Who’s in charge here? Macrophage colony stimulating factor and granulocyte macrophage colony stimulating factor: Competing factors in macrophage polarization

Cytokine 127 (2020) 154939

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Review article

Who’s in charge here? Macrophage colony stimulating factor and granulocyte macrophage colony stimulating factor: Competing factors in macrophage polarization ⁎

Evan Trus, Sameh Basta , Katrina Gee

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Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada

A R T I C LE I N FO

A B S T R A C T

Keywords: GM-CSF M-CSF Macrophage Inflammation Polarization Differentiation Virus infection

Macrophages make up a crucial aspect of the immune system, carrying out a variety of functions ranging from clearing cellular debris to their well-recognized roles as innate immune cells. These cells exist along a spectrum of phenotypes but can be generally divided into proinflammatory (M1) and anti-inflammatory (M2) groups, representing different states of polarization. Due to their diverse functions, macrophages are implicated in a variety of diseases such as atherosclerosis, lupus nephritis, or infection with HIV. Throughout their lifetime, macrophages can be influenced by a wide variety of signals that influence their polarization states, which can affect their function and influence their effects on disease progression. This review seeks to provide a summary of how GM-CSF and M-CSF influence macrophage activity during disease, and provide examples of in vitro research that indicate competition between the two cytokines in governing macrophage polarization. Gaining a greater understanding of the relationship between GM-CSF and M-CSF, along with how these cytokines fit into the larger context of diseases, will inform their use as treatments or targets for treatment in various diseases.

1. Introduction Macrophages form an important part of the immune system, providing innate defense against pathogens and assisting in the activation of an adaptive immune response. These cells constitute a diverse set of different populations each having roles in ontogeny, homeostasis, and healing. As such, they have different phenotypes due to their origin as well as their functions within the tissue. Thus, tissue-resident macrophages are heterogeneous populations that carry out a variety of functions in their respective tissues of origin ranging from cellular debris clearance to immune surveillance [1]. In adults, macrophages differentiate from monocytes upon leaving the bone marrow, spleen, or bloodstream. This is not the only source of macrophages however, as they can also arise from tissue-resident macrophages seeded during embryonic development through differentiation from erythroid-myeloid progenitors in the extra-embryonic yolk sac [1]. Within different tissues macrophages are associated with different identities, such as the microglia of the brain, alveolar macrophages of the lung, and Kupffer cells of the liver. Furthermore, populations of macrophages of different origins are involved in disease states [2,3]. For example, macrophages involved in atherosclerotic lesions primarily arise from monocyte infiltration from the bloodstream [4]. In contrast, macrophages

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influencing obesity originate from a combination of monocyte infiltration as well as from tissue resident populations [5]. Because of the diversity of macrophage populations, it is important to consider how the microenvironment surrounding these cells influences their functions. Historically, macrophages are divided into two generalized groups based on their activation states: pro-inflammatory (M1), and anti-inflammatory (M2) macrophages. As our understanding of macrophage differentiation grew, it became increasingly clear that the M1-M2 dichotomy was a superficial means of defining macrophage phenotype. Scientific consensus has developed that a simple M1-M2 paradigm is not sufficient to appreciate the full range of macrophage phenotype. Instead of “an all-or-nothing” M1 vs M2 definition, it is now understood that there is a spectrum between these two extremes, where macrophages can express M1 and M2 characteristics. New classification systems have been proposed, including the splitting of macrophages into three general groupings: classically activated, regulatory, and woundhealing. The stimuli that drive macrophage differentiation have been well-defined, and it is possible to achieve a range of macrophage phenotypes in culture. This review will focus on two cytokines known to modulate macrophage polarization in particular: macrophage colony stimulating factor (M-CSF) and granulocyte macrophage stimulating

Corresponding authors at: Department of Biomedical and Molecular Sciences, Queen’s University, Botterell Hall, Kingston, ON K7L 3N6, Canada. E-mail addresses: [email protected] (S. Basta), [email protected] (K. Gee).

https://doi.org/10.1016/j.cyto.2019.154939 Received 6 June 2019; Received in revised form 19 November 2019; Accepted 20 November 2019 1043-4666/ © 2019 Elsevier Ltd. All rights reserved.

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been implicated in a variety of diseases, ranging from inflammatory and autoimmune diseases to viral or bacterial infection [26–30]. GM-CSF is another cytokine with a variety of immune functions. GM-CSF is capable of differentiating both granulocytes and macrophages from murine bone marrow progenitor cells. In contrast to MCSF, which is constitutively expressed in circulation, GM-CSF is expressed at low levels in circulation under homeostatic conditions and is induced under inflammatory conditions [31]. GM-CSF is produced by a variety of cells, and signals through its receptor via STAT5, ERK, AKT, and NF-κB [22]. GM-CSF is generally regarded as a proinflammatory cytokine, as such it induces increased production of proinflammatory cytokines such as TNF and IL-6 by monocytes and macrophages. It does not seem to be an essential cytokine for overall macrophage differentiation, as GM-CSF knockout mice have normal amounts of these cells in many tissues [32]. However, it is essential for alveolar macrophage maturation, with knockout mice developing pulmonary alveolar proteinosis as a result [32]. It has been proposed that GM-CSF plays a role in hematopoietic cell differentiation and proliferation while modulating the function of mature hematopoietic cells [22]. Furthermore, in vitro culture of monocytes with GM-CSF has been used as a model for DC differentiation [33]; however, others have reported transcriptome analysis that suggests these cells are heterogeneous may more closely resemble macrophages and consequently are likely a mix of both macrophages and DCs [34–36] (Fig. 1). Moreover, GM-CSF can also be used to culture proinflammatory M1 cells in vitro [37] and is associated with M1 macrophage polarization in vivo [38] (Fig. 1). Much like MCSF, GM-CSF has been implicated in a variety of diseases, and is being explored as both a potential therapy itself and as a potential target for therapy[39]. Because of these contrasting CSF-dependent differentiation states, the opposing effects of M-CSF and GM-CSF on macrophages has been an area of research interest. These effects range from differing expression of cell surface molecules to the expression of different genes in specific disease states [40,41]. Examples of M-CSF and GM-CSF acting in opposition to each other when administered to macrophages have been observed in vitro, resulting in a “hybrid” of differentiation states, capable of producing cytokines associated with differentiation by each CSF [37,42] (Fig. 1). As well, research demonstrating that GM-CSF exerts a stronger “pull” on macrophages than M-CSF, further highlighting the importance of gaining a better understanding of the potential competition between the two cytokines in vivo.

factor (GM-CSF). M-CSF and GM-CSF are tightly regulated cytokines with a plethora of roles in the immune system. Their biology has been extensively reviewed, as have their immune functions [6–13]. M-CSF, also known as Colony Stimulating Factor 1 (CSF-1), is a homeostatic cytokine that exercises a variety of effects on monocytes, macrophages, and bone marrow progenitor cells, among others. Signaling through the MEK, PI3K, and PLC-γ2 pathways, the M-CSF receptor is capable of affecting the proliferation, differentiation, and survival of these cells thanks to its intrinsic tyrosine kinase activity [14]. In terms of macrophage differentiation, this cytokine has been deemed a significant contributor to differentiation and maintenance of macrophage populations in a variety of tissues, as evidenced by the fact that the M-CSF receptor is one of the earliest markers of trophic macrophages during development, and that mice with non-functional M-CSF showed deficiencies in multiple macrophage populations [13,15–18]. Indeed, M-CSF is typically used in vitro to differentiate human monocytes found in circulation into macrophages as well as murine bone marrow or spleen cells into macrophages [19–21]. While M-CSF has the ability to influence a variety of cells, it can also be utilized as a means to generate M2-like macrophages in vitro [22,23] (Fig. 1). There is some debate as to the status of these MCSF derived cells being truly M2-like, with data in human cells showing that M-CSF treated macrophages can still polarize towards an M1 phenotype upon stimulation with IFNγ/LPS [24]. As such, it has been proposed that a “pro-M2” status is a more fitting term for macrophages treated with M-CSF. When considering the in vivo context, M-CSF has a been proposed as a promoter of M2 polarization due to its homeostatic expression coupled with the general M2-like phenotype of resident macrophage populations under normal conditions [1,25]. M-CSF has

2. The impact of M-CSF and GM-CSF on Macrophages in disease 2.1. M-CSF And GM-CSF in autoimmune and inflammatory diseaseassociated macrophages Macrophages play a significant role in the development of many autoimmune and inflammatory diseases, such as arthritis, nephritis, and atherosclerosis. The role of these cells in such conditions has been recently reviewed [43,44]. An abundance of proinflammatory M1 cells have been associated with inflammatory diseases such as atherosclerosis and obesity, and can even signal poor outcomes in these diseases [43]. It has also been demonstrated that aging causes a shift in the M1/M2 balance of macrophages towards M1 differentiation states [44]. Because both M-CSF and GM-CSF play roles in macrophage polarization, they have an impact on autoimmune and inflammatory diseases in which macrophages are centrally involved.

Fig. 1. Overview of relationship between GM-CSF and M-CSF. (1) Murine bone marrow macrophages or human monocytes treated with M-CSF differentiate into macrophages with an M2-like phenotype, while those treated with GM-CSF differentiate into a mixed population of dendritic cells (DC) and M1-like macrophages. (2) In the presence of both M-CSF plus GM-CSF, cells differentiate into macrophages with a gene signature similar to those differentiated in the presence of GM-CSF alone. Polarized cells exhibit plasticity in that cells cultured with M-SCF that are then treated with GM-CSF exhibit a pro-inflammatory phenotype (3), while cells initially cultured with GM-CSF followed by exposure to M-CSF and RANKL differentiated into osteoclasts (4). Furthermore, simultaneous addition of M-CSF plus GM-CSF to cells previously polarized with either M-CSF or GM-CSF also exhibit plasticity (5).

2.1.1. CSFs & arthritis A prime example of the impact of CSFs on inflammatory disease is the involvement of M-CSF in arthritis. M-CSF is known to regulate macrophage development and assist in the differentiation of osteoclasts from macrophages in concert with RANKL [45–47]. Thus, it can be assumed that M-CSF expression in arthritis would affect macrophage differentiation and osteoclastogenesis. The influence of M-CSF in this 2

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the only form of inflammatory kidney disease however, nor is M-CSF the only CSF involved in the progression of disease. It has also been found that GM-CSF is important in murine crescentic glomerulonephritis, as GM-CSF deficient mice were protected from the development of disease, showing lower T-cell recruitment, CD40 + glomerular cells, TNF expression, and IFN-gamma expression when compared to wild type [65].

context could make it a contributor to the imbalance of bone homeostasis and bone erosion by osteoclasts seen in this disease [48]. On their own, M-CSF and RANKL are known to be insufficient for osteoclastogenesis, requiring costimulatory factors to fully activate the process [49]. However, this does not make M-CSF nonessential in this process, as it is needed for RANKL receptor upregulation in pre-osteoclasts [50]. M-CSF can therefore be viewed as an upstream enabler of osteoclastogenesis, and in turn an enabler of the characteristic imbalance of bone homeostasis in arthritis. It thus follows that an overabundance of M-CSF would increase disease severity. In fact, M-CSF administration has been shown to exacerbate collagen induced arthritis in animal models, and that endogenous M-CSF was required for collagen induced arthritis development [51]. M-CSF has also been associated with the production of inflammatory cytokines and the progression of rheumatoid arthritis in the murine model, collagen-induced arthritis [51]. As such, blocking the action of M-CSF in this disease has become an area of interest, seeking to prevent its induction of proinflammatory cytokines and the differentiation of macrophages into osteoclasts that advance disease. In support of this, early experiments with M-CSF deficient mice and mice treated with M-CSF neutralizing antibody showed no progression of collagen induced arthritis, validating M-CSF as a potential therapeutic target for arthritis [51]. More recently, blockade of the M-CSF receptor has been explored as a treatment, demonstrating success as an anti-inflammatory agent [28,52]. Moreover, targeting M-CSF holds potential for decreasing arthritis-associated pain [27,53]. GM-CSF also contributes to the inflammatory sequelae of rheumatoid arthritis. GM-CSF levels are elevated in the synovial fluid of rheumatoid arthritis patients [26,54]. It has also been implicated in the progression of RA pathogenesis through the activation, differentiation and survival of disease associated macrophages [55]. Administration of GM-CSF to treat Felty’s syndrome has been observed to cause flare-ups of arthritis [56]. In animal models of arthritis, GM-CSF receptor signaling has a role in disease activity [57]. Additionally, GM-CSF produced by bone marrow-derived cells is required for the development of collagen induced arthritis, and GM-CSF absence reduced disease severity in the serum-transfer arthritis model [58]. Targeting of GM-CSF for treatment of rheumatoid arthritis has therefore been an area of interest. Blockade of the GM-CSF receptor with the monoclonal GM-CSF receptor alpha monoclonal antibody mavrilimumab has shown promising results, with significant reductions in rheumatoid arthritis activity observed one week after treatment in a phase II clinical trial on human patients that were inadequately responsive to current disease modifying therapies [59].

2.1.3. CSFs & atherosclerosis Another inflammatory disease where the role of M- and GM-CSF and macrophages have drawn research interest is atherosclerosis. The role of macrophages in atherosclerosis has been recently reviewed, as has the role of CSFs [66,67]. Macrophages are inexorably linked to the development and progression of atherosclerosis and are considered the central inflammatory cell in this disease [4]. In the early stages of the disease, monocytes are recruited by the presence of oxidized LDL in the subendothelial space as well as lipoproteins that have undergone modification, rendering them proinflammatory stimuli [4]. These recruited monocytes differentiate into phagocytic cells that ingest the lipoproteins to become foam cells, a type of lipid-rich macrophage which exhibits impaired migration and can secrete proinflammatory cytokines [4]. Foam cells contribute to atherosclerotic lesion development; however the foam cells themselves exhibit a deactivated gene profile, and are not proinflammatory [68,69]. Further development of atherosclerosis is also dependent upon macrophage activity. Macrophages that undergo apoptosis in the lipid core of an atherosclerotic lesion are cleared by other macrophages and phagocytic cells through efferocytosis [70]. While efferocytosis is able to maintain clearance of dead cells temporarily, cell death outpaces the process in chronic lesions, leading to the formation of a necrotic core to the lesion and advancing disease progression [70,71]. There are many subsets of macrophages that have been identified in the context of atherosclerosis. However in general terms, M1 macrophages are considered to be associated with the advancement of atherosclerosis, while M2 macrophages are thought to contribute to plaque stability and the management of apoptotic M1 cells via efferocytosis [66]. The relationship of M-CSF and GM-CSF to atherosclerosis has also been explored due to their influence on macrophage differentiation and polarization. While M-CSF is expressed in healthy arteries, it is elevated in patients with chronic stable coronary heart disease, making it a potential biomarker for the disease [72]. Additionally, GM-CSF is constitutively expressed in arterial smooth muscle and endothelial cells, though this expression is significantly upregulated in atherosclerotic lesions, and can vary as the disease advances [73]. As such, there is evidence that the ratio of GM-CSF and M-CSF can vary over the course of atherosclerotic disease, which may therefore be able to impact the local milieu of stimuli influencing the differentiation and polarization of macrophages in atherosclerotic lesions. Both M- and GM-CSF contribute to disease progression. Mice deficient in M-CSF and apolipoprotein E expression had a 7-fold decrease in atherosclerotic lesion size compared to controls [30]. Meanwhile, GM-CSF promotes intimal macrophage proliferation in nascent atherosclerotic lesions, in addition to increasing inflammatory cytokine production by macrophages [74,75]. However, the roles of these cytokines may change depending on the stage and context of an atherosclerotic lesion. The ability of MCSF to increase monocyte numbers and promote differentiation of proM2 macrophages may be essential to the maintenance of effective efferocytosis, preventing the development of a necrotic core and therefore stalling progression of disease. Such activity would provide a direct counter to GM-CSF promotion of macrophage apoptosis and necrosis in advanced lesions [76]. Therefore it is key to understand the effects that these cytokines have together on macrophages involved in atherosclerosis, as their beneficial or detrimental effects may change depending on their concentration relative to the other CSF or on the stage of disease. In fact, in vitro treatment of macrophages with varying ratios of GM- and M-CSF has revealed that these cytokines act in opposition

2.1.2. CSFs & lupus nephritis Nephritis is also linked to CSF activity, particularly in terms of macrophage differentiation. In mice, higher levels of circulating M-CSF accelerate the progression of lupus nephritis, increasing the number of monocytes in circulation, and skewing these monocytes towards an inflammatory phenotype when they differentiate into macrophages [60]. In the more generalized systemic lupus erythematosus (SLE) disease, M1 macrophages increase disease severity, while M2 macrophages reduce it [61]. M-CSF in mouse models of lupus nephritis was found to expand M1 macrophages, which mediated defective renal repair and contributed to non-resolving inflammation, resulting in a faster onset of lupus nepthritis [62]. Additionally, mice with higher levels of circulating M-CSF showed increased accumulation of intrarenal macrophages which led to more damage to kidney tissue [60]. Given the association of M-CSF with progression of lupus nephritis, this cytokine may serve as a possible biomarker for disease [63]. Furthermore, modulating the balance of proinflammatory M1 macrophages and M2 anti-inflammatory macrophages has been explored as a potential treatment for lupus, using antibodies that selectively induce M2 differentiation and ultimately block autoimmunity in a murine model of spontaneous systemic lupus erythematosus [64]. Lupus nephritis is not 3

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of interest for HIV reservoir elimination [87,88]. It has been determined that M-CSF treatment of macrophages in vitro sensitized HIV-1 infected macrophages to TRAIL-mediated killing, making M-CSF a candidate for treatment to eliminate macrophage reservoirs [89]. Such findings are promising, but highlight the need for greater understanding of M-CSF’s impact in living models and its interactions with other macrophage stimuli such as GM-CSF. The other cytokine of interest to this review, GM-CSF, also has some associations with HIV infection. On its own, high concentrations of GMCSF in the bloodstream have been associated with the prolonged maintenance of CD4 + cell count above 350 cells/µL [90]. With regards to macrophages involved in HIV infection, addition of GM-CSF inhibits HIV replication within monocyte-derived macrophages in vitro [91]. However, a more recent study in cells differentiated with M-CSF or GM-CSF has found no effect of these cytokines on HIV replication in monocyte derived macrophages cultured in vitro [92]. The key difference between these studies is whether GM-CSF was present during differentiation or after cells were allowed to differentiate in culture. As such, it appears that GM-CSF is only able to inhibit the replication of HIV in macrophages that have already differentiated. It is therefore important to explore the effects of this cytokine and any interactions it may have with M-CSF as a possible candidate to modify the progression of HIV disease. Understanding the interactions of GM-CSF and M-CSF in vivo would also be critical to the effective understanding and use of MCSF targeting therapies described previously. GM-CSF itself has also been explored for its potential therapeutic value in preventing HIV infection. This is in contrast to inflammatory and autoimmune diseases where it has often been targeted, rather than administered or promoted. The use of GM-CSF as an adjuvant to a vaccine has shown promise in experimental HIV vaccines, along with those intended for other viral infections such as Ebola and HPV [93,94].

regarding the expression of several genes relevant to atherosclerotic disease [40]. Clearly, both GM-CSF and M-CSF are relevant factors in the development and progression of several inflammatory and autoimmune diseases. The roles each of these cytokines have been studied primarily in vitro and rarely in concert with each other, a complication that may alter our understanding of their roles in these diseases. Further study into the interaction of these cytokines both in vitro and in vivo is essential to the further development of these cytokines as potential targets for treatment or their use as pharmacological treatments. 2.2. M-CSF and GM-CSF macrophages in HIV infection Both M-CSF and GM-CSF are implicated in influencing macrophage responses to viral infection. The effects of these cytokines have been explored in several different viral diseases, but an area of interest for recent research has been their relationship with macrophages in the context of HIV infection. Macrophages themselves are involved in almost every stage of HIV infection, and their relationship has been recently reviewed in detail [77]. In terms of macrophage polarization, macrophages polarized in vitro to an M1 state are able to resist HIV infection at a preintegration stage, while cells polarized to an M2a state in vitro are capable of inhibiting viral replication at a postintegration stage [78]. Given the flexibility of macrophage polarization, with cells able to switch from one polarization state to another with changes in stimulatory factors, HIV may manipulate this to better establish infection. In fact, it has been observed that HIV pushed macrophages to a pro-inflammatory polarization state, even those that held an anti-inflammatory state previous to HIV exposure [79–81]. Such an adaptation would be advantageous to HIV, as switching macrophages from an anti-inflammatory M2 state to a proinflammatory M1 state would provide a more permissive environment for productive HIV replication post-integration in these cells. HIV is not able to influence differentiation with impunity however, as the progression of macrophage involvement in HIV infection is also influenced by the host immune system. A recently reviewed model of macrophage phenotype during HIV infection proposed M1 macrophages are associated with establishment of viral reservoirs which eventually give way to M2 macrophages, halting the expansion of reservoirs, and eventually culminating in IL-10-mediated macrophages deactivation during immune failure at the late stages of HIV infection [82]. Due to the multiple shifts in macrophage polarization and activation over the course of HIV infection, GM-CSF and M-CSF are constituents of the cytokine milieu influencing macrophages in the context of this disease. In the case of M-CSF, production of this cytokine by macrophages is associated with viral replication in vitro [83]. The MCSF−HIV relationship varies from tissue to tissue, at least in the context of macrophage differentiation. For example, HIV-1 Tat protein enhances the M-CSF/RANKL mediated differentiation of monocytes into osteoclasts, making it a factor in the development of HIV related osteoporosis [84]. Another HIV protein, Nef, interferes with M-CSF receptor signaling, and downregulating the expression of the M-CSF receptor via interaction with the endogenous Src tyrosine kinase Hck in vitro [85,86]. In effect, HIV may be playing both sides, using proteins that both enhance or abrogate M-CSF effects, modulating macrophage differentiation and polarization in an advantageous way regardless of the tissue environment. These examples show contexts in which M-CSF plays a role as a possible endogenous countermeasure to the effects of HIV on macrophages in brain tissue, but an unintentional accessory to pathogenesis via monocytic differentiation in bone. Because of these documented interactions of M-CSF and HIV in macrophages, M-CSF has been investigated as a possible target for antiHIV therapy. One of the major obstacles to achieving a cure for HIV has been elimination of viral reservoirs, and because macrophages are among the cells capable of serving as reservoirs, they have been targets

2.3. Summary of M-CSF and GM-CSF contributions to disease As can be seen in Table 1, macrophages and CSFs are involved in a variety of diseases, both inflammatory and viral. Interestingly, it appears that both M-CSF and GM-CSF have similar effects in the context of diseases. Both cytokines are often seen to be elevated in disease contexts, and often both cytokines are associated in disease progression. The similarity of the influences these cytokines have on diseases seems to fly in the face of the established effects of these CSFs in vitro, where M-CSF promotes M2 polarization associated with reducing the progression of inflammatory disease while GM-CSF promotes M1 polarization associated with disease progression. The apparent conflict between the in vitro effects of these cytokines on macrophages and their in vivo effects on diseases highlights the necessity of further study to understand the interplay of these CSFs, especially in the larger context of in vivo models. It should also be noted that the majority of the research extant on the CSFs in the context of diseases presented in this review has been the detection of correlation between CSFs and the disease, rather than detailed mechanistic investigations. This represents a further gap in the knowledge of the roles for M-CSF and GM-CSF in disease states, and highlights the need for the further understanding of the potential that these cytokines have for treatments and for enhancing our understanding of the innate immune system. 3. Potential for an antagonistic or competitive relationship between M-CSF and GM-CSF Evidence of cytokine-cytokine antagonism has long-since been established, examples of which include the anti-inflammatory nature of IL-10 functioning to antagonize proinflammatory cytokines like IL-6 or TNF-α [95–97]. However, the potential for competition or antagonism between the CSFs is relatively understudied. As GM-CSF and M-CSF are both naturally occurring cytokines, monocytes likely encounter both simultaneously while differentiating into macrophages. Previous 4

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Table 1 Overview of M-CSF and GM-CSF in disease. Disease Arthritis

Lupus Nephritis

Atherosclerosis

HIV

M-CSF

GM-CSF

for arthritis development in mouse models [51] • required exacerbates animal models of arthritis[51] • administration of M-CSF/M-CSF receptor beneficial[27,28,51–53] • blockade circulating levels accelerates disease progression [60] • increased M-CSF promotes M1 differentiation while treatment to induce M2 • differentiation is beneficial [62,64] presence in patients with coronary heart disease [72] • elevated • M-CSF deficient mice showed smaller atherosclerotic lesions [30] by macrophages associated with viral replication [83] • production treatment sensitizes infected macrophages to TRAIL-mediated killing • M-CSF in vitro [89]

for arthritis development in mouse models [58] • required causes flare-ups [56] • administration of GM-CSF receptor beneficial [59] • blockade in development of crescentic glomerulonephritis in mice [65] • important • GM-CSF deficient mice protected from disease [65] expressed in arterial tissue, but significantly upregulated • constitutively in lesions [73] deficient mice exhibited lower macrophage apoptosis and necrosis • GM-CSF [76] concentrations in circulation associated with maintenance of • high CD4 + cell count [90] of GM-CSF in vitro prevents HIV replication in macrophages [91] • addition evaluated as a possible vaccine adjuvant for HIV and other viral • being diseases [93,94]

M-CSF [37]. The reverse experiment was also completed with GMBMDMs differentiating over the course of 7 days receiving a similar 16hour treatment with or without M-CSF in a 1:1 ratio with GM-CSF [37]. In M-BMDMs that were exposed to GM-CSF, no changes in CD11c, TLR4, and TLR2 expression were observed [37]. However, the basal mRNA levels of TNF-α, IL-12p40, and IL-23p19 increased, while levels of IL-10 mRNA decreased [37]. In contrast, GM-BMDMs exposed to MCSF had lowered basal levels of TNF-α, IL-12p40, and IL-23p19 mRNA, with a raised level of CCL2 mRNA [37]. As such, it is clear that even before activation by LPS, macrophages exposed to M-CSF and GM-CSF simultaneously display differences in cytokine mRNA expression compared to those treated with the CSF used to differentiate them. Thus, the idea that M-CSF and GM-CSF act in opposition to each other in macrophage differentiation is further supported by these experiments. Fleetwood et al. also found that M-BMDMs treated with GM-CSF that were subsequently activated by LPS stimulation produced greater amounts of TNF-α, IL-12p70, IL-12p40 and IL-23 than untreated MBMDMs [37]. Interestingly, the levels of IL-12p70 and IL-23 produced in the GM-CSF treated M-BMDMs were lower than that of GM-BMDM cells, and the levels of IL-10 and CCL2 production which defined MBMDMs were unaffected by GM-CSF treatment [37]. These findings indicate that GM-CSF does not have a complete dominance over M-CSF, forcing an increase in proinflammatory cytokines while remaining unable to depress anti-inflammatory cytokines when both are present. In contrast, when GM-BMDMs were exposed to M-CSF, production of TNF-α, IL-12p70, and IL-23 decreased, while CCL2 production increased and IL-10 production was not impacted [37]. Such findings complicate the relationship between GM- and M-CSF because M-CSF appears to be able to suppress production of cytokines that defined GMBMDMs, indicating some ability to overcome the effects of GM-CSF driven differentiation. However, M-CSF was only able to increase the production of one cytokine associated with M-CSF differentiation, CCL2, while unable to influence the other, IL-10. In total, a partial dominance of GM-CSF over M-CSF seems to be the case, as GM-CSF was able to increase production of its cytokines unimpeded in M-BMDMs, while M-CSF could not increase all of its associated cytokines in GMBMDMs. Further experiments to define this relationship were conducted by Lacey et al in 2012. These experiments evaluated the gene expression profiles of human and murine cells [42]. To evaluate the effects of CSF co-addition, M-BMDMs were starved of M-CSF overnight and then treated for 16 h with M-CSF, GM-CSF, or both at concentrations of 2500 U/mL and 5 ng/mL respectively. M-BMDMs receiving GM-CSF alone further confirmed the experiments by Fleetwood et al, showing similar gene expression to GM-BMDMs and exhibiting increased gene expression of proinflammatory cytokines such as TNF, IL-6, and IL-12p35 among others [42]. Cells that received both M-CSF and GM-CSF showed

studies have found evidence that the ratio of M-CSF to GM-CSF varies in vivo over the course of inflammatory diseases such as coronary heart disease and angina pectoris, along with infection with pathogens such as M. tuberculosis [29,98,99]. Given the ability of GM-CSF and M-CSF to stimulate an M1 or pro-M2 phenotype in macrophages respectively, it follows that these two cytokines would function in competition, pulling the nascent monocyte/macrophage towards the two different phenotypes simultaneously. When administered individually, GM-CSF and M-CSF have opposing effects on macrophages. In murine macrophages, GM-CSF increased elastase production while M-CSF in contrast inhibited it on a transcriptional level [100]. In one of the more dramatic demonstrations of GM-CSF and M-CSF opposing effects, it was found that GM-CSF derived bone marrow macrophages (GM-BMDM) can be converted to osteoclasts by M-CSF and RANKL just as effectively as their M-CSF derived bone marrow macrophage (M-BMDM) counterparts [101]. This switch to an osteoclast phenotype did require 21 days of culture without GMCSF and in the presence of M-CSF and RANKL [101]. Such a dramatic shift in phenotype indicates that M-CSF is capable of overcoming the effects of GM-CSF with regards to macrophage differentiation. However, these experiments completely removed GM-CSF during the osteoclast differentiation process, so they cannot be viewed as an indicator of the relationship between GM- and M-CSF in vivo. As such, these experiments are a testament to the plasticity of macrophages but are not indicative of the dominance of one CSF over the other in vivo. Additionally, GM-CSF induced MHC II expression while M-CSF inhibits such expression when administered to macrophages on their own [41]. These findings by Willman et al are of particular note because additional experiments where macrophages treated simultaneously with GM-CSF and M-CSF showed that expression levels of MHC II were similar to those of cells that only received M-CSF treatment [41]. These experiments represent some of the earliest evidence of a direct antagonistic relationship between M-CSF and GM-CSF concerning their effects on macrophages. Further investigations examined the nature of macrophage function in the presence of both GM-CSF and M-CSF. As discussed below, experiments conducted by Lacey et al. and Fleetwood et al. investigated the effects of exposing M-CSF derived bone marrow derived macrophages (M-BMDMs) and GM-CSF derived bone marrow derived macrophages (GM-BMDMs) to equal amounts of GM- and MCSF in advance of further stimulation [37,42]. While the aforementioned experiments by Willman et al were among the first to employ the simultaneous treatment of macrophages with GM-CSF and M-CSF, experiments conducted by Fleetwood et al defined the competitive relationship of these two cytokines in detail [37,41]. In these experiments, M-BMDMs differentiated over the course of 7 days were cultured for an additional 16 h with or without GM-CSF while maintaining M-CSF supplementation for a 1:1 ratio of GM-CSF to

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data, it is exceedingly difficult to determine whether murine macrophages would serve as a reliable model for human macrophage differentiation. The effects of simultaneous M-CSF and GM-CSF addition during murine macrophage differentiation has not been explored to the same extent as in human macrophages, nor has the effect of differing ratios of M-CSF and GM-CSF, which also complicates the use of these cells as a model. A further complicating factor in the evaluation of the differing effects of these CSFs on macrophages and their applications to diseases lies in the fact that they are not the only factors exerting influence on macrophages during differentiation and polarization. A prime example of another factor that could shift the balance of this GMCSF vs. M-CSF relationship in an in vivo setting is IL-34, which shares a receptor with M-CSF. Furthermore, elevated IL-34 expression is associated with several inflammatory conditions [102]. Interestingly, IL-34 has been shown to be as efficient as M-CSF at inducing IL-10-producing macrophages in vitro from PBMCs, which could be prevented in the presence of GM-CSF or IFNγ [103]. Differences in the ability of IL-34 vs. M-CSF to polarize macrophages have been highlighted [104], indicating that the two cytokines have non-redundant roles. Given the fact that IL-34 promotes an immunosuppressive M2 macrophage phenotype, and that this is countered by GM-CSF, it is possible that the presence of IL-34 and M-CSF in the context of a disease could complicate efforts to use GM-CSF to alter macrophage phenotype and disease status.

a more complex relationship between the CSFs in terms of gene expression. These cells had a gene expression profile more similar to that of M-BMDMs, with expression of 63% of the genes upregulated by GMCSF reversed by the presence of M-CSF [42]. The narrative of GM-CSF’s partial dominance over M-CSF appears to be strengthened by these results. GM-CSF is able to alter the cytokine production of M-BMDMs towards a more proinflammatory profile, but is unable to achieve a complete conversion of phenotype at least in terms of gene expression. The experiments by Lacey et al provided a wealth of new information on the effects of M-CSF and GM-CSF acting in concert on pre-differentiated murine macrophages. Further study of these effects could potentially be useful in seeking to moderate diseases where M-CSF or GM-CSF have already been implicated. There remain several unanswered questions however, as it is unclear what the effects of both CSFs on GM-BMDMs would be, nor is it known what the impact of CSF co-addition during differentiation would be. Lacey et al provide some data to answer the latter question, albeit in human cells. They demonstrated that cells differentiated in the presence of both GM-CSF and M-CSF at 5 ng/mL and 2500 U/mL respectively had gene expression profiles similar to that of human cells differentiated in GM-CSF alone [42]. The overall effect of co-addition was relatively modest, with only 10% of the differentially expressed genes between M-CSF derived cells and GM-CSF derived cells modulated by both CSFs [42]. These findings reinforce the growing narrative of an incomplete dominance of GM-CSF over M-CSF, with cells being pulled towards a GM-CSF derived state when cultured in both CSFs. These results would seem to suggest a stronger dominance of GM-CSF over MCSF in humans than in mice; however, it should be noted that these experiments are not directly comparable. Murine cells in this study were never subjected to both CSFs during their 7-day differentiation culture, and the M-BMDM-like state of cells treated with both CSFs for 16 h could simply represent the beginning of a transition to a GMBMDM-like state. Further study on both human and murine cells during and post differentiation would help to clarify the similarity, if any, between humans and mice regarding the GM-/M-CSF relationship. An important step has been taken in seeking to define the effects of CSF co-addition during macrophage differentiation. Experiments carried out by Brochériou et al. examined the effects of culturing macrophages in varying ratios of M-CSF and GM-CSF on various genes associated with atherosclerosis and macrophage phenotype [40]. The expression of some genes, such as selenoprotein-1 (SEPP1), stabilin-1 (STAB1), a secreted disintegrin metalloproteinase family member ADAMEC, pro-platelet basic protein (PPBP) and CD163 molecule-like-1 (CD163L1) were found to be significantly impacted by a ratio of MCSF:GM-CSF as little as 10:1, when compared to M-CSF−only culture conditions [40]. Other genes, such as MRC1 and M-CSF were not significantly impacted until M-CSF:GM-CSF ratios were raised to 10:5 or 5:10, respectively [40]. These findings provide clear evidence towards a competitive relationship between M-CSF and GM-CSF, in the in vitro context, and serve to provide some context as to the possible dominance of one CSF over the other. It does not appear that differing ratios of GMCSF to M-CSF have been studied beyond these experiments by Brocheriou et al., however their findings [40] should encourage further study in this area, particularly with regard to the phenotype of macrophages exposed to both CSFs in varying quantities. So far there is early evidence of an incomplete dominance of GMCSF over M-CSF in the polarization of macrophages, and the impacts of these cytokines alone and in tandem are summarized in Fig. 1. It should be noted that studies of co-administration of these CSFs and their possible antagonism or competition have been exclusively conducted in vitro. Thus, the competitive relationship between these cytokines needs to be further investigated in in vivo settings. Such studies will be particularly important to reconcile the differences between the in vitro studies and the documented roles of these cytokines in vivo, where the effects of GM-CSF and M-CSF in disease appear to be similar as discussed above in Section 2.3. Additionally, with the currently available

4. Summary and significance The cytokines GM-CSF and M-CSF have been identified as critical influencers of macrophage development and differentiation. Due to these properties, they also play essential roles in the development of inflammatory and autoimmune disease, along with the pathogenesis of viral infections. As a result, these cytokines and the macrophages they influence are promising targets for therapy in these disease settings. Opposing effects on macrophage differentiation by these two cytokines have been well-documented. However, our understanding is limited by study of these cytokines effects in isolation. Early work with these cytokines acting on macrophages simultaneously shows competition between them, and a possible partial dominance of GM-CSF over M-CSF. Further study to define this relationship, especially using in vivo approaches where the action of these CSFs in the context of other stimuli that impact macrophage polarization, would better inform the impact of the CSF milieu in vivo on macrophage differentiation, and thus disease progression. A better understanding of the M-CSF – GM-CSF relationship will also serve to refine use of these cytokines as treatments or targets for treatment in disease. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by funding from the Faculty of Health Sciences at Queen’s University Spear and Start award to KG and from the Natural Sciences and Engineering Research Council of Canada to SB. Author contributions ET, SB, and KG conceptualized and wrote the review. References [1] L.C. Davies, S.J. Jenkins, J.E. Allen, P.R. Taylor, Tissue-resident macrophages, Nat.

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