Role of the Adiponectin Binding Protein, T-Cadherin (cdh13), in Pulmonary Responses to Subacute Ozone

Adiponectin, an adipose derived hormone with pleiotropic functions, binds to several proteins, including T-cadherin. We have previously reported that adiponectin deficient (Adipo−/−) mice have increased IL-17A-dependent neutrophil accumulation in their lungs after subacute exposure to ozone (0.3 ppm for 72 hrs). The purpose of this study was to determine whether this anti-inflammatory effect of adiponectin required adiponectin binding to T-cadherin. Wildtype, Adipo−/−, T-cadherin deficient (T-cad−/−), and bideficient (Adipo−/−/T-cad−/−) mice were exposed to subacute ozone or air. Compared to wildtype mice, ozone-induced increases in pulmonary IL-17A mRNA expression were augmented in T-cad−/− and Adipo−/− mice. Compared to T-cad−/− mice, there was no further increase in IL-17A in Adipo−/−/T-cad−/− mice, indicating that adiponectin binding to T-cadherin is required for suppression of ozone-induced IL-17A expression. Similar results were obtained for pulmonary mRNA expression of saa3, an acute phase protein capable of inducing IL-17A expression. Comparison of lung histological sections across genotypes also indicated that adiponectin attenuation of ozone-induced inflammatory lesions at bronchiolar branch points required T-cadherin. BAL neutrophils and G-CSF were augmented in T-cad−/− mice and further augmented in Adipo−/−/T-cad−/− mice. Taken together with previous observations indicating that augmentation of these moieties in ozone exposed Adipo−/− mice is partially IL-17A dependent, the results indicate that effects of T-cadherin deficiency on BAL neutrophils and G-CSF are likely secondary to changes in IL-17A, but that adiponectin also acts via T-cadherin independent pathways. Our results indicate that T-cadherin is required for the ability of adiponectin to suppress some but not all aspects of ozone-induced pulmonary inflammation

γδ T Cells Are Required for Pulmonary IL-17A Expression after Ozone Exposure in Mice: Role of TNFα

Ozone is an air pollutant that causes pulmonary symptoms. In mice, ozone exposure causes pulmonary injury and increases bronchoalveolar lavage macrophages and neutrophils. We have shown that IL-17A is important in the recruitment of neutrophils after subacute ozone exposure (0.3 ppm for 24–72 h). We hypothesized that γδ T cells are the main producers of IL-17A after subacute ozone. To explore this hypothesis we exposed wildtype mice and mice deficient in γδ T cells (TCRδ−/−) to ozone or room air. Ozone-induced increases in BAL macrophages and neutrophils were attenuated in TCRδ−/− mice. Ozone increased the number of γδ T cells in the lungs and increased pulmonary Il17a mRNA expression and the number of IL-17A+ CD45+ cells in the lungs and these effects were abolished in TCRδ−/− mice. Ozone-induced increases in factors downstream of IL-17A signaling, including G-CSF, IL-6, IP-10 and KC were also decreased in TCRδ−/− versus wildtype mice. Neutralization of IL-17A during ozone exposure in wildtype mice mimicked the effects of γδ T cell deficiency. TNFR2 deficiency and etanercept, a TNFα antagonist, also reduced ozone-induced increases in Il17a mRNA, IL-17A+ CD45+ cells and BAL G-CSF as well as BAL neutrophils. TNFR2 deficient mice also had decreased ozone-induced increases in Ccl20, a chemoattractant for IL-17A+ γδ T cells. Il17a mRNA and IL-17A+ γδ T cells were also lower in obese Cpefat versus lean WT mice exposed to subacute ozone, consistent with the reduced neutrophil recruitment observed in the obese mice. Taken together, our data indicate that pulmonary inflammation induced by subacute ozone requires γδ T cells and TNFα-dependent recruitment of IL-17A+ γδ T cells to the lung

Role of the Adiponectin Binding Protein, T-Cadherin (Cdh13), in Allergic Airways Responses in Mice

Adiponectin is an adipose derived hormone that declines in obesity. We have previously shown that exogenous administration of adiponectin reduces allergic airways responses in mice. T-cadherin (T-cad; Cdh13) is a binding protein for the high molecular weight isoforms of adiponectin. To determine whether the beneficial effects of adiponectin on allergic airways responses require T-cad, we sensitized wildtype (WT), T-cadherin deficient (T-cad−/−) and adiponectin and T-cad bideficient mice to ovalbumin (OVA) and challenged the mice with aerosolized OVA or PBS. Compared to WT, T-cad−/− mice were protected against OVA-induced airway hyperresponsiveness, increases in BAL inflammatory cells, and induction of IL-13, IL-17, and eotaxin expression. Histological analysis of the lungs of OVA-challenged T-cad−/− versus WT mice indicated reduced inflammation around the airways, and reduced mucous cell hyperplasia. Combined adiponectin and T-cad deficiency reversed the effects of T-cad deficiency alone, indicating that the observed effects of T-cad deficiency require adiponectin. Compared to WT, serum adiponectin was markedly increased in T-cad−/− mice, likely because adiponectin that is normally sequestered by endothelial T-cad remains free in the circulation. In conclusion, T-cad does not mediate the protective effects of adiponectin. Instead, mice lacking T-cad have reduced allergic airways disease, likely because elevated serum adiponectin levels act on other adiponectin signaling pathways

IL-13 Augments Compressive Stress–Induced Tissue Factor Expression in Human Airway Epithelial Cells

Tissue factor (TF) is best known as a cellular initiator of coagulation, but it is also a multifunctional protein that has been implicated in multiple pathophysiologic conditions, including asthma. In the lung, airway epithelial cells express TF, but it is unknown how TF expression is regulated by asthma-associated mediators. We investigated the role of IL-13, a type 2 cytokine, alone and in combination with compressive stress, which mimics asthmatic bronchoconstriction, on TF expression and release of TF-positive extracellular vesicles from primary normal human bronchial epithelial cells. Well-differentiated normal human bronchial epithelial cells were treated with IL-13 and compressive stress, alone and in combination. TF mRNA, protein and activity were measured in the cells and conditioned media. TF was also measured in the bronchoalveolar lavage (BAL) fluid of allergen-challenged mice and patients with asthma. IL-13 and compressive stress increased TF expression, but only compressive stress induced TF-positive extracellular vesicle release. Pretreatment with IL-13 augmented compressive stress–induced TF expression and release. TF protein and activity in BAL fluid were increased in allergen-sensitized and -challenged mice. TF was elevated in the BAL fluid of patients with mild asthma after an allergen challenge. Our in vitro and in vivo data indicate close cooperation between mechanical and inflammatory stimuli on TF expression and release of TF-positive extracellular vesicles in the lungs, which may contribute to pathophysiology of asthma

γδ T Cells Are Required for M2 Macrophage Polarization and Resolution of Ozone-Induced Pulmonary Inflammation in Mice

We examined the role of γδ T cells in the induction of alternatively activated M2 macrophages and the resolution of inflammation after ozone exposure. Wildtype (WT) mice and mice deficient in γδ T cells (TCRδ-/- mice) were exposed to air or to ozone (0.3 ppm for up to 72h) and euthanized immediately or 1, 3, or 5 days after cessation of exposure. In WT mice, M2 macrophages accumulated in the lungs over the course of ozone exposure. Pulmonary mRNA abundance of the M2 genes, Arg1, Retnla, and Clec10a, also increased after ozone. In contrast, no evidence of M2 polarization was observed in TCRδ-/- mice. WT but not TCRδ-/- mice expressed the M2c polarizing cytokine, IL-17A, after ozone exposure and WT mice treated with an IL-17A neutralizing antibody exhibited attenuated ozone-induced M2 gene expression. In WT mice, ozone-induced increases in bronchoalveolar lavage neutrophils and macrophages resolved quickly after cessation of ozone exposure returning to air exposed levels within 3 days. However, lack of M2 macrophages in TCRδ-/- mice was associated with delayed clearance of inflammatory cells after cessation of ozone and increased accumulation of apoptotic macrophages in the lungs. Delayed restoration of normal lung architecture was also observed in TCRδ-/- mice. In summary, our data indicate that γδ T cells are required for the resolution of ozone-induced inflammation, likely because γδ T cells, through their secretion of IL-17A, contribute to changes in macrophage polarization that promote clearance of apoptotic cells

Expression of Sumoylation Deficient Nkx2.5 Mutant in Nkx2.5 Haploinsufficient Mice Leads to Congenital Heart Defects

Nkx2.5 is a cardiac specific homeobox gene critical for normal heart development. We previously identified Nkx2.5 as a target of sumoylation, a posttranslational modification implicated in a variety of cellular activities. Sumoylation enhanced Nkx2.5 activity via covalent attachment to the lysine residue 51, the primary SUMO acceptor site. However, how sumoylation regulates the activity of Nkx2.5 in vivo remains unknown. We generated transgenic mice overexpressing sumoylation deficient mutant K51R (conversion of lysine 51 to arginine) specifically in mouse hearts under the control of cardiac α-myosin heavy chain (α-MHC) promoter (K51R-Tg). Expression of the Nkx2.5 mutant transgene in the wild type murine hearts did not result in any overt cardiac phenotype. However, in the presence of Nkx2.5 haploinsufficiency, cardiomyocyte-specific expression of the Nkx2.5 K51R mutant led to congenital heart diseases (CHDs), accompanied with decreased cardiomyocyte proliferation. Also, a number of human CHDs-associated Nkx2.5 mutants exhibited aberrant sumoylation. Our work demonstrates that altered sumoylation status may underlie the development of human CHDs associated with Nkx2.5 mutants

Convergent evolution of chicken Z and human X chromosomes by expansion and gene acquisition

In birds, as in mammals, one pair of chromosomes differs between the sexes. In birds, males are ZZ and females ZW. In mammals, males are XY and females XX. Like the mammalian XY pair, the avian ZW pair is believed to have evolved from autosomes, with most change occurring in the chromosomes found in only one sex—the W and Y chromosomes1, 2, 3, 4, 5. By contrast, the sex chromosomes found in both sexes—the Z and X chromosomes—are assumed to have diverged little from their autosomal progenitors2. Here we report findings that challenge this assumption for both the chicken Z chromosome and the human X chromosome. The chicken Z chromosome, which we sequenced essentially to completion, is less gene-dense than chicken autosomes but contains a massive tandem array containing hundreds of duplicated genes expressed in testes. A comprehensive comparison of the chicken Z chromosome with the finished sequence of the human X chromosome demonstrates that each evolved independently from different portions of the ancestral genome. Despite this independence, the chicken Z and human X chromosomes share features that distinguish them from autosomes: the acquisition and amplification of testis-expressed genes, and a low gene density resulting from an expansion of intergenic regions. These features were not present on the autosomes from which the Z and X chromosomes originated but were instead acquired during the evolution of Z and X as sex chromosomes. We conclude that the avian Z and mammalian X chromosomes followed convergent evolutionary trajectories, despite their evolving with opposite (female versus male) systems of heterogamety. More broadly, in birds and mammals, sex chromosome evolution involved not only gene loss in sex-specific chromosomes, but also marked expansion and gene acquisition in sex chromosomes common to males and females.National Science Foundation (U.S.)Howard Hughes Medical Institut

Control of Transcription by Cell Size

Cell size increases significantly with increasing ploidy. Differences in cell size and ploidy are associated with alterations in gene expression, although no direct connection has been made between cell size and transcription. Here we show that ploidy-associated changes in gene expression reflect transcriptional adjustment to a larger cell size, implicating cellular geometry as a key parameter in gene regulation. Using RNA-seq, we identified genes whose expression was altered in a tetraploid as compared with the isogenic haploid. A significant fraction of these genes encode cell surface proteins, suggesting an effect of the enlarged cell size on the differential regulation of these genes. To test this hypothesis, we examined expression of these genes in haploid mutants that also produce enlarged size. Surprisingly, many genes differentially regulated in the tetraploid are identically regulated in the enlarged haploids, and the magnitude of change in gene expression correlates with the degree of size enlargement. These results indicate a causal relationship between cell size and transcription, with a size-sensing mechanism that alters transcription in response to size. The genes responding to cell size are enriched for those regulated by two mitogen-activated protein kinase pathways, and components in those pathways were found to mediate size-dependent gene regulation. Transcriptional adjustment to enlarged cell size could underlie other cellular changes associated with polyploidy. The causal relationship between cell size and transcription suggests that cell size homeostasis serves a regulatory role in transcriptome maintenance.National Institutes of Health (U.S.) (grant GM035010)National Institutes of Health (U.S.) (grant GM040266

Activation of an NLRP3 Inflammasome Restricts Mycobacterium kansasii Infection

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium pathogen, whose incidence and prevalence have been increasing in the last decade. M. kansasii can cause pulmonary tuberculosis clinically and radiographically indistinguishable from that caused by Mycobacterium tuberculosis infection. Unlike the widely-studied M. tuberculosis, little is known about the innate immune response against M. kansasii infection. Although inflammasome activation plays an important role in host defense against bacterial infection, its role against atypical mycobacteria remains poorly understood. In this report, the role of inflammasome activity in THP-1 macrophages against M. kansasii infection was studied. Results indicated that viable, but not heat-killed, M. kansasii induced caspase-1-dependent IL-1β secretion in macrophages. The underlying mechanism was found to be through activation of an inflammasome containing the NLR (Nod-like receptor) family member NLRP3 and the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Further, potassium efflux, lysosomal acidification, ROS production and cathepsin B release played a role in M. kansasii-induced inflammasome activation. Finally, the secreted IL-1β derived from caspase-1 activation was shown to restrict intracellular M. kansasii. These findings demonstrate a biological role for the NLRP3 inflammasome in host defense against M. kansasii