Introduction

The gut microbiome exerts a significant influence on the host's cellular energy metabolism during homeostasis and disease, notably due to the production of metabolites (1), such as polyamines (2). Gut dysbiosis and bioenergetic alterations leading to metabolic shifts have been associated with altered cellular responses and are involved in intestinal disorders, such as inflammatory bowel disease (IBD) (3,4). Cadaverine, a gut polyamine derived from L-lysine decarboxylation, has been previously proposed to be transported into mitochondrial inner compartments in basal and induced conditions (5). On the other hand, it seems to be positively regulated in the gut of IBD patients (6). However, the biological significance in both contexts remains still unexplored.

Aims & Methods

This study uncovers the potential immunometabolic role of cadaverine in vitro and during intestinal inflammation. For that, single-cell energetic metabolism by profiling translation inhibition (SCENITH), luminescence and flow cytometry-based assays, Seahorse real-time cell metabolic analysis, metabolomics, RNAseq, and proteomics were applied to explore the role of cadaverine on energy metabolism in purified cells. Antibiotic-induced gut microbiota perturbations and dextran sodium sulfate (DSS)-induced colitis were carried out to further investigate its effect in vivo.

Results

Here we demonstrate that cadaverine enhances adenosine triphosphate (ATP) levels in THP-1 monocytic cell line as well as in human monocytes and macrophages obtained from healthy donors. Those results were associated with increased glucose uptake and utilization as a metabolic fuel and with higher mitochondrial dependence as a metabolic pathway, particularly elevating basal and maximal oxidative phosphorylation (OXPHOS) rates. The effects of cadaverine on mitochondrial functions persisted even in the presence of lipopolysaccharide (LPS) that triggers aerobic glycolysis in monocytes/macrophages. Interestingly, cadaverine presented immunomodulatory activity by increasing interleukin (IL)-10 levels and decreasing tumor necrosis factor (TNF)-α production in classical monocytes while triggering the polarization of macrophages to the M2 anti-inflammatory phenotype. Such immunomodulatory potential appears intricately dependent on mitochondrial respiration since this effect was blocked when OXPHOS was pharmacologically inhibited. Omics analysis reveals that itaconate metabolism and Krebs cycle modulation may be associated with cadaverine immunometabolic activity. Oral administration of cadaverine in antibiotics-induced gut microbiota-depleted mice rescued from both small intestine and colon macrophage energy deficiency. Furthermore, cadaverine also sustained OXPHOS and mitochondrial health features in those cells. As L-lysine decarboxylase genes are mostly expressed by gut Enterobacteriaceae, which in turn are augmented and suggested to be related to IBD, we specifically depleted this bacterial family with colistin in mice and induced colitis with DSS. In this setting cadaverine administration ameliorates disease severity by polarizing colon macrophages towards the M2 phenotype and preserving the overall mitochondrial features, such as mass, mitochondrial membrane potential, and reactive oxygen species, to sustain ATP production by OXPHOS.

Conclusion

Such discoveries at the metabolic level hold promise for identifying novel therapeutic strategies that target cellular energy metabolism and the gut microbiota to modulate monocyte and macrophage responses in IBD.

References

1. Michaudel C, Sokol H. The Gut Microbiota at the Service of Immunometabolism. Cell Metab. 2020 Oct 6;32(4):514-523.
2. Puleston DJ et al. Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation. Cell Metab. 2019 Aug 6;30(2):352-363.e8.
3. Zeng Z et al. Glycolytic Activation of CD14+ Intestinal Macrophages Contributes to the Inflammatory Responses via Exosomal Membrane Tumor Necrosis Factor in Crohn's Disease. Inflamm Bowel Dis. 2024 Jan 5;30(1):90-102.
4. Lloyd-Price et al. Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases. Nature. 2019 May;569(7758):655-662.
5. Toninello A, Dalla Via L, Testa S, Siliprandi D. Electrophoretic polyamine transport in rat liver mitochondria. Amino Acids. 1992 Feb;2(1-2):69-76.
6. Li M, Yang L, Mu C, Sun Y, Gu Y, Chen D, Liu T, Cao H. Gut microbial metabolome in inflammatory bowel disease: From association to therapeutic perspectives. Comput Struct Biotechnol J. 2022 Apr 4;20:2402-2414.

Disclosure

Harry Sokol reports lecture fees, board membership, or consultancy from Carenity, AbbVie, Astellas, Danone, Ferring, Mayoly Spindler, MSD, Novartis, Roche, Tillots, Enterome, BiomX, Biose, Novartis, Takeda, Biocodex and is cofounder of Exeliom Biosciences.

Introduction

Food-induced mucosal reactions have been visualised in the duodenum of patients with irritable bowel syndrome (IBS) using confocal laser endomicroscopy (CLE). The mechanisms underlying these alterations remain elusive but uncontrolled studies have reported symptomatic improvement in patients on a diet excluding foods that triggered acute alterations.

Aims & Methods

Aim: 1/ elucidate mechanisms underlying food-induced, acute mucosal alterations and 2/ assess whether a diet based on CLE results improves symptoms in patients with IBS.
Methods: Randomised, double-blind, controlled, cross-over study. Patients with ROME IV IBS (non-C), underwent CLE with sequential administration of foods (wheat, soy, milk, egg white, fish mix, and nut mix) in a randomised order ≥2 weeks after index gastroscopy. CLE was terminated after acute reactions or after administration of all foods. In case less than three foods were administered, a second exam was scheduled. Biological measures were compared between index endoscopy and CLE with food administration. Patients excluded food(s) that did (=real diet) and food(s) that did not (=sham diet) cause alterations for 4 weeks each in a blinded, cross-over diet intervention. Clinical response rate (=improvement of ≥50 points IBS-SSS) between real and sham diet was compared. Missing data was replaced using an extreme case approach (missing data on the primary endpoint was considered non-response). Healthy controls underwent CLE to assess disease specificity of observed alterations.

Results

Thirty-three patients were included, of which 21 underwent a second CLE. Alterations were observed in all 54 exams (100%) either at baseline (6/54, 11%) or after administration of food. Acute alterations were not associated with altered permeability (transepithelial electrical resistance 25.6 Ωxcm² after CLE vs 24.6 Ωxcm² at baseline; p = 0.6) nor with tryptase release from duodenal biopsies (1.1 µg/mg tissue vs 0.62 µg/mg tissue; p =0.15) . Two patients dropped out during the diet phase (one due to maladherence to study protocol, one due to acute increase of IBS symptoms during washout). Thirty-one patients completed the diet phase: 13 were clinical responders to the real diet (42%) versus 11 responders to sham (36%). The odds ratio (OR) for clinical response to the real diet was 1.33 (95% confidence interval: 0.46-3.84). The trial was terminated prematurely for futility (predetermined futility threshold OR <1.5). There was no difference in symptom evolution between real and sham diet (median change in IBS-SSS real diet: -30 points, median change sham diet: -20 points ; p = 0.7). No differences were observed between real and sham diet regarding change in pain duration (-0.1 vs +0.1, p =0.5) , in pain severity (-0.1 vs -0.1, p = 0.9), in bloating severity (-0.1 vs -0.4, p=0.6) or in flatulence severity (± 0.0 vs -0.2 p=0.5) based on 10cm VAS scales in symptom diaries. Fifteen healthy volunteers underwent CLE with alterations in all 15 (100%) exams at baseline (2/15, 13%) or after food administration. Distribution of alterations among different foods was similar to that in patients.

Conclusion

In this double-blind controlled cross-over trial, trigger foods identified based on CLE responses were not associated with superior clinical improvement following a targeted exclusion diet compared to a sham diet. Acute alterations in healthy controls suggest these alterations are not specific for IBS. The underling physiological or pathophysiological mechanism requires further studies.

Introduction

Inflammatory bowel disease (IBD) is characterised by inflammatory flares with excruciating pain, for which effective analgesics are lacking. Cocaine- and amphetamine-regulated transcript (CART) peptide regulates peripheral pain in mice.¹ However, its role in visceral nociception as well as the underlying molecular signalling mechanism in primary sensory neurons remains elusive. CART is highly expressed in TRPV1-positive colon-projecting sensory neurons.² In addition, the enzyme prohormone convertase 1/3, which is involved in processing CART into its bioactive fragment 55-102, is upregulated in colonic biopsies of IBD patients (internal data), suggesting a potential role for the peptide in colonic pain.

Aims & Methods

Here, we aimed to test if CART would affect firing of the colon-innervating lumbar splanchnic nerve (LSN) as model of visceral nociception and characterise its effect and signalling pathway in sensory neurons innervating the colon. Studies were performed using tissue from male and female C57BL/6J mice euthanised by cervical dislocation and exsanguination in accordance with Schedule 1 of the Animals (Scientific Procedures) Act 1986. Thoracolumbar dorsal root ganglia (DRG) neurons were dissociated and cultured overnight for Ca²⁺ imaging using Fluo-4 AM as previously described.³ Cells were perfused with extracellular solution (ECS) alone or supplemented with either rat CART 55-102, (20-300nM, ECS), thapsigargin (10mM, DMSO), xestospongin C (1mM, ECS), edelfosine (10mM, ECS), capsaicin (10mM, ethanol) or KCl solution (60mM, ddH₂O) for respective challenge or pre-treatment. All conditions were blinded and randomised. Colorectum with attached lumbar splanchnic nerve was carefully removed and mounted on a tissue bath superfused with Krebs buffer as previously described.³ Rat CART 55-102 was superfused at 5ml/min, resulting in a final concentration of 200nM. Statistical analysis was performed using Student´s t test or ANOVA and Dunnett´s multiple comparisons test. Data is displayed as mean ± SEM.

Results

CART elicited a concentration-dependent increase in intracellular Ca²⁺ in DRG neurons, which at the highest concentration tested (300nM) induced a significant increase in intracellular Ca²⁺ in 29.2 ± 2.98% of DRG neurons relative to 8.35 ± 1.25% of vehicle responders (p < 0.0001). 55.05% of CART-responsive neurons were putative nociceptors due to co-sensitivity to capsaicin. Further investigation revealed a statistically significant reduction in the proportion of CART-sensitive neurons compared to CART (300nM) alone following pre-treatment with edelfosine, a phospholipase C inhibitor (10.5 ± 3.88%, p = 0.006), thapsigargin, a SERCA pump inhibitor (7.25 ± 1.78%, p = 0.001), or xestospongin C, an inositol triphosphate receptor inhibitor (6.74± 1.87%, p = 0.006). Furthermore, tissue bath application of CART (200nM) elicited a marked transient increase in ongoing LSN discharge, which increased from a baseline of 12.8 ± 3.9 (spikes/s) to a peak firing of 19.7 ± 6.1 spikes/s (p = 0.0385).

Conclusion

CART stimulates intracellular Ca²⁺ release in capsaicin-sensitive neurons consistent with a role in pain processing. This response was mediated by the release of Ca²⁺ from intracellular stores such as the endoplasmic reticulum and attenuated by inhibitors of the G_q pathway, suggesting activation of a G_q-coupled GPCR. The pronociceptive activity of CART on nerve firing suggests a potential role in colonic pain. Further research is necessary to identify the role of CART in IBD.

References

1. Ohsawa M, Dun SL, Tseng LF, Chang J, Dun NJ. Decrease of hindpaw withdrawal latency by cocaine- and amphetamine-regulated transcript peptide to the mouse spinal cord. Eur J Pharmacol. Jul 7 2000;399(2-3):165-9. doi:10.1016/s0014-2999(00)00374-5
2. McGuire C, Boundouki G, Hockley JRF, et al. Ex vivo study of human visceral nociceptors. Gut. Jan 2018;67(1):86-96. doi:10.1136/gutjnl-2016-311629
3. Higham JP, Bhebhe CN, Gupta RA, et al. Transcriptomic profiling reveals a pronociceptive role for angiotensin II in inflammatory bowel disease. Pain. Jan 29 2024;doi:10.1097/j.pain.0000000000003159

Disclosure

The PhD research is partially funded by Metrion Biosciences.

Introduction

Sodium picosulfate (SP)/magnesium citrate (MC) and polyethylene glycol (PEG) plus ascorbic acid are recommended by Western guidelines as laxative solutions for bowel preparation. The standard formulation for bowel preparation is PEG; however, it requires a large dose and has a distinctive taste that is considered unpleasant. SP/MC requires a small dose and ensures fluid intake because it is administered in another beverage. Therefore, clinical studies have shown that SP/MC is superior to PEG in terms of acceptability.

Aims & Methods

Our aim was to compare the novel bowel cleansing method (test group) consisting of SP/MC with elobixibat hydrate and the standard bowel cleansing method consisting of PEG plus ascorbic acid (standard group) in patients preparing for outpatient colonoscopy. This was a multicenter, single-blind, non-inferiority, randomized controlled trial. Patients 40-69 years of age scheduled for outpatient colonoscopy were enrolled. Patients with a history of abdominal or pelvic surgery, constipation, inflammatory bowel disease, or severe organ dysfunction were excluded. The target number of study participants was 540 (standard group, 270 cases; test group, 270 cases). The primary endpoint was bowel cleansing (Boston Bowel Preparation Scale [BBPS] score ≥6). Secondary endpoints were patient acceptability, adverse events, polyp/adenoma detection rate, number of polyps/adenomas detected, degree of bowel cleansing according to BBPS (BBPS score ≥8), degree of bowel cleansing according to Aronchik scale, and bowel cleansing time. The non-inferiority margin for the primary endpoint was 10%. A study protocol of the present study has already been published in a peer-reviewed journal.

Results

Of 540 participants enrolled, efficacy was analyzed in 524 participants (test group, n=263; standard group, n=261) in the full analysis set. Non-inferiority of the test group was demonstrated for the primary endpoint (BBPS ≥6): 96.2% vs. 97.7% for test and standard group, respectively difference was -1.5%, [95%confidence interval (CI), -4.8 to 1.6%] with P<0.001 for non-inferiority. For the secondary endpoint BBPS≥8 was lower in test group (79.5% vs 91.2%: difference -11.7% [95% CI, -17.8 to -5.8%]). Patient acceptability was superior in the test group in the key questions (1. Was the bowel preparation medication easy to use? 2. Were all bowel preparation medications administered internally? 3. What is your overall impression of the bowel preparation? 4. How did the bowel preparation medicine taste? 5. Would you like to use the same bowel preparation medicine for your next colonoscopy?) Adverse events were similar in both groups.

Conclusion

A new regimen of elobixibat plus sodium picosulfate with magnesium citrate was non-inferior in bowel cleansing and superior in patient’s acceptability compared to split-dose 2-L polyethylene glycol with ascorbic acid.

References

1) Hotta K, Otake Y, Yamaguchi D, et al. Comparison of the efficacy and tolerability of elobixibat plus sodium picosulfate with magnesium citrate and split‑dose 2‑L polyethylene glycol with ascorbic acid for bowel preparation before outpatient colonoscopy: a study protocol for the multicentre, randomised, controlled E‑PLUS trial. BMC Gastroenterol 2024 Feb 3;24(1):61.doi: 10.1186/s12876-024-03146-6.

Disclosure

The primary investigator (Kinichi Hotta) had received research funding from Nippon Chemiphar Co. Ltd.