Volume 23, Issue 3, Pages 561-569 (March 2015) Depletion of Bone Marrow CCSP-Expressing Cells Delays Airway Regeneration  Martha L Bustos, Marco Mura, David Hwang, Olga Ludkovski, Amy P Wong, Armand Keating, Thomas K Waddell  Molecular Therapy  Volume 23, Issue 3, Pages 561-569 (March 2015) DOI: 10.1038/mt.2014.223 Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Bone marrow CCSP+ cells extend survival after ablation of CCSP+ cells in lung. (a) We transplanted female CCtk mice with male wild-type (FVBn; green) or CCtk (purple) bone marrow (BM) cells. Two months later, mice were treated with ganciclovir to eliminate CCSP+ cells in lung and bone marrow. (b) Quantitative RT-PCR for Sry gene to corroborate the presence of male cells in the blood of female recipients 60 days after transplantation. (c) Quantitative RT-PCR to analyze the expression of CCSP in blood cells at 0 and 10 days after ganciclovir treatment. (d) Survival curve after ganciclovir treatment; the control group (blue) consisted in FVBn mice transplanted with FVBn BM. After 12 days of ganciclovir, we analyzed the (e) total cell count and the differential cell count of inflammatory cells (f) in bronchoalveolar lavage (BAL). (g) Quantitative RT-PCR using lung samples to analyze the expression of markers for Clara cells CCSP, Cyp2f2, Pon1, and Aox3; the type II pneumocyte Sftpc and the ciliated cell Foxj1. Confocal microscopic analysis of CCSP (green) in the lung of mice transplanted with (h) CCtk or (i) FVBn BM and graph showing the number of (j) CCSP+ cells per airway in each group. Representative photomicrographs of hematoxylin and eosin stained sections of airways from mice transplanted with (k) CCtk or (l) FVBn bone marrow. Data shown are means ± SD; n = 8 mice per group. Insets are representative isotype staining controls. Scale bar represents 40 μm. *P < 0.05, ***P < 0.0005. Molecular Therapy 2015 23, 561-569DOI: (10.1038/mt.2014.223) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Ablation of bone marrow CCSP+ cells delays lung recovery after naphthalene injury. (a) Female FVBn mice were transplanted with male wild-type (FVBn; green) or CCtk (purple) bone marrow (BM) cells. Sixty days later, mice were treated with ganciclovir to eliminate CCSP+ cells in the bone marrow. Ten days after ganciclovir exposure, mice were injected with naphthalene (NAP) to induce lung injury. (b) Quantitative RT-PCR for Sry gene to corroborate the presence of male cells in the blood of female recipients 60 days after transplantation. (c) Quantitative RT-PCR to analyze the expression of CCSP in blood cells at different time points showing a transient increase in CCSP expression peaking at 2 days of naphthalene injury in the FVBn group, while mice transplanted with CCtk BM didn’t have CCSP+ cells in blood after ganciclovir treatment. Five days after naphthalene injury, the CCtk group had lower levels of oxygen in the arterial blood (d) (PaO2/FiO2 ratio), more inflammatory cells in (e) BAL, with increased numbers of (f) macrophages, (g) lymphocytes, and (h) neutrophils. These mice also showed (i) lower expression of CCSP in the lung, compared to mice transplanted with FVBn BM. Data shown are means ± SD, n = 8 mice per group. *P < 0.05, **P < 0.005, ***P < 0.0005. Molecular Therapy 2015 23, 561-569DOI: (10.1038/mt.2014.223) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Bone marrow CCSP+ cells express epithelial markers and localize in the airways. Confocal microscopic analysis of Clara cell marker CCSP (green) 5 days after lung injury in mice transplanted with (a) CCtk or (b) FVBn bone marrow and (c) graph showing the number of CCSP+ cells per airway in each group. Lung sections from female mice transplanted with male FVBn bone marrow after naphthalene injury. The sections were stained for Y chromosome (red) using FISH and then subjected to immunohistochemistry for (d) CCSP (green) and (e) the pan-cytokeratin epithelial cell marker (yellow). Arrows point to male donor cells. Insets are representative isotype staining controls. Scale bar represents 40 and 10 μm. Data shown are means ± SD; n = 6 mice per group; *P < 0.05. Molecular Therapy 2015 23, 561-569DOI: (10.1038/mt.2014.223) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Administration of CCSP protein improves lung recovery after naphthalene injury. (a) Female FVBn mice were transplanted with male wild-type (FVBn; green) or CCtk (purple) bone marrow cells. Sixty days later, mice were treated with ganciclovir to eliminate CCSP+ cells in the bone marrow. Ten days after ganciclovir exposure, mice were injected with naphthalene (NAP) to induce lung injury and 1 day lather mice were administered CCSP protein or saline transtracheally. Five days after naphthalene injury, mice transplanted with CCtk bone marrow and treated with CCSP protein had a higher level of oxygen in the arterial blood (b) (PaO2/FiO2 ratio) less inflammatory cells in (c) BAL, with reduced numbers of (d) macrophages and (e) neutrophils. These mice also showed (f) increased expression of CCSP, Cyp2f2, Pon1, Aox, and Fmo in the lung, compared to mice transplanted with CCtk BM and administered with saline. Data shown are means ± SD; n = 8 mice per group. *P < 0.05, **P < 0.005, ***P < 0.0005. Molecular Therapy 2015 23, 561-569DOI: (10.1038/mt.2014.223) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Administration of CCSP protein increased the recovery of the airway epithelium after injury. Confocal microscopic analysis of Clara cell marker CCSP (green) 5 days after lung injury in FVBn mice transplanted with CCtk bone marrow and administered with (a) saline or (b) CCSP and (c) graph showing the number of CCSP+ cells per airway in each group. Lung samples from mice transplanted with FVBn bone marrow and administered with (d) saline or (e) CCSP and (f) graph showing the number of CCSP+ cells per airway in each group. Insets are representative isotype staining controls. Scale bar represents 40 μm. Data shown are means ± SD; n = 6 mice per group; **P < 0.005. Molecular Therapy 2015 23, 561-569DOI: (10.1038/mt.2014.223) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions