Cell-free DNA levels associate with COPD exacerbations and mortality.

THE QUESTION ADDRESSED BY THE STUDY: Good biological indicators capable of predicting chronic obstructive pulmonary disease (COPD) phenotypes and clinical trajectories are lacking. Because nuclear and mitochondrial genomes are damaged and released by cigarette smoke exposure, plasma cell-free mitochondrial and nuclear DNA (cf-mtDNA and cf-nDNA) levels could potentially integrate disease physiology and clinical phenotypes in COPD. This study aimed to determine whether plasma cf-mtDNA and cf-nDNA levels are associated with COPD disease severity, exacerbations, and mortality risk. MATERIALS AND METHODS: We quantified mtDNA and nDNA copy numbers in plasma from participants enrolled in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE, n = 2,702) study and determined associations with relevant clinical parameters. RESULTS: Of the 2,128 participants with COPD, 65% were male and the median age was 64 (interquartile range, 59-69) years. During the baseline visit, cf-mtDNA levels positively correlated with future exacerbation rates in subjects with mild/moderate and severe disease (Global Initiative for Obstructive Lung Disease [GOLD] I/II and III, respectively) or with high eosinophil count (≥ 300). cf-nDNA positively associated with an increased mortality risk (hazard ratio, 1.33 [95% confidence interval, 1.01-1.74] per each natural log of cf-nDNA copy number). Additional analysis revealed that individuals with low cf-mtDNA and high cf-nDNA abundance further increased the mortality risk (hazard ratio, 1.62 [95% confidence interval, 1.16-2.25] per each natural log of cf-nDNA copy number). ANSWER TO THE QUESTION: Plasma cf-mtDNA and cf-nDNA, when integrated into quantitative clinical measurements, may aid in improving COPD severity and progression assessment.

Intact mitochondrial function in the setting of telomere-induced senescence.

Mitochondria play essential roles in metabolic support and signaling within all cells. Congenital and acquired defects in mitochondria are responsible for several pathologies, including premature entrance to cellar senescence. Conversely, we examined the consequences of dysfunctional telomere-driven cellular senescence on mitochondrial biogenesis and function. We drove senescence in vitro and in vivo by deleting the telomere-binding protein TRF2 in fibroblasts and hepatocytes, respectively. Deletion of TRF2 led to a robust DNA damage response, global changes in transcription, and induction of cellular senescence. In vitro, senescent cells had significant increases in mitochondrial respiratory capacity driven by increased cellular and mitochondrial volume. Hepatocytes with dysfunctional telomeres maintained their mitochondrial respiratory capacity in vivo, whether measured in intact cells or purified mitochondria. Induction of senescence led to the upregulation of overlapping and distinct genes in fibroblasts and hepatocytes, but transcripts related to mitochondria were preserved. Our results support that mitochondrial function and activity are preserved in telomere dysfunction-induced senescence, which may facilitate continued cellular functions.

Mouse nasal epithelial innate immune responses to Pseudomonas aeruginosa quorum-sensing molecules require taste signaling components.

We previously observed that the human bitter taste receptor T2R38 is an important component of upper respiratory innate defense because it detects acyl homoserine lactone (AHL) quorum-sensing molecules secreted by Gram-negative bacteria. T2R38 activation in human sinonasal epithelial cells stimulates calcium and NO signals that increase mucociliary clearance, the major physical respiratory defense against inhaled pathogens. While mice do not have a clear T2R38 ortholog, they do have bitter taste receptors capable of responding to T2R38 agonists, suggesting that T2R-mediated innate immune mechanisms may be conserved in mice. We examined whether AHLs activate calcium and NO signaling in mouse nasal epithelial cells, and utilized pharmacology, as well as cells from knockout mice lacking important components of canonical taste signal transduction pathways, to determine if AHL-stimulated responses require taste signaling molecules. We found that AHLs stimulate calcium-dependent NO production that increases mucociliary clearance and thus likely serves an innate immune role against Gram-negative bacteria. These responses require PLCß2 and TRPM5 taste signaling components, but not α-gustducin. These data suggest the mouse may be a useful model for further studies of T2R-mediated innate immunity.

Genetic loss or pharmacological blockade of testes-expressed taste genes causes male sterility.

TAS1R taste receptors and their associated heterotrimeric G protein gustducin are involved in sugar and amino acid sensing in taste cells and in the gastrointestinal tract. They are also strongly expressed in testis and sperm, but their functions in these tissues were previously unknown. Using mouse models, we show that the genetic absence of both TAS1R3, a component of sweet and amino acid taste receptors, and the gustducin α-subunit GNAT3 leads to male-specific sterility. To gain further insight into this effect, we generated a mouse model that expressed a humanized form of TAS1R3 susceptible to inhibition by the antilipid medication clofibrate. Sperm formation in animals without functional TAS1R3 and GNAT3 is compromised, with malformed and immotile sperm. Furthermore, clofibrate inhibition of humanized TAS1R3 in the genetic background of Tas1r3(-/-), Gnat3(-/-) doubly null mice led to inducible male sterility. These results indicate a crucial role for these extraoral "taste" molecules in sperm development and maturation. We previously reported that blocking of human TAS1R3, but not mouse TAS1R3, can be achieved by common medications or chemicals in the environment. We hypothesize that even low levels of these compounds can lower sperm count and negatively affect human male fertility, which common mouse toxicology assays would not reveal. Conversely, we speculate that TAS1R3 and GNAT3 activators may help infertile men, particularly those that are affected by some of the mentioned inhibitors and/or are diagnosed with idiopathic infertility involving signaling pathway of these receptors.

Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue.

Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5-expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate (CV) and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5-expressing cells in posterior tongue are a subset of K14-positive epithelial cells. Lineage-tracing experiments using an inducible Cre knockin allele in combination with Rosa26-LacZ and Rosa26-tdTomato reporter strains showed that Lgr5-expressing cells gave rise to taste cells, perigemmal cells, along with self-renewing cells at the bottom of trench areas at the base of CV and foliate papillae. Moreover, using subtype-specific taste markers, we found that Lgr5-expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue.

Soluble extract from the nematode Strongyloides stercoralis induces CXCR2 dependent/IL-17 independent neutrophil recruitment.

Neutrophil recruitment via CXCR2 is required for innate and adaptive protective immunity to the larvae of Strongyloides stercoralis in mice. The goal of the present study was to determine the mechanism of CXCR2-mediated neutrophil recruitment to S. stercoralis. Mice deficient in the receptor for IL-17A and IL-17F, upstream mediators of CXCR2 ligand production, were infected with S. stercoralis larvae; there was no difference in larval survival, neutrophil recruitment, or production of CXCR2 ligands compared with wild type mice. In vivo and in vitro stimulation of neutrophils with S. stercoralis soluble extract resulted in significant neutrophil recruitment. In vitro assays demonstrated that the recruitment functioned through both chemokinesis and chemotaxis, was specific for CXCR2, and was a G protein-coupled response involving tyrosine kinase and PI3K. Finally, neutrophil stimulation with S. stercoralis soluble extract induced release of the CXCR2 ligands MIP-2 and KC from neutrophils, thereby potentially enhancing neutrophil recruitment.

C-terminal processing of GABARAP is not required for trafficking of the angiotensin II type 1A receptor.

OBJECTIVE: GABARAP, a small (117 aa) trafficking protein, binds to the C-terminal, cytoplasmic domain of rat angiotensin type-1A receptor (AT(1)R), the predominant effector of the octapeptide angiotensin II (Ang II) (Cook et al., Circ. Res. 2008;102:1539-47). The objectives of this study were to map the interaction domains of GABARAP and AT(1)R, to determine the effect of GABARAP association on AT(1)R signaling activity, and to determine the importance of post-translational processing of GABARAP on accumulation of AT(1)R on the plasma membrane and its signaling function. RESULTS: Deletion analysis identified two regions within GABARAP necessary for interaction with AT(1)R in yeast two-hybrid assays: 1) a domain comprised of residues 32-51 that is nearly identical to that involved in binding and intracellular trafficking of the GABA(A) receptor and 2) a domain encompassing the C-terminal 21 aa. The GABARAP interaction domain of AT(1)R was delimited to the 15 aa immediately downstream of the last membrane spanning region. Overexpression of GABARAP in rat adrenal pheochromocytoma PC-12 cells increased the cell-surface expression of AT(1)R and Ang II-dependent activation of the cAMP signaling pathway. Residues within AT(1)R necessary for these responses were identified by mutational analysis. In PC-12 cells, GABARAP was constitutively and quantitatively cleaved at the C-terminus peptide bond and this cleavage was prevented by mutation of Gly(116). Wild-type GABARAP and the G116A mutant were, however, equally effective in stimulating AT(1)R surface expression and signaling activity. CONCLUSIONS: GABARAP and AT(1)R interact through discrete domains and this association regulates the cell-surface accumulation and, consequently, ligand-induced function of the receptor. Unlike that observed with the GABA(A) receptor, this regulation is not dependent on C-terminal processing and modification of GABARAP.

Eosinophils utilize multiple chemokine receptors for chemotaxis to the parasitic nematode Strongyloides stercoralis.

Protective innate immunity to the nematode Strongyloides stercoralis requires eosinophils in the parasite killing process. Experiments were performed to determine if an extract of S. stercoralis would trigger eosinophil chemotaxis, and to then compare the chemotactic migration response, including second messenger signals and receptors, to those mechanisms triggered by host chemoattractants. Eosinophils undergo both chemotaxis and chemokinesis to soluble parasite extract in transwell plates. Pretreatment of eosinophils with pertussis toxin, a G protein-coupled receptor inhibitor, inhibited migration of the eosinophils to the parasite extract. Likewise, blocking PI3K, tyrosine kinase, p38 and p44/42 inhibited eosinophil chemotaxis to parasite extract. Furthermore, CCR3, CXCR4 or CXCR2 antagonists significantly inhibited eosinophil chemotaxis to the parasite extract. Molecular weight fractionation of parasite extract revealed that molecules attracting eosinophils were present in several fractions, with molecules greater than 30 kDa being the most potent. Treating the extract with proteinase K or chitinase significantly inhibited its ability to induce chemotaxis, thereby demonstrating that the chemoattractants were both protein and chitin. Therefore, chemoattractants derived from parasites and host species stimulate similar receptors and second messenger signals to induce eosinophil chemotaxis. Parasite extract stimulates multiple receptors on the eosinophil surface, which ensures a robust innate immune response to the parasite.