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Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder caused by recessive mutations in GM-CSF receptor subunit α/β genes ( CSF2RA/ CSF2RB, respectively) characterized by impaired GM-CSF-dependent surfactant clearance by alveolar macrophages (AMs) resulting in alveolar surfactant accumulation and hypoxemic respiratory failure. Because hPAP is caused by CSF2RA mutations in most patients, we created an animal model of hPAP caused by Csf2ra gene disruption ( Csf2ra–/– mice) and evaluated the effects on AMs and lungs. Macrophages from Csf2ra–/– mice were unable to bind and clear GM-CSF, did not exhibit GM-CSF signaling, and had functional defects in phagocytosis, cholesterol clearance, and surfactant clearance. Csf2ra–/– mice developed a time-dependent, progressive lung disease similar to hPAP in children caused by CSF2RA mutations with respect to the clinical, physiological, histopathological, biochemical abnormalities, biomarkers of PAP lung disease, and clinical course. In contrast, Csf2ra+/– mice had functionally normal AMs and no lung disease. Pulmonary macrophage transplantation (PMT) without myeloablation resulted in long-term engraftment, restoration of GM-CSF responsiveness to AMs, and a safe and durable treatment effect that lasted for the duration of the experiment (6 mo). Results demonstrate that homozygous (but not heterozygous) Csf2ra gene ablation caused hPAP identical to hPAP in children with CSF2RA mutations, identified AMs as the cellular site of hPAP pathogenesis in Csf2ra–/– mice, and have implications for preclinical studies supporting the translation of PMT as therapy of hPAP in humans.

Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder caused by recessive mutations in GM-CSF receptor subunit α/β genes (CSF2RA/CSF2RB, respectively) characterized by impaired GM-CSF-dependent surfactant clearance by alveolar macrophages (AMs) resulting in alveolar surfactant accumulation and hypoxemic respiratory failure. Because hPAP is caused by CSF2RA mutations in most patients, we created an animal model of hPAP caused by Csf2ra gene disruption (Csf2ra(–/–) mice) and evaluated the effects on AMs and lungs. Macrophages from Csf2ra(–/–) mice were unable to bind and clear GM-CSF, did not exhibit GM-CSF signaling, and had functional defects in phagocytosis, cholesterol clearance, and surfactant clearance. Csf2ra(–/–) mice developed a time-dependent, progressive lung disease similar to hPAP in children caused by CSF2RA mutations with respect to the clinical, physiological, histopathological, biochemical abnormalities, biomarkers of PAP lung disease, and clinical course. In contrast, Csf2ra(+/–) mice had functionally normal AMs and no lung disease. Pulmonary macrophage transplantation (PMT) without myeloablation resulted in long-term engraftment, restoration of GM-CSF responsiveness to AMs, and a safe and durable treatment effect that lasted for the duration of the experiment (6 mo). Results demonstrate that homozygous (but not heterozygous) Csf2ra gene ablation caused hPAP identical to hPAP in children with CSF2RA mutations, identified AMs as the cellular site of hPAP pathogenesis in Csf2ra(–/–) mice, and have implications for preclinical studies supporting the translation of PMT as therapy of hPAP in humans.

Salamanders have been used as research models for centuries. While they exhibit a wide range of biological features not seen in mammals, none has captivated scientists like their ability to regenerate. Interestingly, axolotl macrophages have emerged as an essential cell population for tissue regeneration. Whether the same is true in other salamanders such as newt species Notophthalmus viridescens, Cynops pyrrhogaster, or Pleurodeles waltl remains to be seen. Unfortunately, regardless of the species, molecular tools to study macrophage function in salamanders are lacking. We propose that the readily available, terminally differentiated peritoneal macrophages from newts or axolotls could be used to validate molecular reagents in the study of macrophage function during tissue regeneration in salamanders.

Lens regeneration in the adult newt illustrates a unique example of naturally occurring cell transdifferentiation. During this process, iris pigmented epithelial cells (iPECs) reprogram into a lens, a tissue that is derived from a different embryonic source. Several methodologies both in vivo and in culture have been utilized over the years to observe this phenomenon. Most recently, Optical Coherence Tomography (OCT) has been identified as an effective tool to study the lens regeneration process in continuity through noninvasive, real-time imaging of the same animal. Described in this chapter are three different methodologies that can be used to observe the newt lens regeneration process both in vivo and ex vivo.

BackgroundAging and regeneration are heavily linked processes. While it is generally accepted that regenerative capacity declines with age, some vertebrates, such as newts, can bypass the deleterious effects of aging and successfully regenerate a lens throughout their lifetime.ResultsHere, we used Optical Coherence Tomography (OCT) to monitor the lens regeneration process of larvae, juvenile, and adult newts. While all three life stages were able to regenerate a lens through transdifferentiation of the dorsal iris pigment epithelial cells (iPECs), an age-related decline in the kinetics of the regeneration process was observed. Consistent with these findings, iPECs from older animals exhibited a delay in cell cycle re-entry. Furthermore, it was observed that clearance of the extracellular matrix (ECM) was delayed in older organisms.ConclusionsCollectively, our results suggest that although lens regeneration capacity does not decline throughout the lifespan of newts, the intrinsic and extrinsic cellular changes caused by aging alter the kinetics of this process. By understanding how aging affects lens regeneration in newts, we can gain important insights for restoring the age-related regeneration decline observed in most vertebrates.

Purpose To establish optical coherence tomography (OCT) as an in vivo imaging modality for investigating the process of newt lens regeneration. Methods Spectral-domain OCT was employed for in vivo imaging of the newt lens regeneration process. A total of 37 newts were lentectomized and followed by OCT imaging over the course of 60 to 80 days. Histological images were obtained at several time points to compare with the corresponding OCT images. Volume measurements were also acquired. Results OCT can identify the key features observed in corresponding histological images based on the scattering differences from various eye tissues, such as the cornea, intact and regenerated lens, and the iris. Lens volume measurements from three-dimensional OCT images showed that the regenerating lens size increased linearly until 60 days post-lentectomy. Conclusions Using OCT imaging, we were able to track the entire process of newt lens regeneration in vivo for the first time. Three-dimensional OCT images allowed us to volumetrically quantify and visualize the dynamic spatial relationships between tissues during the regeneration process. Our results establish OCT as an in vivo imaging modality to track/analyze the entire lens regeneration process from the same animal. Translational relevance Lens regeneration in newts represents a unique example of vertebrate tissue plasticity. Investigating the cellular and morphological events that govern this extraordinary process in vivo will advance our understanding and shed light on developing new therapies to treat blinding disorders in higher vertebrates.

Hereditary pulmonary alveolar proteinosis (PAP) is a genetic lung disease characterized by surfactant accumulation and respiratory failure arising from disruption of GM-CSF signaling. While mutations in either CSF2RA or CSF2RB (encoding GM-CSF receptor α or β chains, respectively) can cause PAP, α chain mutations are responsible in most patients. Pulmonary macrophage transplantation (PMT) is a promising new cell therapy in development; however, no studies have evaluated this approach for hereditary PAP (hPAP) caused by Csf2ra mutations. Here, we report on the preclinical safety, tolerability, and efficacy of lentiviral-vector (LV)-mediated Csf2ra expression in macrophages and PMT of gene-corrected macrophages (gene-PMT therapy) in Csf2ra gene-ablated (Csf2ra(−/−)) mice. Gene-PMT therapy resulted in a stable transgene integration and correction of GM-CSF signaling and functions in Csf2ra(−/−) macrophages in vitro and in vivo and resulted in engraftment and long-term persistence of gene-corrected macrophages in alveoli; restoration of pulmonary surfactant homeostasis; correction of PAP-specific cytologic, histologic, and biomarker abnormalities; and reduced inflammation associated with disease progression in untreated mice. No adverse consequences of gene-PMT therapy in Csf2ra(−/−) mice were observed. Results demonstrate that gene-PMT therapy of hPAP in Csf2ra(−/−) mice was highly efficacious, durable, safe, and well tolerated.

Pulmonary alveolar proteinosis (PAP) is a syndrome of reduced GM-CSF-dependent, macrophage-mediated surfactant clearance, dysfunctional foamy alveolar macrophages, alveolar surfactant accumulation, and hypoxemic respiratory failure for which the pathogenetic mechanism is unknown. Here, we examine the lipids accumulating in alveolar macrophages and surfactant to define the pathogenesis of PAP and evaluate a novel pharmacotherapeutic approach. In PAP patients, alveolar macrophages have a marked increase in cholesterol but only a minor increase in phospholipids, and pulmonary surfactant has an increase in the ratio of cholesterol to phospholipids. Oral statin therapy is associated with clinical, physiological, and radiological improvement in autoimmune PAP patients, and ex vivo statin treatment reduces cholesterol levels in explanted alveolar macrophages. In Csf2rb−/− mice, statin therapy reduces cholesterol accumulation in alveolar macrophages and ameliorates PAP, and ex vivo statin treatment increases cholesterol efflux from macrophages. These results support the feasibility of statin as a novel pathogenesis-based pharmacotherapy of PAP., Pulmonary alveolar proteinosis (PAP) is associated with defective macrophage clearance of surfactant. Here, the authors show that patients with PAP have altered cholesterol-to-phospholipid ratio in their surfactant, and that more importantly, statin therapy and reduction of cholesterol accumulation in macrophages can ameliorate PAP in both humans and mice.

Nonalcoholic fatty liver disease (NAFLD), a metabolic predisposition for development of hepatocellular carcinoma (HCC), represents a disease spectrum ranging from steatosis to steatohepatitis to cirrhosis. Acox1, a rate-limiting enzyme in peroxisomal fatty acid β-oxidation, regulates metabolism, spontaneous hepatic steatosis, and hepatocellular damage over time. However, it is unknown whether Acox1 modulates inflammation relevant to NAFLD pathogenesis or if Acox1-associated metabolic and inflammatory derangements uncover and accelerate potential for NAFLD progression. Here, we show that mice with a point mutation in Acox1 (Acox1Lampe1) exhibited altered cellular metabolism, modified T cell polarization, and exacerbated immune cell inflammatory potential. Further, in context of a brief obesogenic diet stress, NAFLD progression associated with Acox1 mutation resulted in significantly accelerated and exacerbated hepatocellular damage via induction of profound histological changes in hepatocytes, hepatic inflammation, and robust upregulation of gene expression associated with HCC development. Collectively, these data demonstrate that β-oxidation links metabolism and immune responsiveness and that a better understanding of peroxisomal β-oxidation may allow for discovery of mechanisms central for NAFLD progression.

Macrophages are critical to organ structure and function in health and disease. To determine mechanisms by which granulocyte/macrophage-colony stimulating factor (GM-CSF) signaling normally maintains surfactant homeostasis and how its disruption causes pulmonary alveolar proteinosis (PAP), we evaluated lipid composition in alveolar macrophages and lung surfactant, macrophage-mediated surfactant clearance kinetics/dynamics, and cholesterol-targeted pharmacotherapy of PAP in vitro and in vivo. Without GM-CSF signaling, surfactant-exposed macrophages massively accumulated cholesterol ester-rich lipid-droplets and surfactant had an increased proportion of cholesterol. GM-CSF regulated cholesterol clearance in macrophages in constitutive, dose-dependent, and reversible fashion but did not affect phospholipid clearance. PPARγ-agonist therapy increased cholesterol clearance in macrophages and reduced disease severity in PAP mice. Results demonstrate that GM-CSF is required for cholesterol clearance in macrophages, identify reduced cholesterol clearance as the primary macrophage defect driving PAP pathogenesis, and support the feasibility of translating pioglitazone as a novel pharmacotherapy of PAP.

Bone-marrow transplantation is an effective cell therapy but requires myeloablation, which increases infection risk and mortality. Recent lineage-tracing studies documenting that resident macrophage populations self-maintain independently of haematological progenitors prompted us to consider organ-targeted, cell-specific therapy. Here, using granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor-β-deficient (Csf2rb(-/-)) mice that develop a myeloid cell disorder identical to hereditary pulmonary alveolar proteinosis (hPAP) in children with CSF2RA or CSF2RB mutations, we show that pulmonary macrophage transplantation (PMT) of either wild-type or Csf2rb-gene-corrected macrophages without myeloablation was safe and well-tolerated and that one administration corrected the lung disease, secondary systemic manifestations and normalized disease-related biomarkers, and prevented disease-specific mortality. PMT-derived alveolar macrophages persisted for at least one year as did therapeutic effects. Our findings identify mechanisms regulating alveolar macrophage population size in health and disease, indicate that GM-CSF is required for phenotypic determination of alveolar macrophages, and support translation of PMT as the first specific therapy for children with hPAP.

In patients with pulmonary alveolar proteinosis (PAP) syndrome, disruption of granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling is associated with pathogenic surfactant accumulation from impaired clearance in alveolar macrophages.The aim of this study was to overcome these barriers by using monocyte-derived induced pluripotent stem (iPS) cells to recapitulate disease-specific and normal macrophages.We created iPS cells from two children with hereditary PAP (hPAP) caused by recessive CSF2RA(R217X) mutations and three normal people, differentiated them into macrophages (hPAP-iPS-Mφs and NL-iPS-Mφs, respectively), and evaluated macrophage functions with and without gene-correction to restore GM-CSF signaling in hPAP-iPS-Mφs.Both hPAP and normal iPS cells had human embryonic stem cell-like morphology, expressed pluripotency markers, formed teratomas in vivo, had a normal karyotype, retained and expressed mutant or normal CSF2RA genes, respectively, and could be differentiated into macrophages with the typical morphology and phenotypic markers. Compared with normal, hPAP-iPS-Mφs had impaired GM-CSF receptor signaling and reduced GM-CSF-dependent gene expression, GM-CSF- but not M-CSF-dependent cell proliferation, surfactant clearance, and proinflammatory cytokine secretion. Restoration of GM-CSF receptor signaling corrected the surfactant clearance abnormality in hPAP-iPS-Mφs.We used patient-specific iPS cells to accurately reproduce the molecular and cellular defects of alveolar macrophages that drive the pathogenesis of PAP in more than 90% of patients. These results demonstrate the critical role of GM-CSF signaling in surfactant homeostasis and PAP pathogenesis in humans and have therapeutic implications for hPAP.

Hereditary pulmonary alveolar proteinosis (hPAP) is a severe pediatric lung disease caused by mutations in CSF2RA/B (encoding GM-CSF receptor α/β, respectively) without pharmacologic therapy. Disease patho-genesis is mediated by loss of GM-CSF-dependent clearance by alveolar macrophages (AMs) resulting in progressive pulmonary alveolar surfactant accumulation and hypoxemic respiratory failure. We recently reported a conceptually and technically novel, exceptionally efficient gene and cell therapy approach -PMT- as a promising alternative for the existing highly invasive, inefficient whole lung lavage procedure for hPAP. Our preclinical studies revealed that macrophage cell doses from 0.5 to 4 x106/mouse were similarly highly efficacious as therapy of hPAP in Csf2rb-/- mice, an authentic model of human hPAP (Nature, 2014, 514: 450-5). The present study was undertaken to identify a dose-response relationship and to address the hypothesis of an expected ‘trade-off’ between the minimum effective dose and time to treatment effect. We isolated Lineage-, Sca1+, cKit+ hematopoietic stem/progenitors from the bone marrow of wild-type (WT) CD45.1+ mice, differentiated them in vitro into mature macrophages and sorted for highly homogenous CD11cHi, F4/80Hi macrophage population by flow cytometry. We administered cell doses (2.5 x103, 2.5 x104, 2.5 x105, 1 x106 cells/mouse; n=3-4/dose) by PMT to CD45.2+ Csf2rb-/- recipients. At 8 weeks after PMT, therapeutic efficacy was evaluated by measuring the optical density (OD) of bronchoalveolar lavage (BAL turbidity) - an excellent measure of overall PAP disease severity. The CD45.1+ donor macrophages recovered from the BAL were of CD11cHi, F4/80Hi phenotype. BAL turbidity decreased smoothly with increasing PMT cell dose over the entire range but reached significance only at the two highest doses compared to age-matched, untreated Csf2rb-/- controls (OD λ=600 nm = 0.88±0.07 and 0.5±0.08 vs. 2.32±0.18; P

Rationale: Hereditary pulmonary alveolar proteinosis (hPAP) is a disorder caused by recessive mutations in the GM-CSF receptor α or β gene (CSF2RA or CSF2RB), which is characterized by surfactant accumulation and hypoxemic respiratory failure, and for which no pharmacologic therapy exits. Using a murine hPAP model (Csf2rb–/–) phenotypically identical to children with hPAP, we recently reported a new gene/cell transplantation therapy approach – pulmonary macrophage transplantation (PMT) (Nature. 2014, 514: 450), with spontaneous engraftment – without preparative myeloablation, extraordinary therapeutic efficacy, a durable treatment effect, and no safety signal. Interestingly, both GM-CSF and M-CSF are elevated in the lungs of hPAP and provide a possible explanation for the remarkable survival advantage observed for transplanted macrophages compared to endogenous cells. The present study was undertaken to determine the potential relative contribution of these factors to the therapeutic mechanism of action of PMT therapy in preclinical studies in Csf2rb–/– mice.Methods: Csf2rb–/– mice received a single PMT ‘treatment’ – intrapulmonary instillation of wild type (WT) or (as a control) Csfr2b–/– bone marrow derived macrophages (BMDM) (2×106 per mouse) in the absence or presence of a specific M-CSF receptor kinase inhibitor (GW2580) or vehicle. Two months later, therapeutic efficacy was evaluated by measuring both disease severity (bronchoalveolar lavage (BAL) turbidity, BAL surfactant protein D (SP-D) levels) and pulmonary biomarkers of hPAP (BAL GM-CSF, M-CSF levels).Results: We first demonstrated the activity of GW2580 in vivo by measuring splenic macrophage numbers in Csf2rb–/– mice, which were reduced in GW2580-treated compared to untreated mice (2.8±0.2% vs 6.5±0.5%, n=13, 10 /group; p 0.05; all comparisons).Conclusions: Results demonstrate that GM-CSF but not M-CSF contributes to the efficacy of PMT therapy that restores surfactant homeostasis in mice with hPAP.Funding: ATS, CCHMC, NIH UL1TR000077 (TS); NIH R01HL085453, R21HL106134, R01HL118342 (BCT)

Rationale: Hereditary Pulmonary Alveolar Proteinosis (hPAP) is caused by mutations in the CSF2RA or CSF2RB genes (encoding GM-CSF receptor α or β, respectively) leading to defective GM-CSF-dependent surfactant clearance by alveolar macrophages, resulting in severe respiratory failure. Recently, using a validated model of hPAP (Csf2rb−/− mice), we reported a novel Pulmonary Macrophage Transplantation (PMT) approach as a safe, effective, tissue-specific and durable therapy for hPAP disease (Nature. 2014, 514: 450). Our results showed that wild-type bone marrow-derived macrophages (BMDMs) delivered by PMT successfully engraft, proliferate, gradually replace functionally deficient endogenous alveolar macrophages and efficiently clear surfactant in Csf2rb−/−recipient mice resulting in durable hPAP disease correction, in the absence of myeloablation. However, these studies did not exclude the possibility of an expanded myeloid progenitor population as the effector cell in PMT therapy of hPAP. Because the transplanted cells were capable of clearing surfactant, we hypothesized that the therapeutic efficacy of PMT is mediated by mature macrophages without obligate expansion of any myeloid progenitor/stem cell. Methods: BMDMs or alveolar macrophages were obtained from wild-type mice and intrapulmonary administered into Csf2rb−/− mice by PMT (2.5×105 per mouse). The therapeutic efficacy was evaluated at 2 months after PMT by measuring the optical density (OD λ=600 nm) of bronchoalveolar lavage (BAL) turbidity - an excellent measure of overall PAP disease severity. Results: Characterization prior to transplantation showed that BMDMs were highly purified, mature macrophages: they had the morphologic appearance and surface markers of mature macrophages, clonogenic analysis indicated they contained less than 0.005% CFU-GM and no BFU-E, or CFU-GEMM progenitors, and they were able to clear surfactant in vitro. To further increase macrophage purity, BMDMs were sorted by flow cytometer by applying a conservative gating strategy to isolate CD11cHiF4/80Hi macrophages. PMT using these highly uniform, mature CD11cHiF4/80Hi BMDMs into Csf2rb−/− mice showed extraordinary therapeutic efficacy as evidenced by marked reduction of BAL turbidity compared to untreated, age-matched Csf2rb−/− mice (OD = 0.9±0.1 vs 2.3±0.2; n=4; P

Information concerning the late effects of whole-body irradiation has been extensively reviewed (1-9). However, with the exception of a few studies concerning the effects of protracted or almost continuous daily irradiation (10-16), much of the previous work has dealt with the effects of single or moderately fractionated exposures. The data reported in this paper resulted from a series of experiments started in 1954 and designed to study the effects of daily Co60 y-irradiation on the survival, hematology, histology, and pathology of LAF, mice. In this communication we present the tumor and degenerative disease frequency as a function of daily dose, age, and sex at doses ranging from 5 to 56 r/day. Other phases of the program involved daily doses up to 200,000 r/day (17), but, since mice exposed to doses above 56 r/day died before tumors or degenerative diseases could develop, the higher dose levels are not discussed.

Radiosensitivity of ileal crypt cells, to /sup 60/Co gamma radiation, was studied in ground squirrels (Citellus tridecemlineatus) during hibernation, arousal, and the euthermic state. Survival of ileal crypt cells, assayed by the microcolony technique from stained transverse sections of ileum, was greater in animals irradiated in hibernation or 1 hr after initiation of arousal from hibernation. Crypt survival returned to the level of irradiated nonhibernating controls in animals irradiated 3 to 7 hr after initiation of arousal. Over the exposure range of 1500 to 2400 R, the survival of crypt cells for euthermic controls gave a D/sub 0/ = 133 +- 12 R and for animals irradiated in hibernation it gave a D/sub 0/ = 487 +- 92 R. In animals irradiated 1 hr after initiation of arousal, when core temperature is within the range of euthermic controls, crypt survival was almost as high as in the hibernators. These results suggest that the increased resistance of ileal crypt cells in hibernating animals could be due to hypoxia, although not direct evidence for hypoxia in hibernation was established. The changes in mitotic index of ileal crypt cells during hibernation and arousal indicate an alteration in the distribution of cells in themore » phases of the cycle. This change in distribution may also have contributed to the increased radioresistance of hibernators.« less

The mucosal epithelium of the duodenum of BCF1 mice exposed to 220 r/day for 1, 7, 14, 17, and 18 days was studied and compared to that of nonirradiated controls of same age. The generation time of the duodenal crypt cells of mice irradiated for 1, 7, and 14 days, and of the controls, was determined. Since it was found that cells are cold up between the end of synthesis and the completion of tosis, the initial effect may be similar to the effect produced by a single exposure. When exposure is continued. The there is an attempt to compensate for cell loss by a speeding up of the generation cycle which is primarily due to a