COMPOSITIONS AND METHODS FOR LUNG REGENERATION

FIELD

The invention provides compositions and methods tor treating pulmonary conditions and for reducing and/or reversing the negative effects of pulmonary inflammation, exposure to damaging agents or as may be caused by an infectious agent.

BACKGROUND

The pulmonary system provides homeostasis and repair of the king in response to attack by pathogens, toxins, pollutants, and other types of injuries.

Asthma is a non-infectious chronic inflammatory disease of the respiratory system characterized by a reversible airways obstruction. Acute airway obstruction, bronchial hyper- responsiveness and inflammatory state of the bronchial mucosa with increased levels of inflammator mediators, are the most evident phenomenon which characterizes this pathology. Despite the increase in the prescribed anti-asthmatic treatments, the current trends indicate asthma is set to be the most chronic disease in industrialized countries, affecting mostly the children (1.0%) than the adults (15%).

Chronic obstruct ive pulmonary disease (COPD) is the most common of all the respiratory disorders in the world, which embraces several inflammatory pathologies that often co-exist. T he WHO predicts COPD will become the third most common cause of death world over by 2020 accounting 8,4 million Lives. Although asthma for the last 25 years has been managed therapeutically, with a combined bronchodilator and anti-inflammatory therapies, in contrast to this COPD have no effective treatments currently, while the efficacy of the corticosteroids is controversial. Hence, there is an urgent need to develop novel antiinflammatory drags having both the bronchodilatory and anti- inflammatory activity, having applicability to treat both COPD as well as asthma. Thus, the development of therapies for bronc hial asthma has become the major focus of t he pharmaceutical industry in the field of respiratory disorders.

Airway stem cells have been kip heated in the pathology and progression of chronic airway diseases and yet also hold the promise of physiological and ultimately therapeutic repair of damage wrought by these conditions. Chronic airway disease is often regiospecific with allergic rhinitis affecting the sinuses, asthma, cystic fibrosis, and bronchiolitis obliterans in the large conducting tubes such as the bronchi and bronchioles, and COPD, pulmonary fibrosis, and pulmonary hypertension affecting the distal regions of the airways involved in oxygen exchange. A. major focus of airway pa thology therefore is to understand if and ho stem cells initiate repair programs, participate in airway epithelial remodeling seen in chronic conditions, and. mechanisms underlying defects in repair as in pulmonary fibrosis. SUMMARY

The present disclosure is based on the observation thai certain p63 -expressing stem cells in the bronchiolar epithelium undergo rapid proliferation after infection and radiate to inter bronchiolar regions of alveolar ablation. Once there, these cells assemble into discrete, Krt5+ pods and initiate expression of markers typical of alveo li. Gene expression profiles of these pods suggest that they are intermediates in the reconstit.ut.ion of the alveolar-capillary network, such as which may be eradicated by viral infection. The dynamics of this p63~ expressing stem cell in lung regeneration mirrors parallel findings that defined pedigrees of human distal airway stem cells assemble alveoli-like structures in vitro and suggests new therapeutic avenues to acute and chronic airway disease.

To address questions germane to adult stem ceils in general, the inventors isolated and cloned various human airway stem cells from single cells and performed a pedigree-defined analysis of their lineage commitment, developmental potential, and gene expression profiles. These studies reveal three regiospecific stem cell types united by a common, p63 -expressing basal, phenotype and yet distinguished by expression profiles. Stem cell, pedigrees from the distal airways show a remarkable ability in vitro io form either alveoli-like structures or bronchiolar epithelium, while those of proximal a irways beget upper airway ceil types or squamous metaplasia. Pedigree tracking of single cell-derived clones permits the decoding of adult stem cell repertoires to answer basic questions of lineage commitment, developmental plasticity, and their specific roles in repair and disease.

One aspect, of the invention provides a composition comprising a clonal population of airway epithelial stem cells, which cells are keratin 5 (Krt5) positive and p63 positive, can propagate tor at least 10 doublings, and more preferably at least 20, 30, 40, 50 or even 60 doublings, while maintaining a pluripotent phenotype, and can differentiate into airway epithelia.

Another aspect of the invention provides a composition of purified airway epithelial stem cells, which ceils are keratin 5 (Krt5) positive and p(>3 positive, can propagate for at least 10 doublings, and more preferably at least 20, 30, 40, 50 or even 60 doublings, while maintaining a pluripotent phenotype, and can differentiate into airway epithelia. Still another aspect, of the invention provides a composition comprising at least I0 ⁴ (and more preferably 10% 10 ⁶ , 10 10 ^h or even S (f i airway epithelial stem cells, which cells are keratin 5 ( .rt.5) positive and p63 positive, can propagate for at least 1 doublings, and more preferably at least 20, 30, 40, 50 or even 60 doublings, while maintaining a pluripotent phenotype, and can differentiate into airway epitheiia.

In certain embodiments, the airway epithelial stem ceils are nasal epithelial stem cells (NESCs).

In other embodiments, the airway epithelial stem ceils are trachael airway stem cells (TASCs).

In still other embodiments, the airway epithelial stem cells are distal airway stem cell (DASC).

In certain preferred embodiments, the ESC, TASC or DASC cell population can be characterized by the detectable expression of markers, or by the relative levels of one or more niRNA (relative to other stem cells or to normal lung tissue, such as aveoli). Exemplary expression profiles that can be used to distinguish stem cell populations are provided in the examples and figures. To further illustrate, in certain preferred embodiments of DASCs. the stem cells are also characterised as having mR A levels for one or more, and preferably each of PLUNC, SCG.B3A I, GPX2, LTF, SCGB3A2, CYP2F2 and/or GABRP at least two times greater (more preferably three, five, ten or eve twenty times greater) than levels in normal differentiated alveoli cells.

In preferred embodiments, the airway epithelial stem cells are mammalian cells, and even more preferably human cells.

In certain embodiments, the airway epithelial stem cells are isolated from adult tissue. However, it is specifically contemplated that the stem cells can be derived from any source of pluripotent stem ceil, such as embryonic stem cells, induced piuripotency stem (IPS) cells and fetal stem cells, as well as by dedifferenriation of adult tissues, particularly adult lung tissues.

The present invention also provides for the use of the subject airway epithelial stem cells for use in pharmaceutical formulations. For instance, it is specifically contemplated thai the ceils of the present invention can be used in the manufacture of a medicament for promoting lung tissue regeneration and/or slowing the rate of lung tissue degenerat ion. Such compositions will be suitable for use in human patients, i.e., highly pure with respect to the stem cell population and being pyro gen- free.

The disclosure further provides a composition comprising a clonal population of NESC stem cells (which can be isolated from nasal turbinate), wherein the stem cells differentiate into goblet and ciliated ceils. Preferably the composition, with respect to the cellular component, is at least 50 percent stem ceil, more preferably at least 75, 80, 85, 90, 95 or even 99 percent stem cell. The stem cells can be pluripotent, mnltipoteni or oligopotent.

The disclosure further provides a composition comprising a clonal population of TASC stem cells (which can be isolated from tracheobronchial epithelia) that can

differentiate into goblet and ciliated ceils, in certain preferred embodiments, the stem cells are characterized as having an ni NA profile that is positive for expression one or more of TM SS 1 1 D, SPRR1 A, SPRR2C, KRTDAP, TMPR.SS1 I B, CRN and MTI L mRNA, i.e., raRNA present at detectable levels, and more preferably at levels (i.e., relative to actio expression) that are greater than in the tracheobronchial stem cells than in stem cells isolated from distal airway tissue. Preferably the composition, with respect to the cellular component, is at least 50 percent stem cell, more preferably at least 75, 80, 85, 90. 95 or even 99 percent stem cell The stem cells can be pluripotent, multipotent or oligopotent.

The disclosure further provides a composition comprising a clonal population of DASC stem cells {which can be isolated from distal airway tissue), wherein the stem cells differentiate into alveolar type lung cells and/or Clara cells. Preferably the composition, with respect to the cellular component, is at least 50 percent stem cell, more preferably at least 75, 80, 85, 90, 95 or even 99 percent stem cell. The stem cells can be pluripotent, multipotent or oligopotent. In certain preferred embodiments, the stem cells are characterized as having an mRNA profile including expression of GSTA2, GSTA1 , L 03, PPARGC 1A, RPS 15A, ALDH l Ai and SCGBl Ai mRN A at detectable levels, and more preferably at levels (i.e.. relative to act in expression) that are greater than stem cells isolated from tracheobronchial epithelia. Preferably all five genes have an mRN A profile in that range. In certain embodiments, the mRNA transcript profile for the stem cells will also be characterized by detectable levels of KRT5.

The disclosure further provides a method of screening for an agent effective in causing lung regeneration including the steps of providing a population of distal airway stem cells (DASCs), wherein the DASCs are able to differentiate into alveolar type lung cells and/or Clara cells; providing a test agent; and exposing the stem cells to the test agent;

wherein if the test agent to increases viability, growth, migration or differentiation of the DASCs, the test agent is an agent effective in regeneration of lung.

in certain embodiments, the test agent is also contacted with normal ceils or tissue of the lung, and the differential ability, if any, of the test agent to increase the viability, growth, migration or differentiation of the normal cells or tissue is compared to that with the DASC s. in certain embodiments, the DASCs are mammalian DASCs, such as human DASCs or rat DASCs.

The DASCs can be clonal, and can be piuripotent, mu!tipotent or oligopoient. In certain preferred embodiments, the stem cells are characterized as having an mRNA. profile that includes upregulated expression of PLUNC, SCGB3A1, GPX2, LTF, SCGB3A2, CYP2F2 and/or GAB P mRN A in mRNA, which are expressed at least ten or twenty times greater than in normal alveoli. Preferably all seven genes have an mRNA profile in that range. In certain embodiments, the mRNA transcript profile for the stem cells will also be characterized by detectable levels of KRT5.

Another aspect of the invention is based on the observations that DASCs can cause repair and/or remodeling of damaged king tissue in a distal manner, indicating the role of one or more paracrine factors. When compared to NESCs and TASCs, DASCs had a profoundly increased potency for inducing tissue regeneration, further indicating that the secretion of relevant paracrine iaetor(s) were upregulated in the DASCs as compared to the other two stem ceil populations. Accordingly, it is contemplated that the use of one or more therapeutic agents which are derived from the secreted proteins of the DASC population can be used to treat or prevent diseases and disorders involving airway tissue. A list of genes which are upregulated in DASCs is provided in Table I below. Airway therapeutics contemplated by the present invention include polypeptides encoded by the upregulated genes, fragments and analogs of those polypeptides, and agonists or rnimetics of those proteins.

The stem cells or airway therapeutics of the present invention can be used to treat a range of pulmonary condit ions. Such conditions include pulmonary hypertension, neonatal pulmonary hypertension, neonatal bronchopulmonary dysplasia, chronic obstructive pulmonary disease (COPD), acute and chronic bronchitis, emphysema, bronchiolitis, bronchiectasis, radiation pneumonitis, hypersensitivity, pneumonitis, acute inflammatory asthma, acute smoke inhalation, thermal lung injury, allergic asthma, iatrogenic asthma, cystic fibrosis, and alveolar proteinosis, alpha- 1 -protease deficiency, pulmonary

inflammatory disorders, pneumonia, acute respiratory distress syndrome, acute lung injury, idiopathic respiratory distress syndrome, and idiopathic pulmonary fibrosis.

In certain embodiments, compositions of the present invention which may include stem cells and _' Or airway therapeutics can be used as part of methods for treating pulmonary inflammation. Examples of pulmonary inflammation that can be treated include those associated with pulmonary hypertension, neonatal pulmonary hypertension, neonatal bronchopulmonary dysplasia, chronic obstructive pulmonary disease, acute bronchitis. chronic bronchitis, emphysema, bronchiolitis, bronchiectasis, radiation pneumonitis, hypersensitivity; pneumonitis, acute inflammatory asthma, acute smoke inhalation, thermal long injury, allergic asthma, iatrogenic asthma, cystic fibrosis, alveolar proteinosis, alpha- 1 - protease deficiency, pulmonary inflammatory disorders, pneumonia, acute respiratory distress syndrome, acute lung injury, idiopathic respiratory distress syndrome, or idiopathic pulmonary fibrosis.

For instance, the present invention provides a method for causing lung regeneration in a subject in need thereof comprising administering to subject an effective amount of DASCs to the subject.

in certain embodiments, the subject is a mammal, in a preferred embodiment, the mammal is human.

In certain embodiments, the subject suffers from acute airway disease, lung fibrosis or degenerative upper airway disease. In other embodiments, the administration of DASCs treats acute airway disease, lung fibrosis or degenerative upper airway disease.

The disclosure further provides a use of the stem cells and/or airway therapeutics in the manufacture of a medicament for promoting lung tissue regeneration or preventing or slowing lung tissue degeneration.

The disclosure further provides a composition of purified airway epithelial stem cells, which cells are keratin 5 ( Kri5) positive and p63 positive, can propagate for at least 10 doublings, and more preferably at least 20, 30, 40, 50 or even 60 doublings, while

maintaining a pluripotent phenotype, and can differentiate into airway epithelia. In certain embodiments, the composition includes a clonal population of airway epithelial stem cel ls. The airway epithelial stem ceils can be nasal epithelial stem cells (NESCs) (i.e., isolated from nasal turbinate); trachael airway stem cells (TASCs) (i.e., isolated from tracheobronchial epithelia); or distal airway stem cell (DASC) {i.e., isolated from isolated from distal airway tissue). In certain embodiments, the airway epithelial stem cells are mammalian cells. The airway stem epithelial cells can be human cells.

The disclosure further provides a method of screening for an agent which may be used to cause lung regeneration and/or prevent degeneration, including the steps of providing airway epithelial stem cells, which cells are keratin 5 (Krt5) positive and p63 positive, can propagate for at least 10 doublings, and more preferably at least 20, 30, 0, 50 or even 60 doublings, while maintaining a pluripotent phenotype, and can differentiate into airway epithelia; contacting the airway epithelial stem cells with a test agent; and detecting the ability of the test agent to increase viability, growth, migration or differentiation of the airway epithelial stem cells, and/or increased secretion of paracrine factors able to prevent II.,- 13 mediated inflammation and/or remodeling of lung tissue. Wherem if the tes agent increases viability, growth, migration, differentiation and/or paracrine secretion of the airway epithelial stem cells than the test agent may be effective in causing lung regeneration.

in certain embodiments, the airway epithelial stem cells are distal airway stem cell (DASC) isolated from isolated from distal airway tissue. In other embodiments, the test agent is also contacted with nor mal cells or tissue of the lung, and the differential ability, if any, of t he test agent to increa se t he viabilit y, growth, migration or differentiation of the normal cells or tissue is compared to that with the airway epithelial stem ceils. In other embodiments, the airway epithelial stem cells are human cells. In other embodiments, the test agent is selected for farther drug development if the test increases the viability, growth or ability to differentiation of the airway epithelial stem cells by at least 20%.

In certain embodiments, the airway epithelial stem ceils are provided as a clonal population of cells. In other embodiments the test agent is small molecule, carbohydrate, peptide or nucleic acid. The test agent can specifically bind to a cell surface protein on the clonal population of ce lls. The test agent can also be an antibody or antibody mimet ic.

The disclosure further provides a method for causing lung regeneration in a subject in need thereof comprising administering to the subject an effective amount of the medicament described above to the subject to promote lung regeneration in the subject. The subject can be a mammal. Thai mammal can be a human, in certain embodiments, the subject suffers from acute airway disease, rung fibrosis or degenerative upper airway disease. In other embodiments, the administration of airway epithelial stem ceils treats acute airway disease, lung fibrosis or degenerative upper airway disease.

in one aspect, the disclosure pro ides a method for causing lung regeneration, comprising administering to a subject a therapeutic amount of an agent that increases the expression and/or bio logical activity of one or more of the Cluster B genes set forth in Figure 14 or 15, such that the lung is regenerated.

fn another aspect, the disclosure provides a method of identifying a compound useful for regenerating lung tissue, the method comprising administering a test compound to an HI 1 infected mouse and determining the amount of epithelial metaplasia in the presence and absence of the test compound, wherein an increase in the amount of distal airway stem ceils (DASCs) identifies a compound useful for regenerating lung tissue.

The disclosure farther provides a method for causing lung regeneration in a subject in need thereof comprising administering to the subject an effective amount of one or more proteins, or active fragments thereof secreted by airway epithelial stem cells, thereby treating or preventing acute airway disease, king fibrosis or degenerative upper airway disease in the subject. The subject can be a mammal. That mammal can be a human. In certain

embodiments, the subject suffers from acute airway disease, lung fibrosis or degenerative upper airway disease.

The disclosure further provides a method of screening for an agent to promote lung regeneration. The method includes the steps of providing a mammal; administering an agent (such as an EGFR inhibitor and lipopolysacchaiide (LPS)}, thereby inducing symptoms associated with acute lung injury (ALT) in the mammal; and administering the agent to the mamma! with induced ALI ; wherem if the test agent improves the symptoms associated with ALI then the test agent may be effective in causing lung regeneration.

In certain embodiments, the mammal is a rodent. Specifically the rodent can be a mouse, rat or rabbit. In certain embodiments, the EGFR inhibitor is gifitinib. In other embodiments, the EGFR inhibitor is administered daily. Specifically, the EGFR inhibitor can be administered for three days. In certain embodiments, the LPS is administered at between 1 50 and 300 ug per day. Specifically, the LPS can be administered for three days.

In another embodiment, fibrosis is induced in the mamma! prior to administering EGFR inhibitor and lipopolysaecharide (LPS) to the mamma!. This fibrosis can be induced by administering bleomycin. The bleomycin can be administered intxatracheal!y or intraperiioneally.

The disclosure also provides a method of isolating a population of stem cells. The method includes the steps of providing a sample of nasal tracheai or lung tissue; washing the tissue in washing medium; dissociating cells from the tissue: washing the cells in washing medium; resuspending the cells in growth media; filtering the cells; and plating the cells on lethaiiy irradiated 3T3-J2 fibroblasts.

In certain embodiments, the cold washing medium comprises Dulbecco's modified Eagle's medium (DMEM). In other embodiments, the tissue is dissociated by cutting the tissue into pieces and digesting the pieces in digestion medium. The digestion medium comprises DMEM, 5% fetal bovine serum and 2mg ml collagenase. In other embodiments, the growth medium comprises 5 mg/ml insulin, 10 ng/mi F.GF, 2x1 Q ^~9 M

thyronine, 0.4 mg/ml hydrocortisone, 24 mg/ml adenine. 1x1 Q ^"Rt M cholera toxin in

DMEM/Ham's FT 2 3: 1 medium with 10% fetal bovine serum. In other embodiments, the filtering is performed by passing the cells through 70μιη nylon eel! strainer. The disclosure also provides a method of treating chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF) in a subject in need thereof comprising inducing acute lung injury (ALI ) in the subject, thereby treating COPD or IPF by inducing acute injury whic promotes activation of regenerative responses from lung stem ceils, in certain embodiments, the ALI is induced by delivery of one or more inflammatory agents. The delivery can be systemic or localized. In other embodiments, the inflammatory agents are selected from Hpopolysaccharide (LPS), EGFR inhibitors. TNF-a, IL- l a, IL- l b, I L-6. IL-8, and highly controlled bacterial strains (HCBS). The EGFR inhibitor can be gefliinib or erlotinib.

In another embodiment, the method of treating chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF), also includes administering an ablative technology to the subject. In certain embodiments, the ablative technology is radio frequenc ablation (RFA), phofodynamic therapy or cryoablation.

hi another embodiment, the method of treating chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF), also includes administering airway adult stem cells to the subject after, during or before induction of ALL The stem ceils can be exogenously grown. The stem cells can also be allogenic or autologous to the subject. The stem cells can also be TASCs or DASCs. The stem cells can also be isolated from biopsies of the upper and lower airways via bronchoscopy.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows p63 -expressing basal cells in the bronchioles versus deep long of normal mice at 0 and 1 1 dpi .

Figure 2 shows the level of overall p63 protein expression in distal airways.

Figure 3 shows quantification of BrdlI+/ ^' rt5+ cells in the interbronchio!ar regions.

Figure 4 is a schematic of human airways as source of cells for stem ceil cloning. NESCs, nasal epithelial cells; TASCs, tracheobronchial epithelial cells; DASCs, small airway epithelial cells. Left panel, Epithelial cell clones on irradiated Swiss 3T3 ceils. Middle panel p63 immunofluorescence. Right panel Keratin 5 (Krt5) immunofluorescence.

Figure 5 is a comparative heatmap ofNESC, TASC and DASC expression profiles.

Figure 6 is a principle component analysis (PCA) of expression micro arrays.

Figure 7 shows air- liquid interface (ALI) differentiation of NESCs indicated by antibodies to tubulin and mucin 5 A to mark ciliated cells and goblet cells, respectively. Figure 8 shows rat DASCs on 3X3 cells derived from single cell suspension of deep lung tissue. Top left: Phase contrast; Bottom left: hiununofhioreseence with anii-po3 antibodies. Middle panel: Image of imilammar structures produced by rat DASC pedigree- specific lines after 2 i days in 3-D Matrigel culture. The scale bar represents 50 mm.

Figure 9 is a schematic of the differentiation potential of regiospecific airway stem cells derived from multiple in vitro models.

Figure 10 is a Venn diagram depicting differentially expressed genes between TASC and DASC stem cell pedigrees, while the graphics below indie-ate the absolute fold-change in gene expression between TASC and DASC among more than 17,000 informative genes, Figure 1 1 is a table listing differentially expressed genes between TASC and DASC stem eel! pedigrees.

Figure 12 shows a section of 25 dpi lung sta ned with antibodies to rt5, SPC, and coimterstained with DAPF Four regions are demarcated with boxes as laser capture

microdissection, targets (1) S C+ cells in densely infiltrated zones, (2) Krt5 pods. (3) SPC- / rtS" zones with dense infiltrates, and (4) SPC+ cells in normal lung, inset shows PC A of three independent LCM samples corresponding to regions 1-4. The scale bar represents 200 mm.

Figure 13 shows a heatmap of 2205 differentially expressed genes with p value < 0.05 derived from LCM samples of regions 1-4. Gene clusters A-D are indicated. The left panel is a heatmap indicating relative expression of individual genes linked to alveoli in the datasets corresponding to regions 1-4.

Figure 14 shows a gene ontology analysis of gene clusters A-D indicated in heatmap together with associated p values.

Figure 1 is a table listing genes f om the analysis of gene clusters A-D.

DETAILED DESCRIPTION

I. Overview

The present disclosure shows the induction and recovery from an acute respiratory distress-like syndrome in mice infected with sublethal doses of a murine-adapted H1 N1 influenza virus. The disclosure shows that despite extensive damage to airway epithelial tissues, a p63 -expressing population of cells in bronchioles undergoes a massive expansion and dispersion to sites of affected lung parenchyma. These migratory p63 -expressing ceils form discrete foci or "pods" that expand to a size and shape approximating those of alveoli and express genes linked to alveolar function. In parallel studies, three regiospeeifie stem ceils were cloned from human airways demonstrate that one of these, the distal airway stem cell (DASC), has the unique potential of differentiating to alveolar lineages. These p63- expressing ceils participate in alveolar assembly processes modeled by human DASCs in vitro, and therefore represent key features in lung regeneration in response to acute respiratory distress syndrome (ARDS).

Recovery from lung damage that has advanced to ARDS is highly variable and poorly understood at present. Critical questions remain to understand the differential fates of ARDS patients, the potential of lung regenerat ion versus fibrosis, and whether therapies can sway clinical outcomes. This disclosure addresses the recovery from ARDS in mice infected by HI l influenza. The histological evidence suggests a largely complete lung restoration several months following severe influenza infection (this study; arasaraju et al, 2009). in fact the disclosure shows that, unlike bleoniycin-induced A DS, which invariably leads to fibrosis without evidence of regeneration (Moore and Hogaboam, 2008; Hoshino et at, 2009), mice recovering from influenza infections lack detectable lung fibrosis even following viral doses approaching the LD50. These data suggest that considerable regeneration of lung tissue, including complex alveolar-capillary networks, must he acting in this recovery. Indeed for the conducting, upper airways of mice, such as nasal passages, trachea, and bronch i, there are abundant data for regeneration after severe damage involving p63 -expressing basal, cells (Stripp and Reynolds, 2008; Rock et a!,, 2010). Despite this progress in understanding stem ceils of the conducting a irways, definitive evidence for a stem ceil that can contribute to lung regeneration has been more elusive. BronchioaSveolar stem cells, or BASCs (Kim et al, 2005), have been a useful model for such stem cells but have not been cloned nor characterized beyond a limited marker set. More recently, a c- Kit -positive stem cell f om the human airways expressing many markers of embryonic stem (ES) cells has been described to give rise to both epithelial and endothelial components of alveolar capillary complexes in xenograft experiments (Kajstura et al, 201 1 ). The stem cells described herein, are fundamentally different from BASCs or the c-Kit, ES-like stern cells presented earlier and ^• first drew attention as massive numbers of p63 -expressing ceils in the damaged lung parenchyma at the height of influenza- induced damage. p63-expressing cells are not found in normal interstitial lung, and rarely even detected in normal bronchioles. However, p63 ceils increase dramatically in bronchioles in the first several days of influenza infection, and appear in nearby damaged interstitial lung at 1 1 days post infection where they continue proliferation and assemble into pods. Remarkably, the number of clonogenic p63-expressing

l i ceils in the distal airways increases several hundred-fold within seven days of influenza infection, and these cells assume aspects of gene expression patterns see in p63-expressing stem cells in the epidermis during wound repair. Pods containing these cells are almost always found in a radial pattern about a bronchiole that also has p63 ^« expressing cells, and not about bronch io les that lack p63 cells. One interpretation of these data is that bronchioles are the source of these cells, a concept supported by lineage tracing experiments with the Krtl 4~ Cre/Rosa26-stop~LacZ mice.

The efforts to clone and characterize three regiospecific stem cells of the airways has provided a foundation for understanding the nature of th p63 -expressing ceils that comprise the Krt5 pods following influenza infection. The role of p63 -expressing basal cells as stem cells for distal lung was largely discounted because they proved to be rare in the small bronchioles compared with the upper airways. Thus the ease by which immature clones could be generated of p63 -expressing ceils from populations of human disStai airway epithelial cells was surprising. Despite the nearly 99% overlap in gene expression between DASCs and the upper airway TASCs and NESCs, DASCs displayed commitment to a unique program of differentiation that includes alveolar epithelium.

These Kri5 pods are intermediates in alveolar regeneration of influenza-damaged lung, and not a pathogenic pathway akin to bronchiectasis. 11 dpi Kit 5 pods are solid spheres of cells thai develop lumen and expand in size over the next 10 days to form alveoli- like structures. This hollowing and uniiaminar appearance is strikingly similar in timing and appearance to the alveoli- like structures formed by human DASCs in 3-D cultures. These data were further supported by the co-staining of the Krt5 pods with antibodies directed to antigens found exclusively in alveoli such as PDPN and the target of the 1 1B6 monoclonal antibody. Neither of these antibodies reacts with regions of influeirea-damaged lung that lack Kit5 pods.

Perhaps the most intriguing evidence favoring a role for Krt5 pods in alveolar

regeneration was the direct comparison of gene expression profiles of discrete regions of lung at 25dpi. Four regions are evident in these lungs: 1) normal lung marked by ordered alveolar networks, 2) highly infiltrated zones without alveolar markers whatsoever, 3) zones of immune cell infiltration and disordered SPC staining, and 4) clusters of Krt5 pods showing intermediate infiltration. The expression microarray data revealed that the high-density regions (SPC+ or SPC-) showed similar patterns of gene expression marked by inflammation, innate immune functions, and B and T cell profiles. From these data, which are consistent with the high level of infiltration of both regions and the very low expression of alveolar markers such as PDPN or the 1 1 B6 monoclonal antibody, it is likely that both regions are damaged and not undergoing active repair. Regions marked by clusters of KrtS pods, in contrast, had gene expression profiles that showed significant overlap with those of normal regions of interstitial lung. Within the set of genes overlapping between the clusters of KrtS pods and the normal lung were genes common to ATI cells. Interestingly, Gene Ontology analysis showed that these overlapping genes contained genes involved i angiogenesis, endothelin signaling, and aromatic amine degradation, all processes or functions attributed to lung endothelial cells and the formation of new blood vessels. Thus regions occupied by KrtS pods are likely associated with, in addition to ATI cell markers, the initiation of new blood vessels, ft is tempting to speculate that such angiogenesis is related to the need to bring capillary beds into dir ect contact with regenerating alveolar structures.

II. Definitions

The term "agent" includes any substance, molecule, element, compound, entity, or a combination thereof, it includes, but is not limited to, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances

As used herein, the term "RNAi agent" refers to an agent, such as a nucleic acid molecule, that mediates gene-silencing by R A. interference, including, without limitation, small interfering siRNAs, small hairpin RNA (slill A), and micro RNA (miRNA).

The term "cell surface receptor ligand". as used herein, refers to any natural ligan for a cell surface receptor.

The term "antibody" encompasses any antibody (both polyclonal and monoclonal), or fragment thereof, from any animal species. Suitable antibody fragments include, without limitation, single chain antibodies (see e.g.. Bird et al. (1988) Science 242:423-426; and Huston et al (1988) Proe. Natl. Acad. Sci. U.S. A 85:5879-5883, each of which is herein incorporated by reference in its entirety), domain antibodies (see, e.g., U.S. Patent 6.2 1, 1 8; 6,582,915; 6,593,081 ; 6,172,197; 6,696,245, each of which is herein incorporated by reference in its entirety), anobodies (see, e.g., U.S. 6,765,087, which is herein incorporated by reference in its entirety), and UniBodies (see, e.g., W020 7, 59782, which is herein incorporated by reference in its entirety

The term "antibody-like molecule", as used herein, refers to a no n- immunoglobulin protein that has been engineered to bind to a desired antigen. Examples of antibody-like molecules include, without limitation, Adnectins (see. e.g., WO 2009/083804, which is herein incorporated by reference in its entirety), Affibodies (see, e.g., U.S. Patent No. 5,831 ,012, which is herein incorporated by reference in its entirety), DARPins (see, e.g., U.S. Patent Application Publication No. 2004/0132028, which is herein incorporated by reference in its entirety), Aniicaiins (see, e.g., U.S. Patent No. 7,250,297, which is herein incorporated by reference in its entirety), Avimers (see, e.g., U.S. Patent Application Publication Nos.

200610286603, which is herein incorporated by reference in its entirety), and Versabodies (see, e.g.. U.S. Patent Application Publication No. 2007/0191272, which is hereby

incorporated by reference in its entirety).

The term "biological activity ^' " of a gene, as used herein, refers to a -functional activity o f the gene or its protein prod uct in a biological system, e.g., enzymatic activity and transcriptional activity.

The term "biocompatible delivery vehicle", as used herein, refers to any

phyicslogically compatible compound that can carry a drug paySoad, including, without limitation, microcapsules, micropaiticl.es, nanoparticles, and liposomes.

The term "suitable control", as used herein, refers to a measured mR A or protein level (e.g. from a tissue sample not subject to treatmen by an agent), or a reference value that has previously been established.

The term "pluripotent" as used herein, refers to a stem or progenitor cell that is capable of differentiating into any of the three germ layers endoderm, mesoderm or ectoderm.

The term "muStipoient", as used herein, refers to a stem or progenitor cell that is capable of differentiating into multiple l ineages, but not all lineages. Often, muhipotent cells can differentiate into most of the ce ils of a particular lineage, for example, ematopoietic stem cells.

The term "oiigopotent", as used herein, refers to a stem or progenitor cell that ca differentiate into two to five cell types, for example, lymphoid or myeloid stem ceils.

The term "positive", as used herein, refers to the expression of an mRNA or protein in a ce il, wherein the expression is at least 5 percent of the expression of actin in the cell.

The term "negative", as used herein, refers to the expression of an mRNA or protein in a cell, wherem the expression is less than 1 percent of the expression of actin in the cell.

"Pulmonary administration" refers to any mode of administration that delivers a.

pharmaceutically active substance to any surface of the lung. The modes of delivery can include, but are not limited to, those suitable for endotracheal administration, i.e.. generally as a liquid suspension instillate, as a dry powder "dust" or insufflate, or as an aerosol Pulmonary admi istration can be utilized for both local and systemic delivery of pharmaceutically active substances.

"Active agent" refers to a therapeutic or diagnostic compound that is administered to achieve a desired therapeutic or diagnostic result or purpose. Pharmaceutically active agent refers to an agent that is a biologically-active synthetic or natural substance that is useful for treating a medical or veterinary disorder or trauma, preventing a medical or veterinary disorder, or regulating the physiology of a human being or animal. The range of active compounds is considered below.

Ill, Exemplary Embodiments

A. Molecular Signature of Regeneratin Lung Cells.

Transcriptome analysis of UNA derived from a population of cells in regenerating lung led to the remarkable discovery that these cells have a distinct molecular signature. In particular, a number of genes were identified as being upregulated in these cells. Moreover, a subset of these genes (set forth below in Table 1, the sequences of which are each specifically incorporated herein by reference to their respective efSeq Transcript ID numbers) include secret ed proteins that are necessary for the process of regeneration to occ ur in lung. In certain embodiments, these secreted proteins are used as therapeutics to induce lung regeneration. Fable 1 also includes genes that are membrane receptors that are necessary for the process of regeneration to occur in the lung. In certain embodiments, modulators of these membrane receptors are used as therapeutics to induce lung regeneration. Accordingly, the present invention makes use of the identified genes to provide methods and composit ions for diagnosing, monitoring or causing lung regeneration. However, it should be appreciated that such methods and compositions are not limited to diagnosing, monitoring or causing lung regeneration, but can be can be used more generally for diagnosing., monitoring or treating or preventing any disease arising from lung pathology. Such diseases include, without limitation, acute airway disease, influenza, lung fibrosis or degenerative upper airway disease.

Table 1. B. Airway Therapeutics

Another aspect of the invention is based on the observations that DASCs can cause repair and/or remodeling of damaged king tissue in a distal manner, indicating the role of one or more paracrine factors. When compared to NESCs and TASCs, DASCs had a profoundly increased potency for inducing tissue regeneration, further indicating that the secretion of relevant paracrine factor(s) were upregulated in the DASCs as compared to the other two stem ceil populations. Accordingly, it is contemplated that the use of one or more therapeutic agents which are derived from the secreted proteins of the DASC population can be used to treat or prevent diseases and disorders involving airway tissue. A list of genes which are upregulated in DASCs is provided in Table 1 above. The examples below are provided for one of those proteins, PLUNC, but ma equall apply to other proteins encoded by the genes referenced in Table ! above.

In one embodiment, the therapeutic agent includes the secreted protein PLUNC (also referred to as secretory protein in upper respiratory tracts or SPURT), or an active fragment thereof. The human PLUNC cDN A codes for a !eucftie-rich protein of 256 amino acids (GenBank: AF2.14562.1.) which is 72% identical to the murine protein. The full length human PLUNC protein has the following sequence:

MFQTGGLIV ^' YGLLAQTMAQFGGLPVPLDQTLPLNVNPALPLSPTGLAGSLTNALSN

GLLSGGLLGILENLPLLDIL PGGGTSGGLLGGLLGKVTSVTPGLNNIIDIKVTDPQLLE

I JLVQSPDGHRLYVTIPI JIKLQV TPLVGASLL LAVKLDITAEILAV DKQERIHL

VLGDCTHSPGSLQlSLLDGLGPLPiQGLLDSLTGiLNKVLPELVQG VCPLV EVL G LD IT LVHD1 VNM LIHGLQFY ΪΚ V (SEQ ID NO:!)

The PLUNC proteins of the invention also include functional portions or fragments. The length of the fragment is not critical as long as it substantially retains the biological activity of the polypeptide. The therapeutic agent can be the full length protein, or can be a fragment thereof, such as the leucine rich region ranging from L-24 to L-218, or any other fragment or analog thereof which is able to recapitulate the activity of PLUNC for inhibiting IL-1 induced damage in in vitro lung tissue assays. Other illustrative fragments comprise at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more contiguous amino acids of a PLUNC protein. in other embodiments, the fragment comprises no more than about 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, 1.0, 8, 6, or 4 contiguous amino acids of a. PLUNC protein. In one embodiment, the .fragment comprises, consists essentially of, or consists of a sequence from about residue 20 to about residue 41 of human S PLUNC 1 e.g., about residue 22 to about residue 39, or the corresponding sequence (e.g., the approximately 20 amino acids immediately after the signal peptide) from another PLUNC protem.

Likewise, those skilled in the art will appreciate that the present invention also encompasses fusion polypeptides (and polynucleotide sequences encoding the same) comprising a PLUNC protein (or a functional fragment thereof). For example, it may e useful to express the polypeptide (or functional fragment) as a fusion protein that can be recognized by a commercially available antibody (e.g., FLAG motifs) or as a fusion protem that can otherwise be more easily purified (e.g., by addition of a poly-His tail). Additionally, fusion proteins that enhance the stabilit of the polypeptide may be produced, e.g., fusion proteins comprising maltose binding protein (MBP) or glutathione- S -transferase. As another alternative, the fusion protein can comprise a reporter molecule. In other embodiments, the fusion protei can comprise a polypeptide that provides a function or activity thai is the same as or different from the activity of the polypept ide, e.g., a targeting, binding, or enzymatic activity or function.

Likewise, it will be understood that the polypeptides specifically disclosed herein will typically tolerate substitutions in the amino acid sequence and substantially retain biological activity. To identify polypeptides of the invention other than those specifically disclosed herein, amino acid substitutions may be based on any characteristic known in the art, including the relative similarity or differences of the amino acid side-chain substituents, for example, their hydrophobic ity, hydrophilicity, charge, size, and the ^' like.

In identifying amino acid sequences encoding polypeptides other than those specifically disclosed herein, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protem is generally understood in the art (see, Kyle and Doolittle, J. Mol. Biol. 157.105 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its

hydropho icity and charge characteristics ( yte and Doolitiie, id.), these are: iso leucine (+4.5); valine (-H.2); leucine ( 3.8); phenylalanine (-1-2.8); cysteine/cystine (+2.5);

methionine (+1.9); alanine (-H .8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (~ 0.9); tyrosine (-1.3); proline (- 1.6); histidine (-3.2); giutamate (-3.5); giutamine (-3,5);

aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4,5), Accordingly, the hydropathic index of the amino acid (or amino acid sequence) may be considered when modifying the polypeptides specifically disclosed herein.

It is also understood in the art that the substitution of amino ac ids can be made on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 states that the greatest local average

hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophiiicity values have been assigned to amino acid residues: arginine (+3.0); lysine (.+-.3,0); aspartate (+3.0,+-.1); glufamate (+3.0.+-J); serine (+0.3); asparagine (+0,2); giutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.I); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (- 1.5); leucine (- 1.8); isoleucine (- 1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

Thus, the hydrophilic ty of the amino acid (or amino acid sequence) may be considered when identifying additional polypeptides beyond those specifically dis losed herein.

In embodiments of the invention, the polynucleotide encoding the PLUNC protein (or functional fragment) will hybridize to the nucleic acid sequences encoding PLUNC proteins that are known in the art or fragments thereof under standard conditions as known by those skilled in the art and encode a functional polypeptide or functional fragment thereof

For example, hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% formamide with SxDenhardt's solution. 0.5% SDS and IxSSPE at 7°C; conditions represented by a wash stringency of 40-45%

formamide with SxDenhardt's solution, 0.5% SDS. and IxSSPE at 42°C; and conditions represented by a wash stringency of 50% formamide with SxDenhardt's solution, 0.5% SDS and 1 xSSPE at 42°C, respectively) to the polynucleotide sequences encoding the PLUNC protein or functional fragments thereof specifically disclosed herein. See, e.g., Sambrook et aL, Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, Ν,Υ,, 1989), in other embodiments, polynucleotide sequences encoding the PLUNC protein have at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the publicly known nucleic acid sequences (disclosed in GenBank) or functional fragments thereof and encode a functional polypeptide or functional fragment thereof.

Further, it will be appreciated by those skilled in the art thai there can he variability in the polynucleotides that encode the polypeptides (and fragments thereof) of the present invention due to the degeneracy of the genetic code. The degeneracy of the genetic code, which allows different, nucleic acid sequences to code for the same polypeptide, is well known in the literature

Likewise, the polypeptides (and -fragments thereof) of the invention include polypeptides that have at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher amino acid sequence identity with the publicly known polypeptide sequences.

As is known in the art, a number of different programs can be used to ident ify whether a polynucleotide or polypeptide has sequence identity or similarit to a known sequence. Sequence identity or similarity may be determined using standard techniques known in the art, inc luding, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1 81), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 ( 1970), by the search for similarity method of Pearson &. Lipnian, Proc. Natl. Acad. Set. USA. 85:2444 (1988), by computerized

implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the

Wisconsin Genetics Software Package. Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et ai., Nucl. Acid Res. 12:387 (1 84), preferably using the default settings, or by inspection.

An example of a useful algorithm is PILEIIP. PILEIJ P creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments, it can also plot a tree showing the clustering relationships used to create the alignment. PI LEIJP uses a simplification of the progressive alignment method of Feng & Doo little, J. Mol. Evol. 35:351 (1 87); the method is similar to that described by Higgins & Sharp, CABiOS 5:1.51 (1989).

Another example of a. useful algorithm is the BLAST algorithm, described in Altschul et al. J. Mol Biol. 215:403 (1990) and Kariin et aL Proc, Natl. Acad. Sci. USA 90:5873 (1 93). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al, Meth. EnzymoL, 266:460 (1 96);

blast. wustVedu blast README.htmL WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itse lf depending upon the composition, of the particular sequence and composition of the particular database aga inst which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschui et al.,

Nucleic Acids Res, 25:3389 (1997),

A percentage amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

In a similar manner, percent nucleic acid sequence identity with respect to the coding sequence of the polypeptides disc losed herein is defined as the percentage of nucleotide residues in the candidate sequence that are identical with the nucleotides in the

polynucleotide specifically disclosed herein.

The alignment may inc lude the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the polypeptides specifically disclosed herein, it is understood that in one embodiment, the percentage of sequence identity will, be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, sequence identity of sequences shorter than a sequence specifically disclosed herein, will be determined using the number of amino acids in the shorter sequence, in one embodiment, in percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.

in one embodiment, only identities are scored positively (+1 ) and all forms of sequence variation including gaps are assigned a value of "0," which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations.

Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.

in still other embodiments, the invention comprises treating or preventing damage to airway tissue using agents which upregulate the level of expression or secretion of protein shown in Table 1 , To illustrate, TLR2 agonists such as Pam3CSK4 (a synthetic triacylated lipoprotein) and H LM (heai-killed prepara tion of Listeria monocytogenes) can increase PLIJNC expression. Synthetic small molecule agonists ofTLR2 are also well known in the art, suc as described in Guan et al. J Biol Chera, 2010 July 30; 285(31): 23755-23762 and shown below. Accordingly, the invention contemplates the use of TLR2 agonists as

therapeutic agents.

C. Formulation

The proteins, small molecules and other therapeutics of the invention may be formulated into a variety of acceptable compositions. Such pharmaceutical composit ions can be administered to a mammalian host, such as a human patient, in a variety of forms adapted to the chosen route of administration, i.e. , by lavage, orally or parenterally, by intravenous, intramuscular, pulmonary or inhalation routes, in certain preferred embodiments, the airway therapeutic agent is formulated for pulmonary delivery.

in cases where compounds, for example, the therapeutic agent is sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of such compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition satis formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, .alpha.- ketogkttarate, and .alpha. -glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceutically acceptable salts are obtained using standard procedures well known i the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids also are made.

Pharmaceutically acceptable salts of polypeptides inc lude the acid addition salts (formed with the free amino groups o f the polypeptide) that are formed w ith morgan ic acids such as, tor example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxy! groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine and the like.

Thus, the therapeutic agents of the present invention may be systemically

administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food o f the pat ient's diet. For oral therapeutic administration, the compositions may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such

compositions and preparations should contain at least 0.1% of active compound.

The tablets, troches, pills, capsules, and the like may also contain the following; binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated, into sustained-release preparations and devices.

The therapeutic agents may also be administered intravenously or intraperit.onea.lly by infusion or injection. Solutions of the active agents may be prepared in water, optionally mixed, with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use. these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, efhanol, a poiyol {for example, glycerol, propylene glycol, liquid

polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanoL phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can b brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the therapeutic agent(s) in the required amount in the appropriate solvent with various of the other ingredients, enumerated above, as required, followed by filter sterilization, in the case of sterile powders for the preparation o f sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the protease inhibitors, lipase inhibitors or antioxidants plus any additional desired ingredient present in the previously sterile-filtered solutions.

Useful dosages of airway therapeutics of the present invention can be determined by comparing in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and. other animals, to humans are known to the art ; for example, see U.S. Pat. No. 4,938,949.

The amount of airway therapeutic agent, or active salts or derivatives thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the conditio being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator.

An airway therapeutic of the present invention can be delivered directly to the site of interest (the lung) to provide immediate relief of the symptoms of pulmonary inflammation. Such delivery can be by bronchoafveolar lavage, intratracheal administration, inhalation or bolus administration. In these case the surfactant mixture is included.

Procedures for performing pulmonary lavage are available in the art. See, e.g., U.S. Pat. No. 6,013,619. For example, pulmonary lavage can be performed as follows: a) applying gas positive end-expiratory pressure (PEEP) with a ventilator into a lung section of the mamma! at a regulated pressure, preferably from about 4 to 20 cm water;

b) instilling a lavage composition containing dilute surfactant in a pharmaceutically acceptable aqueous medium into one or more lobes or sections of the lung; and

c) removing the resulting pulmonary fluid from the Sung using short intervals of tracheo-bronchial suction, preferably using a negative pressur of about 20 to 100 mm mercury.

Typically, the PEEP is applied for a preselected time period prior to instilling step (b), preferably up to about 30 minutes, and in addition PEEP is typically applied continuously during steps (b) and (c) and for a preselected time period after remo ving step (c), preferably up to about 6 hours.

Delivery by inhalation is described further herein. Alternative delivery means include but are not limited to administration intravenously, orally, by inhalation, by cannulation, intracavitaiSy, intramuscularly, transclermaily, and subcutaneousSy.

Therapeutic compositions of the present invention contain a physiologically tolerable carrier together with an airway therapeutic described herein, dissolved or dispersed therein as an active ingredient. in a. preferred embodiment, the therapeutic composition is not immunogenic when administered to a mamma i or human patient for therapeutic purposes.

The preparation of a pharmacological, composition that contains active ingredients disso lved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically such compositions are prepared as mjeetables either as liquid

solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified.

The active ingredients can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanoS or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredients.

Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition io the active ingredients and water, or contain a buffer such as sodium phosphate at physiological p value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes

1>. Controlled Induction of Acute Lung Injury for COPD and IFF Therapy

A barrier for treating COPD and IPF is the presence of defective lung tissue in the form of sclerotic terminal bronchioles and alveoli in the case of COPD and scar- tissue in pulmonary fibrosis. The simple removal or ablation of this tissue by surgical or ablative technologies (radiofrequency ablation or RFA; photodynamic therapy or PDT; cryoablative therapy or CA T) is often insufficient to trigger stem cell-mediated regeneration. During the regeneration stimulated by influenza- induced A DS, the distal airway stem cells only migrated to interstitial regions of lung with active immune cell infiltrates, inducing an inflammatory response in interstitial regions of lung can both dismantle diseased regions of lung in COPD and fibrosis patients as well as trigger the recruitment of stem cells to mediate the regenerative repair.

Methods to induce King inflammation include the local or systemic application of lipopoiysacciiaride ( LPS), lipid polysaccharide conjugates derived from the outer membranes of Gram-negative bacteria (iJlich et al, 1 1 ; Raetz and Whitfield, 2002 ), These molecules are known to elicit strong inflammatory responses in mammals via a Toil-like receptor 4- (TLR4) dependent process (Jeyaseelan et al, 2005). LPS is thought to be an important mediator of sepsis-induced ARDS in humans, and peritoneal injections of LPS can induce an ARDS-Tike phenomenon in mice marked by a generalized interstitial lung inflammation (Gunther et al, 2001). Interestingly, mice who survive LPS injections recover normal lung fiuiction and histology without fibrosis, further reinforcing the notion that LPS is yielding an ARDS-iike recovery rather than a bleomycin response marked by fibrotic lesions. Thus, LPS represents one means of inducing an ARDS-iike phenomenon that potentially promotes a. regeneration response involving stem cell such as TASCs or DASCs, Additional triggers of ARDS-iike inflammation in the lung include the epidermal growth factor receptor (EGFR) inhibitor Gefitinib (Astrazeneca), which induces acute interstitial lung disease in

approximately 1% of patients (Camus et al, 2004: Tammaro et al. 2006: Takada et al, 201 J ), Significantly, the acute lung damage associated with LPS in mice is greatly

augmented by pretreatment with Gefitinib, with results in extended and enhanced

inflammation (Suzuki et al, 2003; J none et al., 2008). Another trigger could be TNF~CL

In certain embodiments, RFA, PDT, and cryoabiation are used in conjunction with instilling inflammatory signals such as those triggered by LPS, TNF- a (Mukhopadhyay et al., 2006) and other inflammatory triggers to mimic those attending infectious Al l known to promote the recruitment of the regenera tive response.

IV . Exemplification

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustratio of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1. H13S1 influenza infection of mouse airways.

Mice were infected with a murine-adapted H IN! (PR8) influenza A by intratracheal aspiration at viral titers of 125 to 1.x 105 PFU to determine an LD50 of approximately

500PFU. 250PFU was chosen as a dose to induce significant damage without lethality.

Lungs were harvested at multi le dpi and analyzed by histology and viral protein expression. Viral load was estimated by monitoring the expression of the M2 viral ion channel protein by immunofluorescence, which revealed maximal staking at 4dpi and. a loss of M2 signal by 1 1 dpi. However, tissue damage, as measured by the degree of immune cel l infiltration, appeared to peak at 1 ldpi, was reduced at 21dpi, and was largely cleared across the lung by 60dpi. These results are mirrored by weight loss in these animals that reaches an extreme at 10- 12dpi and recovers by day 20. At the cellular level, widespread destruction of all airway epithelial cells was observed at 7dpi resulting in a significant loss of markers tor Clara cells (CC 10) and ciliated cells (aeetylafed α-tubulin, TAp73) in the bronchiolar epithelia and AT2 cells (SPG ) of the alveolar epithelium. Damage to airway epithelium is consistent with observations of viral M2 expression in these cells at tour days post-infection. The peak of dense infiltrates of immune cells (CD45+) corresponds to the dense histological appearance of the lung at 1 1-14dpi.

The H 1N 1 influenza-infected mice show widespread cytopathic effects and extreme weight loss, and yet both these effects are mitigated between 21 -60dpi. Remarkably, these mice recover without the acquisition of lung fibrosis that accompanies the induction of lung damage by bleomycin, suggesting the possibility that epithelial regeneration underlies recover from influenza.

Example 2, Emergence of p63-expressing cells during influenza infection.

Given the identification of p63 -expressing basal, cells as stem cells for nasal and tracheal epithelia of the upper airways (Rock et al., 2009), whether p63-expressing ceils might also participate in lung regeneration following influenza infection was explored. Little evidence was found of p63-expressing basal cells in the bronchioles of norma! mice.

However, by 7dpi, ceils expressing p63 were evident in bronchioles (Figure 1). By 1 ldpi, both p63 cells and Clara cells were found intermingled in the bronchiolar epithelium, and by 21 days most of the bronchiolar epithelium appeared restored at the level of Clara cells while those with p63-expression in the bronchioles were less evident. This rise and fail of p63 expressing cells is reflected in overall p63 protein expression in distal airways (Figure 2). Unexpectedly, p63 -expressing cells were also found in large numbers in the highly damaged lung parenchyma at 1 l dpi. On closer inspection, these p63 -expressing cells in the damage lung appeared to be clustered in small groups. Using other markers of basal cells, such as antibodies to keratin 5 (KrtS), it was evident that the p63 -expressing cells formed discrete clusters or pods (hereafter "KrtS pods") in interstitial lung. On a gross level, KrtS pods were distributed in a concentric pattern about bronchioles. Brdli labeling of proliferating cel ls at 1 idpi revealed robust cell division of the Krt5÷ cells in the bronchioles as well as in the KrtS pods. Direct quantification of BrdU+/Krt5+ cells revealed a progressive decrease in intrabronchioiar regions from 1 1, dpi and an increase in interbroncliioiar regions from 1 1 dpi (Figure 3), The appearance of Krt5+ pods in the peribronchiolar regions of lung parenchyma coincides with the pinnacle of influenza- induced Sung damage. KrtS÷ pods were not observed in the bleo my c in-depe nde nt lung fibrosis model.

Example 3, Molecular analysis of human clonogetiic airway cells.

To identify distal airway stem cells and assess their relationship to stem cells of the upper airways, single cell cloning methods were employed on populations of human epithelial cells derived from nasal turbinate, tracheobronchial epithelia, and distal airway tissue including bronchioles and alveoli (Rheinwald and Green, 1975; Barrandon and Green, 1 87; Senoo et aL 2007). Immature colonies were obtained from approximately 1:500 to 1 :2000 ceils, and all of these stained uniformly for p63 and for keratin 5 (Krt5) (Figure 4). These immature clones were provisionall designated nasal epithelial stem cells (NESCs), tracheal airway stem cells (TASCs), and distal airway stem cells (DASCs). About 80 percent of these clones could be propagated further for fat least ao additional estimated 50 doublings while maintaining an immature phenotype (not shown). Despite the fact that the original starting cell populations were obtained from disparate regions of the airways, the immature stem eel! clones appeared indistinguishable by morphology and staining with basal cell markers. Gene expression datasets from these clones are binned in regiospecific group by unsupervised clustering and by PCA of the whole genome expression patterns despite sharing gene expression of approximately 99 percent of the 17,500 hybridizing genes (Figures 5 and 6).

Example 4. Pedigree tracking of airway stem cells.

The long-term self-renewal potential of the putative human stem cell clones allowed us to isolate independent pedigrees for the analysis of the progeny of a single cell Expansion of these lines yielded abundant immature cells of known pedigree for a range of

differentiation assays to assess lineage potential. For instance, pedigree lines of NESCs were analyzed and posted through multiple differentiation assays. The air-liquid interface model (ALl; Schmidt et aL, 1996) has been a powerful tool in airway epithelial differentiation and supports the differentiation of goblet cell and ciliated cells from immature populations of nasal epithelial cells (Usui et a!., 2000). Significantly, all pedigree-defined lines of NESCs showed similar distributions of goblet cells and ciliated cells in All cultures (Figure 7), supporting the concept the pedigree lines that were developed from single cells in tact have lineage potential ascribed to NESCs. Whole genome expression analysis of immature, pedigree defined lines and ALl-differentiated ceils support this notion and showed an increased expression of genes involved in ciliogenesis (e.g. DYNLRB2, 22.7x;TUBA4b _> 9.8*; and DMAH6, 7,2x; ail p<0.05) and in goblet cell inaction (e.g. MUC! _s 3.8x; MUCI 3, 5.6x; and UC20, 4.5 l x; all p<0.05). Self-assembling sphere (SAS; Jorissen et al. ₅ 1989) cultures of NESCs were also performed, SAS cultures, like the ALl cultures, induce both goblet cell and ciliated cell differentiation from NESCs, and involve the self-assembly of epithelial ceils in conditions where cells cannot adhere to a solid support. NESC pedigrees readily assemble into spheres under such conditions within 24 hours, and efficiently undergo both goblet cell and ciliated cell differentiation over the fo lowing 15 days upon transfer to differentiation media. Unlike the ALl cultures, which stratify into basal ceils and suprabasal differentiated cells, the SAS cultures show a monolayer of ciliated cells and goblet cells polarized to the exterior and lack basal cells altogether. Again, whole genome expression analysis supports the concept that SAS cultures promote ciliogenesis (e.g. increased expression of DYNLRB2, 68.4*; DNAH7, 28.6*; TEKT1 , 25.1 x; ail p<0.05) and goblet ceil formation (increased expression of MUC 15, 6.7*; IJC20, 7.6*; SCGB2A1 , 6.l ^x ; all p<0.05) from NESCs. In contrast to their similar differentiation programs in All and SAS cultures. NESCs behaved very differently in 3-D Matrigel. cultures. NESCs showed robust formation of solid spheres in Matrigel between days 5 and 10, which subsequently hollow with the addition of differentiation media, and by day 21 show immature cells at the periphery and ceils with squamous differentiation towards the lumen. This squamous metaplasia is supported by the comparison between gene expression of undifferentiated NESCs and those differentiated for 21 days in Matrigel, which show strong expression of squamous epithelial genes (e.g. LCE2B, 173.1*; KRT1G, 6.0x; and SPRR2A, 4,5 ; all p<0.05). Similar development of a squamous metaplasia has been seen with the

differentiation of nasal turbinate epithelial cell populations (Endres et al,, 2005), suggesting that pedigree-defined cells reta in the capacit y for this pathwa y of differentiation. Overall gene expression PC A indicates that each of these differentiation assays yields different outcomes marked by a stratified airway epithelium in A Ll cultures, a non-stratified airway epithelium in SAS cultures, and a squamous metaplasia in 3-D Matrigel cultures.

Example 5. DASC pedigrees assemble into alveolar-like structures in vitro. Defined pedigrees of TASCs and DASCs were grown in ALI c ultures to compare their differentiation, potential with that of NESC lines. Like the NESC Sines, the TASC pedigrees showed robust differentiation into ciliated cells and raudn-producing goblei cells during the 21 -day period of ALI culture. In contrast, the DASC pedigrees showed only minor indications of muci expression, rare ciliated ceil formation, and occasional CC10 expression indicative of Clara cells. The TASC ^" lines further differed from the DASC Sines in their degree of stratification in ALI cultures. The TASCs presented a multilayered epithelium with p63-positive basal cells underneath differentiated goblet and ciliated cells, while the DASC pedigrees retain a monolayer appearance over the differentiation period. Gene expression analysis of the ALI cultures of TASC and DASC lineages confirmed these observations, with the differentiated ^" TASC cultures showing high expression of genes involved in ciliogenesis, mucin production, and epithelial stratification compared to DASCs grown in ALI culture. In 3-D atrigei cultures, the TBEC lines, like the NESC pedigrees, underwent squamous metaplasia. Curiously, the DASC pedigree lines also assemble into spheres but ultimately these hollow and collapse into niultispherical structures by day 21. The hollowing effect appears to be the direct consequence of apoptosis as these cells within the spheres show robust activated caspase-3 staining. The multispherica! entities formed from DASC lines do not stratify but appear to be comprised of unilaminar cellular assemblies that express the alveolar type 1 marker PDPN. These same structures are labeled with the 4C 10 monoclonal antibody that recognizes a ca. 300kDa protein specific to human alveoli.

Consistent with the divergent differentiation of the TASC and DASC pedigrees in Matrigei cultures, datasets of who le genome microarrays of the structures assembled in 3-D cultures by these lines showed large differences including a host of genes implicated in alveolar structure or formation, including MGP, SPAC, ANGPT.l , LIMCH 1, CHI3LL KDR, PECAM, A XA3, SLC39A8, E.R FH, PDPN and TSPANS. These findings were supported by wider Gene Set Enrichment Analysis (GSEA), which showed the squamous metaplasia of the TASC Matrigei structures expressed genes associated with epidermal development. In contrast, the DASC-derived structures showed high expression of genes associated with angiogenesis regulation, monocation transport (Matthay et al.. 2002; Eaton et al, 2009). an important physiological function of ATI cells, and synaptic transmission, a possible reflection of the importance of neurogenic control of alveolar function (Safcuraa et al, 2006).

Example 6. Assembly of alveoii-Iike structures conserved in rodent-derived DASC pedigrees, To test whether similar p63 -expressing stem cells could be derived from the deep lung of non-huraan species, multiple immature clones were obtained from distal airways of sk- week-old rats. Defined pedigrees were developed from several of these that were

subsequently grown in 3-D Matrigel cultures. All of the rat pedigree-specific DASC lines formed uniform solid spheres after 10 days of growth that subsequently hollowed over the next 11 days (Figure 8), To determine whether these structures contain proteins linked to alveoli in vivo, a panel of monoc lonal antibodies were generated from mice immunized to rat distal airway tissue. Monoclonal antibodies I3A.1 and 54D1 are specific to rat alveoli and recognize proteins with molecular masses of 45 and 25kDa. respectively. These antibodies. which do not stain rat immature rat DASC clones, efficiently stain the unilaminar structures produced by the DASC pedigree lines. These data suggest that these alveolar-like structures express genes found in alveoli. Further, the DASCs described herein are fully committed to alveolar lineages with additional potential to form Clara, ciliated, and mucin producing cells and therefore distinct from the TASCs and NESCs that are committed to ciliated cells, goblet cells, and as well can undergo squamous metaplasia (Figure 9). Despite these differences in lineage commitment, DASCs show only minor differences in gene expression compared with TASCs and NESCs in the range of 100-200 genes (Figures 1.0 and 1 1 ).

Example 7. influenza-infected lung displays a massive increase in eionogenic basal cells, To probe the basal-like cells observed in infected lung, dissociated distal airway tissues were plated in eionogenic assays. The colonies that arose were uniform in appearance from the infected and control lungs and were composed of small. p63 -expressing immature cells and expressed Krt5. When grown on Matrigel cultures, cells from these colonies form solid spheres that hollow through eel! death to yield unilaminar structures similar to those generated by human DASCs. These unilaminar structures stain for antibodies to aquaporin 5 (Aqp5), a marker of alveoli. A comparison of expression performed on A amplified from individual clones and the Matrigel structures formed from them showed reproducible

differences in gene expression. The immaturity markers ri5 and K.rt l4 are lost in during differentiation in Matrigel, while markers of alveoli, such as Aqp5 and sur factant proteins Sftpai , Sftpb, and Stipe, are all upregulated in these structures. Consistent with the appearance of large numbers of p63-positive cells in lung parenchyma following HI Mi influenza infection, a several hundred-fold increase in p63 -expressing eionogenic cells in infected lungs was observed. Whole genome microarray revealed reproducible differences in 358 genes among three clones from control and infected lungs. One of the genes proved to be keratin 6A

(Krt6a), a known marker of migra ting keratinocyt.es during wound healing in the epidermis ( Wojcik et al, 2000). Expression of this gene alone differentiates between the clones derived from control lung and 12dpi lung. In vivo, Krt6 antibodies differentiate between basallike ceils in KrtS pods of interstitial regions from basal cells of bronchioles whereas KrtS antibodies recognize both these ectopic basal cells and those in the bronchiolar epithelium. GSEA of the expression data sets derived from the distal airway colonies revealed a bias for genes involved in wound healing, tissue development, and regulation of growth in those from infected mice.

Example 8. KrtS pods linked to lung regeneration.

The ability of human distal airway stem ceils to form alveoli-like structures in vitro suggested that the KrtS pods of basal-like cells seen in the influenza-damaged lung were components of a regenerat ive process. Consistent with this, the general appearance of individual KrtS pods at I ldpi was one of tight clusters of cells, while at 15 and 21dpi many of these pods had lumens reminiscent of alveoli. Direct quantification of these KrtS pod "t pes' ^' over time confirmed this notion, suggesting a progression over 10 days from a tight group of KrtS ^' - cells to structures resembling alveoli. Whether the KrtS pods would stain with a new monoclonal antibody generated from mice immunized with human fetal lung designated here as "1 iB6" was explored. 11B6 reacts specifically to alveolar regions of mouse lung but not to bronchioles. Staining of regions of damaged lung containin KrtS pods with the 1 1B6 monoclonal antibody revealed co-localization of the two antigens especially in those pods with larger diameters, suggesting a specificity for those KrtS pods with larger lumens. Quantification of this link between 1 1.B6 and KrtS-*- pods also correlated with dpi. The KrtS pods were also probed with antibodies to PDPN, which shows an alveoli- specific pattern in normal lung, PDPN antibodies also co-localize with KrtS pods.

In assessing the influenza-infected lung f om a panoramic view, three distinct areas were observed; normal appearing lung parenchyma with histologically normal alveoli, damaged regions marked by serai-dense infiltrate and KrtS pods, and damaged regions marked by highly dense infiltrates but without KrtS pods. KrtSr pods were not observed in regions of histologically normal lung tissue. The presence of KrtS pods in iess-denseiy infiltrated regions is that these regions are undergoing regenerative repair and clearing dense immune cell infiltrates in the process, indeed the regions marked by KrtS-f- pods show intermingled, CD45-posilive immune cells in the immediate proximity. Also, within regions of dense infiltrate were bronchioles that lacked Krt5-positive basal cells, whereas bronchioles associated with satellite K.rtS -positive pods also had KrtS-positive basal cells. This observation reflects the possibility that if the infection eradicates the bronchiolar epithelium, it may be incapable of spawning a peribronchiolar population of Krt5 pods.

Example 9, rt5 pods at sites of lung regeneration.

Laser capture microdissection (LCM) was used, to isolate RNA for expression microarray analysis from frozen sections of 25 dpi king regions rich in Krt5 pods, normal or repaired lung, or regions o f high-dens ity infiltrates withoui obvious repair . The regions of high-density infiltrates without obvious alveoli were further divided into regions with and without SPC, a marker of alveolar type II cells (Figure 12). PCA of these datasets revealed that the regions rich in Krt5 pods were most closely related to the regions of normal or repaired lung (Figure 12, inset). This impression is reinforced by a heatmap of differentially expressed genes in these datasets which shows the similarity between the densely infiltrated regions devoid of alveolar markers and the densely infiltrated, SPC - regions marked as

Cluster D genes (Figure 33). Cluster D genes included strong representations by immune cell functions and innate immune signaling consistent with the persistence of a dense infiltration of immune cells ( Figures 14 and 15 ). In contrast, the regions with rt5 pods showed significant, overlap with normal lung in a gene set designated Cluster B including genes involved with angiogenesis and endothelin signaling (Khimji and Rockey, 2010), as well as aromatic amine degradation known to be functionally linked to Sung endothelial cells (Gil I is and Pitt, 1982) (see Figures 14 and 15). Additionally, Cluster B showed an

o verrepresentatton by genes involved in. Wirt, Hedgehog, and nicotinic acetylcholine receptor signaling. Together, these data suggested that the Rrt5 pods are in association wit elements of endothelial cells involved in capillary formation. Moreover, Cluster B genes include an array of alveolar genes not expressed in regions of damaged lung lacking Krt5 pods (marked by the SPO/ .rt.5- or SPC-/K.H5-} (see Figures 14 and 15), including PDPN, eaveolin 2, Aqp5 ₅ and PDGFRa. Cluster C genes, those overrepresenied only in regions non-damaged lung, are characterized by gene sets associated with integrin signaling, angiogenesis, nicotinic acetylcholine receptor signaling, and axon guidance by semaphorins (see Figures 14 and 15). Consistent with the link between Krt5 pods and lung regeneration, robust co-localization of Krt5 and the alveolar-specific monoclonal antibody 1 1 B6 was seen, but no 11 B6 staining of damaged regions regardless of whether they have SPC staining. On the broadest level, these data support the notion that regions with Krt5 pods express genes similar to apparently normal or repaired lung, and very different from regions marked by severe damage. The also suggest a dynamic process involving a host of pathways whose significance for the recovery from ARDS will require extensive empirical validation.

Example 10. l ineage tracing of alveolar progenitors from bronchioles to alveoli.

While clusters of Krt5+ pods always appear in a peribronchiolar pattern in influenza infected lung about bronchioles with Krt5 cells, their origins remained to be established. To test whether the Krt5 cells that appear in the bronchioles are indeed the origins of the parenchymal Krl ^' 5- pods, standard lineage tracing methods based on Tamoxifen dependent LacZ expression driven by Cre recomhinase from the keratin 14 (Krtl4) promoter {Mao et aL 1999; Vasioukhin et aL, 1999} were used. Krtl 4-postive cells are not present in bronchioles prior to infection but become evident at 4 days post-infection as the same ceils expressing Krt5 and increase in numbers through day 9 while remaining restricted to the bronchioles. At or around 1 idpi there appears to be a concerted migration of these

tr5 rtl4 positive cells to interstitial lung and the appearance of Kit 5 pods. Treatment of these mice with daily injections of Tamoxifen at 5, 6, and 7dpi resulted in labeling of both the bronchioles and the rt5+ pods at 25dpi. These data are consistent with the notion that the Krt5+ pods arise from cells that migrate from the bronchioles to local sites of interbronehiolar lung damage.

Example I t, Experimeiitai Procedures.

Animal Models

C57/ I6 adult mice were infected with a sublethal dose of Influenza H INI A/PR/8/34 mouse adapted virus by intratracheal inhalation and longs were harvested at various time points post infection. For BrdU incorporation assays 30 tng/kg BrdU in sterile PBS was administered IP and mice were sacrificed at different time points post injection. All procedures were conducted under lACUC guidelines and approved protocols. For bleomycin experiments, mice were treated with 6U/kg bleomycin by intratracheal instillation and were sacrificed at different time points post treatment to harvest lungs.

Histology and Immunofluorescence

Mice were sacrificed and lungs were inflated and fixed with 4% formaldehyde prior to paraffin embedding. Antibodies included influenza virus A M2 protein (Abeam). p63 (4A.4 clone), alveolar markers (13A1 , 54D1 , 400,1 1 B6 clone), Krt5 (Neotnarkers, Lifespan Biosciences), MucSAc, CC1.0. SPC, Pdpn, Aqp5, Cd45 (Santa Cruz), BrdU (Accurate Chemical), p73 (3A6 clone), Ivl Kril4 ,ΚχίίΟ, rt6 (Covance), pan-Keratin, SMA and acetySated alpha tubulin (Sigma). Appropriate Alexa flour 488 or 594 conjugated secondary antibodies (Invitrogen) were used for IF and Vector Labs ABC kit with DAB substrate (Vector laboratories) were used for 1HC. Murine monoclonal antibodies were generated to human 22 wk fetal lung tissue under ]RB approval using standard methods (Kohler and Milstem, 1975).

Microa/ra ami Bioinformatics

RNAs obtained from LCM and colonies were amplified using the WT Pico RNA Amplification System, WT-Ovation Exon Module and Encore Biotin Module (NuGEN

Technologies) and hybridized onto GeneChip© Mouse Exon 1.0 ST Array.

GeneChip operating software was used to process all the Cel files and calculate probe intensity values. To validate sample qualit probe hybridization ratios were calc lated using Affymetrix Expression Console software. The intensity values were log2 -transformed and imported into the Partek Genomics Suite 6.5(beta). Exons w ere summarized to genes and a I -way ANOV A was performed to identify differentially expressed genes. P values and fold- change were calculated for each analysis. Fieatmaps were generated using Pearson's correlation and Ward's method and Principal Component Analysis was conducted using all probe sets. Pathway analyses were performed using Gene Set Enrichment Analysis (GSEA) software and PANTHER database (Siibramanian et at, 2005; Yuan et al., 2009),

Example 12. Models for acute lung injury.

/. Acute Lung injury

A combination of LPS and EGFR inhibitors is used to induce acute lung injury (ALI) in mice. The LD50 of LPS and EGFR inhibitors are determined. Also, histology and imm whistochemistry for p63, Krt5, and lung marker monoclonal antibodies is tracked in survivors at day 7, day 1 1, and day 21.

EGFR inhibitor (Gifiimib) is dosed on day 1, 2, and 3 with day 3 including 1-300 μ of LPS. The reported LDS0 for LPS is 150-300 g and that the EGFR inhibitor should synergize with the LPS in the induction of ALL Survivors will be sacrificed at day 7, 1 i, and 21 and lungs examined by histology and IRC with various markers to assess the degree of ALI and the extent of king repair and ultimately how much this process compares with king regeneration following influenza infection. 2. All in Chronic Lung Fibrosis

Fibrosis is induced wit intratracheal or intraperitoneal bleomycin. ALI is then induced with sub-LD50 EGFRi +LPS.

3. Administration of Stem Ceils

Exogeneous stem cells are admin istered to determine if they incorporate into regenerating lung following ALI with or without fibrosis. Distal airway stem cells are cloned and expanded in colonies from GFP-expressing mice, and are added via tracheal cannulation.

Example 13, Method of Isolating Stem Cells.

Human nasal, trachea or lung tissue specimens were washed with cold washing medium (DMEM: F12 1 :1 (Gibco), 100 ug/nil pen strep, l OOpg/ml gentamicin)). The samples w ere then cut into small pieces and d igested with digestion medium (DM EM; F12 1: 1,100 ug/ml pen strep, ΙΟΟμα/η Ι gentamicin, 5% fetal bovine serum, 2mg/mi collagenase) at 37 °C for 1 hour. The mixture of cells were spu down and washed by washing buffer three times. The cells were then resuspended in growth medium (5 mg/ml insulin, 10 ng/ml EGF, 2xl( ^)" M 3,3\S-tniodo-L-thyronine, 0.4 mg ml hydrocortisone, 24 mg/ml adenine, lxl0 ^~10 M cholera toxin in DME /Ham's Fl 2 3: 1. medium with 10% fetal bovine serum), filtered through a 70pm nylon cell strainer (BD Falcon) and plated on lethally irradiated 3T3- J2 fibroblast feeders.

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