Introduction

Patients suffering from inflammatory bowel diseases (IBD) manifest an increased risk of developing colitis-associated colon cancer (CAC). Interleukin 22 (IL22) is a cytokine involved in the proliferation and survival of epithelial cells. However, it also correlates with the development of tumoral lesions. IL22 is secreted by different immune cell types, including iNKT cells, tissue resident cells possessing cytotoxic properties in colorectal cancer (CRC). In IBD patients, though, iNKT cells perform both pro-inflammatory or tolerogenic functions in a microbiota-dependent fashion, opening questions on their functional role in CAC development.

Aims & Methods

Immune cell composition and iNKT cell phenotype of inflamed, non-tumor and cancerous tissue lesions of CAC (n=49), IBD (n=20) and sporadic CRC (n=38) patients were analysed in samples obtained from Policlinico Hospital, Milan, and IOV-IRCCS, Padua. High-dimensional single-cell flow cytometry, metagenomics, RNAseq, spatial immunophenotyping and trascriptomic (Cosmx, Nanostring), ex vivo and in vitro experiments were performed to evaluate the phenotype and function of human iNKT cells. Metabolomics was performed to identify microbial-derived molecules involved in iNKT cells activation.Mechanisms were dissected in CAC murine models, either iNKT proficient or deficient.

Results

An increased expression of IL22 and a higher NKT22 infiltration was observed in CAC tissues, as well as an enrichment of Odoribacter, a gram-negative bacteria implicated in tryptophan metabolism. Stimulation of iNKT cells with Odoribacter-derived metabolites induced Ahr-dependent IL22 production by iNKT cells. Metabolomic analyses revealed an increased presence of tryptophan metabolites in the Odoribacter supernatant, promoting NKT22 differentiation both in vitro and in vivo models. Moreover, the administration of Odoribacter Supernatant or of the single purified metabolites were sufficient to increase tumorigenesis in CAC mouse models, but not in iNKT-deficient mice.
Spatial proteomics and trascriptomic analyses of human CAC tissues confirms the microbiota-dependent functional skewing of iNKT cells residing closely to cancer cells.

Conclusion

iNKT cells in CAC patients are the major IL22 producing cells and are induced by the recognition of microbiota-derived metabolites implicated in tryptophan metabolism and Aryl hydrocarbon receptor (AhR) stimulation. NKT22-microbiota interaction in CAC patients plays a potential crucial role in tumor development.

Clinical Case Summary

A 24-year-old male presented with jaundice and mild vertigo after a 10-day prodrome (asthenia, nausea, headache, fever). Ibuprofen (1200 mg/d) was taken for these symptoms only during the first 5 days. Labs revealed acute hepatocellular injury and elevated cholestasis (AST 215, ALT 862, FA 134, GGT 126 IU/L), marked mixed hyperbilirubinemia (Total 13.5 mg/dL, Direct 7.58 mg/dL) and coagulopathy (INR 1.59). Ultrasound showed hepatosplenomegaly. Viral hepatitis (HAV/HBV/HCV/CMV) and autoimmune/metabolic causes were excluded. Epstein-Barr Virus (EBV) serology showed low-titer positive VCA IgM but negative VCA IgG, EBNA IgG, and serum EBV DNA PCR, indicating recent non-active infection. Given ibuprofen timing and high RUCAM score, drug-induced liver injury (DILI) with a hepatocellular pattern was suspected. Liver biopsy confirmed acute hepatitis with lymphomonocytic infiltrate, necrosis and canalicular cholestasis. Despite improving liver function tests post-ibuprofen cessation, he developed worsening vertigo, severe ataxia, nausea, vomiting, hypophonia, necessitating nasogastric feeding/bed confinement. Cerebrospinal fluid (CSF) showed mild mononuclear pleocytosis (16 cells/mm³); a multiplex PCR pathogen panel (including EBV) was negative. Brain MRI/EEG were normal. Post-infectious EBV acute cerebellar ataxia was diagnosed concurrently with DILI, based on timing post-recent EBV, exclusion of active neurological infection (negative CSF PCR, normal imaging), mild pleocytosis, and supported by later IgG seroconversion. IV methylprednisolone treatment, chosen for clinical severity, led to significant neurological improvement within days and eventual recovery after physiotherapy. This case highlights the significant challenges of diagnosis and management of concomitant DILI and recent EBV infection with neurological sequelae, requiring careful differentiation of liver damage patterns (hepatocellular DILI vs cholestatic EBV) and optimal management of neurological symptoms.

References

Illustrative Material References:
- Liver biopsy histology slides (highlighting lymphomonocytic infiltrate, necrosis, and canalicular cholestasis characteristic of DILI).
- Table/Graph showing serial Liver Function Tests (AST, ALT, Bilirubin) over time, depicting injury peak and recovery trend.
- Table summarizing sequential Epstein-Barr Virus (EBV) serology results (VCA IgM, VCA IgG, EBNA IgG) demonstrating seroconversion.
- Table summarizing Cerebrospinal fluid (CSF) analysis results (cell count, protein, glucose, negative multiplex PCR).
- Representative Brain MRI images (axial/sagittal views) confirming absence of structural lesions or encephalitis signs.

Introduction

Growing evidence supports the role of environmental factors, such as diet and lifestyle, in modulating the intestinal microbiota and potentially influencing the symptoms of chronic gastrointestinal disorders. This study aimed to correlate lifestyle habits with colonic microbiota composition in patients with Inflammatory Bowel Disease (IBD) or Irritable Bowel Syndrome (IBS), compared to healthy controls (HCs).

Aims & Methods

38 IBD, 44 IBS and 48 HC were recruited. Fecal microbiota composition was analyzed by sequencing the 16S rDNA V3/V4 region using the Illumina platform. Dietary habits and physical activity levels were assessed using a 7-day food diary, a Food Frequency Questionnaire, and the IPAQ. Urinary indican was measured via colorimetric assay and HPLC, fecal zonulin levels were assessed using ELISA, and selected circulating microRNAs were analyzed in plasma and serum by TaqMan-based quantitative Real-Time PCR.

Results

A significant reduction in dietary fiber intake was observed in both IBD and IBS patients compared to healthy controls (HCs) (p < 0.05). IBS patients engaged in more moderate physical activity than HCs (p < 0.05). Reduced fiber intake was associated with decreased microbial diversity (p < 0.005), compared to adequate fiber consumption. Microbiota analysis revealed a reduction in alpha diversity in IBD patients compared to HCs, particularly in terms of phylogenetic diversity and observed features, while IBS patients showed intermediate values. Beta diversity (weighted and unweighted UniFrac) demonstrated a clear separation among the three groups, with the most pronounced differences observed between IBD and the other conditions. LEfSe analysis identified the highest number of discriminant taxa in HCs, consistent with their greater microbial biodiversity. No clearly pathogenic organisms were identified in either IBD or IBS patients, suggesting that the observed alterations may result from a loss of beneficial commensals rather than the presence of harmful species. Additionally, greater microbial diversity was found in individuals living in rural areas compared to those in urban environments. IBD and IBS patients not adhering to the Mediterranean Diet (MD) showed significantly higher zonulin levels than those following the MD (p < 0.001 and p = 0.003, respectively). Finally, miRNAs involved in inflammatory and epithelial permeability pathways were differentially expressed in both IBD and IBS patients. MiR-24 was significantly dysregulated in IBD (p = 0.01), while miR-923 also showed a significant difference in the same group (p = 0.05). MiR-221 exhibited a non-significant trend toward upregulation in IBS. These findings suggest a potential role for these miRNAs in the molecular mechanisms underlying inflammatory and functional bowel disorders.

Conclusion

The preliminary results of the present study underlined the impact of lifestyle factors, particularly diet and physical activity, on intestinal microbiota composition and barrier function in IBD and IBS. Microbial and molecular markers may support a more personalized approach to these disorders.

References

1. Alfaifi, J. et al; Deep Dive Into MicroRNAs in Inflammatory Bowel Disease. Inflamm. Bowel. Dis. 2023
2. Cena, H. et al; Biodiversity and planetary health: A call for integrated action. Lancet 2024
3. Giammona A et Al: Chronic Gastrointestinal Disorders and miRNA-Associated Disease: An Up-to-Date. Int J Mol Sci 2025
4. Ventham, N.T.et al.; Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease. Nat. Commun. 2016

Introduction

Thiopurines are vital therapeutic agents for inflammatory bowel diseases but often cause severe myelosuppression. The NUDT15 polymorphism, particularly p.Arg139Cys (NUDT15R139C), is strongly linked to severe leukocytopenia and is prevalent in Asian populations. Despite thiopurine use during pregnancy, it remains unclear if NUDT15R139C affects fetal thiopurine sensitivity.

Aims & Methods

The human and murine NUDT15 proteins share 89% homology with the preservation of arginine at codon 139 of human NUDT15 in codon 138 of murine Nudt15. We established a knock-in mouse model with a point mutation (c.412C>T) generating p.Arg138Cys (Nudt15^R138C), mimicking the human phenotype. In the current study, we investigated offspring generated from Nudt15^+/R138C or Nudt15^+/+ female mice that were mated with Nudt15^+/R138C male mice and simultaneously administered with mercaptopurine (MP).To evaluate the effects of thiopurine on the fetus, we evaluated the number of hematopoietic stem cells in the fetal liver, which is the center of fetal hematopoiesis.

Results

In administration with the tolerable MP dose (0.5 mg/kg) for adult Nudt15^+/R138C mice, genotypes of the genotypes of neonatal mice born from breeding between Nudt15^+/R138 male mice and Nudt15^+/R138 female mice were 35 (37.2%) Nudt15^+/+, 59 (62.8%) Nudt15^+/R138C, and 0 Nudt15 ^R138C/R138C. On embryonic day 14.5, Nudt15^R138C/R138C fetal mice showed shorter body lengths and fewer hematopoietic stem cells in the fetal liver than Nudt15^+/+ fetal mice. The number of fetal HSCs was not altered in any of the Nudt15genotypes without MP treatment. However, it was significantly reduced by MP treatment in Nudt15^+/R138C fetal mice (p< 0.01, mean number 1104 in DMSO and 577 in MP) and in Nudt15^R138C/R138C fetal mice (p< 0.01, mean number 927 in DMSO and 402 in MP), but not in Nudt15^+/+ fetal mice (p= 0.39, mean number 1004 in DMSO and 820 in MP). The number of HSCs in Nudt15^R138C/R138C fetal livers was significantly reduced to less than 50% of that in Nudt15^+/+ fetal livers by MP treatment during gestation (p= 0.0089). In administration with the tolerable MP dose (1.0 mg/kg) for Nudt15^+/+ adult mice, genotypes of neonatal mice born from breeding between Nudt15^+/R138 male mice and Nudt15^{++ /}female mice were 23 Nudt15^+/+ and 0 Nudt15^+/R138C. Without MP administration, neonatal mice were born in a Mendelian fashion.

Conclusion

Fetal toxicity may arise if the fetus has more NUDT15 R139C alleles than the mother, despite safe thiopurine doses for the mother. Analysis of the father's NUDT15 genotype may enable safer thiopurine use during pregnancy, emphasizing the importance of considering parental genotypes in therapeutic decisions.