Molecular and biochemical characterization of nematode cofactor independent phosphoglycerate mutases

Phosphoglycerate mutase (PGM, EC 5.4.2.1) catalyzes the isomerization of 3-phosphoglycerate and 2-phosphoglycerate in glycolysis and gluconeogenesis. Two distinct types of PGM exist in nature, one that requires 2,3-bisphosphoglycerate as a cofactor (dPGM) and another that does not (iPGM). The two enzymes are structurally distinct and possess different mechanisms of action. In any particular organism, one form may exist or both. Nematodes possess the iPGM form whereas mammals have dPGM. In the present study, we have cloned and expressed iPGM from Onchocerca volvulus and described the catalytic properties of O. volvulus, Brugia malayi and Caenorhabditis elegans iPGM enzymes. Temperature and pH optima were determined for each enzyme. Like other iPGM enzymes, the activities of the nematode iPGM enzymes were dependent on the presence of divalent ions. Inactivation by EDTA could be restored most effectively by magnesium and manganese ions. Kinetic parameters and specific activities of the various recombinant enzymes were determined. The high similarity in catalytic properties among the enzymes indicates that a single enzyme inhibitor would likely be effective against all nematode enzymes. Inhibition of iPGM activity in vivo may lead to lethality as indicated by RNAi studies in C. elegans. Our results support the development of iPGM as a promising drug target in parasitic nematodes.     

Anti-TNF treatment negatively regulates human CD4+ T-cell activation and maturation in vitro,but does not confer an anergic or suppressive phenotype

TNF-blockade has shown clear therapeutic value in rheumatoid arthritis and other immune-mediated inflammatory diseases, however its mechanism of action is not fully elucidated. We investigated the effects of TNF-blockade on CD4⁺ T cell activation, maturation, and proliferation, and assessed whether TNF-inhibitors confer regulatory potential to CD4⁺ T cells. CyTOF and flow cytometry analysis revealed that in vitro treatment of human CD4⁺ T cells with the anti-TNF monoclonal antibody adalimumab promoted IL-10 expression in CD4⁺ T cells, whilst decreasing cellular activation. In line with this, analysis of gene expression profiling datasets of anti-TNF-treated IL-17 or IFN-γ-producing CD4⁺ T cells revealed changes in multiple pathways associated with cell cycle and proliferation. Kinetics experiments showed that anti-TNF treatment led to delayed, rather than impaired T-cell activation and maturation. Whilst anti-TNF-treated CD4⁺ T cells displayed some hyporesponsiveness upon restimulation, they did not acquire enhanced capacity to suppress T-cell responses or modulate monocyte phenotype. These cells however displayed a reduced ability to induce IL-6 and IL-8 production by synovial fibroblasts. Together, these data indicate that anti-TNF treatment delays human CD4⁺ T-cell activation, maturation, and proliferation, and this reduced activation state may impair their ability to activate stromal cells.     

The <Emphasis Type="Italic">Wolbachia</Emphasis> endosymbiont of <Emphasis Type="Italic">Brugia malayi</Emphasis> has an active phosphoglycerate mutase: a candidate target for anti-filarial therapies

Phosphoglycerate mutases (PGM) interconvert 2- and 3-phosphoglycerate in the glycolytic and gluconeogenic pathways. A putative cofactor-independent phosphoglycerate mutase gene (iPGM) was identified in the genome sequence of the Wolbachia endosymbiont from the filarial nematode, Brugia malayi (wBm). Since iPGM has no sequence or structural similarity to the cofactor-dependent phosphoglycerate mutase (dPGM) found in mammals, it may represent an attractive Wolbachia drug target. In the present study, wBm–iPGM cloned and expressed in Escherichia coli was mostly insoluble and inactive. However, the protein was successfully produced in the yeast Kluyveromyces lactis and the purified recombinant wBm–iPGM showed typical PGM activity. Our results provide a foundation for further development of wBm–iPGM as a promising new drug target for novel anti-filarial therapies that selectively target the endosymbiont.     

The Wolbachia endosymbiont of Brugia malayi has an active pyruvate phosphate dikinase

Genome analysis of the glycolytic/gluconeogenic pathway in the Wolbachia endosymbiont from the filarial parasite Brugia malayi (wBm) has revealed that wBm lacks pyruvate kinase (PK) and may instead utilize the enzyme pyruvate phosphate dikinase (PPDK; ATP:pyruvate, orthophosphate phosphotransferase, EC 2.7.9.1). PPDK catalyses the reversible conversion of AMP, PPi and phosphoenolpyruvate (PEP) into ATP, Pi and pyruvate. The glycolytic pathway of most organisms, including mammals, contains exclusively PK for the production of pyruvate from PEP. Therefore, the absence of PPDK in mammals makes the enzyme an attractive Wolbachia drug target. In the present study, we have cloned and expressed an active wBm-PPDK, thereby providing insight into the energy metabolism of the endosymbiont. Our results support the development of wBm-PPDK as a promising new drug target in an anti-symbiotic approach to controlling filarial infection.     

Presence,function, and regulation of IL-17F-expressing human CD4+ T cells

The pro-inflammatory cytokine IL-17A has been implicated in the immunopathology of inflammatory arthritis. IL-17F bears 50% homology to IL-17A and has recently been suggested to play a role in inflammation. We investigated the induction and cytokine profile of IL-17F⁺ CD4⁺ T cells, and how IL-17F may contribute to inflammation. Upon culture of healthy donor CD4⁺ T cells with IL-1β, IL-23, anti-CD3, and anti-CD28 mAb, both IL-17A and IL-17F-expressing cells were detected. In comparison to IL-17A⁺IL-17F⁻ CD4⁺ T cells, IL-17F⁺IL-17A⁻ and IL-17A⁺IL-17F⁺ CD4⁺ T cells contained lower proportions of IL-10-expressing and GM-CSF-expressing cells and higher proportions of IFN-γ-expressing cells. Titration of anti-CD28 mAb revealed that strong co-stimulation increased IL-17F⁺IL-17A⁻ and IL-17A⁺IL-17F⁺ CD4⁺ T cell frequencies, whereas IL-17A⁺IL-17F⁻ CD4⁺ T cell frequencies decreased. This was partly mediated via an IL-2-dependent mechanism. Addition of IL-17A, IL-17F, and TNF-α to synovial fibroblasts from patients with inflammatory arthritis resulted in significant production of IL-6 and IL-8, which was reduced to a larger extent by combined blockade of IL-17A and IL-17F than blockade of IL-17A alone. Our data indicate that IL-17A and IL-17F are differentially regulated upon T cell co-stimulation, and that dual blockade of IL-17A and IL-17F reduces inflammation more effectively than IL-17A blockade alone.     

Cofactor-independent phosphoglycerate mutase is an essential gene in procyclic form <Emphasis Type="Italic">Trypanosoma brucei</Emphasis>

Glycolysis and gluconeogenesis are, in part, driven by the interconversion of 3- and 2-phosphoglycerate (3-PG and 2-PG) which is performed by phosphoglycerate mutases (PGAMs) which can be cofactor dependant (dPGAM) or cofactor independent (iPGAM). The African trypanosome, Trypanosoma brucei, possesses the iPGAM form which is thought to play an important role in glycolysis. Here, we report on the use of RNA interference to down-regulate the T. brucei iPGAM in procyclic form T. brucei and evaluation of the resulting phenotype. We first demonstrated biochemically that depletion of the steady state levels of iPGM mRNA correlates with a marked reduction of enzyme activity. We further show that iPGAM is required for cell growth in procyclic T. brucei.     

miR-155-overexpressing monocytes resemble HLAhighISG15+ synovial tissue macrophages from patients with rheumatoid arthritis and induce polyfunctional CD4+ T-cell activation

MicroRNAs (miRs) are known to regulate pro-inflammatory effector functions of myeloid cells, and miR dysregulation is implicated in rheumatoid arthritis (RA), a condition characterized by inflammation and destruction of the joints. We showed previously that miR-155 is increased in myeloid cells in RA and induces pro-inflammatory activation of monocytes and macrophages; however, its role at the interface between innate and adaptive immunity was not defined. Here, RNA-sequencing revealed that overexpression of miR-155 in healthy donor monocytes conferred a specific gene profile which bears similarities to that of RA synovial fluid-derived CD14⁺ cells and HLA^highISG15⁺ synovial tissue macrophages, both of which are characterized by antigen-presenting pathways. In line with this, monocytes in which miR-155 was overexpressed, displayed increased expression of HLA-DR and both co-stimulatory and co-inhibitory molecules, and induced activation of polyfunctional T cells. Together, these data underpin the notion that miR-155-driven myeloid cell activation in the synovium contributes not only to inflammation but may also influence the adaptive immune response.