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Transcript of "Bioluminescent Imaging of Histidyl-Transfer RNA Synthetase-Induced Myositis Reveals Early-Phase...
Bioluminescent Imaging of Histidyl-Transfer RNA Synthetase-Induced Myositis Reveals Early-Phase Involvement of NF-kB-Mediated Inflammation
American College of Rheumatology Conference11/16/2014
Nicholas Young, PhD Division of Rheumatology and Immunology
No relevant financial or competing interests specific
to this project to declare
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The conventional function of tRNA synthetases
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The UNconventional function of tRNA synthetases
Autoimmune disease-autoantibody production Anti-tRNA synthetase autoantibodies have been
detected in patient serum autoimmune diseases Anti-Jo-1 autoantibody: histidyl-tRNA synthetase
(HRS;Jo-1) is commonly found in myositis patient serum strongly correlates with disease activity
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Myositis and Anti-Jo-1 autoantibody Autoimmune myositis results in an inflammatory response
targeting muscle tissue as well as extramuscular organs
Anti-Jo-1 autoantibody can actually be used diagnostically Considered a myositis-specific biomarker because of the clear
clinical correlation Found in 20-30% of PM and in 60-70% of those with interstitial lung
disease
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Lab of Dr. Dana Ascherman—previous work IM administration of bacterially expressed murine
histidyl-tRNA synthetase (HRS) induces muscle inflammation
Critical role of the innate immune responses to muscle tissue inflammation TLR2 and TLR4 KO mice phenotypes MyD88 KO
Design a model to look at the role of NFkB in HRS-induced myositis
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in vivo BALB/C-Tg(NFkB-RE-luc)-Xen mouse
Transgenic NFκB-RE-luc mouse
p65 p50
kB site
pNFkB
Luciferase
NF-kB is a transcription factor- controls expression of many genes- plays a crucial role in activation of genes associated with inflammation and an immune response
Experimental set-up
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IM5 mg/mL 70 uLHRS vs. PBS
Xenogen
Time
weeks
luciferin injection
in vivo imaging system (IVIS) 200
HRS
Site of HRS IM injectionused as Region of Interest (ROI)for quantitation
BALB/C-Tg(NFkB-RE-luc)-Xen mouse---2 weeks
Results IM injection of HRS protein induces a significant
inflammatory response in luciferase mice As measured by IVIS photon quantitation at the site of
injection The response subsides by 28 days
HRSPBS
H&Es at 38 days
INSERT HISTOPATHOLOGIC DATA HERE
Results Despite the loss of bioluminescence at 28 days, there still is a
significant inflammatory response histolopathologically (even at 38 days) NFkB activation may play a big role in the initial
autoimmune/inflammatory response, but may not be involved in later/chronic stages of the disease
To resolve this question: Use NFkB inhibitors in this model
Give at the time of HRS injection and at 2 weeks
Nano-emulsified curcumin
NEC suppresses LPS-induced TLR4 and RAGE expression
Our work at EULAR -
Exercise inhibits NF-κB systemically
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Exercise suppresses the expression of LPS-binding receptors on the cell surface
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Conclusions and future directions Luciferse model is a viable platform to assess NFkB-
mediated inflammation in longitudinal analysis NFkB involved in establishment of inflammation, but not
in chronic stages Future work
Further characterizing NFkB in this response Test efficacy of NEC to reduce inflammation
Exercise may still be the best medicine!!
Acknowledgements
Wael Jarjour, MDLai-Chu Wu, PhDWilliam Willis, PhDBenjamin Kaffenberger, MDAlexandra FriedmanMichael BrussMark GardnerAmanda KiblerJeff HamptonGiancarlo Valiente
The Ohio State University Wexner Medical Center, Columbus, OHDepartment of Internal MedicineDivision of Rheumatology and Immunology
Department of PharmacologyComprehensive Cancer Center
The Ohio State University College of Veterinary Medicine
Zhongfa Liu, PhDYu Cao, PhD
Center for Biostatistics
Xiaokui Mo, PhD
Thomas Rosol, PhDBrad Bolon, DVM, MS, PhD
Sudha Agarwal, PhDAlisa D. Blazek Derrick M. Knapik
Department of Orthopedic SurgeryThe Biomechanics and Tissue Engineering Laboratory, College of Dentistry
The University of Miami Health System, Miami, FLDana P. Ascherman, MD
Extra stuff
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We have generated ZAS3 knockout (KO) mice to study its
physiological function
The most obvious phenotype of ZAS3 KO mice are high bone mass
Adult ZAS3 KO mice have higher bone mass
IkB
p65 p50
IkB
p65 p50
Genes
TLR4
IKK complexp
degradation
kB site
pNFkB
RAGE
LPS
TLR2
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Healthy-media Vs. Healthy-E2 1.34343 0.140597
SLE-media Vs. SLE-E2 1.39908 0.0301386*Healthy-E2 Vs. SLE-E2 1.0208 0.898485
Gene chip analysis of ZAS3 in PBMCs with estrogen treatment
Fold change in expression p-value
Gene Titlehuman immunodeficiency virus type I enhancer binding protein 3
Probeset ID--233884_atGene Symbol--HIVEP3
PBMCs of SLE patients and healthy subjects were purified and treated with estrogen for 48 hours.Growth media was hormone-deficient and consisted of 5% charcoal-stripped FBS supplemented RPMI. After 48 hours, RNA was isolated via Ficoll centrifugation and RNA was purified. Samples given Microarray center for genomic analysis.
Exercise inhibits LPS-induced activation of NF-κB systemically
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2 hrs 24 hrs
ControlExercise
LPSLPS + Exercise
Tota
l bod
y lu
x-N
F-κB
B
iolu
min
esce
nce
(pix
els
x 10
9 )
2
6
0
4
1
2
3
0
** *
**
*
Molecular targets of curcuminMany well-characterized molecular targets
Curcumin is significantly more soluble in NEC
Curcumin in water
Curcumin in NEC
0.5 mg/ml 100 mg/ml
Experimental set-up
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Inject 25 µg LPS in the right paw
Treatments1. Control 2. Exercise only3. LPS only4. LPS + Exercise*
luciferin injection 2 hours
or 24 hrs
IVIS
* = treadmill walking 8 m/min 45 min
Bioluminescence in IVIS
N = 10/group
Xenogen
ZAS3 is a potent immuno-regulatory transcription factor
0
2
4
6
8
10
T0 T24
WTKO
phot
ons
(E+0
9)
**
0
1
2
3
4
5
6
0
5
10
15
20
25
30
Healthy SLE
p= 0.04 *
-ZAS3
-actin
SLE Healthy
Healthy SLE
p=0.005 *
Healthy SLE
LPS
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ZAS3 Immunohistochemistry
HRS-induced myositis