Nitric Oxide 42 (2014) 99–153
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Nitric Oxide j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i o x
Abstracts from the 2014 International Nitric Oxide Society Conference, held June 16–20, in Cleveland, USA Abstracts – oral presentations Oral 1711-1. Nitric oxide alters hyaluronan deposition by cultured airway smooth muscle cells and correlates with hyaluronan levels in asthmatics
Oral 1711-2. S-nitrosoglutathione reductase deﬁciency impairs fungal clearance through s-nitrosylation of pattern recognition receptor dectin-1
Alana Majors a, Ahila Subramanian a, Lisa Ruple a, Ritu Chakravarti b, Suzy Comhair a, Rachel Leahy a, Serpil Erzurum a, Dennis Stuehr c, Mark Aronica a a Cleveland Clinic Foundation b Cleveland Clinic c Department of Pathobiology, Cleveland Clinic, Cleveland, OH 44195, USA
Nitric oxide is elevated in many allergic asthmatics and correlates with inﬂammation and worsening asthma but the consequences of elevated NO are largely not known. Alterations of the extracellular matrix, including changes in hyaluronan (HA), are key features in asthma and contribute to the pathogenesis but it is not known how NO may affect HA deposition. We examined the effects of NO on cultured murine airway smooth muscle cells (ASMCs). Untreated ASMCs deposit relatively small amounts of HA into the matrix. Treatment with NO donors (0– 500 micromolar NOC-18, NOC 12 or GSNO, 0–50 micromolar NOC-2, or 0–125 micromolar SNAP) for up to 23 hrs dramatically alters HA deposition. Neither nitrate nor nitrite had an effect on HA deposition. Aﬃnity histochemistry demonstrates that HA is present in the form of unique, cable-like structures in the NOtreated cultures. The effect of NO on HA appears to be independent of the activation of soluble guanylate cyclase. HA cable formation is rapid and can be detected after only 1 hr of NO treatment. The cable structures serve as an attachment site for immune cells. Interactions with the HA produced by NO-treated SMCs may modulate immune cells and perpetuate the inﬂammatory response. HA was quantiﬁed in human bronchial alveolar lavage ﬂuid (n = 24) and NO levels were measured in exhaled breath. Bivariate linear regression analysis demonstrated a statistically signiﬁcant, positive correlation (R-squared = 0.221, p = 0.0487) between HA and exhaled NO. Together these results demonstrate that elevated NO levels in asthmatics may contribute to the upregulation of HA in the inﬂamed airway. Further understanding the relationship between NO, HA deposition and leukocyte retention may lead to the development of novel therapies for patients with asthma, particularly those that are refractory to the current, standard methods of treatment. This work was supported by the National Heart Lung and Blood Institute P01 HL081064 (M.A.A. & S.C.E). Keywords: Nitric oxide; Hyaluronan; Asthma; Smooth muscle cells; Extracellular matrix.
Changjiang Guo, James Gow, Yujie Yang, Mohan Govendraj, Andrew Gow Department of Pharmacology & Toxicology, Rutgers University, Piscataway, NJ 08854, USA
Recognition of fungal surface β-glucan by pattern recognition receptor dectin-1 is a critical process for fungal clearance in the lung. In humans, persistent fungal infection is observed in individuals with particular dectin-1 polymorphism. We have recently identiﬁed that NO modiﬁes critical cysteines in surfactant protein-D to disassemble and alter protein function. There is a hydrophobic S-nitrosylation motif present in surfactant protein-D that is also present in dectin-1. We hypothesized that dectin-1 can be modiﬁed by nitrosative stress potentially leading to impairment of fungal clearance. Dectin-1 was S-nitrosylated by L-nitrosocysteine (L-SNOC) in vitro as determined by biotinswitch assay. Incubation of murine macrophages (Raw264.7 cells) with GSNO reduced the surface dectin-1 expression by ﬂowcytometric analysis. The reduction of dectin-1 is due to formation of SNO-dectin-1 and disruption of dectin-1 complex. GSNO also induces dectin-1 shedding from the cell surface. S-nitrosoglutathione reductase (GSNOR) degrades the SNO moiety and in GSNOR−/− mice there is an altered SNO pool. Bronchi alveolar lavage (BAL) and alveolar macrophages were harvested from wild-type (WT), GSNOR+/− and GSNOR−/−. SNOdectin-1 was found in the BAL from GSNOR+/− and GSNOR−/− but not in WT mice. The levels of dectin-1 in the BAL of GSNOR+/− and GSNOR−/− are much higher than those of WT, suggesting the shedding of dectin-1 from alveolar macrophages from these mice. Flow cytometric analysis of dectin-1 on alveolar macrophages reveals that GSNOR+/− and GSNOR−/− have lower levels of dectin-1 on the macrophage surface. Alveolar macrophages from GSNOR−/− mice produce lower levels of interleukin-1beta (IL-1β) and IL-6 by curdlan, an agonist of dectin-1, further proving the dysfunction of dectin-1. In addition, the BAL from GSNOR−/− mice inhibits phagocytic activity of Raw264.7 cells. These data show that nitrosative stress affects alveolar macrophages through direct modiﬁcation of pattern recognition receptors, causing shedding from the surface and impairment of fungal clearance from the lung. This work was supported by NIH HL086621. Keywords: S-nitrosylation; Dectin-1; Fungal clearance; GSNOR.