Volume 18, Issue 9, Pages 1706-1713 (September 2010) Macrophages Expressing Heme Oxygenase-1 Improve Renal Function in Ischemia/Reperfusion Injury  David A Ferenbach, Vasudev Ramdas, Nishrin Spencer, Lorna Marson, Ignacio Anegon, Jeremy Hughes, David C Kluth  Molecular Therapy  Volume 18, Issue 9, Pages 1706-1713 (September 2010) DOI: 10.1038/mt.2010.100 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Adenoviral transduction results in expression of HO-1 protein in Mϕ. (a) Western blotting demonstrates potent induction of HO-1 protein expression in BMDM following increasing multiplicity of infection with Ad-HO-1. (b) HO-1 transduction was associated with increased bioactivity as shown by degradation of heme to bilirubin (n = 8/group). BMDM, bone marrow–derived Mϕ. Molecular Therapy 2010 18, 1706-1713DOI: (10.1038/mt.2010.100) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Transduction of HO-1 in Mϕ results in altered responses to classical activating stimuli. Transduction with Ad-HO-1 results in significantly reduced (a) Mϕ NO and (b) TNFα production, with augmented (c) IL-10 in response to stimulation with IFNγ+LPS (n = 9/group; all P < 0.001 versus all groups by analysis of variance). Molecular Therapy 2010 18, 1706-1713DOI: (10.1038/mt.2010.100) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Transduction of HO-1 in Mϕ results in increased phagocytosis of apoptotic cells in vitro. Transduction with Ad-HO-1 results in (a) augmented phagocytosis of apoptotic cells (ACs) (n = 9/group; P = 0.0032) and (b) increased phagocytic index (n = 9/group; P = 0.0093). Phagocytosis was quantified visually using colocalization of Lysotracker Red–labeled Mϕ and CM-Green-labeled apoptotic cells to confirm presence of the apoptotic cells within a phagolysosome. Photomicrographs of Mϕ following a 30-minute interaction with apoptotic cells [(c) control unmodified Mϕ, (d) Ad-HO-1 Mϕ; phagocytosed ACs shown with white arrows]. BMDM, bone marrow–derived Mϕ. Molecular Therapy 2010 18, 1706-1713DOI: (10.1038/mt.2010.100) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Intravenously administered Mϕ home to the injured kidney after IRI. (a) IV administered Mϕ localize selectively to the injured kidney compared to the contralateral uninjured control (P = 0.0048, n = 6/group). (b) Maximal localization is seen at 1 hour after injection. PKH+ Mϕ can be visualized within the interstitium of the injured renal medulla at (c) 1 and (d) 24 hours (original magnification: ×200, blue: DAPI nuclear stain, green: tissue autofluorescence, red: PKH26+ Mϕ). Medullary F4/80+ cell counts (mean per ×400 field) are augmented in animals receiving IV Mϕ. (e) Exogenously delivered Mϕ were identified by PKH+ fluorescence, and comprise between 35 and 58% of total medullary Mϕ post-IRI. IRI, ischemia/reperfusion injury. Molecular Therapy 2010 18, 1706-1713DOI: (10.1038/mt.2010.100) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Treatment with Ad-HO-1 transduced Mϕ results in functional protection after IRI at comparable levels of acute tubular necrosis. (a) Cell therapy with Ad-HO-1 transduced Mϕ after experimental IRI has no impact on severity of tubular necrosis but (b) results in significant preservation of renal function 24 hours after injury (all groups n = 9–12; P < 0.05). IRI, ischemia/reperfusion injury. Molecular Therapy 2010 18, 1706-1713DOI: (10.1038/mt.2010.100) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Animals treated with Ad-HO-1 transduced Mϕ exhibit increased clearance of renal platelet deposition. Treatment with HO-1 expressing Mϕ has no effect on immediate levels of platelet deposition compared with control Mϕ at 1 hour post-IRI, while resulting in significant reduction in platelet deposition at 24 hours post-IRI [(a) Adβgal Mϕ at 1 hour; (b) Ad-HO-1 Mϕ at 1 hour, (c) Adβgal Mϕ at 24 hours; (d) Ad-HO-1 Mϕ at 24 hours] demonstrated in frozen sections stained with CD41 mAb (original magnification: ×200). (e) Mean area of platelet staining was quantified by image analysis and expressed as % hpf positive for CD41. HO-1, heme oxygenase-1; IRI, ischemia/reperfusion injury. Molecular Therapy 2010 18, 1706-1713DOI: (10.1038/mt.2010.100) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Proposed schema of renal dysfunction in the aftermath of IRI. Dotted arrows indicate pathways potentially ameliorated by actions of HO-1 expressing Mϕ. IRI, ischemia/reperfusion injury. Molecular Therapy 2010 18, 1706-1713DOI: (10.1038/mt.2010.100) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions