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