UNIVERSITY OF FLORIDA
Grant: $219,750 - National Institutes of Health - Jul. 17, 2009
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Award Description: Sjögren's syndrome (SjS) is a systemic autoimmune disease characterized primarily by salivary and lacrimal gland dysfunction. As with many autoimmune connective tissue diseases, there exists a sexual dimorphism in SjS with women affected 10- to 20-times more frequently than men, suggesting a role for sex hormones in disease susceptibility or progression. Even though SjS is generally not considered a lethal disease in the absence of B cell lymphoma formation, patients have an increasingly diminished quality of life as the disease progresses. One fascinating feature of SjS autoimmunity in both humans and animal models is the formation of germinal-like centers in the salivary and lacrimal glands, referred to as lymphocytic foci (LF). Although LF and LF scores are important criteria for clinical disease and depict the level of leukocytic infiltrations of the exocrine glands, the scores often do not correlate with the severity of disease. Until recently, it was thought that LF were comprised mostly of B lymphocytes and CD4+ TH1 T helper cells. However, we recently reported that LF contain significant numbers of CD4+ TH17 memory T cells plus IL-23-producing macrophages and/or dendritic cells. Recent work has shown that IL-27, the cytokine that apparently regulates TH17 activity, is expressed at very low levels in the exocrine glands, suggesting that the TH17/IL-17/ IL-23 system may be inappropriately up-regulated in the exocrine glands of SjS patients, as well as our SjS-mouse model, C57BL/6.NOD-Aec1Aec2. To better define the role of LF in SjS and determine whether the cell populations present within LF, especially the CD4+ TH17 memory T cells, are involved in the autoimmune response inducing exocrine glandular dysfunction, we have proposed to investigate the temporal nature of the leukocyte populations forming LF in salivary glands and determine the inter-relationship between CD4+ TH17 memory T cells and its regulatory cytokine, IL-27. Goals of the proposed studies. The proposed studies utilize our C57BL/6.NOD-Aec1Aec2 mouse model of SjS. SjS-like disease of the C57BL/6.NOD-Aec1Aec2 mouse has been well-characterized and closely mimics SjS in humans. The parental strain, C57BL/6J, represents an excellent and appropriate control. The current studies are being carried out under protocol 200801756, approved by the University of Florida's IACUC. The two Specific Aims of this study, and their sub-aims, are: Aim 1: Characterize the IL-23 secreting and CD4+ TH17 T cell populations infiltrating the salivary glands during development of SjS-like disease in C57BL/6.NOD-Aec1Aec2 mice. Sub-aim 1.A. Define the temporal changes in leukocyte compositions of salivary gland LF defining the appearance of CD4+ TH17 memory T lymphocytes in relationship to development of SjS-like disease. Sub-aim 1.B. Determine the role of the IL-23 / TH17 / IL-17 system in the formation of LF during development and onset of SjS-like disease in C57BL/6.NOD-Aec1Aec2 mice. Aim 2: Determine the possible regulatory potential of IL-27 on the CD4+ TH17 T cell populations for preventing development of SjS in the C57BL/6.NOD-Aec1Aec2 mouse model. Sub-aim 2.A. Identify the temporal expression of IL-27 and its signaling pathway(s) during the development of SjS. Sub-aim 2.B. Examine the potential of IL-27 to suppress TH17 cell-associated functions in-vitro: Sub-aim 2.C. Examine the feasibility for suppression of SjS-like disease using rAAV-Il27 vectors. The hypotheses being tested are: Hypothesis 1: IL-23-secreting monocytes and CD4+ TH17 cells are major cell populations infiltrating the submandibular glands and are responsible for glandular pathology and dysfunction. Hypothesis 2: IL-27 is an effector cytokine that specifically targets and suppresses development of SjS in our animal model, most likely through its regulation of pathogenic CD4+ TH17 cells.
Project Description: AIM-1 A Temporal changes in LF lcompositions-Characterization of LF cell populations will be carried out on mice at the ages 4, 8,12,16,20 & 24 weeks. The mice to be used are being raised. B Role of the IL-23/TH17/IL-17 system in LF-One facet of these studies will be generating Il23 & Il17 gene knock-out (KO) mice. We calculate that these KO lines won’t be available for at least 1 year. Until then, we’re constructing the IL23- & IL17-expressing vectors needed to carry out planned gene therapy studies. In this regard, we’ve obtained a functional IL17 –Ad5 vector to be used to initiate SjS-like disease in C57BL/6J mice, establishing the basis for the same studies in the KO mice. AIM-2 A Temporal expression of IL-27 & its signaling pathway(s)- Studies are initiated to define the presence of IL-27 & its pathway-related molecules in the salivary glands of SjS-susceptible C57BL/6.NOD-Aec1Aec2 mice using rt-PCR and panels of commercially available antibodies. To date, majority of these associated molecules are up-regulated at the 16 week time period. B Potential of IL-27 to suppress TH17 cell-associated functions- Studies have been initiated to explore the potential of IL-27 cytokine to suppress the activity of the pathogenic TH17 cell population. Lymphocytes isolated from spleens & draining lymph nodes of C57BL/6.NOD-Aec1Aec2 and C57BL/6 mice have been used, results indicating that IL-27 can suppress the differentiation of the Th17 cells in vitro. C Suppression of SjS-like disease using rAAV-Il27 vectors-To complement the in vitro studies, we will determine if IL-27 can suppress TH17 cell activity in-vivo. Construction & testing of a rAAV2-Il27 vector has begun. The constructed vector has been sequenced for fidelity and shown to produce both IL-27 EBI3 & p28 subunits. Next we will generate enough vector for the proposed in vivo studies
Jobs Summary: Biological Scientist -- will serve as Laboratory Manager and is responsible for maintaining a laboratory environment that optimizes the conduct of the on-going studies. With respect to the specific experiments, he is responsible for the construction of the IL23, IL17 and IL27 plasmids to be used in the production of the AAV viral vectors. Master's student -- This position is involved in studies looking at the functional activities of the IL27 and IL23 cytokines. OPS Technical -- This position is carrying out much of the immunohistochemical analyses defining the cell populations within LF of the exocrine glands. (Total jobs reported: 2)
Project Status: Less Than 50% Completed
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