Dual TLR2/9 Recognition of Herpes Simplex Virus Infection Is Required for Recruitment and Activation of Monocytes and NK Cells and Restriction of Viral Dissemination to the Central Nervous System.

The importance of TLR2 and TLR9 in the recognition of infection with herpes simplex virus (HSV) and HSV-caused diseases has been described, but some discrepancies remain concerning the benefits of these responses. Moreover, the impact of TLR2/9 on innate and adaptive immune responses within relevant mucosal tissues has not been elucidated using natural mucosal infection model of HSV. Here, we demonstrate that dual TLR2/9 recognition is essential to provide resistance against mucosal infection with HSV via an intravaginal route. Dual TLR2/9 ablation resulted in the highly enhanced mortality with exacerbated symptoms of encephalitis compared with TLR2 or TLR9 deficiency alone, coinciding with highly increased viral load in central nervous system tissues. TLR2 appeared to play a minor role in providing resistance against mucosal infection with HSV, since TLR2-ablated mice showed higher survival rate compared with TLR9-ablated mice. Also, the high mortality in dual TLR2/9-ablated mice was closely associated with the reduction in early monocyte and NK cell infiltration in the vaginal tract (VT), which was likely to correlate with low expression of cytokines and CCR2 ligands (CCL2 and CCL7). More interestingly, our data revealed that dual TLR2/9 recognition of HSV infection plays an important role in the functional maturation of TNF-α and iNOS-producing dendritic cells (Tip-DCs) from monocytes as well as NK cell activation in VT. TLR2/9-dependent maturation of Tip-DCs from monocytes appeared to specifically present cognate Ag, which effectively provided functional effector CD4+ and CD8+ T cells specific for HSV Ag in VT and its draining lymph nodes. TLR2/9 expressed in monocytes was likely to directly facilitate Tip-DC-like features after HSV infection. Also, dual TLR2/9 recognition of HSV infection directly activated NK cells without the aid of dendritic cells through activation of p38 MAPK pathway. Taken together, these results indicate that dual TLR2/9 recognition plays a critical role in providing resistance against mucosal infection with HSV, which may involve a direct regulation of Tip-DCs and NK cells in VT. Therefore, our data provide a more detailed understanding of TLR2/9 role in conferring antiviral immunity within relevant mucosal tissues after mucosal infection with HSV.

TGF-ß signaling in endothelial cells, but not neuroepithelial cells, is essential for cerebral vascular development.

The various organs of the body harbor blood vessel networks that display unique structural and functional features; however, the mechanisms that control organ-specific vascular development and physiology remain mostly unknown. In the developing mouse brain, αvß8 integrin-mediated TGF-ß activation and signaling is essential for normal blood vessel growth and sprouting. Whether integrins activate TGF-ß signaling pathways in vascular endothelial cells (ECs), neural cells, or both, has yet to be determined. Here, we have generated and characterized mice in which TGF-ß receptors are specifically deleted in neuroepithelial cells via Nestin-Cre, or in ECs via a novel Cre transgenic strain (Alk1(GFPCre)) in which Cre is expressed under control of the endogenous activin receptor-like kinase 1 (Alk1) promoter. We report that deletion of Tgfbr2 in the neuroepithelium does not impact brain vascular development. In contrast, selective deletion of the Tgfbr2 or Alk5 genes in ECs result in embryonic lethality because of brain-specific vascular pathologies, including blood vessel morphogenesis and intracerebral hemorrhage. These data reveal for the first time that αvß8 integrin-activated TGF-ßs regulate angiogenesis in the developing brain via paracrine signaling to ECs.

Real-time imaging of de novo arteriovenous malformation in a mouse model of hereditary hemorrhagic telangiectasia.

Arteriovenous malformations (AVMs) are vascular anomalies where arteries and veins are directly connected through a complex, tangled web of abnormal arteries and veins instead of a normal capillary network. AVMs in the brain, lung, and visceral organs, including the liver and gastrointestinal tract, result in considerable morbidity and mortality. AVMs are the underlying cause of three major clinical symptoms of a genetic vascular dysplasia termed hereditary hemorrhagic telangiectasia (HHT), which is characterized by recurrent nosebleeds, mucocutaneous telangiectases, and visceral AVMs and caused by mutations in one of several genes, including activin receptor-like kinase 1 (ALK1). It remains unknown why and how selective blood vessels form AVMs, and there have been technical limitations to observing the initial stages of AVM formation. Here we present in vivo evidence that physiological or environmental factors such as wounds in addition to the genetic ablation are required for Alk1-deficient vessels to develop to AVMs in adult mice. Using the dorsal skinfold window chamber system, we have demonstrated for what we believe to be the first time the entire course of AVM formation in subdermal blood vessels by using intravital bright-field images, hyperspectral imaging, fluorescence recordings of direct arterial flow through the AV shunts, and vascular casting techniques. We believe our data provide novel insights into the pathogenetic mechanisms of HHT and potential therapeutic approaches.

Genetic ablation of the BMPR2 gene in pulmonary endothelium is sufficient to predispose to pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare but fatal lung disease of diverse origins. PAH is now further subclassified as idiopathic PAH, familial PAH, and associated PAH varieties. Heterozygous mutations in BMPR2 can be detected in 50% to 70% of patients with familial PAH and 10% to 40% of patients with idiopathic PAH. Although endothelial cells have been suspected as the cellular origin of PAH pathogenesis, no direct in vivo evidence has been clearly presented. The present study was designed to investigate whether endothelial Bmpr2 deletion can predispose to PAH. METHODS AND RESULTS: The Bmpr2 gene was deleted in pulmonary endothelial cells using Bmpr2 conditional knockout mice and a novel endothelial Cre transgenic mouse line. Wide ranges of right ventricular systolic pressure were observed in mice with heterozygous (21.7 to 44.1 mm Hg; median, 23.7 mm Hg) and homozygous (20.7 to 56.3 mm Hg; median, 27 mm Hg) conditional deletion of Bmpr2 in pulmonary endothelial cells compared with control mice (19.9 to 26.7 mm Hg; median, 23 mm Hg) at 2 to 7 months of age. A subset of mice with right ventricular systolic pressure >30 mm Hg exhibited right ventricular hypertrophy and an increase in the number and wall thickness of muscularized distal pulmonary arteries. In the lungs of these mice with high right ventricular systolic pressure, the expression of proteins involved in the pathogenesis of PAH such as serotonin transporter and tenascin-C was elevated in distal arteries and had a high incidence of perivascular leukocyte infiltration and in situ thrombosis. CONCLUSIONS: Conditional heterozygous or homozygous Bmpr2 deletion in pulmonary endothelial cells predisposes mice to develop PAH.

The effect of essential oils of dietary wormwood (Artemisia princeps), with and without added vitamin E, on oxidative stress and some genes involved in cholesterol metabolism.

Wormwood (Artemisia princeps) due to the abundance of antioxidant in its essential oils (EO), has been used as a traditional drug and health food in Korea. Oxidative stress plays an important role in the etiology of atherosclerosis thus antioxidative chemicals improves hepatic lipid metabolism partly by reducing oxysterol formation. The antioxidant activity was assessed using two methods, human low-density lipoprotein (LDL) oxidation and the anti-DPPH free radical assays. It was found that the antioxidant activity of EO with vitamin E higher than EO alone. To study mechanisms accounting for the antiatherosclerotic properties of this wormwood EO, we examined the expression of key genes in cholesterol metabolism such as the LDL receptor, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and sterol regulatory element binding proteins. The induction was increased up to twofold at 0.05 mg/mL of EO treatment in HepG2 cells for 24h. When EO (0.2 mg/mL) was co-incubated with vitamin E, interestingly, the LDL receptor was dramatically induced by 5-6-folds. HMG-CoA reductase did not change. However, treatment with the higher concentration resulted in cytotoxicity. Our data suggest that wormwood EO with vitamin E may be anti-atherogenic due to their inhibition of LDL oxidation and upregulation of the LDL receptor.

The PRETTY FEW SEEDS2 gene encodes an Arabidopsis homeodomain protein that regulates ovule development.

The PRETTY FEW SEEDS2 gene encodes a homeodomain protein that regulates ovule development. In peptide alignments spanning the homeodomain and the WOX domain, PFS2 shared 95% amino acid identity with the PRESSED FLOWER and WUSCHEL proteins. In the pfs2-1 allele, the integuments display morphological abnormalities and 95% of the embryo sacs fail to develop properly, which results in reduced fecundity. PFS2 transcripts were most abundant in developing ovules, which accounts for the ovule phenotype in pfs2 mutants. In addition, PFS2 transcripts were present in developing primordia and differentiating organs, but, interestingly, they were absent during cell maturation. Ectopic PFS2 expression interfered with differentiation of primordia from meristems. For most plants, this resulted in fasciated stems, altered phyllotaxy, a cessation of primordia differentiation, or a combination of these. In the plants that made ovules, ectopic PFS2 expression blocked megaspore mother cell differentiation and often impeded polarized growth of the outer integument. PFS2 activity altered AGAMOUS expression, which accounts for some of the gain- and loss-of-function phenotypes. Based on analyses presented here, PFS2 affects either ovule patterning or differentiation.

The phenotype of Arabidopsis ovule mutants mimics the morphology of primitive seed plants.

In seed plants, the ovule is the female reproductive structure, which surrounds and nourishes the gametophyte and embryo. This investigation describes the PRETTY FEW SEEDS2 (PFS2) locus, which regulates ovule patterning. The pfs2 mutant exhibited developmental defects in the maternal integuments and gametophyte. This mutation was inherited as a maternal trait, indicating that gametophyte defects resulted from ovule patterning aberrations. Specifically, the boundary between the chalaza and the nucellus, two regions of the ovule primordia, shifted towards the distal end of pfs2 ovule primordia. Results indicated that the PFS2 locus could: (i) be involved in the development of either the nucellus or the chalaza; or (ii) establish a boundary between these two regions. Examination of genetic interactions of the pfs2 mutation with other well-characterized ovule loci indicates that this locus affects integument morphogenesis. Interestingly, the pfs2 inner no outer and pfs2 strubbelig double mutants had inner integuments that appeared similar to their ancestral precursor. The fossil record indicates that the inner integument evolved by fusion of sterilized sporangia or branches around a central megasporangium. The question of whether the structures observed in these double mutants are homologous or merely analogous to the ancestral precursors of the inner integument is discussed.