The Pruett lab is involved in research on the mechanisms of lethality in systemic infections known as sepsis. Sepsis is the 10th leading cause of death in the U.S., and mortality rates have improved little during the last 40 years. The causes of death in sepsis are related to systemic inflammatory response syndrome, which can decrease blood pressure and cause shock and inappropriate blood clotting leading to organ failure. However, the molecular basis and regulation of these events is not fully understood. In collaboration with Drs. Bindu Nanduri and Minny Bhatty, we are taking a systems biology approach in an attempt to determine sets of molecules that might be targeted to diminish the lethal effects of sepsis and to understand the time dependence and programming of lethal outcomes.
The Pruett lab also studies the effects of drugs and chemicals on the immune system, particularly innate immunity mediated through toll-like receptors (TLRs). In particular, we have grants from the National Institutes of Health (currently in unfunded extensions) to study the effects of ethanol in a binge drinking model and sodium methyldithiocarbamate, the 3rd most abundantly used pesticide in the U.S. Both of these compounds have anti-inflammatory effects and decrease resistance to sepsis, but the mechanisms of action seem to be quite different. In collaboration with Ed Lewis and Bindu Nanduri, we are using techniques such as circular dichroism to directly measure the effects of ethanol on conformational changes in the TLR3 protein induced by its ligand. We found distinctive evidence for major effects, indicating for the first time that at least some of the effects of ethanol on the immune system are mediated directly by ethanol, which prevents ligand-induced conformational change that is necessary for cellular signaling and subsequent immunological responses.
We are also involved in collaborating with Dr. Changhe Yuan in the Department of Computer Science and Engineering to conduct research on the quantitative prediction of resistance to infection on the basis of changes in individual immunological parameters. At present, we know that elimination or near elimination of one or more immunological parameters (e.g., Th depletion in AIDS) can cause clinically important decreases in resistance to infection. However, most immunosuppressive agents are not highly selective but decrease several immunological functions partially. There is currently no way to predict the effects this will have on resistance to infection. We are using machine learning programs to identify patterns in the National Toxicology Program’s immunotoxicology database that will allow prediction of host resistance.
Mice treated with ethanol in a model for binge drinking in humans have decreased activation of NF-kB, a transcription factor that is critical for inflammatory responses. The mice in both rows in the photograph were anesthetized and treated with an inflammatory stimulus; mice in the bottom row were also treated with ethanol. These mice have a reporter gene that causes emission of light when NF-kB is activated, and light emission is indicated by the colors shown. It is interesting that light emission in the region occupied by the liver (shown in the mouse farthest to the right in each group) is almost completely eliminated in ethanol treated mice, which is consistent with the higher concentration of ethanol and its metabolites in the liver than in other organs.
Cells were obtained by peritoneal lavage from mice in an Escherichia coli model of sepsis. In 2A, cells from untreated mice are shown; 2B, cells from mice harvested 18 hr after E. coli infection; 2C, cells from mice 18 hours after E. coli infection preceded by ethanol administration. Ethanol substantially decreased the clearance of bacteria.
Microarray analysis was performed using peritoneal cells from mice treated with sodium methyldithiocarbamate, the third most abundantly used pesticide in the U.S. The results indicate that several genes are associated with protection from oxidative stress.
Microarray analysis was used to demonstrate that ethanol inhibits the induction of innate immunity by poly I:C (an artificial analog of double stranded viral RNA) by inhibiting expression of genes associated with an amplification loop. Inhibition of anti-viral immunity in this system is consistent with decreased resistance to viruses documented in humans who consume excessive amounts of ethanol.
Exogenous corticosterone at concentrations designed to mimic concentrations observed during stress responses affects the expression of a number of different cellular markers in the spleen and thymus. A, B: Spleen from a control rat (H&E and immunohistochemistry [IHC] for CD79a, 100 X). C, D: Spleen from a treated rat with decreased size and cellularity of the B-cell areas, including marginal zone (mz) (H&E and IHC for CD79a). E: Thymus from a treated rat, showing increased numbers of macrophages containing apoptotic cell debris (H&E, 200 X). F: Thymus from a control rat, showing the normal density of apoptotic cells (IHC for cleaved caspase-3, 200 X). G: Thymus from the same rat as in E, showing markedly increased positivity for cleaved caspase-3 (IHC for cleaved caspase-3, 200 X). H: Thymus from a control rat, showing the normal size and cellularity of cortex and medulla (H&E, 100 X). I: Thymus from a treated rat, showing marked thinning and depletion of lymphocytes in the cortical areas (H&E, 100 X).