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.

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.

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.

Retinoic acid signaling is essential for airway smooth muscle homeostasis.

Airway smooth muscle (ASM) is a dynamic and complex tissue involved in regulation of bronchomotor tone, but the molecular events essential for the maintenance of ASM homeostasis are not well understood. Observational and genome-wide association studies in humans have linked airway function to the nutritional status of vitamin A and its bioactive metabolite retinoic acid (RA). Here, we provide evidence that ongoing RA signaling is critical for the regulation of adult ASM phenotype. By using dietary, pharmacologic, and genetic models in mice and humans, we show that (a) RA signaling is active in adult ASM in the normal lung, (b) RA-deficient ASM cells are hypertrophic, hypercontractile, profibrotic, but not hyperproliferative, (c) TGF-ß signaling, known to cause ASM hypertrophy and airway fibrosis in human obstructive lung diseases, is hyperactivated in RA-deficient ASM, (d) pharmacologic and genetic inhibition of the TGF-ß activity in ASM prevents the development of the aberrant phenotype induced by RA deficiency, and (e) the consequences of transient RA deficiency in ASM are long-lasting. These results indicate that RA signaling actively maintains adult ASM homeostasis, and disruption of RA signaling leads to aberrant ASM phenotypes similar to those seen in human chronic airway diseases such as asthma.

Prenatal retinoid deficiency leads to airway hyperresponsiveness in adult mice.

There is increasing evidence that vitamin A deficiency in utero correlates with abnormal airway smooth muscle (SM) function in postnatal life. The bioactive vitamin A metabolite retinoic acid (RA) is essential for formation of the lung primordium; however, little is known about the impact of early fetal RA deficiency on postnatal lung structure and function. Here, we provide evidence that during murine lung development, endogenous RA has a key role in restricting the airway SM differentiation program during airway formation. Using murine models of pharmacological, genetic, and dietary vitamin A/RA deficiency, we found that disruption of RA signaling during embryonic development consistently resulted in an altered airway SM phenotype with markedly increased expression of SM markers. The aberrant phenotype persisted postnatally regardless of the adult vitamin A status and manifested as structural changes in the bronchial SM and hyperresponsiveness of the airway without evidence of inflammation. Our data reveal a role for endogenous RA signaling in restricting SM differentiation and preventing precocious and excessive SM differentiation when airways are forming.

Trinucleotide repeat containing 6a (Tnrc6a)-mediated microRNA function is required for development of yolk sac endoderm.

Tnrc6 family members (Tnrc6a/b/c) are key components of the RNA-induced silencing complex in microRNA (miRNA)-mediated gene suppression. Here, we show that Tnrc6a, also known as GW182, is selectively expressed in the yolk sac endoderm and that gene trap disruption of GW182 leads to growth arrest and apoptosis. We found that targets of miRNAs highly expressed in the yolk sac are significantly derepressed in GW182(gt/gt) mutant mice, although levels of miRNAs are not altered. Specifically, growth arrest and apoptosis phenotype are associated with significant derepression of Cdkn1a (p21), Cdkn1c (P27), Lats1, Lats2, Rb1, Rbl, Bim, and Pten, known targets of miRNAs from miR-17/20/93/106 clusters highly expressed in yolk sac endoderm. Together, these data strongly suggest that GW182 is an essential functional component in the RNA-induced silencing complex for miRNA-mediated gene silencing in vivo, and selectively regulation of miRNA activity plays an important role in the proper development of yolk sac.

miR-29 is a major regulator of genes associated with pulmonary fibrosis.

MicroRNAs (miRNA) are small regulatory RNAs that control gene expression by translational suppression and destabilization of target mRNAs. There is increasing evidence that miRNAs regulate genes associated with fibrosis in organs, such as the heart, kidney, liver, and the lung. In a large-scale screening for miRNAs potentially involved in bleomycin-induced fibrosis, we found expression of miR-29 family members significantly reduced in fibrotic lungs. Analysis of normal lungs showed the presence of miR-29 in subsets of interstitial cells of the alveolar wall, pleura, and at the entrance of the alveolar duct, known sites of pulmonary fibrosis. miR-29 levels inversely correlated with the expression levels of profibrotic target genes and the severity of the fibrosis. To study the impact of miR-29 down-regulation in the lung interstitium, we characterized gene expression profiles of human fetal lung fibroblast IMR-90 cells in which endogenous miR-29 was knocked down. This confirmed the derepression of reported miR-29 targets, including several collagens, but also revealed up-regulation of a large number of previously unrecognized extracellular matrix-associated and remodeling genes. Moreover, we found that miR-29 is suppressed by transforming growth factor (TGF)-ß1 in these cells, and that many fibrosis-associated genes up-regulated by TGF-ß1 are derepressed by miR-29 knockdown. Interestingly, a comparison of TGF-ß1 and miR-29 targets revealed that miR-29 controls an additional subset of fibrosis-related genes, including laminins and integrins, independent of TGF-ß1. Together, these strongly suggest a role of miR-29 in the pathogenesis of pulmonary fibrosis. miR-29 may be a potential new therapeutic target for this disease.

A retinoic acid-dependent network in the foregut controls formation of the mouse lung primordium.

The developmental abnormalities associated with disruption of signaling by retinoic acid (RA), the biologically active form of vitamin A, have been known for decades from studies in animal models and humans. These include defects in the respiratory system, such as lung hypoplasia and agenesis. However, the molecular events controlled by RA that lead to formation of the lung primordium from the primitive foregut remain unclear. Here, we present evidence that endogenous RA acts as a major regulatory signal integrating Wnt and Tgfbeta pathways in the control of Fgf10 expression during induction of the mouse primordial lung. We demonstrated that activation of Wnt signaling required for lung formation was dependent on local repression of its antagonist, Dickkopf homolog 1 (Dkk1), by endogenous RA. Moreover, we showed that simultaneously activating Wnt and repressing Tgfbeta allowed induction of both lung buds in RA-deficient foreguts. The data in this study suggest that disruption of Wnt/Tgfbeta/Fgf10 interactions represents the molecular basis for the classically reported failure to form lung buds in vitamin A deficiency.