Multiple Sclerosis is a disease characterized by inflammation and demyelination in the central nervous system. Experimental Autoimmune Encephalomyelitis (EAE) is a mouse model used to study MS. Previous studies have shown that EAE can be induced by helper T cells of both the Th17 (IL-17 producing) and Th1 (IFNg-producing) lineages. In classic EAE induced by Th1 cells, inflammation is observed predominantly in the spinal cord, however in our model of atypical EAE, induced by transfer of Th17-skewed cells, inflammation is observed in both the brain and spinal cord. This atypical model is closer to the disease course of MS in humans, because only rarely do MS patients have spinal cord inflammation without accompanying brain inflammation. Using mice that lack IL-17RA, a protein required for IL-17 signal transduction, we can study the mechanism of IL-17-induced inflammation in the brain versus the spinal cord. In our model we have shown that IL-17 promotes neutrophil recruitment to the brain, but is not required for neutrophil recruitment to the spinal cord. The presence of neutrophils in the spinal cord in IL-17 receptor knockout mice suggests some other mechanism must be used to recruit neutrophils in the spinal cord. Using RT-PCR we have been looking at the role of other factors (IFN-g, CXCL5, CXCL2, CXCL1, GMCFS, and IL-1b) in recruiting neutrophils in spinal cord independent of IL-17.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes gastroenteritis. Components of the outer and inner membrane, characteristic of Gram-negative bacteria, contribute to Salmonella’s mechanisms to avoid detection and killing by the host immune system. The anionic bacterial cell surface is disrupted by cationic antimicrobial proteins (CAMP) that increase outer membrane (OM) permeability and access to the inner membrane. To resist CAMP killing Salmonella activates the PhoPQ two-component system, which is essential for pathogenesis. Because many PhoPQ-regulated enzymes are dispensable during infection, we conducted a forward genetic screen that identified new components of the PhoPQ regulon involved in maintaining the OM permeability barrier. Sequencing revealed mutations in genes encoding a variety of membrane proteins including transporters, phospholipid and lipopolysaccharide biosynthetic enzymes and efflux pumps, as well as regulatory proteins. Amongst the results, yejM mutants expressed the highest levels of membrane damage. YejM is an inner membrane protein with a periplasmic enzymatic domain that is critically involved in maintaining the OM permeability barrier. Structural alignments suggest that YejM is a member of the alkaline phosphatase superfamily. Attempts at full length deletions of yejM were unsuccessful suggesting the transmembrane domain is essential for viability, whereas periplasmic deletion mutants were viable but expressed varied levels of OM damage. Complementation with periplasmic deletion mutants and plasmids carrying the periplasmic domain rescued the OM permeability defect. Using structural models generated by a Protein Homolog/analogy Recognition Engine (PHYRE), we located putative metal binding sites involved in catalysis. To investigate the enzymatic mechanism, specific metal-binding residues were mutated via Polymerase Chain Reaction. We aim to determine if these residues contribute to the OM permeability defect of the yejM mutant Salmonella.

T lymphocytes protect against the universe of pathogens by generating a diverse repertoire of antigen receptors called T cell receptors (TCRs). Each individual T cell expresses TCRs with a single antigen specificity. The induction of self-tolerance is crucial to the health of the individual because it culls the T cell population of those cells that bear self-antigen reactive receptors. Such autoreactive T cells are the driving force behind autoimmune diseases. One mechanism for inducing self-tolerance is TCR revision, by which peripheral T cells replace self-reactive TCRs with newly generated, non-autoreactive TCRs. This replacement results in cell rescue and involves cleaving DNA, which requires implementation of many controls to protect genome stability. Cells undergoing TCR revision appear to be localized in the spleen and lymph nodes inside a confined microenvironment known as the germinal center (GC), and this constrainment may provide a means for controlling this process. Given the rarity of revising T cells, my project focused on potential means for increasing the frequency of cells undergoing TCR revision using as a model system TCR transgenic mice, in which the majority of T cells express TCRs of the same antigen specificity. We hypothesized that expanding the GC T cell compartment would increase the frequency of cells undergoing TCR revision. I carried out injections of lipopolysaccharide, sheep red blood cells, and anti-CD40 antibody into TCR transgenic mice in an effort to induce GC formation. I then analyzed the accumulation of post-revision and revising T cell populations by flow cytometry. Use of any of these agents to increase TCR revision would facilitate the isolation of post-revision and revising T cells and allow further investigation into the mechanistic details of this tolerance process. Our goal is to understand how the challenging balance between having a diverse T cell repertoire and avoiding autoimmunity is maintained.