Developmental control of macrophage function

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ScienceDirect Developmental control of macrophage function Johnny Bonnardel1,2 and Martin Guilliams1,2 The combination between novel fate-mapping tools and singlecell RNA-sequencing technology has revealed the presence of multiple macrophage progenitors. This raises the fascinating possibility that what was once perceived as immense functional plasticity of macrophages could in fact come down to separate macrophage subsets performing distinct functions because of their differential cellular origin. The question of macrophage plasticity versus macrophage heterogeneity is broader than the difference between macrophages of embryonic or adult hematopoietic origin and is particularly relevant in the context of inflammation. In this manuscript, we review the potential impact of cellular origin on the function of macrophages. We also highlight the need for novel ‘functional fate-mapping’ tools that would reveal the history of the functional state of macrophages, rather than their cellular origin, in order to finally study their true plasticity in vivo.

Addresses 1 Laboratory of Myeloid Cell Ontogeny and Functional Specialisation, VIB Centre for Inflammation Research, Ghent 9052, Belgium 2 Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Corresponding authors: Bonnardel, Johnny ([email protected]), Guilliams, Martin ([email protected])

Current Opinion in Immunology 2018, 50:64–74 This review comes from a themed issue on Innate immunity Edited by Randolph

may differ in function. It has been recently proposed that circulating monocytes consist of multiple subsets with distinct functional capacities [4,5]. If one now takes liver fibrosis as example then one obtains two different views based on the ‘macrophage plasticity model’ or the ‘macrophage heterogeneity model’. Kupffer cells are the main resident macrophages in the steady-state liver and these cells have been shown to possess the capacity to self-maintain for months in the liver [6–8] and to participate to hepatic iron recycling [9]. It is believed that Kupffer cells produce fibrogenic factors that exacerbate fibrosis and this is a typical example of potential pathogenic plasticity of macrophages [10]. But if the macrophages driving liver fibrosis are in fact derived from distinct precursors than the resident Kupffer cells, then this could also come down to heterogeneity among the liver macrophage pool between bona fide ‘resident’ Kupffer cells of embryonic origin and pro-fibrotic monocytederived macrophages recruited during fibrosis. The macrophage heterogeneity model is supported by the recent identification of a pro-fibrotic monocyte subset that has been termed segregated-nucleus-containing atypical monocytes (SatM) [5]. The possibility of pathogenic macrophage precursors opens interesting therapeutic avenues because it implies that blocking the recruitment of these cells could dampen inflammation. We here review the evidence for both macrophage plasticity and macrophage heterogeneity and speculate about the implications for future research.

Distinct embryonic macrophage precursors https://doi.org/10.1016/j.coi.2017.12.001 0952-7915/ã 2017 Published by Elsevier Ltd.

Introduction Macrophages have been involved in steady-state function of organs, tissue development and tissue repair, but also in defense against pathogens, chronic inflammation, fibrosis and cancer [1]. The fact that one same cell is involved in all these biological processes has given rise to the idea that macrophages would be exceptionally plastic and adapt extremely well to their micro-environment to perform a vast array of cellular functions [2]. The recent identification of distinct macrophage precursors by the combination of genetic fate-mapping tools [3] and the single-cell RNA-sequencing technology has offered an alternative perspective where macrophages of distinct cellular origin Current Opinion in Immunology 2018, 50:64–74

Many resident macrophages have a predominant embryonic origin [6–8,11–16]. Distinct but overlapping waves of embryonic precursors have been described [3] and potentially include four waves: firstly, primitive hematopoiesis in the yolk-sac giving rise to primitive macrophages; secondly, yolk-sac ‘early’ EMPs giving rise to pre-Macs that colonize the distinct embryonic tissues and develop into resident macrophages without passing through a monocytic intermediate [7,8,15]; thirdly, yolk-sac ‘late’ EMPs migrating to the fetal liver and giving rise to fetal liver monocytes that in turn colonize all developing organs (except the brain) [6]; and finally, HSC-derived monocytes giving rise to resident macrophages before birth [17]. Importantly, as organs continue to grow during the neonatal period HSC-derived monocytes may continue to enter the pool of resident macrophages in the first weeks of life in organs that are accessible such as the liver [18,19], the heart [20] or the dermis [21,22]. We have proposed that macrophage precursors have an almost identical potential to develop into self-maintaining resident macrophages but that these cells compete for a www.sciencedirect.com

Does macrophage ontogeny matter? Bonnardel and Guilliams 65

restricted number of niches and that niche availability and accessibility are the main factors determining the origin of resident macrophages [19]. This theoretical model is supported by recent reports revealing that peritubular testis macrophages and T-cell zone macrophages develop after birth from HSC-derived monocytes but self-maintain for months [23,24]. The separation of macrophages based on their ontogeny into self-maintaining ‘resident’ macrophages of embryonic origin and shortlived ‘passenger’ macrophages of HSC-derived monocytes, as we [25] and others [26] have proposed, therefore does not hold well when one incorporates tissues that still develop after birth such as the lymph nodes, in which the segregation between the T cell zone and the B cell zone only happens after birth [27], or the testis, which only matures when spermatogenesis is initiated in the first weeks after birth [28]. The transcriptional profile of resident macrophages derived from distinct cellular origins seems not to differ significantly. When macrophages were depleted by whole body irradiation the macrophages derived from the newly transplanted bone-marrow cells acquired a similar transcriptomic and epigenetic landscape [29]. When we transferred distinct macrophage precursors into the empty alveolar macrophage niche of GM-CSF-deficient mice all precursors differentiated into alveolar macrophages that could self-maintain for a year, were fully functional in that they rescued the mice from alveolar proteinosis and had a very similar gene-expression profile [30]. Similar findings were reported in mice of which the lungs had been protected from irradiation [31]. Finally, depletion of Kupffer cells by clodronate liposomes [32] or diphtheria toxin in a mouse model where Kupffer cells specifically express the diphtheria toxin receptor [18], revealed that monocyte-derived Kupffer cells acquire a similar gene-expression profile as their embryonic counterparts and display equivalent functional capacities. Note that expression of a few genes correlated with embryonic versus HSC-derived monocyte origin [18,32]. However, we have recently performed singlecell RNA-Sequencing of mouse Kupffer cells and have not found subsets of Kupffer cells that are linked to these ontogenically related genes. Kupffer cells are thus relatively homogeneous and while the Kupffer cell pool consists of cells derived from all progenitor waves, including yolk sac-derived macrophages [7,8,15], fetal liver monocytes [6] and HSC-derived monocytes [17,18,33], they all acquire a similar functional specialization and gene-expression profile. We can at this stage however not exclude that functionally important epigenetic changes linked to the distinct cellular origin on the cells may subsist (see below).

Distinct adult macrophage precursors Circulating monocytes derived from bone-marrow HSCs are not homogeneous. First monocytes can be separated www.sciencedirect.com

into Ly-6ChiCD43lowCX3CR1int classical monocytes and Ly-6ClowCD43hiCX3CR1hi patrolling monocytes [34,35]. The Ly-6Clow patrolling monocytes can be regarded as the terminally differentiated resident phagocyte population from the blood stream [36]. These cells develop from Ly-6Chi classical monocytes through a NR4A1-dependent [35,37,38]; KLF2-dependent [38] and C/EBPbdependent pathway [39]. The continuum between Ly-6Chi and Ly-6Clow monocytes was recently mapped by single-cell RNA-Sequencing [39]. This revealed two distinct developmental pathways within the Ly-6Cint population: a C/EBPb-NR4A1-dependent pathway towards Ly-6ClowMHCIIlow patrolling monocytes and a C/EBPb-NR4A1-independent developmental pathway towards MHCII+CD209a+ monocytes. The latter MHCII+CD209a+ monocytes were independently identified as precursors of monocyte-derived dendritic cells (mo-DCs) by single-cell RNA-Sequencing [4]. This is based on the fact that MHCII+CD209a+ monocytes acquire higher MHCII expression upon stimulation with GM-CSF in vitro and in vivo. This population is also more sensitive to PU.1 haplo-insufficiency than Ly-6ChiMHCIIlowCD209alow classical monocytes. Whether these cells are also derived from the cMop is unknown. The contribution of MHCII+CD209a+ monocytes to in vivo antigen-presentation has also not been demonstrated. In contrast to MHCII+CD209a+ monocytes, Ly-6ChiMHCIIlowCD209alow classical monocytes give rise to iNOS+MHCIIlow macrophages when infected with Listeria monocytogenes in vitro. In vivo infection however gives rise to iNOS+MHCIIhi macrophages that have been termed Tip-DCs (for TNF and iNOS producing DCs, [40]). The antigen-presentation capacity of this population is unclear but mice lacking Tip-DCs develop comparable T cell responses, implying that these cells are not crucial for antigen-presentation. Whether Tip-DCs derive from Ly-6ChiMHCIIlowCD209alow classical monocytes and/or from MHCII+CD209a+ monocytes in vivo has not been directly investigated, but these cells have been proposed to derive from Ly-6ChiMHCIIlowCD209alow classical monocytes due to their lack of CD209a and their increased presence in PU.1 haplo-insufficient mice [4]. In humans it was recently reported that monocytes can differentiate into macrophage-like and DC-like cells and that this decision is controlled by MAFB and IRF4 [41]. However, whether there are subsets of human monocytes that are more prone to differentiate into mo-DCs is currently unknown. In fact, when the authors applied single-cell RNA-Seq they did not identify a monocyte subset that would be precommitted to acquire a mo-DC phenotype and they propose that macrophage development would represent the default pathway but that environmental stimuli such as IL-4-receptor-signaling or aryl hydrocarbon receptor-signaling can push monocytes towards a mo-DC fate [41]. What about MHCIIhi macrophages in the mouse intestine or the Current Opinion in Immunology 2018, 50:64–74

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dermis? Would they derive preferentially from Ly6ChiMHCIIlowCD209alow classical monocytes or from MHCII+CD209a+ monocytes? More in vivo research will be required to understand whether distinct monocyte subsets are indeed programmed for a predefined function by their differential development or whether these cells will quickly adapt when recruited to tissues so that their functional capacities are mainly imprinted by local cues rather than by their ontogeny. An additional subset of monocytes was recently described. These CD11bhiLy-6ClowF4/80lowCeacam1+Msr1+ cells were termed segregated-nucleus-containing atypical monocytes (SatM) and were proposed to drive fibrosis [5]. These cells were also reported to be C/EBPb-dependent but proposed to be distinct from patrolling monocytes in that they derive from granulocyte-macrophage progenitors (GMPs) and not from the classical monocyte-macrophage/DC progenitors (MDP) to cMop to Ly-6Chi developmental pathway [5]. Further work will be required to establish the relationship between SatMs and other monocytes subsets. If the SatM would represent a significant fraction of CD11bhiLy-6Clow monocytes in circulation of steady-state animals then one would expect to find these cells back as a separate entity in the single-cellRNA-Sequencing data and this was not reported [39]. However, it could be that production of SatM is low in the steady-state but increases during fibrosis as a result of emergency myelopoiesis. The possibility that monocytes may also derive from GMPs without passing by the MDP-cMop pathway and that this may increase during inflammation is interesting as it may yield monocytes that are functionally distinct and may share characteristics with neutrophils, as was proposed for the SatM [5]. Severe infections have been shown to result in emergency myelopoiesis [42,43], which is often associated with an increased production of monocytes. This could be due to increased levels of M-CSF in circulation as MCSF signaling has been shown to drive monocyte production by HSCs [44]. Elevated levels of GM-CSF can also boost the production of myeloid cells from the bonemarrow. Indeed, the Cheers lab has reported that GMCSF-deficient mice have a decreased presence of recruited (presumably monocyte-derived) macrophages upon L. monocytogenes [45] and Mycobacterium avium infections [46]. It is also noteworthy that GM-CSF has been proposed to boost the production of MHCII+CD209a+ monocytes with mo-DC properties [4], implying that increased levels of cytokines in circulation may not only boost the number of recruited monocytes but may also favor a particular functional subset of monocytes. Similarly, Toxoplasma infection is associated with an increase in monocyte production in the bone-marrow [47]. Interestingly, as these cells display higher expression of MHCII it could well be that Current Opinion in Immunology 2018, 50:64–74

this represents an expansion of the MHCII+CD209a+ monocytes subset with a more mo-DC-like profile. Elevated levels of IFN-g have been shown to underlie this increase in MHCII+ monocytes [47]. It has been shown that IFN-g shifts the GMP towards monocyte production as the expense of neutrophil production. This was proposed to work through the induction of IRF8, a transcription factor known to be important for the monocyte development upstream of the cMop stage [48]. Indeed, mice lacking IRF8 display an increased neutrophil production in the bone-marrow [49,50]. Seeing the recent reports demonstrating important functional heterogeneity among monocytes [4,5,39], it will be interesting to see how inflammation influences the development of specific monocytes subsets and through which molecular mechanisms this occurs.

Plasticity versus ontogeny defined heterogenity Macrophages have long been considered very plastic cells but the presence of multiple embryonic and adult macrophage progenitors may provide an alternative explanation. We would like to discuss two extreme scenarios that are not strictly mutually exclusive (Figure 1). On one hand, we would envisage that plasticity is indeed immense in the macrophage lineage and that imprinting by the tissue or by the inflammatory micro-environment will negate any differences present in the distinct precursors cells, yielding a relatively homogeneous macrophage population. Of course distinct micro-environments within a given tissue would yield very distinct macrophages that are completely adapted to the local signals they receive. On the other hand, we consider the possibility that cellular origin may control an important part of the functional specialization of macrophages. This is particularly plausible during inflammation where restricted plasticity of terminally differentiated macrophages but important plasticity of recruited monocytes and developmental intermediate cells would imply that resident cells and recruited precursors react differently to inflammatory signals. Note that this implies that bonemarrow monocyte-derived resident macrophages that developed in the steady-state weeks before the onset of inflammation would be functionally very different from bone-marrow monocyte-derived macrophages developing during inflammation. In the following sections we will go over a couple of inflammation models per organ and discuss whether the current data support macrophage plasticity or whether their is macrophage heterogeneity linked to the distinct cellular origin. But first we will discuss functional heterogeneity among steady-state macrophage populations.

Effect of cellular origin on steady-state macrophage function If one takes liver-resident Kupffer cells, then the various fate-mapping system find some contribution of www.sciencedirect.com

Does macrophage ontogeny matter? Bonnardel and Guilliams 67

Figure 1 PLASTICITY MODEL

Yolk-Sac Mac

Fetal Liver Mono

ONTOGENY MODEL

Bone-Marrow Mono

Yolk-Sac Mac

Fetal Liver Mono

Bone-Marrow Mono

SteadyState

SteadyState Tissue imprinting converts into identical resident Macs Identical gene expression profile for all resident Macs Identical epigenetic landscape for all resident Macs Identical functions for all resident Macs

Yolk-Sac Mac

Fetal Liver Mac

Bone-Marrow Mac

Recruited Mono

Tissue imprinting converts into similar resident Macs Relativemy similar gene expression profile for all resident Macs Important epigenetic differences linked to ontogeny remain Some differential functions of distinct resident Macs

Yolk-Sac Mac

Fetal Liver Mac

Bone-Marrow Mac

Recruited Mono

Inflamed tissue

Inflamed tissue Inflammation converts into inflammatory macrophages Identical gene expression profile Identical epigenetic landscape Identical inflammatory functions

Yolk-Sac Mac

Fetal Liver Mac

Bone-Marrow Mac

Recruited Mac

Inflammation acts differently on distinct cells Recruited monocytes differentiate into inflammatory Macs Resident macs have restricted plasticity, as compared to recruited Mono Epigenetic differences induce differential responses of Resident Macs Differential inflammatory functions for distint Mac populations

Yolk-Sac Mac

Fetal Liver Mac

Bone-Marrow Mac

Recruited Mac

Recovery

Recovery Return to healthy tissue reverts Macs into resident Macs Identical gene expression profile for all resident Macs Minor epigenetic differences among resident Macs Identical functions for Macs, no long term effect of inflammation

Healthy tissue does not completely revert Macs to resident Macs Differential gene expression profile for all resident Macs Important epigenetic differences linked to inflammation & ontogeny Differential functions due to long term effect of inflammation & ontogeny

Current Opinion in Immunology

On the left, a model based on immense plasticity of macrophages. Imprinting by the tissue or by the inflammatory micro-environment negates any differences present in the distinct precursors cells, yielding a relatively homogeneous macrophage population. On the right, a model based on functional heterogeneity among macrophages based on their cellular origin. Bone-marrow monocyte-derived resident macrophages that developed in the steady-state weeks before the onset of inflammation would be functionally close to resident macrophages of embryonic origin even if some differences remain. Bone-marrow monocyte-derived macrophages developing during inflammation would be very different because these cells are more impacted by the inflammation, as precursors would be more plastic than terminally differentiated cells. The epigenetic inflammatory imprinting would be highest in the monocyte-derived macrophages the have developed during inflammation.

yolk-sac-derived macrophages [7,8,51], fetal-liver-derived monocytes [6] and HSC-derived monocytes to the adult Kupffer cell pool [17,33]. There is currently not much evidence for distinct functional capacities of steady-state Kupffer cells derived from distinct cellular origins. Other steady-state tissue-resident macrophages comprised of a mix of embryonic and adult HSC origins include at least splenic macrophages [6–8,33], peritoneal macrophages [52], cardiac macrophages [11,20] and dermal macrophages [21,22] and, to our knowledge, in none of these organs there has been a important difference reported in the capacity to perform tissue-specific accessory functions www.sciencedirect.com

between steady-state macrophages of distinct cellular origin. This is probably due to the fact that when the distinct precursors engraft stably in the respective macrophage niches they undergo such thorough imprinting by their microenvironment that the original differences are dampened until the terminally differentiated and selfmaintaining macrophages are transcriptionally and functionally equivalent, regardless of their origin. Note however that steady-state macrophages of distinct cellular origins may carry epigenetic differences that are not visible in their transcription profile. Some genes may possess ‘poised’ enhancers in macrophages derived from Current Opinion in Immunology 2018, 50:64–74

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bone-marrow monocytes but be inactive in macrophages of embryonic origin (or vice versa). A poised enhancer often reflects that this particular gene was at some point active in the precursors. The distinct macrophage precursors possess strikingly distinct gene expression profiles [30] as exemplified by much higher expression of IL-10 transcript in yolk-sac macrophages as compared to bonemarrow monocytes or fetal liver monocytes. Initiation of gene expression is often induced by binding of transcription factor into enhancers that are poised for activity [53]. This might therefore imply that even if Kupffer cells express only low levels of IL-10 in the steady-state liver (we did not pick up any cells expressing high levels of IL10 by single-cell RNA-Seq), yolk-sac derived Kupffer cells might theoretically express IL-10 more rapidly or to higher levels upon stimulation as their IL-10 enhancers were poised and more readily inducible. This is in fact not unthinkable since significant differences in epigenetic landscape were found between steady-state Kupffer cells and bone-marrow derived Kupffer cells that colonize the liver after irradiation [29]. Of course one could also envisage that these differences could in part be due to the irradiation. We are currently repeating these experiments utilizing our Clec4F-DTR mice that permit specific depletion of Kupffer cells by a single injection of diphtheria toxin [18]. Note also that it should be evaluated whether these potential epigenetic differences subsist with time or whether such ontogeny related differences only impact macrophage function for a relatively short amount of time after the differentiation into resident macrophages.

Effect of cellular origin on macrophage function during inflammation Intestinal inflammation

Steady-state intestinal macrophages produce low levels of TNF upon TLR triggering, but macrophages recruited during DSS colitis maintain their capacity to produce TNF [54]. This could of course be due to immense plasticity of macrophages and be explained by adaptation of the recruited macrophages to an inflammatory environment but it should be noted that the resident intestinal macrophages that were present before the DSS administration did not increase their TNF expression. One could therefore consider that monocytes recruited during colitis may consist of a particular subset of monocytes that has an increased capacity to produce TNF. As Toxoplasma infection induces the production of bone-marrow monocytes that are functionally distinct from steady-state monocytes [47], it could perfectly be possible that DSS induced the generation of monocytes more prone to produce TNF. Alternatively, one may consider that while monocytes as precursors have an immense plasticity and rapidly react to the danger signals induced by the DSS, terminally differentiated macrophages have lost an important part of their plasticity and will therefore not be able to completely adapt to the DSS environment. In Current Opinion in Immunology 2018, 50:64–74

this case, the differential TNF production might have nothing to do with ontogeny but would rather reflect the decrease of plasticity often associated with terminal differentiation in many cell types. Liver inflammation

Paracetamol-induced liver injury results in massive recruitment of monocytes to the liver [55–58]. These monocytes are short-lived, disappear within a week and are transcriptionally very distinct from resident Kupffer cells [56]. Kupffer cells analyzed on the third day post paracetamol-induced liver injury did not change their gene-expression profile, suggesting minimal plasticity of resident Kupffer cells during inflammation. It should however be noted that the first 48 h of paracetamolinduced liver injury are associated with important death of Kupffer cells. It could therefore be that a fraction of the Kupffer cells do react to the liver inflammation, get activated and die, leaving only Kupffer cells that have not been activated by the inflammation behind on the third day post paracetamol-induced liver injury. This macrophage disappearing reaction is common and in fact poorly understood [59]. The reason why recruited monocytes do not engraft in the Kupffer cell pool during paracetamol-induced liver injury is unknown. Listeria infection is also associated to important Kupffer cell disappearance but results in massive conversion of recruited monocytes into Kupffer cells that reside in the liver for at least one month [60]. Whether these monocyte-derived Kupffer cells differ transcriptionally from embryonic Kupffer cells is unknown. Injection of a high number of aged red blood cells also induces Kupffer cell death due to iron overload and conversion of monocytes into monocyte-derived Kupffer cells. These cells could self-maintain for more than one month but disappeared from the liver eventually [9]. Similar findings were observed in a mouse model of non-alcoholic steatohepatitis (NASH) [61]. The reason for this late disappearance is unknown. The monocyte-derived Kupffer cells were profiled one week after the injection of aged red blood cells and showed differentially expressed genes such as high Ccr2 expression and low Timd4 expression. It should however be noted that in our Kupffer cell DTRmodel these genes are rapidly changing with time and by 30 days post Kupffer cell depletion only a few genes remain differentially expressed between resident Kupffer cells and recruited monocyte-derived Kupffer cells. As such, it may well be that many differences observed at day 7 post aged red blood cell injection will not persist for prolonged periods. Pulmonary inflammation

Monocytes recruited to the alveolar space during the influenza infection were initially reported to not engraft efficiently in the alveolar macrophage pool [16]. A recent study however found significant engraftment of monocytes in the alveolar macrophage pool during influenza www.sciencedirect.com

Does macrophage ontogeny matter? Bonnardel and Guilliams 69

infection [62]. Moreover, monocytes differentiate into long-lived alveolar macrophages during gammaherpesvirus [63]. It should be noted that the alveolar macrophages that derive from monocytes during inflammation express a distinct surface expression profile with lower Siglec-F and higher CD11b. This may explain why these cells were missed in earlier studies. It could also be that monocyte engraftment depends on the strength of resident macrophage depletion and whether the niche is sufficiently emptied to give a chance to incoming monocytes to colonize it and this is probably dependent on the viral dose, the virulence of the strain and the disease severity, which may vary from one laboratory to another. Importantly, the gammaherpesvirus-induced alveolar macrophages are functionally distinct from steady-state macrophages and protect from allergic asthma for weeks after the viral infection. Again at this stage it is difficult to understand why these macrophages are functionally different. The resident alveolar macrophage pool is almost completely wiped out during gammaherpesvirus infection with 90% of the alveolar macrophages being derived from bone-marrow monocytes after the infection. However, it is not known whether the few resident macrophages also changed their functional characteristics due to the viral infection. In that case the change in macrophage function would have nothing to do with ontogeny and any macrophage present during the viral infection would be converted into a functionally distinct cell due to high plasticity of the macrophage lineage. But why are these cells not reverting to the steady-state macrophage profile after the infection if the macrophages are so plastic? Maybe because the lung environment is permanently altered by the viral infection? The authors demonstrate that gammaherpesvirus infection is associated with the presence of MHCII+Sca1+ monocytes in the blood that produce high levels of IL-10. These monocytes are reminiscent of the monocytes produced during Toxoplasma infection [47]. They might already be programmed for regulatory functions even before reaching the lung. Note that this might not be linked to a bonemarrow origin per se but to the type of monocyte (subset) produced during infection or to cytokines produced in the bone-marrow during the viral infection that induce a particular activation state in these cells. Alternatively, the resident macrophages present before the infection might, as terminally differentiated cells, not be as plastic while the recruited monocytes adapt completely to the viral infected lungs as plastic precursors and acquire a distinct regulatory activation profile. However, as they become terminally differentiated macrophages, they may lose their plasticity properties and consequently stay locked in this virus-imprinted regulatory stage. Misharin et al. have recently tracked the effect of fibrosis on the transcriptional profile of alveolar macrophages [62]. Lung fibrosis induced massive engraftment of bone marrow monocytes in the alveolar macrophage pool. www.sciencedirect.com

Importantly, these fibrosis-induced bone marrow-derived macrophages persisted for months in the lungs. The latter macrophages and resident macrophages isolated from fibrotic lungs shared a common fibrosis-induced transcriptional profile of more than 1800 genes that were not expressed in steady-state alveolar macrophages. Strikingly, only 300 genes were differentially expressed between resident macrophages during fibrosis versus steady-state resident macrophages, but not shared with bone-marrow-derived alveolar macrophages that develop during fibrosis. However, bone marrow-derived alveolar macrophages that developed during fibrosis expressed an additional set of 1900 genes that were not expressed by resident alveolar macrophages isolated from the same fibrotic lungs. This additional set of genes could represent genes linked to their bone-marrow origin. In fact, we could even raise the possibility that the fibrosis-induced bone marrow-derived alveolar macrophages are derived from the newly described pro-fibrotic SatM monocytes derived from the GMP and that this set of genes is linked to their distinct developmental origin. Alternatively, these genes may be induced locally in the lungs and linked to higher plasticity of monocyte precursors as compared to the resident terminally differentiated macrophages. In fact since only 300 genes were associated with the embryonic resident origin we would argue that the majority of the 1900 genes expressed in monocytederived alveolar macrophages will not per se be linked to ontogeny itself, but will be the result of the fact that these cells have differentiated into alveolar macrophages during inflammation. Indeed, the transient state of the cells undergoing differentiation into alveolar macrophages may allow much stronger pro-fibrotic imprinting during inflammation. Note also that both populations of alveolar macrophages reverted to a profile close to the steady-state resident alveolar macrophage transcriptional profile during the resolution of fibrosis, again demonstrating some plasticity of terminally differentiated macrophages and emphasizing that the pro-fibrotic state may also be transient in monocyte-derived alveolar macrophages. However, it might still be that important epigenetic differences would remain that may, upon a second lung inflammation, lead to a differential response of the distinct alveolar macrophage populations. Brain inflammation

When microglia were depleted in a DTR-based mouse model in absence of irradiation, these cells repopulated from a local brain population, which probably represent few microglia that had not been depleted [64]. However, when the mice were irradiated then microglia were derived from peripheral bone-marrow derived cells, presumably circulating monocytes. The gene-expression profile of microglia derived from remaining CNS cells was compared to the microglia derived from bone-marrow precursors, and this revealed a striking difference between the two populations. These genes may be Current Opinion in Immunology 2018, 50:64–74

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directly linked to the distinct origin of these cells but it cannot be excluded that these differences reflect the impact of irradiation on the brain cells which in turn will influence the profile of the bone-marrow derived microglia. Using a mouse glioma model, the Joyce laboratory recently studied the impact of ontogeny on the geneexpression profile of resident microglia and recruited bone-marrow-derived macrophages infiltrating brain tumors [65]. Both populations had a shared tumor gene signature implying that even resident macrophages retain some plasticity and adapt to the tumor environment. Bone-marrow-derived macrophages had also an additional set of genes that differed from tumor-associated microglia. This could be linked to their bone-marrow origin or to or higher plasticity of recruited monocytes as precursors. The reason why we believe the second hypothesis is more likely is because a majority of the genes differentially expressed between bone marrowderived macrophages and resident microglia were tumor model specific and did not correspond to the genes identified using the DTR-based microglia depletion model [64]. However, there was indeed a core set of about 150 genes that seemed strictly linked to the bone-marrow origin. But this may also be just the tip of the iceberg if one considers the possibility of much bigger differences present at the epigenetic level. It should also be noted that similar findings were reported in EAE models [66,67]. Here the authors also studied the recovery period and found that microglia partially reverted their gene-expression profile to the steady-state profile. Two things remain unknown: are there additional epigenetic traces remaining in the macrophages due to their passed inflamed state, and did the activated microglia truly revert to the steady-state or did activated microglia die and did the microglia pool repopulate from the microglia that had not been impacted too heavily by the disease? We could indeed envisage important heterogeneity within the microglia pool. Recently, the Amit team and the Schwartz team joined forces to study the effect of Alzheimer’s disease on microglia using singlecell RNA-Seq technology [68]. This revealed heterogeneity within the microglia pool with some of the cells still representing steady-state microglia and some of the cells that were localized near the plaques that had acquired a transcriptional profile linked to Alzheimer’s disease. It could well be that, upon return to homeostasis, microglia adapted to the diseased environment have lost their capacity to revert to a steady-state profile as well as the capacity to repopulate the brain. Importantly, neurological diseases are also linked to changes in microglia geneexpression in humans [69]. Pancreatic cancer

Pancreatic macrophages can be divided in two main subsets based on their localization. Macrophages Current Opinion in Immunology 2018, 50:64–74

populating the islets of Langerhans are derived from bone-marrow precursors but, once established, possess the capacity to self-maintain [70]. These cells react to circulating LPS and have high expression of TNF and IL1-b in the steady-state [71]. Macrophages in the interacinar stroma are partially derived from yolk-sac-derived precursors and also possess self-maintaining capacities [70]. These cells express markers associated to the alternative activation of macrophages such as Mgl1, Mgl2, Arg1 and Fizz1 (although we are not in favor of the M1/ M2 nomenclature [72,73]). Interestingly, depletion of pancreatic macrophages by irradiation leads to repopulation of the distinct macrophage populations from bonemarrow precursors and these cells acquired the same differential expression of genes linked to the localization in the steady-state pancreas, again demonstrating the strength of imprinting by the micro-environment. However, study of a mouse model of pancreatic ductal adenocarcinoma suggests that cellular origin may have important effects on macrophage function during cancer. Pancreas cancer was associated with an increase of the pancreatic macrophage population through two distinct mechanisms: expansion of the resident macrophage population (which itself is composed of both bone-marrow derived and embryonic derived macrophages with comparable self-maintaining capacity, [70]) and recruitment of circulating monocytes that differentiate into tumorassociated macrophages. Depletion of circulating monocytes using CCR2-inhibitors did not alter tumor burden. Depletion of resident macrophages before tumor inoculation by the injection of CSF1-neutralizing antibodies in combination with clodronate-liposomes, however, did dampen tumor burden. Moreover, injection of antiCSF1R embryonically, which efficiently depletes embryonic macrophages [6], also diminished the pancreatic tumor burden. This suggests a specific role for embryonic-derived resident macrophages in tumor growth. However, it should be noted that bone-marrow-derived resident macrophages, which make up more than half of the pancreatic macrophage population, may in fact function in the same way than their embryonic counterparts. It has not been demonstrated that depletion of bone-marrow-derived resident macrophages would not have the same effect. In addition, it is not inconceivable that depletion of all macrophages during embryogenesis may have long-term effects on tissues since macrophages may actively participate to embryonic tissue development [1]. Transcriptional profiling of embryonic and bonemarrow-derived resident macrophages in the steady-state pancreas yielded an almost identical profile [74], as has been observed for other tissue-resident macrophages in the steady-state [18,30,31]. However, comparison between embryonic macrophages and bone-marrow derived macrophages in pancreatic tumors yielded an important difference in gene expression profile. Indeed it was found that embryonic macrophages displayed higher expression of genes involved in fibrosis. It should www.sciencedirect.com

Does macrophage ontogeny matter? Bonnardel and Guilliams 71

however be noted that in this study the bone marrowderived macrophages are a mix composed of a major fraction of recently recruited bone-marrow-derived macrophages and a minor fraction of resident self-maintaining macrophages of bone-marrow origin that developed long before the onset of pancreatic cancer. As the latter where not sequenced separately, their genetic profile will have been diluted by the newly recruited bone-marrow macrophages that develop during cancer. We hypothesize that the resident macrophages of bonemarrow origin will rather cluster together with their embryonic resident counterparts (and share similar expression of pro-fibrotic genes) than with the bonemarrow derived macrophages recruited during cancer because we believe that long-term residency in the pancreas will have a bigger effect on the gene expression profile and function than the bone-marrow origin itself. Alternatively, it could well be that the pro-fibrotic genes were poised in the embryonic macrophages predisposing these macrophages to pro-fibrotic function upon cancer development. It will therefore be fascinating to compare the epigenetic profile of embryonic and bone marrowderived resident macrophages in the steady-state pancreas to verify whether these genes already showed a distinct chromatin structure and enhancer landscape before cancer development.

Do these cells die and disappear from the pool? Is there an important epigenetic imprinting during inflammation? Is this imprinting the same for resident macrophages of various cellular origins and for macrophages that developed during inflammation? To answer these questions we will need much better ‘ontogeny fate mapping’ tools that fate-map entire populations and not a few percentages of the cells as is often the case. Moreover, we will require ‘functional fate-mapping’ tools that would reveal the history of the functional state of macrophages. The recently developed NOS2-CRE mouse model should, for example, allow to track macrophages that have undergone activation and have expressed iNOS as was recently demonstrated for microglia [75]. In our view, we will only be able to truly understand the plasticity of macrophages in vivo once we will have developed better ontogeny fatemapping and functional fate-mapping tools and we are actively developing these tools in our laboratory.

Conclusion

Papers of particular interest, published within the period of review, have been highlighted as:

It is clear from the different inflammation models that resident macrophages do react to inflammatory triggers. There is thus definitely a case for macrophage plasticity. It should also be noted that in many cases the resident macrophages that react most to the inflammatory triggers may die and disappear from the macrophage pool. This could be a mechanism to avoid that macrophages with an inflammatory profile self-maintain in tissues and induce prolonged collateral tissue damage. In the models where macrophages that develop during inflammation could be separated from resident macrophages present before inflammation there are many differences in gene-expression profile. This could be strictly linked to their bonemarrow origin and potentially even to the development from a specific subset of monocytes. However, until now it has not been possible to compare the gene-expression profile of monocyte-derived resident macrophages and monocyte-derived macrophages recruited during inflammation. This would permit to distinguish between genes strictly linked to the bone-marrow origin and genes induced due to the fact that these cells develop in an inflammatory environment. Indeed we hypothesize that precursors and cells in a transient developmental intermediate stage will be more malleable and will be more susceptible to inflammatory imprinting than terminally differentiated cells. Few studies have been able to study what happens to macrophages of distinct origin during the recovery phase. Do the macrophages that acquired an inflammatory profile revert to their steady-state profile? www.sciencedirect.com

Acknowledgements This work was supported by the European Research Council (ERC grant to MG), the Fonds Wetenschappelijk Onderzoek Vlaanderen (FWO postdoc grant to JB, and FWO research grants to MG) and Horizon 2020 (Marie Curie CIG to MG).

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