1. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 THE ROLE OF MACROPHAGES IN FIBROSIS MICHAEL R. BLACKBURN, PhD LUNG REMODELING AND REGENERATION FRIDAY NOVEMBER 13, 2015

2. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 THE DIVERSITY OF MACROPHAGES ©2015 Pulmonary Fibrosis Foundation. All rights reserved.2 web.mit.edu - generated in bone marrow - specialized function in tissues Brock, T.G. Cayman Chemicals - diverse activation and function in host defense and disease - roles in wound healing and tissue remodeling

3. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 MACROPHAGES IN FIBROSIS ©2015 Pulmonary Fibrosis Foundation. All rights reserved.3 CD206 Arg-1 AAM Wynn & Murray. Nature Medicine. Vol 18: (2012).

4. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 Adenosine Adenosine Receptors (A 1 R, A 2A R, A 2B R, A 3 R) Cell Injury Hypoxia Pro-InflammatoryAnti-Inflammatory ? (tissue destruction/remodeling) (pro-fibrotic) (tissue protection) (barrier function) (wound healing) ©2015 Pulmonary Fibrosis Foundation. All rights reserved.4 Hypothesis: Adenosine signaling is too much of a good thing in pulmonary fibrosis.

5. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 A 2B R Signaling - pro and anti-inflammatory features - widely distributed and induced following injury (hypoxia) - low affinity for adenosine - G  s - pro- and anti-fibrotic features Hypothesis: A 2B R signaling contributes to the progression of pulmonary fibrosis. - pro-fibrotic activities on pulmonary cells - elevated in patients with chronic lung disease - tissue protective (vascular/epithelial barrier function) ©2015 Pulmonary Fibrosis Foundation. All rights reserved.5

6. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 In Vivo Antagonism of the A 2B R in Mice Exposed to Bleomycin CVT-6883 aka GS-6201 ©2015 Pulmonary Fibrosis Foundation. All rights reserved.6 Day 1Day 15 Day33 Intra-peritoneal Bleomycin Fibrosis Hypertension CVT-6883/ GS-6201 GS-6201 Diet

7. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 A 2B R Antagonism in Bleomycin-Induced Lung Injury saline + CVT-6883 bleomycin + CVT-6883bleomycin + vehicle Sun et al. (2006) J. Clin. Invest. 116, 2173 ©2015 Pulmonary Fibrosis Foundation. All rights reserved.7

8. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 Reduced Bleomycin-Induced Lung Fibrosis in A 2B R Knockout Mice Zhou et al. (2011) J. Immunol. 186, 1097 ©2015 Pulmonary Fibrosis Foundation. All rights reserved.8

9. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 A 2B R Signaling and IL-6 Levels Induction of fibrotic genes ©2015 Pulmonary Fibrosis Foundation. All rights reserved.9

10. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 A2B-DEPENDENT EXPRESSION OF IL-6 ©2015 Pulmonary Fibrosis Foundation. All rights reserved. 10 Attenuated pulmonary fibrosis is associated with decreased IL-6 production in alveolar macrophages of A 2B R knockout mice. Zhou et al. (2011) J. Immunol. 186, 1097 ELISA Immunostain

11. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 A 2B R Signaling and Alternatively Activated (M2) Macrophages lavage cell pellets primary lung macrophage cultures ©2015 Pulmonary Fibrosis Foundation. All rights reserved. 11

12. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 DECREASED IL-6 IN A2B CONDITIONAL KNOCKOUT MICE ©2015 Pulmonary Fibrosis Foundation. All rights reserved. 12 Karmouty-Quintana et al. (2015) FASEB J. 29, 50

13. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 13 Karmouty-Quintana et al. (2015) FASEB J. 29, 50 ©2015 Pulmonary Fibrosis Foundation. All rights reserved. DECREASED FIBROSIS IN A2B CONDITIONAL KNOCKOUT MICE

14. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 A 2B R and IL-6 in IPF ©2015 Pulmonary Fibrosis Foundation. All rights reserved.14

15. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 A 2B R-Dependent Expression of IL-6 in IPF staining ©2015 Pulmonary Fibrosis Foundation. All rights reserved.15

16. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 GS-6201 primary macrophages from IPF patients CD206 A 2B R-Dependent Expression of IL-6 in IPF ©2015 Pulmonary Fibrosis Foundation. All rights reserved.16 - Potential approach for targeted response to A2B antagonist in clinical trials

17. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 Working Model ©2015 Pulmonary Fibrosis Foundation. All rights reserved. 17 - A2B adenosine receptor is elevated on M2 cells - M2 A2B plays a role in IL-6 production a fibrosis - A2B antagonism attenuates active lung fibrosis - A2B is a potential target for IPF therapy - M2 macrophages are prevalent in lung fibrosis Conclusions

18. PFF Teal = 0+160+175 MAIN COLORS PFF Green = 120+162+47 Light Green = 193+216+47 Red = 242+102+73 HIGHLIGHT COLORS Light Grey = 220+220+210 Dark Grey = 100+98+76 Black = 0+0+0 Jose Molina Dewan Zeng (Gilead Scientific) Luiz Belardinelli (Gilead Scientific) Thuy Le Funding Tingting Weng Hongyan Zhong (Gilead Scientific) Harry Karmouty-Quintana Blackburn Lab Collaborators Jonathan Davies Mathias Loebe Luis Garcia Raquel Bunge Brian Bruckner Methodist Hospital Ning Chen Holger Eltzschig (UC Denver) RO1-HL070952-12 PO1-HL114457-01 Jay Murthy (UTHealth) Richard Castriota (UTHealth) Soma Jyothula (UTHealth) Kemly Philips Frank Lou Kelly Volcik Yang Zhou Gilead National Heart, Lung, and Blood Institute