Muc5b is required for airway defence.

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

Deletion of c-FLIP from CD11bhi Macrophages Prevents Development of Bleomycin-induced Lung Fibrosis.

Idiopathic pulmonary fibrosis is a progressive lung disease with complex pathophysiology and fatal prognosis. Macrophages (MΦ) contribute to the development of lung fibrosis; however, the underlying mechanisms and specific MΦ subsets involved remain unclear. During lung injury, two subsets of lung MΦ coexist: Siglec-Fhi resident alveolar MΦ and a mixed population of CD11bhi MΦ that primarily mature from immigrating monocytes. Using a novel inducible transgenic system driven by a fragment of the human CD68 promoter, we targeted deletion of the antiapoptotic protein cellular FADD-like IL-1ß-converting enzyme-inhibitory protein (c-FLIP) to CD11bhi MΦ. Upon loss of c-FLIP, CD11bhi MΦ became susceptible to cell death. Using this system, we were able to show that eliminating CD11bhi MΦ present 7-14 days after bleomycin injury was sufficient to protect mice from fibrosis. RNA-seq analysis of lung MΦ present during this time showed that CD11bhi MΦ, but not Siglec-Fhi MΦ, expressed high levels of profibrotic chemokines and growth factors. Human MΦ from patients with idiopathic pulmonary fibrosis expressed many of the same profibrotic chemokines identified in murine CD11bhi MΦ. Elimination of monocyte-derived MΦ may help in the treatment of fibrosis. We identify c-FLIP and the associated extrinsic cell death program as a potential pathway through which these profibrotic MΦ may be pharmacologically targeted.

Phagocytosis of microparticles by alveolar macrophages during acute lung injury requires MerTK.

Microparticles are a newly recognized class of mediators in the pathophysiology of lung inflammation and injury, but little is known about the factors that regulate their accumulation and clearance. The primary objective of our study was to determine whether alveolar macrophages engulf microparticles and to elucidate the mechanisms by which this occurs. Alveolar microparticles were quantified in bronchoalveolar fluid of mice with lung injury induced by LPS and hydrochloric acid. Microparticle numbers were greatest at the peak of inflammation and declined as inflammation resolved. Isolated, fluorescently labeled particles were placed in culture with macrophages to evaluate ingestion in the presence of endocytosis inhibitors. Ingestion was blocked with cytochalasin D and wortmannin, consistent with a phagocytic process. In separate experiments, mice were treated intratracheally with labeled microparticles, and their uptake was assessed though microscopy and flow cytometry. Resident alveolar macrophages, not recruited macrophages, were the primary cell-ingesting microparticles in the alveolus during lung injury. In vitro, microparticles promoted inflammatory signaling in LPS primed epithelial cells, signifying the importance of microparticle clearance in resolving lung injury. Microparticles were found to have phosphatidylserine exposed on their surfaces. Accordingly, we measured expression of phosphatidylserine receptors on macrophages and found high expression of MerTK and Axl in the resident macrophage population. Endocytosis of microparticles was markedly reduced in MerTK-deficient macrophages in vitro and in vivo. In conclusion, microparticles are released during acute lung injury and peak in number at the height of inflammation. Resident alveolar macrophages efficiently clear these microparticles through MerTK-mediated phagocytosis.

Cell Origin Dictates Programming of Resident versus Recruited Macrophages during Acute Lung Injury.

Two populations of alveolar macrophages (AMs) coexist in the inflamed lung: resident AMs that arise during embryogenesis, and recruited AMs that originate postnatally from circulating monocytes. The objective of this study was to determine whether origin or environment dictates the transcriptional, metabolic, and functional programming of these two ontologically distinct populations over the time course of acute inflammation. RNA sequencing demonstrated marked transcriptional differences between resident and recruited AMs affecting three main areas: proliferation, inflammatory signaling, and metabolism. Functional assays and metabolomic studies confirmed these differences and demonstrated that resident AMs proliferate locally and are governed by increased tricarboxylic acid cycle and amino acid metabolism. Conversely, recruited AMs produce inflammatory cytokines in association with increased glycolytic and arginine metabolism. Collectively, the data show that even though they coexist in the same environment, inflammatory macrophage subsets have distinct immunometabolic programs and perform specialized functions during inflammation that are associated with their cellular origin.

Selective and inducible targeting of CD11b+ mononuclear phagocytes in the murine lung with hCD68-rtTA transgenic systems.

During homeostasis two distinct macrophage (Mø) populations inhabit the lungs: tissue Mø (often called interstitial Mø) and resident alveolar Mø (resAMø). During acute lung inflammation, monocytes from the circulation migrate to areas of injury where they mature into a third Mø population: recruited Mø. Resident AMø uniquely express low levels of CD11b and high levels of CD11c. In comparison, recruited Mø and tissue Mø express high levels of CD11b and low levels of CD11c. It is likely that these three Mø subpopulations play distinct roles in injury and disease states; however, tools with which to individually target or track these populations are lacking. Here we demonstrate the utility of an hCD68-rtTA transgenic system for specific, robust, and inducible targeting of CD11b(+) recruited Mø and tissue Mø in the murine lung with negligible activation in resAMø. Using hCD68rtTA-GFP reporter mice, we show both during homeostasis and inflammation that administration of doxycycline induces tet-On reporter expression in recruited Mø and tissue Mø but not in resident AMø. We further demonstrate how hCD68-rtTA can be effectively combined with tet-On Cre to target these same recMø and tissue Mø. Accordingly, the hCD68-rtTA system is a powerful new tool that can be used for lineage tracing, fate mapping, and gene deletion in a variety of murine models, thereby enabling sophisticated investigation of the unique role of these CD11b(+) Mø during lung heath and disease.

The Causal Role of IL-4 and IL-13 in Schistosoma mansoni Pulmonary Hypertension.

RATIONALE: The etiology of schistosomiasis-associated pulmonary arterial hypertension (PAH), a major cause of PAH worldwide, is poorly understood. Schistosoma mansoni exposure results in prototypical type-2 inflammation. Furthermore, transforming growth factor (TGF)-ß signaling is required for experimental pulmonary hypertension (PH) caused by Schistosoma exposure. OBJECTIVES: We hypothesized type-2 inflammation driven by IL-4 and IL-13 is necessary for Schistosoma-induced TGF-ß-dependent vascular remodeling. METHODS: Wild-type, IL-4(-/-), IL-13(-/-), and IL-4(-/-)IL-13(-/-) mice (C57BL6/J background) were intraperitoneally sensitized and intravenously challenged with S. mansoni eggs to induce experimental PH. Right ventricular catheterization was then performed, followed by quantitative analysis of the lung tissue. Lung tissue from patients with schistosomiasis-associated and connective tissue disease-associated PAH was also systematically analyzed. MEASUREMENTS AND MAIN RESULTS: Mice with experimental Schistosoma-induced PH had evidence of increased IL-4 and IL-13 signaling. IL-4(-/-)IL-13(-/-) mice, but not single knockout IL-4(-/-) or IL-13(-/-) mice, were protected from Schistosoma-induced PH, with decreased right ventricular pressures, pulmonary vascular remodeling, and right ventricular hypertrophy. IL-4(-/-)IL-13(-/-) mice had less pulmonary vascular phospho-signal transducer and activator of transcription 6 (STAT6) and phospho-Smad2/3 activity, potentially caused by decreased TGF-ß activation by macrophages. In vivo treatment with a STAT6 inhibitor and IL-4(-/-)IL-13(-/-) bone marrow transplantation also protected against Schistosoma-PH. Lung tissue from patients with schistosomiasis-associated and connective tissue disease-associated PAH had evidence of type-2 inflammation. CONCLUSIONS: Combined IL-4 and IL-13 deficiency is required for protection against TGF-ß-induced pulmonary vascular disease after Schistosoma exposure, and targeted inhibition of this pathway is a potential novel therapeutic approach for patients with schistosomiasis-associated PAH.

Fas ligand-expressing lymphocytes enhance alveolar macrophage apoptosis in the resolution of acute pulmonary inflammation.

Apoptosis of alveolar macrophages and their subsequent clearance by neighboring phagocytes are necessary steps in the resolution of acute pulmonary inflammation. We have recently identified that activation of the Fas death receptor on the cell surface of macrophages drives macrophage apoptosis. However, the source of the cognate ligand for Fas (FasL) responsible for induction of alveolar macrophage apoptosis is not defined. Given their known role in the resolution of inflammation and ability to induce macrophage apoptosis ex vivo, we hypothesized that T lymphocytes represented a critical source of FasL. To address this hypothesis, C57BL/6J and lymphocyte-deficient (Rag-1(-/-)) mice were exposed to intratracheal lipopolysaccharide to induce pulmonary inflammation. Furthermore, utilizing mice expressing nonfunctional FasL, we adoptively transferred donor lymphocytes into inflamed lymphocyte-deficient mice to characterize the effect of lymphocyte-derived FasL on alveolar macrophage apoptosis in the resolution of inflammation. Herein, evidence is presented that lymphocytes expressing FasL enhance alveolar macrophage apoptosis during the resolution of LPS-induced inflammation. Moreover, lymphocyte induction of alveolar macrophage apoptosis results in contraction of the alveolar macrophage pool, which occurs in a FasL-dependent manner. Specifically, FasL-expressing CD8(+) T lymphocytes potently induce alveolar macrophage apoptosis and contraction of the alveolar macrophage pool. Together, these studies identify a novel role for CD8(+) T lymphocytes in the resolution of acute pulmonary inflammation.

Kinetics of the angiogenic response in lung endothelium following acute inflammatory injury with bleomycin.

PURPOSE/AIM: Angiogenesis is a central component of normal wound healing but it has not been fully characterized in lung repair following acute inflammatory injury. The current literature lacks vital information pertaining to the extent, timing, and location of this process. This information is necessary for examining mechanisms that drive normal lung repair in resolving acute inflammatory injury. The goal of our study was to formally characterize lung angiogenesis over a time course of bleomycin-induced lung injury. MATERIALS AND METHODS: Female C57BL/6 mice age 8-12 weeks were treated with a single dose of intratracheal bleomycin. Total lung endothelial cells were quantified with flow cytometry 0, 7, 14, 21, and 28 days following bleomycin administration, and endothelial cell replication was assessed using bromodeoxyuridine (BrdU) incorporation. RESULTS: Endothelial cell replication was maximal 14 days after bleomycin administration, while total lung endothelial cells peaked at day 21. Tissue analysis with stereology was performed to measure total lung vascular surface area in bleomycin at day 21 relative to controls and demonstrated a trend toward increased vasculature in the bleomycin group. CONCLUSIONS: Angiogenesis begins shortly after injury in the bleomycin model and leads to an expansion in the lung endothelial cell population that peaks at day 21. This study offers the first longitudinal examination of angiogenesis following acute inflammatory lung injury induced by bleomycin. Information provided in this study will be vital for further investigating mechanisms of angiogenesis in both normal and abnormal lung repair.

Circulating hematopoietic progenitor cells are decreased in COPD.

RATIONALE: Bone marrow derived progenitor cells participate in the repair of injured vessels. The lungs of individuals with emphysema have reduced alveolar capillary density and increased endothelial apoptosis. We hypothesized that circulating levels of endothelial and hematopoietic progenitor cells would be reduced in this group of patients. OBJECTIVES: The goal of this study was to measure circulating levels of endothelial progenitor cells (EPCs) and hematopoietic progenitor cells (HPCs) in subjects with COPD and to determine if progenitor levels correlated with disease severity and the presence of emphysema. METHODS: Peripheral blood mononuclear cells were isolated from 61 patients with COPD and 32 control subjects. Levels of EPCs (CD45(dim) CD34+) and HPCs (CD45(+) CD34(+) VEGF-R2(+)) were quantified using multi-parameter flow cytometry. Progenitor cell function was assessed using cell culture assays. All subjects were evaluated with spirometry and CT scanning. MEASUREMENTS AND MAIN RESULTS: HPC levels were reduced in subjects with COPD compared to controls, whereas circulating EPC levels were similar between the two groups. HPC levels correlated with severity of obstruction and were lowest in subjects with severe emphysema. These associations remained after correction for factors known to affect progenitor cell levels including age, smoking status, the use of statin medications and the presence of coronary artery disease. The ability of mononuclear cells to form endothelial cell colony forming units (EC-CFU) was also reduced in subjects with COPD. CONCLUSIONS: HPC levels are reduced in subjects with COPD and correlate with emphysema phenotype and severity of obstruction. Reduction of HPCs may disrupt maintenance of the capillary endothelium, thereby contributing to the pathogenesis of COPD.

Macrophages mediate lung inflammation in a mouse model of ischemic acute kidney injury.

Serum IL-6 is increased in acute kidney injury (AKI) and inhibition of IL-6 reduces AKI-mediated lung inflammation. We hypothesized that circulating monocytes produce IL-6 and that alveolar macrophages mediate lung inflammation after AKI via chemokine (CXCL1) production. To investigate systemic and alveolar macrophages in lung injury after AKI, sham operation or 22 min of renal pedicle clamping (AKI) was performed in three experimental settings: 1) systemic macrophage depletion via diphtheria toxin (DT) injection to CD11b-DTR transgenic mice, 2) DT injection to wild-type mice, and 3) alveolar macrophage depletion via intratracheal (IT) liposome-encapsulated clodronate (LEC) administration to wild-type mice. In mice with AKI and systemic macrophage depletion (CD11b-DTR transgenic administered DT) vs. vehicle-treated AKI, blood monocytes and lung interstitial macrophages were reduced, renal function was similar, serum IL-6 was increased, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In wild-type mice with AKI administered DT vs. vehicle, serum IL-6 was increased. In mice with AKI and alveolar macrophage depletion (IT-LEC) vs. AKI with normal alveolar macrophage content, blood monocytes and lung interstitial macrophages were similar, alveolar macrophages were reduced, renal function was similar, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In conclusion, administration of DT in AKI is proinflammatory, limiting the use of the DTR-transgenic model to study systemic effects of AKI. Mice with AKI and either systemic mononuclear phagocyte depletion or alveolar macrophage depletion had reduced lung inflammation and lung CXCL1, but increased lung capillary leak; thus, mononuclear phagocytes mediate lung inflammation, but they protect against lung capillary leak after ischemic AKI. Since macrophage activation and chemokine production are key events in the development of acute lung injury (ALI), these data provide further evidence that AKI may cause ALI.

Fas determines differential fates of resident and recruited macrophages during resolution of acute lung injury.

RATIONALE: During acute lung injury (ALI) the macrophage pool expands markedly as inflammatory monocytes migrate from the circulation to the airspaces. As inflammation resolves, macrophage numbers return to preinjury levels and normal tissue structure and function are restored. OBJECTIVES: To determine the fate of resident and recruited macrophages during the resolution of ALI in mice and to elucidate the mechanisms responsible for macrophage removal. METHODS: ALI was induced in mice using influenza A (H1N1; PR8) infection and LPS instillation. Dye labeling techniques, bone marrow transplantation, and surface immunophenotyping were used to distinguish resident and recruited macrophages during inflammation and to study the role of Fas in determining macrophage fate during resolving ALI. MEASUREMENTS AND MAIN RESULTS: During acute and resolving lung injury from influenza A and LPS, a high proportion of the original resident alveolar macrophages persisted. In contrast, recruited macrophages exhibited robust accumulation in early inflammation, followed by a progressive decline in their number. This decline was mediated by apoptosis with local phagocytic clearance. Recruited macrophages expressed high levels of the death receptor Fas and were rapidly depleted from the airspaces by Fas-activating antibodies. In contrast, macrophage depletion was inhibited in mice treated with Fas-blocking antibodies and in chimeras with Fas-deficient bone marrow. Caspase-8 inhibition prevented macrophage apoptosis and delayed the resolution of ALI. CONCLUSIONS: These findings indicate that Fas-induced apoptosis of recruited macrophages is essential for complete resolution of ALI.

Mapping Macrophage Polarization and Origin during the Progression of the Foreign Body Response to a Poly(ethylene glycol) Hydrogel Implant.

Poly(ethylene glycol) (PEG) hydrogels hold promise for in vivo applications but induce a foreign body response (FBR). While macrophages are key in the FBR, many questions remain. This study investigates temporal changes in the transcriptome of implant-associated monocytes and macrophages. Proinflammatory pathways are upregulated in monocytes compared to control monocytes but subside by day 28. Macrophages are initially proinflammatory but shift to a profibrotic state by day 14, coinciding with fibrous capsule emergence. Next, this study assesses the origin of macrophages responsible for fibrous encapsulation using wildtype, C-C Motif Chemokine Receptor 2 (CCR2)-/- mice that lack recruited macrophages, and Macrophage Fas-Induced Apoptosis (MaFIA) mice that enable macrophage ablation. Subpopulations of recruited and tissue-resident macrophages are identified. Fibrous encapsulation proceeds in CCR2-/- mice similar to wildtype mice. However, studies in MaFIA mice indicate that macrophages are necessary for fibrous capsule formation. These findings suggest that macrophage origin impacts the FBR progression and provides evidence that tissue-resident macrophages and not the recruited macrophages may drive fibrosis in the FBR to PEG hydrogels. This study demonstrates that implant-associated monocytes and macrophages have temporally distinct transcriptomes in the FBR and that profibrotic pathways associated with macrophages may be enriched in tissue-resident macrophages.

Neutrophil transfer of miR-223 to lung epithelial cells dampens acute lung injury in mice.

Intercellular transfer of microRNAs can mediate communication between critical effector cells. We hypothesized that transfer of neutrophil-derived microRNAs to pulmonary epithelial cells could alter mucosal gene expression during acute lung injury. Pulmonary-epithelial microRNA profiling during coculture of alveolar epithelial cells with polymorphonuclear neutrophils (PMNs) revealed a selective increase in lung epithelial cell expression of microRNA-223 (miR-223). Analysis of PMN-derived supernatants showed activation-dependent release of miR-223 and subsequent transfer to alveolar epithelial cells during coculture in vitro or after ventilator-induced acute lung injury in mice. Genetic studies indicated that miR-223 deficiency was associated with severe lung inflammation, whereas pulmonary overexpression of miR-223 in mice resulted in protection during acute lung injury induced by mechanical ventilation or by infection with Staphylococcus aureus Studies of putative miR-223 gene targets implicated repression of poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) in the miR-223-dependent attenuation of lung inflammation. Together, these findings suggest that intercellular transfer of miR-223 from neutrophils to pulmonary epithelial cells may dampen acute lung injury through repression of PARP-1.

TGF-ß activation by bone marrow-derived thrombospondin-1 causes Schistosoma- and hypoxia-induced pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is an obstructive disease of the precapillary pulmonary arteries. Schistosomiasis-associated PAH shares altered vascular TGF-ß signalling with idiopathic, heritable and autoimmune-associated etiologies; moreover, TGF-ß blockade can prevent experimental pulmonary hypertension (PH) in pre-clinical models. TGF-ß is regulated at the level of activation, but how TGF-ß is activated in this disease is unknown. Here we show TGF-ß activation by thrombospondin-1 (TSP-1) is both required and sufficient for the development of PH in Schistosoma-exposed mice. Following Schistosoma exposure, TSP-1 levels in the lung increase, via recruitment of circulating monocytes, while TSP-1 inhibition or knockout bone marrow prevents TGF-ß activation and protects against PH development. TSP-1 blockade also prevents the PH in a second model, chronic hypoxia. Lastly, the plasma concentration of TSP-1 is significantly increased in subjects with scleroderma following PAH development. Targeting TSP-1-dependent activation of TGF-ß could thus be a therapeutic approach in TGF-ß-dependent vascular diseases.

Tracheal dysplasia precedes bronchial dysplasia in mouse model of N-nitroso trischloroethylurea induced squamous cell lung cancer.

Squamous cell lung cancer (SCC) is the second leading cause of lung cancer death in the US and has a 5-year survival rate of only 16%. Histological changes in the bronchial epithelium termed dysplasia are precursors to invasive SCC. However, the cellular mechanisms that cause dysplasia are unknown. To fill this knowledge gap, we used topical application of N-nitroso-tris chloroethylurea (NTCU) for 32 weeks to induce squamous dysplasia and SCC in mice. At 32 weeks the predominant cell type in the dysplastic airways was Keratin (K) 5 and K14 expressing basal cells. Notably, basal cells are extremely rare in the normal mouse bronchial epithelium but are abundant in the trachea. We therefore evaluated time-dependent changes in tracheal and bronchial histopathology after NTCU exposure (4, 8, 12, 16, 25 and 32 weeks). We show that tracheal dysplasia occurs significantly earlier than that of the bronchial epithelium (12 weeks vs. 25 weeks). This was associated with increased numbers of K5+/K14+ tracheal basal cells and a complete loss of secretory (Club cell secretory protein expressing CCSP+) and ciliated cells. TUNEL staining of NTCU treated tissues confirmed that the loss of CCSP+ and ciliated cells was not due to apoptosis. However, mitotic index (measured by bromodeoxyuridine incorporation) showed that NTCU treatment increased proliferation of K5+ basal cells in the trachea, and altered bronchial mitotic population from CCSP+ to K5+ basal cells. Thus, we demonstrate that NTCU-induced lung epithelial dysplasia starts in the tracheal epithelium, and is followed by basal cell metaplasia of the bronchial epithelium. This analysis extends our knowledge of the NTCU-SCC model by defining the early changes in epithelial cell phenotypes in distinct airway locations, and this may assist in identifying new targets for future chemoprevention studies.

The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

Development and characterization of a lung-protective method of bone marrow transplantation in the mouse.

Allogeneic bone marrow transplantation is a common method used to study the contribution of myeloid and lymphoid cell populations in murine models of disease. The method requires lethal doses of radiation to ablate the bone marrow. Unintended consequences of radiation include organ injury and inflammatory cell activation. The goal of our study was to determine the degree to which bone marrow transplantation alters lungs and to develop a system to protect the lungs during radiation. C57BL/6 mice were subjected to total body irradiation with 900cGy and then transplanted with bone marrow from green fluorescent protein (GFP) expressing mice. Resultant chimeras exhibited a significant decline in alveolar macrophage numbers within 72h, modest influx of neutrophils in the lungs at 14days, and repopulation of the lungs by alveolar macrophages of bone marrow origin by 28days. Neutrophil influx and alveolar macrophage turnover were prevented when 1cm thick lead shields were used to protect the lungs during radiation, such that 8weeks after transplantation less than 30% of alveolar macrophages were of donor origin. Lung-shielded mice achieved a high level of bone marrow engraftment with greater than 95% of circulating leukocytes expressing GFP. In addition, their response to intratracheal lipopolysaccharide was similar to non-transplanted mice. We describe a model whereby lead shields protect resident cell populations in the lungs from radiation during bone marrow transplantation but permit full bone marrow engraftment. This system may be applicable to other organ systems in which protection from radiation during bone marrow transplantation is desired.

Tetracyclines that promote SMN2 exon 7 splicing as therapeutics for spinal muscular atrophy.

There is at present no cure or effective therapy for spinal muscular atrophy (SMA), a neurodegenerative disease that is the leading genetic cause of infant mortality. SMA usually results from loss of the SMN1 (survival of motor neuron 1) gene, which leads to selective motor neuron degeneration. SMN2 is nearly identical to SMN1 but has a nucleotide replacement that causes exon 7 skipping, resulting in a truncated, unstable version of the SMA protein. SMN2 is present in all SMA patients, and correcting SMN2 splicing is a promising approach for SMA therapy. We identified a tetracycline-like compound, PTK-SMA1, which stimulates exon 7 splicing and increases SMN protein levels in vitro and in vivo in mice. Unlike previously identified molecules that stimulate SMN production via SMN2 promoter activation or undefined mechanisms, PTK-SMA1 is a unique therapeutic candidate in that it acts by directly stimulating splicing of exon 7. Synthetic small-molecule compounds such as PTK-SMA1 offer an alternative to antisense oligonucleotide therapies that are being developed as therapeutics for a number of disease-associated splicing defects.