An Examination of the Mucosa-Associated Microbiome and Mucosal Defenses in Complicated Versus Uncomplicated Diverticulitis
Restricted (Penn State Only)
- Author:
- Portolese, Austin
- Graduate Program:
- Biomedical Sciences
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- March 15, 2024
- Committee Members:
- Walter Alex Koltun, Thesis Advisor/Co-Advisor
Gregory Steven Yochum, Committee Member
Lisa M Shantz, Program Head/Chair
Prashant Nighot, Committee Member - Keywords:
- Microbiome
Diverticulitis - Abstract:
- Diverticulitis is inflammation of otherwise benign outpouching of the colonic mucosa through the muscular wall, and although it affects almost two million Americans each year, the pathophysiology of this disease is poorly understood. We hypothesized that dysbiosis of the mucosal-associated microbiome contributes to the pathophysiology of diverticulitis. To test this hypothesis, we analyzed the microbiome in surgically resected colonic tissue segments from a cohort of diverticulitis patients. Within this cohort of 100 patients, half the patients had uncomplicated disease, disease marked by multiple bouts of inflammation and symptoms without progression to full thickness perforation or abscess, while the other half had complicated disease, with abscess or perforation warranting surgical intervention. We evaluated 100 patient tissue samples obtained at the time of left-sided colectomy from patients with diverticulitis. We analyzed tissue from the site of disease and adjacent healthy appearing tissue from each patient. From isolated DNA, from full-thickness tissue specimens, we performed 16S rRNA gene sequencing based on the V4 hypervariable region to perform taxonomic identification of the mucosa-associated microbiome. We found an increased relative abundance of sulfur-reducing microbes at the site of disease in patients with complicated diverticulitis compared to uncomplicated diverticulitis. We also identified increased abundances of sulfur-metabolizing microbes at the site of disease in patients with complicated diverticulitis compared to adjacent normal appearing tissue in the same patients. These findings implicate the role of sulfur-reducing microbes and their metabolite hydrogen sulfide in the development of complicated diverticulitis. We then turned our attention to evaluating a bilayer of heavily glycosylated mucin proteins lining the colonic epithelium which form a protective barrier from direct contact with the luminal microbiota. Mucin 2 (MUC2) is the major mucin constituent of this mucus layer in the colon. Because we identified microbial dysbiosis in diverticulitis, we next evaluated whether differences in MUC2 were evident in the colonic segments in a patient cohort. Using a subset of 30 patients from the aforementioned cohort we evaluated MUC2 expression using RT-qPCR and tested for the presence of MUC2 using immunohistochemistry. We identified decreased MUC2 expression at the site of diverticulitis with evidence of a field effect extending to the adjacent normal tissue in complicated diverticulitis. Despite an increased abundance of goblet cells, which secrete MUC2, our gene expression findings were confirmed with an absence of MUC2 at the site of diverticulitis on immunohistochemistry. These findings suggest increased barrier breakdown at the site of diverticulitis and therefore increased interactions between luminal microbes and the underlying mucosa due to partial loss of the mucin bilayer. Together, our findings indicate that microbiome dysbiosis contributes to the pathophysiology of diverticulitis. The microbiome then provides a target for possible future diagnostics and therapeutics. Appropriately identified and selected patients may be candidates for probiotics or other interventions to alter the microbiome and mitigate this disease.