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Transcript of Functional Effects of TGF-beta1 on Mesenchymal Stem Cell Mobilization in Cockroach Allergen Induced...
Functional Effects of TGF-beta1 on Mesenchymal Stem Cell Mobilization in Cockroach Allergen
Induced Asthma
Pei-Song Gao, MD, PhD
Division of Allergy & Clinical Immunology
Johns Hopkins University School of Medicine
Cockroach Allergy and Asthma
In the US, cockroach allergy is most prevalent in urban areas and inner cities (17-41%).
23-60% of asthmatics who live in urban areas are allergic to cockroaches (Gruchalla et al. JACI 2005).
Cockroach sensitization and exposure is an important risk factor for developing asthma (Rosenstreich et al NEJM 1997).
Cockroach-specific immunotherapy for asthma is being conducted under the NIAID-funded Inner-City Asthma Initiatives (ICACII, 2009-2013) (Togias et al. JACI 2010).
Bla g1 DNA vaccine has been suggested to be effective in the treatment of allergic airway inflammation in mouse model (Zhou et al. Allergy 2012).
JACI 2011;128(2):284-92 e7
Among all tested allergens, sensitization to cockroach allergen was more commonamong children (cases and controls) living in the HAPNs than LAPNs (23.7% vs 10.8%).
HAPN: high asthma prevalence neighborhood
LAPN: low asthma prevalence neighborhood
Household incomeOlder apartment
Asthma
Allergic sensitization
Allergen exposure(cockroach, mouse, dust mite)
HYPOTHESIS
Unifying Concept for Understanding TH2-cell Sensitization
Hammad et al. Nature Reviews Immunology 2008
TGF-1
Other cell types: fibrocytes?, MSCs?
(I)
(III)(II)
(B)
(A)
(C)(D)
Cockroach Allergen Exposure, Together with Environmental Chemicals, Contributes to the development of Asthma
Hypothesis
TGF-1 (?Eosinophils, fibroblasts…)
MSCs in Epithelial Repair in Asthma
Epithelium-Conditioned Medium (ECM) Prepared by Cockroach Extract (CRE) Challenged Epithelium Induces MSC Migration
PTFE - membranemedium BEC
Un-challenged
MSCs
DMEM
Un-challenged ECM
Challenged ECM
CREChallenged
Migrated MSCs
Filter
DMEM Un-
challenged
CRE-
challenged
*
No
of
mig
rate
d c
ell
s
Air-liquid interface Transwell assays Migration
A B
*
*
DMEM
CRE-challe
nged
Un-
chal
leng
ed
-
IgG
TGFβ
1 Ab
SDF1α
Ab
CRE-challenged
Un-
challe
nged
Un-
chal
leng
ed
-
0.5
µM
1.0
µM
2.0
µM
SB505124
CRE-challenged
**
*
* **
C
No
of
mig
rate
d c
ells
TG
Fβ
1 (
pg
/ml)
No
of
mig
rate
d c
ells
TGF-β1 Signaling in ECM-Induced MSC Migration
*
TGF-β1, ng/ml
**
*
20.0
10.0 5.0
2.5
DMEM
ECM
TGF-β1 Induced MSC Migration in a Dose-Dependent Manner
Saline
Saline
CRE
CRE
**
**
Saline CREB
C
D
E **
Establishment of a Cockroach Allergen-Induced Asthma Mouse Model:
A: Protocol for mouse models of asthmaB:H&E stained sections C: PAS stained sections D: Dense peribronchial infiltrates scored by the number of infilitrates.E: Serum levels of active TGFβ1. **P<0.01.
24h
CRE, i.p CRE, i.n. Sacrifice
70 2221Day 23A
A B
C
MSCs in Lungs of Cockroach Extract (CRE)-Challenged Mice
A: Nestin+ cells in airway epithelial cells from mice after CRE challenge. B: Nestin+ cells in airway epithelial cells from saline treated mice.C: Nestin+ cells were also observed in the airway sub-epithelial region of human patients with allergic asthma.
MSCs are increased in airway after allergen sensitization and challengeMSCs are increased in airway after allergen sensitization and challenge
A B
PBS CRE
P-S
mad
2/3
C
PBSPBS CRE CRE
D
****
Increased Activation of TGFβ1 Signaling in Lung Tissue of Allergic Asthma
Characterization of Mouse GFP+ MSCs
CD
29
CD
45
Sca1
CD11b
A B
CREMedium
αSMA
DAPI
Merge
GFP+ Sca-1+CD29+
CD45– CD11b–
αSMA
NestinαSMA
Alizarin red Oil-red Toluidine blue
Control Differentiated DifferentiatedControl
C
Increased Activation of TGFβ1 Signaling in CRE-treated MSCs
24h
CRE/alumSensitization, i.p
CRE Challenge, i.n.
Sacrifice
70 222120Day
TGFβ Ab, i.p 0.25 mg/mouse
23
CREIgG
CERTGFβ1 Ab
GFP+-MSCs
GFP+-MSCsGFP+-MSCs
CRETGFβ Ab
PBS
GFP+-MSCs (2x106, i.v.)
CRE ** *
GFP--MSCs
MSCs Mobilize to the Lungs from Peripheral Blood through TGFβ1
A
B C
A B
*
**
PBS CRE+TGFβ1 AbCRE+IgG1
D
C
*
TGFβ1 Neutralizing Antibody Inhibits Airway Inflammation
GFP+ cells and Cytokines in the Airways of CRE-Challenged or Saline-Treated Nes-GFP mice
PBS CRE+TGFβ1 Ab
CRE+IgG1
PBS CREIgG1
CRETGFβ1 Ab
***A B
CD4+CD25+Foxp3+ Cells from CRE-Challenged Mice Treated with TGFβ1 Antibody
Med CRE
Med
Med
CRE
CREMSC
MSC
MSCCREMSC
CREMSC
CREMSC
****
** **
A B
C
Med CRE MSC CREMSC
D
**
Modulatory effects of MSCs on CRE-induced T responses in vitro
MSCs are accumulated in lung tissue of CRE-challenged mice and asthmatic patients.
TGFβ1 signaling is activated in lung tissue of allergic asthma.
TGFβ1 mediates MSCs migration induced by human epithelium conditioned medium.
TGFβ1 mediates the recruitment of MSCs to the lungs in asthma.
TGFβ1 limits the allergic inflammation in mouse models of asthma.
MSCs modulate T responses to CRE in vitro.
Summary
Determine the role of active TGFβ1 in the recruitment of MSCs to the lung in asthma.
Track the lineage commitment/differentiation of recruited MSCs in lungs
Investigate the role of MSCs in CRE-induced allergic inflammation (CRE-MSCs-ILCs)
Ongoing Research Studies
Genetic Marking Strategy for in vivo Analysis of Individual Nestin+ Cells
Nestin/GFP
Nestin/GFP
C D E
FSC
SS
C
CD45
FS
C
Thy1.2
lin
GATA3
RO
Rg
t
CD45+ 65%CD45+lin-Thy1.2+ 1.62%
ILC3 7.2%
ILC2 69.8%
No
. of
ILC
2 ce
lls(x
104
)
A
B C **
**N
o. o
f IL
C3
cells
(x10
3)
PBS PBSCRE CRE
Increased Innate Lymphoid Cells (ILCs)-2 and 3 in CRE Induced Mouse Model
Acknowledgments
Division of Allergy & Clincal Division of Allergy & Clincal ImmunologyImmunology
Yufeng ZhouTing XuBeverly PlunkettAllen MeyersShau-Ku Huang
Funded by
NIH Grants (Peisong Gao): 1R21AI109062 1RO1ES021739
Funded by
NIH Grants (Peisong Gao): 1R21AI109062 1RO1ES021739
Department of Orthopedic Department of Orthopedic SurgerySurgery
Lingling XianChangjun LiZhuang CuiMei WanXu Cao