Spatial Metabolomics Reveals Localized Impact of Influenza Virus Infection on the Lung Tissue Metabolome.

The influenza virus (IAV) is a major cause of respiratory disease, with significant infection increases in pandemic years. Vaccines are a mainstay of IAV prevention but are complicated by IAV's vast strain diversity and manufacturing and vaccine uptake limitations. While antivirals may be used for treatment of IAV, they are most effective in early stages of the infection, and several virus strains have become drug resistant. Therefore, there is a need for advances in IAV treatment, especially host-directed therapeutics. Given the spatial dynamics of IAV infection and the relationship between viral spatial distribution and disease severity, a spatial approach is necessary to expand our understanding of IAV pathogenesis. We used spatial metabolomics to address this issue. Spatial metabolomics combines liquid chromatography-tandem mass spectrometry of metabolites extracted from systematic organ sections, 3D models, and computational techniques to develop spatial models of metabolite location and their role in organ function and disease pathogenesis. In this project, we analyzed serum and systematically sectioned lung tissue samples from uninfected or infected mice. Spatial mapping of sites of metabolic perturbations revealed significantly lower metabolic perturbation in the trachea compared to other lung tissue sites. Using random forest machine learning, we identified metabolites that responded differently in each lung position based on infection, including specific amino acids, lipids and lipid-like molecules, and nucleosides. These results support the implementation of spatial metabolomics to understand metabolic changes upon respiratory virus infection. IMPORTANCE The influenza virus is a major health concern. Over 1 billion people become infected annually despite the wide distribution of vaccines, and antiviral agents are insufficient to address current clinical needs. In this study, we used spatial metabolomics to understand changes in the lung and serum metabolome of mice infected with influenza A virus compared to uninfected controls. We determined metabolites altered by infection in specific lung tissue sites and distinguished metabolites perturbed by infection between lung tissue and serum samples. Our findings highlight the utility of a spatial approach to understanding the intersection between the lung metabolome, viral infection, and disease severity. Ultimately, this approach will expand our understanding of respiratory disease pathogenesis.

Inter-species Variabilities of Droplet Transport, Size Change, and Deposition in Human and Rat Respiratory Systems: An In Silico Study.

To speculate on human responses from animal studies, scale-up factors (body weight, lung volume, or lung surface area ratios) are currently used to extrapolate aerosol lung deposition from animal to human. However, those existing scale-up methods between animals and humans neglected two important inter-subject variability factors: (1) the effect of anatomical differences in respiratory systems from mouth/nose to peripheral lungs between human and rat, and (2) the effect of spatial distributions and temporal evolutions of temperature and relative humidity (RH) on droplet size change dynamics between the two species. To test the above-mentioned inter-species variability effects on droplet fates in pulmonary routes and generate correlations as a precise scale-up method for lung deposition estimation, this study simulated the transport of pure-water droplets in both human and Sprague-Dawley (SD) rat respiratory systems. Employing an experimentally validated Euler-Lagrange based Computational Fluid-Particle Dynamics (CFPD) model, simulations were performed for droplets with Stk/Fr between 8.36×10-5 and 1.25×10-2. Droplets were inhaled through human and rat nostrils with resting breathing conditions. Numerical results indicate that RH becomes uniformly distributed in rat airways sooner than in human airways, which significantly influences droplet size change dynamics and the resultant trajectories in pulmonary paths. Using the Stokes-Froude dimensionless number group (i.e., Stk/Fr) as the independent variable, the regional deposition fractions and evaporation fractions in both rat and human respiratory systems collapsed into unified correlations. The correlations can be used as a new rat-to-human scale-up method, estimating the lung depositions with consideration of anatomical differences. Furthermore, the necessity to employ realistic RH and temperature boundary conditions at airway walls was also confirmed for the accurate prediction of droplet size change using CFPD. Employing idealized boundary conditions leads the droplets to evaporate slower and deposit more than using realistic RH and temperature boundary conditions.

Xylosyltransferase 2 deficiency and organ homeostasis.

In this paper we characterize the function of Xylosyltransferase 2 (XylT2) in different tissues to investigate the role XylT2 has in the proteoglycan (PG) biochemistry of multiple organs. The results show that in all organs examined there is a widespread and significant decrease in total XylT activity in Xylt2 knock out mice (Xylt2-/-). This decrease results in increased organ weight differences in lung, heart, and spleen. These findings, in addition to our previous findings of increased liver and kidney weight with loss of serum XylT activity, suggest systemic changes in organ function due to loss of XylT2 activity. The Xylt2-/- mice have splenomegaly due to enlargement of the red pulp area and enhanced pulmonary response to bacterial liposaccharide. Tissue glycosaminoglycan composition changes are also found. These results demonstrate a role of XylT2 activity in multiple organs and their PG content. Because the residual XylT activity in the Xylt2-/- is due to xylosyltransferase 1 (XylT1), these studies indicate that both XylT1 and XylT2 have important roles in PG biosynthesis and organ homeostasis.

The NuRD chromatin-remodeling complex enzyme CHD4 prevents hypoxia-induced endothelial Ripk3 transcription and murine embryonic vascular rupture.

Physiological hypoxia can trigger transcriptional events that influence many developmental processes during mammalian embryogenesis. One way that hypoxia affects transcription is by engaging chromatin-remodeling complexes. We now report that chromodomain helicase DNA binding protein 4 (CHD4), an enzyme belonging to the nucleosome remodeling and deacetylase (NuRD) chromatin-remodeling complex, is required for transcriptional repression of the receptor-interacting protein kinase 3 (Ripk3)-a critical executor of the necroptosis cell death program-in hypoxic murine embryonic endothelial cells. Genetic deletion of Chd4 in murine embryonic endothelial cells in vivo results in upregulation of Ripk3 transcripts and protein prior to vascular rupture and lethality at midgestation, and concomitant deletion of Ripk3 partially rescues these phenotypes. In addition, CHD4 binds to and prevents acetylation of the Ripk3 promoter in cultured endothelial cells grown under hypoxic conditions to prevent excessive Ripk3 transcription. These data demonstrate that excessive RIPK3 is detrimental to embryonic vascular integrity and indicate that CHD4 suppresses Ripk3 transcription when the embryonic environment is particularly hypoxic prior to the establishment of fetal-placental circulation at midgestation. Altogether, this research provides new insights into regulators of Ripk3 transcription and encourages future studies into the mechanism by which excessive RIPK3 damages embryonic blood vessels.

Adipose tissue loss and lipodystrophy in xylosyltransferase II deficient mice.

BACKGROUND/OBJECTIVES: The cellular and extracellular matrix (ECM) interactions that regulate adipose tissue homeostasis are incompletely understood. Proteoglycans (PGs) and their sulfated glycosaminoglycans (GAGs) provide spatial and temporal signals for ECM organization and interactions with resident cells by impacting growth factor and cytokine activity. Therefore, PGs and their GAGs could be significant to adipose tissue homeostasis. The purpose of this study was to determine the role of ECM sulfated GAGs in adipose tissue homeostasis. METHODS: Adipose tissue and metabolic homeostasis in mice deficient in xylosyltransferase 2 (Xylt2-/-) were examined by histologic analyses, gene expression analyses, whole body fat composition measurements, and glucose tolerance test. Adipose tissue inflammation and adipocyte precursors were characterized by flow cytometry and in vitro culture of mesenchymal stem cells. RESULTS: Xylt2-/- mice have low body weight due to overall reductions in abdominal fat deposition. Histologically, the adipocytes are reduced in size and number in both gonadal and mesenteric fat depots of Xylt2-/- mice. In addition, these mice are glucose intolerant, insulin resistant, and have increased serum triglycerides as compared to Xylt2 + / + control mice. Furthermore, the adipose tissue niche has increased inflammatory cells and enrichment of proinflammatory factors IL6 and IL1ß, and these mice also have a loss of adipose tissue vascular endothelial cells. Lastly, xylosyltransferease-2 (XylT2) deficient mesenchymal stem cells from gonadal adipose tissue and bone marrow exhibit impaired adipogenic differentiation in vitro. CONCLUSIONS: Decreased GAGs due to the loss of the key GAG assembly enzyme XylT2 causes reduced steady state adipose tissue stores leading to a unique lipodystrophic model. Accumulation of an adipocytic precursor pool of cells is discovered indicating an interruption in differentiation. Therefore, adipose tissue GAGs are important in the homeostasis of adipose tissue by mediating control of adipose precursor development, tissue inflammation, and vascular development.

MicroRNA-26b Modulates the NF-κB Pathway in Alveolar Macrophages by Regulating PTEN.

NF-κB is one of the best-characterized transcription factors, providing the link between early membrane-proximal signaling events and changes in many inflammatory genes. MicroRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. In this study, we evaluated the role of miR-26b in the LPS-induced inflammatory response in bovine alveolar macrophages (bAMs). LPS stimulation of bAMs upregulated miR-26b at 1 h and downregulated it at 6 and 36 h. Overexpression of miR-26b in bAMs enhanced the LPS-induced mRNA expression of proinflammatory cytokines and chemokines, including TNF-α, IL-1ß, IL-8, and IL-10, but it directly inhibited that of IL-6. A similar trend was observed for the release of these cytokines and chemokines from bAMs. miR-26b directly bound the 3'-untranslated region of PTEN, leading to the reduction of PTEN protein in bAMs. miR-26b also enhanced the LPS-induced NF-κB signaling pathway, as revealed by increased NF-κB transcriptional activity and phosphorylation of p65, IκBα, IκB kinase, and Akt. Moreover, PTEN silencing increased the LPS-induced mRNA expression of TNF-α, IL-1ß, IL-6, IL-8, and IL-10 and upregulated the NF-κB pathway. Taken together, we conclude that miR-26b participates in the inflammatory response of LPS-stimulated bAMs by modulating the NF-κB pathway through targeting PTEN.

PDGFRß signalling regulates local inflammation and synergizes with hypercholesterolaemia to promote atherosclerosis.

Platelet-derived growth factor (PDGF) is a mitogen and chemoattractant for vascular smooth muscle cells (VSMCs). However, the direct effects of PDGF receptor ß (PDGFRß) activation on VSMCs have not been studied in the context of atherosclerosis. Here we present a new mouse model of atherosclerosis with an activating mutation in PDGFRß. Increased PDGFRß signalling induces chemokine secretion and leads to leukocyte accumulation in the adventitia and media of the aorta. Furthermore, PDGFRß(D849V) amplifies and accelerates atherosclerosis in hypercholesterolemic ApoE(-/-) or Ldlr(-/-) mice. Intriguingly, increased PDGFRß signalling promotes advanced plaque formation at novel sites in the thoracic aorta and coronary arteries. However, deletion of the PDGFRß-activated transcription factor STAT1 in VSMCs alleviates inflammation of the arterial wall and reduces plaque burden. These results demonstrate that PDGFRß pathway activation has a profound effect on vascular disease and support the conclusion that inflammation in the outer arterial layers is a driving process for atherosclerosis.

Homozygosity for frameshift mutations in XYLT2 result in a spondylo-ocular syndrome with bone fragility, cataracts, and hearing defects.

Heparan and chondroitin/dermatan sulfated proteoglycans have a wide range of roles in cellular and tissue homeostasis including growth factor function, morphogen gradient formation, and co-receptor activity. Proteoglycan assembly initiates with a xylose monosaccharide covalently attached by either xylosyltransferase I or II. Three individuals from two families were found that exhibited similar phenotypes. The index case subjects were two brothers, individuals 1 and 2, who presented with osteoporosis, cataracts, sensorineural hearing loss, and mild learning defects. Whole exome sequence analyses showed that both individuals had a homozygous c.692dup mutation (GenBank: NM_022167.3) in the xylosyltransferase II locus (XYLT2) (MIM: 608125), causing reduced XYLT2 mRNA and low circulating xylosyltransferase (XylT) activity. In an unrelated boy (individual 3) from the second family, we noted low serum XylT activity. Sanger sequencing of XYLT2 in this individual revealed a c.520del mutation in exon 2 that resulted in a frameshift and premature stop codon (p.Ala174Profs(∗)35). Fibroblasts from individuals 1 and 2 showed a range of defects including reduced XylT activity, GAG incorporation of (35)SO4, and heparan sulfate proteoglycan assembly. These studies demonstrate that human XylT2 deficiency results in vertebral compression fractures, sensorineural hearing loss, eye defects, and heart defects, a phenotype that is similar to the autosomal-recessive disorder spondylo-ocular syndrome of unknown cause. This phenotype is different from what has been reported in individuals with other linker enzyme deficiencies. These studies illustrate that the cells of the lens, retina, heart muscle, inner ear, and bone are dependent on XylT2 for proteoglycan assembly in humans.

Regulation of myofibroblast differentiation by miR-424 during epithelial-to-mesenchymal transition.

Idiopathic pulmonary fibrosis (IPF) is one of the most common and severe interstitial lung diseases. Epithelial-to-mesenchymal transition (EMT) is a process whereby epithelial cells undergo transition to a mesenchymal phenotype. This process has been shown to contribute to IPF. MicroRNAs (miRNAs) are small non-coding RNAs of 18-24 nucleotides in length which regulate gene expression. Several studies have implicated miRNAs in EMT; however, specific miRNAs that regulate EMT in IPF have not yet been identified. In this study, we identified 6 up-regulated and 3 down-regulated miRNAs in a human lung epithelial cell EMT model using miRNA microarray and real-time PCR. Overexpression of one of these up-regulated miRNAs, miR-424, increased the expression of α-smooth muscle actin, an indicator of myofibroblast differentiation, but had no effects on the epithelial or mesenchymal cell markers. miR-424 enhanced the activity of the TGF-ß signaling pathway, as demonstrated by a luciferase reporter assay. Further experiments showed that miR-424 decreased the protein expression of Smurf2, a negative regulator of TGF-ß signaling, indicating that miR-424 exerts a forward regulatory loop in the TGF-ß signaling pathway. Our results suggest that miR-424 regulates the myofibroblast differentiation during EMT by potentiating the TGF-ß signaling pathway, likely through Smurf2.

Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice.

Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (-/-) mice. Mice of both genotypes (n=5-6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemical changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82-95% at 48h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 µM) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20-23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner.

Xylosyltransferase II is a significant contributor of circulating xylosyltransferase levels and platelets constitute an important source of xylosyltransferase in serum.

Circulating glycosyltransferases including xylosyltransferases I (XylT1) and II (XylT2) are potential serum biomarkers for various diseases. Understanding what influences the serum activity of these enzymes as well as the sources of these enzymes is important to interpreting the significance of alterations in enzyme activity during disease. This article demonstrates that in the mouse and human the predominant XylT in serum is XylT2. Furthermore, that total XylT levels in human serum are approximately 200% higher than those in plasma due in part to XylT released by platelets during blood clotting in vitro. In addition, the data from Xylt2 knock-out mice and mice with liver neoplasia show that liver is a significant source of serum XylT2 activity. The data presented suggest that serum XylT levels may be an informative biomarker in patients who suffer from diseases affecting platelet and/or liver homeostasis.

Polycystic disease caused by deficiency in xylosyltransferase 2, an initiating enzyme of glycosaminoglycan biosynthesis.

The basic biochemical mechanisms underlying many heritable human polycystic diseases are unknown despite evidence that most cases are caused by mutations in members of several protein families, the most prominent being the polycystin gene family, whose products are found on the primary cilia, or due to mutations in posttranslational processing and transport. Inherited polycystic kidney disease, the most prevalent polycystic disease, currently affects approximately 500,000 people in the United States. Decreases in proteoglycans (PGs) have been found in tissues and cultured cells from patients who suffer from autosomal dominant polycystic kidney disease, and this PG decrease has been hypothesized to be responsible for cystogenesis. This is possible because alterations in PG concentrations would be predicted to disrupt many homeostatic mechanisms of growth, development, and metabolism. To test this hypothesis, we have generated mice lacking xylosyltransferase 2 (XylT2), an enzyme involved in PG biosynthesis. Here we show that inactivation of XylT2 results in a substantial reduction in PGs and a phenotype characteristic of many aspects of polycystic liver and kidney disease, including biliary epithelial cysts, renal tubule dilation, organ fibrosis, and basement membrane abnormalities. Our findings demonstrate that alterations in PG concentrations can occur due to loss of XylT2, and that reduced PGs can induce cyst development.

Biosynthesis of chondroitin and heparan sulfate in chinese hamster ovary cells depends on xylosyltransferase II.

Xylosyltransferase (XylT) catalyzes the initial enzymatic reaction in the glycosaminoglycan assembly pathway for proteoglycan biosynthesis. Its activity is thought to be rate-limiting. Two xylosyltransferases have been found using genomic analyses, and one of these, XylT1, has been shown to have xylosyltransferase activity. On the other hand, the less studied XylT2 in recombinant form lacks xylosyltransferase activity and has no known function. Wild-type Chinese hamster ovary cells express abundant Xylt2 mRNA levels and lack detectable Xylt1 mRNA levels. Analysis of a previously described Chinese hamster ovary cell xylosyltransferase mutant (psgA-745) shows that it harbors an Xylt2 nonsense mutation and fails to assemble glycosaminoglycans onto recombinant biglycan. Transfection of this cell line with a murine Xylt2 minigene results in the production of recombinant chondroitin sulfate-modified biglycan core protein and restoration of fibroblast growth factor binding to cell surface-associated heparan sulfate. Expression analyses on 10 different human transformed cell lines detect exclusive XYLT2 expression in two and co-expression of XYLT1 and XYLT2 in the others but at disparate ratios where XYLT2 expression is greater than XYLT1 in most cell lines. These results indicate that XylT2 has a significant role in proteoglycan biosynthesis and that cell type may control which family member is utilized.

Complex control of mouse apolipoprotein B gene expression revealed by targeted duplication.

An elevated plasma level of apolipoprotein B-containing lipoproteins is a risk factor for atherosclerotic cardiovascular disease. Subtle genetic abnormalities in gene expression including an increased expression of the APOB gene may play an important role in determining overall risk. In an attempt to increase mouse Apob expression, we used gene targeting and duplicated approximately 65 kb of genomic DNA containing the Apob locus in its natural genomic position in mice. While we successfully generated mice carrying the Apob gene duplication, the amount of the total Apob mRNA was not increased in their liver. In the intestine, total Apob mRNA was reduced to half of the wild-type mice. Plasma lipids in the Apob duplication mice were not altered. Expression analyses showed that the proximal Apob gene in the duplicated locus was preferentially expressed in both tissues suggesting a limitation of tissue-specific enhancer function. The previously characterized distant intestinal control element was not duplicated, explaining the unequal ratio of intestinal Apob expression. While the existence of an additional liver-specific enhancer element is unknown, our findings suggest the presence of an additional enhancer outside the duplicated region, and that Apob gene expression is more complicated than previously thought.

ApoB-48 and apoB-100 differentially influence the expression of type-III hyperlipoproteinemia in APOE*2 mice.

Apolipoprotein E (apoE) is essential for the clearance of plasma chylomicron and VLDL remnants. The human APOE locus is polymorphic and 5-10% of APOE*2 homozygotes exhibit type-III hyperlipoproteinemia (THL), while the remaining homozygotes have less than normal plasma cholesterol. In contrast, mice expressing APOE*2 in place of the mouse Apoe (Apoe(2/2) mice) are markedly hyperlipoproteinemic, suggesting a species difference in lipid metabolism (e.g., editing of apolipoprotein B) enhances THL development. Since apoB-100 has an LDLR binding site absent in apoB-48, we hypothesized that the Apoe(2/2) THL phenotype would improve if all Apoe(2/2) VLDL contained apoB-100. To test this, we crossed Apoe(2/2) mice with mice lacking the editing enzyme for apoB (Apobec(-/-)). Consistent with an increase in remnant clearance, Apoe(2/2). Apobec(-/-) mice have a significant reduction in IDL/LDL cholesterol (IDL/LDL-C) compared with Apoe(2/2) mice. However, Apoe(2/2).Apobec(-/-) mice have twice as much VLDL triglyceride as Apoe(2/2) mice. In vitro tests show the apoB-100-containing VLDL are poorer substrates for lipoprotein lipase than apoB-48-containing VLDL. Thus, despite a lowering in IDL/LDL-C, substituting apoB-48 lipoproteins with apoB-100 lipoproteins did not improve the THL phenotype in the Apoe(2/2).Apobec(-/-) mice, because apoB-48 and apoB-100 differentially influence the catabolism of lipoproteins.