Kashyap, Purna

Abstract Background As the acute phase of the COVID-19 pandemic subsides, an increasing number of individuals are recognized as having long COVID, affecting over 65 million people worldwide. Recent studies have suggested a potential link between long COVID and alterations in the gut microbiome. In this study, we examined the gut microbiome of a large outpatient cohort during acute phase of infection, with the ultimate goal of determining predictors of long-term COVID-19 health outcomes. Methods We conducted a study of 394 COVID-19 patients with positive SARS-CoV-2 testing from November 2020 to September 2021 at a multi-site healthcare system. Long COVID was defined as the persistence of symptoms for four weeks or more after illness onset. DNA extraction was performed using the MoBio PowerSoil Pro DNA isolation kit. Samples were sequenced using shotgun metagenomics. Stool SARS-CoV-2 RNA was tested using qRT-PCR of the E gene, and stool ELISA was performed for calprotectin levels. Standard biostatistical tools in R (v 4.2.3) were used for statistical analysis. Results Of the 394 COVID-19 patients, 84 (21%) had long COVID, with fatigue being the most common persistent symptom (Table 1). No significant differences were seen in age, BMI, and antibiotic use. Those with long COVID were more likely to be female, had higher Charlson's comorbidity scores, more severe index illnesses. However, the two groups had no difference in SARS-CoV-2 stool PCR positivity or stool calprotectin levels (Table 2). Principal coordinate analysis of Bray Curtis dissimilarities revealed that patients with long COVID had a distinct microbial composition compared with those without long COVID during the acute phase of infection (ANOSIM R2= 0.005, P= 0.01). Categorizing patients by number of long COVID symptoms showed significant differences among groups (ANOSIM R2=0.1, P=0.007), mainly between patients with >3 symptoms and those with 0-1 symptoms (Bonferroni, P= 0.018). Alpha diversity was lower in patients with > 3 symptoms, although this was not statistically significant (Shannon diversity, two-way-ANOVA, P=0.051). Figure 1 Conclusion Long COVID patients show distinct gut microbial composition during the acute phase of infection, suggesting a potential role in predicting long-term COVID health outcomes. Disclosures John C. O'Horo, Sr., MD, MPH, Janssen Pharmaceuticals.: Grant/Research Support|nference: Grant/Research Support Purna C. Kashyap, MD, Intrinsic Medicine: Advisor/Consultant|Novome Biotechnologies: Advisor/Consultant|Pendulum Therapeutics: Advisor/Consultant

The human gastrointestinal (GI) tract is home to trillions of microorganisms. There is dynamic interaction between the gut microbiota and the human host, but how gut microbiota modulates GI physiology is largely unknown. A recent study from our laboratory showed that human gut microbiota increases host serotonin (5‐hydroxytryptamine, 5‐HT) biosynthesis. 5‐HT is an important neurotransmitter and a paracrine messenger in the GI tract that increases intestinal secretion primarily through 5‐HT 3 and 5‐HT 4 receptors. In this study we investigated whether gut microbiota modulate 5‐HT 3 R and 5‐HT 4 R expression and host secretory response to 5‐HT. Methods We used proximal colon mucosa‐submucosa preparations from germ free (GF) and humanized (ex‐GF colonized with human bacteria for 4–5 weeks; HM) male mice. The change in short circuit current (ΔIsc) was determined in response to increasing concentrations of 5‐HT (0.003–300μM) on the submucosal side either alone or in the presence of 5‐HT 3 R antagonist ondansetron (100nM) in an Ussing chamber. Receptor expression was quantified using rt‐PCR (normalized to L32) and western blot (normalized to GAPDH). Immunohistochemistry (IHC) was used to determine localization of 5‐HT 3 R in the paraformaldehyde‐fixed 10μm sections from proximal colonic epithelium. Differences between groups were tested using Mann‐Whitney U (Mdn; IQ range) with P ‐value lower than 0.05 considered significant. Results 5‐HT induced ΔIsc was significantly higher in GF than HM mice (218; 184–274 vs 163; 132–182 μA/cm 2 , n=6, P = 0.04). To determine if this change was due to an alteration in 5‐HTR, we quantified receptor expression. We found significantly higher 5‐HT 3 R mRNA expression (1.82; 1.08–2.80 vs 1.00; 0.33–1.38, n=8–9, P = 0.046) in GF compared to HM mice, but no change in 5‐HT 4 R expression. 5‐HT 3 R protein expression was also significantly higher in GF mice compared to HM (4.62; 1.06–6.14 vs 1.00; 1–1.3, n= 11–12, P = 0.01) mice. There was a significantly higher ΔIsc in GF compared to HM (87.1; 51.7–149 vs 37.0; 19.5–44 μA/cm 2 , n=5, P = 0.02) mice, following application of 2‐methyl 5‐hydroxytryptamine hydrochloride (selective 5‐HT 3 R agonist) suggesting increased receptor expression augments secretory response. Furthermore, ondansetron significantly blocked 5‐HT induced maximum ΔIsc in GF mice (201; 189–260 vs 117.6; 92–149 μA/cm 2 , n=6–8, P = 0.001) but not in HM mice (163; 132–182 vs 120; 107–150 μA/cm 2 , n=6, P = 0.24). To determine the expression of 5‐HT 3 R in mouse colon mucosa‐submucosa we used 5‐HT 3 –tagged GFP mice. IHC identified 5‐HT 3 R‐GFP positive cells in the mouse colonic epithelium. To ascertain if the significant difference in 5‐HT‐induced ΔIsc between GF and HM mice (188; 152–200 vs 125; 99–139 μA/cm 2 , n=5, P = 0.008) is driven by epithelial 5‐HT 3 R, we measured the response in the presence of TTX (block neuronal transmission). TTX (500 nM) had no effect of ondansetron inhibition of 5‐HT‐induced ΔIsc, suggesting a role for epithelial 5HT 3 R. Conclusion Gut microbial colonization decreases host secretory response to 5‐HT by downregulating 5‐HT 3 R expression. Epithelial 5‐HT 3 R may be important in gut microbiota modulation of intestinal secretion Support or Funding Information NIH K08 DK

Pathogenesis of Irritable Bowel Syndrome (IBS) is multifactorial, and gut microbes are sought to play a role in development and progression of disease. In this study we examined how constipation‐predominant Irritable Bowel Syndrome (IBS‐C) microbiota affect host intestinal transit and signaling pathways. To determine IBS‐C dysbiosis effects on the host, we introduced fecal microbiota from constipated mice to germ‐free recipients. Recipient mice of IBS‐C microbiota developed delayed GI transit time when compared to controls. Bacterial DNA analysis by HiSeq sequencing showed significant increase in Bacteroides uniformis , Bacteroides ovatus and Parabacteroides distasonis species from Bacteroidetes phylum in constipated mice. Fecal microbiota metabolic potential was measured with tetrazolium dye assay and showed significant differences between recipients of control and IBS‐C microbiota. Microbial metabolites such as short chain fatty acids (SCFA) are known to alter intestinal transit. SCFA levels in cecal contents of recipeints were measured by GC‐MS, and showed lower butyrate, propionate and acetate concentration in IBS‐C mice when compared to controls. To determine contractile properties of intestine after introduction of control and IBS‐C microbiota, we stimulated colonic rings with carbachol and KCl, and observed hypercontractility in IBS‐C microbiota recipients. To determine mechanism for delayed GI transit we analyzed colon tissues in fecal microbiota recipients by qRT‐PCR, and determined dysregulated neuromediator pathways. Overall, these results suggest that IBS‐C dysbiosis contributes to development of delayed transit time through microbial metabolite production and changes in colonic contractile properties. Support or Funding Information R01 DK097268 and T32 DK007074

Abstract Background Patients with ulcerative colitis and total abdominal proctocolectomy with ileal pouch-anal anastomosis have a 50% risk of pouchitis and a 5% to 10% risk of chronic pouchitis. Aims The goal of the study was to compare pouch microbiota and stool bile acid composition in patients with chronic pouchitis, chronic pouchitis and primary sclerosing cholangitis, and normal pouch. Methods Patients with ulcerative colitis and ileal pouch-anal anastomosis were recruited from March 20, 2014, to August 6, 2019, and categorized into normal pouch, chronic pouchitis, and chronic pouchitis/primary sclerosing cholangitis groups. Stool samples were subjected to bile acid quantification and 16S rRNA gene sequencing. Statistical comparisons of absolute bile acid abundance and pouch microbiota α-diversity, β-diversity, and taxa abundance were performed among the patient groups. Results A total of 51 samples were analyzed. Both α-diversity (P = .01, species richness) and β-diversity (P = .001) significantly differed among groups. Lithocholic acid was significantly lower in patients with chronic pouchitis/primary sclerosing cholangitis than in those with chronic pouchitis (P = .01) or normal pouch (P = .03). Decreased α-diversity was associated with an increased primary to secondary bile acid ratio (P = .002), which was also associated with changes in β-diversity (P = .006). Conclusions Pouch microbiota α- and β-diversity differed among patients with normal pouch, chronic pouchitis, and chronic pouchitis/primary sclerosing cholangitis. Lithocholic acid level and primary to secondary bile acid ratio were highly associated with pouch microbiota richness, structure, and composition. These findings emphasize the associations between pouch microbiota and bile acid composition in dysbiosis and altered metabolism, suggesting that secondary bile acids are decreased in chronic pouchitis.

Diet is an important determinant of health and consequently is often implicated in the development of disease, particularly gastrointestinal (GI) diseases, given the high prevalence of meal-related symptoms. The mechanisms underlying diet-driven pathophysiology are not well understood, but recent studies suggest that gut microbiota may mediate the effect of diet on GI physiology. In this review, we focus primarily on two distinct GI diseases where the role of diet has been best studied: irritable bowel syndrome and inflammatory bowel disease. We discuss how the concurrent and sequential utilization of dietary nutrients by the host and gut microbiota determines the eventual bioactive metabolite profiles in the gut and the biological effect of these metabolites on GI physiology. We highlight several concepts that can be gleaned from these findings, such as how distinct effects of an individual metabolite can influence diverse GI diseases, the effect of similar dietary interventions on multiple disease states, and the need for extensive phenotyping and data collection to help make personalized diet recommendations.

INTRODUCTION: Intragastric balloons (IGBs) are a safe and effective treatment for obesity. However, limited knowledge exists on the underlying biological changes with IGB placement. METHODS: This single-institution study was part of an adjustable IGB randomized controlled trial. Subjects with obesity were randomized in a 2 is to 1 ratio to 32 weeks of IGB with diet/exercise counseling (n = 8) vs counseling alone (controls, n = 4). Diet/exercise counseling was continued for 24 weeks post-IGB removal to assess weight maintenance. We used mass spectrometry for nontargeted plasma lipidomics analysis and 16S rRNA sequencing to profile the fecal microbiome. RESULTS: Subjects with IGBs lost 15.5% of their body weight at 32 weeks vs 2.59% for controls ( P < 0.05). Maintenance of a 10.5% weight loss occurred post-IGB explant. IGB placement, followed by weight maintenance, led to a −378.9 μM/L reduction in serum free fatty acids compared with pre-IGB (95% confidence interval: 612.9, −145.0). This reduction was mainly in saturated, mono, and omega-6 fatty acids when compared with pre-IGB. Polyunsaturated phosphatidylcholines also increased after IGB placement (difference of 27 μM/L; 95% confidence interval: 1.1, 52.8). Compared with controls, saturated and omega-6 free fatty acids (linoleic and arachidonic acids) were reduced after IGB placement. The fecal microbiota changed post-IGB placement and weight maintenance vs pre-IGB ( P < 0.05). Further analysis showed a possible trend toward reduced Firmicutes and increased Bacteroidetes post-IGB and counseling, a change that was not conclusively different from counseling alone. DISCUSSION: IGB treatment is associated with an altered fecal microbiome profile and may have a better effect on obesity-related lipidome than counseling alone.

Irritable bowel syndrome is a globally prevalent disorder categorized into constipation, diarrhea or mixed (IBS‐C, IBD‐D, IBS‐M) based on the predominant stool form/frequency. While the gut microbiome is recognized as an important factor underlying the pathophysiology of IBS, the specific mechanisms remain an area of active investigation. In our recent longitudinal multi‐omics study in IBS patients, we found bacteria‐derived hypoxanthine was significantly decreased in stool from IBS‐C patients. The aim of this study was to determine if IBS‐C relevant molecular pathways are affected by hypoxanthine. We hypothesized that hypoxanthine acts on enterochromaffin (EC) cells to stimulate serotonin (5‐HT) release, where 5‐HT is an important determinant of gastrointestinal (GI) transit time. We used Ca 2+ imaging in QGP cells (EC model), T84 cells (colonocyte model), primary cells and organoids from NeuroD1‐cre;GCaMP5‐tdTomato mice [NeuroD1 is a marker for enteroendocrine cells (EECs) which express calcium indicator GCaMP in these mice] in the absence or presence of receptor antagonists, and in ex vivo colon preparations with intact innervation from E2aCre:GCaMP6s mice (GCaMP is expressed in neuronal and non‐neuronal cells), to test the effect of hypoxanthine. The physiologic relevance of hypoxanthine signaling was assessed by measuring whole gut transit time. We found hypoxanthine increased Ca 2+ influx in QGP cells [using Cal520 (qualitative) and Fura‐2AM (quantitative)], primary EECs and EECs within organoids (previous research showed that the majority of these EECs are EC cells), but not in T84 cells. Hypoxanthine‐evoked Ca 2+ response was absent in Ca 2+ ‐free medium (n=6‐8, t‐test, P <0.0001), inhibited by G αi/o ‐GPCR antagonist (pertussis toxin; n=8‐14, t‐test, P <0.01)), Transient Receptor Potential Cation Channel Subfamily C Member 4 (TRPC4) antagonist (ML204; n=14‐15, t‐test, P <0.0001) and TRPC4 siRNA (n=5‐21, t‐test, P <0.01), and was accompanied by increased 5‐HT release (measured using 5‐HT biosensor). In ex vivo experiments, luminal application of hypoxanthine activated a subset of epithelial cells (likely EC cells) followed by subsets of neurons (latency to response onset, n=4, Mann‐Whitney, P <0.0001), as well as movement of the imaging field, which indicates smooth muscle contraction/relaxation. Our in vitro findings were confirmed by in vivo experiments showing that germ‐free mice colonized with hypoxanthine‐producing bacteria had faster whole gut transit and higher stool 5‐HT level compared to hypoxanthine‐consuming bacteria (n=7‐8, t‐test, P <0.05). In summary, hypoxanthine increases Ca 2+ influx specifically in EC cells by activating a signaling cascade involving a G αi/o ‐GPCR and TRPC4 channels, which results in 5‐HT release. The delay in activation of myenteric neurons and motor activity suggests that EC cell 5‐HT release likely induces neurogenic motor patterns and may underlie the shorter GI transit time seen with increased bacterial hypoxanthine production. While the current empirically designed probiotics have proven ineffective in IBS, our findings will allow development of novel mechanism‐based probiotic therapies for IBS‐C using hypoxanthine‐producing bacteria.