We have demonstrated that LTBR-pathway blockade has therapeutic effects in NOD mice, a popular model of Sjogren's syndrome. Affymetrix analysis, ELISA and q-PCR analyses showed that CXCL13 increased with disease progression in glands, but was reduced ~5-fold by LTBR-pathway inhibition. Our own preliminary data and a recent report that LTass2 expressed by DC drives most if not all of the LTBR-dependent events in lymph nodes [1] suggest that DC play a critical role in induction of HEV, CXCL13 and gland pathology in Sjogren's syndrome. Until now the evidence connecting DC, HEV, CXCL13 and disease progression is only circumstantial but herein we propose to deplete DC, or to silence LT-ass2 expression by DC, and thus definitively verify or refute this concept. We also will determine if dendritic cells are the direc source of CXCL13, a chemokine well known to drive ectopic lymphoid follicles and B-lymphocyte accumulation in diseased glands of mice and man, and evaluate the result of CXCL13 neutralization on disease progression. Our primary goal is to determine definitively whether or not expression of LTass2 by a dendritic cell subset orchestrates much of the pathology in exocrine glands that is associated with reduced gland function and altered gene expression.
Aim 1. Determine if direct neutralization of CXCL13 might serve as a therapy in Sjogren's syndrome by interrupting ectopic follicle formation and B-cell accumulation in diseased glands. A neutralizing monoclonal antibody will be used to treat NOD mice in a therapeutic manner. Histology, immunolocalization studies, real- time PCR, Affymetrix chip analyses, direct measurement of the rate of salivary and lacrimal gland secretions and ocular integrity scores will be used to assess the efficacy of direct CXCL13 ablation as a therapeutic target in the NOD mouse model of Sjogren's syndrome. Off target effects on immune responses will be examined.
Aim 2. Determine the sources of CXCL13 in lacrimal glands using CXCL13-GFP mice backcrossed to NOD background and/or in situ hybridization and isolation of infiltrating cells by cell-sorting with a FacsAria III. Determine the kinetics, localization, abundance and phenotype of cd11c+ dendritic cell populations of glands of NOD during disease development and progression. Isolation by fluorescent cell sorting, multicolor FACS analyses, real-time PCR, ELISA assays, multicolor fluorescence microscopy and other methods will be employed to define DC populations present in diseased glands at various stages of disease and determine if these dendritic cells are a direct source of CXCL13 and express LTass2, the ligand for LTBR.
Aim 3. Determine whether depletion of all CD11c DC or DC-specific ablation of certain gene products (lymphotoxin-alpha, CXCL13) alters the onset or progression of disease or alters the size and nature of the leukocyte infiltrates in in salivary and lacrimal glands in transgenic NOD mouse strains we will create by backcrossing. We will utilize existing mouse strains, namely (1) CD11c-diptheria toxin receptor-NOD mice (NOD.FVB-Tg(Itgax-DTR/EGFP)57Lan/JdkJ), (2) NOD mice, and (3) bone marrow chimeras of various NOD- background transgenic mouse strains. We will create strains in 1.5 years by Simple Sequence Length Polymorphism-assisted speed-backcrossing (cd11c-DTR/NOD, LTalpha-/-/NOD, CXCL13-/-/NOD). These will be used to determine whether dendritic cells provide critical signals via the lymphotoxin-beta receptor pathway to drive HEV development, leukocyte accumulation, chemokine production and gland hypofunction.

Public Health Relevance

Women's health issues: All Veterans that are eligible for health care are eligible for diagnostic eye and vision care services. As the number of women Veterans increases, so will the care required for diseases that predominately affect women. Sjogren's syndrome is as prevalent as rheumatoid arthritis, affecting ~1% of the population. Women contract the disease ~9 times more frequently than men. The insights gained from this study (autoimmune dry-eye disease) will help advance the development of disease modifying therapies for Sjogren's syndrome, and other related diseases. Although it cannot be tested in currently existing animal models, the treatments being examined here may also impact the increased risk of leukemia associated with Sjogren's syndrome. Translational potential: The pre-clinical trials proposed will facilitate translation of novel therapies such as specific chemokine receptor inhibitors for this and other diseases (helicobacter gastritis, diverticulitis).

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
5I01BX001797-02
Application #
8967088
Study Section
Immunology A (IMMA)
Project Start
2014-10-01
Project End
2017-09-30
Budget Start
2015-10-01
Budget End
2016-09-30
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
White River Junction VA Medical Center
Department
Type
DUNS #
053404034
City
White River Junction
State
VT
Country
United States
Zip Code