how animal models inform potential therapies for immune...
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How animal models inform potential therapies for immune dysfunction in PANDAS/PANS
Dritan Agalliu, Ph.D. Departments of Neurology, Pathology & Cell Biology
and Pharmacology Columbia Translational Neuroscience Initiative
Columbia University Medical Center
Disclosures
Consultant for Hughes, Hubbard and Reed LLP
Funding : NIH / NHLBI R01HL116995 NIH / NIMH NIMH R01MH112849 NIH / NCTSA (CUMC CAMPRII – BASIC) International OCD Foundation Newport Equities LLC (private donation) PANDAS Network
Infection, autoimmunity and behavior: The quest for a link
Autoantibodies disrupt communication between neurons
NMDAR NMDARAMPAR
glutamate
D1R D1RD2R
dopamine
Access to the brain? Access to the brain?
The blood-brain barrier: an important gatekeeper between the blood and the central nervous system
• Maintains brain homeostasis • Limits CNS entry of
– pathogens – immune cells – drugs
Evans blue dye
Abnormal blood-brain barrier function is a prominent feature of many CNS diseases
Multiple sclerosis
Bruce Trapp
• Acute traumatic injury
• Stroke
• Exposure to toxic or hypertonic conditions
• Brain infections
• Autoimmune diseases - Multiple sclerosis - Autoimmune Encephalitis?
• Neurodegenerative diseases • Alzheimer’s • Huntington’s • Parkinson’s
Acute ischemic stroke
Yu et al. (2015)
Engelhardt & Ransohoff, 2012 Trends Immunol Huppert, J. et al., 2010 FASEB Okada & Khoury, 2012 JCI Risau W. et al., 1990 JCB
Hypothesis 1: Destruction of tight junctions between endothelial cells
Zhang, D. et al., 2012 Brain Behav Immun Abuqayyas & Balthasar, 2012 Mol Pharmaceutics Okun, E. et al., 2010 Neuromol Med Diamond, B. et al., 2013 Ann Rev Immunol
Hypothesis 2: Selective transport of antibodies from endothelial cells into the brain
Group A β-hemolytic Streptococcus pyogenes causes a plethora of autoimmune diseases
Implicated in autoimmunity Scarlet fever
Rheumatic fever
Glomerulonephritis
Sydenham’s chorea PANDAS
heart
kidney
Skin (viral toxin)brain
Neurological symptoms in SC and PANDAS
Chorea Hypotonia
Cardiac involvement Hyperactivity Obsessions
time
sym
ptom
sev
erity
Choreiform movements Tics
Severe separation anxiety Urinary frequency
Food refusal / anorexia
Emotional lability OCD-like symptoms
Contamination fears
SC PANDAS
S. pyogenes activates both arms of the immune system: humoral (antibodies) and cellular (Th17 cells).
S. pyogenes
Generation of Th17 cells after multiple intranasal infections
% o
f CD
4+ 2
W T
cel
ls
with
Cyt
okin
e ph
enot
ype
Perc
enta
ge o
f T
cells
IFNγ
Dileepan et al., (2011). PLoS Pathogens
Patients’ serum autoantibodies bind neurons and activate neuronal signaling
100%C
aMK
II A
ctiv
atio
n
PANDAS non-PANDASSC
p < 0.0001
CaM kinase
TH-(P)
Dopamine
Dopamine release Unwanted movement
Autoantibody
Kirvan, C. et al. (2006), J Neuroimmunol Kirvan, C. et al. (2003), Nat Med
Autoantibodies can bind post-synaptic D1R and D2R receptors
D1R D1RD2R
SC: D2R, D1R or ratio of D2R/D1R titers correlate with symptoms
PANDAS: D2R; Mixed reportsdopamine
Ben-Pazi, H. et al., 2012 J Mol Neurosci Ben-Pazi, H. et al., 2013 PLoS One Brilot, F. et al., 2011 Neurology Dale, R. C. et al., 2012 Brain Kirvan, C. et al., 2006 J Neuroimm Kirvan, C. et al., 2003 Nat Med
Access to the brain?
Outline
1. What is the role of S. pyogenes-specific Th17 lymphocytes in post-infectious basal ganglia encephalitis?
2. Are Th17 lymphocytes necessary for the development of disease in the brain?
GAS or PBS
Intranasal
Weekly i.n. GAS or PBS
i.v. tracer, sac
Experiment day 0 7 14 21 28 30
A novel rodent model to understand the impact of the immune system on the brain after S. pyogenes infections
GAS T cells
Dileepan T et al., (2011) PLoS Pathogens; Dileepan T., Smith E. et al., 2016 J Clin Invest
Hypothesis: Dysregulated Th17 immune response to S. pyogenes infections is key to understanding “autoimmune” complications associated with this pathogen.
Immune pathology
Normal immune response
protection
Th17 cellular immune response:
a balance between protection and disease
0
125
250
375
500
Bregma 4.28 Bregma 3.2 Bregma 2.46 Bregma 1.54 Bregma 0.26 Bregma -0.94 Bregma -2.46
Naïve 1 Inf 6H 4 Inf 6H
Aver
age
num
ber o
f C
D4+
T-C
ells
/ 1
2µm
S. pyogenes-specific CD4+ T lymphocytes are present in the brain after multiple intranasal infections.
*** ***
** *
Dileepan T., Smith E. et al., 2016 J Clin Invest
Does the route of GAS infections determine entry of Th17 cells into the brain?
PTx i.v.
GAS or PBS CFA emulsion GAS or PBS
Subcutaneous Intranasal
Weekly i.n. GAS or PBS
sac
Experiment day 0 7 14 21 28 30
Biweekly s.c. emulsion
sac
Experiment day 0 14 28 30
Platt M. et al. (unpublished)
Only intranasal GAS infections promote entry of Th17 cells into the brain
Platt M. et al. (unpublished)
The route of inoculation determines immune outcome
Subcutaneous Intranasal
T cell phenotype Th1 Th17, Th1
T cells in the CNS Few Many
Microglia activation Some Robust
Glomerular synapse loss
N/A Degraded
Anti-GAS titer High High
BBB permeability Adjuvants High
Behavioral deficits Perseveration Motor deficits
Mild motor deficits
Platt M. et al. (unpublished)
S. pyogenes CD4+ T cells in the brain are associated with neuroinflammation
Dileepan T., Smith E. et al., 2016 J Clin Invest
Do Th17 cells in the brain affect the integrity of blood vessels?
GAS or PBS
Intranasal
Weekly i.n. GAS or PBS
i.v. tracer, sac
Experiment day 0 7 14 21 28 30
Biocytin-TMR
Dileepan T., Smith E. et al., 2016 J Clin Invest
The integrity of the blood-brain barrier is compromised after multiple S. pyogenes infections.
Naive
GAS-6H
Dileepan T., Smith E. et al., 2016 J Clin Invest
LH
OT
GAS-specific Th17 lymphocytes may disrupts blood-brain barrier tight junctions
Dileepan T., Smith E. et al., 2016 J Clin Invest
Are Th17 cells present in tonsils from PANDAS children?
• S. pyogenes subtypes may be important for disease pathogenesis
• Genetic risk factors for SC and PANDAS
• Repeated infections with S. pyogenes or other pathogens that generate Th17 cells.
• Presence of both Th17 cells and autoantibodies is important for disease pathogenesis
Dileepan T., Smith E. et al., 2016 J Clin Invest
Novel pathway for T cell entry into the CNS
Dileepan T., Smith E. et al., 2016 J Clin Invest
Outline
1. What is the role of S. pyogenes-specific Th17 lymphocytes in post-infectious basal ganglia encephalitis?
2. Are Th17 lymphocytes necessary for the development of disease in the brain?
Visualizing the presence of Th17 lymphocytes in the brain using RORγt+/GFP mice
Martin Lipp & Gerd Müller, Lymphoid organogenesis: getting the green light from RORt Nature Immunology 5, 12 - 14 (2004) doi:10.1038/ni0104-12
GAS or PBS
Intranasal
Weekly i.n. GAS or PBS
sac
Experiment day 0 7 14 21 28 30
Platt M. et al. (unpublished)
DAPI GFP CD4
Th17 cells are a subset of total CD4+ T lymphocytes in the CNS after multiple S. pyogenes infections
~32.5% Th17To
tal C
D4+
T c
ells
per
12
um O
B s
ectio
n
0
175
350
525
700
WT HET
Perc
enta
ge la
bele
d Th
17
cells
in O
B
0%
11%
23%
34%
45%
WT HET
DAPI GFP CD4
RORγt+/GFP
Platt M. et al. (unpublished)
RORγtc-/- mice have fewer CD4+ T cells in the brain after multiple S. pyogenes infections
n=5
n=8
n=8
Platt M. et al. (unpublished)
Neuroinflammation is present in the brain in the absence of Th17 cells after multiple infections
n.s.
PBS GAS ROR -/-GAS ROR+/-
Platt M. et al. (unpublished)
Blood-brain barrier is still damaged in the absence of Th17 lymphocytes after multiple S. pyogenes infections
PBS GAS ROR-/-GAS ROR+/-
IgG
Glu
t1Ig
G G
lut1
n.s.
n.s.
Platt M. et al. (unpublished)
PBS GAS ROR-/-GAS ROR+/-
Th17 cells are not required to damage synapses after multiple S. pyogenes infections
Platt M. et al. (unpublished)
S. pyogenes-infected mice have blunted olfactory perception regardless of the presence or absence of Th17 cells
Platt M. et al. (unpublished)
Th17 and Th1 cells are present in the CNS after multiple GAS infections
How can OCD/PANDAS research in animals inform clinical diagnosis and treatment?
• Test whether cytokine profiles characteristic of a Th17/Th1 phenotype are present in CSF from PANDAS patients.
• A “signature profile” for autoimmunity to ascertain diagnosis of PANDAS/PANS should be established.
• Develop novel, dynamic contrast-enhanced MRI imaging tools to detect blood-brain barrier dysfunction during periods of disease flares.
• Develop new immunotherapies targeted against Th17/Th1 cells, in conjunction with IVIG or plasmapheresis
Cutforth T., Agalliu, D et al., 2016 J Future Neurology
Acknowledgements
University of Minnesota, Minneapolis Paul Patrick Cleary, Ph.D. Thamotharapail Dileepan, V.D.M., Ph.D.
Agalliu Laboratory Maryann Platt Tyler Cutforth, Ph.D. Erica Smith Ph.D. Charlotte Wayne Lauren Cuje M.S. Nicole Ampatey Sarah Chaudry
NIH / NHLBI R01HL116995 NIH / NIMH NIMH R01MH112849 NIH / NCTSA (CUMC CAMPRII International OCD Foundation Newport Equities LLC (private donation) PANDAS Network