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Background: Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. It is highly adapted to intracellular replication and manipulates host cell functions like vesicle trafficking and mRNA translation to its own advantage. However, it is still unknown to what extent microRNAs (miRNAs) are involved in the Legionella-host cell interaction.
Methods: WT and MyD88-/- murine bone marrow-derived macrophages (BMM) were infected with L. pneumophila, the transcriptome was analyzed by high throughput qPCR array (microRNAs) and conventional qPCR (mRNAs), and mRNA-miRNA interaction was validated by luciferase assays with 3´-UTR mutations and western blot.
Results: L. pneumophila infection caused a pro-inflammatory reaction and significant miRNA changes in murine macrophages. In MyD88-/- cells, induction of inflammatory markers, such as Ccxl1/Kc, Il6 and miR-146a-5p was reduced. Induction of miR-125a-3p was completely abrogated in MyD88-/- cells. Target prediction analyses revealed N-terminal asparagine amidase 1 (NTAN1), a factor from the n-end rule pathway, to be a putative target of miR-125a-3p. This interaction could be confirmed by luciferase assay and western blot.
Conclusion: Taken together, we characterized the miRNA regulation in L. pneumophila infection with regard to MyD88 signaling and identified NTAN1 as a target of miR-125a-3p. This finding unravels a yet unknown feature of Legionella-host cell interaction, potentially relevant for new treatment options.
Background: Inflammation, particularly cytokine release, contributes to epileptogenesis by influencing the cerebral tissue remodeling and neuronal excitability that occurs after a precipitating epileptogenic insult. While several cytokines have been explored in this process, release kinetics are less well investigated. Determining the time course of cytokine release in the epileptogenic zone is necessary for precisely timed preventive or therapeutic anti-inflammatory interventions. Methods: Hippocampal extracellular levels of six cytokines and chemokines (IL-1β, IL-6, IL-10, CCL2, CCL3, and CCL5) were quantified at various time points during epileptogenesis in a rat model of mesial temporal lobe epilepsy with hippocampal sclerosis (mTLE-HS) using microdialysis (MD). Results: The analysis of microdialysates demonstrated consistent elevation at all time points during epileptogenesis for IL-1β and IL-10. IL-10 release was maximal on day 1, IL-1β release peaked at day 8. No correlation between local hippocampal IL-1β concentrations and IL-1β blood levels was found. Conclusion: The release kinetics of IL-1β are consistent with its established pro-epileptogenic properties, while the kinetics of IL-10 suggest a counter-regulatory effect. This proof-of-concept study demonstrates the feasibility of intraindividual longitudinal monitoring of hippocampal molecular inflammatory processes via repetitive MD over several weeks and sheds light on the kinetics of hippocampal cytokine release during epileptogenesis.
Parkinson's disease (PD) is a frequent neurodegenerative process in old age. Accumulation and aggregation of the lipid-binding SNARE complex component α-synuclein (SNCA) underlies this vulnerability and defines stages of disease progression. Determinants of SNCA levels and mechanisms of SNCA neurotoxicity have been intensely investigated. In view of the physiological roles of SNCA in blood to modulate vesicle release, we studied blood samples from a new large pedigree with SNCA gene duplication (PARK4 mutation) to identify effects of SNCA gain of function as potential disease biomarkers. Downregulation of complexin 1 (CPLX1) mRNA was correlated with genotype, but the expression of other Parkinson's disease genes was not. In global RNA-seq profiling of blood from presymptomatic PARK4 indviduals, bioinformatics detected significant upregulations for platelet activation, hemostasis, lipoproteins, endocytosis, lysosome, cytokine, Toll-like receptor signaling and extracellular pathways. In PARK4 platelets, stimulus-triggered degranulation was impaired. Strong SPP1, GZMH and PLTP mRNA upregulations were validated in PARK4. When analysing individuals with rapid eye movement sleep behavior disorder, the most specific known prodromal stage of general PD, only blood CPLX1 levels were altered. Validation experiments confirmed an inverse mutual regulation of SNCA and CPLX1 mRNA levels. In the 3′-UTR of the CPLX1 gene we identified a single nucleotide polymorphism that is significantly associated with PD risk. In summary, our data define CPLX1 as a PD risk factor and provide functional insights into the role and regulation of blood SNCA levels. The new blood biomarkers of PARK4 in this Turkish family might become useful for PD prediction.
Diffuse invasion of the surrounding brain parenchyma is a major obstacle in the treatment of gliomas with various therapeutics, including anti-angiogenic agents. Here we identify the epi-/genetic and microenvironmental downregulation of ephrinB2 as a crucial step that promotes tumour invasion by abrogation of repulsive signals. We demonstrate that ephrinB2 is downregulated in human gliomas as a consequence of promoter hypermethylation and gene deletion. Consistently, genetic deletion of ephrinB2 in a murine high-grade glioma model increases invasion. Importantly, ephrinB2 gene silencing is complemented by a hypoxia-induced transcriptional repression. Mechanistically, hypoxia-inducible factor (HIF)-1α induces the EMT repressor ZEB2, which directly downregulates ephrinB2 through promoter binding to enhance tumour invasiveness. This mechanism is activated following anti-angiogenic treatment of gliomas and is efficiently blocked by disrupting ZEB2 activity. Taken together, our results identify ZEB2 as an attractive therapeutic target to inhibit tumour invasion and counteract tumour resistance mechanisms induced by anti-angiogenic treatment strategies.