Functional Effects of TGF-beta1 on Mesenchymal Stem Cell Mobilization in Cockroach Allergen Induced Asthma

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1 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

2 Cockroach Allergy and Asthma In the US, cockroach allergy is most prevalent in urban areas and inner cities (17-41%) % 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, ) (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).

3 HYPOTHESIS Household income Older apartment Among all tested allergens, sensitization to cockroach allergen was more common among children (cases and controls) living in the HAPNs than LAPNs (23.7% vs 10.8%). Allergen exposure (cockroach, mouse, dust mite) Allergic sensitization Asthma HAPN: high asthma prevalence neighborhood LAPN: low asthma prevalence neighborhood JACI 2011;128(2): e7

4 Unifying Concept for Understanding TH2-cell Sensitization (II) (III) (I) TGF-β1 Other cell types: fibrocytes?, MSCs? Hammad et al. Nature Reviews Immunology 2008

5 Hypothesis Cockroach Allergen Exposure, Together with Environmental Chemicals, Contributes to the development of Asthma (B) (A) (C) (D)

6 MSCs in Epithelial Repair in Asthma TGF-β1 (?Eosinophils, fibroblasts )

7 Epithelium-Conditioned Medium (ECM) Prepared by Cockroach Extract () Challenged Epithelium Induces MSC Migration medium PTFE - membrane BEC ECM MSCs Migrated MSCs Unchallenged DMEM Un-challenged ECM No of migrated cells * Challenged Filter Challenged ECM Air-liquid interface Transwell assays Migration

8 TGF-β1 Signaling in ECM-Induced MSC Migration A B C * * * * ** ** TGFβ1 (pg/ml) No of migrated cells No of migrated cells -challenged SB challenged

9 TGF-β1 Induced MSC Migration in a Dose-Dependent Manner No. of migrated cells ** * * TGF-β1, ng/ml

10 Establishment of a Cockroach Allergen-Induced Asthma Mouse Model: A Day h, i.p, i.n. Sacrifice B Saline D ** H&E Score C PAS E TGFβ1, ng/ml Saline Saline ** A: Protocol for mouse models of asthma B: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.

11 MSCs in Lungs of Cockroach Extract ()-Challenged Mice A B C A: Nestin+ cells in airway epithelial cells from mice after 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 challenge

12 Increased Activation of TGFβ1 Signaling in Lung Tissue of Allergic Asthma A ** B ** PBS PBS C PBS D P-Smad 2/3

13 Characterization of Mouse GFP + MSCs A GFP + Sca-1 + CD29 + CD45 CD11b B Alizarin red Oil-red Toluidine blue αsma αsma Nestin Control Differentiated Control Differentiated CD29 C αsma Medium Sca1 DAPI CD45 Merge CD11b

14 Increased Activation of TGFβ1 Signaling in -treated MSCs

15 MSCs Mobilize to the Lungs from Peripheral Blood through TGFβ1 A GFP + -MSCs (2x10 6, i.v.) Day /alum Sensitization, i.p TGFβ Ab, i.p 0.25 mg/mouse 23 Challenge, i.n. 24h Sacrifice B GFP - -MSCs GFP + -MSCs C PBS ** * IgG GFP + -MSCs CER TGFβ1 Ab GFP + -MSCs TGFβ Ab

16 TGFβ1 Neutralizing Antibody Inhibits Airway Inflammation A B ** * * C PBS +IgG1 +TGFβ1 Ab Eos H&E D

17 GFP + cells and Cytokines in the Airways of -Challenged or Saline-Treated Nes-GFP mice A Nes-MSC Cs PBS +IgG 1 +TGFβ1 Ab Total no. of MSCs B ** * PBS IgG1 TGFβ1 Ab

18 CD4 + CD25 + Foxp3 + Cells from -Challenged Mice Treated with TGFβ1 Antibody

19 Modulatory effects of MSCs on -induced T responses in vitro A ** ** B Med MSC Med MSC MSC MSC C ** D * * ** Med MSC MSC Med MSC MSC

20 Summary MSCs are accumulated in lung tissue of -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 in vitro.

21 Ongoing Research Studies 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 -induced allergic inflammation (-MSCs-ILCs)

22 Genetic Marking Strategy for in vivo Analysis of Individual Nestin + Cells C D E Nestin/GFP Nestin/GF P

23 Increased Innate Lymphoid Cells (ILCs)-2 and 3 in Induced Mouse Model A SSC FSC CD45+ 65% lin CD45+lin- Thy % RORgt ILC3 7.2% ILC2 69.8% FSC CD45 Thy1.2 GATA3 B No. of ILC2 cells (x10 4 ) ** C No. of ILC3 cells (x10 3 ) ** PBS PBS

24 Acknowledgments Division of Allergy & Clincal Immunology Yufeng Zhou Ting Xu Beverly Plunkett Allen Meyers Shau-Ku Huang Department of Orthopedic Surgery Lingling Xian Changjun Li Zhuang Cui Mei Wan Xu Cao Funded by NIH Grants (Peisong Gao): 1R21AI RO1ES021739