Age-related patterns of microbial dysbiosis in multiplex inflammatory bowel disease families.

OBJECTIVE: IBD is characterised by dysbiosis, but it remains unclear to what extent dysbiosis develops in unaffected at-risk individuals. To address this, we investigated age-related patterns of faecal and serum markers of dysbiosis in high-risk multiplex IBD families (two or more affected first-degree relatives). DESIGN: Faecal and serum samples were collected from multiplex IBD and control families (95 IBD, 292 unaffected, 51 controls). Findings were validated in independent cohorts of 616 and 1173 subjects including patients with IBD, infants born to mothers with IBD and controls. 16S rRNA gene sequencing and global untargeted metabolomics profiling of faeces and serum were performed. RESULTS: Microbial and metabolomic parameters of dysbiosis progressively decreased from infancy until age 8. This microbial maturation process was slower in infants born to mothers with IBD. After age 15, dysbiosis steadily increased in unaffected relatives throughout adulthood. Dysbiosis was accompanied by marked shifts in the faecal metabolome and, to a lesser extent, the serum metabolome. Faecal and serum metabolomics dysbiosis indices were validated in an independent cohort. Dysbiosis was associated with elevated antimicrobial serologies but not with faecal calprotectin. Dysbiosis metrics differentiated IBD from non-IBD comparably to serologies, with a model combining calprotectin, faecal metabolomics dysbiosis index and serology score demonstrating highest accuracy. CONCLUSION: These findings support that dysbiosis exists as a pre-disease state detectable by faecal and serum biomarkers for IBD risk prediction. Given the expansion of disease-modifying agents and non-invasive imaging, the indices developed here may facilitate earlier diagnoses and improved management in at-risk individuals.

Subchronic inhalation exposure to ultrafine particulate matter alters the intestinal microbiome in various mouse models.

Exposure to ultrafine particles (UFPs) has been associated with multiple adverse health effects. Inhaled UFPs could reach the gastrointestinal tract and influence the composition of the gut microbiome. We have previously shown that oral ingestion of UFPs alters the gut microbiome and promotes intestinal inflammation in hyperlipidemic Ldlr-/- mice. Particulate matter (PM)2.5 inhalation studies have also demonstrated microbiome shifts in normolipidemic C57BL/6 mice. However, it is not known whether changes in microbiome precede or follow inflammatory effects in the intestinal mucosa. We hypothesized that inhaled UFPs modulate the gut microbiome prior to the development of intestinal inflammation. We studied the effects of UFP inhalation on the gut microbiome and intestinal mucosa in two hyperlipidemic mouse models (ApoE-/- mice and Ldlr-/- mice) and normolipidemic C57BL/6 mice. Mice were exposed to PM in the ultrafine-size range by inhalation for 6 h a day, 3 times a week for 10 weeks at a concentration of 300-350 µg/m3.16S rRNA gene sequencing was performed to characterize sequential changes in the fecal microbiome during exposures, and changes in the intestinal microbiome at the end. PM exposure led to progressive differentiation of the microbiota over time, associated with increased fecal microbial richness and evenness, altered microbial composition, and differentially abundant microbes by week 10 depending on the mouse model. Cross-sectional analysis of the small intestinal microbiome at week 10 showed significant changes in α-diversity, ß-diversity, and abundances of individual microbial taxa in the two hyperlipidemic models. These alterations of the intestinal microbiome were not accompanied, and therefore could not be caused, by increased intestinal inflammation as determined by histological analysis of small and large intestine, cytokine gene expression, and levels of fecal lipocalin. In conclusion, 10-week inhalation exposures to UFPs induced taxonomic changes in the microbiome of various animal models in the absence of intestinal inflammation.

ZIP8 A391T Crohn's Disease-Linked Risk Variant Induces Colonic Metal Ion Dyshomeostasis, Microbiome Compositional Shifts, and Inflammation.

BACKGROUND: The pathogenesis of Crohn's disease involves genetic and environmental factors, with the gut microbiome playing a crucial role. The Crohn's disease-associated variant rs13107325 in the SLC39A8 gene results in an A391T substitution in the ZIP8 metal ion transporter and has previously been linked to alterations in the colonic microbiome in variant carriers. We hypothesized that the A391T substitution alters metal ion homeostasis in the colonic mucosal-luminal interface, thereby inducing dysbiosis which may promote intestinal inflammation. METHODS: To evaluate this hypothesis, we generated a SLC39A8 A393T mouse model (matching human A391T). We first examined trace element abundance in the colonic mucosal epithelium and lumen of homozygous A393T and wild-type (WT) mice to determine if the variant affected metal distribution. We also performed 16S rRNA gene sequencing on colon samples at 2 months, 3-4 months, and 12 months of age, and conducted histological scoring of colon tissue collected from 5-month and 10-month old mice. RESULTS: Consistent with an effect of the variant on ZIP8 function, homozygous A393T mice exhibited increased cobalt in the colonic mucosa, but reduced iron, zinc, manganese, cobalt, copper, and cadmium in the colonic lumen. 16S rRNA gene sequencing of colon samples revealed variant-linked effects on microbiome beta diversity in 2-month-, 3-4-month-, and 12-month-old mice. Histological scoring showed spontaneous intestinal inflammation in 10-month but not in 5-month-old mice. Lastly, predicted pathway analysis of the microbiome samples revealed differential enrichment of iron-, zinc-, and cobalt-dependent pathways in A393T mice compared to wild-type controls. CONCLUSION: These results suggest that the variant in SLC39A8 primarily restricts metal availability to the microbiota, resulting in compositions that can adapt to the environment and that A393T-linked dysbiosis occurs prior to the onset of inflammation. This study paves the way for future studies investigating risk variants as microbiome-disease modifiers.

Ambient Particulate Matter Induces In Vitro Toxicity to Intestinal Epithelial Cells without Exacerbating Acute Colitis Induced by Dextran Sodium Sulfate or 2,4,6-Trinitrobenzenesulfonic Acid.

Inflammatory bowel disease (IBD) is an immunologically complex disorder involving genetic, microbial, and environmental risk factors. Its global burden has continued to rise since industrialization, with epidemiological studies suggesting that ambient particulate matter (PM) in air pollution could be a contributing factor. Prior animal studies have shown that oral PM10 exposure promotes intestinal inflammation in a genetic IBD model and that PM2.5 inhalation exposure can increase intestinal levels of pro-inflammatory cytokines. PM10 and PM2.5 include ultrafine particles (UFP), which have an aerodynamic diameter of <0.10 µm and biophysical and biochemical properties that promote toxicity. UFP inhalation, however, has not been previously studied in the context of murine models of IBD. Here, we demonstrated that ambient PM is toxic to cultured Caco-2 intestinal epithelial cells and examined whether UFP inhalation affected acute colitis induced by dextran sodium sulfate and 2,4,6-trinitrobenzenesulfonic acid. C57BL/6J mice were exposed to filtered air (FA) or various types of ambient PM reaerosolized in the ultrafine size range at ~300 µg/m3, 6 h/day, 3-5 days/week, starting 7-10 days before disease induction. No differences in weight change, clinical disease activity, or histology were observed between the PM and FA-exposed groups. In conclusion, UFP inhalation exposure did not exacerbate intestinal inflammation in acute, chemically-induced colitis models.

High Perceived Stress is Associated With Increased Risk of Ulcerative Colitis Clinical Flares.

BACKGROUND & AIMS: Although perceived stress (PS) has been associated with symptomatic flares in inflammatory bowel disease, clinical and physiological measures associated with perceived stress and flare are not known. The aim of this study was to identify physiological factors associated with perceived stress in ulcerative colitis (UC) subjects, and their relationship with flare. METHODS: Patients with UC in clinical remission (Simple Colitis Clinical Activity Index [SCCAI] score <5) underwent clinical and behavioral assessments, morning salivary cortisol measurements, autonomic nervous system activity testing (heart rate variability, electrodermal activity) at baseline with patient-reported SCCAI every 2 weeks over 1 to 2 years and fecal calprotectin at time of flare. Clinical flares (SCCAI ≥5) and biochemical flares (SCCAI ≥5 with fecal calprotectin ≥250 µg/g) were evaluated. RESULTS: One hundred ten patients with UC were enrolled, with mean follow-up of 65.6 weeks. Patients with UC with higher and lower PS were determined. Although the high PS group had 3.6 times higher odds of a clinical flare than the low PS group, no significant differences in biochemical flares were observed between the low and high PS groups. The high vs low PS group differed in tonic sympathetic arousal as indexed by significantly greater baseline electrodermal activity (4.3 vs 3.4 microsiemens; P = .026) in the high PS group, but not in terms of heart rate variability and morning cortisol levels. Increased fecal calprotectin was associated with cardioautonomic measures, suggesting lower parasympathetic activity. CONCLUSIONS: Increased PS assessed at baseline is associated with tonic sympathetic arousal and greater odds of clinical flares in patients with UC.

Oxidized phospholipids cause changes in jejunum mucus that induce dysbiosis and systemic inflammation.

We previously reported that adding a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to a Western diet (WD) ameliorated systemic inflammation. To determine the mechanism(s) responsible for these observations, Ldlr-/- mice were fed chow, a WD, or WD plus Tg6F. We found that a WD altered the taxonomic composition of bacteria in jejunum mucus. For example, Akkermansia muciniphila virtually disappeared, while overall bacteria numbers and lipopolysaccharide (LPS) levels increased. In addition, gut permeability increased, as did the content of reactive oxygen species and oxidized phospholipids in jejunum mucus in WD-fed mice. Moreover, gene expression in the jejunum decreased for multiple peptides and proteins that are secreted into the mucous layer of the jejunum that act to limit bacteria numbers and their interaction with enterocytes including regenerating islet-derived proteins, defensins, mucin 2, surfactant A, and apoA-I. Following WD, gene expression also decreased for Il36γ, Il23, and Il22, cytokines critical for antimicrobial activity. WD decreased expression of both Atoh1 and Gfi1, genes required for the formation of goblet and Paneth cells, and immunohistochemistry revealed decreased numbers of goblet and Paneth cells. Adding Tg6F ameliorated these WD-mediated changes. Adding oxidized phospholipids ex vivo to the jejunum from mice fed a chow diet reproduced the changes in gene expression in vivo that occurred when the mice were fed WD and were prevented with addition of 6F peptide. We conclude that Tg6F ameliorates the WD-mediated increase in oxidized phospholipids that cause changes in jejunum mucus, which induce dysbiosis and systemic inflammation.

Colonic mucosal microbiota is associated with bowel habit subtype and abdominal pain in patients with irritable bowel syndrome.

Mucosal microbiota differ significantly from fecal microbiota and may play a different role in the pathophysiology of irritable bowel syndrome (IBS). The aims of this study were to determine if the composition of mucosal microbiota differed between IBS, or IBS bowel habit (BH) subtypes, and healthy controls (HCs). Sigmoid colon mucosal biopsies were obtained from 97 Rome-positive patients with IBS (28% IBS-constipation, 38% IBS-diarrhea, 24% IBS-mixed, and 10% IBS-unsubtyped) and 54 HCs, from which DNA was extracted. 16S rRNA gene sequencing and microbial composition analysis were performed. Group differences in α and ß diversity and taxonomic level differences were determined using linear regression while controlling for confounding variables. IBS BH subtype was associated with microbial α diversity (P = 0.0003) with significant differences seen in the mucosal microbiota of IBS-constipation versus IBS-diarrhea (P = 0.046). There were no significant differences in α or ß diversity in the mucosal microbiota of IBS versus HCs (P = 0.29 and 0.93, respectively), but metagenomic profiling suggested functional differences. The relative abundance of Prevotella_9 copri within IBS was significantly correlated with increased abdominal pain (r = 0.36, P = 0.0003), which has not been previously reported in IBS. Significant differences in the mucosal microbiota were present within IBS BH subtypes but not between IBS and HCs, supporting the possibility of IBS BH subtype-specific pathogenesis. Increased Prevotella copri may contribute to symptoms in patients with IBS.NEW & NOTEWORTHY Gut mucosal microbiota differs significantly from fecal microbiota in irritable bowel syndrome (IBS) and may play a different role in its pathophysiology. Investigation of colonic mucosal microbiota in the largest cohort of patients with IBS and healthy controls accounting for confounding variables, including diet demonstrated significant differences in mucosal microbiota between IBS bowel habit subtypes but not between IBS and healthy controls. In addition, the study reported gut microbiota is associated with abdominal pain in patients with IBS.

Effect of Exclusion Diets on Symptom Severity and the Gut Microbiota in Patients With Irritable Bowel Syndrome.

BACKGROUND & AIMS: Altered fecal microbiota have been reported in irritable bowel syndrome (IBS), although studies vary, which could be owing to dietary effects. Many IBS patients may eliminate certain foods because of their symptoms, which in turn may alter fecal microbiota diversity and composition. This study aimed to determine if dietary patterns were associated with IBS, symptoms, and fecal microbiota differences reported in IBS. METHODS: A total of 346 IBS participants and 170 healthy controls (HCs) completed a Diet Checklist reflecting the diet(s) consumed most frequently. An exclusion diet was defined as a diet that eliminated food components by choice. Within this group, a gluten-free, dairy-free, or low fermentable oligosaccharides, disaccharides, monosaccharides, and polyols diet was further defined as restrictive because they often are implicated in reducing symptoms. Stool samples were obtained from 171 IBS patients and 98 HCs for 16S ribosomal RNA gene sequencing and microbial composition analysis. RESULTS: Having IBS symptoms was associated with consuming a restrictive diet (27.17% of IBS patients vs 7.65% of HCs; odds ratio, 3.25; 95% CI, 1.66-6.75; P value = .006). IBS participants on an exclusion or restrictive diet reported more severe IBS symptoms (P = .042 and .029, respectively). The composition of the microbiota in IBS patients varied depending on the diet consumed. IBS participants on an exclusion diet had a greater abundance of Lachnospira and a lower abundance of Eubacterium (q value, <.05), and those on a restrictive diet had a lower abundance of Lactobacillus (q value, <.05). CONCLUSIONS: Restrictive diets likely are consumed more by IBS patients than HCs to reduce GI symptom severity. Dietary patterns influence the composition of the fecal microbiota and may explain some of the differences between IBS and HCs.

Susceptibility to epilepsy after traumatic brain injury is associated with preexistent gut microbiome profile.

OBJECTIVE: We examined whether posttraumatic epilepsy (PTE) is associated with measurable perturbations in gut microbiome. METHODS: Adult Sprague Dawley rats were subjected to lateral fluid percussion injury (LFPI). PTE was examined 7 months after LFPI, during 4-week continuous video-electroencephalographic monitoring. 16S ribosomal RNA gene sequencing was performed in fecal samples collected before LFPI/sham-LFPI and 1 week, 1 month, and 7 months thereafter. Longitudinal analyses of alpha diversity, beta diversity, and differential microbial abundance were performed. Short-chain fatty acids (SCFAs) were measured in fecal samples collected before LFPI by liquid chromatography with tandem mass spectrometry. RESULTS: Alpha diversity changed over time in both LFPI and sham-LFPI subjects; no association was observed between alpha diversity and LFPI, the severity of post-LFPI neuromotor impairments, and PTE. LFPI produced significant changes in beta diversity and selective changes in microbial abundances associated with the severity of neuromotor impairments. No association between LFPI-dependent microbial perturbations and PTE was detected. PTE was associated with beta diversity irrespective of timepoint vis-à-vis LFPI, including at baseline. Preexistent fecal microbial abundances of four amplicon sequence variants belonging to the Lachnospiraceae family (three enriched and one depleted) predicted the risk of PTE, with area under the curve (AUC) of .73. Global SCFA content was associated with the increased risk of PTE, with AUC of .722, and with 2-methylbutyric (depleted), valeric (depleted), isobutyric (enriched), and isovaleric (enriched) acids being the most important factors (AUC = .717). When the analyses of baseline microbial and SCFA compositions were combined, AUC to predict PTE increased to .78. SIGNIFICANCE: Whereas LFPI produces no perturbations in the gut microbiome that are associated with PTE, the risk of PTE can be stratified based on preexistent microbial abundances and SCFA content.

Duodenal Microbiome and Serum Metabolites Predict Hepatocellular Carcinoma in a Multicenter Cohort of Patients with Cirrhosis.

BACKGROUND: Hepatocellular carcinoma (HCC) is rapidly increasing in the U.S. and is a leading cause of mortality for patients with cirrhosis. Discovering novel biomarkers for risk stratification of HCC is paramount. We examined biomarkers of the gut-liver axis in a prospective multicenter cohort. METHODS: Patients with cirrhosis without a history of HCC were recruited between May 2015 and March 2020 and prospectively followed at 3 tertiary care hospitals in Los Angeles. Microbiome analysis was performed on duodenal biopsies and metabolomic analysis was performed on serum samples, collected at the time of enrollment. Optimal microbiome-based survival analysis and Cox proportional hazards regression analysis were used to determine microbiota and metabolite associations with HCC development, respectively. RESULTS: A total of 227 participants with liver cirrhosis contributed a total of 459.58 person-years of follow-up, with 14 incident HCC diagnoses. Male sex (HR = 7.06, 95% CI = 1.02-54.86) and baseline hepatic encephalopathy (HE, HR = 4.65, 95% CI = 1.60-13.52) were associated with developing HCC over follow-up. Adjusting for age, sex, baseline HE, and alkaline phosphatase, an increased risk of HCC were observed for participants with the highest versus lowest three quartiles for duodenal Alloprevotella (HR = 3.22, 95% CI = 1.06-9.73) and serum taurocholic acid (HR = 6.87, 95% CI = 2.32-20.27), methionine (HR = 9.97, 95% CI = 3.02-32.94), and methioninesulfoxide (HR = 5.60, 95% CI = 1.84-17.10). Being in the highest quartile for Alloprevotella or methionine had a sensitivity and specificity for developing HCC of 85.71% and 60.56%, respectively, with an odds ratio of 10.92 (95% CI = 2.23-53.48). CONCLUSION: Alloprevotella and methionine, methioninesulfoxide, and taurocholic acid predicted future HCC development in a high-risk population of participants with liver cirrhosis.

The intestinal microbiota as a predictor for antidepressant treatment outcome in geriatric depression: a prospective pilot study.

OBJECTIVES: (1) To investigate if gut microbiota can be a predictor of remission in geriatric depression and to identify features of the gut microbiota that is associated with remission. (2) To determine if changes in gut microbiota occur with remission in geriatric depression. DESIGN: Secondary analysis of a parent randomized placebo-controlled trial (NCT02466958). SETTING: Los Angeles, CA, USA (2016-2018). PARTICIPANTS: Seventeen subjects with major depressive disorder, over 60 years of age, 41.2% female. INTERVENTION: Levomilacipran (LVM) or placebo. MEASUREMENTS: Remission was defined by Hamilton Depression Rating Scale score of 6 or less at 12 weeks. 16S-ribosomal RNA sequencing based fecal microbiota composition and diversity were measured at baseline and 12 weeks. Differences in fecal microbiota were evaluated between remitters and non-remitters as well as between baseline and post-treatment samples. LVM and placebo groups were combined in all the analyses. RESULTS: Baseline microbiota showed no community level α-diversity or ß-diversity differences between remitters and non-remitters. At the individual taxa level, a random forest classifier created with nine genera from the baseline microbiota was highly accurate in predicting remission (AUC = .857). Of these, baseline enrichment of Faecalibacterium, Agathobacter and Roseburia relative to a reference frame was associated with treatment outcome of remission. Differential abundance analysis revealed significant genus level changes from baseline to post-treatment in remitters, but not in non-remitters. CONCLUSIONS: This is the first study demonstrating fecal microbiota as a potential predictor of treatment response in geriatric depression. Our findings need to be confirmed in larger prospective studies.

Development of the infant gut microbiome predicts temperament across the first year of life.

Perturbations to the gut microbiome are implicated in altered neurodevelopmental trajectories that may shape life span risk for emotion dysregulation and affective disorders. However, the sensitive periods during which the microbiome may influence neurodevelopment remain understudied. We investigated relationships between gut microbiome composition across infancy and temperament at 12 months of age. In 67 infants, we examined if gut microbiome composition assessed at 1-3 weeks, 2, 6, and 12 months of age was associated with temperament at age 12 months. Stool samples were sequenced using the 16S Illumina MiSeq platform. Temperament was assessed using the Infant Behavior Questionnaire-Revised (IBQ-R). Beta diversity at age 1-3 weeks was associated with surgency/extraversion at age 12 months. Bifidobacterium and Lachnospiraceae abundance at 1-3 weeks of age was positively associated with surgency/extraversion at age 12 months. Klebsiella abundance at 1-3 weeks was negatively associated with surgency/extraversion at 12 months. Concurrent composition was associated with negative affectivity at 12 months, including a positive association with Ruminococcus-1 and a negative association with Lactobacillus. Our findings support a relationship between gut microbiome composition and infant temperament. While exploratory due to the small sample size, these results point to early and late infancy as sensitive periods during which the gut microbiome may exert effects on neurodevelopment.

A randomized, phase 1, placebo-controlled trial of APG-157 in oral cancer demonstrates systemic absorption and an inhibitory effect on cytokines and tumor-associated microbes.

BACKGROUND: Although curcumin's effect on head and neck cancer has been studied in vitro and in vivo, to the authors' knowledge its efficacy is limited by poor systemic absorption from oral administration. APG-157 is a botanical drug containing multiple polyphenols, including curcumin, developed under the US Food and Drug Administration's Botanical Drug Development, that delivers the active components to oromucosal tissues near the tumor target. METHODS: A double-blind, randomized, placebo-controlled, phase 1 clinical trial was conducted with APG-157 in 13 normal subjects and 12 patients with oral cancer. Two doses, 100 mg or 200 mg, were delivered transorally every hour for 3 hours. Blood and saliva were collected before and 1 hour, 2 hours, 3 hours, and 24 hours after treatment. Electrocardiograms and blood tests did not demonstrate any toxicity. RESULTS: Treatment with APG-157 resulted in circulating concentrations of curcumin and analogs peaking at 3 hours with reduced IL-1ß, IL-6, and IL-8 concentrations in the salivary supernatant fluid of patients with cancer. Salivary microbial flora analysis showed a reduction in Bacteroidetes species in cancer subjects. RNA and immunofluorescence analyses of tumor tissues of a subject demonstrated increased expression of genes associated with differentiation and T-cell recruitment to the tumor microenvironment. CONCLUSIONS: The results of the current study suggested that APG-157 could serve as a therapeutic drug in combination with immunotherapy. LAY SUMMARY: Curcumin has been shown to suppress tumor cells because of its antioxidant and anti-inflammatory properties. However, its effectiveness has been limited by poor absorption when delivered orally. Subjects with oral cancer were given oral APG-157, a botanical drug containing multiple polyphenols, including curcumin. Curcumin was found in the blood and in tumor tissues. Inflammatory markers and Bacteroides species were found to be decreased in the saliva, and immune T cells were increased in the tumor tissue. APG-157 is absorbed well, reduces inflammation, and attracts T cells to the tumor, suggesting its potential use in combination with immunotherapy drugs.

Metformin alters the duodenal microbiome and decreases the incidence of pancreatic ductal adenocarcinoma promoted by diet-induced obesity.

Pancreatic ductal adenocarcinoma (PDAC)'s growing incidence has been linked to the rise in obesity and type 2 diabetes mellitus. In previous work, we have shown that metformin can prevent the increased incidence of PDAC in a KrasG12D mouse model subjected to a diet high in fat and calories (HFCD). One potential way that metformin can affect the host is through alterations in the gut microbiome. Therefore, we investigated microbial associations with PDAC development and metformin use in the same mouse model. Lox-Stop-Lox Kras G12D/+ (LSL-Kras G12D/+); p48-Cre (KC) mice were given control diet, HFCD, or HFCD with 5 mg/mL metformin in drinking water for 3 mo. At the end of the 3 mo, 16S rRNA sequencing was performed to characterize microbiome composition of duodenal mucosal, duodenal luminal, and cecal luminal samples. KC mice on an HFCD demonstrated depletion of intact acini and formation of advanced pancreatic intraepithelial neoplasia. This effect was completely abrogated by metformin treatment. HFCD was associated with significant changes in microbial composition and diversity in the duodenal mucosa and lumen, much of which was prevented by metformin. In particular, Clostridium sensu stricto was negatively correlated with percent intact acini and seemed to be inhibited by the addition of metformin while on an HFCD. Administration of metformin eliminated PDAC formation in KC mice. This change was associated with significant microbial changes in both the mucosal and luminal microbiome of the duodenum. This suggests that the microbiome may be a potential mediator of the chemopreventive effects of metformin.NEW & NOTEWORTHY Pancreatic ductal adenocarcinoma (PDAC)'s growing incidence has been linked to the rise in obesity and type 2 diabetes mellitus. Administration of metformin eliminated PDAC formation in KC mice with diet-induced obesity. This change was associated with significant microbial changes in both the mucosal and luminal microbiome of the duodenum. This suggests that the microbiome may be a potential mediator of the chemopreventive effects of metformin.

Ceragenin CSA13 Reduces Clostridium difficile Infection in Mice by Modulating the Intestinal Microbiome and Metabolites.

BACKGROUND & AIMS: Clostridium difficile induces intestinal inflammation by releasing toxins A and B. The antimicrobial compound cationic steroid antimicrobial 13 (CSA13) has been developed for treating gastrointestinal infections. The CSA13-Eudragit formulation can be given orally and releases CSA13 in the terminal ileum and colon. We investigated whether this form of CSA13 reduces C difficile infection (CDI) in mice. METHODS: C57BL/6J mice were infected with C difficile on day 0, followed by subcutaneous administration of pure CSA13 or oral administration of CSA13-Eudragit (10 mg/kg/d for 10 days). Some mice were given intraperitoneal vancomycin (50 mg/kg daily) on days 0-4 and relapse was measured after antibiotic withdrawal. The mice were monitored until day 20; colon and fecal samples were collected on day 3 for analysis. Blood samples were collected for flow cytometry analyses. Fecal pellets were collected each day from mice injected with CSA13 and analyzed by high-performance liquid chromatography or 16S sequencing; feces were also homogenized in phosphate-buffered saline and fed to mice with CDI via gavage. RESULTS: CDI of mice caused 60% mortality, significant bodyweight loss, and colonic damage 3 days after infection; these events were prevented by subcutaneous injection of CSA13 or oral administration CSA13-Eudragit. There was reduced relapse of CDI after administration of CSA13 was stopped. Levels of CSA13 in feces from mice given CSA13-Eudragit were significantly higher than those of mice given subcutaneous CSA13. Subcutaneous and oral CSA13 each significantly increased the abundance of Peptostreptococcaceae bacteria and reduced the abundance of C difficile in fecal samples of mice. When feces from mice with CDI and given CSA13 were fed to mice with CDI that had not received CSA13, the recipient mice had significantly increased rates of survival. CSA13 reduced fecal levels of inflammatory metabolites (endocannabinoids) and increased fecal levels of 4 protective metabolites (ie, citrulline, 3-aminoisobutyric acid, retinol, and ursodeoxycholic acid) in mice with CDI. Oral administration of these CSA13-dependent protective metabolites reduced the severity of CDI. CONCLUSIONS: In studies of mice, we found the CSA13-Eudragit formulation to be effective in eradicating CDI by modulating the intestinal microbiota and metabolites.

Microbiome and bile acid profiles in duodenal aspirates from patients with liver cirrhosis: The Microbiome, Microbial Markers and Liver Disease Study.

AIM: Cirrhosis is a leading cause of death worldwide, yet there are no well-established risk stratifying tools for lethal complications, including hepatocellular carcinoma (HCC). Patients with liver cirrhosis undergo routine endoscopic surveillance, providing ready access to duodenal aspirate samples that could be a source for identifying novel biomarkers. The aim of this study was to characterize the microbiome and bile acid profiles in duodenal aspirates from patients with liver cirrhosis to assess the feasibility of developing biomarkers for HCC risk stratification. METHODS: Thirty patients with liver cirrhosis were enrolled in the Microbiome, Microbial Markers, and Liver Disease study between May 2015 and March 2017. Detailed clinical and epidemiological data were collected at baseline and at 6-monthly follow-up visits. Duodenal aspirate fluid was collected at baseline for microbial characterization using 16S ribosomal RNA sequencing and bile acid quantification using mass spectroscopy. RESULTS: Alcohol-related cirrhosis was associated with reductions in the Bacteroidetes phylum, particularly Prevotella (13-fold reduction), and expansion of Staphylococcus (13-fold increase), compared to hepatitis C virus-related cirrhosis. Participants with hepatic encephalopathy (HE) had less microbial diversity compared to patients without HE (P < 0.05), and were characterized by expansion of Mycobacterium (45-fold increase) and Gram-positive cocci including Granulicatella (3.1-fold increase), unclassified Planococcaceae (3.3-fold increase), and unclassified Streptococcaceae (4.5-fold increase). Non-Hispanic White patients had reduced microbial richness (P < 0.01) and diversity (P < 0.05), and increased levels of conjugated ursodeoxycholic acid (glycoursodeoxycholic acid and tauroursodeoxycholic acid, P < 0.05) compared to Hispanic patients. CONCLUSION: Microbial profiles of duodenal aspirates differed by cirrhosis etiology, HE, and Hispanic ethnicity.

NK cells are biologic and biochemical targets of 6-mercaptopurine in Crohn's disease patients.

NK cells, which contribute to immune defense against certain viral infections and neoplasia, are emerging as modifiers of chronic immunologic diseases including transplant rejection and autoimmune diseases. Immunobiology and genetic studies have implicated NK cells as a modifier of Crohn's disease, a condition often treated with thiopurine agents such as 6-mercaptopurine (6-MP). Here, we demonstrate that thiopurines mediate NK cell apoptosis via a caspase 3 and 9 inclusive pathway, and that this process is triggered by thiopurine-mediated inhibition of Rac1. We also show that CD patients in clinical remission maintained on 6-MP have decreased NK cell Rac1 activity, and decreased NK cell numbers in their intestinal biopsies. These observations suggest that thiopurine targeting of NK cells may be a previously unappreciated therapeutic action of these agents in IBD.

High-protein diet improves sensitivity to cholecystokinin and shifts the cecal microbiome without altering brain inflammation in diet-induced obesity in rats.

High-protein diet (HPD) curtails obesity and/or fat mass, but it is unknown whether it reverses neuroinflammation or alters glucose levels, CCK sensitivity, and gut microbiome in rats fed a Western diet (WD)-induced obesity (DIO). Male rats fed a WD (high fat and sugar) for 12 wk were switched to a HPD for 6 wk. Body composition, food intake, meal pattern, sensitivity to intraperitoneal CCK-8S, blood glucose, brain signaling, and cecal microbiota were assessed. When compared with a normal diet, WD increased body weight (9.3%) and fat mass (73.4%). CCK-8S (1.8 or 5.2 nmol/kg) did not alter food intake and meal pattern in DIO rats. Switching to a HPD for 6 wk reduced fat mass (15.7%) with a nonsignificantly reduced body weight gain, normalized blood glucose, and decreased feeding after CCK-8S. DIO rats on the WD or switched to a HPD showed comparable microbial diversity. However, in HPD versus WD rats, there was enrichment of 114 operational taxonomic units (OTUs) and depletion of 188 OTUs. Of those, Akkermansia muciniphila (enriched on a HPD), an unclassified Clostridiales, a member of the RF39 order, and a Phascolarctobacterium were significantly associated with fat mass. The WD increased cytokine expression in the hypothalamus and dorsal medulla that was unchanged by switching to HPD. These data indicate that HPD reduces body fat and restores glucose homeostasis and CCK sensitivity, while not modifying brain inflammation. In addition, expansion of cecal Akkermansia muciniphila correlated to fat mass loss may represent a potential peripheral mechanism of HPD beneficial effects.

Surgically Induced Changes in Gut Microbiome and Hedonic Eating as Related to Weight Loss: Preliminary Findings in Obese Women Undergoing Bariatric Surgery.

OBJECTIVE: Weight loss surgery results in significant changes in the anatomy, function, and intraluminal environment of the gastrointestinal tract affecting the gut microbiome. Although bariatric surgery results in sustained weight loss, decreased appetite, and hedonic eating, it is unknown whether the surgery-induced alterations in gut microbiota play a role in the observed changes in hedonic eating. We explored the following hypotheses: (1) laparoscopic sleeve gastrectomy (LSG) results in changes in gut microbial composition; (2) alterations in gut microbiota are related to weight loss; (3) alterations in gut microbiome are associated with changes in appetite and hedonic eating. METHODS: Eight obese women underwent LSG. Their body mass index, body fat mass, food intake, hunger, hedonic eating scores, and stool samples were obtained at baseline and 1-month postsurgery. 16S ribosomal RNA gene sequencing was performed on stool samples. DESeq2 changes in microbial abundance. Multilevel-sparse partial least squares discriminant analysis was applied to genus-level abundance for discriminative microbial signatures. RESULTS: LSG resulted in significant reductions in body mass index, food intake, and hedonic eating. A microbial signature composed of five bacterial genera discriminated between pre- and postsurgery status. Several bacterial genera were significantly associated with weight loss (Bilophila, q = 3E-05; Faecalibacterium q = 4E-05), lower appetite (Enterococcus, q = 3E-05), and reduced hedonic eating (Akkermansia, q = .037) after surgery. CONCLUSIONS: In this preliminary analysis, changes in gut microbial abundance discriminated between pre- and postoperative status. Alterations in gut microbiome were significantly associated with weight loss and with reduced hedonic eating after surgery; however, a larger sample is needed to confirm these findings.

Dysregulation of CLOCK gene expression in hyperoxia-induced lung injury.

Hyperoxic acute lung injury (HALI) is characterized by inflammation and epithelial cell death. CLOCK genes are master regulators of circadian rhythm also implicated in inflammation and lung diseases. However, the relationship of CLOCK genes in hyperoxia-induced lung injury has not been studied. This study will determine if HALI alters CLOCK gene expression. To test this, wild-type and NALP3(-/-) mice were exposed to room air or hyperoxia for 24, 48, or 72 h. In addition, mice were exposed to different concentrations of hyperoxia (50, 75, or 100% O2) or room air for 72 h. The mRNA and protein levels of lung CLOCK genes, based on quantitative PCR and Western blot analysis, respectively, and their target genes are significantly elevated in mice exposed to hyperoxia compared with controls. Alterations in CLOCK genes are associated with increased inflammatory markers in bronchoalveolar lavage fluid of hyperoxic mice compared with controls. Histological examination of mice lungs exposed to hyperoxia show increased inflammation and alveolar congestion compared with controls. Our results indicate sequential increase in CLOCK gene expression in lungs of mice exposed to hyperoxia compared with controls. Additionally, data suggest a dose-dependent increase in CLOCK gene expression with increased oxygen concentrations. To validate if the expression changes related to CLOCK genes are indeed associated with inflammation, NALP3(-/-) was introduced to analyze loss of function in inflammation. Western blot analysis showed significant CLOCK gene downregulation in NALP3(-/-) mice compared with wild-type controls. Together, our results demonstrate that hyperoxia-mediated lung inflammation is associated with alterations in CLOCK gene expression.