Effects of ART on Oral Epithelial Cell Biology

Human. immun.odefi~iency' virus (HIV) infects millions of humans annually and is a leading cause of death worldwld~ .. Th~s unique viral pathogen attack~ the immune system and renders its host ineffective in fighting opp~rtUnlStlC .dlsease. In .the. abse~ce of .medlcal therapy, death usually results within 10 years of infection. The introduction of ~omblnatl~n antlretrovlral (ART) drugs has significantly diminished morbidity and mortality. ~nfortunately, ART IS ~ot ~valla~le to most of the globally-infected population, and ART must be taken for the life of. the h~st to maintain their benefits. Despite the benefits, long term use of ART has clearly been assoclat~d with m~ny well documented adverse effects on systemic tissues (e.g., dyslipidemia, mitochondrial dysfunction, exocnne pathology) .. Howe~er, studie~. that address the effects of ART on mucosal epithelial stru~ture, substructu~es and function, which are cntlcal for the homeostatic interaction of the host with its environment are lacking. Ou.rgoal is to el~cidate th.e.effects of ART on.the function of oral soft tissues, focusing on epithelial cell biology. This knowledge ISa r~qul.slte f?r underst~ndlng the long-term implications of ART on the critical importance of the mucosa and epithelium In controlling oral opportunistic infections in HIV infected individuals. The G.ENERA~ HYPteasome pathways, epithelial cell homeostasis, and parameters of Innate Immunity, resulting In deleterious alterations of the oral mucosa and the epithelial and It IS and/or proliferation critical to delineate the effects of ART on oral 1 epithelial cells. The proposed studies are designed Healthoforalmucosaand to provide insight into the cellular alterations abilitytorespond toviralinfections imposed by ART that can influence epithelial Fig. 1. PutativepathwaysforART effectsonoralepithelium. responses to infection by oral pathogens associated with AIDS-related oral complications. To address these issues, we have established a strong collaborative team of clinician scientists who are experts in the fields of infectious disease, oral medicine and oral pathology together with basic research scientists who have extensive understanding of the proteosome and mucosal immunity. Our research team has a competitive edge for studying the long term effects of ART on oral health, since during the past decade our team has investigated molecular and biochemical aspects of oral mucosal epithelium, innate defenses, the relationships of cytokines in oral fluids with oral health and disease, and oral viral-host cell biology. By bringing together these experts, we are well positioned to study the interactions between ART and oral epithelial cells that contribute to the state of oral mucosal health. Our specific aims are: Specific Aim 1. Examine the effect of ART therapy on the proteasome-proteolytic pathway in oral epithelial cells. The hypothesis to be tested is select ART drugs will alter the ubiquitin-proteasome pathw~y consistent with decreased ability of the epithelial cells to maintain normal homeostasis of message and protein degradation and recycling. Proteasome function will be analyzed in oral epithelial cells at three progressive levels: 1) the effects of ART on the individual peptidase activities of the proteasome, 2) the effect of ART on purified human proteasomes, and 3) the effect of ART on the turnover of established intracellular proteasome substrates. Specific Aim 2. Determine if ART alters the innate immune responses of. ?ral epithelial cell~. O.ur hypothesis is that select ART drugs will modify the expression of pattern. rec?gmtlon receptors. on epl~hehal cells as well as the signal transduction from ligand engagement resulting In altered production of Innate imm~ne molecules by the cells and the ability of the epithelial cell to respond to microbial infection. The effects of ART on TLR signaling in oral epithelial cells will be determined by stimulating cultures of oral epith~lial cells with ligands for TLR2, TLR3 and TLR4 and measuring the expression of key pro-in~ammatory chem?klnes and cytokines. To assess whether ART drugs affect intracellular signalin~ p~thways tnggere? by TLR hgands, we will analyze the impact of ART drugs on TLR-induced NF-KB activation. These studies should offer ~e~ insights of the potential impact of ART on innate immune function against different classes of oral opportunistic pathogens. Specific Aim 3. Determine the effects of ART on epithelial cell differentiation. We will test the hypothesis that different ART drugs vary in their ability to influence oral epithelial cell homeostasis by assessing cellular differentiation. In cell monolayers, transforming growth factor beta 1 (TGFj31) will be used to induce differentiation, and the effect of ART on cellular differentiation will be assessed using immunocytochemistry and enzyme linked immunosorbent assay (ELISA). Organotypic raft cultures will be used to examine the anatomic relationships of ART's influence on cellular differentiation. These studies are of great importance because alterations in processes that regulate cellular differentiation could have long term consequences for the health of the oral cavity. Specifically, we anticipate that the findings will provide insight into how human papillomavirus (HPV) utilizes signals to commandeer cellular machinery for dysregulated differentiation and/or proliferation of oral epithelial cells.