Myosin Skews Effector Immune Cells of Scurfy Mice to Target Muscles in an Adoptive Transfer Model of Myositis

Nicholas A. Young1, Rahul Sharma2, Alexandra Friedman1, Benjamin Kaffenberger1, Brad Bolon1, and Wael N. Jarjour1

1Wexner Medical Center at The Ohio State University, Columbus, Ohio 2University of Virginia Health System, Charlottesville, VA

No Conflicts of Interest

Background

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Autoimmune myositis results in an inflammatory response targeting muscle tissue

Pathogenesis remains unclear Standard treatment includes non-specific

immunosuppressive therapyTargeted therapies could be made if the disease

mechanism was better understood Dermatomyositis patients have reduced

Regulatory Tcell (Treg) populations Tregs could suppress this inflammatory

response

Synaptotagmin 7 (Syt7) Member of the synaptotagmin family of

membrane trafficking proteins Involved in membrane resealing Mutation to Syt7 causes the abnormal release of

intercellular proteins Syt7 knockout mice develop myositis

Presumably due to endogenous muscle tissue antigen exposure from “leaky” cells

• activation and expansion of autoreactive lymphocytes

Preliminary work---Syt7

Preliminary work---FoxP3 knockout (scurfy mice) are Treg deficient

Gene product is scurfin X-linked gene encoding a transcription factor

imperative in the maturation and activation of Tregs

So these mice are Treg deficient Tregs suppress autoreactive cells in the

peripheral immune system FoxP3 knockout results in multi-organ

inflammation into a few target organs in male mice

No response to muscle tissue The mice succumb to this inflammation before

5 weeks of age

FoxP3-/y Treg knockout (scurfy) mouse

Forkhead box P3 (FoxP3)

IP transfer of autoimmune responses against multiple organs/tissues into RAG1-/- recipients

Not only were the target organs of the FoxP3 knockout mouse inflamed But 7 additional organs had an inflammatory response

Very mild muscle tissue inflammation

There are autoimmune lymphocytes against organs that were spared from autoimmune attack in the FoxP3 knockout mice

A greater repertoire of autoreactive cells exists in this mouse

Additional preliminary data suggested that autoreactive cells to muscle also existed This then served as the model for further work

Preliminary work---FoxP3 knockout adoptive transfers into adult RAG1 -/- mice

Hypotheses

Abnormal release of intracellular proteins can result in robust autoimmune myositis when combined with the absence of Tregs

Tregs can suppress the pathogenesis of autoimmune inflammation of muscle tissue

Project GoalsDesign an animal model of autoimmune myositis to:

Explore disease mechanism Identify possible self-antigens that could drive this

autoimmune response Characterize infiltrate

Examine role of Tregs See if Tregs could suppress target organ inflammation

Analysis of inflammationHISTOPATHOLOGY SCORING CRITERIA

Score Criteria

Skeletal Muscle: Inflammation (H&E-stained slide)0 Within normal limits

essentially no inflammatory cells between fibersany inflammatory cells are isolated, not clustered

1 Minimal few inflammatory cells in the fascia between fibersinflammatory cells are isolated or in very small clusters (< 10 cells)

2 Mildfew inflammatory cells are located in the fascia between myofibersinflammatory cells typically are arranged in small clusters (15 to 25 cells)

3 Moderate inflammatory foci are fairly common between fibersinflammatory cells typically are arranged in short rows or modest clusters (30 to 45 cells)

4 Marked inflammatory foci are numerousinflammatory cells typically are arranged in long rows and large clusters (> 50 cells)

Lesion scores were assigned in a coded (“blinded”) fashion using the 10x objective

Analysis of inflammation

Syt

7 -/

-S

yt 7

+/-

Week4 8 12 17+

His

topa

thol

ogy

scor

e

Ave strong intensityIM IMinj non-inj homogenate

CD4--Tcells 377661.2 1122126 69100CD8--Tcells 163976.1 1027996 6252.8F4/80--Macrophages 226820.3 702672.6 10181.8B220--Bcells 0 0 0

SD strong intensityIM IMinj non-inj homogenate

CD4--Tcells 261189.3 467778.8 15308.6253CD8--Tcells 68405.15 454180.2 2538.56078F4/80--Macrophages 152379.5 534542 13801.8185B220--Bcells 0 0 0

Syt7 knockout mice have mild muscle tissue inflammation that subsides with time

*

At 4 weeks

Generation of double knockout (DKO) (Syt 7/FoxP3) mice

Syt7+/- FoxP3+/y

X

FoxP3 –/y Syt7-/-

males

Syt7+/- Foxp3+/- females

FoxP3 –/y Syt7 +/-

FoxP3 +/y

Syt7 +/-

Will the absence of Tregs lead to greater muscle tissue inflammation in Syt 7-induced myositis?

KODKO

FoxP3 KO Syt 7-/- DKO mouse

Treg/Syt7 double knockout (DKO) mice develop autoimmune myositis—4weeks

supp 4wks 4wksscurfy Treg Syt het Syt ko dko

0 1 0 2 21 * 1 1 31 * 0 2 31 * 0 2 20 * * * 3

* * * * ** * * * ** * * * ** * * * ** * * * *

0.6 1 0.25 1.75 2.60.547723 #DIV/0! 0.5 0.5 0.547723

0.028834

0

0.5

1

1.5

2

2.5

3

3.5

DKOKODKO AveKO AVE

His

topa

thol

ogy

scor

e

FoxP3 KO FoxP3 KOSyt7 -/+ Syt7 -/-

FoxP3 KO Syt 7 -/-

FoxP3 KO Syt 7 +/-

KO

DKO

KO DKO

*

CD3T cells B220

B cells

IP Adoptive transfer into RAG1 -/- mice

Rag1 -/-

Lymph node preparation

FoxP3 –/y Syt7 -/-

FoxP3 –/y Syt7 +/-

or

Can this double knockout of Syt and FoxP3 induce myositis in Rag1 mouse adoptive transfer model?

KO

DKO

0

1

2

3

4KODKOKO AveDKO AVE

IPadoptive transfer

into RAG1 -/-

His

topa

thol

ogy

scor

e

AdoptiveTransfer

KO DKO

FoxP3 KO FoxP3 KOSyt7 -/+ Syt7 -/-

FoxP3 KO Syt 7 -/-

FoxP3 KO Syt 7 +/-

KO

DKO

FoxP3/Syt7 double knockout adoptive transfer into RAG1 -/- mice induces even more robust

autoimmune myositis

*

FoxP3/Syt7 double knockout adoptive transfer into RAG1 -/- mice has primarily a CD4+ infiltrate

CD4--Tcells 261189.3 467778.8 15308.6253 4367.94 646067.6CD8--Tcells 68405.15 454180.2 2538.56078 5396.437 155713.6F4/80--Macrophages 152379.5 534542 13801.8185 688.0432 243180.2B220--Bcells 0 0 0 0 0

0

200000

400000

600000

800000

1000000

1200000

CD4--Tcells CD8--Tcells F4/80--Macrophages

B220--Bcells

IM-injected muscleIM-non-injected muscle

IP+muscle tissue homogenate

IP+myosin

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

CD4--Tcells CD8--Tcells F4/80--Macrophages B220--Bcells

IM-noninjected muscle

IM-injected muscle

DKO-RAG1

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

CD4--Tcells CD8--Tcells F4/80--Macrophages B220--Bcells

FoxP3 KO Syt 7 -/-

Pix

el in

tens

ity

*

T cells CD4

T cells CD8

B cells B220

Macrophages

F 4/80

T cells CD4

T cells CD8

B cells B220

Macrophages

F 4/80

16

WT B6 mice

IP Adoptive transfer into RAG1 -/- mice with and without muscle tissue preparation

Will endogenous tissue antigens induce myositis?

Rag1 -/-

Lymph node preparation

FoxP3 –/y

Lymph node preparation

Muscle tissue preparationor

purified myosin protein

0

1

2

3

4 IPIP+homogenateIP+myosinAverage

IPIP + musclehomogenate

IP + myosinprotein

Intracellular muscle tissue antigens can stimulate an inflammatory response

His

topa

thol

ogy

scor

e

*

*

IP IP + musclehomogenate

IP + myosinprotein

IP +

myo

sin

prot

ein

T cells CD4

T cells CD8

B cells B220

MacrophagesF 4/80

IP +

mus

cle

hom

ogen

ate

Pix

el in

tens

ity

Pix

el in

tens

ity

0

1000000

2000000

3000000

4000000

5000000

6000000

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000 IP + myosinprotein

IP + musclehomogenate

muscle antigens stimulate an inflammatory response primarily of CD4+ cells

T cells CD4

T cells CD8

B cells B220

MacrophagesF 4/80

T cells CD4

T cells CD8

* *

MacrophagesF 4/80

B cells B220

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Rag1 -/-

Lymph node preparation

FoxP3 –/ySyt7 -/-

Lymph node preparation

Treg cells

Can Tregs prevent myositis in the adoptive transfer Rag1 model?

WTmice

IP Adoptive transfer into RAG1 -/- mice with and without purified Treg addition

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

- Treg+ Treg

Pix

el in

tens

ity

Histopathology score = 1*

Histopathology score = 3

+ Treg

- Treg

Treg supplementation suppresses inflammation induced by Treg/Syt7 double knockout adoptive transfer into RAG1 -/- mice

Conclusions

Abnormal exposure or release of intracellular proteins can cause autoimmune myositis Our results indicate that myosin is at least one self-antigen that

can stimulate this response priming autoreactive effector T cells

This muscle tissue infiltrate consists largely of T cells (both CD4 and CD8) Few to no B cells This highlights the pathogenic role of CD4 cells in the disease

Depletion of Tregs cause an inflammatory myositis Very mild inflammation observed in syt7 -/- mouse (which has

Tregs) Very robust inflammatory response seen when combined with

Treg deficiency Addition of Tregs can suppress this response

Study Implications and Future Work

Animal models for polymyositis and dermatomyositis are rare

Can be used as a model to study any autoimmune disease that involves abnormal release of cellular protein

Muscle tissue cells are subjected to: Mechanical stress Drug injury

This leads to cell death, which can contribute to abnormal release of intracellular muscle tissue antigens

Future investigations will focus on drug injury (statin-induced myopathy)

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Acknowledgements

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Principal Investigator: Dr. Wael Jarjour

Research Associate: Alex Friedman

Medical Student Researcher: Dr. Ben Kaffenberger

Dr. Norma Andrews: (Synaptotagmin VII+/- females)

Collaborator:Dr. Rahul Sharma

The Ohio State University Medical Center, Columbus, OH

University of Virginia Health System, Charlottesville, VA

Undergraduate Researcher: Nitish Aggarwal

The Ohio State UniversityDepartment of Veterinary Bioscience

Veterinary Pathologist:Dr. Brad Bolon

University of MarylandCollege Park, MD

Great thanks to the ACR for the invitation to speak at the conference this year

Questions?

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