In the intestinal tract, the first line of defense against pathogenic microorganisms is the layer of epithelial cells that line its extensive mucosal surface. These cells provide a physical barrier to infection and play critical roles at mucosal immune sites by detecting and eliminating enteric pathogens, including the human immunodeficiency virus (HIV) and Salmonella enterica. The intestinal mucosa contains, in addition to epithelial cells, an extensive nervous system capable of pre-programmed behavior, and it houses the largest lymphocyte population in the body;these three cell types communicate with each other through molecular signals which modulate inflammation and coordinate mucosal defense responses to infection. Opioid abuse is an important co-factor in host susceptibility to HIV and other mucosal pathogens, but its impact on mucosal host defense is relatively unknown. Opioid drugs may impair neuro-immuno-epithelial interactions at immune inductive and effector sites in the intestine. The proposed experiments will test the general hypotheses that opioids impair the immune responses of epithelial cells and lymphoid cells in the intestinal mucosa through actions mediated by neuronal and extraneuronal opioid receptors, and that opioid neuroimmune signaling is enhanced by mucosal inflammation.
In Specific Aim 1, we will determine and compare the effects of opioids on proinflammatory cytokine release and changes in opioid receptor expression before and after inflammation in porcine intestinal epithelial cells in culture and from explants of Peyer's patch and absorptive mucosae through measurements of secreted cytokines and their mRNA expression. We will also assess opioid actions on intestinal epithelial cell wound healing by electric cell-substrate impedance sensing.
In Specific Aim 2, we will compare the morphological interrelationships between opioid receptors and opioid peptides in epithelial cells, neurons and leukocytes of Peyer's patch and absorptive mucosae by immunocytochemical and in situ hybridization methods. We will also examine opioid effects on an epithelial-neuronal co-culture from porcine intestine through measurements of cytokine release and changes in neuronal excitability evoked by Salmonella infection. Finally, we will characterize changes in the ligand affinity and G protein coupling coupling of specific opioid binding sites in neural membranes from inflamed and uninflamed Peyer's patches by radioligand binding techniques.
In Specific Aim 3, we will characterize delta-opioid receptors mediating chemokine-induced chemotaxis of intestinal T lymphocytes, and determine the effects of opioids on neurally-mediated secretory immunoglobulin-A release from intestinal mucosa explants. The results from this multi-faceted transdisciplinary project will provide a fundamental understanding of how opioid drugs act to alter mucosal defense function. Moreover, they will unveil new drug targets for the modulation of mucosal immune responses to HIV and other gut pathogens as well as oral vaccines offering protection from these microbes.
The goal of the proposed transdisciplinary investigation is to elucidate the mechanisms by which opioid drugs of abuse disrupt the interplay between enteric neurons, epithelial cells and immunocytes at sites of mucosal immunity, which are key targets for infection by enteropathogens like S. typhimurium and the human immunodeficiency virus (HIV). Our experiments will provide new and significant information on the ability of opioids to influence mucosal responses evoked by Salmonella typhimurium, an enteroinvasive zoonotic pathogen and potential HIV vaccine vector. The results that we obtain in this multi-faceted project will advance our understanding of how opioid drugs act to alter mucosal defense function. In addition, they will reveal new drug targets for the modulation of mucosal immune responses to enteric pathogens including HIV, and provide new information contributing to the development of oral vaccines based on bacterial vectors that are designed to confer protection against HIV infection in gut-associated lymphoid tissue, the major body reservoir for this virus.
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