Introduction

Micronutrient deficiency is an underestimated problem in the westernized population with unclear effects on the manifestation of acute or chronic disease. Westernized lifestyle instigates disrupted gut-microbial crosstalk and chronic low-grade inflammation¹. Tryptophan (Trp) is an essential amino acid used for de novo synthesis of cellular nicotinamide adenine dinucleotide (NAD). We have previously shown increased Trp degradation in several chronic inflammatory disorders, including IBD^2,3. Whether Trp deficiency is a widespread phenomenon in the western population is unknown. Here, we tested the hypothesis that Trp deficiency is frequent in the western population and associates with increased overall disease severity in internal-medicine and especially gastrointestinal (GI) diseases.

Aims & Methods

To screen the micronutrient status of the healthy population, we assessed quantitative amino acid levels in n=15.188 healthy controls (HC) with a commercially available home-care dry blood spot test for targeted metabolomics (cohort 1). Self-reported symptoms were assessed with a digital questionnaire. To validate Trp deficiency (defined as the lowest Trp quartile) in acute internal-medicine diseases (cohort 2), we measured serum Trp by HPLC in HC (n=291) and patients presenting in the emergency ward of the University Hospital Schleswig-Holstein, Kiel (n=4349, categorized by ICD10; thereof GI diseases n=516 (K00-K93: diseases of the digestive system); 10/2023-09/2024). Next to laboratory and clinical assessment at presentation, disease courses were followed up digitally for at least 6 months. Statistical analyses were performed with linear mixed models (LMM) and logistic regression correcting for age and sex at birth; predictive performance was evaluated via area under the receiver-operating-characteristic (AUROC; R version 4.4.1).

Results

In cohort 1, 39.3% of HC presented with Trp deficiency. Trp deficiency significantly inversely correlated with the amount of self-reported symptoms, such as e.g. GI symptoms. Validating these findings in internal-medicine and a subgroup of GI diseases in cohort 2, we found significantly reduced serum Trp compared to HC (mean Trp 56.58±10.84µM vs. 45.06±16.65 (all disease groups), 45.21±20.69 (GI diseases)), especially when the serum inflammation marker C-reactive protein was not elevated (<5 mg/l). Here, Trp deficiency was observed in 1.4% of HC, while it was strongly increased in all disease groups (26.6%) and GI diseases (30.2%). Using LMM, we found that patients that were hospitalized, admitted to the ICU or died within the follow-up period had a significantly lower Trp compared to patients with favourable outcomes (outpatient/no ICU/alive). Patients with Trp at the lowest compared to the highest quartile underwent longer hospitalisation and ICU treatment (all diseases: 3.17/0.37 days (d) longer; GI diseases: 3.91/0.48d longer) as well as a higher number of in-hospital procedures (all diseases: 1.47 more; GI diseases: 3.3 more). The predictive value of Trp for complicated outcomes (hospitalisation, hospitalisation >7d, ICU treatment, death) as assessed by AUROC was low-moderate (all diseases: AUC 0.69, 0.67, 0.61, 0.72; GI diseases: AUC 0.61, 0.6, 0.54 and 0.62, respectively).

Conclusion

We report that Trp deficiency is frequent in the westernized population and associates with more severe disease courses. Our data suggests Trp degradation along the de novo NAD synthesis pathway as an overarching metabolic hallmark of chronic low-grade inflammation and a risk factor for severe disease courses of internal-medicine and GI diseases.

References

1. Christ, A. & Latz, E. The Western lifestyle has lasting effects on metaflammation. Nature Reviews Immunology vol. 19 267–268 Preprint at https://doi.org/10.1038/s41577-019-0156-1 (2019).
2. Harris, D. M. M. et al. Tryptophan degradation as a systems phenomenon in inflammation – an analysis across 13 chronic inflammatory diseases. EBioMedicine 102, 105056 (2024).
3. Nikolaus, S. et al. Increased Tryptophan Metabolism Is Associated With Activity of Inflammatory Bowel Diseases. Gastroenterology 153, (2017).

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

The role of the gut microbiota in inflammatory bowel disease (IBD) pathogenesis is widely acknowledged, with numerous bacteria identified as contributing factors. Little however is known about the part viruses play – a likely major component in the structuring and functioning of the gut microbiota. Understanding how viruses interact with the gut microbiota and host cells in the context of IBD could facilitate innovative treatment approaches and personalized interventions.

Aims & Methods

We explore the colonic and upper intestinal virome in patients with IBD compared with general population (GP) controls. Faecal (colonic) and effluent (upper intestinal) derived shotgun metaviromes of 100 IBD individuals (UC = 50, CD = 50) and 50 IBD individuals with an ileostomy, respectively were compared to faecal derived metaviromes of 50 GP individuals. Metaviromes were generated using a combination of shotgun methods and viral particle enrichment, allowing for identification of both eukaryotic and prokaryotic, dsDNA, ssDNA and RNA viruses. Viral diversity, composition and prevalence were compared by calculating Shannon diversity, Bray-Curtis distances and performing logistic regression analyses. P-values were adjusted using false discovery rate (FDR) to correct for multiple testing, where relevant; FDR<0.05 was considered significant.

Results

Similar to bacterial diversity in IBD versus GP samples, we see a significantly lower viral diversity in CD and UC colonic samples compared with the GP, and an even lower diversity when comparing upper intestinal samples to colonic samples (FDR<0.05). Comparing viral populations between groups, we observe that, whilst there is evident interindividual variability within groups, distinct clustering between colonic and upper intestinal samples can be seen, with colonic IBD samples clustering slightly more towards upper intestinal samples. When comparing viral group prevalences between GP and IBD colonic samples, 83 out of 260 viral groups present in IBD-UC samples are absent in the GP samples and 125 out of 302 viral groups present in the GP were absent in IBD-UC. For IBD-CD samples these numbers are 79 out of 248 and 133 out of 302, respectively. The top 3 viral groups that positively associated with IBD-UC samples are: one unknown phage group (predicted bacterial host: Parasutterella spp.), a group containing Lactobacillus phages (bacterial host: Lactobacillus spp.) and a group containing Lactococcus phages (bacterial host: primarily Lactococcus spp). The top 3 viral groups that positively associated with IBD-CD samples are: the same unknown phage group as for IBD-UC, and two other unknown phage groups (predicted bacterial hosts: primarily Enterocloster & Bacteroides spp, respectively).

Conclusion

This first comprehensive description of the gut virome across multiple gastrointestinal sites shows specific alterations in IBD. Many viral groups differentially present in the IBD intestine infect bacteria often associated with gut health and significantly reduced in IBD patients. Dysregulated bile acid signaling has also been implicated in the pathogenesis of IBD, with bacterial dysbiosis being linked to its cause. In IBD populations, we observe phages with the host E.bolteae – a bacteria increased in dysbiosis due to their potential to metabolise bile acids. We speculate that alterations in the gut virome composition are key events that disrupt the equilibrium between gut microbiota and host, promoting a pro-inflammatory state and driving IBD pathogenesis. The next steps will be to investigate the mechanisms underlying these complex interactions.

Disclosure

RKW unrestricted grants: Takeda, Johnson and Johnson, Tramedico, Ferring. Consultant: Takeda