Brucella Cyclic β-1,2-Glucan Plays a Critical Role in the Induction of Splenomegaly in Mice

Brucella, the etiological agent of animal and human brucellosis, is a bacterium with the capacity to modulate the inflammatory response. Cyclic β-1,2-glucan (CβG) is a virulence factor key for the pathogenesis of Brucella as it is involved in the intracellular life cycle of the bacteria. Using comparative studies with different CβG mutants of Brucella, cgs (CβG synthase), cgt (CβG transporter) and cgm (CβG modifier), we have identified different roles for this polysaccharide in Brucella. While anionic CβG is required for bacterial growth in low osmolarity conditions, the sole requirement for a successful Brucella interaction with mammalian host is its transport to periplasmic space. Our results uncover a new role for CβG in promoting splenomegaly in mice. We showed that CβG-dependent spleen inflammation is the consequence of massive cell recruitment (monocytes, dendritics cells and neutrophils) due to the induction of pro-inflammatory cytokines such as IL-12 and TNF-α and also that the reduced splenomegaly response observed with the cgs mutant is not the consequence of changes in expression levels of the characterized Brucella PAMPs LPS, flagellin or OMP16/19. Complementation of cgs mutant with purified CβG increased significantly spleen inflammation response suggesting a direct role for this polysaccharide.     

Helix α4 of the Bacillus thuringiensis Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation

Helix α4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins'' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix α4 amino acid residues have previously been shown to cause substantial reductions in the protein''s pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix α4 is involved mainly in the postbinding steps of pore formation.During the last few decades, the insecticidal toxins produced by Bacillus thuringiensis have been used increasingly in the forms of formulated sprays and transgenic plants for the highly focused biological control of insect pests (29). At the same time, the mechanism by which these proteins form pores in the apical membrane of midgut epithelial cells of targeted insects has been studied extensively (7, 29). In the case of the three-domain Cry toxins, specificity is mostly attributable to their capacity to bind to certain proteins located on the surface of the intestinal membrane through specific segments of domains II and III, composed mainly of β sheets (16, 27). On the other hand, membrane insertion and pore formation are thought to occur through elements of domain I, composed of a bundle of six amphipathic α-helices surrounding the highly hydrophobic helix α5 (17, 20).Several lines of evidence indicate that helices α4 and α5 play a particularly important role in these processes (3). Spectroscopic studies with synthetic peptides corresponding to domain I helices revealed that α4 and α5 have the greatest propensity for insertion into artificial membranes, although insertion and pore formation were most efficient when α4 and α5 were connected by a segment corresponding to the α4-α5 loop of the toxin (13, 14). A particularly large number of single-site mutations with altered amino acids from these helices, which lead to a strong reduction in the toxicity and pore-forming ability of the toxin, have been characterized (2, 9, 10, 15, 18, 23, 25, 30, 31, 33). Finally, a site-directed chemical modification study has provided strong evidence that α4 lines the lumens of the pores formed by the toxin (23).Recent studies have established that toxin activity is especially sensitive to modifications not only in the charged residues of α4 (31) but in most of its hydrophilic residues (15). Furthermore, the loss of activity of most of these mutants did not result from an altered selectivity or size of the pores but from a reduced pore-forming capacity of the toxin (15, 31). In order to better understand the role of α4 in the mechanism of pore formation, the present study was carried out with a series of previously characterized Cry1Aa mutants in which most of the residues from this helix were replaced by cysteines (15). By subjecting these mutants to circular dichroism (CD), protease sensitivity, pore formation inhibition, and electrophoretic mobility analyses, our data suggest that the mutations in α4 which alter the pore-forming ability of Cry1Aa do so mainly by preventing the proper oligomerization or membrane insertion of the toxin.     

Receptor-Associated Protein (RAP) Plays a Central Role in Modulating Aβ Deposition in APP/PS1 Transgenic Mice

Background

Receptor associated protein (RAP) functions in the endoplasmic reticulum (ER) to assist in the maturation of several membrane receptor proteins, including low density lipoprotein receptor-related protein (LRP) and lipoprotein receptor 11 (SorLA/LR11). Previous studies in cell and mouse model systems have demonstrated that these proteins play roles in the metabolism of the amyloid precursor protein (APP), including processes involved in the generation, catabolism and deposition of β-amyloid (Aβ) peptides.

Methodology/Principal Findings

Mice transgenic for mutant APPswe and mutant presenilin 1 (PS1dE9) were mated to mice with homozygous deletion of RAP. Unexpectedly, mice that were homozygous null for RAP and transgenic for APPswe/PS1dE9 showed high post-natal mortality, necessitating a shift in focus to examine the levels of amyloid deposition in APPswe/PS1dE9 that were hemizygous null for RAP. Immunoblot analysis confirmed 50% reductions in the levels of RAP with modest reductions in the levels of proteins dependent upon RAP for maturation [LRP trend towards a 20% reduction ; SorLA/LR11 statistically significant 15% reduction (p<0.05)]. Changes in the levels of these proteins in the brains of [APPswe/PS1dE9](+/−)/RAP(+/−) mice correlated with 30–40% increases in amyloid deposition by 9 months of age.

Conclusions/Significance

Partial reductions in the ER chaperone RAP enhance amyloid deposition in the APPswe/PS1dE9 model of Alzheimer amyloidosis. Partial reductions in RAP also affect the maturation of LRP and SorLA/LR11, which are each involved in several different aspects of APP processing and Aβ catabolism. Together, these findings suggest a central role for RAP in Alzheimer amyloidogenesis.     

A Critical Role of the mTOR/eIF2α Pathway in Hypoxia-Induced Pulmonary Hypertension

Enhanced proliferation of pulmonary arterial vascular smooth muscle cells (PASMCs) is a key pathological component of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Mammalian targeting of rapamycin (mTOR) signaling has been shown to play a role in protein translation and participate in the progression of pulmonary hypertension. Eukaryotic translation initiation factor-2α (eIF2α) is a key factor in regulation of cell growth and cell cycle, but its role in mTOR signaling and PASMCs proliferation remains unknown. Pulmonary hypertension (PH) rat model was established by hypoxia. Rapamycin was used to treat rats as an mTOR inhibitor. Proliferation of primarily cultured rat PASMCs was induced by hypoxia, rapamycin and siRNA of mTOR and eIF2α were used in loss-of-function studies. The expression and activation of eIF2α, mTOR and c-myc were analyzed. Results showed that mTOR/eIF2α signaling was significantly activated in pulmonary arteries from hypoxia exposed rats and PASMCs cultured under hypoxia condition. Treatment with mTOR inhibitor for 21 days attenuated vascular remodeling, suppressed mTOR and eIF2α activation, inhibited c-myc expression in HPH rats. In hypoxia-induced PASMCs, rapamycin and knockdown of mTOR and eIF2α by siRNA significantly abolished proliferation and increased c-myc expression. These results suggest a critical role of the mTOR/eIF2αpathway in hypoxic vascular remodeling and PASMCs proliferation of HPH.     

A Critical Role for PDGFRα Signaling in Medial Nasal Process Development

The primitive face is composed of neural crest cell (NCC) derived prominences. The medial nasal processes (MNP) give rise to the upper lip and vomeronasal organ, and are essential for normal craniofacial development, but the mechanism of MNP development remains largely unknown. PDGFRα signaling is known to be critical for NCC development and craniofacial morphogenesis. In this study, we show that PDGFRα is required for MNP development by maintaining the migration of progenitor neural crest cells (NCCs) and the proliferation of MNP cells. Further investigations reveal that PI3K/Akt and Rac1 signaling mediate PDGFRα function during MNP development. We thus establish PDGFRα as a novel regulator of MNP development and elucidate the roles of its downstream signaling pathways at cellular and molecular levels.     

TAK1-ECSIT-TRAF6 Complex Plays a Key Role in the TLR4 Signal to Activate NF-κB

ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) is known as a multifunctional regulator in different signals, including Toll-like receptors (TLRs), TGF-β, and BMP. Here, we report a new regulatory role of ECSIT in TLR4-mediated signal. By LPS stimulation, ECSIT formed a high molecular endogenous complex including TAK1 and TRAF6, in which ECSIT interacted with each protein and regulated TAK1 activity, leading to the activation of NF-κB. ECSIT-knockdown THP-1 (ECSIT^KD THP-1) cells exhibited severe impairments in NF-κB activity, cytokine production, and NF-κB-dependent gene expression, whereas those were dramatically restored by reintroduction of wild type (WT) ECSIT gene. Interestingly, ECSIT mutants, which lack a specific interacting domain for either TAK1 or TRAF6, could not restore these activities. Moreover, no significant changes in both NF-κB activity and cytokine production induced by TLR4 could be seen in TAK1^KD or TRAF6^KD THP-1 cells transduced by WT ECSIT, strongly suggesting the essential requirement of TAK1-ECSIT-TRAF6 complex in TLR4 signaling. Taken together, our data demonstrate that the ECSIT complex, including TAK1 and TRAF6, plays a pivotal role in TLR4-mediated signals to activate NF-κB.     

Differential roles of Sirt1 in HIF-1α and HIF-2α mediated hypoxic responses

Hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) determine cancer cell fate under hypoxia. Despite the similarities of their structures, HIF-1α and HIF-2α have distinct roles in cancer growth under hypoxia, that is, HIF-1α induces growth arrest whereas HIF-2α promotes cell growth. Recently, sirtuin 1 (Sirt1) was reported to fine-tune cellular responses to hypoxia by deacetylating HIF-1α and HIF-2α. Yet, the roles of Sirt1 in HIF-1α and HIF-2α functions have been controversial. We here investigated the precise roles of Sirt1 in HIF-1α and HIF-2α regulations. Immunological analyses revealed that HIF-1α K674 and HIF-2α K741 are acetylated by PCAF and CBP, respectively, but are deacetylated commonly by Sirt1. In the Gal4 reporter systems, Sirt1 was found to repress HIF-1α activity constantly in ten cancer cell-lines but to regulate HIF-2α activity cell type-dependently. Moreover, Sirt1 determined cell growth under hypoxia depending on HIF-1α and HIF-2α. Under hypoxia, Sirt1 promoted cell proliferation of HepG2, in which Sirt1 differentially regulates HIF-1α and HIF-2α. In contrast, such an effect of Sirt1 was not shown in HCT116, in which Sirt1 inactivates both HIF-1α and HIF-2α because conflicting actions of HIF-1α and HIF-2α on cell growth may be offset. Our results provide a better understanding of the roles of Sirt1 in HIF-mediated hypoxic responses and also a basic concept for developing anticancer strategy targeting Sirt1.     

Critical Role of Toll-Like Receptor 9 in Morphine and Mycobacterium tuberculosis–Induced Apoptosis in Mice

Background

Although it is established that opioid and Mycobacterium tuberculosis are both public health problems, the mechanisms by which they affect lung functions remain elusive.

Methodology/Principal Findings

We report here that mice subjected to chronic morphine administration and M. tuberculosis infection exhibited significant apoptosis in the lung in wild type mice as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Morphine and M. tuberculosis significantly induced the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, deficiency in TLR9 significantly inhibited the morphine and M. tuberculosis induced apoptosis in the lung. In addition, chronic morphine treatment and M. tuberculosis infection enhanced the levels of cytokines (TNF-α, IL-1β, and IL-6) in wild type mice, but not in TLR9 knockout (KO) mice. The bacterial load was much lower in TLR9 KO mice compared with that in wild type mice following morphine and M. tuberculosis treatment. Morphine alone did not alter the bacterial load in either wild type or TLR9 KO mice. Moreover, administration of morphine and M. tuberculosis decreased the levels of phosphorylation of Akt and GSK3β in the wild type mice, but not in TLR9 KO mice, suggesting an involvement of Akt/GSK3β in morphine and M. tuberculosis-mediated TLR9 signaling. Furthermore, administration of morphine and M. tuberculosis caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type mice, but not in TLR9 KO mice, indicating a role of Bcl-2 family in TLR9-mediated apoptosis in the lung following morphine and M. tuberculosis administration.

Conclusions/Significance

These data reveal a role for TLR9 in the immune response to opioids during M. tuberculosis infection.     

PLC-γ1 Signaling Plays a Subtype-Specific Role in Postbinding Cell Entry of Influenza A Virus

Host signaling pathways and cellular proteins play important roles in the influenza viral life cycle and can serve as antiviral targets. In this study, we report the engagement of host phosphoinositide-specific phospholipase γ1 (PLC-γ1) in mediating cell entry of influenza virus H1N1 but not H3N2 subtype. Both PLC-γ1-specific inhibitor and short hairpin RNA (shRNA) strongly suppress the replication of H1N1 but not H3N2 viruses in cell culture, suggesting that PLC-γ1 plays an important subtype-specific role in the influenza viral life cycle. Further analyses demonstrate that PLC-γ1 activation is required for viral postbinding cell entry. In addition, H1N1, but not H3N2, infection leads to the phosphorylation of PLC-γ1 at Ser 1248 immediately after infection and independent of viral replication. We have further shown that H1N1-induced PLC-γ1 activation is downstream of epidermal growth factor receptor (EGFR) signaling. Interestingly, both H1N1 and H3N2 infections activate EGFR, but only H1N1 infection leads to PLC-γ1 activation. Taking our findings together, we have identified for the first time the subtype-specific interplay of host PLC-γ1 signaling and H1N1 virus that is critical for viral uptake early in the infection. Our study provides novel insights into how virus interacts with the cellular signaling network by demonstrating that viral determinants can regulate how the host signaling pathways function in virally infected cells.     

HIF-1α protein is upregulated in HIF-2α depleted cells via enhanced translation

The hypoxia-inducible factors HIF-1 and HIF-2 are primarily regulated via stabilization of their respective α-subunits under hypoxic conditions. Previously, compensatory upregulation of one HIF-α-subunit upon depletion of the other α-subunit was described, yet the underlying mechanism remained elusive. Here we provide evidence that enhanced HIF-1α protein expression in HIF-2α knockdown (k/d) cells neither results from elevated HIF-1α mRNA expression, nor from increased HIF-1α protein stability. Instead, we identify enhanced HIF-1α translation as molecular mechanism. Moreover, we found elevated levels of the RNA-binding protein HuR and provide evidence that HuR is critical for the compensatory HIF-1α regulation in HIF-2α k/d cells.     

A Specific PfEMP1 Is Expressed in P. falciparum Sporozoites and Plays a Role in Hepatocyte Infection

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HIF-1α Inhibition Reduces Nasal Inflammation in a Murine Allergic Rhinitis Model

Background

Hypoxia-inducible factor 1α (HIF-1α) is an important regulator of immune and inflammatory responses. We hypothesized that nasal allergic inflammation is attenuated by HIF-1α inhibition and strengthened by HIF-1α stabilization.

Objective

To elucidate the role of HIF-1α in a murine model of allergic rhinitis (AR).

Methods

Mice were pretreated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2) or the HIF-1α inducer cobalt chloride (CoCl₂) in an established AR murine model using ovalbumin (OVA)-sensitized BALB/c mice. HIF-1α and vascular endothelial growth factor (VEGF) expression in nasal mucosa was measured and multiple parameters of allergic responses were evaluated.

Results

HIF-1α and VEGF levels were locally up-regulated in nasal mucosa during AR. Inflammatory responses to OVA challenge, including nasal symptoms, inflammatory cell infiltration, eosinophil recruitment, up-regulation of T-helper type 2 cytokines in nasal lavage fluid, and serum OVA-specific IgE levels were present in the OVA-challenged mice. 2ME2 effectively inhibited HIF-1α and VEGF expression and attenuated the inflammatory responses. Stabilization of HIF-1α by CoCl₂ facilitated nasal allergic inflammation. HIF-1α protein levels in nasal airways correlated with the severity of AR in mice.

Conclusions

HIF-1α is intimately involved in the pathogenesis of nasal allergies, and the inhibition of HIF-1α may be useful as a novel therapeutic approach for AR.