Pei-Song Gao, MD, PhD

1. Functional Effects of TGF-beta1 on Mesenchymal Stem Cell Mobilization inCockroach 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%). • 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).

3. 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%). HYPOTHESIS Household income Older apartment Allergen exposure (cockroach, mouse, dust mite) Allergic sensitization HAPN: high asthma prevalence neighborhood LAPN: low asthma prevalence neighborhood Asthma JACI 2011;128(2):284-92 e7

4. Unifying Concept for Understanding TH2-cell Sensitization (II) (III) (I) TGF-b1 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) (D) (C) (A)

6. MSCs in Epithelial Repair in Asthma TGF-b1 (?Eosinophils, fibroblasts…)

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

8. A B C * TGF-β1 Signaling in ECM-Induced MSC Migration * * No of migrated cells No of migrated cells ** TGFβ1 (pg/ml) DMEM * - CRE-challenged IgG Un- challenged ** Un-challenged SDF1α Ab TGFβ1 Ab CRE-challenged - 0.5 µM 1.0 µM 2.0 µM Un-challenged SB505124 CRE-challenged

9. TGF-β1 Induced MSC Migration in a Dose-Dependent Manner No. of migrated cells * 5.0 2.5 ECM 20.0 DMEM 10.0 TGF-β1, ng/ml ** *

10. Establishment of a Cockroach Allergen-Induced Asthma Mouse Model: A Score 23 24h Saline CRE Day B D 0 21 22 7 ** Saline CRE Sacrifice CRE, i.n. CRE, i.p H&E E C ** ** Saline CRE TGFβ1, ng/ml PAS 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 (CRE)-Challenged Mice A B C 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 challenge

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

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

14. Increased Activation of TGFβ1 Signaling in CRE-treated MSCs

15. MSCs Mobilize to the Lungs from Peripheral Blood through TGFβ1 GFP+-MSCs (2x106, i.v.) GFP+-MSCs A 23 Day 20 0 21 22 7 24h TGFβ Ab, i.p 0.25 mg/mouse CRE/alum Sensitization, i.p CRE Challenge, i.n. Sacrifice GFP--MSCs B C PBS CRE ** * CRE IgG CER TGFβ1 Ab GFP+-MSCs GFP+-MSCs CRE TGFβ Ab

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

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

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

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

20. Summary • 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.

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 CRE-induced allergic inflammation (CRE-MSCs-ILCs)

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

23. Increased Innate Lymphoid Cells (ILCs)-2 and 3 in CRE Induced Mouse Model A CD45+lin-Thy1.2+ 1.62% ILC3 7.2% CD45+ 65% ILC2 69.8% RORgt C B SSC lin FSC No. of ILC2 cells (x104) No. of ILC3 cells (x103) ** ** GATA3 FSC CD45 Thy1.2 CRE PBS PBS CRE

24. Acknowledgments Department of Orthopedic Surgery Lingling Xian Changjun Li Zhuang Cui Mei Wan Xu Cao Division of Allergy & Clincal Immunology Yufeng Zhou Ting Xu Beverly Plunkett Allen Meyers Shau-Ku Huang • Funded by • NIH Grants (Peisong Gao): • 1R21AI109062 • 1RO1ES021739