AIM2 senses Brucella abortus DNA in dendritic cells to induce IL-1ß secretion, pyroptosis and resistance to bacterial infection in mice.

Absent in melanoma 2 (AIM2) is a sensor of cytosolic dsDNA and is responsible for the activation of inflammatory and host immune responses to DNA viruses and intracellular bacteria. AIM2 is a member of the hematopoietic interferon-inducible nuclear proteins with a 200 amino-acid repeat (HIN200) family, containing a pyrin domain (PYD) at the N-terminus. Several studies have demonstrated that AIM2 is responsible for host defense against intracellular bacteria such as Francisella tularensis, Listeria monocytogenes and Mycobacerium tuberculosis. However, the role of AIM2 in host defenses against Brucella is poorly understood. In this study, we have shown that AIM2 senses Brucella DNA in dendritic cells to induce pyroptosis and regulates type I IFN. Confocal microscopy of infected cells revealed co-localization between Brucella DNA and endogenous AIM2. Dendritic cells from AIM2 KO mice infected with B. abortus showed impaired secretion of IL-1ß as well as compromised caspase-1 cleavage. AIM2 KO mice displayed increased susceptibility to B. abortus infection in comparison to wild-type mice, and this susceptibility was associated with defective IL-1ß production together with reduced IFN-γ responses. In summary, the increased bacterial burden observed in vivo in AIM2 KO animals confirmed that AIM2 is essential for an effective innate immune response against Brucella infection.

GLP overexpression is associated with poor prognosis in Chronic Lymphocytic Leukemia and its inhibition induces leukemic cell death.

Background Heterodimeric methyltransferases GLP (EHMT1/KMT1D) and G9a (EHMT2/KMT1C) are two closely related enzymes that promote the monomethylation and dimethylation of histone H3 lysine 9. Dysregulation of their activity has been implicated in several types of human cancer. Patients and methods Here, in order to investigate whether GLP/G9a exerts any impact on Chronic Lymphocytic Leukemia (CLL), GLP/G9a expression levels were assessed in a cohort of 50 patients and the effects of their inhibition were verified for the viability of CLL cells. Also, qRT-PCR was used to investigate the transcriptional levels of GLP/G9a in CLL patients. In addition, patient samples were classified according to ZAP-70 protein expression by flow cytometry and according to karyotype integrity by cytogenetics analysis. Finally, a selective small molecule inhibitor for GLP/G9a was used to ascertain whether these methyltransferases influenced the viability of MEC-1 CLL cell lineage. Results mRNA analysis revealed that CLL samples had higher levels of GLP, but not G9a, when compared to non-leukemic controls. Interestingly, patients with unfavorable cytogenetics showed higher expression levels of GLP compared to patients with favorable karyotypes. More importantly, GLP/G9a inhibition markedly induced cell death in CLL cells. Conclusion Taken together, these results indicate that GLP is associated with a worse prognosis in CLL, and that the inhibition of GLP/G9a influences CLL cell viability. Altogether, the present data demonstrate that these methyltransferases can be potential markers for disease progression, as well as a promising epigenetic target for CLL treatment and the prevention of disease evolution.

Brucella abortus Triggers a cGAS-Independent STING Pathway To Induce Host Protection That Involves Guanylate-Binding Proteins and Inflammasome Activation.

Immunity against microbes depends on recognition of pathogen-associated molecular patterns by innate receptors. Signaling pathways triggered by Brucella abortus DNA involves TLR9, AIM2, and stimulator of IFN genes (STING). In this study, we observed by microarray analysis that several type I IFN-associated genes, such as IFN-ß and guanylate-binding proteins (GBPs), are downregulated in STING knockout (KO) macrophages infected with Brucella or transfected with DNA. Additionally, we determined that STING and cyclic GMP-AMP synthase (cGAS) are important to engage the type I IFN pathway, but only STING is required to induce IL-1ß secretion, caspase-1 activation, and GBP2 and GBP3 expression. Furthermore, we determined that STING but not cGAS is critical for host protection against Brucella infection in macrophages and in vivo. This study provides evidence of a cGAS-independent mechanism of STING-mediated protection against an intracellular bacterial infection. Additionally, infected IFN regulatory factor-1 and IFNAR KO macrophages had reduced GBP2 and GBP3 expression and these cells were more permissive to Brucella replication compared with wild-type control macrophages. Because GBPs are critical to target vacuolar bacteria, we determined whether GBP2 and GBPchr3 affect Brucella control in vivo. GBPchr3 but not GBP2 KO mice were more susceptible to bacterial infection, and small interfering RNA treated-macrophages showed reduction in IL-1ß secretion and caspase-1 activation. Finally, we also demonstrated that Brucella DNA colocalizes with AIM2, and AIM2 KO mice are less resistant to B. abortus infection. In conclusion, these findings suggest that the STING-dependent type I IFN pathway is critical for the GBP-mediated release of Brucella DNA into the cytosol and subsequent activation of AIM2.

Zika Virus Promotes Neuronal Cell Death in a Non-Cell Autonomous Manner by Triggering the Release of Neurotoxic Factors.

Zika virus (ZIKV) has recently caused a worldwide outbreak of infections associated with severe neurological complications, including microcephaly in infants born from infected mothers. ZIKV exhibits high neurotropism and promotes neuroinflammation and neuronal cell death. We have recently demonstrated that N-methyl-d-aspartate receptor (NMDAR) blockade by memantine prevents ZIKV-induced neuronal cell death. Here, we show that ZIKV induces apoptosis in a non-cell autonomous manner, triggering cell death of uninfected neurons by releasing cytotoxic factors. Neuronal cultures infected with ZIKV exhibit increased levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and glutamate. Moreover, infected neurons exhibit increased expression of GluN2B and augmented intracellular Ca2+ concentration. Blockade of GluN2B-containing NMDAR by ifenprodil normalizes Ca2+ levels and rescues neuronal cell death. Notably, TNF-α and IL-1ß blockade decreases ZIKV-induced Ca2+ flux through GluN2B-containing NMDARs and reduces neuronal cell death, indicating that these cytokines might contribute to NMDAR sensitization and neurotoxicity. In addition, ZIKV-infected cultures treated with ifenprodil exhibits increased activation of the neuroprotective pathway including extracellular signal-regulated kinase and cAMP response element-binding protein, which may underlie ifenprodil-mediated neuroprotection. Together, our data shed some light on the neurotoxic mechanisms triggered by ZIKV and begin to elucidate how GluN2B-containing NMDAR blockade can prevent neurotoxicity.

Aberrant levels of SUV39H1 and SUV39H2 methyltransferase are associated with genomic instability in chronic lymphocytic leukemia.

Chromosomal alterations are commonly detected in patients with chronic lymphocytic leukemia (CLL) and impact disease pathogenesis, prognosis, and progression. Telomerase expression (hTERT), its activity and the telomere length are other important predictors of survival and multiple outcomes in CLL. SUV39H and SUV420H enzymes are histone methyltransferases (HMTases) involved in several cellular processes, including regulation of telomere length, heterochromatin organization, and genome stability. Here, we investigated whether SUV39H1, SUV39H2, SUV420H1, SUV420H2, and hTERT are associated with genomic instability of CLL. SUV39H (1/2), SUV420H (1/2), and hTERT expression was determined in 59 CLL samples by real time PCR. In addition, ZAP-70 protein expression was evaluated by Flow Cytometry and patients' karyotype was defined by Cytogenetic Analysis. Low expression of SUV39H1 was associated with the acquisition of altered and complex karyotypes. Conversely, high expression of SUV39H2 correlated with cytogenetic abnormalities in CLL patients. The pattern of karyotypic alterations differed in samples with detectable or undetectable hTERT expression. Furthermore, hTERT expression in CLL showed a correlation with transcript levels of SUV39H2, which, in part, can explain the association between SUV39H2 expression and cytogenetic abnormalities. Moreover, SUV39H1 correlated with SUV420H1 expression while SUV420H2 was associated with all other investigated HMTases. Our data show that the differential expression of SUV39H1 and SUV39H2 is associated with genomic instability and that the modulation of these HMTases can be an attractive approach to prevent CLL evolution. Environ. Mol. Mutagen. 58:654-661, 2017. © 2017 Wiley Periodicals, Inc.

Orchestrated activation of mGluR5 and CB1 promotes neuroprotection.

The metabotropic glutamate receptor 5 (mGluR5) and the cannabinoid receptor 1 (CB1) exhibit a functional interaction, as CB1 regulates pre-synaptic glutamate release and mGluR5 activation increases endocannabinoid synthesis at the post-synaptic site. Since both mGluR5 and CB1 promote neuroprotection, we delineated experiments to investigate a possible link between CB1 and mGluR5 activation in the induction of neuroprotection using primary cultured corticostriatal neurons. We find that either the pharmacological blockade or the genetic ablation of either mGluR5 or CB1 can abrogate both CB1- and mGluR5-mediated neuroprotection against glutamate insult. Interestingly, decreased glutamate release and diminished intracellular Ca(2+) do not appear to play a role in CB1 and mGluR5-mediated neuroprotection. Rather, these two receptors work cooperatively to trigger the activation of cell signaling pathways to promote neuronal survival, which involves MEK/ERK1/2 and PI3K/AKT activation. Interestingly, although mGluR5 activation protects postsynaptic terminals and CB1 the presynaptic site, intact signaling of both receptors is required to effectively promote neuronal survival. In conclusion, mGluR5 and CB1 act in concert to activate neuroprotective cell signaling pathways and promote neuronal survival.

Mutant Brucella abortus membrane fusogenic protein induces protection against challenge infection in mice.

Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies.

Overexpression of EZH2 associates with a poor prognosis in chronic lymphocytic leukemia.

EZH2, a histone methyltransferase, is overexpressed in several human tumors, but whether it exerts any impact in chronic lymphocytic leukemia (CLL) remains unknown. We used real time PCR to investigate the expression profile of EZH1 and EZH2 in 59 CLL patients, 10 samples of purified B-cells from healthy donors and 12 normal adult tissues. EZH2 was overexpressed in CLL patients and correlates with high white blood cell count, ZAP-70 expression and chromosomal abnormalities. EHZ1 expression does not correlate with CLL progression. EZH2 overexpression is related to a poor prognosis of CLL and could be a useful tool to assess its aggressiveness.

Sm10.3, a member of the micro-exon gene 4 (MEG-4) family, induces erythrocyte agglutination in vitro and partially protects vaccinated mice against Schistosoma mansoni infection.

BACKGROUND: The parasitic flatworm Schistosoma mansoni is a blood fluke that causes schistosomiasis. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Numerous antigens that are expressed at the interface between the parasite and the mammalian host have been assessed. Among the most promising molecules are the proteins present in the tegument and digestive tract of the parasite. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we evaluated the potential of Sm10.3, a member of the micro-exon gene 4 (MEG-4) family, for use as part of a recombinant vaccine. We confirmed by real-time PCR that Sm10.3 was expressed at all stages of the parasite life cycle. The localization of Sm10.3 on the surface and lumen of the esophageal and intestinal tract in adult worms and lung-stage schistosomula was confirmed by confocal microscopy. We also show preliminary evidence that rSm10.3 induces erythrocyte agglutination in vitro. Immunization of mice with rSm10.3 induced a mixed Th1/Th2-type response, as IFN-γ, TNF-α, and low levels of IL-5 were detected in the supernatant of cultured splenocytes. The protective effect conferred by vaccination with rSm10.3 was demonstrated by 25.5-32% reduction in the worm burden, 32.9-43.6% reduction in the number of eggs per gram of hepatic tissue, a 23.8% reduction in the number of granulomas, an 11.8% reduction in the area of the granulomas and a 39.8% reduction in granuloma fibrosis. CONCLUSIONS/SIGNIFICANCE: Our data suggest that Sm10.3 is a potential candidate for use in developing a multi-antigen vaccine to control schistosomiasis and provide the first evidence for a possible role for Sm10.3 in the blood feeding process.

Par3/Bazooka and phosphoinositides regulate actin protrusion formation during Drosophila dorsal closure and wound healing.

Effective wound closure mechanisms are essential for maintenance of epithelial structure and function. The repair of wounded epithelia is primarily driven by the cells bordering the wound, which become motile after wounding, forming dynamic actin protrusions along the wound edge. The molecular mechanisms that trigger wound edge cells to become motile following tissue damage are not well understood. Using wound healing and dorsal closure in Drosophila, we identify a direct molecular link between changes in cell-cell adhesion at epithelial edges and induction of actin protrusion formation. We find that the scaffolding protein Par3/Bazooka and the lipid phosphatase Pten are specifically lost from cell-cell junctions at epithelial edges. This results in a localized accumulation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which promotes the formation of actin protrusions along the epithelial edge. Depleting PIP3 results in defective epithelial closure during both dorsal closure and wound healing. These data reveal a novel mechanism that directly couples loss of epithelial integrity to activation of epithelial closure.

An insight into the sialome of Glossina morsitans morsitans.

BACKGROUND: Blood feeding evolved independently in worms, arthropods and mammals. Among the adaptations to this peculiar diet, these animals developed an armament of salivary molecules that disarm their host's anti-bleeding defenses (hemostasis), inflammatory and immune reactions. Recent sialotranscriptome analyses (from the Greek sialo = saliva) of blood feeding insects and ticks have revealed that the saliva contains hundreds of polypeptides, many unique to their genus or family. Adult tsetse flies feed exclusively on vertebrate blood and are important vectors of human and animal diseases. Thus far, only limited information exists regarding the Glossina sialome, or any other fly belonging to the Hippoboscidae. RESULTS: As part of the effort to sequence the genome of Glossina morsitans morsitans, several organ specific, high quality normalized cDNA libraries have been constructed, from which over 20,000 ESTs from an adult salivary gland library were sequenced. These ESTs have been assembled using previously described ESTs from the fat body and midgut libraries of the same fly, thus totaling 62,251 ESTs, which have been assembled into 16,743 clusters (8,506 of which had one or more EST from the salivary gland library). Coding sequences were obtained for 2,509 novel proteins, 1,792 of which had at least one EST expressed in the salivary glands. Despite library normalization, 59 transcripts were overrepresented in the salivary library indicating high levels of expression. This work presents a detailed analysis of the salivary protein families identified. Protein expression was confirmed by 2D gel electrophoresis, enzymatic digestion and mass spectrometry. Concurrently, an initial attempt to determine the immunogenic properties of selected salivary proteins was undertaken. CONCLUSIONS: The sialome of G. m. morsitans contains over 250 proteins that are possibly associated with blood feeding. This set includes alleles of previously described gene products, reveals new evidence that several salivary proteins are multigenic and identifies at least seven new polypeptide families unique to Glossina. Most of these proteins have no known function and thus, provide a discovery platform for the identification of novel pharmacologically active compounds, innovative vector-based vaccine targets, and immunological markers of vector exposure.