Flt3⁺ macrophage precursors commit sequentially to osteoclasts, dendritic cells and microglia

The mononuclear phagocyte system encompasses a widely distributed family of related cells exhibiting highly specialized functions such as macrophages, osteoclasts, dendritic cells, and microglia. Resident macrophages, found in most organs and connective tissues, serve as professional phagocytes, removing pathogens or apoptotic cells [1]. Microglia represents a unique category of mononuclear phagocytes distributed throughout the central nervous system (CNS) parenchyma in both white and grey matter [2]. Microglial cells share a number of immunological markers with other mononuclear phagocytes, yet they present a unique ramified morphology, which is best characterized by electron microscopy [3]. Besides their role as immune effectors of the CNS, microglial cells exert non-immunological functions, including production of neurotrophic factors and glutamate uptake [4, 5]. Dendritic cells (DCs) are specialized in capturing antigens and initiating immune response through naive T-cell activation [6], and are also implicated in maintaining tolerance to self-antigen [7]. The diverse functions of DCs in immune regulation are dictated by the instructions they received during innate immune responses to different pathogens, but DC response may be also lineage-dependent as distinct myeloid and lymphoid DC lineages have been recently identified [8]. Osteoclasts are multinucleated, adherent, bone-resorbing cells found in the bone vicinity. They play an essential role in bone remodelling, as well as in regulating calcium homeostasis [9].

Functional and phenotypic heterogeneity within the mononuclear phagocyte system may reveal differentiation plasticity of a common progenitor, but the developmental pathways leading to macrophages, osteoclasts, DCs, or microglia are still unclear. Cell transfer experiments have established that alveolar macrophages in the lung, and Kupfer cells in the liver, derive from mature monocytes [10]. Late monocyte precursors have been shown to differentiate into osteoclasts in response to macrophage colony-stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL) [9, 11]. Similarly, DCs could be derived from circulating human monocytes following stimulation with GM-CSF and interleukin-4 [12], or from human CD34⁺ myeloid progenitors using GM-CSF and tumor necrosis factor-α (TNFα) [13]. Furthermore, bone marrow progenitors were recently identified through their ability to differentiate into DCs or osteoclasts, depending on whether RANKL was combined to granulocyte-macrophage colony stimulating-factor (GM-CSF) or M-CSF, respectively [14]. Whereas microglia has been well characterized in vivo and in vitro, little is known about microglial precursors, and attempts to induce in vitro microglial differentiation of mature or immature monocytes have been unsuccessful [3, 15]. However, recent in vivo studies have shown that microglia and other CNS resident cells (macroglia and neurons) are continuously renewed from as yet uncharacterized progenitors originating from the adult bone marrow which are, themselves or their progeny, able to cross the blood-brain barrier and give rise to mature microglia [16, 17]. Altogether, data from the literature plead for the myeloid origin of macrophages, osteoclasts, DCs, and microglia. However, a global approach proved necessary for delineating lineage relationships within the mononuclear phagocyte system. In particular, learning how cellular diversity is generated in this cellular system is of great importance regarding cellular or gene therapy protocols.

Here we describe ex vivo expansion of murine myeloid cells in response to Flt3 ligand (FL) and investigate their response to cytokines inducing differentiation towards macrophages (M-CSF), osteoclasts (M-CSF plus RANKL), dendritic cells (GM-CSF plus TNFα), or microglia (glial-cell conditioned medium: GCCM). Results support a model based on the sequential commitment of Flt3⁺ bone-marrow progenitors.

The culture system described here is to our knowledge the first to enable mass selective production of pure osteoclasts, pure CD8α^- DCs, and pure macrophages, thus providing a unique tool to study primary cells from the mononuclear phagocyte system. Indeed, bone marrow from a single mouse (7–10 × 10⁷ cells) currently yields 1–2 × 10⁷ osteoclasts, 2–3 × 10⁸ DCs, or 1–2 × 10⁸ macrophages. In particular, this protocol will facilitate investigations on genetically modified cells obtained from transgenic or knockout mice, or following retrovirus-mediated cell transfer.

CFSE monitoring of cell division demonstrated that most day 6-FL cells are precursors of day 8-FL cells. Both cell types were shown to be macrophage precursors endowed with osteoclast or prominent DC differentiation potential, respectively. As both proliferation and cell mortality were limited or absent during the differentiation process (not shown), we conclude that osteoclast or DC differentiation of FL cells was not preceded by preferential expansion of distinct precursor sub-populations. Thus we propose that Flt3⁺ macrophage precursors commit sequentially to osteoclast and DC differentiation, possibly through time-dependent expression of intracellular factors. Consistent with this model, Miyamoto et al. have recently demonstrated that M-CSF and GM-CSF regulate differentiation of osteoclasts and DC from a common precursor whether they express c-Fos or not, respectively [14]. It remains to be shown whether FL-expanded osteoclast precursors are identical or not to those previously described by Arai et al. [11].

The protocol described in the present paper also represents a powerful tool for studying microglia differentiation control mechanisms. It now becomes possible to determine which soluble factors in GCCM induce microglia differentiation. In addition to macrophages and microglia, day 11-FL cells could also differentiate to DCs (30 to 40%), though less efficiently than day 8-FL cells (> 90%). It was recently suggested that native microglia are uncommitted precursors of DC and macrophages [23]. However, it is not clear whether this results from microglia plasticity or de-differentiation to an uncommitted precursor. In that respect, day 11-FL cells, that have no microglia characteristics, may represent a valuable model to delineate the relationship between DC and microglia pathways.

Facilitating in vitro studies on microglia differentiation may help to shed some light on several physiopathological processes. Indeed, microglia is the principal immunoeffector cell located in the CNS parenchyma, and its involvement in pathogenesis has been established for numerous neurological conditions including stroke, trauma and Alzheimer's disease [24]. Furthermore, bone marrow-derived progenitors committed to microglial differentiation should be ideally suited for CNS-targeted gene therapy since they may be able to migrate from blood to brain through an intact blood-brain barrier [16, 17, 25].

The unique DC properties of either eliciting immunity or ensuring self-tolerance [7], make them candidates for enhancing responses against foreign antigens in vaccination protocols [26] and against tumor antigens in cell immunotherapy [27], as well as making them targets for the treatment of autoimmunity and allograft rejection [6]. Whether mature or immature DCs should be targeted in these various fields is a matter of debate that could be solved with the two culture conditions described, with and without the use of TGFβ. These last years, substantial progress were made to characterize distinct subsets of DCs, based on differential phenotype and function, both in humans and mice. In mice, all DC subsets described so far express CD11c [28]. CD8α homodimer expression now appears to be more related to maturation state than lineage origin [8]. Flow cytometry analysis of spleen cells from mice treated with FL for 9 days reveals five pre-DC or DC subpopulations that occur in distinct microenvironments of the spleen [29]: two putative immature populations, CD11b⁺ Ly6C⁺ Gr-1⁺ (named A, B), and three CD11c⁺ DC populations distinguished by CD8α and CD11b expressions (named C, D, E). Although phenotypic characterization of these DCs does not exactly fit to the in vitro FL-derived DCs presented in this work, latter cells are closely related to spleen DCs of myeloid origin, referred to as population C by Pulendran et al.[29].

Plasmacytoid DCs, also called interferon producing cells (IPC) were recently characterized in humans and mice [30‐33]. Both myeloid and plasmacytoid DCs were clearly expanded by FL in vivo, with or without GM-CSF mobilization [33]. Recently, Gilliet et al. demonstrated that plasmacytoid DC precursors are differentially regulated by FL and GM-CSF [20]. Though cellular phylogeny between Flt3+ progenitors and DCs become more and more clear, the relationships between Flt3⁺ bone-marrow progenitors (0.3% of total bone marrow cells), lymphoid progenitors and myeloid progenitors remains to be clarified. Concerning functional aspects, DC-dependent T-cell polarizing activity (Th1-, Th2-, Th3- or Tr-type) is not related to lineage origin, but rather depends on the stimuli used to activate these cells, probably due to their equipment and subsequent transduction of pathogen associated molecular pattern receptors, such as Toll receptor family [34].

Based on the present study and data from the literature, we propose a model where early Flt3⁺ progenitors commit themselves to a sequential differentiation program proceeding through narrow windows; each time window corresponds to expression of a specific differentiation potential (Fig. 8). A first phase of selection (hatched line), likely through apoptotic death of Flt3^- cells, would lead to Flt3⁺ cell expansion (plain line). Each time window may permit generation of different types of precursors able to differentiate into osteoclasts, then into DCs, and finally, for 25% of day 11-FL cells, into microglia cells. Moreover, FL cells can differentiate to macrophages in response to M-CSF whatever the duration of primary culture in FL.

Data obtained with transgenic FL-mice and FL deficient mice support a major role of FL in DC development [35]. In contrast, development of DC lineages is almost normal in lymphoid organs of mice deficient for GM-CSF or GM-CSF receptor [36]. It would be interesting to test whether DC differentiation from FL cultivated cells is dependent on the presence of GM-CSF, or whether it could be obtained in the presence of proinflammatory cytokines or bacteria-derived products, as recently shown [19]. Indeed, an attractive hypothesis would be that DCs represent the default cell type in the progeny of Flt3+ progenitors, unless appropriate microenvironment stimulation is added to differentiate progenitors to osteoclasts (M-CSF and RANKL), macrophages (M-CSF) or microglia (GCCM). Nevertheless, the picture emerging from our study is that diversity within the mononuclear phagocyte system is generated from a continuum of macrophage precursors whose differentiation potential might change as a function of relative ageing and cytokine environment. Whether this sequential commitment process is cell-autonomous or depends on paracrine/autocrine loops remains to be determined. However, in vivo experiments have clearly shown that tissue macrophages may differentiate into lymph-node DCs when exposed to a phagocytic stimuli [37]. Moreover, microglia could be induced to acquire specific DC or macrophage markers when exposed to GM-CSF or M-CSF, respectively [23]. Thus, mature macrophages, DCs, or microglia, may activate latent differentiation programs under appropriate stimuli. We have shown here that macrophage precursors show more limited differentiation potential, raising the attractive possibility that mature cells cells of the mononuclear phagocyte system are more capable of differentiation plasticity than their precursors.