AS) approach by Oxford Nanopore Technologies (ONT) providing microbial signal enrichment by aborting unwanted host DNA sequencing. The NEBNext and QIAamp kits proved to be effective in shotgun metagenomic sequencing studies, as they efficiently reduced host DNA contamination, resulting in 24% and 28% bacterial DNA sequences, respectively, compared to <1% in the AllPrep controls. Additional optimization steps using further detergents and bead-beating steps improved the efficacy of less efficient protocols but not of the QIAamp kit. In contrast, ONT AS increased the overall number of bacterial reads resulting in a better bacterial metagenomic assembly with more bacterial contigs with greater completeness compared to non-AS approaches. Additionally, AS also allowed for the recovery of antimicrobial resistance markers and the identification of plasmids, demonstrating the potential utility of AS for targeted sequencing of microbial signals in complex samples with high amounts of host DNA. However, ONT AS resulted in relevant shifts in the observed bacterial abundance, including 2 to 5 times more Escherichia coli reads. Furthermore, a modest enrichment of Bacteroides fragilis and Bacteroides thetaiotaomicron was also observed with AS. Overall, this study provides insight into the efficacy and limitations of various methods for reducing host DNA contamination in human intestinal samples to improve the utility of metagenomic sequencing.

Intestinal dysbiosis as an intraoperative predictor of septic complications: evidence from human surgical cohorts and preclinical models of peritoneal sepsis.

Major surgery exposes the intestinal microbiota to inflammatory and antibiotic stressors, which alter the microbiota composition of the intestinal lumen and fecal contents. However, it is not sufficiently understood, if such dysbiosis develops already during surgery and if alterations in microbiota may be the cause of surgical complications. End-of-surgery composition of the microbiota in the rectum was assessed in 41 patients undergoing either rectal or duodenopancreatic resection and was compared to baseline before surgery using 16S-rRNA sequencing. A subset of patients developed severe dysbiosis at the end of surgery, which was characterized by an overgrowth of the Proteobacteria phylum that includes the facultative pathogen E. coli. To test if dysbiosis impacts on surgical outcomes, dysbiosis was modeled in mice by a single oral administration of vancomycin prior to cecal ligation and puncture. Dysbiosis was associated with impaired post-surgical survival, dysregulation of the host's immune response, elevated bacterial virulence and reduced bacterial metabolism of carbon sources. In conclusion, dysbiosis can be detected already at the end of surgery in a fraction of patients undergoing major surgery. Modelling surgery-associated dysbiosis in mice using single-shot administration of vancomycin induced dysbiosis and resulted in elevated mortality.

Immune activation and inflammation in lactating women on combination antiretroviral therapy: role of gut dysfunction and gut microbiota imbalance.

Introduction: Combination antiretroviral therapy (cART) effectively controls HIV; however, chronic low-level viremia and gut microbiota dysbiosis remain significant drivers of gut and systemic inflammation. In this study, we explored the relationship between gut microbiota composition, intestinal inflammation, microbial translocation, and systemic inflammation in women on cART in Sub-Saharan Africa. Methods: We conducted a study in HIV-infected and HIV-uninfected lactating women followed up at 6 weeks and 6 months postpartum in Harare, Zimbabwe. We used 16S ribosomal Ribonucleic Acid (rRNA) sequencing and MesoScale Discovery V-Plex assays to examine the gut microbiome and to quantify plasma inflammatory biomarkers, respectively. In addition, we measured fecal calprotectin, plasma lipopolysaccharide-binding protein (LBP), and soluble cluster of differentiation 14 (sCD14) by enzyme-linked immunosorbent assay to assess gut inflammation, microbial translocation, and monocyte/macrophage activation. Results: A group of 77 lactating women were studied, of which 35% were HIV-infected. Fecal calprotectin levels were similar by HIV status at both follow-up time points. In the HIV-infected group at 6 weeks postpartum, fecal calprotectin was elevated: median (interquartile range) [158.1 µg/g (75.3-230.2)] in women who had CD4+ T-lymphocyte counts <350 cells/µL compared with those with ≥350 cells/µL [21.1 µg/g (0-58.4)], p = 0.032. Plasma sCD14 levels were significantly higher in the HIV-infected group at both 6 weeks and 6 months postpartum, p < 0.001. Plasma LBP levels were similar, but higher levels were observed in HIV-infected women with elevated fecal calprotectin. We found significant correlations between fecal calprotectin, LBP, and sCD14 with proinflammatory cytokines. Gut microbial alpha diversity was not affected by HIV status and was not affected by use of antibiotic prophylaxis. HIV significantly affected microbial beta diversity, and significant differences in microbial composition were noted. The genera Slackia and Collinsella were relatively more abundant in the HIV-infected group, whereas a lower relative abundance of Clostriduim sensu_stricto_1 was observed. Our study also found correlations between gut microbial taxa abundance and systemic inflammatory biomarkers. Discussion and conclusion: HIV-infected lactating women had increased immune activation and increased microbial translocation associated with increased gut inflammation. We identified correlations between the gut inflammation and microbial composition, microbial translocation, and systemic inflammation. The interplay of these parameters might affect the health of this vulnerable population.