(T1430-10-65) Novel Biomarkers for Inflammatory Bowel Disease

Purpose: Inflammatory bowel disease (IBD) is a complex and chronic disorder characterized by inflammation in gastrointestinal tract (GIT) and affects nearly 1.8 million people across North America [1]. The exact cause is unknown, however multiple factors including gut dysbiosis, proinflammatory foods, and stress have been associated with IBD pathogenesis [2]. Diagnosis of IBD involves invasive procedures like colonoscopy, ileoscopy or biopsy to visually confirm the signs of inflammation. Non-invasive inflammatory biomarkers like C-reactive protein and fecal calprotectin have limitations of being non-specific and their levels can vary due to inflammation outside of GIT [3]. Thus, monitoring disease progression and drug response remains challenging. In this study, we aim to identify novel biomarkers related to IBD progression. Short Chain Fatty Acids (SCFAs), mainly acetate, propionate and butyrate are produced in intestinal lumen by gut bacteria through fermentation of carbohydrates and dietary fiber. SCFAs activate genes coding for epithelial tight junctions in intestinal mucosa to maintain epithelial barrier integrity. SCFAs also regulate inflammation by inducing anti-inflammatory cytokine IL-10 [4-6]. Eicosanoids are pro-inflammatory mediators which also play important role in maintaining a healthy mucosal barrier layer. Prostaglandins (PGs) are cytoprotective in nature and increase secretion in colon to speed-up toxin removal, thereby contributing to diarrhea in IBD [7]. Changes in the concentration of SCFAs and eicosanoids due to gut dysbiosis can be a potential cause of inflammation of the mucosal layer in GIT and lead to systemic symptoms. We hypothesize that gut dysbiosis will change the concentrations of SCFAs and eicosanoids in IBD and hence, can be used as non-invasive biomarkers for mucosal barrier layer integrity, disease progression and drug response. We evaluated serum, colon and stool SCFA and eicosanoid concentrations in a mouse model of IBD to assess disease progression and therapeutic intervention.

Methods: Mice were grouped as healthy, colitis, and colitis with treatment (TX) (n=5 per group). IBD was induced in control and TX group by administrating 100 µL Citrobacter rodentium (5x10⁸ CFU) by oral gavage. The TX group was administered with 30 mg/kg polymeric hydroxychloroquine (PCQ) per day until Day 8 when mice were sacrificed and serum, feces and colon were harvested. Using previously developed and validated UPLC-MS/MS assays, serum and fecal SCFAs and eicosanoids were quantitated. The SCFA assay quantitated acetic acid, propionic acid, butyric acid, 2-methyl butyric acid, isobutyric acid, valeric acid, capric acid and isocapric acid over the range of 0.01-100 µM [8]. A total of 66 different eicosanoid metabolites were assessed, with the assay linear over the range of 0.01-1 ng/mL [9]. MetaboAnalyst 5.0 was used for identifying statistically significant differences in SCFA and eicosanoid concentrations as per healthy, infected and TX groups.

Results: A total of 15 mice (5 in each arm) were evaluated. Induction of colitis in mice resulted in increased serum concentrations of SCFAs and eicosanoids compared to healthy controls. Treatment intervention with PCQ resulted in decrease in SCFA and eicosanoid concentrations back to baseline level (Figure 1). Specifically, serum concentrations of isobutyric acid and acetic acid (SCFAs) and PGE1, PGF1 and 8-HEPE (eicosanoids) increased significantly compared to healthy mice on induction of IBD (Figure 2). Serum concentrations of 22 eicosanoids were significantly higher in colitis mice (red class) compared to healthy mice (green class) (Figure 2). The comparison for 22 serum eicosanoids concentrations between infected mice (red class) and post-treatment (green class) with PCQ are shown in the heatmap (Figure 3.) Eicosanoid concentrations were significantly lower in mice receiving treatment with PCQ compared to untreated colitis mice (Figure 3). Following treatment, isobutyric acid and acetic acid (SCFAs) and PGE1, PGF1 and 8-HEPE levels decreased significantly in serum and were similar to baseline values observed in control mice.

Conclusion: Infectious colitis resulted in increased concentrations of pro-inflammatory lipid mediators eicosanoids and SCFAs in serum. Clear distinction between serum SCFA and eicosanoid concentrations were observed between healthy and colitis groups, as well as between colitis and TX mice. The differences observed in serum concentrations of SCFAs and eicosanoids with respect to diseased conditions will be useful in understanding the etiology, potential pathways and novel drug targets associated with inflammation in IBD. Quantitation of these novel biomarkers may also help monitor progression of IBD, response to therapy and risk of relapse.

References: 1. Crohn's and Ulcerative disease: know the difference? 11/14/2022 [cited 2023 01/23/2023]; Available from: https://www.webmd.com/ibd-crohns-disease/crohns-disease/crohns-colitis-difference.
2. Adolph, T.E., et al., The metabolic nature of inflammatory bowel diseases. Nature Reviews Gastroenterology & Hepatology, 2022. 19(12): p. 753-767.
3. How is IBD diagnosed? ; Available from: https://www.crohnscolitisfoundation.org/what-is-ibd/diagnosing-ibd.
4. Parada Venegas, D., et al., Short Chain Fatty Acids (SCFAs)-Mediated Gut Epithelial and Immune Regulation and Its Relevance for Inflammatory Bowel Diseases. Frontiers in Immunology, 2019. 10.
5. Parada Venegas, D., et al., Short Chain Fatty Acids (SCFAs)-Mediated Gut Epithelial and Immune Regulation and Its Relevance for Inflammatory Bowel Diseases. Front Immunol, 2019. 10: p. 277.
6. Salomon, J., et al., Dysbiosis and Intestinal Barrier Dysfunction in Pediatric Congenital Heart Disease Is Exacerbated Following Cardiopulmonary Bypass. JACC Basic Transl Sci, 2021. 6(4): p. 311-327.
7. Wallace, J.L., Eicosanoids in the gastrointestinal tract. Br J Pharmacol, 2019. 176(8): p. 1000-1008.
8. Trivedi, N., et al. A Concise Review of Liquid Chromatography-Mass Spectrometry-Based Quantification Methods for Short Chain Fatty Acids as Endogenous Biomarkers. International Journal of Molecular Sciences, 2022. 23, DOI: 10.3390/ijms232113486.
9. Chhonker, Y.S., et al., Simultaneous Quantitation of Lipid Biomarkers for Inflammatory Bowel Disease Using LC-MS/MS. Metabolites, 2021. 11(2).

Acknowledgements:This work was partially supported by grant 5R01DK124095 to DO and ABS. The authors declare no conflict of interest.

Figure 1: PCA plot showing comparison of serum concentrations between infected (red) and post-treatment (green) group for SCFAs (left panel) and eicosanoids (right panel)

Figure 2: Heatmap of SCFAs (left panel) and eicosanoids (right panel) analyzed by LC-MS/MS. Statistically significant difference (p < 0.05) was observed in SCFAs isobutyric acid and acetic acid concentrations between healthy (green panel) and infected mice (red panel). 22 eicosanoids (11-HDHA, 10-HDHA, 12-HEPE, 8-HEPE, 12-HETE, 8-HETE, Docosahexaenoic acid, 15-HEDE, 11-HEDE, PGF-1, 15-HETE, 12-oxo-ETE, 13(S)-HOTrE, LTB4, PGJ2, 13,14-DiOH-15-Keto, PGE1, 7-HDHA, 15-HEPE, 11-HEPE, 4-HDHA, 6-KetoPGF1) were significantly increased during infection compared to healthy mice.

Figure 3: Heatmap of eicosanoids analyzed by LC-MS/MS. Statistically significant difference (p < 0.05) was observed for 22 eicosanoids (PGE1, PGF1, 8-HEPE, 12-HEPE, 13(S)-HOTrE, 13,14-DiOH-15-Keto, 7-HDHA, 11-HDHA, 10-HDHA, 15-OxoETE, 4-HDHA, 9-OxoEDE, 11-HEPE, 15-HETE, 5-OxoETE, 11-HETE, 15-HEDE, 11-HEDE, Docosahexaenoic acid, 12-HETE, 8-HETE, 8-HDHA) between PCQ-treated (green panel) and infected mice (red panel).