Introduction

The gut virome has been established to hold a key role in the pathogenesis of Crohn’s disease (CD), belonging to the Inflammatory Bowel Disease (IBD) class. Indeed, some years ago Norman and colleagues described the expansion of Caudovirales as associated with CD pathogenesis and coupled with decreased bacterial diversity¹.

Aims & Methods

Therefore, to further explore this aspect we exploited the IBD TaMMAframework², to find which viral family was highly abundant in the CD colon by comparison with the healthy control. Moreover, to investigate the role of this family in the different mucosal cell compartments, CD- and healthy-derived intestinal biopsies were digested to obtain a cell suspension undergoing flow cytometry cell sorting (FACS) to isolate immune and non-immune cells according to cell population-specific markers. Specifically, CD8 and CD4 T cells, B cells, macrophages, dendritic cells, epithelium, endothelium, and fibroblasts were obtained and underwent transcriptomics and metatranscriptomics.

Results

We found that the Autographiviridae, belonging to the class of Caudovirales was highly abundant in the CD colon by comparison with the healthy control, confirming Norman study. Interestingly, the gene ontology (GO) revealed that the sole CD cell populations showing impaired biological response to viral insults by comparison with the healthy were the dendritic cells, fibroblasts, and epithelial cells suggesting that in CD mucosa only specific cell compartments were impacted by viral entities. Furthermore, by metatranscriptomics analysis we identified the CD dendritic cells and endothelial cells to retain a higher abundance of the Autographiviridae family by comparison with the healthy, indicating that the virome dysbiosis in these cells may underlie CD pathogenesis by altering their biological functions. Consistently, the heatmap of differentially regulated genes in CD versus healthy dendritic cells displayed downregulation of the transcripts key for dendritic cell-directed immune response.

Conclusion

Similar results have been recently proposed by our group to explain ulcerative colitis (UC) pathogenesis, where we identified a virome-derived protein, belonging to the Hepadnaviridae family, to directly disrupt the epithelial barrier and guide the development of intestinal inflammation³. Therefore, given the growing evidence of the viral entities as triggers for IBD, these data are promising and may pave the way to the discovery of viruses directly involved in CD pathogenesis that can represent a target for the development of novel strategies of intervention, ultimately ameliorating the CD management.

References

1. Norman, J. M. et al. Disease-specific alterations in the enteric virome in inflammatory bowel disease. Cell 160, 447–460 (2015).
2. Massimino, L. et al. The Inflammatory Bowel Disease Transcriptome and Metatranscriptome Meta-Analysis (IBD TaMMA) framework. Nat. Comput. Sci. 1, 511–515 (2021).
3. Massimino, L. et al. Gut virome-colonising Orthohepadnavirus genus is associated with ulcerative colitis pathogenesis and induces intestinal inflammation in vivo. Gut (2023) doi:10.1136/gutjnl-2022-328375.

Introduction

Crohn's disease (CD) has been increasingly linked to imbalances in the gut microbiota, notably through dysregulation in the gut virome. Particularly, elevated levels of Caudovirales have been observed in CD patients. Among these, the Proteus virus Isfahan species is abundant in CD, suggesting it might trigger an autoimmune response through molecular mimicry.

Aims & Methods

The aim of this study is to investigate whether Proteus virus Isfahan proteins share homology with human proteins, potentially initiating autoimmune mechanisms in CD. Utilizing the IBD Transcriptome and Metatranscriptome Meta-Analysis (TaMMA) framework, we analyzed cell populations from intestinal biopsies of CD patients and healthy controls. Immune and non-immune cells were isolated using flow cytometry and underwent transcriptomic analysis. Additionally, we examined the homology of viral proteins to human proteins using NCBI BLAST and iPBA. In vitro experiments involved coculturing T cells with immune cells overexpressing Proteus virus Isfahan protein to explore immune responses.

Results

Our results confirmed increased levels of Proteus virus Isfahan, in CD patients, especially in dendritic cells and macrophages. Gene ontology analysis indicated compromised immune responses in dendritic cells and enhanced viral-response pathways in CD macrophages. Notably, the viral protein gp82 showed structural homology with human dCMP deaminase, supporting the molecular mimicry hypothesis. These findings highlight Proteus virus Isfahan as a potential activator of autoreactive T cells, exacerbating CD's autoimmunity.

Conclusion

This evidence suggests that Proteus virus Isfahan may play a significant role in CD pathogenesis through molecular mimicry, where its protein's similarity to human proteins could trigger immune misrecognition, fostering autoimmunity and perpetuating inflammation in CD. Ongoing experiments aim to further elucidate these interactions, potentially identifying novel therapeutic targets.

Introduction

Over 50% of Crohn’s disease (CD) patients develop intestinal fibrosis, often resulting in strictures. Although anti-inflammatory therapies have improved disease management, fibrotic complications continue to represent a major unmet clinical challenge. Emerging evidence suggests that the gut microbiota may contribute to fibrosis independently of inflammation¹, yet the microbial signatures associated with different stages of fibrosis remain poorly defined.

Aims & Methods

We performed a meta-analysis of RNA-seq data using the IBD TaMMA framework² on fibrotic CD and healthy tissue. Mucosal fibroblasts, epithelial, and endothelial cells were isolated via FACS and profiled transcriptomically. Epithelial-derived organoids and co-culture systems were used to model host–microbe interactions. Fibroblasts stimulated with Roseomonas mucosa lysates (10 µg/mL) were assessed for fibrosis markers (α-SMA, vimentin, FAP) by immunofluorescence and qRT-PCR (ACTA2, PDPN, FAP).

Results

Gene ontology analysis revealed enrichment of antimicrobial immune responses during fibrosis progression. Microbial profiling indicated a consistent enrichment of Roseomonas mucosa in CD samples across fibroblasts, epithelial, endothelial, and immune cell compartments. Notably, fibroblasts stimulated with R. mucosa lysates showed increased expression of fibrosis markers compared to controls.

Conclusion

These findings suggest that Roseomonas mucosa or its components may influence the transcriptional programming of mucosal cells and contribute to fibrosis development in CD. Ongoing spatial transcriptomics and RNA-scope analyses aim to define the spatial and temporal dynamics of microbial–host interactions in fibrotic tissue, advancing our understanding of microbiota-driven fibrosis.

References

1.  Watanabe, D. & Kamada, N. Contribution of the gut microbiota to intestinal fibrosis in crohn’s disease. Front Med (Lausanne) 9, 826240 (2022).
2. Massimino, L. et al. The Inflammatory Bowel Disease Transcriptome and Metatranscriptome Meta-Analysis (IBD TaMMA) framework. Nat. Comput. Sci. 1, 511–515 (2021).

Introduction

Colitis-associated carcinoma (CAC) is a serious complication of long-standing ulcerative colitis (UC) and Crohn’s disease (CD). While the mutational landscape of CAC has been extensively characterised, its transcriptomic landscape remains incompletely defined. To investigate the tumour ecology and molecular mechanisms underpinning CAC development, we utilised FFPE tissues from human CAC patients and colon organoids. Through the integration of immunology-focused bulk gene expression profiling and spatial transcriptomics, we derived a spatially resolved gene signature of CAC at single-cell resolution.

Aims & Methods

To comprehensively profile the CAC tumour immune microenvironment, we generated a targeted bulk RNA expression dataset (Nanostring Immunology v2) from FFPE samples of 40 patients with IBD or CAC, and inflammation-free healthy controls (10 per group). We observed robust upregulation of SPP1 (encoding osteopontin, OPN) and FN1 (Fibronectin-1) in CAC. To expand these findings, we applied spatial transcriptomics (Xenium) on 5μm FFPE sections from CAC (10 patients), UC (3 patients), and healthy controls (3 patients), enabling spatial and single-cell resolution of SPP1 and its receptor expression. We further mapped ligand–receptor interactions to specific cell types, revealing distinct SPP1-mediated signalling in CAC. These results were validated at the protein level using whole-section FFPE confocal microscopy.

Results

Immunology gene expression profiling (Nanostring) revealed strong upregulation of SPP1 (osteopontin, OPN) in colitis-associated cancer (CAC) compared to ulcerative colitis (UC) and Crohn’s disease (CD). Spatial transcriptomics (Xenium) demonstrated spatial heterogeneity of SPP1, primarily arising from CD68+ macrophages in CAC, as validated by OPN/CD68 co-immunostaining. Ligand-receptor analysis identified SPP1 as a key outgoing signal from macrophages, engaging CD44 and integrin receptors on T cells. In immune subsets, CD4+ T cells showed upregulation of FOXP3 and CTLA4, indicating a regulatory T cell phenotype enriched in CAC. CD8A+ T cells displayed upregulation of GZMK, alongside downregulated JAK-STAT signalling and upregulated LDHA, hypoxia and glycolysis hallmark pathways, suggesting metabolic reprogramming and impaired effector function in the hypoxic CAC microenvironment.

Conclusion

We identified a CAC-specific upregulation of SPP1 at both RNA and protein levels, expressed by CD68 macrophages. Spatially, SPP1/OPN+ macrophages and CD8+ T cells were mutually exclusive, suggesting a role for OPN in driving T cell exclusion and immunosuppression. Ligand-receptor analysis highlighted SPP1-CD44/integrin interactions in immune modulation. CD4+ T cells showed a regulatory phenotype, while CD8+ T cells showed metabolic reprogramming and functional impairment. These features define a CAC-specific immune landscape and a gene signature with potential to detect pre-malignant changes.

Introduction

Eukaryotic-targeting viruses have recently attracted considerable interest in gastrointestinal diseases such as ulcerative colitis (UC), a chronic inflammatory condition of the colonic tract, and colorectal cancer (CRC)^1–3, the third most commonly diagnosed malignancy and the fourth leading cause of cancer death in the world⁴. Both UC and CRC share several factors influencing their etiogenesis, including intestinal dysbiosis^{2, 5–7}. Our group has recently published a work pinpointing a gut virome-associated Orthohepadnaviridae protein, namely Hepatitis B protein X (HBx) to correlate with UC pathogenesis and promote intestinal inflammation in mice by disrupting the epithelial barrier and shaping the gut mucosal immune environment, independently of the microbiota³. Moreover, we showed that HBx is able to induce DNA damage-related biological processes and active biological processes related to the Wnt pathway³ known to be involved in colorectal carcinogenesis⁸.

Aims & Methods

Based on this evidence we hypothesize that HBx may fill the gap existing between intestinal inflammation and CRC onset by stimulating pro-inflammatory and/or pro-carcinogenic effects.
To verify this hypothesis, we address the following Aims:
1. To evaluate the presence of HBx-transcript in CRC-derived mucosal samples
2. To evaluate the pro-carcinogenic properties of HBx in vitro
3. To investigate the role of HBx in CRC onset and development in vivo
HBx positivity of CRC patient-derived gut mucosa was assessed by RT-PCR and Sanger.
Multidimensional flow cytometry analysis was performed on HBx-overexpressing Caco-2 cells, an in vitro model of the gut barrier.
C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx-encoding plasmids or the control. Multidimensional flow cytometry analysis and transcriptomic analysis were performed on colonic samples from HBx-treated and control animals.

Results

Metatranscriptomic analysis revealed that HBx is significantly abundantly present in CRC-derived mucosal samples compared with healthy individuals, strengthening the link between a virome protein-induced inflammation and tumor development in patients.
Moreover, HBx overexpression in Caco-2 cells directly provokes DNA damage, as assessed by the significant increase in the number of γH2AX+ cells, a marker of double-strand DNA breaks which identifies genomic instability that can potentially contribute to cancer initiation and progression⁹.
Furthermore, we already showed that HBx was able to induce colitis-like symptoms in mice after 15-20 days-long administration³. Interestingly, experiments conducted in C57BL/6 mice at longer time points (from 40 to 60 days-long observation) revealed that animals developed polypoid structures in the colonic mucosa, characterized by decreased abundance of T cells, including effector T cells and NK T cells¹⁰. Of note, in vivo HBx modulated the expression of genes involved in the regulation of transcription and activation of the proto-oncogene PIM3¹¹, further supporting the role of a viral protein on CRC onset.

Conclusion

This study introduces unconventional and innovative insights for giving CRC studies a new direction for the scientific and clinical investigations, looking at the gut virome as an active component and not as a mere bystander entity of the microbiota, ultimately promoting a new standpoint in the CRC studies and management in the short-term future.

References

1. T. Zuo et al., Gut. 68, 1169–1179 (2019).
2. L. Massimino, S. Lovisa, L. Antonio Lamparelli, S. Danese, F. Ungaro, Comput. Struct. Biotechnol. J. 19, 16–28 (2021).
3. L. Massimino et al., Gut (2023), doi:10.1136/gutjnl-2022-328375.
4. E. Morgan et al., Gut. 72, 338–344 (2023).
5. F. Ungaro, S. D’Alessio, S. Danese, Cancers (Basel). 12 (2020), doi:10.3390/cancers12082060.
6. C. Fiorentini et al., Int. J. Mol. Sci. 21 (2020), doi:10.3390/ijms21176201.
7. F. Ungaro, L. Massimino, S. D’Alessio, S. Danese, United European Gastroenterol. J. 7, 999–1007 (2019).
8. E. M. Schatoff, B. I. Leach, L. E. Dow, Curr. Colorectal Cancer Rep. 13, 101–110 (2017).
9. J. Gantchev et al., J. Cell Commun. Signal. 16, 159–177 (2022).
10. S. Aristin Revilla, O. Kranenburg, P. J. Coffer, Front. Immunol. 13, 903564 (2022).
11. N. Mukaida, Y.-Y. Wang, Y.-Y. Li, Cancer Sci. 102, 1437–1442 (2011).

Introduction

Recent evidence suggests that appendectomy is associated with a reduced risk of developing ulcerative colitis (UC) or even the risk of flare after UC is diagnosed, thus suggesting a potential involvement of the appendix in UC pathogenesis. Interestingly, several studies support the role of the appendix in the maintenance of the intestinal microbiome and gut immunity1. We hypothesize that the appendix in UC may function as a reservoir for dysbiotic microbiota capable of promoting and re-establishing dysbiosis that could fuel inflammation in the colon in patients with UC.

Aims & Methods

The current study aimed to define the tissue-associated microbiota of the UC appendix, and investigate the changes compared to non-UC appendix. Thirty-seven patients operated on for UC or acute appendicitis were recruited. Appendicular wall tissue samples were obtained, inflammatory cells were isolated and analyzed by flow cytometry, and total DNA was extracted from the whole tissue. The V3-V4 region of the 16S rRNA gene was sequenced utilizing a MiSeq Platform (Illumina, USA). Sequences were processed using the QIIME22 pipeline, the taxonomy was assigned using the Greengenes3 database, and statistical analysis was performed in R.

Results

Twenty-seven samples were analyzed from 37 patients, 19 from UC, and 8 from acute appendicitis patients. Analyses revealed Firmicutes was the most proportionally abundant bacterial phylum in the UC appendix (mean=72.6%), followed by Proteobacteria (16%). Analysis of alpha diversity revealed no significant difference in terms of species richness, Shannon, and Pielou indices between UC and non-UC appendix (p=0.9;p=0.087;p=0.26). The PERMANOVA test revealed that UC appendix communities were significantly different from non-UC appendix communities (p=0.015). We identified 5 differentially abundant species in the study's samples, all of these significantly decreased in UC appendix vs. non-UC appendix: Fusobacterium unknown species (p=4.36x10-4), Pseudoramibacter unknown species (p=4.46x10-3), Streptococcus anginosus (p=2.67x10-2), Mogibacterium unknown species (p=2.67x10-2) and Actinomyces hyovaginalis (p=3.64x10-2). Fusobacterium, Pseudoramibacter, and Mogibacterium were also differentially abundant at the genus level (p=3.10x10-4; p=3.17x10-3; p=2.53x10-2). The expression of CD86 on activated dendritic cells (CD1a+HLAdr+CD86+) was significantly higher in UC patients than in appendicitis patients (p=0.049) and in UC patients CD1a+HLAdr+CD86+ cells rate directly correlated with disease activity as expressed by Harvey Bradshaw Index (rho=-0.513, p=0.025). The abundance of Fusobacterium unknown species inversely correlated with CD1a+HLAdr+CD86+ mean fluorescence intensity (rho=-0.466, p=0.047).

Conclusion

These preliminary data showed that specific taxa are significantly less present in the UC appendix compared to the non-UC appendix, suggesting that a distinctive UC appendix microbiota exists. Taxa with altered abundance may contribute to UC pathogenesis. These data may provide microbial signatures useful for guiding both the treatment and diagnosis of UC.

References

1. Arjomand Fard N, Armstrong H, Perry T, Wine E. Appendix and Ulcerative Colitis: a Key to Explaining the Pathogenesis and Directing Novel Therapies? Inflamm Bowel Dis. 2023;29(1):151–160. doi:10.1093/ibd/izac106.
2. Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F, Bai Y. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature biotechnology. 2019 Aug;37(8):852-7.
3. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Applied and environmental microbiology. 2006 Jul;72(7):5069-72.