Jesus Paez-Cortez

Notch3-Jagged signaling controls the pool of undifferentiated airway progenitors

Basal cells are multipotent airway progenitors that generate distinct epithelial cell phenotypes crucial for homeostasis and repair of the conducting airways. Little is known about how these progenitor cells expand and transition to differentiation to form the pseudostratified airway epithelium in the developing and adult lung. Here, we show by genetic and pharmacological approaches that endogenous activation of Notch3 signaling selectively controls the pool of undifferentiated progenitors of upper airways available for differentiation. This mechanism depends on the availability of Jag1 and Jag2, and is key to generating a population of parabasal cells that later activates Notch1 and Notch2 for secretory-multiciliated cell fate selection. Disruption of this mechanism resulted in aberrant expansion of basal cells and altered pseudostratification. Analysis of human lungs showing similar abnormalities and decreased NOTCH3 expression in subjects with chronic obstructive pulmonary disease suggests an involvement of NOTCH3-dependent events in the pathogenesis of this condition. Highlighted article: In the adult mouse lung, Notch3 identifies a population of parabasal cells and regulates the balance between basal and parabasal progenitor pools.

A Shh/miR-206/BDNF cascade coordinates innervation and formation of airway smooth muscle.

Dysfunctional neural control of airway smooth muscle (ASM) is involved in inflammatory diseases, such as asthma. However, neurogenesis in the lung is poorly understood. This study uses mouse models to investigate developmental mechanisms of ASM innervation, a process that is highly coordinated with ASM formation during lung branching morphogenesis. We show that brain-derived neurotrophic factor (BDNF) is an essential ASM-derived signal for innervation. Although BDNF mRNA expression is temporally dissociated with ASM formation and innervation, BDNF protein is coordinately produced through post-transcriptional suppression by miR-206. Using a combination of chemical and genetic approaches to modulate sonic hedgehog (Shh) signaling, a pathway essential for lung branching and ASM formation, we show that Shh signaling blocks miR-206 expression, which in turn increases BDNF protein expression. Together, our work uncovers a functional cascade that involves Shh, miR-206 and BDNF to coordinate ASM formation and innervation.

An NT4/TrkB-dependent increase in innervation links early-life allergen exposure to persistent airway hyperreactivity

Children who are exposed to environmental respiratory insults often develop asthma that persists into adulthood. In this study, we used a neonatal mouse model of ovalbumin (OVA)‐induced allergic airway inflammation to understand the long‐term effects of early childhood insults on airway structure and function. We showed that OVA sensitization and challenge in early life led to a 2‐fold increase in airway smooth muscle (ASM) innervation (P<0.05) and persistent airway hyperreactivity (AHR). In contrast, OVA exposure in adult life elicited short‐term AHR without affecting innervation levels. We found that postnatal ASM innervation required neurotrophin (NT)‐4 signaling through the TrkB receptor and that early‐life OVA exposure significantly elevated NT4 levels and TrkB signaling by 5‐ and 2‐fold, respectively, to increase innervation. Notably, blockade of NT4/TrkB signaling in OVA‐exposed pups prevented both acute and persistent AHR without affecting baseline airway function or inflammation. Furthermore, biophysical assays using lung slices and isolated cells demonstrated that NT4 was necessary for hyperreactivity of ASM induced by early‐life OVA exposure. Together, our findings show that the NT4/TrkB‐dependent increase in innervation plays a critical role in the alteration of the ASM phenotype during postnatal growth, thereby linking early‐life allergen exposure to persistent airway dysfunction.—Aven, L., Paez‐Cortez, J., Achey, R., Krishnan, R., Ram‐Mohan, S., Cruikshank, W. W., Fine, A., Ai X. An NT4/TrkB‐dependent increase in innervation links early‐life allergen exposure to persistent airway hyperreactivity. FASEB J. 28, 897–907 (2014). www.fasebj.org

Endothelial GATA-6 Deficiency Promotes Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a chronic and progressive disease characterized by pulmonary vasculopathy with elevation of pulmonary artery pressure, often culminating in right ventricular failure. GATA-6, a member of the GATA family of zinc-finger transcription factors, is highly expressed in quiescent vasculature and is frequently lost during vascular injury. We hypothesized that endothelial GATA-6 may play a critical role in the molecular mechanisms underlying endothelial cell (EC) dysfunction in PAH. Here we report that GATA-6 is markedly reduced in pulmonary ECs lining both occluded and nonoccluded vessels in patients with idiopathic and systemic sclerosis-associated PAH. GATA-6 transcripts are also rapidly decreased in rodent PAH models. Endothelial GATA-6 is a direct transcriptional regulator of genes controlling vascular tone [endothelin-1, endothelin-1 receptor type A, and endothelial nitric oxide synthase (eNOS)], pro-inflammatory genes, CX3CL1 (fractalkine), 5-lipoxygenease-activating protein, and markers of vascular remodeling, including PAI-1 and RhoB. Mice with the genetic deletion of GATA-6 in ECs (Gata6-KO) spontaneously develop elevated pulmonary artery pressure and increased vessel muscularization, and these features are further exacerbated in response to hypoxia. Furthermore, innate immune cells including macrophages (CD11b(+)/F4/80(+)), granulocytes (Ly6G(+)/CD45(+)), and dendritic cells (CD11b(+)/CD11c(+)) are significantly increased in normoxic Gata6-KO mice. Together, our findings suggest a critical role of endothelial GATA-6 deficiency in development and disease progression in PAH.

Activation Dynamics and Signaling Properties of Notch3 Receptor in the Developing Pulmonary Artery

Notch3 signaling is fundamental for arterial specification of systemic vascular smooth muscle cells (VSMCs). However, the developmental role and signaling properties of the Notch3 receptor in the mouse pulmonary artery remain unknown. Here, we demonstrate that Notch3 is expressed selectively in pulmonary artery VSMCs, is activated from late fetal to early postnatal life, and is required to maintain the morphological characteristics and smooth muscle gene expression profile of the pulmonary artery after birth. Using a conditional knock-out mouse model, we show that Notch3 receptor activation in VSMCs is Jagged1-dependent. In vitro VSMC lentivirus-mediated Jagged1 knockdown, confocal localization analysis, and co-culture experiments revealed that Notch3 activation is cell-autonomous and occurs through the physical engagement of Notch3 and VSMC-derived Jagged1 in the interior of the same cell. Although the current models of mammalian Notch signaling involve a two-cell system composed of a signal-receiving cell that expresses a Notch receptor on its surface and a neighboring signal-sending cell that provides membrane-bound activating ligand, our data suggest that pulmonary artery VSMC Notch3 activation is cell-autonomous. This unique mechanism of Notch activation may play an important role in the maturation of the pulmonary artery during the transition to air breathing.

Airway Contractility in the Precision-Cut Lung Slice after Cryopreservation

An emerging tool in airway biology is the precision-cut lung slice (PCLS). Adoption of the PCLS as a model for assessing airway reactivity has been hampered by the limited time window within which tissues remain viable. Here we demonstrate that the PCLS can be frozen, stored long-term, and then thawed for later experimental use. Compared with the never-frozen murine PCLS, the frozen-thawed PCLS shows metabolic activity that is decreased to an extent comparable to that observed in other cryopreserved tissues but shows no differences in cell viability or in airway caliber responses to the contractile agonist methacholine or the relaxing agonist chloroquine. These results indicate that freezing and long-term storage is a feasible solution to the problem of limited viability of the PCLS in culture.

A new approach for the study of lung smooth muscle phenotypes and its application in a murine model of allergic airway inflammation.

Phenotypes of lung smooth muscle cells in health and disease are poorly characterized. This is due, in part, to a lack of methodologies that allow for the independent and direct isolation of bronchial smooth muscle cells (BSMCs) and vascular smooth muscle cells (VSMCs) from the lung. In this paper, we describe the development of a bi-fluorescent mouse that permits purification of these two cell populations by cell sorting. By subjecting this mouse to an acute allergen based-model of airway inflammation that exhibits many features of asthma, we utilized this tool to characterize the phenotype of so-called asthmatic BSMCs. First, we examined the biophysical properties of single BSMCs from allergen sensitized mice and found increases in basal tone and cell size that were sustained ex vivo. We then generated for the first time, a comprehensive characterization of the global gene expression changes in BSMCs isolated from the bi-fluorescent mice with allergic airway inflammation. Using statistical methods and pathway analysis, we identified a number of differentially expressed mRNAs in BSMCs from allergen sensitized mice that code for key candidate proteins underlying changes in matrix formation, contractility, and immune responses. Ultimately, this tool will provide direction and guidance for the logical development of new markers and approaches for studying human lung smooth muscle.

Continuous IL-23 stimulation drives ILC3 depletion in the upper GI tract and, in combination with TNFα, induces robust activation and a phenotypic switch of ILC3

Mutations in the Interleukin (IL)-23/IL-23 receptor loci are associated with increased inflammatory bowel disease (IBD) susceptibility, and IL-23 neutralization has shown efficacy in early clinical trials. To better understand how an excess of IL-23 affects the gastrointestinal tract, we investigated chronic systemic IL-23 exposure in healthy wildtype mice. As expected, IL-23 exposure resulted in early activation of intestinal type 3 innate lymphoid cells (ILC3), followed by infiltration of activated RORγt+ T helper cells. Surprisingly, however, sustained IL-23 stimulus also dramatically reduced classical ILC3 populations within the proximal small intestine, and a phenotypically distinct T-bet expressing ILC3 population emerged. TNFα neutralization, a widely used IBD therapy, reduced several aspects of the IL-23 driven ILC3 response, suggesting a synergy between IL-23 and TNFα in ILC3 activation. In vitro studies supported these findings, revealing previously unappreciated effects of IL-23 and TNFα within the intestine.