Age-Related Dopaminergic Innervation Augments T Helper 2-Type Allergic Inflammation in the Postnatal Lung.

Young children are more susceptible to developing allergic asthma than adults. As neural innervation of the peripheral tissue continues to develop after birth, neurons may modulate tissue inflammation in an age-related manner. Here we showed that sympathetic nerves underwent a dopaminergic-to-adrenergic transition during post-natal development of the lung in mice and humans. Dopamine signaled through a specific dopamine receptor (DRD4) to promote T helper 2 (Th2) cell differentiation. The dopamine-DRD4 pathway acted synergistically with the cytokine IL-4 by upregulating IL-2-STAT5 signaling and reducing inhibitory histone trimethylation at Th2 gene loci. In murine models of allergen exposure, the dopamine-DRD4 pathway augmented Th2 inflammation in the lungs of young mice. However, this pathway operated marginally after sympathetic nerves became adrenergic in the adult lung. Taken together, the communication between dopaminergic nerves and CD4+ T cells provides an age-related mechanism underlying the susceptibility to allergic inflammation in the early lung.

Lung dopaminergic nerves facilitate the establishment of TH2 resident memory cells in early life.

BACKGROUND: Allergic asthma develops from allergen exposure in early childhood and progresses into adulthood. The central mediator of progressive allergic asthma is allergen-specific, TH2-resident memory cells (TRMs). Although the crosstalk between nerves and immune cells plays an established role in acute allergic inflammation, whether nerves facilitate the establishment of TH2-TRMs in the immature lung following early life allergen exposure is unknown. OBJECTIVES: The aim of this study was to identify nerve-derived signals that act in TH2 effector cells to regulate the tissue residency in the immature lung. METHODS: Following neonatal allergen exposure, allergen-specific TH2-TRMs were tracked temporally and spatially in relationship to developing sympathetic nerves in the lung. Functional mediators of dopamine signaling in the establishment of TH2-TRMs were identified by in vitro bulk RNA-sequencing of dopamine-treated TH2 cells followed by in vivo assessment of candidate genes using adoptive transfer of TH2 cells with viral gene knockdown. RESULTS: This study found that sympathetic nerves produce dopamine and reside in proximity to TH2 effector cells during the contraction phase following neonatal allergen exposure. Dopamine signals via DRD4 on TH2 cells to elevate IL2RA and epigenetically facilitate type 2 cytokine expression. Blockade of dopamine-DRD4 signaling following neonatal allergen exposure impairs lung residence of TH2 cells and ameliorates anamnestic inflammation in adults. CONCLUSIONS: These results demonstrate that maturing sympathetic nerves enable a dopamine-enriched lung environment in early life that promotes the establishment of allergen-specific TH2-TRMs. The dopamine-DRD4 axis may provide a therapeutic target to modify allergic asthma progression from childhood to adulthood.

Stem Cells in Lung Injury and Repair.

In this review, we summarize the recent literature on the biology of endogenous stem cells in adult lung injury repair. We focus on in vivo studies in mice with an emphasis on data generated using cell-specific Cre-dependent lineage-tracing systems. These studies provide new information on the identification of lung stem cells, their hierarchical relationships, the plasticity of their behavior in different types of injury, and the molecular signals that control their fates. Although most of this work has been on epithelial hierarchies, we expect that further development of robust genetic tools will foster meaningful investigations into how nonepithelial cell populations are controlled during lung injury repair in adults. The ultimate challenge will be to translate these findings to the pathogenesis and treatment of human lung diseases.

Derivation of lung mesenchymal lineages from the fetal mesothelium requires hedgehog signaling for mesothelial cell entry.

Recent studies have shown that mesothelial progenitors contribute to mesenchymal lineages of developing organs. To what extent the overlying mesothelium contributes to lung development remains unknown. To rigorously address this question, we employed Wt1(CreERT2/+) mice for high-fidelity lineage tracing after confirming that Cre recombinase was mesothelial specific and faithfully recapitulated endogenous Wilms' tumor 1 (Wt1) gene expression. We visualized WT1(+) mesothelial cell entry into the lung by live imaging and identified their progenies in subpopulations of bronchial smooth muscle cells, vascular smooth muscle cells and desmin(+) fibroblasts by lineage tagging. Derivation of these lineages was only observed with Cre recombinase activation during early lung development. Using loss-of-function assays in organ cultures, and targeted mesothelial-restricted hedgehog loss-of-function mice, we demonstrated that mesothelial cell movement into the lung requires the direct action of hedgehog signaling. By contrast, hedgehog signaling was not required for fetal mesothelial heart entry. These findings further support a paradigm wherein the mesothelium is a source of progenitors for mesenchymal lineages during organogenesis and indicate that signals controlling mesothelial cell entry are organ specific.

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.

Experience-dependent versus experience-independent postembryonic development of distinct groups of zebrafish olfactory glomeruli.

Olfactory glomeruli are innervated with great precision by the axons of different olfactory sensory neuron types and act as functional units in odor information processing. Approximately 140 glomeruli are present in each olfactory bulb of adult zebrafish; these units consist of either highly stereotypic large glomeruli or smaller anatomically indistinguishable glomeruli. In the present study, we investigated developmental differences among these types of glomeruli. We observed that 10 large and individually identifiable glomeruli already developed before hatching, at 72 h after fertilization, in configurations that resembled their mature organization. However, the cross-sectional area of these glomeruli increased throughout larval development, and they eventually comprised the largest units in postlarval olfactory bulbs. In contrast, small and anatomically indistinguishable glomeruli formed only after hatching, apparently by segregating from five larger precursors that were identifiable during embryonic development. The differentiation of these small glomeruli proceeded with conspicuous variation in number and arrangement, both among larvae and between olfactory bulbs of the same individuals. To determine factors that might contribute to this variability, we investigated the effects of olfactory enrichment on the development of amino acid-responsive lateral glomeruli, which include both large and small units. Larvae reared in an amino acid-enriched environment had normal large lateral glomeruli, but the small lateral glomeruli were more numerous and displayed reduced cross-sectional areas compared with glomeruli in control animals. Our results suggest that large and small glomeruli mature via distinct developmental processes that may be differentially influenced by sensory experience.

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.

Adiponectin attenuates lipopolysaccharide-induced acute lung injury through suppression of endothelial cell activation.

Adiponectin (APN) is an adipose tissue-derived factor with anti-inflammatory and vascular protective properties whose levels paradoxically decrease with increasing body fat. In this study, APN's role in the early development of ALI to LPS was investigated. Intratracheal LPS elicited an exaggerated systemic inflammatory response in APN-deficient (APN(-/-)) mice compared with wild-type (wt) littermates. Increased lung injury and inflammation were observed in APN(-/-) mice as early as 4 h after delivery of LPS. Targeted gene expression profiling performed on immune and endothelial cells isolated from lung digests 4 h after LPS administration showed increased proinflammatory gene expression (e.g., IL-6) only in endothelial cells of APN(-/-) mice when compared with wt mice. Direct effects on lung endothelium were demonstrated by APN's ability to inhibit LPS-induced IL-6 production in primary human endothelial cells in culture. Furthermore, T-cadherin-deficient mice that have significantly reduced lung airspace APN but high serum APN levels had pulmonary inflammatory responses after intratracheal LPS that were similar to those of wt mice. These findings indicate the importance of serum APN in modulating LPS-induced ALI and suggest that conditions leading to hypoadiponectinemia (e.g., obesity) predispose to development of ALI through exaggerated inflammatory response in pulmonary vascular endothelium.

Influenza Induces Lung Lymphangiogenesis Independent of YAP/TAZ Activity in Lymphatic Endothelial Cells.

The lymphatic system consists of a vessel network lined by specialized lymphatic endothelial cells (LECs) that are responsible for tissue fluid homeostasis and immune cell trafficking. The mechanisms for organ-specific LEC responses to environmental cues are not well understood. We found robust lymphangiogenesis during influenza A virus infection in the adult mouse lung. We show that the number of LECs increases 2-fold at 7 days post-influenza infection (dpi) and 3-fold at 21 dpi, and that lymphangiogenesis is preceded by lymphatic dilation. We also show that the expanded lymphatic network enhances fluid drainage to mediastinal lymph nodes. Using EdU labeling, we found that a significantly higher number of pulmonary LECs are proliferating at 7 dpi compared to LECs in homeostatic conditions. Lineage tracing during influenza indicates that new pulmonary LECs are derived from preexisting LECs rather than non-LEC progenitors. Lastly, using a conditional LEC-specific YAP/TAZ knockout model, we established that lymphangiogenesis, fluid transport and the immune response to influenza are independent of YAP/TAZ activity in LECs. These findings were unexpected, as they indicate that YAP/TAZ signaling is not crucial for these processes.

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.

Maintenance and repair of the lung endothelium does not involve contributions from marrow-derived endothelial precursor cells.

Lung endothelium is believed to be a quiescent tissue with the potential to exhibit rapid and effective repair after injury. Endothelial progenitor cells derived from the bone marrow have been proposed as one source of new endothelial cells that may directly contribute to pulmonary endothelial cell homeostasis and repair. Here we use bone marrow transplantation models, using purified hematopoietic stem cells (HSCs) or unfractionated whole marrow, to assess engraftment of cells in the endothelium of a variety of tissues. We find scant evidence for any contribution of bone marrow-derived cells to the pulmonary endothelium in the steady state or after recovery from hyperoxia-induced endothelial injury. Although a rare population of CD45-/CD31+/VECadherin+ bone marrow-derived cells, originating from HSCs, can be found in lung tissue after transplantation, these cells are not readily found in anatomic locations that define the pulmonary endothelium. Moreover, by tracking transplanted bone marrow cells obtained from donor transgenic mice containing endothelial lineage-selective reporters (Tie2-GFP), no contribution of bone marrow-derived cells to the adult lung, liver, pancreas, heart, and kidney endothelium can be detected, even after prolonged follow-up periods of 11 months or after recovery from hyperoxic pulmonary endothelial injury. Our findings argue against any significant engraftment of bone marrow-derived cells in the pulmonary vascular endothelium.

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.

Expression regulation and function of heparan sulfate 6-O-endosulfatases in the spermatogonial stem cell niche.

Glial cell line-derived neurotrophic factor (GDNF) is a heparan sulfate (HS)-binding factor. GDNF is produced by somatic Sertoli cells, where it signals to maintain spermatogonial stem cells (SSCs) and reproduction. Here, we investigate the roles of extracellular HS 6-O-endosulfatases (Sulfs), Sulf1 and Sulf2, in the matrix transmission of GDNF from Sertoli cells to SSCs. Although Sulfs are not required for testis formation, Sulf deficiency leads to the accelerated depletion of SSCs, a testis phenotype similar to that of GDNF+/- mice. Mechanistically, we show that Sulfs are expressed in GDNF-producing Sertoli cells. In addition, reduced Sulf activity profoundly worsens haplo-deficient GDNF phenotypes in our genetic studies. These findings establish a critical role of Sulfs in promoting GDNF signaling and support a model in which Sulfs regulate the bioavailability of GDNF by enzymatically remodeling HS 6-O-desulfation to release GDNF from matrix sequestration. Further, Sertoli cell-specific transcriptional factor Wilm's tumor 1 (WT1) directly activates the transcription of both Sulf1 and Sulf2 genes. Together, our studies not only identify Sulfs as essential regulators of GDNF signaling in the SSC niche, but also as direct downstream targets of WT1, thus establishing a physiological role of WT1 in Sertoli cells.

Airway basal stem cells generate distinct subpopulations of PNECs.

Pulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.

State-dependent mechanisms of LTP expression revealed by optical quantal analysis.

The expression mechanism of long-term potentiation (LTP) remains controversial. Here we combine electrophysiology and Ca(2+) imaging to examine the role of silent synapses in LTP expression. Induction of LTP fails to change p(r) at these synapses but instead mediates an unmasking process that is sensitive to the inhibition of postsynaptic membrane fusion. Once unmasked, however, further potentiation of formerly silent synapses leads to an increase in p(r). The state of the synapse thus determines how LTP is expressed.

Adiponectin deficiency: a model of pulmonary hypertension associated with pulmonary vascular disease.

Adiponectin (APN) is an adipocyte-derived factor that exists at high concentrations in serum and has anti-inflammatory and systemic vascular-protective properties. In this study, we investigated the role of APN in pulmonary vascular homeostasis. We found that APN localizes to the luminal side of blood vessels in lung and acts in vitro to block TNF-alpha-induced E-selectin upregulation in pulmonary artery endothelial cells. Targeted deletion of the APN gene in mice leads to a vascular phenotype in lung characterized by E-selectin upregulation and age-dependent increases in perivascular inflammatory cell infiltration and pulmonary arterial pressures. Taken together, these findings demonstrate an important role for APN in lung vascular homeostasis and suggest that APN-deficient states may contribute to the pathogenesis of inflammatory pulmonary vascular disease and to the development of pulmonary hypertension.

Launching invasive, first-in-human trials against Parkinson's disease: ethical considerations.

The decision to initiate invasive, first-in-human trials involving Parkinson's disease presents a vexing ethical challenge. Such studies present significant surgical risks, and high degrees of uncertainty about intervention risks and biological effects. We argue that maintaining a favorable risk-benefit balance in such circumstances requires a higher than usual degree of confidence that protocols will lead to significant direct and/or social benefits. One critical way of promoting such confidence is through the application of stringent evidentiary standards for preclinical studies. We close with a series of recommendations for strengthening the internal and external validity of preclinical studies, reducing their tendency toward optimism and publication biases, and improving the knowledge base used to design and evaluate preclinical studies.

Optical Quantal Analysis.

Understanding the mechanisms by which long-term synaptic plasticity is expressed remains an important objective in neuroscience. From a physiological perspective, the strength of a synapse can be considered a consequence of several parameters including the probability that a presynaptic action potential (AP) evokes the release of neurotransmitter, the mean number of quanta of transmitter released when release is evoked, and the mean amplitude of a postsynaptic response to a single quantum. Various methods have been employed to estimate these quantal parameters from electrophysiological recordings; such "quantal analysis" has been used to support competing accounts of mechanisms of expression of long-term plasticity. Because electrophysiological recordings, even with minimal presynaptic stimulation, can reflect responses arising at multiple synaptic sites, these methods are open to alternative interpretations. By combining intracellular electrical recording with optical detection of transmission at individual synapses, however, it is possible to eliminate such ambiguity. Here, we describe methods for such combined optical and electrical monitoring of synaptic transmission in brain slice preparations and illustrate how quantal analyses thereby obtained permit more definitive conclusions about the physiological changes that underlie long-term synaptic plasticity.

Transcriptional landscape of pulmonary lymphatic endothelial cells during fetal gestation.

The genetic programs responsible for pulmonary lymphatic maturation prior to birth are not known. To address this gap in knowledge, we developed a novel cell sorting strategy to collect fetal pulmonary lymphatic endothelial cells (PLECs) for global transcriptional profiling. We identified PLECs based on their unique cell surface immunophenotype (CD31+/Vegfr3+/Lyve1+/Pdpn+) and isolated them from murine lungs during late gestation (E16.5, E17.5, E18.5). Gene expression profiling was performed using whole-genome microarrays, and 1,281 genes were significantly differentially expressed with respect to time (FDR q < 0.05) and grouped into six clusters. Two clusters containing a total of 493 genes strongly upregulated at E18.5 were significantly enriched in genes with functional annotations corresponding to innate immune response, positive regulation of angiogenesis, complement & coagulation cascade, ECM/cell-adhesion, and lipid metabolism. Gene Set Enrichment Analysis identified several pathways coordinately upregulated during late gestation, the strongest of which was the type-I IFN-α/ß signaling pathway. Upregulation of canonical interferon target genes was confirmed by qRT-PCR and in situ hybridization in E18.5 PLECs. We also identified transcriptional events consistent with a prenatal PLEC maturation program. This PLEC-specific program included individual genes (Ch25h, Itpkc, Pcdhac2 and S1pr3) as well as a set of chemokines and genes containing an NF-κB binding site in their promoter. Overall, this work reveals transcriptional insights into the genes, signaling pathways and biological processes associated with pulmonary lymphatic maturation in the fetal lung.