aberrant muscle antigen exposure in mice is sufficient to cause myositis in a treg cell-deficient...
TRANSCRIPT
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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