GM-CSF and M-CSF (CSF-1) can enhance macrophage lineage numbers as well as modulate their differentiation and function. Of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. What the critical actions are of these CSFs on macrophages during inflammatory reactions are unknown. To address this issue, adherent macrophages (GM-BMM and BMM) were first derived from murine bone marrow precursors by GM-CSF and M-CSF, respectively, and stimulated in vitro with LPS to measure secreted cytokine production, as well as NF-κB and AP-1 activities. GM-BMM preferentially produced TNF-α, IL-6, IL-12p70, and IL-23 whereas, conversely, BMM generated more IL-10 and CCL2; strikingly the latter population could not produce detectable IL-12p70 and IL-23. Following LPS stimulation, GM-BMM displayed rapid IκBα degradation, RelA nuclear translocation, and NF-κB DNA binding relative to BMM, as well as a faster and enhanced AP-1 activation. Each macrophage population was also pretreated with the other CSF before LPS stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and NF-κB activity. Thus, GM-CSF and M-CSF demonstrate, at the level of macrophage cytokine production, different and even competing responses with implications for their respective roles in inflammation, including a possible dampening or suppressive role for M-CSF in certain circumstances.

GM-CSF and M-CSF (CSF-1) induce different phenotypic changes in macrophage lineage populations. The nature, extent, and generality of these differences were assessed by comparing the responses to these CSFs, either alone or in combination, in various human and murine macrophage lineage populations. The differences between the respective global gene expression profiles of macrophages, derived from human monocytes by GM-CSF or M-CSF, were compared with the differences between the respective profiles for macrophages, derived from murine bone marrow cells by each CSF. Only 17% of genes regulated differently by these CSFs were common across the species. Whether a particular change in relative gene expression is by direct action of a CSF can be confounded by endogenous mediators, such as type I IFN, IL-10, and activin A. Time-dependent differences in cytokine gene expression were noted in human monocytes treated with the CSFs; in this system, GM-CSF induced a more dramatic expression of IFN-regulated factor 4 (IRF4) than of IRF5, whereas M-CSF induced IRF5 but not IRF4. In the presence of both CSFs, some evidence of “competition” at the level of gene expression was observed. Care needs to be exercised when drawing definitive conclusions from a particular in vitro system about the roles of GM-CSF and M-CSF in macrophage lineage biology.

M-CSF and GM-CSF are mediators involved in regulating the numbers and function of macrophage lineage populations and have been shown to contribute to macrophage heterogeneity. Type I IFN is an important mediator produced by macrophages and can have profound regulatory effects on their properties. In this study, we compared bone marrow-derived macrophages (BMM) and GM-CSF-induced BMM (GM-BMM) from wild-type and IFNAR1^−/− mice to assess the contribution of endogenous type I IFN to the phenotypic differences between BMM and GM-BMM. BMM were capable of higher constitutive IFN-β production, which contributed significantly to their basal transcriptome. Microarray analysis found that of the endogenous type I IFN-regulated genes specific to either BMM or GM-BMM, 488 of these gene alterations were unique to BMM, while only 50 were unique to GM-BMM. Moreover, BMM displayed enhanced basal mRNA levels, relative to GM-BMM, of a number of genes identified as being dependent on type I IFN signaling, including Stat1, Stat2, Irf7, Ccl5, Ccl12, and Cxcl10. As a result of prior type I IFN “priming,” upon LPS stimulation BMM displayed increased activation of the MyD88-independent IRF-3/STAT1 pathways compared with GM-BMM, which correlated with the distinct cytokine/chemokine profiles of the two macrophage subsets. Furthermore, the autocrine type I IFN signaling loop regulated the production of the M1 and M2 signature cytokines, IL-12p70 and IL-10. Collectively, these findings demonstrate that constitutive and LPS-induced type I IFN play significant roles in regulating the differences in phenotype and function between BMM and GM-BMM.

Porphyromonas gingivalis is one of the bacterial species most closely associated with periodontitis and can shed large numbers of outer membrane vesicles (OMVs), which are increasingly thought to play a significant role in bacterial virulence and pathogenicity. Macrophages are amongst the first immune cells to respond to bacteria and their products, so we sought to directly compare the response of macrophages to P. gingivalis or its purified OMVs. Macrophages stimulated with OMVs produced large amounts of TNF, IL-12p70, IL-6, IL-10, IFN and nitric oxide compared to cells infected with P. gingivalis, which produced very low levels of these mediators. Both P. gingivalis and OMVs induced a shift in macrophage metabolism from oxidative phosphorylation (OXPHOS) to glycolysis, which was supported by enhanced lactate release, decreased mitochondrial oxygen consumption with reduced spare respiratory capacity, as well as increased mitochondrial reactive oxygen species (ROS) production. Corresponding to this metabolic shift, gene expression analysis of macrophages infected with P. gingivalis or stimulated with OMVs revealed a broad transcriptional upregulation of genes critical to glycolysis and a downregulation of genes associated with the TCA cycle. Upon examination of inflammasome signalling and pyroptosis it was found that P. gingivalis did not activate the inflammasome in macrophages as the mature forms of caspase-1, IL-1β and IL-18 were not detected and there was no extracellular release of lactate dehydrogenase (LDH) or 7-AAD staining. In comparison, macrophages stimulated with OMVs potently activated caspase-1, produced large amounts of IL-1β, IL-18, released LDH and were positive for 7-AAD indicative of pyroptotic cell death. These data directly quantitate the distinct effects of P. gingivalis and its OMVs on macrophage inflammatory phenotype, mitochondrial function, inflammasome activation and pyroptotic cell death that may have potential implications for their roles in chronic periodontitis.

GM-CSF was originally defined by its ability to generate in vitro granulocyte and macrophage colonies from bone marrow precursor cells. Apart from its physiological role in the control of alveolar macrophage development, it now appears more likely that its major role lies in its ability to govern the properties of the more mature myeloid cells of the granulocyte and macrophage lineages, particularly during host defence and inflammatory reactions. Recent evidence is summarized below for a key role for GM-CSF in inflammatory and autoimmune diseases, making it therefore worthy of consideration for targeting. Such evidence includes disease exacerbation following its administration and amelioration of disease in animal models by GM-CSF gene targeting or by anti-GM-CSF antibody blockade. This review summarizes the evidence supporting a major role for GM-CSF in inflammation and autoimmunity and its functions as major regulator governing granulocyte and macrophage lineage populations at all stages of maturation.

Data from preclinical and clinical studies have demonstrated that granulocyte macrophage colony-stimulating factor (GM-CSF) can function as a key proinflammatory cytokine. However, therapies that directly target GM-CSF function could lead to undesirable side effects, creating a need to delineate downstream pathways and mediators. In this work, we provide evidence that GM-CSF drives CCL17 production by acting through an IFN regulatory factor 4–dependent (IRF4-dependent) pathway in human monocytes, murine macrophages, and mice in vivo. In murine models of arthritis and pain, IRF4 regulated the formation of CCL17, which mediated the proinflammatory and algesic actions of GM-CSF. Mechanistically, GM-CSF upregulated IRF4 expression by enhancing JMJD3 demethylase activity. We also determined that CCL17 has chemokine-independent functions in inflammatory arthritis and pain. These findings indicate that GM-CSF can mediate inflammation and pain by regulating IRF4-induced CCL17 production, providing insights into a pathway with potential therapeutic avenues for the treatment of inflammatory diseases and their associated pain.