Chronic TNF in the aging microenvironment exacerbates Tet2-loss-of-function myeloid expansion.

Somatic mutations in the TET2 gene occur more frequently with age, imparting an intrinsic hematopoietic stem cell (HSC) advantage and contributing to a phenomenon termed clonal hematopoiesis of indeterminate potential (CHIP). Individuals with TET2-mutant CHIP have a higher risk of developing myeloid neoplasms and other aging-related conditions. Despite its role in unhealthy aging, the extrinsic mechanisms driving TET2-mutant CHIP clonal expansion remain unclear. We previously showed an environment containing TNF favours TET2-mutant HSC expansion in vitro. We therefore postulated that age-related increases in TNF also provide an advantage to HSCs with TET2-mutations in vivo. To test this hypothesis, we generated mixed bone marrow chimeric mice of old wild-type (WT) and TNF-/- genotypes reconstituted with WT CD45.1+ and Tet2-/-CD45.2+ HSCs. We show that age-associated increases in TNF dramatically increased the expansion of Tet2-/-cells in old WT recipient mice, with strong skewing towards the myeloid lineage. This aberrant myelomonocytic advantage was mitigated in old TNF-/- recipient mice, suggesting that TNF signalling is essential for the expansion Tet2-mutant myeloid clones. Examination of human rheumatoid arthritis patients with clonal hematopoiesis revealed that hematopoietic cells carrying certain mutations, including in TET2, may be sensitive to reduced TNF bioactivity following blockade with adalimumab. This suggests that targeting TNF may reduce the burden of some forms of CHIP. To our knowledge, this is the first evidence to demonstrate that TNF has a causal role in driving TET2-mutant CHIP in vivo. These findings highlight TNF as a candidate therapeutic target to control TET2-mutant CHIP.

Neutrophil-mediated innate immune resistance to bacterial pneumonia is dependent on Tet2 function.

Individuals with clonal hematopoiesis of indeterminate potential (CHIP) are at increased risk of aging related health conditions and all-cause mortality, but whether CHIP affects risk of infection is much less clear. Using UK Biobank data, we revealed a positive association between CHIP and incident pneumonia in 438,421 individuals. We show that inflammation enhanced pneumonia risk, as CHIP carriers with a hypomorphic IL6 receptor polymorphism were protected. To better characterize the pathways of susceptibility, we challenged hematopoietic Tet Methylcytosine Dioxygenase 2-knockout (Tet2-/-) and floxed control mice (Tet2fl/fl) with Streptococcus pneumoniae. As with human CHIP carriers, Tet2-/- mice had hematopoietic abnormalities resulting in the expansion of inflammatory monocytes and neutrophils in peripheral blood. Yet, these cells were insufficient in defending against S. pneumoniae and resulted in increased pathology, impaired bacterial clearance, and higher mortality in Tet2-/- mice. We delineated the transcriptional landscape of Tet2-/- neutrophils and found that, while inflammation-related pathways were upregulated in Tet2-/- neutrophils, migration and motility pathways were compromised. Using live-imaging techniques, we demonstrated impairments in motility, pathogen uptake, and neutrophil extracellular trap (NET) formation by Tet2-/- neutrophils. Collectively, we show that CHIP is a risk factor for bacterial pneumonia related to innate immune impairments.

Fasting increases microbiome-based colonization resistance and reduces host inflammatory responses during an enteric bacterial infection.

Reducing food intake is a common host response to infection, yet it remains unclear whether fasting is detrimental or beneficial to an infected host. Despite the gastrointestinal tract being the primary site of nutrient uptake and a common route for infection, studies have yet to examine how fasting alters the host's response to an enteric infection. To test this, mice were fasted before and during oral infection with the invasive bacterium Salmonella enterica serovar Typhimurium. Fasting dramatically interrupted infection and subsequent gastroenteritis by suppressing Salmonella's SPI-1 virulence program, preventing invasion of the gut epithelium. Virulence suppression depended on the gut microbiota, as Salmonella's invasion of the epithelium proceeded in fasting gnotobiotic mice. Despite Salmonella's restored virulence within the intestines of gnotobiotic mice, fasting downregulated pro-inflammatory signaling, greatly reducing intestinal pathology. Our study highlights how food intake controls the complex relationship between host, pathogen and gut microbiota during an enteric infection.

Early life environmental exposures have a minor impact on the gut ecosystem following a natural birth.

A growing body of evidence suggests that the environment is an important source of colonizing bacteria for the gastrointestinal tract of C-section delivered infants, who undergo multiple birth-related interventions; however, the extent to which environmental microbes impact vaginally delivered infants remains unclear. Here we investigated the impact of rural and urban environmental exposures on microbial establishment and immunity in vaginally delivered mice. We simulated rural and urban home environments by adding soil types to cages from breeding to weaning. Our aims were to determine the impact of rural and urban soil exposures on the gut microbiome in young mice and to understand whether these changes persisted into adulthood. Host immune cytokines and microbial short-chain fatty acids were quantified to understand the impact on immunity. We found that early-life soil exposure had a minor effect on the richness of the neonatal gut microbiota contributing 5% and 9% variation in the bacterial community structure between mice during early-life and adulthood, respectively. Exposure to urban soil increased Clostridiaceae and propionic acid which persisted into adulthood. While soil exposure had a limited effect on the gut taxa, systemic cytokine and chemokine profiles were altered in adulthood. The findings presented here show that unlike in C-section deliveries previously reported, environmental exposures following a natural birth have a limited impact on the gut microbial taxa but potentially play an important role in immune-mediated disease susceptibility later in life.

Maternal Intake of Dietary Fat Pre-Programs Offspring's Gut Ecosystem Altering Colonization Resistance and Immunity to Infectious Colitis in Mice.

SCOPE: The transgenerational impact of dietary fat remains unclear. Here, the role of maternal fat consumption as a modulator of gut microbial communities and infectious disease outcomes in their offspring is explored. METHODS AND RESULTS: C57BL/6 mice are fed isocaloric high-fat diets throughout breeding, gestation and lactation. Diets contained either milk fat (MF), olive oil (OO) or corn oil (CO), with or without fish oil. The pups born to maternally exposed mice are weaned on to chow and raised into adulthood. At 8 weeks, the offsprings are either euthanized for colonic 16S rRNA analysis or challenged with the enteric pathogen, Citrobacter rodentium. Maternal CO exposure resulted in unique clustering of bacterial communities in offspring compared with MF and OO. Diets rich in CO reduced survival in offspring challenged with C. rodentium. The addition of fish oil did not improve mortality caused by CO and worsened disease outcomes when combined with OO. Unlike the unsaturated diets, MF is protective with and without fish oil. CONCLUSIONS: Overall, these data reveal that maternal intake of fatty acids do have transgenerational impacts on their offspring's bacteriome and enteric infection risk. Based on this study, saturated fats should be included in maternal diets.

Physical Activity Shapes the Intestinal Microbiome and Immunity of Healthy Mice but Has No Protective Effects against Colitis in MUC2-/- Mice.

The interactions among humans, their environment, and the trillions of microbes residing within the human intestinal tract form a tripartite relationship that is fundamental to the overall health of the host. Disruptions in the delicate balance between the intestinal microbiota and host immunity are implicated in various chronic diseases, including inflammatory bowel disease (IBD). There is no known cure for IBD; therefore, novel therapeutics targeting prevention and symptom management are of great interest. Recently, physical activity in healthy mice was shown to be protective against chemically induced colitis; however, the benefits of physical activity during or following disease onset are not known. In this study, we examine whether voluntary wheel running is protective against primary disease symptoms in a mucin 2-deficient (Muc2-/- ) lifelong model of murine colitis. We show that 6 weeks of wheel running in healthy C57BL/6 mice leads to distinct changes in fecal bacteriome, increased butyrate production, and modulation in colonic gene expression of various cytokines, suggesting an overall primed anti-inflammatory state. However, these physical activity-derived benefits are not present in Muc2-/- mice harboring a dysfunctional mucosal layer from birth, ultimately showing no improvements in clinical signs. We extrapolate from our findings that while physical activity in healthy individuals may be an important preventative measure against IBD, for those with a compromised intestinal mucosa, a commonality in IBD patients, these benefits are lost.IMPORTANCE Perturbation in the gut microbial ecosystem has been associated with various diseases, including inflammatory bowel disease. Habitual physical activity, through its ability to modulate the gut microbiome, has recently been shown to prophylactically protect against chemically induced models of murine colitis. Here, we (i) confirm previous reports that physical activity has limited but significant effects on the gut microbiome of mice and (ii) show that such changes are associated with anti-inflammatory states in the gut, such as increased production of beneficial short-chain fatty acids and lower levels of proinflammatory immune markers implicated in human colitis; however, we also show that (iii) these physical activity-derived benefits are completely lost in the absence of a healthy intestinal mucus layer, a hallmark phenotype of human colitis.

Fish oil supplementation reduces maternal defensive inflammation and predicts a gut bacteriome with reduced immune priming capacity in infants.

Habitual supplementation of fish oil is thought to provide benefits to the developing infant; however, the effects on infant microbial establishment and immune development are unknown. A 6-month observational cohort study was conducted where 47 out of 91 women self-administered dietary fish oil during breastfeeding. Infant stool and mothers' breast milk were collected each month over 6 months. Gas chromatography was used to quantify breast milk fatty acids and high-throughput sequencing was used to assess the infant fecal microbiota. Immune markers and parent-reported questionnaires were used to assess infant immunity and health up to 2 years. Our results reveal that fish oil supplementation decreased secretory immunoglobulin A and increased IL-10 production in lactating women along with increased breast milk eicosapentaenoic acid, and this corresponded to increased abundances of fecal Bifidobacterium and Lactobacillus spp. in their infants. Docosahexaenoic acid levels in breast milk aligned with decreases in infant gut bacterial richness and the predicted bacterial phenotypes suggested that fish oil lowers commensal traits involved in pathogen colonization resistance. Despite this, there were no differences in sickness incidence in toddlers. This study revealed that fish oil associates with decreases in breast milk defensive inflammatory responses and corresponds with infant fecal microbiota with anti-inflammatory potential.

Human behavior, not race or geography, is the strongest predictor of microbial succession in the gut bacteriome of infants.

Colonization of the gastrointestinal tract with microorganisms during infancy represents a critical control point for shaping life-long immune-mediated disease susceptibility. Abnormal colonization or an imbalance of microbes, termed dysbiosis, is implicated in several diseases. Consequently, recent research has aimed at understanding ways to manipulate a dysbiotic microbiome during infancy to resemble a normal, healthy microbiome. However, one of the fundamental issues in microbiome research is characterizing what a "normal" infant microbiome is based on geography, ethnicity and cultural variations. This review provides a comprehensive account of what is currently known about the infant microbiome from a global context. In general, this review shows that the influence of cultural variations in feeding practices, delivery modes and hygiene are the biggest contributors to microbial variability. Despite geography or race, all humans have similar microbial succession during infancy.

Influence of sulfonated and diet-derived human milk oligosaccharides on the infant microbiome and immune markers.

Human milk oligosaccharides (HMOs) promote the development of the neonatal intestinal, immune, and nervous systems and has recently received considerable attention. Here we investigated how the maternal diet affects HMO biosynthesis and how any diet-induced HMO alterations influence the infant gut microbiome and immunity. Using capillary electrophoresis and MS-based analyses, we extracted and measured HMOs from breast milk samples and then correlated their levels with results from validated 24-h diet recall surveys and breast milk fatty acids. We found that fruit intake and unsaturated fatty acids in breast milk were positively correlated with an increased absolute abundance of numerous HMOs, including 16 sulfonated HMOs we identified here in humans for the first time. The diet-derived monosaccharide 5-N-glycolyl-neuraminic acid (Neu5Gc) was unambiguously detected in all samples. To gain insights into the potential impact of Neu5Gc on the infant microbiome, we used a constrained ordination approach and identified correlations between Neu5Gc levels and Bacteroides spp. in infant stool. However, Neu5Gc was not associated with marked changes in infant immune markers, in contrast with sulfonated HMOs, whose expression correlated with suppression of two major Th2 cytokines, IL-10 and IL-13. The findings of our work highlight the importance of maternal diet for HMO biosynthesis and provide as yet unexplored targets for future studies investigating interactions between HMOs and the intestinal microbiome and immunity in infants.

Dietary Fatty Acids and Host-Microbial Crosstalk in Neonatal Enteric Infection.

Human milk is the best nutritional choice for infants. However, in instances where breastfeeding is not possible, infant formulas are used as alternatives. While formula manufacturers attempt to mimic the performance of human breast milk, formula-fed babies consistently have higher incidences of infection from diarrheal diseases than those breastfed. Differences in disease susceptibility, progression and severity can be attributed, in part, to nutritional fatty acid differences between breast milk and formula. Despite advances in our understanding of breast milk properties, formulas still present major differences in their fatty acid composition when compared to human breast milk. In this review, we highlight the role of distinct types of dietary fatty acids in modulating host inflammation, both directly and through the microbiome-immune nexus. We present evidence that dietary fatty acids influence enteric disease susceptibility and therefore, altering the fatty acid composition in formula may be a potential strategy to improve infectious outcomes in formula-fed infants.

Gut Mucosal Proteins and Bacteriome Are Shaped by the Saturation Index of Dietary Lipids.

The dynamics of the tripartite relationship between the host, gut bacteria and diet in the gut is relatively unknown. An imbalance between harmful and protective gut bacteria, termed dysbiosis, has been linked to many diseases and has most often been attributed to high-fat dietary intake. However, we recently clarified that the type of fat, not calories, were important in the development of murine colitis. To further understand the host-microbe dynamic in response to dietary lipids, we fed mice isocaloric high-fat diets containing either milk fat, corn oil or olive oil and performed 16S rRNA gene sequencing of the colon microbiome and mass spectrometry-based relative quantification of the colonic metaproteome. The corn oil diet, rich in omega-6 polyunsaturated fatty acids, increased the potential for pathobiont survival and invasion in an inflamed, oxidized and damaged gut while saturated fatty acids promoted compensatory inflammatory responses involved in tissue healing. We conclude that various lipids uniquely alter the host-microbe interaction in the gut. While high-fat consumption has a distinct impact on the gut microbiota, the type of fatty acids alters the relative microbial abundances and predicted functions. These results support that the type of fat are key to understanding the biological effects of high-fat diets on gut health.

Effects of Azithromycin on Behavior, Pathologic Signs, and Changes in Cytokines, Chemokines, and Neutrophil Migration in C57BL/6 Mice Exposed to Dextran Sulfate Sodium.

Here we characterized the murine dextran sulfate sodium (DSS) model of acute colitis. Specifically, we evaluated azithromycin and metronidazole treatment regimens to assess their effects on animal wellbeing, pathologic changes, barrier function, cytokine and chemokine profiles, and neutrophil migration in colon tissue. Azithromycin treatment significantly reduced the severity of colitis, as assessed through body weight change, water consumption, macroscopic lesions, and animal behaviors (activity level, climbing, and grooming), but did not alter food consumption or feeding behavior. Mucosal barrier function (evaluated by using FITC-labeled dextran) was decreased after DSS exposure; azithromycin did not significantly alter barrier function in mice with colitis, whereas metronidazole exacerbated the colitis-related deficit in barrier function. In addition, metronidazole appeared to exacerbate disease as assessed through water consumption and animal behaviors (overall activity, climbing, grooming, and drinking) but had no effect on weight loss, macroscopic lesions, or eating behavior. Pathologic changes were typical for DSS treatment. Antibiotic treatment resulted in reduced levels of proinflammatory cytokines and chemokines and decreased neutrophil adhesion and emigration in DSS-exposed mice. The results highlight the importance of clinical and behavioral assessments in addition to laboratory evaluation as tools to evaluate animal welfare and therapeutic efficacy in disease models. Data from this study suggest that azithromycin may convey some benefits in the mouse DSS colitis model through modulation of the immune response, including neutrophil migration into tissues, whereas metronidazole may exacerbate colitis.

Dietary Lipid Type, Rather Than Total Number of Calories, Alters Outcomes of Enteric Infection in Mice.

Dietary lipids modulate immunity, yet the means by which specific fatty acids affect infectious disease susceptibility remains unclear. Deciphering lipid-induced immunity is critical to understanding the balance required for protecting against pathogens while avoiding chronic inflammatory diseases. To understand how specific lipids alter susceptibility to enteric infection, we fed mice isocaloric, high-fat diets composed of corn oil (rich in n-6 polyunsaturated fatty acids [n-6 PUFAs]), olive oil (rich in monounsaturated fatty acids), or milk fat (rich in saturated fatty acids) with or without fish oil (rich in n-3 PUFAs). After 5 weeks of dietary intervention, mice were challenged with Citrobacter rodentium, and pathological responses were assessed. Olive oil diets resulted in little colonic pathology associated with intestinal alkaline phosphatase, a mucosal defense factor that detoxifies lipopolysaccharide. In contrast, while both corn oil and milk fat diets resulted in inflammation-induced colonic damage, only milk fat induced compensatory protective responses, including short chain fatty acid production. Fish oil combined with milk fat, unlike unsaturated lipid diets, had a protective effect associated with intestinal alkaline phosphatase activity. Overall, these results reveal that dietary lipid type, independent of the total number of calories associated with the dietary lipid, influences the susceptibility to enteric damage and the benefits of fish oil during infection.

Positron emission tomography and functional characterization of a complete PBR/TSPO knockout.

The evolutionarily conserved peripheral benzodiazepine receptor (PBR), or 18-kDa translocator protein (TSPO), is thought to be essential for cholesterol transport and steroidogenesis, and thus life. TSPO has been proposed as a biomarker of neuroinflammation and a new drug target in neurological diseases ranging from Alzheimer's disease to anxiety. Here we show that global C57BL/6-Tspo(tm1GuWu(GuwiyangWurra))-knockout mice are viable with normal growth, lifespan, cholesterol transport, blood pregnenolone concentration, protoporphyrin IX metabolism, fertility and behaviour. However, while the activation of microglia after neuronal injury appears to be unimpaired, microglia from (GuwiyangWurra)TSPO knockouts produce significantly less ATP, suggesting reduced metabolic activity. Using the isoquinoline PK11195, the ligand originally used for the pharmacological and structural characterization of the PBR/TSPO, and the imidazopyridines CLINDE and PBR111, we demonstrate the utility of (GuwiyangWurra)TSPO knockouts to provide robust data on drug specificity and selectivity, both in vitro and in vivo, as well as the mechanism of action of putative TSPO-targeting drugs.