Non-Hematopoietic MLKL Protects Against Salmonella Mucosal Infection by Enhancing Inflammasome Activation.

The intestinal mucosal barrier is critical for host defense against pathogens infection. Here, we demonstrate that the mixed lineage kinase-like protein (MLKL), a necroptosis effector, promotes intestinal epithelial barrier function by enhancing inflammasome activation. MLKL-/- mice were more susceptible to Salmonella infection compared with wild-type counterparts, with higher mortality rates, increased body weight loss, exacerbated intestinal inflammation, more bacterial colonization, and severe epithelial barrier disruption. MLKL deficiency promoted early epithelial colonization of Salmonella prior to developing apparent intestinal pathology. Active MLKL was predominantly expressed in crypt epithelial cells, and experiments using bone marrow chimeras found that the protective effects of MLKL were dependent on its expression in non-hematopoietic cells. Intestinal mucosa of MLKL-/- mice had impaired caspase-1 and gasdermin D cleavages and decreased interleukin (IL)-18 release. Moreover, administration of exogenous recombinant IL-18 rescued the phenotype of increased bacterial colonization in MLKL-/- mice. Thus, our results uncover the role of MLKL in enhancing inflammasome activation in intestinal epithelial cells to inhibit early bacterial colonization.

Decidual Stromal Cell Necroptosis Contributes to Polyinosinic-Polycytidylic Acid-Triggered Abnormal Murine Pregnancy.

Infectious agents can reach the placenta either via the maternal blood or by ascending the genito-urinary tract, and then initially colonizing the maternal decidua. Decidual stromal cells (DSCs) are the major cellular component of the decidua. Although DSCs at the maternal-fetal interface contribute to the regulation of immunity in pregnancy in the face of immunological and physiological challenges, the roles of these DSCs during viral infection remain ill defined. Here, we characterized the response of DSCs to a synthetic double-stranded RNA molecule, polyinosinic-polycytidylic acid [poly(I:C)], which is a mimic of viral infection. We demonstrated that both transfection of cells with poly(I:C) and addition of extracellular (non-transfected) poly(I:C) trigger the necroptosis of DSCs and that this response is dependent on RIG-I-like receptor/IPS-1 signaling and the toll-like receptor 3/TIR-domain-containing adapter-inducing interferon-ß pathway, respectively. Furthermore, following poly(I:C) challenge, pregnant mixed lineage kinase domain-like protein-deficient mice had fewer necrotic cells in the mesometrial decidual layer, as well as milder pathological changes in the uterine unit, than did wild-type mice. Collectively, our results establish that necroptosis is a contributing factor in poly(I:C)-triggered abnormal pregnancy and thereby indicate a novel therapeutic strategy for reducing the severity of the adverse effects of viral infections in pregnancy.

Cirtical role for Salmonella effector SopB in regulating inflammasome activation.

OBJECTIVE: Salmonella is known to evolve many mechanisms to avoid or delay inflammasome activation which remain largely unknown. In this study, we investigated whether the SopB protein critical to bacteria virulence capacity was an effector that involved in the regulation of inflammasome activation. METHODS: BMDMs from NLRC4-, NLRP3-, caspase-1/-11-, IFI16- and AIM2-deficient mice were pretreated with LPS, and subsequently stimulated with a series of SopB-related strains of Salmonella, inflammasome induced cell death, IL-1ß secretion, cleaved caspase-1 production and ASC speckle formation were detected. RESULTS: We found that SopB could inhibit host IL-1ß secretion, caspase-1 activation and inflammasome induced cell death using a series of SopB-related strains of Salmonella; however the reduction of IL-1ß secretion was not dependent on sensor that contain PYD domain, such as NLRP3, AIM2 or IFI16, but dependent on NLRC4. Notably, SopB specifically prevented ASC oligomerization and the enzymatic activity of SopB was responsible for the inflammasome inhibition. Furthermore, inhibition of Akt signaling induced enhanced inflammasome activation. CONCLUSIONS: These results revealed a novel role in inhibition of NLRC4 inflammasome for Salmonella effector SopB.

Genipin inhibits NLRP3 and NLRC4 inflammasome activation via autophagy suppression.

Inflammasomes are cytoplasmic, multiprotein complexes that trigger caspase-1 activation and IL-1ß maturation in response to diverse stimuli. Although inflammasomes play important roles in host defense against microbial infection, overactive inflammasomes are deleterious and lead to various autoinflammatory diseases. In the current study, we demonstrated that genipin inhibits the induction of IL-1ß production and caspase-1 activation by NLRP3 and NLRC4 inflammasomes. Furthermore, genipin specifically prevented NLRP3-mediated, but not NLRC4-mediated, ASC oligomerization. Notably, genipin inhibited autophagy, leading to NLRP3 and NLRC4 inflammasome inhibition. UCP2-ROS signaling may be involved in inflammasome suppression by genipin. In vivo, we showed that genipin inhibited NLRP3-dependent IL-1ß production and neutrophil flux in LPS- and alum-induced murine peritonitis. Additionally, genipin provided protection against flagellin-induced lung inflammation by reducing IL-1ß production and neutrophil recruitment. Collectively, our results revealed a novel role in inhibition of inflammatory diseases for genipin that has been used as therapeutics for centuries in herb medicine.

Cytosolic double-stranded DNA induces nonnecroptotic programmed cell death in trophoblasts via IFI16.

The mechanisms underlying the immune defense by trophoblasts against pathogens remain ill defined. We demonstrated that placental cell death was increased upon in vivo exposure to Listeria monocytogenes. The death of infected cells is an important host innate defense mechanism. Meanwhile, double-stranded DNA (dsDNA) derived from intracellular bacteria or dsDNA viruses is emerging as a potent pathogen-associated molecular pattern recognized by host cells. We sought to characterize trophoblast death in response to cytosolic dsDNA challenge. Our results showed that dsDNA induced caspase-dependent and -independent cell death in human trophoblasts. However, necroptosis, a cell death pathway independent of caspase, could not be induced by dsDNA treatment, even in the presence of exogenously expressed RIPK3. L. monocytogenes-derived genomic DNA triggered a similar cell death pattern. Moreover, the cell death in response to dsDNA was IFI16 dependent. These data suggest that cytosolic dsDNA induces nonnecroptotic cell death in trophoblasts via IFI16, and this could contribute to placental barrier against infection.

Liquiritigenin prevents Staphylococcus aureus-mediated lung cell injury via inhibiting the production of α-hemolysin.

Staphylococcus aureus is a significant Gram-positive bacterium that is associated with a broad spectrum of diseases ranging from minor skin infections to lethal pneumonia, endocarditis, and toxinoses. α-Hemolysin is one of the most important exotoxins that contribute to the pathogenesis of S. aureus infections. Liquiritigenin is one of the most significant active components in licorice. In this study, hemolysis, western blot, and real-time reverse transcription-PCR assays were performed to investigate the impact of liquiritigenin on the production of S. aureus α-hemolysin. The results showed that low concentrations of liquiritigenin remarkably decreased S. aureus α-hemolysin production in a dose-dependent manner. Using live/dead cell staining and lactate dehydrogenase assays, we found that liquiritigenin could protect human lung cells (A549) from α-hemolysin-mediated injury. The data indicated that this compound could potentially be useful in developing drugs aiming at staphylococcal α-hemolysin.

Inhibition of α-toxin production by subinhibitory concentrations of naringenin controls Staphylococcus aureus pneumonia.

Staphylococcal pneumonia provoked by methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening infection in which α-toxin is an essential virulence factor. In this study, we investigate the influence of naringenin on α-toxin production and further assess its therapeutic performance in the treatment of staphylococcal pneumonia. Remarkably, the expression of α-toxin was significantly inhibited when the organism was treated with 16 µg/ml of naringenin. When studied in a mouse model of S. aureus pneumonia, naringenin could attenuate the symptoms of lung injury and inflammation in infected mice. These results suggest that naringenin is a promising agent for treatment of S. aureus infection.

[Chemical constituents from the fruits of Kadsura marmorata].

OBJECTIVE: To study the chemical constituents from the fruits of Kadsura marmorata. METHODS: The chemical constituents were isolated and purified by chromatography on silica gel, Sephadex LH-20 column and HPLC. RESULTS: 9 compounds were isolated and identified as 4,5-dihydroxy-3-methoxybiphenyl (I), eriobofuran (II), 3beta, 16beta-dihydroxy urs-2-ene (III), 2alpha, 3beta, 6beta, 23-tetrahydroxy urs-12,18-dien-28-oic acid (IV), 2alpha,3beta,23-trihydroxy urs-12-en-28-oic acid (V), rutin (VI), 2-ethylhexanoic acid (VII), 2-monolaurin (VIII), glyceryl monoricinoleate (IX) on the basis of NMR and EI-MS spectroscopic analysis. CONCLUSION: All these compounds are isolated from this genus for the first time.

Glycyrrhetinic acid protects mice from Staphylococcus aureus pneumonia.

In the present study, the antimicrobial activity of glycyrrhetinic acid (GA) against Staphylococcus aureus, and its influence on the production of S. aureus alpha-haemolysin (Hla) were investigated, along with the in vivo activity of GA against S. aureus-induced pneumonia. GA could not inhibit the growth of S. aureus, but the secretion of Hla by S. aureus was significantly inhibited by low concentrations of GA in a dose-dependent manner. Furthermore, in vivo data show that GA provides protection against staphylococcal pneumonia in a murine model system.

Allicin reduces the production of α-toxin by Staphylococcus aureus.

Staphylococcus aureus causes a broad range of life-threatening diseases in humans. The pathogenicity of this micro-organism is largely dependent upon its virulence factors. One of the most extensively studied virulence factors is the extracellular protein α-toxin. In this study, we show that allicin, an organosulfur compound, was active against S. aureus with MICs ranged from 32 to 64 µg/mL. Haemolysis, Western blot and real-time RT-PCR assays were used to evaluate the effects of allicin on S. aureus α-toxin production and on the levels of gene expression, respectively. The results of our study indicated that sub-inhibitory concentrations of allicin decreased the production of α-toxin in both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in a dose-dependent manner. Furthermore, the transcriptional levels of agr (accessory gene regulator) in S. aureus were inhibited by allicin. Therefore, allicin may be useful in the treatment of α-toxin-producing S. aureus infections.

Immunomodulatory effects of cinobufagin isolated from Chan Su on activation and cytokines secretion of immunocyte in vitro.

The objective of this study was to evaluate the immunomodulatory effects of cinobufagin (CBG) isolated from Chan Su (Venenum Bufonis) in vitro. In this paper, our results show that CBG significantly stimulated cell proliferation of splenocytes and peritoneal macrophages (PMΦ) and markedly enhanced the phagocytic activation of PMΦ. CBG also significantly increased CD4(+)CD8(+) double-positive T-cell populations and the percentage of S-phase cells of splenic lymphocytes. The levels of several Th1 cytokines, including interferon-γ and tumor necrosis factor-α, are significantly increased after CBG treatment, whereas the levels of the Th2 cytokine interleukin-4 and interleukin-10 are significantly decreased. As a result, the ratio of Th1/Th2 also increased. Taken together, these results indicated that CBG had potential immune system regulatory effects and suggested that this compound could be developed as a novel immunotherapeutic agent to treat immune-mediated diseases such as cancer.

Peppermint oil decreases the production of virulence-associated exoproteins by Staphylococcus aureus.

The present study aimed to evaluate the antimicrobial activity of peppermint oil against Staphylococcus aureus, and further investigate the influence of peppermint oil on S. aureus virulence-related exoprotein production. The data show that peppermint oil, which contained high contents of menthone, isomenthone, neomenthol, menthol, and menthyl acetate, was active against S. aureus with minimal inhibitory concentrations (MICs) ranging from 64-256 µg/mL, and the production of S. aureus exotoxins was decreased by subinhibitory concentrations of peppermint oil in a dose-dependent manner. The findings suggest that peppermint oil may potentially be used to aid in the treatment of S. aureus infections.

Influence of magnolol on the secretion of alpha-toxin by Staphylococcus aureus.

In this study we investigated the antimicrobial activity of magnolol on Staphylococcus aureus. The minimal inhibitory concentrations of magnolol against 31 S. aureus strains ranged from 4-32 microg/mL. In addition, hemolysin assays, Western blotting, and real-time RT-PCR were performed to investigate the effect of magnolol on alpha-toxin secretion by both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). The results indicated that sub-inhibitory concentrations of magnolol dose-dependently inhibited the transcription of hla (the gene encoding alpha-toxin) in S. aureus, resulting in a reduction of alpha-toxin secretion and, thus, hemolytic activities.

(E)-N'-(2,5-Dimethoxy-benzyl-idene)-2,4-dihydroxy-benzohydrazide.

In the title compound, C(16)H(16)N(2)O(5), the dihedral angle between the two benzene rings is 4.2 (2)° and an intra-molecular O-H⋯O hydrogen bond generates an S(6) ring. In the crystal, mol-ecules are linked into layers lying parallel to the bc plane by O-H⋯O and N-H⋯O hydrogen bonds.

Immunomodulatory effects of proanthocyanidin A-1 derived in vitro from Rhododendron spiciferum.

The objective of this study was to evaluate the immunomodulatory effects of proanthocyanidin A-1 (PAA-1) from leaves of Rhododendron spiciferum (Ericaceae). In vitro tests showed that PAA-1 stimulated cell proliferation of splenocytes and peritoneal macrophages significantly enhanced the cytotoxicity of natural killer (NK) cells and increased CD4(+) and CD8(+) cell populations. PAA-1 also regulated the expression of Th1- and Th2-related cytokines. Moreover, this study showed that PAA-1 exhibited a significant effect on NBT dye reduction and lysosomal enzyme activity responses in macrophages, indicating effective phagocytic activation. These results revealed that PAA-1 exhibits immunomodulatory activity without a clear dose response.

A novel threefold-interpenetrating primitive cubic network based on a dinuclear Zn2 node.

In the mixed-ligand metal-organic polymeric compound poly[[mu(2)-1,4-bis(imidazol-1-yl)benzene](mu(2)-terephthalato)dizinc(II)], [Zn(2)(C(8)H(4)O(4))(2)(C(12)H(10)N(4))](n) or [Zn(2)(bdc)(2)(bib)](n) [H(2)bdc is terephthalic acid and bib is 1,4-bis(imidazol-1-yl)benzene], the asymmetric unit contains one Zn(II) ion, with two half bdc anions and one half bib molecule lying around inversion centers. The Zn(II) ion is in a slightly distorted tetrahedral environment, coordinated by three carboxylate O atoms from three different bdc anions and by one bib N atom. The crystal structure is constructed from the secondary building unit (SBU) [Zn(2)(CO(2))(2)N(2)O(2)], in which the two metal centers are held together by two bdc linkers with bis(syn,syn-bridging bidentate) bonding modes. The SBU is connected by bdc bridges to form a two-dimensional grid-like (4,4)-layer, which is further pillared by the bib ligand. Topologically, the dinuclear SBU can be considered to be a six-connected node, and the extended structure exhibits an elongated primitive approximately cubic framework. The three-dimensional framework possesses a large cavity with dimensions of approximately 10 x 13 x 17 A in cross-section. The potential porosity is filled with mutual interpenetration of two identical equivalent frameworks, generating a novel threefold interpenetrating network with an alpha-polonium topology [Abrahams, Hoskins, Robson & Slizys (2002). CrystEngComm, 4, 478-482].

Isolation of two new prenylflavonols from Epimedium brevicornum and their effects on cytokine production in vitro.

In an attempt to search for bioactive natural products, two new prenylflavonols, 2''-hydroxy-3''-en-anhydroicaritin (1) and 2''-hydroxy-beta-anhydroicaritin (2), were isolated from the Chinese medicinal herb Epimedium brevicornum. Their structures were determined by 1D and 2D NMR spectroscopic analysis. The effects of compounds 1 and 2 on cytokine production in vitro were investigated. Compound 1 could significantly downregulate tumor necrosis factor-alpha (TNF-alpha) production and increase interleukin-10 (IL-10) production. These results suggested that compound 1 may have anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated mouse macrophages. In addition, the biogenetic relationships among compounds 1 and 2 are discussed.

(E)-N'-(3,4-Dimethoxy-benzyl-idene)-2,4-dihydroxy-benzohydrazide methanol solvate.

The title compound, C(16)H(16)N(2)O(5)·CH(3)OH, was obtained from a condensation reaction of 3,4-dimethoxy-benzaldehyde and 2,4-dihydroxy-benzohydrazide. The non-H atoms of the Schiff base mol-ecule are approximately coplanar (r.m.s. deviation = 0.043 Å) and the dihedral angle between the two benzene rings is 1.6 (1)°. The mol-ecule adopts an E configuration with respect to the C=N double bond. An intra-molecular O-H⋯O hydrogen bond is observed. The Schiff base and methanol mol-ecules are linked into a two-dimensional network parallel to (10) by inter-molecular N-H⋯O, O-H⋯N and O-H⋯O hydrogen bonds.

(E)-4-Bromo-N'-(2-nitro-benzyl-idene)benzohydrazide.

The title compound, C(14)H(10)BrN(3)O(3), was obtained by a condensation reaction between 2-nitro-benzaldehyde and 4-bromo-benzohydrazide. The dihedral angle between the two benzene rings is 4.1 (2)°. The mol-ecule displays an E configuration about the C=N bond. In the crystal, mol-ecules are linked into a chain along [100] by inter-molecular N-H⋯O hydrogen bonds.