Thus, we can postulate that calcium homeostasis upon oxidative challenge should also be impacted in our cellular models, confirming the emerging consensus around the cross-talk between these two cellular events. The most important and innovative a part of our study was verification of the hypothesis that physiologically relevant signals modulating monocyte/macrophage function (in our case, activation of the pattern recognition receptor TLR2 as a canonical example of pathogen-triggered signalling) can contribute to antioxidant defence development. levels even upon differentiated macrophage-like cells, mainly related to thioredoxin-linked antioxidant enzymes. cultured cell lines, a mainstay of modern experimental biology, are especially helpful for investigating basic biochemical and genetic mechanisms in a relatively isolated and well-characterised, but still physiologically relevant setting. Therefore, they are commonly used in studies on the impact of external factors on cellular homeostatic mechanisms, including redox homeostasis, the delicate balance between pro-oxidant and anti-oxidant activities that ensures not only survival of oxidatively respiring cells, but also strong resistance to environmental oxidative stress [4C6]. Specifically, the availability of immortal, clonal cell lines of the monocytic lineage made it possible to study monocyte and macrophage function in molecular detail. Among the most commonly used and physiologically relevant models of this type, the THP-1 A-804598 cell line  is usually a gold standard for studying early stages of monocyte differentiation, while the more mature Mono Mac 6 cell line  allows the study of mechanisms emerging in more developed macrophages. Oxidative stress is usually prevalent in the innate immune system, derived both from endogenous sources (oxidative burst in immune cells) and the cellular microenvironment (enhanced reactive oxidant production at contamination and/or inflammation sites). Since this oxidative response is usually central to efficient anti-microbial action and reactive oxidants are important direct toxins against infectious microorganisms, the presence of oxidative stress must be considered physiological for immune cells, especially macrophages which must be present at the very site of immune response [9C12]. Therefore, antioxidant resistance is crucial for survival and correct function of monocytes and macrophages, and their redox homeostasis is known to be A-804598 both strong and tightly regulated, although molecular mechanisms of this regulation are still obscure [13C14]. Redox homeostasis in mammalian cells is usually mediated mainly by a number of enzymatic and non-enzymatic mechanisms for removal of potentially dangerous reactive oxidant molecules. While the level of many small-molecule, cell-permeable antioxidants (e.g. ascorbate or vitamin E) is usually regulated predominantly at the level of whole organism, each individual cell autonomously regulates the Mouse monoclonal to BRAF expression of intracellular antioxidant enzymes and peptide (thiol) antoxidants . Among the thiol antioxidants, some are genetically expressed (thioredoxin) and some are biochemically synthesised (glutathione), but all exert their function with help of a plethora of accessory enzymes (reductases, peroxidases etc.), which together form distinct antioxidant systems to facilitate safe electron transfer [16C17]. While it is usually expected that redox homeostasis evolves together with changing cell fate during differentiation of monocytes and macrophages, it is important to assess this phenomenon also with regard to actual immune activity, i.e. functional activation of both monocytes and macrophages upon stimulation for immune response. In innate immunity, the central triggering mechanism for cellular activation are pattern recognition receptors, especially from the Toll-like receptor (TLR) family [18, 19]. The impact of TLR signalling on redox homeostasis is usually acknowledged in various cell types around the phenotype level, but it is sometimes difficult to directly identify the molecular mechanisms responsible for enhanced resistance to oxidants [20, 21]. One of the most important TLR family members is usually TLR2, a pattern recognition receptor for bacterial lipoproteins and lipopeptides. It is expressed at relatively high levels A-804598 on the surface of monocytes and macrophages  A-804598 and mediates a large number of mostly proinflammatory interactions between microbial components and the innate immune system. The conversation of pathogens with TLR2 results in activation of NF-B and release of IL-1, IL-6, IL-8, IL-10, IL-12p40, TNF- and nitric oxide from human monocytes and macrophages [23C26]. TLR2 stimulation induces the expression of phagocytic receptors and results in enhanced phagocytosis of bacteria by macrophages . TLR2 activity is crucial e.g. for cell line models of the monocyte-macrophage differentiation axis to study the evolution of redox homeostasis mechanisms along this axis, but also to verify the capability of these mechanisms to react to infectious challenge (in the form of activation of TLR2) at various points along the differentiation continuum. At the basis of our experimental design is an orthogonal approach to differentiation and activation: we compare the response to TLR2 ligand in.