Generation of CNS1 deficient mice

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Allergy, atopy and asthma Nov. 17, 2008
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Immune modules Helper T cells activate discrete
cell populations
Extracellular bacteria and fungi
Facultative and obligate intracellular organisms
Helminths and biting insects
IL-35
IL-22
DeFranco, Locksley, Robertson, Immunity- The
Immune Response in Infectious and Inflammatory
Disease, New Science Press, 2007. http//www.new-s
cience press.com/browse/immunity/info
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Ana - phylaxis Backwards Protection
Richet 1913 Nobel Prize
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Allergy
An adverse immune response to environmental
antigens (allergens).
Operational definition IgE
Manifestations Anaphylaxis, Eczema, Hayfever,
Asthma
Insect venoms Pollens Mite feces Animal
danders Food Haptenylated drugs (penicillin)
Many Allergens Acquired Via the Mucosa
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IgE Reagin or Reaginic Antibodies PCA
Passive Cutaneous Anaphylaxis
Serum (IgE) (Ishizaka, 1966)
Atopic to Antigen X
aIgE
Non-Atopic
Antigen X
Immediate Hypersensitivity
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Stages of Allergy - I.
Early Phase Reaction - minutes
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Stages of Allergy - II.
Late Phase Reaction - hours
a4
CCR8
TNF
Th2
PGD2
IL-4 IL-13
IL-5
IL-3 IL-9
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Stages of Allergy - III.
Chronic Phase Reaction - wks - yrs
1. Subepithelial Fibrosis
Toxic Granules
2. Smooth Muscle Hypertrophy
3. Mucosal and Mast Cell Hyperplasia
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FceRI Key Player

IgE Kd 10-10 M
a
gg
b
Syk
P
P
Lyn
PI(3)K
P
LAT
AA
PLA-2
Btk/Itk
PIP3
GADS/SLP-76
PLCg1
VAV
Ca
Ras
Rab
MAPK
PGD2 LTC4
Granule Exocytosis
Cytoskeleton
TNF IL-4
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Mouse Human
Mouse IgG1/FcgRIII on mast cells can trigger
pathway (not present on human mast cells)
Human APC, Eosinophils
ag2 FceRI
a
gg
IgE-linked antigen presentation in human
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Mast Cell Granules
Histamine Vasodilator Edema Heparin
Bonds constituents (negative charge) Neutral
Serine Proteases Tryptase All Chymase
Carboxypeptidase Cathepsin G
C.T. gt mucosal
Knock-out no mast cell granules
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Myeloid Cell Eicosanoids
Ca
IgE
AA
FLAP
LTA4
LTC4 synthase in asthma
5-LO
Mast Cells Eosinophils Basophils AAM?
PLA2
LTC4
LTA4 Hydrolase
PMN M?
Transpeptidase
LTB4
LTD4
chemoattractant
Dipeptidase
(SRS-A)
CysLT1,CysLT2
LTE4
BLT1 BLT2
Smooth Muscle Contraction Endothelial NO Mucus
Secretion
Cell Recruitment
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Leukotriene Receptors
BLT1 LTB4 (Kd0.5 nM) Gene duplication Myeloid,
lymphoid cells Tissue 14q
(h) recruitment BLT2 LTB4 (Kd23 nM),
More widely
other eicosanoids CysLT1 LTD4gtC4E4
X (h) PBL, lung
smooth muscle, Vascular
lung macrophages, relaxation smooth


small intestine
muscle contraction cell
activation CysLT2 LTC4LTD4gtE4 13q14 (h)
PBL, lung, heart, brain Cell
activation
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Mast Cell PGD2
Ca
IgE
in asthma
AA
COX
PGD2
PLA-2
DP1 Kd 1.5 nM (airway epi, basophils,smooth
muscle lung, GI)
CRTh2 (DP2) Kd 2.5 nM (Th2, eo, baso,lung
smooth muscle, GI)
Mouse ko lung inflammation, bronchial
hyperreactivity Mouse ko skin allergic
hypersensitivity
Bronchoconstriction Chemokines
(Eotaxins, MCPs, MDC)
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Eosinophil Granules
Specific Primary
Major Basic Protein Eosinophil Cationic
Protein Eosinophil Peroxidase Eosinophil-derived
Neurotoxin Lysophospholipase (Charcot-Leyden
crystals)
CYTOTOXICITY (multi-system toxicity in patients
with idiopathic HES)
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Chemokines and Auto-amplification
Mf
IL-4 IL-13 ?PGD2
MDC
CCR4 Basophils, Th2 (some)
Epithel. cells
MCP 2,3,4
Eotaxins 1,2,3
RANTES
MEC
CCR3 - eosinophils, mast cells, basophils, Th2
(some)
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Asthma
Prevalence gt300 million worldwide 1/6 U.S.
children Cost in U.S. gt 6 billion/yr ing
Developed gt Nondeveloped Countries
Hygiene Hypothesis Reflects developmental
evolution of mucosal/regulatory immune system,
possibly through effects on establishing the
commensal flora ?Worms, ectoparasites,
childhood infections
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Asthma an epidemic in the absence of
infection Inverse relationship between
infections and immune disorders
Bach JF. 2002. N Engl J Med. 347911.
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The global epidemic of asthma
Masoli et al., The global burden of asthma
executive summary of the GINA dissemination
committee report. Allergy 59469-78, 2004

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Global Burden of Type 2-associated Immunopathology
Developed countries Allergy, asthma
gt300 million worldwide 1 in 8-12 U.S.
children gt6 billion/yr U.S. Hygiene
Hypothesis Default type 2 response to common
environmental antigens - mites, pollens, danders,
etc. - in absence of inflammatory exposures in
early life - Treg deficiency?
Developing countries Worms - intestinal
nematodes (gt2 billion) tissue filaria (gt120
million) tapeworms (gt2 million) schistosoma
(gt200 million) Liver failure, epilepsy, bladder
cancer, elephantiasis, blindness, anemia
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A disease of persistent Th2-associated airway
inflammation
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Stereotyped gene expression in Th2-associated
immunity
Lewis CC et al. Disease-specific gene expression
profiling in multiple models of lung disease.
AJRCCM 177376, 2008.
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Wenzel S et al., Lancet 3701422-31, 2007.
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Life cycle of Nippostrongylus brasiliensis
Day 4 jejunum
Day 2 lung
Day 9
CD4 T cells, IL-4/13, IL-4Ra, Stat6
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Lung IL-4 cells during helminth infection
naive
infected
CD4
GFP
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Cytokine production in 4get x KN2 mice in vivo
Uninfected
Lung PP
mesLN Spleen
K Mohrs et al, Immunity 23419-29, 2005
GFP
Heligomosomoides infection, 2 wks
Systemic dissemination of GFP-positive
(cytokine-competent) cells
Local induction of GFP/huCD2-positive
(cytokine-secreting) cells
huCD2
GFP
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T cells and basophils are IL-4 secreting cells
KN2 x 4get mice
Primed
Naive
Activated
Basophil activation in involved tissues
IL-4 secretion
IL-4-secreting cells in lung
Brandon Sullivan
IL-4 secretion
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(No Transcript)
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Focal aggregates of arginase-1 aaM? in d9
Nippo-infected lung
Infected
Uninfected
Lung 4x, DAPI YFP
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IL-4/IL13-dependent genes in allergic
immunopathology
Vertebrate chitinase-like family
David Voehringer, Tiffany Reese, David Erle,
Xiaozhu Huang
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Vertebrate chitinase and chi-lectin family Gene
duplication at two syntenic loci
AP Bussink et al., Evolution of mammalian
chitinase(-like) members of family 18 glycosyl
hydrolases. Genetics 177959-70 2007.
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Chitin
Chitinases

• Abundant biopolymer (2nd to cellulose)
• Fungi, crustaceans, insects, worms
• Not present in vertebrates

• Enzymes that degrade chitin
• Conserved in plants, parasites, humans
• AMCase, chitotriosidase

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Chitin in the helminth feeding organ
Zhang et al., Develop Biol 285330, 2005
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Staining N. brasiliensis with a fluorescent CBD
probe
Eggs
Pharynyx
Steve Van Dyken
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Chitin induces eosinophil and basophil recruitment
3 hr 6 hr 24 hr
72 hr
Red Chitin-binding probe Green GFP (IL-4
expression) Blue DAPI
TA Reese et al., Nature 44792, 2007
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Chitin induces aaM? in tissues
attenuated by over-expression of AMCase
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Chitin and ?-glucans in Aspergillus hyphae
Chs Chitinase Qzm quantizyme
(?-glucanase) CBD chitin-binding domain
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Insoluble constituents of Aspergillus hyphae
recruit innate immune cells to the lung
MeanSEM n3 plt0.01 plt0.001
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Attenuation of lung eosinophil recruitment by
fungal hyphae by enzymatic digestion or
transgenic chitinase expression
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Is chitin recognition in the lung due to its
particulate nature?
Chitin
3 hrs
24 hrs
6 hrs
48 hrs
Red Chitin-binding probe Green GFP
(4get) Blue DAPI
Polystyrene beads
Orange Polystyrene Green GFP (4get) Blue
DAPI
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Particle recruitment of innate IL-4/IL-13-producin
g cells to the lung
Basophils
Total cells
Eosinophils
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Does chitin play a role in human asthma?
Snow crab processors asthma Cartier A et
al., Prevalence of crab asthma in crab plant
workers in Newfoundland and Labrador. Internat J
Circumpolar Health 2004, 63 Suppl 2333-6. Ortega
HG, et al., Respiratory symptoms among crab
processing workers in Alaska epidemiological
and environmental assessment. Am J Ind Med 2001,
39598-607. Malo JL et al., Patterns of
improvement in spirometry, bronchial
hyperresponsiveness and specific IgE antibody
levels after cessation of exposure in
occupational asthma caused by snow-crab
processing. Am Rev Respir Dis 1988,
138807-812. CDC, Asthma-like illness among
crab-processing workers - Alaska. MMWR 1982,
3195-96.
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Common human AMCase variants segregate with
asthma risk
Glycoside Hydrolase Family 18 Domain
Active Site
Signal Peptide
Chitin Binding Domain
Hinge
1 2 3 4 5 6 7
8
Asn Asp Arg Gly Arg Ile Phe Gly
Non-protective
1
Asn Asp Arg Gly Arg Val Phe Val
Non-protective
2
Asn Asp Arg Gly Arg Val Ser Val
Non-protective
3
Asthma Protection
Asp Asn Met Gly Arg Val Ser Val
Seibold M et al., J Biol Chem 28419650, 2009
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AMCase variants display disparate chitinase
activities in vitro
Seibold M et al., J Biol Chem 28419650, 2009
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BF Dickey. 2007. Exoskeletons and exhalation.
New Engl J Med 3572082.
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Asthma Triad
1. Reversible episodes airway obstruction (
mucus, eosinophils, T cells with IL-4, IL-5,
IL-13, GM-CSF, etc.) 2. Chronic airway
inflammation ( T cells, eosinophils with
type 2 cytokines chronic subepithelial,
epithelial changes with mucus cell
hyperplasia) 3. Bronchial hyperreactivity with
provocative agents
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Asthma Genetics
1. Polygenic, but few. 2. Dyzygotic 15
concordance Monozygotic 75 concordance 3.
Linkage Studies
Site Proposed Candidate
Disease 13q14 CysLT2 Atopy
5q31-33 Type 2 cytokine cluster Asthma,
atopy 16p12 IL-4Ra Atopy X
IL-13RA1, IL-13RA2 IgE, Asthma 11q
FceRI b Atopy 12q13-14 STAT6
Atopy 3q25 Bcl6 Atopy
Recent ADAM33 (metalloproteinase), DPP10
(dipeptidyl peptidase), PHF11 (Txn factor), GPRA
(orphan GPCR in epithelial cells), AMCase
(Stat6-inducible chitinase)
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Genome wide associations

• 21 genes implicated through SNP associations,
  all with low predictive value.
• ORMDL3 transcript levels altered but multiple
  other candidate genes.

AJ Rogers et al. 2009. Assessing the
reproducibility of asthma candidate gene
associations, using genome-wide data. Am J Resp
Crit Care Med 1791084, 2009.
Epidemiologic associations
Severe rhinovirus or RSV infection in first 1-3
yrs of life. Chr 17q21 SNPs associated with
passive smoking. Inverse relationship with
bacteria in first 3-6 months of life.
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The hygiene hypothesis
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Mouse 'Asthma'
OVA
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Accumulation of Th2, eosinophils and basophils in
lung tissues of OVA-sensitized mice
Airway Hyperreactivity
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Mouse Asthma Th2-associated disease
Resistant IL-4 ko, IL-13 ko, IL-4Ra ko,
STAT6 ko, dominant neg. GATA-3, T cell
deficient Spontaneous Lung IL-4 Tg, IL-13 Tg,
IL-5 Tg, IL-9 Tg, TSLP Tg No Effect No B
cells, no IgE, no eosinophils, no mast cells
Caveat Measure acute changes,
not chronic remodeling
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Eosinophils and airway remodeling
Less subepithelial and interstitial collagen
deposition in sensitized mice lacking eosinophils
(Humbles et al., Science 3051776, 2004)
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Therapy of human asthma
In clinic Steroids 5-LO inhibitors (LTA4
synthesis blockers) CysLTI antagonists (LTD4
blockers) Monoclonal humanized anti-IgE mAb
(E25) In testing Anti-IL-5 (decrease eos,
decrease collagen, little change airway
hyperreactivity) Suplatast tosilate
(suppresses IL-4, IL-5 from Th2 cells) CDP840,
phosphodiesterase type 4 inhibitor Soluble IL-4
receptor, IL-4/13 analog inhibitor OX40L
blockade anti-TNF (steroid resistant asthma),
IL-17 inhibitors
Hendeles L, et al. 2004. Evaluation of cytokine
modulators for asthma. Pediatric Resp Rev 5
(suppl A), S107-12.
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Humanized anti-IgE
Mouse anti-IgE
Human IgG1 k
Mutate Complement-fixing domain
98 Human (3 aa difference)
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Humanized anti-IgE Trial
Anti-IgE (2.5 - 5 mg/kg) Every 2 wks x 20
wks Placebo
300 pts.
Results 1. IgE gt95 within 1 hr (gt1000 ng/ml
10 ng/ml) 2. Sx, drug use 3.
IgE remained low many weeks after stopping
infusion 4. FceRI expression 5. No
antibodies.
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Allergen
DC maturation and OX40L expression
TSLP
Th2 cells
IL-25
IL-33
Mast cell/basophil activation
Enigmacyte activation
Cytokines, chemokines, eicosanoids, vasoactive
amines
IL-13, IL-5
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J Immunol 169443, 2002
J Exp Med 2031105, 2006
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Live (DAPI)
IL-4/13 competent
Eosinophils (4get, SSChi, SigF)
Basophils (4get, IgE, DX5)
Cells
IL-4/13 competent IL-4 secreting
Th2 cells (4get, CD4)
4getCD4-
IL-4/13 competent IL-4/13 secreting
Enigmacytes (4get, SSClo CD4-, IgE- DX5-)
IL-4/13 competent IL-13 secreting
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Working model for the organization of allergic
immunity
TSLP, IL-25, IL-33
OX-40L
DC
IL-4
IL-13, IL-5
Tissue
Lymph node
IL-4, IL-13, IL-5, IL-9, IL-3 Eosinophil
recruitment Mast cell activation Basophil
expansion AAM? activation
Memory
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The End