Monitoring the Sialome on Human Immune Cells.

The sialome or display of sialic acids on the surface of human immune cells can vary according to immune response and activation state. Here, human peripheral blood mononuclear cells (PBMCs) were isolated and activated with anti-CD3 antibody and the cell surface sialome was quantified using a combination of click chemistry, confocal microscopy and flow cytometry techniques. Carbohydrate click chemistry was used to detect and measure the incorporation of an azido-m65odified sialic acid precursor molecule, N-acetylmannosamine (ManNaz) sugar into the PBMC surface sialome. Incorporation of sialic acid into the PBMC glycocalyx was visualized using copper-catalyzed click conjugation of Alexa 488 alkyne and confocal microscopy and further quantified using flow cytometry. The use of these methods indicate that regulating the sialome content on the surface of activated immune cells may be monitored during immunomodulatory responses and anti-inflammatory therapies.

Osteoarthritis-associated basic calcium phosphate crystals induce pro-inflammatory cytokines and damage-associated molecules via activation of Syk and PI3 kinase.

The pro-inflammatory cytokines, TNFα, IL-1 and IL-18, amplify cartilage destruction associated with osteoarthritis (OA). Current data suggest that basic calcium phosphate (BCP) crystals are potent drivers of inflammatory mediator and matrix metalloprotease expression in the OA joint. It has previously been demonstrated that synovial macrophages play a role in initiating and driving BCP-induced inflammation. However, the molecular mechanisms by which BCP crystals exert their effects remain unclear. Here we demonstrate that exposure of macrophages to BCP crystals leads to activation of Syk and PI3 kinase. Furthermore, we show that production of pro-inflammatory cytokines and phosphorylation of the downstream kinase, ERK, are suppressed following treatment with Syk and PI3 kinase inhibitors. Finally, we demonstrate that treatment of macrophages with BCP crystals induces the production of the damage-associated molecule, S100A8, in a Syk dependent manner. We therefore identify Syk and PI3 kinase as potential novel targets for the treatment of BCP-related pathologies.