Date of Award
Master of Science (MS)
Microbiology and Immunology
Introduction: Burn injury remains a prominent clinical problem. Patients suffering from burns often succumb to secondary infectious complications leading to sepsis and widespread tissue injury ending in Multiple Organ Dysfunction. However, the mechanism behind the onset of these extraneous symptoms following burn injury is not fully understood. The integrity of the gut barrier is of critical importance as it harbors the largest bacterial reservoir in the body. Following burn injury, we observed a breakdown of the gut barrier resulting in increased gut leakiness and bacterial translocation. Under homeostatic conditions, heat shock proteins (HSPs) stabilize tight junction proteins. Particularly, HSP72 is shown to play a role in stabilizing tight junctional complexes of the blood brain barrier. Since tight junction proteins are responsible for maintaining gut barrier integrity, we examined the effect of burn injury on the heat shock response via HSPs and the claudin and occludin family of tight junction proteins.
Hypothesis: Burn injury suppresses heat shock protein expression, which leads to alterations in tight junction proteins contributing to increased intestinal permeability and bacterial translocation after injury.
Methods: Male C57BL/6 (10-12 week old) mice were anesthetized and administered an ~20% total body surface area dorsal scald burn using 85⁰C water for ~7-9 seconds and resuscitated with 1mL normal saline. The small and large intestines were harvested 4 hours, one, and three days following burn injury and processed for isolation of intestinal epithelial cells (IEC). IEC HSP25, 72, and 90 mRNA expression was examined by q-RTPCR along with claudin-4, claudin-8, and occludin. Protein levels of HSP25 and 72 and then claudin-4 and claudin-8 were examined by ELISA and immunofluorescence staining respectively.
Results: We found significant decreases in HSP25, 72, and 90 expression (25%, 85%, and 51% respectively) in IECs harvested from the small intestine one day post burn injury compared to sham controls. This accompanied a significant decrease in claudin-4 and -8 expression (54% and 49%) in small intestine IECs of burn animals one day after burn compared to sham controls. At the protein level, burn injury resulted in a decrease of 85% in HSP25 four hours after burn injury and significant decreases in HSP72 four (51%) and three days (46%) in small intestine IECs compared to sham controls. Upon assessment of immunofluorescence staining of TJ proteins one day after burn injury, we observed significant decreases in claudin-4 levels, but claudin-8 immunofluorescence staining is still in progress. To discourage biases, we are currently expanding our immunofluorescence staining of tight junction proteins to include more tissue sections.
Analysis of expression HSP25, 72, and 90 in large intestine IECS one day after burn injury resulted in a significant decrease in all three HSPs 4 hours of 44%, 79%, and 33% respectively after injury compared to sham. This significant decrease in HSP72 expression (81%) persisted to one day post burn injury. No significant changes in the expression of the tight junction proteins claudin-4, -8 or occludin were observed large intestine IECs. Protein levels of HSP25 mimicked mRNA expression with significant decreases in HSP25 (71%) in large intestine IECs four hours after burn injury. Yet, HSP72 levels post burn injury were significantly down in large intestine IECs on both one (63%) and three days (46%) compared to sham controls. Immunofluorescence staining of large intestine tissue sections for claudin-4 and claudin-8 is being done at the present time.
We attempted to induce HSPs in our murine model of burn injury with the hypothesis that if HSPs stabilize TJ proteins, up-regulating HSPs after burn injury could potentially restore the decrease in TJ proteins we observe and bring back normal barrier function. We examined whether treatment of mice with mesalamine (5-ASA), an agent used in vitro to up-regulate HSPs, influences the expression of HSPs after burn injury. We found that mice treated with 100mg/kg 5-ASA at time of resuscitation, did not up-regulate HSPs in either small or large intestine IECs, but it did significantly restore claudin-4 and trends toward restoration of occludin expression in small intestine IECs one day after burn injury. Furthermore, 5-ASA significantly reduced the small intestine IEC pro-inflammatory cytokines IL-18 (62% increase one day after burn and back to sham levels with 5-ASA) and IL-6 (34% increase back to sham levels one day post burn) in IECs after burn injury. There was a trend towards a reduction in IL-6 and the pro-inflammatory cytokine KC in large intestine IECs with 5-ASA treatment one day following burn injury. Interestingly, there was a significant decrease (63%) in the pro-inflammatory chemokine MCP-1 following 5-ASA treatment one day after burn injury in large intestine IECs.
Additionally, we found that 5-ASA reduces intestinal permeability back to that of sham controls one day after burn injury. Treatment with 5-ASA also allows for more efficient intestinal transit one day after burn injury.
Conclusion: Our data suggest that burn results in significant decreases in HSPs in both small and large intestinal epithelial cells, which correlates with a significant decreases in levels of the tight junction proteins, claudin 4,8 and occludin. Although 5-ASA did not up-regulate HSP expression after burn injury, it did normalize claudin 4 and 8 expression and reduced IL-18 and IL-6 levels after burn injury in the small intestine. Levels of IL-6 and KC after burn injury in large intestine IECs are trending towards that of sham with 5-ASA treatment, but levels of MCP-1 did normalize as a result of treatment. 5-ASA also allowed for a complete reduction in the increased intestinal permeability we observe after burn injury and a partial restoration of intestinal peristalsis one day after injury. Together, these findings suggest that 5-ASA-mediated protective effects on the gut barrier integrity appears to be independent of HSPs.
Cannon, Abigail Rhea, "Role of Heat Shock Proteins in Maintenance of the Gut Barrier Following Burn Injury" (2015). Master's Theses. 3127.
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Copyright © 2015 Abigail Rhea Cannon