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

2

3

The conventional function of tRNA synthetases

4

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

5

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

6

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

7

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

9

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

17

Exercise suppresses the expression of LPS-binding receptors on the cell surface

13

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

21

22

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

125

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

27

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

30

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

32

ZAS3 Immunohistochemistry

HRS-induced myositis