Date of Award
Summer 8-15-2018
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Immune dysregulation has been implicated in pediatric autoimmune disease
pathogenesis, but the underlying mechanisms still remain poorly understood. We explored
immune cell signaling dysregulation in two different pediatric autoimmune diseases:
polyarticular juvenile idiopathic arthritis (JIA), a disease characterized by pain, swelling, and
limited range of motion in five or more joints and juvenile dermatomyositis (JDM), a disease
characterized by pathognomonic skin rashes and proximal muscle weakness. Both polyarticular
JIA and JDM can be controlled with newer biologic medications, but a substantial fraction of
patients still experiences refractory disease courses. To explore potential immune cell signaling
dysregulation, mass cytometry was performed on peripheral blood mononuclear cells (PBMCs)
from treatment-naïve patient, remission patient, and control blood samples. Cryopreserved
samples were thawed, rested, stimulated with different cytokines (polyarticular JIA) or a cocktail
of stimuli over a time course (JDM), fixed, barcoded, stained with surface marker antibodies,
methanol permeabilized, stained with antibodies for intracellular molecules, and analyzed on a
CyTOF2/Helios mass cytometer.
No differences in immune cell signaling were detected at baseline or after 15 minutes of
IL-6 stimulation between treatment-naïve polyarticular JIA patients and controls. With IFN�
stimulation, treatment-naïve polyarticular JIA patient classical monocytes and naïve CD4 T cells
more strongly phosphorylated STAT1 and/or STAT3 than those from controls. These stratifying
cell populations were heterogeneous. Enhanced IFN� responsiveness in naïve CD4 T cells was
associated with elevated levels of JAK1 and SOCS1.
Examination of the JDM data set at 0, 3, and 15 minutes with a cocktail of multiple
stimuli with Significance Analysis of Microarrays (SAM) yielded 292 results with a false
discovery rate (FDR) of less than 5%. As a large number of results is difficult to manually
interpret, Citrus paired with LASSO feature selection was also employed to model differences in
immune cell signaling between treatment-naïve JDM patients and controls over three time
points. LASSO selected 12 Citrus features (signaling molecule in specific immune cell subset);
10 out of 12 were in PLC�2 phosphorylation with 4 out of 12 in NK cell PLC�2
phosphorylation. NK cell PLC�2 phosphorylation was lower in treatment-naïve JDM patients in
comparison to controls across all analyzed time points. Decreased treatment-naïve JDM patient
NK cell PLC�2 phosphorylation was associated with lower levels of calcium flux in NK cells
upon receptor crosslinking in comparison to a healthy control.
While similar analytical approaches were applied to the polyarticular JIA and JDM
cohorts, different stimulation protocols were utilized. However, quantitative analytical
techniques revealed previously undescribed signaling abnormalities in both diseases. These
signaling differences provide a mechanism for tofactinib (a JAK inhibitor) treatment in a clinical
trial for polyarticular JIA and a potential new target for therapy in JDM. In addition, these
signaling differences may be valuable for confirmation of polyarticular JIA and JDM diagnoses,
as well as biomarkers to predict which treatment-naïve patients may respond to a given therapy
with expansion of the original models.
Language
English (en)
Chair
Anthony R. French
Committee Members
Lori Setton, Kristen Naegle, Todd Fehniger, Stephen Oh,
Comments
Permanent URL: https://doi.org/10.7936/q14t-sm67