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1.  Airway Epithelial Progenitors Are Region Specific and Show Differential Responses to Bleomycin-Induced Lung Injury 
Stem cells (Dayton, Ohio)  2012;30(9):1948-1960.
Mechanisms that regulate regional epithelial cell diversity and pathologic remodeling in airways are poorly understood. We hypothesized that regional differences in cell composition and injury-related tissue remodeling result from the type and composition of local progenitors. We used surface markers and the spatial expression pattern of an SFTPC-GFP transgene to subset epithelial progenitors by airway region. Green fluorescent protein (GFP) expression ranged from undetectable to high in a proximal-to-distal gradient. GFPhi cells were subdivided by CD24 staining into alveolar (CD24neg) and conducting airway (CD24low) populations. This allowed for the segregation of three types of progenitors displaying distinct clonal behavior in vitro. GFPneg and GFPlow progenitors both yielded lumen containing colonies but displayed transcriptomes reflective of pseudostratified and distal conducting airways, respectively. CD24lowGFPhi progenitors were present in an overlapping distribution with GFPlow progenitors in distal airways, yet expressed lower levels of Sox2 and expanded in culture to yield undifferentiated self-renewing progeny. Colony-forming ability was reduced for each progenitor cell type after in vivo bleomycin exposure, but only CD24lowGFPhi progenitors showed robust expansion during tissue remodeling. These data reveal intrinsic differences in the properties of regional progenitors and suggest that their unique responses to tissue damage drive local tissue remodeling. Disclosure of potential conflicts of interest is found at the end of this article.
doi:10.1002/stem.1150
PMCID: PMC4083019  PMID: 22696116
Regional; Progenitor; Epithelium; Lung; Bleomycin
2.  Surfactant Protein–C Chromatin-Bound Green Fluorescence Protein Reporter Mice Reveal Heterogeneity of Surfactant Protein C–Expressing Lung Cells 
The regeneration of alveolar epithelial cells is a critical aspect of alveolar reorganization after lung injury. Although alveolar Type II (AT2) cells have been described as progenitor cells for alveolar epithelia, more remains to be understood about how their progenitor cell properties are regulated. A nuclear, chromatin-bound green fluorescence protein reporter (H2B-GFP) was driven from the murine surfactant protein–C (SPC) promoter to generate SPC H2B-GFP transgenic mice. The SPC H2B-GFP allele allowed the FACS-based enrichment and gene expression profiling of AT2 cells. Approximately 97% of AT2 cells were GFP-labeled on Postnatal Day 1, and the percentage of GFP-labeled AT2 cells decreased to approximately 63% at Postnatal Week 8. Isolated young adult SPC H2B-GFP+ cells displayed proliferation, differentiation, and self-renewal capacity in the presence of lung fibroblasts in a Matrigel-based three-dimensional culture system. Heterogeneity within the GFP+ population was revealed, because cells with distinct alveolar and bronchiolar gene expression arose in three-dimensional cultures. CD74, a surface marker highly enriched on GFP+ cells, was identified as a positive selection marker, providing 3-fold enrichment for AT2 cells. In vivo, GFP expression was induced within other epithelial cell types during maturation of the distal lung. The utility of the SPC H2B-GFP murine model for the identification of AT2 cells was greatest in early postnatal lungs and more limited with age, when some discordance between SPC and GFP expression was observed. In adult mice, this allele may allow for the enrichment and future characterization of other SPC-expressing alveolar and bronchiolar cells, including putative stem/progenitor cell populations.
doi:10.1165/rcmb.2011-0403OC
PMCID: PMC3604082  PMID: 23204392
alveolar Type II cells; SPC; epithelial; GFP; differentiation
3.  RORγt Modulates Macrophage Recruitment during a Hydrocarbon Oil-Induced Inflammation 
PLoS ONE  2013;8(11):e79497.
Hydrocarbon oils are often utilized as adjuvants in vaccines. In response to naturally occurring hydrocarbon oils, inflammation is initiated and persists with the continuous recruitment of immune cells such as macrophages and neutrophils. However, the mechanism underlying the chronic inflammation in response to hydrocarbon oils is not fully defined. In this study, we revealed an essential role of retinoid-related orphan receptor gamma t (RORγt) in sustaining the recruitment of macrophages following pristane treatment. RORγt absence resulted in the incompetent formation of mesenteric oil granulomas which may associate to a reduction in the migration of macrophages into the mesentery during pristane-induced inflammation. This is at least partially dependent on the expression of the monocyte chemoattractant protein-1 (MCP-1) in the mesentery and the decrease in the macrophage reservoir in the spleen. However, the absence of RORγt had no impact on the recruitment of neutrophils to the mesentery after pristane treatment. Our data uncovered an important role of RORγt in the recruitment of macrophages during hydrocarbon oil-induced chronic inflammation.
doi:10.1371/journal.pone.0079497
PMCID: PMC3829825  PMID: 24260235
4.  Activation of the Canonical Bone Morphogenetic Protein (BMP) Pathway during Lung Morphogenesis and Adult Lung Tissue Repair 
PLoS ONE  2012;7(8):e41460.
Signaling by Bone Morphogenetic Proteins (BMP) has been implicated in early lung development, adult lung homeostasis and tissue-injury repair. However, the precise mechanism of action and the spatio-temporal pattern of BMP-signaling during these processes remains inadequately described. To address this, we have utilized a transgenic line harboring a BMP-responsive eGFP-reporter allele (BRE-eGFP) to construct the first detailed spatiotemporal map of canonical BMP-pathway activation during lung development, homeostasis and adult-lung injury repair. We demonstrate that during the pseudoglandular stage, when branching morphogenesis progresses in the developing lung, canonical BMP-pathway is active mainly in the vascular network and the sub-epithelial smooth muscle layer of the proximal airways. Activation of the BMP-pathway becomes evident in epithelial compartments only after embryonic day (E) 14.5 primarily in cells negative for epithelial-lineage markers, located in the proximal portion of the airway-tree, clusters adjacent to neuro-epithelial-bodies (NEBs) and in a substantial portion of alveolar epithelial cells. The pathway becomes activated in isolated E12.5 mesenchyme-free distal epithelial buds cultured in Matrigel suggesting that absence of reporter activity in these regions stems from a dynamic cross-talk between endoderm and mesenchyme. Epithelial cells with activated BMP-pathway are enriched in progenitors capable of forming colonies in three-dimensional Matrigel cultures.
As lung morphogenesis approaches completion, eGFP-expression declines and in adult lung its expression is barely detectable. However, upon tissue-injury, either with naphthalene or bleomycin, the canonical BMP-pathways is re-activated, in bronchial or alveolar epithelial cells respectively, in a manner reminiscent to early lung development and in tissue areas where reparatory progenitor cells reside. Our studies illustrate the dynamic activation of canonical BMP-pathway during lung development and adult lung tissue-repair and highlight its involvement in two important processes, namely, the early development of the pulmonary vasculature and the management of epithelial progenitor pools both during lung development and repair of adult lung tissue-injury.
doi:10.1371/journal.pone.0041460
PMCID: PMC3423416  PMID: 22916109
5.  c-Kit Is Essential for Alveolar Maintenance and Protection from Emphysema-like Disease in Mice 
Rationale: Previously, we demonstrated a candidate region for susceptibility to airspace enlargement on mouse chromosome 5. However, the specific candidate genes within this region accounting for emphysema-like changes remain unrecognized. c-Kit is a receptor tyrosine kinase within this candidate gene region that has previously been recognized to contribute to the survival, proliferation, and differentiation of hematopoietic stem cells. Increases in the percentage of cells expressing c-Kit have previously been associated with protection against injury-induced emphysema.
Objectives: Determine whether genetic variants of c-Kit are associated with spontaneous airspace enlargement.
Methods: Perform single-nucleotide polymorphism association studies in the mouse strains at the extremes of airspace enlargement phenotype for variants in c-Kit tyrosine kinase. Characterize mice bearing functional variants of c-Kit compared with wild-type controls for the development of spontaneous airspace enlargement. Epithelial cell proliferation was measured in culture.
Measurements and Main Results: Upstream regulatory single-nucleotide polymorphisms in the divergent mouse strains were associated with the lung compliance difference observed between the extreme strains. c-Kit mutant mice (KitW-sh/W-sh), when compared with genetic controls, developed altered lung histology, increased total lung capacity, increased residual volume, and increased lung compliance that persist into adulthood. c-Kit inhibition with imatinib attenuated in vitro proliferation of cells expressing epithelial cell adhesion molecule.
Conclusions: Our findings indicate that c-Kit sustains and/or maintains normal alveolar architecture in the lungs of mice. In vitro data suggest that c-Kit can regulate epithelial cell clonal expansion. The precise mechanisms that c-Kit contributes to the development of airspace enlargement and increased lung compliance remain unclear and warrants further investigation.
doi:10.1164/rccm.201007-1157OC
PMCID: PMC3136992  PMID: 21471107
genetic; tyrosine kinase; SASH; chronic obstructive pulmonary disease; aging
6.  Functional Analysis of Two Distinct Bronchiolar Progenitors during Lung Injury and Repair 
Air spaces of the mammalian lung are lined by a specialized epithelium that is maintained by endogenous progenitor cells. Within bronchioles, the abundance and distribution of progenitor cells that contribute to epithelial homeostasis change as a function of maintenance versus repair. It is unclear whether functionally distinct progenitor pools or a single progenitor cell type maintain the epithelium and how the behavior is regulated in normal or disease states. To address these questions, we applied fractionation methods for the enrichment of distal airway progenitors. We show that bronchiolar progenitor cells can be subdivided into two functionally distinct populations that differ in their susceptibility to injury and contribution to repair. The proliferative capacity of these progenitors is confirmed in a novel in vitro assay. We show that both populations give rise to colonies with a similar dependence on stromal cell interactions and regulation by TGF-β. These findings provide additional insights into mechanisms of epithelial remodeling in the setting of chronic lung disease and offer hope that pharmacologic interventions may be developed to mitigate tissue remodeling.
doi:10.1165/rcmb.2010-0098OC
PMCID: PMC3135841  PMID: 20656948
bronchiolar stem cell; Clara; progenitor; fractionation; epithelium
7.  Distinct Granuloma Responses in C57BL/6J and BALB/cByJ Mice in Response to Pristane 
Summary
Granuloma formation is an inflammatory response of the host against invading pathogens or indigestible substances. We generated mesenteric oil granulomas by injecting pristane into the peritoneal cavity of mice, and oil granuloma formation in C57BL/6J and BALB/cByJ mice was compared. The formation and kinetics of oil granulomas were distinct between the two strains. In C57BL/6J mice, injected pristane induced oil granuloma formation at both the mesenteric centers (MG) and margins (SG). MG was resolving by 11 weeks, whereas SG persisted. In BALB/cByJ mice, MG developed slower but persisted longer than in C57BL/6J mice, whereas SG resolved sooner than in C57BL/6J mice. Injection of India ink revealed that phagocytes were mainly localized to the SG in C57BL/6J mice, but were diffusely located in both MG and SG of BALB/cByJ mice. SG cells expressed more monocyte chemotactic protein-1 mRNA than MG cells in C57BL/6J mice, but there was no difference in MCP-1 expression between the MG and SG in BALB/cByJ mice. These observations suggest that the recruitment of inflammatory leukocytes under the direction of chemokines differentiates the patterns of granuloma responses to pristane in C57BL/6J and BALB/cByJ mice.
doi:10.1111/j.1365-2613.2010.00725.x
PMCID: PMC2974958  PMID: 20681981
oil granuloma; strain; pristane; MCP-1
8.  Bronchiolar Progenitor Cells 
A comprehensive appreciation of mechanisms regulating epithelial maintenance and repair in pulmonary airways is fundamental to our understanding of tissue remodeling and dysfunction in chronic lung disease. This review provides an update on current concepts that have emerged from recent work in the field of airway epithelial repair and progenitor cell biology. New models to investigate the behavior of lung epithelial progenitor cells have provided fresh insights into their regulation and organization, and help to clarify their roles in normal maintenance and repair. Emerging technologies for the fractionation and culture of lung epithelial cells also provide opportunities to investigate the behavior and regulation of progenitor cell subsets in controlled systems. These advances hold promise for development of new strategies to modulate epithelial cell behavior and to effect tissue repair in the setting of lung disease.
doi:10.1513/pats.200907-078RM
PMCID: PMC3266052  PMID: 19934356
progenitor; bronchiolar epithelium; stem cell; transit-amplifying cell
9.  Effects of Acute and Chronic Inflammation on B-Cell Development and Differentiation 
Recently, our understanding of hematopoiesis and the development of the immune system has fundamentally changed, leading to significant discoveries with important clinical relevance. Hematopoiesis, once described in terms of irreversible and discrete developmental branch points, is now understood to exist as a collection of alternative developmental pathways capable of generating functionally identical progeny. Developmental commitment to a particular blood-cell lineage is gradually acquired and reflects both cell intrinsic and extrinsic signals. Chief among the extrinsic factors are the environmental cues of hematopoietic microenvironments that comprise specific “developmental niches” that support hematopoietic stem and progenitor cells. Most of this new understanding comes from the study of normal, steady-state hematopoiesis, but there is ample reason to expect that special developmental and/or differentiative mechanisms operate in response to inflammation. For example, both stem and progenitor cells are now known to express Toll-like receptors that can influence hematopoietic cell fates in response to microbial products. Likewise, pro-inflammatory cytokines mobilize hematopoietic stem cells to peripheral tissues. In this Perspective, we review inflammation’s effects on central and extramedullary B lymphopoiesis and discuss the potential consequences of peripheral B-cell development in the context of systemic autoimmune diseases.
doi:10.1038/jid.2008.286
PMCID: PMC2778726  PMID: 19148216

Results 1-9 (9)