Society: AGA

Background: Through shotgun metagenomic sequencing, we identified the depletion of probiotic species Lactobacillus acidophilus in stools of mice with non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC). Here, we aimed to investigate the prophylactic potential and protective mechanism of L. acidophilus against NAFLD-HCC.

Methods: NAFLD-HCC tumorigenesis models were established in mice with a single injection of diethylnitrosamine (DEN) at day 14 and feeding of high-fat high-cholesterol (HFHC) diet for 8 months, or choline-deficient high-fat diet (CD-HFD) for 6 months. Orthotopic NAFLD-HCC xenografts were established in mice with intrahepatic injection of mouse HCC cell line Hepa 1-6 and feeding of HFHC diet for 4 months. Metabolomic profiling of stool, portal vein serum and liver tissue of mice, and culture medium of L. acidophilus (LA-CM) were evaluated by non-targeted and targeted liquid chromatography-mass spectrometry. Human NAFLD-HCC cell lines (HKCI-2, HKCI-10) and mouse NAFLD-HCC organoids were used for bio-functional assays of L. acidophilus and its metabolite. RNA-sequencing was performed on NAFLD-HCC cells treated with candidate metabolite.

Results: Oral gavage of L. acidophilus suppressed NAFLD-HCC tumorigenesis in mice induced by DEN and HFHC diet, and also in orthotopic mouse xenografts. L. acidophilus was undetectable in liver tissues but was detected in stools of L. acidophilus-treated mice, implicating that the antitumor function of L. acidophilus is attributed to its products. Co-culture with LA-CM significantly reduced viability (P<0.0001) and proliferation (P<0.01) of human NAFLD-HCC cells, and suppressed mouse NAFLD-HCC organoid growth (P<0.0001). The tumor-suppressing effect of L. acidophilus was attributed to its non-protein small molecules with a molecular weight of <3kDa. By metabolomic profiling, valeric acid which is a short-chain fatty acid, was identified as the top differential metabolite in LA-CM, and its upregulation was confirmed in stool and portal vein serum of L. acidophilus-treated mice. Oral gavage of valeric acid significantly suppressed tumor number (P<0.01) and tumor load (P<0.05) in DEN-treated mice fed with HFHC diet, accompanied with improved gut barrier integrity. This was confirmed in DEN-treated mice fed with CD-HFD diet. Valeric acid also reduced viability (P<0.01) and proliferation (P<0.001) of human NAFLD-HCC cells, and inhibited mouse NAFLD-HCC organoid growth (P<0.0001). Mechanistically, valeric acid bound to G protein-coupled receptor (GPR)41 and GPR43 of hepatocytes to induce their expression, which in turn inhibiting the oncogenic Rho-GTPase pathway to ablate NAFLD-HCC development.

Conclusion: L. acidophilus protects against hepatocarcinogenesis by secreting antitumor metabolite valeric acid. Probiotic supplementation is therefore a potential prophylactic of NAFLD-HCC.

Background
Malnutrition amongst under-fives remains common in resource-poor countries and is resistant to current interventions. New opportunities have emerged to target environmental enteric dysfunction, which refers to the abnormal gut structure and function that compromises nutrition and growth in early life. Dietary supplements of pro- or synbiotics may build the resilience of the gut microbiome against environmental factors and boost colonization resistance against ingested pathogens.
Methods
We conducted an open label, randomized, controlled trial to assess whether administering pro/synbiotics to infants in western Kenya improved gut health and reduced systemic inflammation. Newborns less than four days old were enrolled at the Homa Bay County Teaching and Referral Hospital, western Kenya. They were randomly allocated, stratified by HIV exposure, in a 1:1:1:1 ratio to one of 4 study arms to receive either
Labinic synbiotic^[1]; Biofloratech Ltd, Walton-on-Thames, UK
Lab4b synbiotic^[2]; Cultech Ltd., Port Talbot, UK
Lab4b probiotic (as above)
No supplement
Interventions were given daily for ten days and then weekly until six months of age. Biomarkers of systemic inflammation (plasma alpha 1-acid glycoprotein (AGP) concentration, a biomarker of longer-term systemic inflammation; primary outcome), gut health and growth (secondary outcomes) were measured in blood and stool samples collected at 6 weeks, 3, and 6 months.
Results
Between 28 October, 2020 and 13 January, 2022, 600 newborns were enrolled. Urban vs. rural residence, place and mode of delivery, sex, gestational age, birthweight, HIV exposure, antibiotic exposure and socio-economic and hygiene/sanitation variables at recruitment were similar in the four study arms.
Median plasma AGP concentration, increased progressively at 3 and 6 months in the controls. This increase was almost completely abrogated in all the intervention arms (Figure). At 6 months, plasma AGP was raised (>1g/L) in 56/134 (41.8%) infants in the control arm but in <1.5% infants in each of the intervention arms (P<0.001). Gut health biomarkers were significantly higher at 3 and/or 6 months in the control arm than in any intervention arm. Growth hormones were slightly, but significantly, lower at 6 months in the control arm than in any intervention arm (Table).
Conclusion
Pro/synbiotics offer a novel approach to improving gut health and reducing systemic inflammation in young infants in a low-resource setting.

[1] BENEO Orafti Synergy1; 50 oligofructose:50 FOS; 200mg + Lactobacillus acidophilus NCFM, Bifidobacterium infantis Bi-26 and Bifidobacterium bifidum Bb-06; 5 x 10⁹organisms/day

[2] long-chain fructo-oligosaccharide 150mg + Lactobacillus salivarius CUL61, Lactobacillus paracasei CUL08, Bifidobacterium animalis subspecies lactis CUL34 and Bifidobacterium bifidum CUL20; 10¹⁰ organisms/day

<b>Median plasma alpha 1-acid glycoprotein</b> <b>concentration per study arm and visit</b>

Median plasma alpha 1-acid glycoprotein concentration per study arm and visit

<b>Summary of biomarkers of systemic inflammation, gut health and growth in control vs. intervention arms</b>

Summary of biomarkers of systemic inflammation, gut health and growth in control vs. intervention arms

Background & Aims: Lactococcus lactis (L. lactis) was identified to be depleted in the stool of colorectal cancer (CRC) patients in contrast to healthy subjects by fecal shotgun metagenomic sequencing, suggesting its potential protective role in the pathogenesis of CRC and microbiota interaction. Here, we aim to clarify the antitumorigenic role of L. lactis in colorectal tumorigenesis.

Methods: We isolated L. lactis bacterium from stool samples of healthy subject and its complete genome sequence was obtained by PacBio 3^rd generation sequencing. The anti-tumor effects of L. lactis was assessed in patient-derived organoids, and in two murine models of colorectal tumorigenesis (transgenic Apc^min/+ mouse model and AOM-DSS carcinogen-induced CRC mouse model). The effect of L. lactis on the gut microbiome alteration was assessed by shotgun metagenome sequencing. The bio-function of L. lactis conditioned medium (LL-CM) was assessed in CRC cell lines (HCT116 and HT29) and normal colonic epithelial cell (NCM460). Fraction separation of LL-CM with mini-pore filters defines the approximate protein size and the functional component in LL-CM was identified by liquid chromatography-mass spectrometry (LC-MS/MS) analysis.

Results: Oral administration of L. lactis significantly reduced tumor number (P<0.05) and size (P<0.05), along with reduced Ki-67 cells and increased TUNEL-positive cells, compared to E. coli MG1655 and plain culture broth control in AOM/DSS-induced CRC mice. This was confirmed in Apc^min/+ CRC mice. A significant shift of fecal microbial profiling revealed the enrichment of known probiotics, Lactobacillus johnsonii and Limosilactobacillus reuteri, and depletion of pathogenic bacteria, Olsenella uli and Bacteroides eggerthii, in L. lactis treated mice. Co-culturing CRC cells (HCT116 and HT29) with LL-CM decreased cell viability and lowered colony formation, but this was not observed in NCM460 cells. Besides, coincubation of the LL-CM drastically retarded the growth of cultured human-derived CRC organoids (P<0.05). Digesting LL-CM with protease K or heating LL-CM to inactivate protein could abolish the tumor-suppressive effect of LL-CM, indicating the functional fraction in LL-CM should be protein. With further investigation into the fractions of LL-CM, we found that the fraction size >100kDa was identified to exert an anti-CRC effect. Alpha-mannosidase was identified as the critical protein produced by L. lactis by mass spectrometry screening. The tumor suppressive effects of alpha-mannosidase was confirmed in CRC cell lines.

Conclusion: L. lactis protects against colorectal tumorigenesis in mice through restoring the healthy microbial composition. The anti-tumor effect of L. lactis is facilitated by its generated beneficial protein alpha-mannosidase. L. lactis might serve as a novel prophylactic for CRC prevention.

Introduction
Diverticulitis is among the most common gastrointestinal disorders and is responsible for millions of healthcare encounters yearly in the US. However, the pathogenesis of diverticulitis respecting the gut microbiome remains largely unknown. We aimed to better understand dysbioses in diverticulitis including potential functional implications.

Methods
We conducted a nested case-control study within the Nurses’ Health Study II, a prospective cohort that enrolled 116,429 female nurses in 1989 and followed biennially through mailed questionnaires to collect updated lifestyle and disease information. Among 14,992 women who returned a stool sample during 2019-2021, we identified 121 women who reported a case of diverticulitis requiring antibiotics or hospitalizations and matched each to a diverticulitis-free control according to age, race, and month of stool collection. We performed shotgun metagenomic sequencing, coupled with untargeted liquid chromatography–mass spectrometry metabolomic profiling, of stool samples from patients with diverticulitis and controls.

Results
Overall microbial community structure and metabolomic profiles differed in diverticulitis versus controls (Fig 1A). Species-specific alterations in diverticulitis explained these overall changes, such as enriched pro-inflammatory Ruminococcus gnavus (Fig 1B/C). Analogously, highly prioritized metabolomic features altered in diverticulitis included enrichments of N-Acetylglutamine (uremic toxin) and 1,7-dimethyluric acid that has been linked to circulating C-reactive protein, as well as depletions of ceramides (key cellular signaling molecules and important in inflammation), indolin-2-one (anti-inflammatory), and 7alpha-hydroxy-4-cholesten-3-one (a marker for bile acid malabsorption). Combining these measurements, we detected covarying microbial and metabolic features relevant to diverticulitis (Fig 2). For example, the pathobiont Bilophila wadsworthia, known to expand with bile acid dysmetabolism during a Western pattern diet, was associated with bile acids both inversely (e.g., alpha-muricholic acid) and positively (e.g., isoallolithocholic acid) in diverticulitis. Finally, microbial composition modulated the protective association between dietary fiber and diverticulitis. A greater fiber intake was associated with a more significant reduction in diverticulitis risk in individuals with high PCo1, corresponding with low Faecalibacterium prausnitzii and Prevotella copri, whereas fiber was not protective with high F. prausnitzii or P. copri carriage.

Conclusion
Our findings provide a first-of-its-kind platform for understanding novel perturbations in inflammation-associated microbial composition and metabolomic activity during diverticulitis. These results may inform potential microbial-based diagnostic and therapeutic targets and personalized intervention for diverticulitis.

Background: Probiotic Limosilactobacillus reuteri DSM 17938 (DSM 17938) has been shown to prevent experimental necrotizing enterocolitis (NEC), a model developed in newborn mice or rats that are subjected to acute maternal separation stress, cow-milk based formula feeding, and brief recurrent hypoxia with cold stress. The NEC model features intestinal inflammation with reduced regulatory T cells (Tregs) and increased proinflammatory effector T cells (Teffs). The immune modulatory mechanisms of DSM 17938 in the intestinal mucosa of NEC include promoting tolerogenic dendritic cells (DCs) to educate naïve CD4⁺T cells to differentiate into Tregs, which inhibit inflammatory Teffs.

Objective: To analyze whether DSM 17938 can educate Tregs to improve their tolerogenic potency during experimental NEC development.

Methods: We isolated T cells (Tregs and Teffs) from "donor" mice expressing congenic marker Thy1.2 fed with either DSM 17938 in fresh media (10⁷ CFU) (LR-educated Treg) or control media (naïve Treg) daily for 2 weeks. The cells were adoptively transferred (AT) by intraperitoneal injection (5 × 10⁵ cells/mouse) to newborn recipient mice expressing Thy1.1 (on d5). Thy1.1 Mice were then exposed to NEC procedures for 4 days (NeoStress). We evaluated NEC histological scores, endogenous and exogenous Tregs, activated Teffs and DCs in the intestine by flow cytometry.

Results: We found that B6.PL-Thy1a/CyJ (Thy1.1) mice were resistant to experimental NEC. None of the mice in the NeoStress group (with AT of Tregs or without AT of Tregs) exhibited histological findings typical of NEC. However, the mice with AT of Teffs showed NEC-like histological injury in 14% of NeoStress mice. For Treg analysis, we observed that (a) neonatal stress significantly reduced the % of Foxp3⁺Tregs in the intestine; (b) AT of Tregs (either naïve or LR-educated Foxp3⁺Tregs) to NeoStress mice significantly increased the % of Tregs in the intestine; (c) most importantly, LR-educated Tregs were able to increase the % of Tregs in the intestine in NeoStress mice more than AT of naïve Tregs; and (d) even though the majority of intestinal Tregs following AT were endogenous Tregs, LR-educated Tregs were identified at a significantly higher % than naïve Tregs in the intestine of NeoStress mice. In addition, (e) AT of LR-educated Tregs reduced pro-inflammatory CD44⁺Foxp3^-NonTregs and changed DC specific markers associated with inflammation and T cell activation in the intestine of NeoStress mice.

Conclusions: Adoptive transfer of Tregs promotes the generation and/or migration of endogenous Tregs into the intestine of recipient mice. Importantly, probiotic-educated Tregs are more effective than naïve Tregs for adoptive transfer of immune tolerance. This finding may be relevant to beneficial effects of lactobacilli in human babies.

Background: Carnobacterium maltaromaticum was previously identified to be depleted in the gut of patients with CRC by shotgun metagenomics. Here, we examined the potential anti-CRC effect of C. maltaromaticum.
Methods: The abundance of C. maltaromaticum in the human stool samples was measured by qPCR. The tumor-suppressive effect of C. maltaromaticum was evaluated in two murine intestinal tumorigenesis models – Apc^min/+ mice and azoxymethane-injected mice. Ovariectomy or orchiectomy was performed in female or male Apc^min/+ mice, respectively, to investigate the sex-specific anti-CRC effect of C. maltaromaticum. The colonization of C. maltaromaticum on colonic tissues was confirmed by qPCR and fluorescence in situ hybridization. Biotinylation-based far-Western, pull-down assays, and bimolecular fluorescence complementation were used to identify and confirm the binding of the host and bacteria surface receptors. Metabolomic profile was acquired by high-performance liquid chromatography-mass spectrometry. Host genes regulated by C. maltaromaticum were identified by RNA sequencing. Metabolic cross-feeding between C. maltaromaticum and other bacteria was demonstrated by in vitro fecal fermentation model. The vitamin D receptor (VDR)-dependency of the anti-CRC effect of C. maltaromaticum was confirmed by the pharmacological blockade of VDR with TEI-9647 in Apc^min/+ mice.
Results: C. maltaromaticum is depleted in stool samples of patients with CRC in a female-specific manner. Administration of C. maltaromaticum reduced intestinal tumor formation in two murine CRC models in a female-specific manner. Estrogen deprivation in female Apc^min/+ mice abolished the tumor-suppressive effect of C. maltaromaticum, while the anti-CRC effect of C. maltaromaticum was restored upon 17β-estradiol replacement in the ovariectomized-female mice. Also, the reduction in tumor number and tumor size by C. maltaromaticum was observed in male Apc^min/+ mice after feminization. Mechanistically, estrogen increased the attachment and colonization of C. maltaromaticum via increasing the colonic expression of SLC3A2 that binds to DD-CPase of this bacterium. Metabolomic and transcriptomic profiling unveiled the increased gut abundance of vitamin D-related metabolites and the mucosal activation of VDR signaling in C. maltaromaticum-gavaged mice in a gut microbiome- and VDR-dependent manner. In vitro fermentation system confirmed the metabolic cross-feeding of C. maltaromaticum with Faecalibacterium prausnitzii to convert C. maltaromaticum-produced 7-dehydrocholesterol into vitamin D for activating the host VDR signaling.
Conclusion: C. maltaromaticum colonizes the gut in an estrogen-dependent manner and acts along with other microbes to augment the intestinal vitamin D production to activate the host VDR for suppressing CRC.

Siu-Kin Ng

Appearances