Introduction

As common precursors to colorectal cancer (CRC), polyps and adenomas in the large intestine may develop into malignancy over time through a cascade known as the "normal-polyp-adenoma-carcinoma" sequence. Colorectal cancer (CRC) is the third most common cancer and the leading cause of cancer-related death worldwide^​, with an estimated increase to 3.2 million new cases and 1.6 million deaths by 2040^​. The high mortality of CRC is largely due to the late diagnosis, uncomplete molecular understanding of pathological progression, and limited treatment options for patients with advanced disease stage. Therefore, the identification of novel biomarkers at early (pre-cancerous) phase, thorough elucidation of pathological mechanisms, and development of effective therapies is crucial for CRC diagnosis and treatment.

Aims & Methods

Here we have utilized 10× genomics scRNA-seq platform to perform a high-resolution transcriptomic analysis of three normal colorectal tissues, two samples with inflammatory polyps, two specimens with adenomatous polyps and three CRC samples.

Results

By analyzing the scRNA-seq data of the ten human colorectal samples, which represent the four phases of colorectal pathogenesis, in combination with validation by immunofluorescence staining using expanded clinical samples, and together with verification using large scale TCGA and Kaplan–Meier data, we revealed a dynamic molecular landscape and for the first time demonstrated the importance of ligand-receptor genes, transcription factors and genes encoding multiple immunoglobulins and heat shock proteins driving the colorectal carcinogenesis.
Besides the heterogeneity of (sub-) cell types and difference of the transcriptomic profiling across the four stages, one of the most interesting and novel findings of this study is that the expression of ligand-receptor genes can be used to determine the cell types and even cell sub-types, at least for the colorectal tissues collected in this study, not the expressions of other gene sets, such as cell surface receptor signaling pathways, cell-cell signaling, transcription factors, cytokines, cell activation factors, cell signaling factors, or extracellular matrisome, implying that ligand-receptor pairs are fundamentally important for the cell type determination and cell-cell communications in colorectal tissues.
We found that some genes encoding immunoglobulin members are highly expressed in the T cells (IGHA1, IGHA2, IGKC and IGLC2) and in the fibroblasts (IGHA1, IGHA2, IGLC1, IGLC2, IGKC and JCHAIN) from normal colorectal tissues, while some genes encoding heat shock proteins are highly expressed in T cells (HSP90AA1, HSP90AB1, HSPA1B, HSPD1, HSPE1 and HSPH1) and plasma cells (HSP90AA1, HSPA1A, HSPA1B, HSPA6, HSPB1 and HSPD1) from abnormal colorectal samples, especially in the ones with adenomatous polyps.
Furthermore, IgG plasma cells can be grouped into two sub-types, characteristic of high expression of immunoglobulin-encoding genes IGKC and IGLC1/IGLC2, respectively. In addition, the six clusters of IgA plasma cells can also be characterized based on the combinatory expression level of some immunoglobulin-encoding genes (IGHA1, IGHA2, IGKC, IGLC1, IGLC2 and IGLC3). In summary, our analyses suggested the potential distinct functional importance of immunoglobulins and heat shock proteins in immunity and microenvironment during the colorectal malignancy.

Conclusion

Our transcriptome data provides valuable insights and a useful resource for better understanding the molecular pathological landscape in CRC to achieve more effective diagnosis and treatments.

References

Siegel, R.L., Wagle, N.S., Cercek, A., Smith, R.A., and Jemal, A. (2023). Colorectal cancer statistics, 2023. CA Cancer J Clin 73, 233-254. 10.3322/caac.21772.
2. Morgan, E., Arnold, M., Gini, A., Lorenzoni, V., Cabasag, C.J., Laversanne, M., Vignat, J., Ferlay, J., Murphy, N., and Bray, F. (2023). Global burden of colorectal cancer in 2020 and 2040: incidence and mortality estimates from GLOBOCAN. Gut 72, 338-344. 10.1136/gutjnl-2022-327736.
3. Huck, M.B., and Bohl, J.L. (2016). Colonic Polyps: Diagnosis and Surveillance. Clin Colon Rectal Surg 29, 296-305. 10.1055/s-0036-1584091.
4. He, D.G., Chen, X.J., Huang, J.N., Chen, J.G., Lv, M.Y., Huang, T.Z., Lan, P., and He, X.S. (2022). Increased risk of colorectal neoplasia in inflammatory bowel disease patients with post-inflammatory polyps: A systematic review and meta-analysis. World J Gastrointest Oncol 14, 348-361. 10.4251/wjgo.v14.i1.348.
5. Rudy, D.R., and Zdon, M.J. (2000). Update on colorectal cancer. Am Fam Physician 61, 1759-1770, 1773-1754.
6. Heumos, L., Schaar, A.C., Lance, C., Litinetskaya, A., Drost, F., Zappia, L., Lucken, M.D., Strobl, D.C., Henao, J., Curion, F., et al. (2023). Best practices for single-cell analysis across modalities. Nat Rev Genet 24, 550-572. 10.1038/s41576-023-00586-w.

Introduction

Canopy FGF signaling regulator 3 (CNPY3) is a protein predominantly localized in the endoplasmic reticulum. Its expression is increased in several cancers, including colorectal cancer (CRC). However, how CNPY3 affects CRC progression and the function of CNPY3 translocation to the nucleus in CRC is not clear.

Aims & Methods

This study aimed to investigate the role of CNPY3 in the development of CRC. In vitro, the impacts of CNPY3 on CRC growth were evaluated by 5-Ethynyl-2′-deoxyuridine staining, colony formation, wound healing, transwell migration, and apoptosis assay. Tumor xenograft mouse models and orthotopic mouse models of colorectal cancer metastasis were used to assess in vivo. Furthermore, the underlying mechanism of CNPY3 in CRC was revealed by RT-qPCR, western blot, LC-MS/MS analysis, immunofluorescence staining, coimmunoprecipitation, GST-pulldown, confocal microscopy, ubiquitination assays, and rescue experiments.

Results

CNPY3 was significantly upregulated in CRC tissue and cell lines, correlating with their poor prognosis. Functional experiments demonstrated that CNPY3 enhances CRC proliferation, migration, invasion, and tumor growth. Notably, our investigation reveals a nucleus translocation of cnpy3 by activation of nuclear localization sequences through protein conformational changes. Meanwhile, nuclear localization of CNPY3 promotes the binding of TRIM21 and p53 and forms a CNPY3-TRIM21-p53 complex, thereby promoting the ubiquitination-proteasomal degradation of wild-type p53 protein, inhibiting the related genes of the TP53 signaling pathway, and promoting apoptosis of tumor cells. In vivo and in vitro, inhibiting the degradation of p53 was able to suppress CNPY3 in cancer-promoting tumors, whereas the knockdown of CNPY3 in p53^-/- HCT116 cells did not significantly inhibit CRC.

Conclusion

CNPY3, after nuclear translocated, binds to TRIM21 and p53 and stabilizes the TRIM21–p53 interaction. This CNPY3–TRIM21–p53 complex facilitates TRIM21‐mediated p53 ubiquitinoylation and causes p53 proteasome degradation. Collectively, these findings support that CNPY3, an oncogene with CRC progression and poor prognosis, serves as a biomarker and therapeutic target correlated to suppress p53 function.

References

1. Wakabayashi Y, Kobayashi M, Akashi-Takamura S, et al. A protein associated with toll-like receptor 4 (PRAT4A) regulates cell surface expression of TLR4. J Immunol. 2006;177(3):1772-9.
2.
3. Xiao L, Li XX, Chung HK, Kalakonda S, et al. RNA-Binding Protein HuR Regulates Paneth Cell Function by Altering Membrane Localization of TLR2 via Post-transcriptional Control of CNPY3. Gastroenterology. 2019;157(3):731-743.
4. Ghait M, Husain RA, Duduskar SN, et al. The TLR-chaperone CNPY3 is a critical regulator of NLRP3-inflammasome activation. Eur J Immunol. 2022;52(6):907-923.
5. Zhou YQ, Bao TS, Xie JX, et al. The SLITRK4-CNPY3 axis promotes liver metastasis of gastric cancer by enhancing the endocytosis and recycling of TrkB in tumour cells. Cell Oncol (Dordr). 2023;46(4):1049-1067.
6. Reddy BA, van der Knaap JA, Bot AG, et al. Nucleotide biosynthetic enzyme GMP synthase is a TRIM21-controlled relay of p53 stabilization. Mol Cell. 2014;53(3):458-70.
7. Liu J, Zhang C, Xu D, et al. The ubiquitin ligase TRIM21 regulates mutant p53 accumulation and gain of function in cancer. J Clin Invest. 2023;133(6):e164354.

Introduction

Cendakimab is a recombinant, humanized, high-affinity, neutralizing monoclonal antibody that binds to interleukin 13 (IL-13) and blocks interaction with both its receptors, IL-13Rα1 and IL-13Rα2. IL-13 is a cytokine implicated in the pathogenesis of atopic dermatitis (AD) and is thought to be a key driver of the complex pathophysiology of eosinophilic esophagitis (EoE). IL-13 signals through both IL-13Rα1 and IL-13Rα2 to regulate cellular and molecular pathways across a variety of cell types. The binding of IL-13 to IL-13Rα1 promotes a Type 2 immune response and eosinophil recruitment, whereas the binding of IL-13 to IL-13Rα2 is believed to play a role in fibrosis and epithelial tissue remodeling. Several biomarkers associated with Type 2 inflammatory diseases are known to be regulated by IL-13. Eotaxin-3, a key biomarker, plays a central role in attracting eosinophils to the site of inflammation. Periostin, an extracellular matrix protein linked to activated fibroblasts, is implicated in tissue remodeling and fibrosis. Another enzyme governed by IL-13 is matrix metalloproteinase-12 (MMP-12)—this enzyme contributes to tissue remodeling and breakdown of extracellular matrix components. Here, we evaluate the pharmacodynamic effects of IL-13 inhibition with cendakimab on the key serum biomarkers eotaxin-3, periostin, and MMP-12 in patients with AD, with a focus on exploring potential implications for EoE.

Aims & Methods

In this phase 2, multicenter, global, randomized, double-blinded, placebo-controlled, parallel-group study (NCT04800315), 220 adults with moderate-to-severe AD received cendakimab 360 mg Q2W, 720 mg QW, 720 mg Q2W, or placebo for 16 weeks. Blood samples were collected, and a panel of biomarkers were assessed. The current analysis focuses on the assessment of serum levels of eotaxin-3, periostin, and MMP-12 using Proximity Extension Assay pre- and post-treatment on day 1 (baseline), at week 1 and week 2, and then biweekly until week 16. The least square mean percent change from baseline at week 16 for each treatment group was evaluated.

Results

For all 3 doses of cendakimab, there was a significant decrease from baseline in the levels of eotaxin-3, periostin, and MMP-12 as early as week 1 post-treatment and continued through week 16 of cendakimab treatment (Table). Compared with placebo, cendakimab resulted in significantly greater reductions in these biomarker levels through week 16.

Conclusion

Cendakimab decreased levels of key inflammatory and remodeling biomarkers over 16 weeks of treatment in patients with AD. These findings suggest that cendakimab modulates pathways of inflammation, fibrosis, and tissue remodeling in AD and add to the existing clinical evidence of its modulation in other Type 2 inflammatory diseases such as EoE.
​
Table. Least square mean percent change from baseline in key serum biomarker levels at week 16 in patients with AD

Disclosure

ESD served as a consultant for Abbott, Abbvie, Adare/Ellodi, Aimmune, Akesobio, Alfasigma, ALK, Allakos, Amgen, Aqilion, Arena/Pfizer, Aslan, AstraZeneca, Avir, Biorasi, Calypso, Celgene/Receptos/ Bristol Myers Squibb, Celldex, Eli Lilly, EsoCap, Eupraxia, Dr. Falk Pharma, Ferring, GlaxoSmithKline, Gossamer Bio, Holoclara, Invea, Knightpoint, Landos, LucidDx, Morphic, Nexstone Immunology/Uniquity, Nutricia, Parexel/Calyx, Phathom, Regeneron, Revolo, Robarts/Alimentiv, Salix, Sanofi, Shire/Takeda, Target RWE, Upstream Bio, received research funding from Adare/Ellodi, Allakos, Arena/Pfizer, AstraZeneca, Eupraxia, Ferring, GSK, Meritage, Miraca, Nutricia, Celgene/Receptos/ Bristol Myers Squibb, Regeneron, Revolo, Shire/Takeda, and received educational grant from Allakos, Aqilion, Holoclara, and Invea. JL, SH, LG, CDO, and CC are employees and stockholders of Bristol Myers Squibb. JS served as consultant for Abbvie, Alamar, Aldena, Amgen, AObiome, Apollo, Arcutis, Arena, Asana, Aslan, Attovia, BioMX, Biosion, Bodewell, Boehringer-Ingelheim, Bristol Myers Squibb, Cara, Castle Biosciences, Celgene, Connect Biopharma, Corevitas, Dermavant, Eli Lilly, FIDE, Galderma, GlaxoSmithKline, Incyte, Invea, Kiniksa, Leo Pharma, Merck, My-Or Diagnostics, Nektar, Novartis, Optum, Pfizer, RAPT, Recludix, Regeneron, Sandoz, Sanofi-Genzyme, Shaperon, TARGET-RWE, Teva, Union, UpToDate, participated on a data safety monitoring board or advisory board for Biosion and Sanofi, received grants or contracts from Galderma, Incyte, Pfizer, received honoraria from Abbvie, Eli Lilly, Leo Pharma, Pfizer, Regeneron, Sanofi-Genzyme.

Introduction

Lifestyle factors are significant contributors to fatigue, which affects approximately 45% of patients with Inflammatory Bowel Disease (IBD) in remission.

Aims & Methods

In this study, we evaluated the effect of a digital multimodal lifestyle intervention on fatigue in patients with IBD. Patients with IBD in biochemical remission (fecal calprotectin <150 mg/kg) were enrolled in a multicenter, non-randomized, controlled interventional study. The intervention group participated in a 12-month lifestyle program—provided by the non-profit organization Voeding Leeft—that included digital group meetings with a nutritionist and a lifestyle coach focusing on gradual and sustainable behavior change regarding nutrition, physical activity, sleep, and stress management. In addition, the program encouraged patients to exercise more self-control over their personal health. Following the initial six months, attendance at online sessions was not mandatory; however, patients could attend online sessions every six to 12 weeks to stay motivated and foster the lifestyle habits they acquired throughout the course. Patients in the control group received standard care that included an informational leaflet about the potential causes of fatigue in IBD, the leaflet also included basic lifestyle information and advice to exercise more, eat a healthier diet, stop smoking, reduce alcohol intake, improve sleeping habits, and manage stress. The allocation to a study group was determined by the availability within the group and participant’s ability to attend the scheduled intervention. Clinical data and patient-reported outcomes were collected. Fatigue was measured with the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F); any increase in FACIT-F after six months was considered a positive response to the intervention. The Minimal Clinically Important Difference (MCID) for fatigue was at least a four-point increase in the FACIT-F score¹. Inverse probability treatment weighting was used to correct confounding by indication.

Results

Thirty-six patients in the intervention group and 32 in the control group were compared. After six months, more patients in the intervention group (82.1% of patients) than in the control group (54.2% of patients) experienced improvement in fatigue, P = 0.029. Likewise, significantly more patients in the intervention group (71.4% of patients) than the control group (33.3% of patients) achieved a MCID regarding fatigue; P = 0.006. The improvements in fatigue were sustained throughout the study period in the intervention group. Acceptance of the health status was a significant factor for improvement in fatigue (β 7.813, SE 1.908, P < 0.001). Compared to the control group, quality of life improved in the intervention group; these patients gained 0.05 more quality-adjusted life years throughout the study. Last but not least, significant improvements in dietary quality were observed as early as three months after the baseline in the intervention but not the control group; these improvements in the intervention group were sustained throughout the study period.

Conclusion

Multimodal lifestyle intervention improves fatigue, quality of life, and dietary quality in patients with IBD who are in remission. Acceptance of the health status appears essential for fatigue improvement; instruments evaluating acceptance could help to personalize treatment and maximize its effectiveness.

References

1. Cella D, Yount S, Sorensen M, et al. Validation of the Functional Assessment of Chronic Illness Therapy Fatigue Scale relative to other instrumentation in patients with rheumatoid arthritis. J Rheumatol 2005;32:811-9.

Disclosure

AEvdMdJ received unrestricted research grants from Galapagos, Norgine, Vedanta, Nestle, including speaker’s fees from Galapagos, Tramedico, Takeda, Ferring, and Janssen Pharmaceuticals. PWV has received speaker fee from Galapagos and Takeda and served on the advisory board of Lilly, Galapagos, Janssen, BMS, Takeda, and Abbvie. RL has received travel expenses from Galapagos and Cablon Medical and served on the advisory board at Cablon Medical. NW and MJHB are employed by the non-profit organization Voeding Leeft and were a part of the team that guided participants in the intervention group. All other authors have no conflicts of interest to declare.

Introduction

Poor quality of sleep has detrimental effects on job performance, accident rates and health risks, with a potential link to the gut microbiome composition. First pilot trials have indicated beneficial effects of probiotic modulation of the microbiome on quality of sleep.

Aims & Methods

We conducted a randomized, double-blind, placebo-controlled trial testing the effects of a probiotic intervention on quality of sleep, quality of life and intestinal microbiome.
In total, 130 volunteers with impaired quality of sleep (PSQI>5) were included in the study. They were randomized in a 1:1 ratio to a 28-day intervention with either a multispecies probiotic (Omnibiotic-Stress Repair) or a placebo. Participants completed validated questionnaires to estimate quality of sleep (PSQI), quality of life (SF12, GIQLI) and perceived stress (PSQ20) before, during and after the intervention. Furthermore, stool samples for 16S rRNA gene sequencing were collected before and after the intervention. Ninety-four participants finished the study and could be included in the analysis.

Results

The 50 participants (88.6% female, 41.2±10.6 years old) analysed in the probiotic group and 44 participants (88.0% female, 40.1±10.7 years old) in the placebo group were well comparable at baseline, including the initial PSQI-score (10.1±2.7 vs. 10.5±2.6). After two weeks of intervention, both groups showed a comparable improvement in their PSQI-scores. After four weeks of intervention, the improvement of the PSQI score was significantly more pronounced in the probiotic group compared to the placebo group (6.8±2.9 vs. 7.7±3.1; p=0.036), mainly due to improved sleep efficiency and latency. The intervention did not influence quality of life or perceived stress. Ingested bacteria were partially recovered in the microbiome, causing a slight shift in beta diversity in the probiotic group, while alpha diversity and the overall composition of the microbiome remained unaffected.

Conclusion

In conclusion, the intervention with a multispecies probiotic was able to improve quality of sleep of participants with poor quality of sleep beyond an observable placebo-effect. Participants reported falling asleep more quickly and spending less time awake in bed. This well-powered RCT confirms that the probiotic modulation of the intestinal microbiome can be effective in alleviating sleep disturbances.

Disclosure

VS received research funding and speaker honoraria from Institut Allergosan (Graz, Austria) and Winclove Probiotics (Amsterdam, The Netherlands).

Introduction

Fatigue is a prevalent and debilitating symptom among patients with Crohn’s disease (CD) or ulcerative colitis (UC). However, due to its subjective nature and the lack of effective management strategies in CD/UC, fatigue often goes under-recognized and under-treated.¹ The Communicating Needs and Features of IBD Experiences (CONFIDE) study explored patient and healthcare professional (HCP) perspectives on the experiences and impact of CD/UC-related symptoms in Europe (France, Germany, Italy, Spain, and UK), the United States (US) and Japan.

Aims & Methods

Online, quantitative, cross-sectional surveys were conducted separately among patients with moderate-to-severe CD/UC and HCPs who make prescribing decisions for patients. Moderate-to-severe CD/UC were defined using criteria based on previous treatment, steroid use, and/or hospitalization. Descriptive statistics were used here to summarize the European and US data related to fatigue.

Results

A total of 547 European (55.4% male, mean age: 38.0 years) and 215 US (54.9% male, 40.9 years) patients with CD and 556 European (57.4% male, 38.9 years) and 200 US (61.5% male, 40.4 years) patients with UC completed the surveys. The proportions of patients who received advanced therapies at the time of survey completion were 63.4% and 58.1% among European and US patients with CD. Among patients with UC, the percentages were 54.1% and 76.5% of European and US patients.

Overall, 34.2% European and 35.8% US patients with CD, and 20.9% European and 27.5% US patients with UC reported currently experiencing fatigue (in the past month). Similar results were observed among patients receiving advanced therapies: CD (Europe: 34.3%; US: 36.0%); UC (Europe: 19.9%; US: 25.5%). Fatigue was ranked among the top five symptoms with greatest impact on patients by 44.5% European and 52.9% US patients with CD, and 55.2% European and 37.5% US patients with UC. Among patients who reported currently experiencing fatigue, 29.9% European and 36.4% US patients with CD, and 22.4% European and 40.0% US patients with UC discussed it with their HCPs at every appointment. Among patients who did not discuss fatigue with their HCPs at every appointment, at least two-thirds indicated that they would like to discuss it more frequently: CD (Europe: 75.6%; US: 67.3%); UC (Europe: 74.4%; US: 66.7%).

Surveys were completed by 503 European (70.8% male) and 200 US (78.0% male) HCPs, of which 92.5% were gastroenterologists/internal medicine practitioners. A minority of HCPs reported fatigue among the top five symptoms with the greatest impact on patients: CD (Europe: 33.9%; US: 22.0%); UC (Europe: 22.7%; US: 11.5%). More than half of HCPs reported proactively discussing fatigue at a routine appointment with patients: CD (Europe: 69.4%, US: 59.5%); UC (Europe: 64.6%, US: 59.0%). Among HCPs not proactively discussing fatigue, the most common reason was that they expected patients to bring it up if it is an issue: CD (Europe: 39.0%, US: 30.9%); UC (Europe: 37.1%, US: 32.9%).

Conclusion

For both patients with CD/UC, fatigue was a common and impactful symptom, despite the use of advanced therapies. A substantial proportion of patients experiencing fatigue who did not discuss it at every HCP appointment, reported a desire to discuss fatigue more frequently. Although more than half of HCPs reported proactive discussion of fatigue with their patients, some HCPs expected patients to bring it up. This indicates a communication gap between patients and HCPs and highlights the underappreciation of the impact of fatigue in CD/UC.

References

1. Farrell D, et al. (2020) Cochrane Database Syst Rev. 4(4), CD012005.

Disclosure

Remo Panaccione, Consultant for: Abbott, AbbVie, Abbivax, Alimentiv (formerly Robarts), Amgen, AnaptysBio, Arena Pharmaceuticals, AstraZeneca, Biogen, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Celltrion, Cosmos Pharmaceuticals, Eisai, Elan, Eli Lilly, Ferring, Galapagos, Fresenius Kabi, Genentech, Gilead Sciences, Glaxo-Smith Kline, JAMP Bio, Janssen, Merck, Mylan, Novartis, Oppilan Pharma, Organon, Pandion Pharma, Pendopharm, Pfizer, Progenity, Prometheus Biosciences, Protagonist Therapeutics, Roche, Sandoz, Satisfai Health, Shire, Sublimity Therapeutics, Spyre Therapeutics, Takeda Pharmaceuticals, Theravance Biopharma, Trellus, Union Biopharma, Viatris, Ventyx, UCB; Speaker’s Fees for: AbbVie, Amgen, Arena Pharmaceuticals, Bristol-Myers Squibb, Celgene, Eli Lilly, Ferring, Fresenius Kabi, Gilead Sciences, Janssen, Merck, Organon, Pfizer, Roche, Sandoz, Shire, Takeda Pharmaceuticals; Advisory Boards for: AbbVie, Alimentiv (formerly Robarts), Amgen, Arena Pharmaceuticals, AstraZeneca, Biogen, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Eli Lilly, Ferring, Fresenius Kabi, Genentech, Gilead Sciences, Glaxo-Smith Kline, JAMP Bio, Janssen, Merck, Mylan, Novartis, Oppilan Pharma, Organon, Pandion Pharma, Pfizer, Progenity, Protagonist Therapeutics, Roche, SandozShire, Sublimity Therapeutics, Takeda Pharmaceuticals, Ventyx.
Stefan Schreiber, Consultancy fees: AbbVie, Arena, Biogen, Bristol-Myers Squibb, Celgene, Celltrion, Falk, Fresenius, Gilead Sciences, IMAB, Janssen, Eli Lilly and Company, Hikma, MSD, Mylan, Pfizer Inc, Protagonist, Provention Bio, Takeda, Ventyx, and Theravance.
Simon Travis, Grants: AbbVie, ECCO, Eli Lilly and Company, Helmsley Trust International, International Organization for the Study of Inflammatory Bowel Disease, Norman Collision Foundation, Pfizer, Takeda, UCB Pharma, UKIERI, Vifor Pharma; Honoraria: Ferring Pharmaceuticals, Takeda, Eli Lilly and Company, Bristol Myers Squibb, Janssen, Violicom; Travel grants: AbbVie, Eli Lilly and Company, Janssen, Pfizer, Takeda, Ferring Pharmaceuticals, Amgen, Biogen; Consultancy fees: AbbVie, Allergan, Amgen, Apexian, Bioclinica, Biogen, Bristol Myers Squibb, ChemoCentryx, Cosmo Pharmaceuticals, Endpoint Health, Eli Lilly and Company, Enterome, Equillium, Ferring Pharmaceuticals, Genentech/Roche, GlaxoSmithKline, Immunocore, Immunometabolism, Janssen, Merck, Merck Sharp & Dohme, Mestag Therapeutics, Novartis, Pfizer, Protagonist Therapeutics, Proximagen, Receptos, Roche, Sanofi, Satisfai Health, Sensyne Health, Sorriso Pharmaceuticals, Takeda, UCB Pharma, VHsquared, Vifor Pharma.
Marla C. Dubinsky, Consultancy fees: AbbVie, Abivax, Astra Zeneca, Boehringer Ingelheim International GmbH, Bristol Myers Squibb, Celgene Corporation, Eli Lilly and Company, F. Hoffman-La Roche Ltd, Genentech Inc., Gilead Sciences, Janssen, LLC, Merck, Pfizer, Prometheus Biosciences, Takeda Pharmaceuticals USA, Inc., and UCB; Stock options: TrellusHealth Inc.; Research support: AbbVie, Janssen, LLC, Pfizer, and Prometheus Labsa; and licensing fees: Takeda.
Toshifumi Hibi, Personal fees: Abbvie GK, EA Pharma, Janssen Pharmaceutical K.K, JIMRO, Mitsubishi-Tanabe, Mochida Pharmaceutical, Pfizer Japan, Sandoz K.K, Takeda Pharmaceutical, Zeria Pharmaceutical, Celltrion Healthcare Japan, Eli Lilly Japan, Gilead Sciences, Miyarisan Pharmaceutical, Alfresa Pharma Corporation, Kyorin Pharmaceutical; Grants: JIMRO, Mochida Pharmaceutical, Zeria Pharmaceutical, Miyarisan Pharmaceutical, Alfresa Pharma Corporation, and Kyorin Pharmaceutical.
Christian Atkinson, Employment: Adelphi Real World.
David Rubin, Grant support: Takeda; Consultant: Abbvie, Altrubio, Apex, Avalo Therapeutics, Bristol-Myers Squibb, Buhlmann Diagnostics Corp, Celgene, Connect BioPharma, Intouch Group, Iterative Health, Janssen Pharmaceuticals, Lilly, Pfizer, Samsung Neurologica, and Takeda. Board of Trustees: Crohn’s & Colitis Foundation; Board of Directors: Cornerstones Health.
Alison Potts Bleakman, Theresa Hunter Gibble, Tommaso Panni, Cem Kayhan, Eoin J. Flynn, and Angelo D. Favia are employees and stockholders of Eli Lilly and Company.

Introduction

Late onset neonatal sepsis (LOS) is a leading cause of mortality in preterm infants, affecting up to 15% of preterm neonates and a mortality rate of ~15%¹. Clinical symptoms of LOS are nonspecific and the diagnosis is therefore often established in an advanced stage of infection, negatively impacting prognosis, illustrating the need for novel preclinical biomarkers². This study aimed to identify predictive biomarkers for LOS in preterm infants using faecal metagenomics and targeted metabolomics. Functional implication of identified biomarker on preterm neonatal barrier function was assessed in foetal intestinal organoid models.

Aims & Methods

In a Dutch multicentre study, including 9 neonatal intensive care units, faecal samples and clinical data were collected daily from birth to onset of S. aureus and E. coli LOS in preterm neonates (<30wks gestation) and matched to healthy controls by gestational and postnatal age, mode of birth, antibiotics exposure and birth centre (n=10 E. coli, n=10 S. aureus). 16S sequencing and shotgun metagenomics was performed to characterise fecal microbiota, as well as targeted HPLC MS/MS to determine tryptophan (Trp) metabolites of serotonin, kynurenine and indole pathway up to 7 days before LOS diagnosis.
In vitro effect of Indole-3-aldehyde (I3A) and heat inactivated culture medium (CDM) of E. coli clinical isolates was assessed human foetal intestinal organoid models. Tight junction and AhR expression was determined in 3D organoids by RTqPCR on TJP-2, CLDN-1,-4,-10, OCLN, AHR and CYP1A1. Exposed 2D organoid monolayers were assessed by longitudinal trans epithelial electrical resistance (TEER) and wound healing assay³. Consecutively, effect of I3A on intestinal barrier function during infection with E. coli clinical isolate was assessed by barrier translocation and longitudinal TEER⁴.

Results

The metagenomic faecal relative abundance of E.coli was significantly higher 4-5 days before onset of clinical symptoms of E.coli LOS, compared to matched controls (P=0.045, 95% CI cont. -0.47-1.72%, LOS = 17.21-101.3%). In contrast, no significant difference in abundance of faecal S. aureus was detected in neonates prior to S. aureus LOS. Further analysis indicated altered genetic abundance of Trp related genes in the E.coli LOS metagenome 4-5 days prior to LOS diagnosis. Of detected metabolites, Tryptamine was significantly decreased (adj.P=0.03), Indole (adj.P=0.002), Indole-3-sulfate (adj.P=0.02) and I3A (adj.P <0.0001) were significantly elevated prior to E. coli LOS.
3D fetal intestinal organoid experiments showed significantly increased CYP1A1 expression in organoids exposed to I3A at concentration measured in faeces (100µM). A trend of increased tight junction expression was observed, indicating potential effect of I3A on intestinal barrier prior to LOS. TEER measurements showed increased barrier resistance and decreased proliferation of I3A exposed 2D organoids. Additionally, infection of I3A exposed organoid monolayers with clinically isolated E. coli showed indication of more rapid and increased barrier disruption.

Conclusion

This research indicates an increase in faecal relative abundance of E. coli, but not of S. aureus prior to LOS onset, and a functional difference mediated by Trp metabolism and microbially produced I3A. In vitro analysis of I3A effect on barrier integrity, proliferation and E. coli infection suggest altered barrier function preceding LOS. These results indicate relevance of I3A as novel preclinical biomarker as well as a functional metabolite potentially influencing the preterm intestinal barrier.

References

1. Granger C, Dermyshi E, Roberts E, Beck LC, Embleton N, Berrington J. Necrotising enterocolitis, late-onset sepsis and mortality after routine probiotic introduction in the UK. Arch Dis Child Fetal Neonatal Ed. 2022 Jul;107(4):352-358. doi: 10.1136/archdischild-2021-322252. Epub 2021 Aug 25. PMID: 34433587.
2. Kurul S, Fiebig K, Flint RB, Reiss IKM, Küster H, Simons SHP, Voller S, Taal HR. Knowledge gaps in late-onset neonatal sepsis in preterm neonates: a roadmap for future research. Pediatr Res. 2022 Jan;91(2):368-379. doi: 10.1038/s41390-021-01721-1. Epub 2021 Sep 8. PMID: 34497356.
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