Macrophages are an integral part of the innate immune system and key players in pathogen clearance and tissue remodelling. Both functions are accomplished by a pivotal network of different macrophage subtypes, including proinflammatory M1 and anti-inflammatory M2 macrophages. Previously, our laboratory identified the transcription factor interferon regulatory factor 5 (IRF5) as the master regulator of the M1 macrophage polarisation. IRF5 was found to be highly expressed in human M1 compared to M2 macrophages. Furthermore, IRF5 dictates the expression of proinflammatory genes such as IL12b and IL23a whilst repressing anti-inflammatory genes like IL10. Here we show that murine bone marrow derived macrophages differentiated in vitro with GM-CSF are also characterised by high levels of 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 the in vitro results in an in vivo model of antigen-induced arthritis, emphasising their physiological relevance. Thus, we establish the species-invariant role of IRF5 in controlling the inflammatory macrophage phenotype both in vitro and in in 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 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 the in vitro differentiation of lineage-defined macrophages. In general, these methods utilise M-CSF (macrophage colony stimulating factor; CSF-1) to differentiate bone marrow derived progenitors, followed by priming with various stimuli. Addition of interferon-γ 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
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