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

4. Unifying Concept for Understanding TH2-cell Sensitization Hammad et al. Nature Reviews Immunology 2008 TGF-  1 Other cell types: fibrocytes?, MSCs? (I) (III)(II)

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

6. TGF-  1 (?Eosinophils, fibroblasts…) MSCs in Epithelial Repair in Asthma

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

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

9. * No. of migrated cells TGF-β1, ng/ml ** * 20.0 10.0 5.02.5 DMEM ECM TGF-β1 Induced MSC Migration in a Dose-Dependent Manner

10. Score Saline CRE ** SalineCRE H&E PAS B C D E TGF β 1, ng/ml ** Establishment of a Cockroach Allergen-Induced Asthma Mouse Model: 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. 24h CRE, i.p CRE, i.n. Sacrifice 7 02221 Day 23 A

11. AB 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 challenge

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

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

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

15. 24h CRE/alum Sensitization, i.p CRE Challenge, i.n. Sacrifice 7 02221 20 Day TGFβ Ab, i.p 0.25 mg/mouse 23 CRE IgG CER TGFβ1 Ab GFP + -MSCs CRE TGFβ Ab PBS GFP + -MSCs (2x10 6, i.v.) CRE *** GFP - -MSCs MSCs Mobilize to the Lungs from Peripheral Blood through TGFβ1 A BC

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

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

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

19. MedCRE Med CRE MSC CRE MSC CRE MSC CRE MSC *** AB C MedCREMSCCRE MSC D ** Modulatory effects of MSCs on CRE-induced T responses in vitro

20.  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

21.  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

22. Genetic Marking Strategy for in vivo Analysis of Individual Nestin + Cells Nestin/GF P C DE

23. FSC SSC CD45 FSC Thy1.2 lin GATA3 RORgt CD45+ 65% CD45+lin- Thy1.2+ 1.62% ILC3 7.2% ILC2 69.8% No. of ILC2 cells (x10 4 ) A B C ** No. of ILC3 cells (x10 3 ) PBS CRE Increased Innate Lymphoid Cells (ILCs)-2 and 3 in CRE Induced Mouse Model

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