Animals and Antigen-Induced Arthritis == For this study wild type mice were bred on a C57Bl/6 background. IRF5 mRNA and protein and express proinflammatory cytokines upon LPS stimulation. These macrophages display characteristic expression of M1-marker MHC II but lack the M2-marker CD206. Significantly, we develop intracellular staining of IRF5- expressing macrophages and utilise it to recapitulate thein vitroresults in anin vivomodel of antigen-induced arthritis, emphasising their physiological relevance. Thus, we establish the species-invariant role of IRF5 in controlling the inflammatory macrophage phenotype bothin vitroand inin vivo. == 1. Introduction == Macrophages are immune cells involved in recognition of pathogenic stimuli and the initiation and resolution of inflammation. They can adapt to various different environmental signals giving rise to several subtypes with distinct functions [1]. These subtypes can be classified as M1 (classically activated) and Atazanavir M2 (alternatively activated) macrophages. In addition, there are several phenotypes associated with M2 macrophages, for example, M2-like or tumour associated macrophages [2]. M1 macrophages secrete high levels of IL-12 and IL-23 but low levels of IL-10, whereas M2 macrophages secrete low levels of IL-12 and IL-23 but high levels of IL-10 [3]. Several reports have described thein vitrodifferentiation of lineage-defined macrophages. In general, these methods utilise M-CSF (macrophage colony stimulating factor; CSF-1) to differentiate bone marrow Atazanavir derived progenitors, followed by priming with various stimuli. Addition of interferon-followed by lipopolysaccharide (LPS) stimulation has been used to acquire M1 macrophages whereas addition of IL-4 or IL-13 without LPS yields M2 macrophages [3]. Another established method uses GM-CSF (granulocyte/macrophage colony stimulating factor) in order to generate M1 macrophages or alternatively M-CSF treatment for M2 differentiation, usually followed by LPS challenge for both subtypes [4,5]. In the physiological situation, M-CSF is detected in low steady state levels whereas GM-CSF has been shown to be increased upon stimulation with inflammatory stimuli, such as IL-1, TNF, or LPS [6,7]. Macrophages are also known to play a key role in autoimmune diseases such as rheumatoid arthritis (RA), a degenerative disease characterised by joint inflammation and bone destruction [8]. At the site of inflammation, macrophages are present in high numbers and it has been found that depletion ameliorates disease severity [911]. More specifically, M1 macrophages contribute to RA pathogenesis by secreting proinflammatory cytokines and thereby taking Atazanavir part in the Th1/Th17 response [12,13]. Distinct macrophage subtypes are not only characterised by their differences in cytokine release but also display differential expression of key transcription factors. Recently, we identified the transcription factor interferon regulatory factor 5 (IRF5) as the major regulator of proinflammatory M1 macrophage polarisation [14]. IRF5 directly induces the expression of Atazanavir proinflammatory cytokines such as IL-6, IL-12b, and IL-23a whilst repressing transcription of anti-inflammatory cytokines such as IL-10 [14,15]. IRF5 is involved in various inflammatory processes such as the type I interferon response to virus infection and pathogen recognition receptor signalling [16]. Upon viral infection, IRF5 is phosphorylated and thereby translocated to the nucleus where it binds to the regulatory regions of its target genes [17]. Nonviral stimulation of toll-like receptors (TLR) including TLR4, 7, and 9 also leads to activation of IRF5 [16]. Moreover, polymorphisms in theIRF5gene have been found to associate with RA [18,19]. Despite the major role IRF5 plays in macrophage activation, it has rarely been used to track inflammatory macrophages in disease. In this study, we aim to characterise murine macrophages and IRF5 expression in bothin vitroandin vivomodels of inflammation. We therefore used the murine model of antigen-induced arthritis (AIA) in which mice are immunised with methylated BSA (mBSA) prior to intra-articular injection of mBSA in one knee, leading to localised inflammation and a Th17 response [20,21]. First, we analysedin vitrodifferentiated macrophages regarding their IRF5 expression, LPS response, and surface receptor expression. We then used flow cytometry to label intracellular IRF5 in both thein vitromacrophages and those derived from the affected knee of the AIA mouse model. == 2. Material and Methods == == 2.1. Animals and Antigen-Induced Arthritis == For this study wild type mice were bred on a C57Bl/6 background. The experimental animal procedures used in this work were approved by the Kennedy Institute of Rheumatology Ethics Committee Rabbit Polyclonal to CATD (L chain, Cleaved-Gly65) and the UK Home Office. We induced arthritis as described previously; briefly, at day zero, mice were sedated using inhaled isoflurane anaesthesia and subsequently immunised with 100g of mBSA emulsified in Atazanavir 0.2 mL of complete Freund’s adjuvant, administered intra-dermally at the base of the tail. At day seven, we induced arthritis by means of an intraarticular injection of mBSA (200g in 10L of sterile PBS), or PBS alone using a sterile.