CRISPR/Cas9-based double-strand oligonucleotide insertion strategy corrects metabolic abnormalities in murine glycogen storage disease type-Ia.

Glycogen storage disease type-Ia (GSD-Ia), characterized by impaired blood glucose homeostasis, is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). Using the G6pc-R83C mouse model of GSD-Ia, we explored a CRISPR/Cas9-based double-strand DNA oligonucleotide (dsODN) insertional strategy that uses the nonhomologous end-joining repair mechanism to correct the pathogenic p.R83C variant in G6pc exon-2. The strategy is based on the insertion of a short dsODN into G6pc exon-2 to disrupt the native exon and to introduce an additional splice acceptor site and the correcting sequence. When transcribed and spliced, the edited gene would generate a wild-type mRNA encoding the native G6Pase-α protein. The editing reagents formulated in lipid nanoparticles (LNPs) were delivered to the liver. Mice were treated either with one dose of LNP-dsODN at age 4 weeks or with two doses of LNP-dsODN at age 2 and 4 weeks. The G6pc-R83C mice receiving successful editing expressed ~4% of normal hepatic G6Pase-α activity, maintained glucose homeostasis, lacked hypoglycemic seizures, and displayed normalized blood metabolite profile. The outcomes are consistent with preclinical studies supporting previous gene augmentation therapy which is currently in clinical trials. This editing strategy may offer the basis for a therapeutic approach with an earlier clinical intervention than gene augmentation, with the additional benefit of a potentially permanent correction of the GSD-Ia phenotype.

Mouse phospholipid phosphatase 6 regulates dendritic cell cholesterol, macropinocytosis, and allergen sensitization.

Lipid phosphate phosphatases are a family of enzymes with diverse cellular metabolic functions. Phospholipid phosphatase 6 (PLPP6) is a regulator of cellular polyisoprenyl phosphates; however, its in vivo functions remain to be determined. Here, mouse PLPP6 was characterized to possess similar catalytic properties as the human enzyme. Plpp6 knockout mice (Plpp6 -/- ) were generated and displayed decreased airway allergen sensitization, pointing to a role for PLPP6 in the early events of lung allergic responses. Dendritic cell (DC) responses were investigated and endocytosis of allergen via macropinocytosis was decreased in Plpp6 -/- DCs that had lower cholesterol content. When reversed by cholesterol loading, the DC macropinocytosis defect is corrected. Adoptive transfer of Plpp6 -/- DCs to wild-type mice during sensitization was sufficient to decrease allergen-induced responses. Together, our findings have identified PLPP6 as a pivotal regulator of DC cholesterol content and macropinocytosis, cellular mechanisms that are important for pathologic responses in allergen-induced lung inflammation.

15-Epi-lipoxin A4 inhibits human neutrophil superoxide anion generation by regulating polyisoprenyl diphosphate phosphatase 1.

Regulation of leukocyte activation is critical to limit unintended tissue injury during acute inflammation. On neutrophil activation, polyisoprenyl diphosphate phosphatase 1 (PDP1) rapidly converts presqualene diphosphate to presqualene monophosphate to facilitate cell activation. Lipoxins are potent anti-inflammatory mediators for neutrophils, yet their counterregulatory signaling mechanisms remain to be determined. 15-Epi-lipoxin A4 (15-epi-LXA4) blocked agonist-initiated association of the nicotinamide adenine dinucleotide phosphate oxidase components p47(PHOX) and p22(PHOX) in human neutrophils. 15-Epi-LXA4 (0.1-100 nM) inhibited neutrophil superoxide anion (O2(-)) generation in a concentration- and ALX/FPR2 receptor-dependent manner that was disrupted by PDP1-specific antibodies. In differentiated HL60 cells, a myeloid cell line, agonist-initiated O2(-) generation was inhibited by PDP1 siRNA. Recombinant human PDP1 was directly phosphorylated in vitro by select protein kinase C (PKC) isoforms, including PKCßII. When neutrophils were exposed to formyl-methionyl-leucyl-phenylalanine (fMLP), PKCßII was rapidly phosphorylated and physically associated with PDP1. Agonist-initiated conversion of neutrophil presqualene diphosphate to presqualene monophosphate was blocked by PKCßII inhibition. Neutrophil exposure to 15-epi-LXA4 attenuated fMLP triggered PKCßII phosphorylation and its interactions with PDP1. Together, these findings indicate that PDP1 serves an integral signaling role in neutrophil proinflammatory responses and as a target for counter-regulatory mediators.

Resolvin D1 and aspirin-triggered resolvin D1 promote resolution of allergic airways responses.

Asthma is a disease of airway inflammation that in most cases fails to resolve. The resolution of inflammation is an active process governed by specific chemical mediators, including D-series resolvins. In this study, we determined the impact of resolvin D1 (RvD1) and aspirin-triggered RvD1 (AT-RvD1) on the development of allergic airway responses and their resolution. Mice were allergen sensitized, and RvD1, AT-RvD1 (1, 10, or 100 ng), or vehicle was administered at select intervals before or after aerosol allergen challenge. RvD1 markedly decreased airway eosinophilia and mucus metaplasia, in part by decreasing IL-5 and IκBα degradation. For the resolution of established allergic airway responses, AT-RvD1 was even more efficacious than RvD1, leading to a marked decrease in the resolution interval for lung eosinophilia, decrements in select inflammatory peptide and lipid mediators, and more rapid resolution of airway hyperreactivity to methacholine. Relative to RvD1, AT-RvD1 resisted metabolic inactivation by macrophages, and AT-RvD1 significantly enhanced macrophage phagocytosis of IgG-OVA-coated beads in vitro and in vivo, a new proresolving mechanism for the clearance of allergen from the airways. In conclusion, RvD1 and AT-RvD1 can serve as important modulators of allergic airway responses by decreasing eosinophils and proinflammatory mediators and promoting macrophage clearance of allergen. Together, these findings identify D-series resolvins as potential proresolving therapeutic agents for allergic responses.

Resolution of Toll-like receptor 4-mediated acute lung injury is linked to eicosanoids and suppressor of cytokine signaling 3.

The purpose of this study was to investigate roles for Toll-like receptor 4 (TLR4) in host responses to sterile tissue injury. Hydrochloric acid was instilled into the left mainstem bronchus of TLR4-defective (both C3H/HeJ and congenic C.C3-Tlr4(Lps-d)/J) and control mice to initiate mild, self-limited acute lung injury (ALI). Outcome measures included respiratory mechanics, barrier integrity, leukocyte accumulation, and levels of select soluble mediators. TLR4-defective mice were more resistant to ALI, with significantly decreased perturbations in lung elastance and resistance, resulting in faster resolution of these parameters [resolution interval (R(i)); ∼6 vs. 12 h]. Vascular permeability changes and oxidative stress were also decreased in injured HeJ mice. These TLR4-defective mice paradoxically displayed increased lung neutrophils [(HeJ) 24×10(3) vs. (control) 13×10(3) cells/bronchoalveolar lavage]. Proresolving mechanisms for TLR4-defective animals included decreased eicosanoid biosynthesis, including cysteinyl leukotrienes (80% mean decrease) that mediated CysLT1 receptor-dependent vascular permeability changes; and induction of lung suppressor of cytokine signaling 3 (SOCS3) expression that decreased TLR4-driven oxidative stress. Together, these findings indicate pivotal roles for TLR4 in promoting sterile ALI and suggest downstream provocative roles for cysteinyl leukotrienes and protective roles for SOCS3 in the intensity and duration of host responses to ALI.

Molecular circuits of resolution in airway inflammation.

Inflammatory diseases of the lung are common, cause significant morbidity, and can be refractory to therapy. Airway responses to injury, noxious stimuli, or microbes lead to leukocyte recruitment for host defense. As leukocytes respond, they interact with lung resident cells and can elaborate specific mediators that are enzymatically generated from polyunsaturated fatty acids via transcellular biosynthesis. These bioactive, lipid-derived, small molecules serve as agonists at specific receptors and are rapidly inactivated in the local environment. This review will focus on the biosynthesis, receptors, cellular responses, and in vivo actions of lipoxins, resolvins, and protectins as exemplary molecular signaling circuits in the airway that are anti-inflammatory and proresolving.

Activation of polyisoprenyl diphosphate phosphatase 1 remodels cellular presqualene diphosphate.

Polyisoprenyl diphosphates play diverse and vital roles in cell function in health and disease. The counter-regulatory lipid signaling molecule, presqualene diphosphate (PSDP), is rapidly converted to its monophosphate form (PSMP) upon cell activation [Levy, B. D., Petasis, N. A., and Serhan, C. N. (1997) Nature 389, 985-990]. The first PSDP phosphatase was recently identified and named polyisoprenyl diphosphate phosphatase 1 (PDP1) [Fukunaga, K., et al. (2006) J. Biol. Chem. 281, 9490-9497]. Here, we present evidence that PDP1 displays properties of a lipid phosphate phosphatase/phosphotransferase with distinct substrate preference for PSDP. Cell activation with PMA increased PSDP phosphatase activity in a concentration-dependent manner, and Western analysis suggested that PDP1 is directly phosphorylated by protein kinase C. Cellular PSDP phosphatase activity was also induced by the receptor-mediated agonists insulin and TNF-alpha. To address PDP1's contribution to cellular PSDP phosphatase activity, HEK293 cells were established that stably expressed PDP1 siRNA, leading to a 60% decrease in the level of PDP1 RNA, and concomitant decreases in PDP1 protein and PMA-initiated PSDP phosphatase activity. HEK293 cells harboring the PDP1 siRNA construct also displayed a marked decrease in the extent of PMA-initiated conversion of cellular PSDP to PSMP. Together, these findings are the first to indicate that PDP1 is activated during cell responses to soluble stimuli to convert PSDP to PSMP. Moreover, they provide evidence that PDP1 can serve as a new checkpoint for polyisoprenyl phosphate remodeling during cell activation.

Chemical mediators and the resolution of airway inflammation.

Asthma pathobiology is remarkable for chronic airway inflammation that fails to spontaneously resolve. No curative therapy is currently available. A growing body of evidence indicates that, in health, inflammation resolution is an active process orchestrated by specific chemical mediators that are elaborated to restore tissue homeostasis. Activated cell membranes release polyunsaturated fatty acids from phospholipids for enzymatic conversion to biologically active mediators with profound regulatory effects on innate and adaptive immunity. Some of these mediators carry anti-inflammatory and pro-resolving actions that are transduced in a cell-type specific manner via specific recognition sites that initiate regulatory intracellular signals, such as presqualene diphosphate remodeling, to limit pro-phlogistic cell activation. Some of these counter-regulatory lipid mediators have been identified in the airway during asthma and defects in their production are associated with disease severity. In this review, we describe the biosynthesis and bioactions of pro-resolving chemical mediators and provide examples of select mediators and their structural analogs with particular relevance to asthma.

The fruitless gene is required for the proper formation of axonal tracts in the embryonic central nervous system of Drosophila.

The fruitless (fru) gene in Drosophila melanogaster is a multifunctional gene that has sex-specific functions in the regulation of male sexual behavior and sex-nonspecific functions affecting adult viability and external morphology. While much attention has focused on fru's sex-specific roles, less is known about its sex-nonspecific functions. We have examined fru's sex-nonspecific role in embryonic neural development. fru transcripts from sex-nonspecific promoters are expressed beginning at the earliest stages of neurogenesis, and Fru proteins are present in both neurons and glia. In embryos that lack most or all fru function, FasII- and BP102-positive axons have defasciculation defects and grow along abnormal pathways in the CNS. These defects in axonal projections in fru mutants were rescued by the expression of specific UAS-fru transgenes under the control of a pan-neuronal scabrous-GAL4 driver. Our results suggest that one of fru's sex-nonspecific roles is to regulate the pathfinding ability of axons in the embryonic CNS.