Arf GTPase-activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor.

Arf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), which are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs; however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport.

Burkholderia pseudomallei-loaded cells act as a Trojan horse to invade the brain during endotoxemia.

Neurologic melioidosis occurs in both human and animals; however, the mechanism by which the pathogen Burkholderia pseudomallei invades the central nervous system (CNS) remains unclear. B. pseudomallei-loaded Ly6C cells have been suggested as a putative portal; however, during melioidosis, lipopolysaccharide (LPS) can drive disruption of the blood-brain barrier (BBB). This study aims to test whether the Trojan horse-like mechanism occurs during endotoxemia. The expression levels of cerebral cytokines, chemokines and cell adhesion molecules; the activation of astrocytes, microglia and endothelial cells; and the increased vascular permeability and brain-infiltrating leukocytes were evaluated using B. pseudomallei, B. thailandensis, B. cenocepacia and B. multivorans LPS-induced brains. Accordingly, different degrees of BBB damage in those brains with endotoxemia were established. The B. multivorans LPS-induced brain exhibited the highest levels of disruptive BBB according to the above mediators/indicators. Into these distinct groups of endotoxemic mice, B. pseudomallei-loaded Ly6C cells or free B. pseudomallei were adoptively transferred at equal bacterial concentrations (103 CFU). The bacterial load and number of cases of meningeal neutrophil infiltration in the brains of animals treated with B. pseudomallei-loaded Ly6C cells were higher than those in brains induced by free B. pseudomallei in any of the endotoxemic groups. In particular, these results were reproducible in B. multivorans LPS-induced brains. We suggest that B. pseudomallei-loaded cells can act as a Trojan horse and are more effective than free B. pseudomallei in invading the CNS under septic or endotoxemic conditions even when there is a high degree of BBB disruption.

Expression of cerebral serotonin related to anxiety-like behaviors in C57BL/6 offspring induced by repeated subcutaneous prenatal exposure to low-dose lipopolysaccharide.

Prenatal exposure to lipopolysaccharide (LPS), which likely occurs due to infection or contact with environmental allergens during pregnancy, is a proposed risk factor that induces anxiety- and autism spectrum disorder-like behaviors in offspring. However, the molecular and behavioral changes in offspring after maternal immune activation have not been completely identified. We hypothesized that a subcutaneous injection of LPS in a pregnant mouse would induce changes in cerebral serotonin (5-HT) in parallel to the appearance of anxiety-like behaviors in the dam's offspring. After LPS injections (total, 100 µg/Kg), the time spent in the central region during the open field test and the number of times that the mice moved between the light and dark boxes and between the open and closed arms on the elevated plus maze test revealed anxiety-like behaviors in offspring at 5, 6 and 9 weeks of age. The mRNA expression levels of tph2 (5-HT synthesizing enzyme) and slc6a4 (5-HT transporter) were down-regulated in both adolescent (5 weeks of age) and adult (8 weeks of age) brains. Immunohistochemistry revealed that the numbers and sizes of tph2-expressing cells were notably decreased in the raphe nuclei of the midbrain of adults. Moreover, compared with controls (phosphate-buffered saline-treated offspring), the cerebral 5-HT concentration at adolescence and adulthood in LPS-induced offspring was significantly decreased. We concluded that maternal immune activation induced by exposure to a low dose of LPS decreased cerebral 5-HT levels in parallel to the down-regulation of the tph2 and slc6a4 genes and in conjunction with anxiety-like behaviors in offspring.

Involvement of L-selectin expression in Burkholderia pseudomallei-infected monocytes invading the brain during murine melioidosis.

The development of neurologic melioidosis was linked to the elicitation of Burkholderia pseudomallei-infected L-selectinhiCD11b+ BALB/c cells in our previous study. However, whether monocytic L-selectin (CD62L, encoded by the sell gene) is a key factor remains uncertain. In the present study, after establishing multi-organ foci via hematogenous routes, we demonstrated that B. pseudomallei GFP steadily persisted in blood, splenic, hepatic and bone marrow (BM) Ly6C monocytes; however, the circulating CD16/32+CD45hiGFP+ brain-infiltrating leukocytes (BILs) derived from the blood Ly6C monocytes were expanded in BALB/c but not in C57BL/6 bacteremic melioidosis. Consistent with these results, 60% of BALB/c mice but only 10% of C57BL/6 mice exhibited neurologic melioidosis. In a time-dependent manner, B. pseudomallei invaded C57BL/6 BM-derived phagocytes and monocytic progenitors by 2 d. The number of Ly6C+CD62L+GFP+ inflamed cells that had expanded in the BM and that were ready for emigration peaked on d 21 post-infection. Hematogenous B. pseudomallei-loaded sell+/+Ly6C monocytes exacerbated the bacterial loads and the proportion of Ly6C+GFP+ BILs in the recipient brains compared to sell-/- infected Ly6C cells when adoptively transferred. Moreover, a neutralizing anti-CD62L antibody significantly depleted the bacterial colonization of the brain following adoptive transfer of B. pseudomallei-loaded C57BL/6 or BALB/c Ly6C cells. Our data thus suggest that Ly6C+CD62L+ infected monocytes served as a Trojan horse across the cerebral endothelium to induce brain infection. Therefore, CD62L should be considered as not only a temporally elicited antigen but also a disease-relevant leukocyte marker during the development of neurologic melioidosis.

A comparison of the immunological potency of Burkholderia lipopolysaccharides in endotoxemic BALB/c mice.

Lipopolysaccharide is one of the virulence factors of the soil-borne pathogens Burkholderia pseudomallei, B. thailandensis, B. cenocepacia and B. multivorans, which cause septic melioidosis (often in B. pseudomallei infections but rarely in B. thailandensis infections) or cepacia syndromes (commonly in B. cenocepacia infections but rarely in B. multivorans infections). The inflammatory responses in Burkholderia LPS-induced endotoxemia were evaluated in this study. Prior to induction, the conserved structures and functions of each purified LPS were determined using electrophoretic phenotypes, the ratios of 3-hydroxytetradecanoic to 3-hydroxyhexadecanoic acid and endotoxin units. In an in vitro assay, cytokine expression of myeloid differentiation primary response gene 88 and Toll/IL-1 receptor domain containing adapter-inducing INF-ß-dependent signaling-dependent signaling differed when stimulated by different LPS. Endotoxemia was induced in mice by s.c. injection as evidenced by increasing serum concentrations of 3-hydroxytetradecanoic acid and the septic prognostic markers CD62E and ICAM-1. During endotoxemia, splenic CD11b+ I-A+ , CD11b+ CD80+ , CD11b+ CD86+ and CD11b+ CD11c+ subpopulations increased. After induction with B. pseudomallei LPS, there were significant increases in splenic CD49b NK cells and CD14 macrophages. The inflamed CD11b+ CCR2+ , CD11b+ CD31+ , CD11b+ CD14+ , resident CD11b+ CX3 CR1+ and progenitor CD11b+ CD34+ cells showed delayed increases in bone marrow. B. multivorans LPS was the most potent inducer of serum cytokines and chemokines, whereas B. cenocepacia LPS induced relatively low concentrations of the chemokines MIP-1α and MIP-1ß. Endotoxin activities did not correlate with the virulence of Burkholderia strains. Thus factors other than LPS and/or other mechanisms of low activity LPS must mediate the pathogenicity of highly virulent Burkholderia strains.

Two Genome Sequences of Klebsiella pneumoniae Strains with Sequence Type 23 and Capsular Serotype K1.

Here, we report the whole-genome sequences of Klebsiella pneumoniae ED2 and ED23, isolated, respectively, from bacteremic patients with liver abscesses (ED2) and patients with primary liver abscess and metastatic meningitis (ED23). Both strains were of multilocus sequence type 23 with capsule serotype K1.

The up-regulation of heme oxygenase-1 expression in human gingival fibroblasts stimulated with nicotine.

BACKGROUND: Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. Heme oxygenase-1 (HO-1) is known as a stress-inducible protein and functions as an antioxidant enzyme. There is limited information on the expression of HO-1 in smoking-associated periodontal disease. OBJECTIVES: The aim of the present study was to investigate the effects of nicotine on the expression of HO-1 protein in cultured human gingival fibroblasts in vitro and further to compare HO-1 expression in gingival tissues obtained from cigarette smokers and non-smokers in vivo. METHODS: Western blot assay was used to investigate the effects on human gingival fibroblasts exposed to nicotine. In addition, antioxidants catalase, superoxide dismutase (SOD), and N-acetyl-l-cysteine (NAC) were added to test how they modulated the effects on nicotine-induced HO-1 expression. Gingival biopsies taken from the flap surgery of 20 male patients with periodontal disease (10 cigarette smokers and 10 non-smokers) were examined by immunohistochemistry. RESULTS: The exposure of quiescent human gingival fibroblasts to 10 mm nicotine resulted in the induction of HO-1 protein expression in a time-dependent manner (p < 0.05). The addition of glutathione (GSH) precursor NAC inhibited the nicotine-induced HO-1 protein expression (p < 0.05). However, SOD and catalase did not decrease the nicotine-induced HO-1 protein expression (p > 0.05). The results from immunohistochemistry demonstrated that HO-1 expression was significantly higher in cigarette smokers (p < 0.05). HO-1 was noted in the basal layers of epithelium, inflammatory cells, and fibroblasts in specimens from cigarette smoking. CONCLUSIONS: Taken together, these results suggest that HO-1 expression is significantly up-regulated in gingival tissues from cigarette smokers, and nicotine may, among other constituents, be responsible for the enhanced HO-1 expression in vivo. The regulation of HO-1 expression induced by nicotine is critically dependent on the intracellular GSH concentration.