Specific gut pathobionts escape antibody coating and are enriched during flares in patients with severe Crohn's disease.

OBJECTIVE: Patients with Crohn's disease (CD) exhibit great heterogeneity in disease presentation and treatment responses, where distinct gut bacteria and immune interactions may play part in the yet unresolved disease aetiology. Given the role of antibodies in the barrier defence against microbes, we hypothesised that gut bacterial antibody-coating patterns may influence underlying disease-mediated processes. DESIGN: Absolute and relative single and multicoating of gut bacteria with IgA, IgG1, IgG2, IgG3 and IgG4 in patients with CD and healthy controls were characterised and compared with disease activity. IgG2-coated and non-coated taxa from patients with severe CD were identified, profiled for pathogenic characteristics and monitored for enrichment during active disease across cohorts. RESULTS: Patients with severe CD exhibited higher gut bacterial IgG2-coating. Supervised clustering identified 25 bacteria to be enriched in CD patients with high IgG2-coating. Sorting, sequencing and in silico-based assessments of the virulent potential of IgG2-coated and bulk stool bacteria were performed to evaluate the nature and pathogenicity of IgG2-coated and non-coated bacteria. The analyses demonstrated IgG2-coating of both known pathogenic and non-pathogenic bacteria that co-occurred with two non-coated pathobionts, Campylobacter and Mannheimia. The two non-coated pathobionts exhibited low prevalence, rarely coincided and were strongly enriched during disease flares in patients with CD across independent and geographically distant cohorts. CONCLUSION: Distinct gut bacterial IgG2-coating was demonstrated in patients with severe CD and during disease flares. Co-occurrence of non-coated pathobionts with IgG2-coated bacteria points to an uncontrolled inflammatory condition in severe CD mediated via escape from antibody coating by two gut pathobionts.

IgG and IgM cooperate in coating of intestinal bacteria in IgA deficiency.

Immunoglobulin A (IgA) is acknowledged to play a role in the defence of the mucosal barrier by coating microorganisms. Surprisingly, IgA-deficient humans exhibit few infection-related complications, raising the question if the more specific IgG may help IgM in compensating for the lack of IgA. Here we employ a cohort of IgA-deficient humans, each paired with IgA-sufficient household members, to investigate multi-Ig bacterial coating. In IgA-deficient humans, IgM alone, and together with IgG, recapitulate coating of most bacterial families, despite an overall 3.6-fold lower Ig-coating. Bacterial IgG coating is dominated by IgG1 and IgG4. Single-IgG2 bacterial coating is sparse and linked to enhanced Escherichia coli load and TNF-α. Although single-IgG2 coating is 1.6-fold more prevalent in IgA deficiency than in healthy controls, it is 2-fold less prevalent than in inflammatory bowel disease. Altogether we demonstrate that IgG assists IgM in coating of most bacterial families in the absence of IgA and identify single-IgG2 bacterial coating as an inflammatory marker.

Oral administration of helminth fluid modulates distinct tuft cell and immune-metabolic cues linked to reduced body fat.

Intestinal tuft cells have been shown to induce type 2 immune responses during viable parasite infections, but whether oral supplementation with a parasitic exudate is able to promote type 2 immune responses that have been shown to positively regulate obesogenic metabolic processes is yet unresolved. High-fat fed mice were gavaged with pseudocoelomic fluid (PCF) derived from the helminth Ascaris suum or saline thrice a week during weeks 5-9, followed by examination of intestinal tuft cell activity, immune, and metabolic parameters. Helminth PCF upregulated expression of distinct genes in small intestinal tuft cells, including genes involved in regulation of RUNX1 and organic cation transporters. Helminth PCF also enhanced levels of innate lymphoid cells in the ileum, and eosinophils in epididymal white adipose tissue (eWAT). Network analyses revealed two distinct immunometabolic cues affected by oral helminth PCF in high-fat fed mice: one coupling the small intestinal tuft cell responses to the fat-to-lean mass ratio and a second coupling eosinophils in eWAT to general regulation of body fat mass. Our findings point to specific mechanisms by which oral supplementation with helminth PCF may translate into systems-wide effects linking to reduced body and fat mass gain in mice during high-fat feeding.

AlphaFold2 enables accurate deorphanization of ligands to single-pass receptors.

Secreted proteins play crucial roles in paracrine and endocrine signaling; however, identifying novel ligand-receptor interactions remains challenging. Here, we benchmarked AlphaFold as a screening approach to identify extracellular ligand-binding pairs using a structural library of single-pass transmembrane receptors. Key to the approach is the optimization of AlphaFold input and output for screening ligands against receptors to predict the most probable ligand-receptor interactions. Importantly, the predictions were performed on ligand-receptor pairs not used for AlphaFold training. We demonstrate high discriminatory power and a success rate of close to 90 % for known ligand-receptor pairs and 50 % for a diverse set of experimentally validated interactions. These results demonstrate proof-of-concept of a rapid and accurate screening platform to predict high-confidence cell-surface receptors for a diverse set of ligands by structural binding prediction, with potentially wide applicability for the understanding of cell-cell communication.

Profiling the Atopic Dermatitis Epidermal Transcriptome by Tape Stripping and BRB-seq.

Tape stripping is a non-invasive skin sampling technique, which has recently gained use for the study of the transcriptome of atopic dermatitis (AD), a common inflammatory skin disorder characterized by a defective epidermal barrier and perturbated immune response. Here, we performed BRB-seq-a low cost, multiplex-based, transcriptomic profiling technique-on tape-stripped skin from 30 AD patients and 30 healthy controls to evaluate the methods' ability to assess the epidermal AD transcriptome. An AD signature consisting of 91 differentially expressed genes, specific for skin barrier and inflammatory response, was identified. The gene expression in the outermost layers, stratum corneum and stratum granulosum, of the skin showed highest correlation between tape-stripped skin and matched full-thickness punch biopsies. However, we observed that low and highly variable transcript counts, probably due to low RNA yield and RNA degradation in the tape-stripped skin samples, were a limiting factor for epidermal transcriptome profiling as compared to punch biopsies. We conclude that deep BRB-seq of tape-stripped skin is needed to counteract large between-sample RNA yield variation and highly zero-inflated data in order to apply this protocol for population-wide screening of the epidermal transcriptome in inflammatory skin diseases.

Pot-pollen supplementation reduces fasting glucose and modulates the gut microbiota in high-fat/high-sucrose fed C57BL/6 mice.

Pot-pollen is a mixture of pollen and nectar from flowers combined with salivary substances of stingless bees, which together are fermented inside cerumen pots. As pot-pollen is rich in polyphenols, we hypothesized that dietary ingestion could modulate obesity, glucose metabolism, and the gut microbiota in an animal model of diet-induced obesity. Male C57BL/6J mice were fed a low-fat/low-sucrose diet (LF/LS), a HF/HS diet or a HF/HS diet containing 0.1% pot-pollen (HF/HS-PP) for 12 weeks. In HF/HS-fed mice, pot-pollen supplementation decreased fasting blood glucose and increased glucose-stimulated insulin secretion without modifying weight gain, body composition, glucose tolerance, and insulin sensitivity. Intake of pot-pollen resulted in changes of the gut microbiota, including a decrease in the abundance of the Rikenellaceae RC9 gut group and Lactobacillus, and an increase in the abundance of Romboutsia. Correlations between genus abundances and metabolic changes in response to supplementation indicated that the gut microbiota contributed to the positive effects of pot-pollen ingestion on fasting glucose. Pot-pollen supplementation-associated changes in the gut microbiota composition correlated with the lowering of fasting glucose levels without modulating weight gain.

Small Intestinal Tuft Cell Activity Associates With Energy Metabolism in Diet-Induced Obesity.

Little is known about the involvement of type 2 immune response-promoting intestinal tuft cells in metabolic regulation. We here examined the temporal changes in small intestinal tuft cell number and activity in response to high-fat diet-induced obesity in mice and investigated the relation to whole-body energy metabolism and the immune phenotype of the small intestine and epididymal white adipose tissue. Intake of high fat diet resulted in a reduction in overall numbers of small intestinal epithelial and tuft cells and reduced expression of the intestinal type 2 tuft cell markers Il25 and Tslp. Amongst >1,700 diet-regulated transcripts in tuft cells, we observed an early association between body mass expansion and increased expression of the gene encoding the serine protease inhibitor neuroserpin. By contrast, tuft cell expression of genes encoding gamma aminobutyric acid (GABA)-receptors was coupled to Tslp and Il25 and reduced body mass gain. Combined, our results point to a possible role for small intestinal tuft cells in energy metabolism via coupled regulation of tuft cell type 2 markers and GABA signaling receptors, while being independent of type 2 immune cell involvement. These results pave the way for further studies into interventions that elicit anti-obesogenic circuits via small intestinal tuft cells.

Systems-wide effects of short-term feed deprivation in obese mice.

While prolonged fasting induces significant metabolic changes in humans and mice, less is known about systems-wide metabolic changes in response to short-term feed deprivation, which is used in experimental animal studies prior to metabolic challenge tests. We here performed a systems biology-based investigation of connections between gut bacterial composition and function, inflammatory and metabolic parameters in the intestine, liver, visceral adipose tissue, blood and urine in high-fat fed, obese mice that were feed deprived up to 12 h. The systems-wide analysis revealed that feed deprivation linked to enhanced intestinal butyric acid production and expression of the gene encoding the pro-thermogenic uncoupling protein UCP1 in visceral adipose tissue of obese mice. Ucp1 expression was also positively associated with Il33 expression in ileum, colon and adipose tissue as well as with the abundance of colonic Porphyromonadaceae, the latter also correlating to cecal butyric acid levels. Collectively, the data highlighted presence of a multi-tiered system of inter-tissue communication involving intestinal, immune and metabolic functions which is affected by feed deprivation in obese mice, thus pointing to careful use of short-feed deprivation in metabolic studies using obese mice.

Response of the Human Milk Microbiota to A Maternal Prebiotic Intervention is Individual and Influenced by Maternal Age.

Maternal bacteria are shared with infants via breastfeeding. Prebiotics modulate the gut microbiota, promoting health benefits. We investigated whether the maternal diet supplementation with a prebiotic (fructooligosaccharides, FOS) could influence the milk microbiota. Twenty-eight lactating women received 4.5 g of fructooligosaccharides + 2 g of maltodextrin (FOS group) and twenty-five received 2 g of maltodextrin (placebo group) for 20 days. Breast-milk samples were taken before and after the intervention. The DNA from samples was used for 16S rRNA sequencing. No statistical differences between the groups were found for the bacterial genera after the intervention. However, the distances of the trajectories covered by paired samples from the beginning to the end of the supplementation were higher for the FOS group (p = 0.0007) indicating greater changes in milk microbiota compared to the control group. Linear regression models suggested that the maternal age influenced the response for FOS supplementation (p = 0.02). Interestingly, the pattern of changes to genus abundance upon supplementation was not shared between mothers. We demonstrated that manipulating the human milk microbiota through prebiotics is possible, and the maternal age can affect this response. .

The Human Milk Microbiota is Modulated by Maternal Diet.

Human milk microorganisms contribute not only to the healthy development of the immune system in infants, but also in shaping the gut microbiota. We evaluated the effect of the maternal diet during pregnancy and during the first month of lactation on the human milk microbiota in a cross-sectional study including 94 healthy lactating women. Microbiota composition was determined by 16S rDNA profiling and nutrient intake assessed through food questionnaires. Thirteen genera were present in at least 90% of all samples, with three genera present in all samples: Streptococcus, Staphylococcus, and Corynebacterium. Cluster analysis indicated two distinct compositions: one marked by a high abundance of Streptococcus (cluster 1), and other by a high abundance of Staphylococcus (cluster 2). A global association with milk microbiota diversity was observed for vitamin C intake during pregnancy (p = 0.029), which was higher for cluster 2 individuals (cluster 2 median = 232 mg/d; cluster 1 = 175 mg/d; p = 0.02). Positive correlations were found between Bifidobacterium in the milk and intake of polyunsaturated and linoleic fatty acids during the lactation period (p < 0.01). We show that maternal diet influences the human milk microbiota, especially during pregnancy, which may contribute in shaping the gut microbiota.

Interplay between food and gut microbiota in health and disease.

Numerous microorganisms colonize the human gastrointestinal tract playing pivotal roles in relation to digestion and absorption of dietary components. They biotransform food components and produce metabolites, which in combination with food components shape and modulate the host immune system and metabolic responses. Reciprocally, the diet modulates the composition and functional capacity of the gut microbiota, which subsequently influence host biochemical processes establishing a system of mutual interaction and inter-dependency. Macronutrients, fibers, as well as polyphenols and prebiotics are strong drivers shaping the composition of the gut microbiota. Especially, short-chain fatty acids produced from ingested fibers and tryptophan metabolites are key in modulating host immune responses. Since reciprocal interactions between diet, host, and microbiota are personal, understanding this complex network of interactions calls for novel use of large datasets and the implementation of machine learning algorithms and artificial intelligence. In this review, we aim to provide a base for future investigations of how interactions between food components and gut microbiota may influence or even determine human health and disease.

Effects of Gliadin consumption on the Intestinal Microbiota and Metabolic Homeostasis in Mice Fed a High-fat Diet.

Dietary gluten causes severe disorders like celiac disease in gluten-intolerant humans. However, currently understanding of its impact in tolerant individuals is limited. Our objective was to test whether gliadin, one of the detrimental parts of gluten, would impact the metabolic effects of an obesogenic diet. Mice were fed either a defined high-fat diet (HFD) containing 4% gliadin (n = 20), or a gliadin-free, isocaloric HFD (n = 20) for 23 weeks. Combined analysis of several parameters including insulin resistance, histology of liver and adipose tissue, intestinal microbiota in three gut compartments, gut barrier function, gene expression, urinary metabolites and immune profiles in intestinal, lymphoid, liver and adipose tissues was performed. Mice fed the gliadin-containing HFD displayed higher glycated hemoglobin and higher insulin resistance as evaluated by the homeostasis model assessment, more hepatic lipid accumulation and smaller adipocytes than mice fed the gliadin-free HFD. This was accompanied by alterations in the composition and activity of the gut microbiota, gut barrier function, urine metabolome, and immune phenotypes within liver and adipose tissue. Our results reveal that gliadin disturbs the intestinal environment and affects metabolic homeostasis in obese mice, suggesting a detrimental effect of gluten intake in gluten-tolerant subjects consuming a high-fat diet.

A safflower oil based high-fat/high-sucrose diet modulates the gut microbiota and liver phospholipid profiles associated with early glucose intolerance in the absence of tissue inflammation.

SCOPE: Omega-6 (n-6) PUFA-rich diets are generally considered obesogenic in rodents. Here, we examined how long-term intake of a high-fat/high-sucrose (HF/HS) diet based on safflower oil affected metabolism, inflammation, and gut microbiota composition. METHODS AND RESULTS: We fed male C57BL/6J mice a HF/HS diet based on safflower oil-rich in n-6 PUFAs-or a low-fat/low-sucrose diet for 40 wk. Compared to the low-fat/low-sucrose diet, intake of the safflower-based HF/HS diet only led to moderate weight gain, while glucose intolerance developed at week 5 prior to signs of inflammation, but concurrent with increased levels of linoleic acid and arachidonic acid in hepatic phospholipids. Intake of the HF/HS diet resulted in early changes in the gut microbiota, including an increased abundance of Blautia, while late changes coincided with altered inflammatory profiles and increased fasting plasma insulin. Analysis of immune cells in visceral fat and liver revealed no differences between diets before week 40, where the number of immune cells decreased in the liver of HF/HS-fed mice. CONCLUSION: We suggest that a diet-dependent increase in the n-6 to omega-3 (n-3) PUFA ratio in hepatic phospholipids together with gut microbiota changes contributed to early development of glucose intolerance without signs of inflammation.

Depletion of regulatory T cells leads to an exacerbation of delayed-type hypersensitivity arthritis in C57BL/6 mice that can be counteracted by IL-17 blockade.

Rodent models of arthritis have been extensively used in the elucidation of rheumatoid arthritis (RA) pathogenesis and are instrumental in the development of therapeutic strategies. Here we utilise delayed-type hypersensitivity arthritis (DTHA), a model in C57BL/6 mice affecting one paw with synchronised onset, 100% penetrance and low variation. We investigate the role of regulatory T cells (Tregs) in DTHA through selective depletion of Tregsand the role of IL-17 in connection with Tregdepletion. Given the relevance of Tregsin RA, and the possibility of developing Treg-directed therapies, this approach could be relevant for advancing the understanding of Tregsin inflammatory arthritis. Selective depletion of Tregswas achieved using aFoxp3-DTR-eGFPmouse, which expresses the diphtheria toxin receptor (DTR) and enhanced green fluorescent protein (eGFP) under control of theFoxp3gene. Anti-IL-17 monoclonal antibody (mAb) was used for IL-17 blockade. Numbers and activation of Tregsincreased in the paw and its draining lymph node in DTHA, and depletion of Tregsresulted in exacerbation of disease as shown by increased paw swelling, increased infiltration of inflammatory cells, increased bone remodelling and increased production of inflammatory mediators, as well as increased production of anti-citrullinated protein antibodies. Anti-IL-17 mAb treatment demonstrated that IL-17 is important for disease severity in both the presence and absence of Tregs, and that IL-17 blockade is able to rescue mice from the exacerbated disease caused by Tregdepletion and caused a reduction in RANKL, IL-6 and the number of neutrophils. We show that Tregsare important for the containment of inflammation and bone remodelling in DTHA. To our knowledge, this is the first study using theFoxp3-DTR-eGFPmouse on a C57BL/6 background for Tregdepletion in an arthritis model, and we here demonstrate the usefulness of the approach to study the role of Tregsand IL-17 in arthritis.

Intrauterine Exposure to Paracetamol and Aniline Impairs Female Reproductive Development by Reducing Follicle Reserves and Fertility.

Studies report that fetal exposure to paracetamol/acetaminophen by maternal consumption can interfere with male reproductive development. Moreover, recent biomonitoring data report widespread presence of paracetamol in German and Danish populations, suggesting exposure via secondary (nonpharmaceutical) sources, such as metabolic conversion from the ubiquitous industrial compound aniline. In this study, we investigated the extent to which paracetamol and aniline can interfere with female reproductive development. Intrauterine exposure to paracetamol by gavage of pregnant dams resulted in shortening of the anogenital distance in adult offspring, suggesting that fetal hormone signaling had been disturbed. Female offspring of paracetamol-exposed mothers had ovaries with diminished follicle reserve and reduced fertility. Fetal gonads of exposed animals had also reduced gonocyte numbers, suggesting that the reduced follicle count in adults could be due to early disruption of germ cell development. However, ex vivo cultures of ovaries from 12.5 days post coitum fetuses showed no decrease in proliferation or expression following exposure to paracetamol. This suggests that the effect of paracetamol occurs prior to this developmental stage. Accordingly, using embryonic stem cells as a proxy for primordial germ cells we show that paracetamol is an inhibitor of cellular proliferation, but without cytotoxic effects. Collectively, our data show that intrauterine exposure to paracetamol at levels commonly observed in pregnant women, as well as its precursor aniline, may block primordial germ cell proliferation, ultimately leading to reduced follicle reserves and compromised reproductive capacity later in life.