Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohn's Disease.

Background and Aims: We previously identified small molecules predicted to reverse an ileal gene signature for future Crohn's Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA). Methods: Induced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs. Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. Results: iPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation of fibroblast activation genes including ACTA2 and COL1A1 , and an increase in HIO collagen content. ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. Conclusions: ETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model may be used in future untargeted screens for small molecules to treat refractory CD.

The Intestinal Microbiome Predicts Weight Loss on a Calorie-Restricted Diet and Is Associated With Improved Hepatic Steatosis.

Background: The microbiome has been shown in pre-clinical and epidemiological studies to be important in both the development and treatment of obesity and metabolic associated fatty liver disease (MAFLD). However, few studies have examined the role of the microbiome in the clinical response to calorie restriction. To explore this area, we performed a prospective study examining the association of the intestinal microbiome with weight loss and change in hepatic steatosis on a calorie-restricted diet. Methods: A prospective dietary intervention study of 80 overweight and obese participants was performed at the Greater West Los Angeles Veterans Affair Hospital. Patients were placed on a macronutrient standardized diet for 16 weeks, including 14 weeks of calorie restriction (500 calorie deficit). Body composition analysis by impedance, plasma lipid measurements, and ultrasound elastography to measure hepatic steatosis were performed at baseline and week 16. Intestinal microbiome composition was assessed using 16S rRNA gene sequencing. A per protocol analysis was performed on all subjects completing the trial (n = 46). Results: Study completers showed significant reduction in weight, body mass index, total cholesterol, low density lipoprotein, and triglyceride. Subjects who lost at least 5% of their body weight had significantly greater reduction in serum triglyceride and hepatic steatosis than those with <5% body weight loss. Enterococcus and Klebsiella were reduced at the end of the trial while Coprococcus and Collinsella were increased. There were also significant baseline microbiome differences between patients who had at least 5% weight loss as compared to those that did not. Lachnoclostridium was positively associated with hepatic steatosis and Actinomyces was positively associated with hepatic steatosis and weight. Baseline microbiome profiles were able to predict which patients lost at least 5% of their body weight with an AUROC of 0.80. Conclusion: Calorie restriction alters the intestinal microbiome and improves hepatic steatosis in those who experience significant weight loss. Baseline microbiome differences predict weight loss on a calorie-restricted diet and are associated with improvement in hepatic steatosis, suggesting a role of the gut microbiome in mediating the clinical response to calorie restriction.

Specimen Collection and Analysis of the Duodenal Microbiome.

Shifts in the microbiome have been correlated with the physiology and pathophysiology of many organ systems both in humans and in mouse models. The gut microbiome has been typically studied through fecal specimen collections. The ease of obtaining fecal samples has resulted in many studies that have revealed information concerning the distal luminal gastrointestinal tract. However, few studies have addressed the importance of the microbiome in the proximal gut. Given that the duodenum is a major site for digestion and absorption, its microbiome is relevant to nutrition and liver disease and warrants further investigation. Here we detail a novel method for sampling the proximal luminal and mucosal gut microbiome in human subjects undergoing upper endoscopy by obtaining duodenal aspirate and biopsies. Specimen procurement is facile and unaffected by artifacts such as patient preparatory adherence, as might be the case in obtaining colonic samples during colonoscopy. The preliminary results show that the luminal and mucosal microbiomes differ significantly, which is likely related to environmental conditions and barrier functions. Therefore, a combination of duodenal aspirate and biopsies reveal a more comprehensive picture of the microbiome in the duodenum. Biopsies are obtained from the descending and horizontal segments of the duodenum, which are anatomically close to the liver and biliary tree. This is important in studying the role of bile acid biology and the gut-liver axis in liver disease. Biopsies and aspirate can be used for 16S ribosomal RNA sequencing, metabolomics, and other similar applications.

Gut microbiome profiles associated with steatosis severity in metabolic associated fatty liver disease.

Aim: The microbiome has been shown to be pivotal in the development of metabolic associated fatty liver disease (MAFLD). Few have examined the relationship of the microbiome specifically with steatosis grade. Therefore, our aim was to characterize the association of the microbiome with MAFLD steatosis severity while adjusting for metabolic comorbidities including diabetes. Methods: We enrolled patients with MAFLD at the West Los Angeles Veterans Affair Hospital. All patients underwent ultrasound elastography, fasting serum collection, and fecal sampling for 16S sequencing. We examined the associations of microbial diversity and composition with advanced steatosis, defined as a CAP score of ≥ 300 dB/m, with or without the presence of metabolic comorbidities. Results: Seventy-five patients were enrolled. African American were less likely to have advanced steatosis than either Hispanics or Whites (P = 0.001). Patients with more advanced steatosis had higher fasting serum triglyceride (192.6 ± 157.1 mg/dL vs. 122.5 ± 57.4 mg/dL), HbA1c (6.7% ± 1.4% vs. 6.1% ± 0.8%), transaminases, and were more likely to have metabolic syndrome (52.4% vs. 24.2%, P = 0.02). Advanced steatosis and diabetes were associated with altered microbial composition. Bacteroides was negatively associated with advanced steatosis while Megasphaera was positively associated with steatosis. Akkermansia was negatively associated with diabetes, while Anaerostipes and Parabacteroides were positively associated with diabetes. Conclusion: Diabetes and metabolic syndrome are associated with hepatic steatosis severity in MAFLD patients and both advanced steatosis and comorbid diabetes are independently associated with microbiome changes. These results provide insight into the role of the gut microbiome in MAFLD associated with metabolic syndrome.

A High Protein Calorie Restriction Diet Alters the Gut Microbiome in Obesity.

BACKGROUND: High protein calorie restriction diets have shown clinical efficacy for obesity, but the mechanisms are not fully known. The intestinal microbiome is a mediator of obesity and preclinical data support an effect of high protein diet (HPD) on the gut microbiome of obesity, but there are few studies in humans. METHODS: To address this, we conducted a dietary intervention trial of 80 overweight and obese subjects who were randomized to a calorie-restricted high protein diet (HPD) (30% calorie intake) or calorie-restricted normal protein diet (NPD) (15%) for 8 weeks. Baseline dietary intake patterns were assessed by the Diet History Questionnaire III. Longitudinal fecal sampling was performed at baseline, week 1, week 2, week 4, week 6, and week 8, for a total of 365 samples. Intestinal microbiome composition was assessed by 16S rRNA gene sequencing. RESULTS: At baseline, microbial composition was associated with fiber and protein intake. Subjects on the HPD showed a significant increase in microbial diversity as measured by the Shannon index compared to those on the NPD. The HPD was also associated with significant differences in microbial composition after treatment compared to the NPD. Both diets induced taxonomic shifts compared to baseline, including enrichment of Akkermansia spp. and Bifidobacterium spp. and depletion of Prevotella spp. Conclusion: These findings provide evidence that weight loss diets alter the gut microbiome in obesity and suggest differential effects of HPDs compared to NPDs which may influence the clinical response to HPD.