Semester

Fall

Date of Graduation

2021

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Gordon Meares

Committee Member

Candice Brown

Committee Member

Werner Geldenhuys

Committee Member

Rosana Schafer

Committee Member

James Simpkins

Abstract

Sepsis is a disorder that targets the microcirculation with significant effects at blood-tissue barriers such as the blood-brain barrier (BBB) and gut-vascular barrier (GVB). Intestinal barrier dysfunction is thought to be one of the most important contributors to multi-organ dysfunction in sepsis. As a common site of infection, the impaired gut allows the dissemination of bacteria, which exacerbates the disease pathophysiology, and can lead to long-term morbidity and mortality. The mechanisms that influence altered barrier permeability in the GVB in sepsis and other inflammatory disorders are not well understood. Tissue-nonspecific alkaline phosphatase (TNAP) enzyme activity, which has been shown to be highly expressed on brain microvascular endothelial cells (BMECs), is a viable therapeutic target. TNAP activity is recognized as generally anti-inflammatory in sepsis-associated acute kidney injury (AKI) through mechanisms not well understood, and its role in health and disease at various tissue barriers has yet to be explored. One critical barrier to studying TNAP has been a lack of specific pharmacological and genetic tools. Therefore, we first sought to examine the effects of pharmacological and genetic manipulation of TNAP in experimental sepsis using novel techniques. Our central hypothesis was that pharmacological or genetic depletion of TNAP enzyme activity would result in exaggerated barrier permeability, increased inflammation, and impaired gut homeostasis in experimental sepsis. Initially, a genetic mouse model with over-expression of TNAP on endothelial cells (i.e., VE-cKO) revealed that TNAP-overexpression resulted in better clinical scores and increased survival at 48-hours post-sepsis. Additionally, mice with genetic deletion of TNAP on endothelial cells (i.e., VE-cKO) were used to determine a role for TNAP in GVB homeostasis. We found that VE-cKO septic mice display increased bacterial burden and increased parameters of gut dysbiosis at 8 days post-CLP, including a decreased Firmicutes:Bacteroidetes (F:B) ratio, decreased relative antimicrobial expression, and, at 3 days post-CLP, decreased levels of short-chain fatty acids (SCFAs). At 24 hours post-CLP, permeability to molecules ranging from 625 Da to 69 kDa is increased in VE-cKO ileum compared to Alplfl/fl littermate controls. The differences in permeability between intestinal segments demonstrates that TNAP’s enzyme activity may impact each region using different mechanisms. Ultimately, our results have shown that endothelial TNAP is protective during sepsis via two potential mechanisms: (1) decreasing bacterial burden and gut dysbiosis by 8 days post-CLP, and (2) decreasing intestinal permeability at 24 hours post-CLP. This work demonstrates a protective role for endothelial TNAP against GVB dysfunction which may be applicable to a number of inflammatory diseases.

Embargo Reason

Publication Pending

Download

Share

COinS