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Biotechnology Applications
The 1950’s
GM Firebird III
Public Health - Sanitation
Public Health - Vaccination
Polio
Allergy and Asthma Epidemic in Developing Countries
Devereux G. 2006. Nature Rev Immunol 6;869-874.
Hygiene Hypothesis
early childhood infections inhibit the tendency to develop allgergic disease
Helminth Infections & Allergies: Inverse Correlation
Helminth infections are associated with a decreased
incidence of allergic disease in industrialized areas
% Population*
Helminth
Infection**
Allergic Disease***
Globally
~ 25%
~ 2%
United States
< 1%
~ 13%
* United States Census (www.census.gov)
** Clin Exp Immunol, 2002. 128(3): p. 398-404
*** Lancet, 1998. 351(9111): p. 1225-32
The Hygiene Hypothesis
Hygiene Hypothesis - Infection history makes a difference
Many Helminth Species Have an Obligate Lung Stage
Filariasis
Schisto
ASCARIS
Malaria
HOOKWORM
Geohelminths
SCHISTOSOMA
INFECTION
Skin penetration
L3 larvae enter circulation
Reach lungs within a day
PULMONARY PHASE
L3 molt into L4 Larvae
Spend less than 48hrs in lung
Crawl/coughed up trachea
Swallowed down esophagus
ADULT PHASE
L4 reach small intestine
Molt into adult stage worms
Patency by day 7
RESOLUTION
Normal BALB/c will expel the
worms within 11 days
SCID maintain infection
Outline
•
Part I: Innate responses to Nb in the
lungs - Gene array, PCR
•
Part II: Persistent alterations in the
immune environment of the lung Flow cytometry
•
Part III: Molecular basis for
helminth-induced dampening of
inflammation
Innate Immune Responses to Nb in the Lung
Experimental Design
Major Gene Array Platforms
Bead Array
Illuminia
Spotted Array
Agilent
GeneChip
Affymetrix
General Scheme Affymetrix GeneChip Array
DNA
RNA
Protein
>5,500,000
features per chip
Image Courtesy of Bioteach
Innate Source of Th2 Cytokines in the Lung
lung
intestine
lung
intestine
Nb Infection Innately Induced AAM Genes
lung
intestine
lung
intestine
Polymerase Chain Reaction - PCR
Thermal
Vents
Thermus aquaticus
T. aquaticus
DNA Polymerase
30 rounds of amplification = >1,000,000,000 molecules
Real Time RT PCR results
Alternatively Activated Macrophage
Alternatively
Activated
Macrophages
T Cell Help is Required for AAAM Maintenance
Observations:
Nb induces cellular/molecular changes through
day 12 PI
Helminth-induced suppression of reactivity to
secondary challenge (Wohlleben et al. 2004; Wilson
et al. 2005; Yang et al. 2006)
Hypotheses:
• Helminth infection results in permanent
alterations to the lung’s immunological
environment
Nb Induces an Altered Lung Environment
Experimental Design
2-4
13-15
36
Granular Macrophages Contain Surfactant
day 36 PI
Flow cytometry
antibody-based
detection of
surface molecules
Day 36 PI AMs are CD11c+, F4/80+, Mrc1mid & MHC IIhi
day 36 PI
Surface Phenotype of CD11c+ AAAM
Day
0
2
4
8
13
MHC I
CD11c+ AAAM up-regulate:
MHC I & II
PDL-1
CD80/CD86
CD40
MHC II
PDL-I
PDL-2
CD80
CD86
CD1d
CD40
ICAM-1
CD1d
AAAM are Highly Phagocytic
Nb+ 0.02
Nb-
0.07
Ova-
CD11c APC
CD11c APC
22.5
9.2
Ova+*
CD11c APC
CD11c APC
3.5
5.2
Ova+*
CytoB
Ova-Alexa 448 at 2 μg/ml
for 30 min
CD11c APC
CD11c APC
CytoB - cytochalasin B
at 800 μg/ml
CD11c+ AM Expression Profile
Up-regulated >2-fold
AAM (arg1, fizz1,
Ym1/2, trem2b)
Scavenger
Receptors
(clecsf5/6/8/10, marco,
day 2 PI
mgl1/2, msr1/2)
Chemokines
(ccl4/8/9/11/12/17/24)
Cell Cycle
Lipid Metabolism
(ppar gamma, cd68,
apobec1, ch25h)
194
797
76
day 4 PI
(1,674)
(606)
277
59
524
392
day 8 PI
(1,252)
Altered Lung Environment:
Hypotheses:
• The Nb-altered lung environment
results in a modulation in responses
to secondary challenges - HDM
allergen challenge
• Gene array, PCR, histology, lung
function
Similar Numbers, Different Genes are Up-regulated
Nb-Altered Lung Down-Regulates Many More Genes
Nb-Altered Lung Decreases Airway Hyperresponsiveness
Altered Lung Environment:
Dampened Responses to Allergen
Key Findings:
• Persistent changes resulted in a reduction in
the overall inflammatory response to allergen
challenge in the lungs of Nb-infected animals
• Distinct gene expression profiles in the lungs
of NbHDM vs HDM mice
• Lower fold-increase of transcription of Th1 &
Th2 cytokines in NbHDM mice
• Resistance in the airways is significantly
depressed in the lungs of NbHDM mice
Hygiene Hypothesis
early childhood infections inhibit the tendency to develop allgergic disease
Alternatively Activated Alveolar Macrophages
(PDL 1/2 ?)
CD1d
?
IL-6
NKT
IL-21
?
Acknowledgments
Scott Lab
Josh Reece
Mark Siracusa
Kiwon Park
Sarah Coffman
Ellen Tarr
JHU
Sabra Klein
Mike Overstreet
Fidel Zavala
Mark Soloski
Bill Spannhake
Wayne Mitzner
Jon Fallica
Doug Norris
Vern Carruthers
Brian Schofield
Paul Fallon
Theresa Southard
Cory Brayton
JHU Gene Array Core Facility
Anne Jedlicka
Meg Mintz
Jason Bailey
Amanda Dziedzic
Collaborators
Joe Urban (USDA)
Andrea Keane-Myers (NIH)
Jamie Lee (Mayo Clinic)
Funding / Support
NIH NHLBI (U01 HL66623)
NIH Training Grant (T32AI007417)
Hegner, Cort Root Memorial Scholarship
Martin Frobisher Fellowship