Differential modularity of the mammalian Engrailed 1 enhancer network directs sweat gland development.

Enhancers are context-specific regulators of expression that drive biological complexity and variation through the redeployment of conserved genes. An example of this is the enhancer-mediated control of Engrailed 1 (EN1), a pleiotropic gene whose expression is required for the formation of mammalian eccrine sweat glands. We previously identified the En1 candidate enhancer (ECE) 18 cis-regulatory element that has been highly and repeatedly derived on the human lineage to potentiate ectodermal EN1 and induce our species' uniquely high eccrine gland density. Intriguingly, ECE18 quantitative activity is negligible outside of primates and ECE18 is not required for En1 regulation and eccrine gland formation in mice, raising the possibility that distinct enhancers have evolved to modulate the same trait. Here we report the identification of the ECE20 enhancer and show it has conserved functionality in mouse and human developing skin ectoderm. Unlike ECE18, knock-out of ECE20 in mice reduces ectodermal En1 and eccrine gland number. Notably, we find ECE20, but not ECE18, is also required for En1 expression in the embryonic mouse brain, demonstrating that ECE20 is a pleiotropic En1 enhancer. Finally, that ECE18 deletion does not potentiate the eccrine phenotype of ECE20 knock-out mice supports the secondary incorporation of ECE18 into the regulation of this trait in primates. Our findings reveal that the mammalian En1 regulatory machinery diversified to incorporate both shared and lineage-restricted enhancers to regulate the same phenotype, and also have implications for understanding the forces that shape the robustness and evolvability of developmental traits.

Repeated mutation of a developmental enhancer contributed to human thermoregulatory evolution.

Humans sweat to cool their bodies and have by far the highest eccrine sweat gland density among primates. Humans' high eccrine gland density has long been recognized as a hallmark human evolutionary adaptation, but its genetic basis has been unknown. In humans, expression of the Engrailed 1 (EN1) transcription factor correlates with the onset of eccrine gland formation. In mice, regulation of ectodermal En1 expression is a major determinant of natural variation in eccrine gland density between strains, and increased En1 expression promotes the specification of more eccrine glands. Here, we show that regulation of EN1 has evolved specifically on the human lineage to promote eccrine gland formation. Using comparative genomics and validation of ectodermal enhancer activity in mice, we identified a human EN1 skin enhancer, hECE18. We showed that multiple epistatically interacting derived substitutions in the human ECE18 enhancer increased its activity compared with nonhuman ape orthologs in cultured keratinocytes. Repression of hECE18 in human cultured keratinocytes specifically attenuated EN1 expression, indicating this element positively regulates EN1 in this context. In a humanized enhancer knock-in mouse, hECE18 increased developmental En1 expression in the skin to induce the formation of more eccrine glands. Our study uncovers a genetic basis contributing to the evolution of one of the most singular human adaptations and implicates multiple interacting mutations in a single enhancer as a mechanism for human evolutionary change.

Cutting Edge: Role of NK Cells and Surfactant Protein D in Dendritic Cell Lymph Node Homing: Effects of Ozone Exposure.

The roles of NK cells, surfactant protein D (SP-D), and IFN-γ, as well as the effect of ozone (O3) inhalation, were studied on recirculation of pulmonary dendritic cells (DC) to the mediastinal lymph nodes. O3 exposure and lack of SP-D reduced NK cell IFN-γ and lung tissue CCL21 mRNA expression and impaired DC homing to the mediastinal lymph nodes. Notably, addition of recombinant SP-D to naive mononuclear cells stimulated IFN-γ release in vitro. Because NKp46, a glycosylated membrane receptor, was necessary for dose-dependent SP-D binding to NK cells in vitro and DC migration in vivo, we speculate that SP-D may constitutively stimulate IFN-γ production by NK cells, possibly via NKp46. This mechanism could then initiate the IFN-γ/IL-12 feedback circuit, a key amplifier of DC lymph node homing. Inhibition of this process during an acute inflammatory response causes DC retention in the peripheral lung tissue and contributes to injury.

Properdin Contributes to Allergic Airway Inflammation through Local C3a Generation.

Complement is implicated in asthma pathogenesis, but its mechanism of action in this disease remains incompletely understood. In this study, we investigated the role of properdin (P), a positive alternative pathway complement regulator, in allergen-induced airway inflammation. Allergen challenge stimulated P release into the airways of asthmatic patients, and P levels positively correlated with proinflammatory cytokines in human bronchoalveolar lavage (BAL). High levels of P were also detected in the BAL of OVA-sensitized and challenged but not naive mice. Compared with wild-type (WT) mice, P-deficient (P(-/-)) mice had markedly reduced total and eosinophil cell counts in BAL and significantly attenuated airway hyperresponsiveness to methacholine. Ab blocking of P at both sensitization and challenge phases or at challenge phase alone, but not at sensitization phase alone, reduced airway inflammation. Conversely, intranasal reconstitution of P to P(-/-) mice at the challenge phase restored airway inflammation to wild-type levels. Notably, C3a levels in the BAL of OVA-challenged P(-/-) mice were significantly lower than in wild-type mice, and intranasal coadministration of an anti-C3a mAb with P to P(-/-) mice prevented restoration of airway inflammation. These results show that P plays a key role in allergen-induced airway inflammation and represents a potential therapeutic target for human asthma.

Group 2 innate lymphoid cells mediate ozone-induced airway inflammation and hyperresponsiveness in mice.

BACKGROUND: Asthmatic patients are highly susceptible to air pollution and in particular to the effects of ozone (O3) inhalation, but the underlying mechanisms remain unclear. OBJECTIVE: Using mouse models of O3-induced airway inflammation and airway hyperresponsiveness (AHR), we sought to investigate the role of the recently discovered group 2 innate lymphoid cells (ILC2s). METHODS: C57BL/6 and BALB/c mice were exposed to Aspergillus fumigatus, O3, or both (3 ppm for 2 hours). ILC2s were isolated by means of fluorescence-activated cell sorting and studied for Il5 and Il13 mRNA expression. ILC2s were depleted with anti-Thy1.2 mAb and replaced by means of intratracheal transfer of ex vivo expanded Thy1.1 ILC2s. Cytokine levels (ELISA and quantitative PCR), inflammatory cell profile, and AHR (flexiVent) were assessed in the mice. RESULTS: In addition to neutrophil influx, O3 inhalation elicited the appearance of eosinophils and IL-5 in the airways of BALB/c but not C57BL/6 mice. Although O3-induced expression of IL-33, a known activator of ILC2s, in the lung was similar between these strains, isolated pulmonary ILC2s from O3-exposed BALB/c mice had significantly greater Il5 and Il13 mRNA expression than C57BL/6 mice. This suggested that an altered ILC2 function in BALB/c mice might mediate the increased O3 responsiveness. Indeed, anti-Thy1.2 treatment abolished but ILC2s added back dramatically enhanced O3-induced AHR. CONCLUSIONS: O3-induced activation of pulmonary ILC2s was necessary and sufficient to mediate asthma-like changes in BALB/c mice. This previously unrecognized role of ILC2s might help explain the heightened susceptibility of human asthmatic airways to O3 exposure.

IFN-γ-induced JAK/STAT, but not NF-κB, signaling pathway is insensitive to glucocorticoid in airway epithelial cells.

Although the majority of patients with asthma are well controlled by inhaled glucocorticoids (GCs), patients with severe asthma are poorly responsive to GCs. This latter group is responsible for a disproportionate share of health care costs associated with asthma. Recent studies in immune cells have incriminated interferon-γ (IFN-γ) as a possible trigger of GC insensitivity in severe asthma; however, little is known about the role of IFN-γ in modulating GC effects in other clinically relevant nonimmune cells, such as airway epithelial cells. We hypothesized that IFN-γ-induced JAK/STAT-associated signaling pathways in airway epithelial cells are insensitive to GCs and that strategies aimed at inhibiting JAK/STAT pathways can restore steroid responsiveness. Using Western blot analysis we found that all steps of the IFN-γ-induced JAK/STAT signaling pathway were indeed GC insensitive. Transfection of cells with reporter plasmid showed IFN-γ-induced STAT1-dependent gene transcription to be also GC insensitive. Interestingly, real-time PCR analysis showed that IFN-γ-inducible genes (IIGs) were differentially affected by GC, with CXCL10 being GC sensitive and CXCL11 and IFIT2 being GC insensitive. Further investigation showed that the differential sensitivity of IIGs to GC was due to their variable dependency to JAK/STAT vs. NF-κB signaling pathways with GC-sensitive IIGs being more NF-κB dependent and GC-insensitive IIGs being more JAK/STAT dependent. Importantly, transfection of cells with siRNA-STAT1 was able to restore steroid responsiveness of GC-insensitive IIGs. Taken together, our results show the insensitivity of IFN-γ-induced JAK/STAT signaling pathways to GC effects in epithelial cells and also suggest that targeting STAT1 could restore GC responsiveness in patients with severe asthma.

Sweat gland development requires an eccrine dermal niche and couples two epidermal programs.

Eccrine sweat glands are indispensable for human thermoregulation and, similar to other mammalian skin appendages, form from multipotent epidermal progenitors. Limited understanding of how epidermal progenitors specialize to form these vital organs has precluded therapeutic efforts toward their regeneration. Herein, we applied single-nucleus transcriptomics to compare the expression content of wild-type, eccrine-forming mouse skin to that of mice harboring a skin-specific disruption of Engrailed 1 (En1), a transcription factor that promotes eccrine gland formation in humans and mice. We identify two concurrent but disproportionate epidermal transcriptomes in the early eccrine anlagen: one that is shared with hair follicles and one that is En1 dependent and eccrine specific. We demonstrate that eccrine development requires the induction of a dermal niche proximal to each developing gland in humans and mice. Our study defines the signatures of eccrine identity and uncovers the eccrine dermal niche, setting the stage for targeted regeneration and comprehensive skin repair.

Ectodysplasin Signaling through XEDAR Is Required for Mammary Gland Morphogenesis.

XEDAR is a member of the TNF receptor subfamily and a mediator of the ectodysplasin (EDA) pathway. EDA signaling plays evolutionarily conserved roles in the development of the ectodermal appendage organ class, which includes hair, eccrine sweat glands, and mammary glands. Loss-of-function sequence variants of EDA, which encodes the two major ligand isoforms, EDA-A1 and EDA-A2, result in X-linked hypohidrotic ectodermal dysplasia characterized by defects in two or more types of ectodermal appendages. EDA-A1 and EDA-A2 signal through the receptors EDAR and XEDAR, respectively. Although the contributions of the EDA-A1/EDAR signaling pathway to EDA-dependent ectodermal appendage phenotypes have been extensively characterized, the significance of the EDA-A2/XEDAR branch of the pathway has remained obscure. In this study, we report the phenotypic consequences of disrupting the EDA-A2/XEDAR pathway on mammary gland differentiation and growth. Using a mouse Xedar knockout model, we show that Xedar has a specific and temporally restricted role in promoting late pubertal growth and branching of the mammary epithelium that can be influenced by genetic background. Our findings implicate Xedar in ectodermal appendage development and suggest that the EDA-A2/XEDAR signaling axis contributes to the etiology of EDA-dependent mammary phenotypes.

The effect of lipoprotein-associated phospholipase A2 deficiency on pulmonary allergic responses in Aspergillus fumigatus sensitized mice.

BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2)/platelet-activating factor acetylhydrolase (PAF-AH) has been implicated in the pathogenesis of cardiovascular disease. A therapeutic targeting of this enzyme was challenged by the concern that increased circulating platelet activating factor (PAF) may predispose to or increase the severity of the allergic airway response. The aim of this study was to investigate whether Lp-PLA2 gene deficiency increases the risk of PAF and IgE-mediated inflammatory responses in vitro and in vivo using mouse models. METHODS: Lp-PLA2-/- mice were generated and back crossed to the C57BL/6 background. PAF-AH activity was measured using a hydrolysis assay in serum and bronchoalveolar lavage (BAL) samples obtained from mice. Aspergillus fumigatus (Af)-specific serum was prepared for passive allergic sensitization of mice in vivo and mast cells in vitro. ß- hexosaminidase release was studied in bone marrow derived mast cells sensitized with Af-specific serum or DNP-IgE and challenged with Af or DNP, respectively. Mice were treated with lipopolysaccharide (LPS) and PAF intratracheally and studied 24 hours later. Mice were sensitized either passively or actively against Af and were studied 48 hours after a single intranasal Af challenge. Airway responsiveness to methacholine, inflammatory cell influx in the lung tissue and BAL, immunoglobulin (ELISA) and cytokine (Luminex) profiles were compared between the wild type (WT) and Lp-PLA2-/- mice. RESULTS: PAF-AH activity was reduced but not completely abolished in Lp-PLA2-/- serum or by in vitro treatment of serum samples with a high saturating concentration of the selective Lp-PLA2 inhibitor, SB-435495. PAF inhalation significantly enhanced airway inflammation of LPS treated WT and Lp-PLA2-/- mice to a similar extent. Sensitized WT and Lp-PLA2-/- bone-marrow derived mast cells released ß-hexosaminidase following stimulation by allergen or IgE crosslinking to equivalent levels. Wild type and Lp-PLA2-/- mice responded to passive or active allergic sensitization by significant IgE production, airway inflammation and hyperresponsiveness after Af challenge. BAL cell influx was not different between these strains while IL-4, IL-5, IL-6 and eotaxin release was attenuated in Lp-PLA2-/- mice. There were no differences in the amount of total IgE levels in the Af sensitized WT and Lp-PLA2-/- mice. CONCLUSIONS: We conclude that Lp-PLA2 deficiency in C57BL/6 mice did not result in a heightened airway inflammation or hyperresponsiveness after PAF/LPS treatment or passive or active allergic sensitization and challenge.

Ozone Inhalation Attenuated the Effects of Budesonide on Aspergillus fumigatus-Induced Airway Inflammation and Hyperreactivity in Mice.

Inhaled glucocorticoids form the mainstay of asthma treatment because of their anti-inflammatory effects in the lung. Exposure to the air pollutant ozone (O3) exacerbates chronic airways disease. We and others showed that presence of the epithelial-derived surfactant protein-D (SP-D) is important in immunoprotection against inflammatory changes including those induced by O3 inhalation in the airways. SP-D synthesis requires glucocorticoids. We hypothesized here that O3 exposure impairs glucocorticoid responsiveness (including SP-D production) in allergic airway inflammation. The effects of O3 inhalation and glucocorticoid treatment were studied in a mouse model of allergic asthma induced by sensitization and challenge with Aspergillus fumigatus (Af) in vivo. The role of O3 and glucocorticoids in regulation of SP-D expression was investigated in A549 and primary human type II alveolar epithelial cells in vitro. Budesonide inhibited airway hyperreactivity, eosinophil counts in the lung and bronchoalveolar lavage (BAL) and CCL11, IL-13, and IL-23p19 release in the BAL of mice sensitized and challenged with Af (p < 0.05). The inhibitory effects of budesonide were attenuated on inflammatory changes and were completely abolished on airway hyperreactivity after O3 exposure of mice sensitized and challenged with Af. O3 stimulated release of pro-neutrophilic mediators including CCL20 and IL-6 into the airways and impaired the inhibitory effects of budesonide on CCL11, IL-13 and IL-23. O3 also prevented budesonide-induced release of the immunoprotective lung collectin SP-D into the airways of allergen-challenged mice. O3 had a bi-phasic direct effect with early (<12 h) inhibition and late (>48 h) activation of SP-D mRNA (sftpd) in vitro. Dexamethasone and budesonide induced sftpd transcription and translation in human type II alveolar epithelial cells in a glucocorticoid receptor and STAT3 (an IL-6 responsive transcription factor) dependent manner. Our study indicates that O3 exposure counteracts the effects of budesonide on airway inflammation, airway hyperreactivity, and SP-D production. We speculate that impairment of SP-D expression may contribute to the acute O3-induced airway inflammation. Asthmatics exposed to high ambient O3 levels may become less responsive to glucocorticoid treatment during acute exacerbations.