Schaefers, Matthew Michael2012-07-192012-07-192011-01https://hdl.handle.net/11299/128290University of Minnesota Ph.D. dissertation. January 2011. Major: Experimental & Clinical Pharmacology. Advisor: Marnie L. Peterson. 1 computer file (PDF); xii, 203 pages, appendix P. 198-203.Staphylococcus aureus is a significant human pathogen that causes a wide range of diseases from skin and soft tissue infections, pneumonia, osteomyelitis, to toxic shock syndrome (TSS). S. aureus initiates infections at skin and mucosal surfaces by producing a multitude of virulence factors, including superantigens (SAgs). SAgs are exotoxins that enhance the ability of S. aureus to cause infection by dysregulation of the host’s immune system. Numerous Staphylococcal SAgs have been identified including the staphylococcal enterotoxins (SEA- SEU) and Toxic Shock Syndrome Toxin-1 (TSST-1). SAgs cause a wide range of diseases such as food-poisoning, atopic dermatitis, and TSS by relatively unknown mechanisms of action on mucosal surfaces. The ability of SAgs to non-specifically cross-link T-cells and antigen presenting cells (APC), which results in a cytokine storm and toxic shock syndrome have been studied extensively. However, interactions of SAgs with mucosal surfaces remain poorly understood. TSST-1, the most common cause of menstrual TSS (mTSS), induces proinflammatory cytokines from vaginal epithelial cells These proinflammatory effects are hypothesized to contribute to the progression of mTSS by disrupting the permeability barrier of the vaginal mucosa directly and by causing a migration of neutrophils, macrophages and lymphocytes to the site of infection. The aims of this thesis were to 1) characterize TSST-1’s mechanism of action on human vaginal epithelial cells (HVEC), leading to the induction of proinflammatory cytokines, including the identification of the TSST-1 HVEC receptor and residues of TSST-1 critical for HVEC interactions, and to 2) evaluate curcumin, an anti-inflammatory compound, as an anti-TSS mucosal therapeutic. HVEC were exposed to TSST-1, and cytokine expression levels were determined by real-time reverse transcription polymerase chain reaction (PCR) and multiplex cytokine assay. IL-6, IL-8, MIP-3α, and TNF- α transcripts were up-regulated (1.5- to 12-fold), with corresponding increases in protein expression. TSST-1 activated an NF-κB luciferase reporter in HVEC, suggesting that NF-κB is a downstream target of TSST-1 signaling. Previous studies have suggested that major histocompatibility complex class II molecules (MHC II) could serve as the epithelial receptors for SAgs, and when activated, induce cytokines. Flow cytometry and Western blotting of HVEC did not detect MHC II molecules. These data suggest that MHC II is not the HVEC surface receptor responsible for induction of cytokines by TSST-1 and another undefined SAg epithelial receptor present on the surface of HVEC is implicated. A dodecapeptide region (TSST-1 amino acids F119 to D130) that is relatively conserved among SAgs has been implicated in SAg penetration of the epithelium. Single amino acid mutations were constructed in TSST-1 amino acids D120 to D130. All mutants maintained superantigenicity similar to wild type toxin. TSST-1 mutants induced IL-8 from HVEC; however, three toxin mutants (S127A, T128A, and D130A) induced lower levels of IL-8 compared to wild type TSST-1. These toxin mutants were administered intravenously to rabbits and all three were 100% lethal. When administered vaginally to rabbits, D130A toxin was nonlethal, while wild type TSST-1 was 100% lethal. Residue D130 may contribute to toxin binding to an epithelial receptor that allows it to penetrate the vaginal mucosa, induce cytokines/chemokines from epithelial cells, and cause TSS. CD40 was explored as a candidate HVEC receptor for TSST-1, based on a previous study which described synergistic activity of CD40 with MHC II for TSST-1 to induce cytokines from monocytes. HVEC (by flow cytometry) and ex vivo human ectocervical tissue (by immunohistochemistry) both expressed CD40. The biological role of CD40 in TSST-1-induced cytokine production was evaluated by reducing CD40 expression in HVEC by stable transfection with small hairpin RNA (shRNA) plasmids by 60% on the protein level. Surprisingly, CD40 shRNA-expressing HVEC produced more IL-8 in response to TSST-1 compared to irrelevant shRNA expressing HVEC. TSST-1 also bound to HVEC expressing shRNA more than HVEC expressing irrelevant shRNA. These data suggest that CD40 is not the HVEC receptor for TSST-1 that is responsible for induction of proinflammatory cytokines. Curcumin, a component of the spice turmeric, is a compound that has been used in traditional medicine therapies for 4,000 years. Curcumin was evaluated as a potential anti-TSST-1 agent by targeting the host mucosal response to TSST-1 and S. aureus. Curcumin inhibited S. aureus exoprotein- or live S. aureus-induced IL-8 production in an ex vivo porcine vaginal tissue model. The importance of TSST-1-induced inflammation of the vaginal mucosa in TSS disease progression was tested by using curcumin to prevent TSS in a rabbit vaginal model. Curcumin co-administrated with TSST-1 intravaginally significantly reduced lethality of TSST-1 by 60% (5 of 8 rabbits survived whereas 0 of 8 rabbits survived in TSST-1 controls, p <0.05). In addition, TNF-α was undetectable from serum or vaginal tissue of curcumin-treated rabbits that survived. These data demonstrate the importance of local inflammation in the progression of TSS, and curcumin as a potential anti-TSS agent. These studies describe the importance of TSST-1 interactions with vaginal epithelial cells in disease progression to TSS. The mechanism of TSST-1 activation of proinflammatory cytokines was determined to be through the NF-κB pathway with TSST-1 amino acid D130 being critical for induction of cytokines from epithelial cells. However, the NF-κB pathway was not induced by TSST-1 binding to MHC II molecules or CD40 suggesting the role of an unidentified epithelial receptor. These studies also determined that curcumin inhibits S. aureus exoprotein-induced cytokine response in ex vivo vaginal porcine tissue; and when administered intravaginally, curcumin partially prevents TSS in a rabbit model of TSS, demonstrating the importance of local inflammation caused by TSST-1 in progression of TSS. These studies were conducted to develop novel therapies for the prevention and treatment of superantigen-mediated diseases.en-USCurcuminCytokinesNF-kappa BStaphylococcus aureusTSST-1Vaginal EpithelialExperimental & Clinical PharmacologyThesis or Dissertation