Interleukin-1 mediates ischaemic brain injury via distinct actions on endothelial cells and cholinergic neurons.

The cytokine interleukin-1 (IL-1) is a key contributor to neuroinflammation and brain injury, yet mechanisms by which IL-1 triggers neuronal injury remain unknown. Here we induced conditional deletion of IL-1R1 in brain endothelial cells, neurons and blood cells to assess site-specific IL-1 actions in a model of cerebral ischaemia in mice. Tamoxifen treatment of IL-1R1 floxed (fl/fl) mice crossed with mice expressing tamoxifen-inducible Cre-recombinase under the Slco1c1 promoter resulted in brain endothelium-specific deletion of IL-1R1 and a significant decrease in infarct size (29%), blood-brain barrier (BBB) breakdown (53%) and neurological deficit (40%) compared to vehicle-treated or control (IL-1R1fl/fl) mice. Absence of brain endothelial IL-1 signalling improved cerebral blood flow, followed by reduced neutrophil infiltration and vascular activation 24 h after brain injury. Conditional IL-1R1 deletion in neurons using tamoxifen inducible nestin-Cre mice resulted in reduced neuronal injury (25%) and altered microglia-neuron interactions, without affecting cerebral perfusion or vascular activation. Deletion of IL-1R1 specifically in cholinergic neurons reduced infarct size, brain oedema and improved functional outcome. Ubiquitous deletion of IL-1R1 had no effect on brain injury, suggesting beneficial compensatory mechanisms on other cells against the detrimental effects of IL-1 on endothelial cells and neurons. We also show that IL-1R1 signalling deletion in platelets or myeloid cells does not contribute to brain injury after experimental stroke. Thus, brain endothelial and neuronal (cholinergic) IL-1R1 mediate detrimental actions of IL-1 in the brain in ischaemic stroke. Cell-specific targeting of IL-1R1 in the brain could therefore have therapeutic benefits in stroke and other cerebrovascular diseases.

Characterization of a conditional interleukin-1 receptor 1 mouse mutant using the Cre/LoxP system.

IL-1 is a key cytokine known to drive chronic inflammation and to regulate many physiological, immunological, and neuroimmunological responses via actions on diverse cell types of the body. To determine the mechanisms of IL-1 actions as part of the inflammatory response in vivo, we generated a conditional IL-1 receptor 1 (IL-1R1) mouse mutant using the Cre/LoxP system (IL-1R1(fl/fl) ). In the mutant generated, exon 5, which encodes part of the extracellular-binding region of the receptor, is flanked by LoxP sites, thereby inactivating the two previously described functional IL-1R1 gene transcripts after Cre-mediated recombination. Using keratin 14-Cre driver mice, new IL-1R1 deficient (-/-) mice were subsequently generated, in which all signaling IL-1 receptor isoforms are deleted ubiquitously. Furthermore, using vav-iCre driver mice, we deleted IL-1 receptor isoforms in the hematopoietic system. In these mice, we show that both the IL-17 and IL-22 cytokine response is reduced, when mice are challenged by the helminth Trichuris muris. We are currently crossing IL-1R1(fl/fl) mice with different Cre-expressing mice in order to study mechanisms of acute and chronic inflammatory diseases.

Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against Mycobacterium tuberculosis.

In contrast with most bacteria, which harbour a single inosine monophosphate dehydrogenase (IMPDH) gene, the genomic sequence of Mycobacterium tuberculosis H37Rv predicts three genes encoding IMPDH: guaB1, guaB2 and guaB3. These three genes were cloned and expressed in Escherichia coli to evaluate functional IMPDH activity. Purified recombinant Mt-GuaB2, which uses inosine monophosphate as a substrate, was identified as the only active GuaB orthologue in M. tuberculosis and showed optimal activity at pH 8.5 and 37 °C. Mt-GuaB2 was inhibited significantly in vitro by a panel of diphenyl urea-based derivatives, which were also potent anti-mycobacterial agents against M. tuberculosis and Mycobacterium smegmatis, with MICs in the range of 0.2-0.5 µg ml(-1). When Mt-GuaB2 was overexpressed on a plasmid in trans in M. smegmatis, a diphenyl urea analogue showed a 16-fold increase in MIC. Interestingly, when Mt-GuaB orthologues (Mt-GuaB1 and 3) were also overexpressed on a plasmid in trans in M. smegmatis, they also conferred resistance, suggesting that although these Mt-GuaB orthologues were inactive in vitro, they presumably titrate the effect of the inhibitory properties of the active compounds in vivo.

Flavonoid inhibitors as novel antimycobacterial agents targeting Rv0636, a putative dehydratase enzyme involved in Mycobacterium tuberculosis fatty acid synthase II.

Flavonoids comprise a large group of bioactive polyphenolic plant secondary metabolites. Several of these possess potent in vivo activity against Escherichia coli and Plasmodium falciparum, targeting enzymes involved in fatty acid biosynthesis, such as enoyl-ACP-reductase, beta-ketoacyl-ACP reductase and beta-hydroxyacyl-ACP dehydratase. Herein, we report that butein, isoliquirtigenin, 2,2',4'-trihydroxychalcone and fisetin inhibit the growth of Mycobacterium bovis BCG. Furthermore, in vitro inhibition of the mycolic-acid-producing fatty acid synthase II (FAS-II) of Mycobacterium smegmatis suggests a mode of action related to those observed in E. coli and P. falciparum. Through a bioinformatic approach, we have established the product of Rv0636 as a candidate for the unknown mycobacterial dehydratase, and its overexpression in M. bovis BCG conferred resistance to growth inhibition by butein and isoliquirtigenin, and relieved inhibition of fatty acid and mycolic acid biosynthesis in vivo. Furthermore, after overexpression of Rv0636 in M. smegmatis, FAS-II was less sensitive to these inhibitors in vitro. Overall, the data suggest that these flavonoids are inhibitors of mycobacterial FAS-II and in particular Rv0636, which represents a strong candidate for the beta-hydroxyacyl-ACP dehydratase enzyme of M. tuberculosis FAS-II.