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The physiology of foamy phagocytes in multiple sclerosis

The physiology of foamy phagocytes in multiple sclerosis Multiple sclerosis (MS) is a chronic disease of the central nervous system characterized by massive infiltration of immune cells, demyelination, and axonal loss. Active MS lesions mainly consist of macrophages and microglia containing abundant intracellular myelin remnants. Initial studies showed that these foamy phagocytes primarily promote MS disease progression by internalizing myelin debris, presenting brain-derived autoantigens, and adopting an inflammatory phenotype. However, more recent studies indicate that phagocytes can also adopt a beneficial phenotype upon myelin internalization. In this review, we summarize and discuss the current knowledge on the spatiotemporal physiology of foamy phagocytes in MS lesions, and elaborate on extrinsic and intrinsic factors regulating their behavior. In addition, we discuss and link the physiology of myelin-containing phagocytes to that of foamy macrophages in other disorders such atherosclerosis. Keywords: Macrophage, Microglia, Polarization, Neuroinflammation, Remyelination, Multiple sclerosis Introduction microglia, play an essential role in the pathogenesis of MS Macrophages are mononuclear phagocytes that reside in [14, 141]. Until recently, phagocytes were regarded to pri- every tissue of the body in which they play a crucial role marily cause lesion progression by releasing inflammatory in maintaining tissue homeostasis. They fulfill this task and toxic mediators that negatively impact neuronal and by interacting with microorganisms, remodeling tissue, oligodendrocyte integrity [152, 188], internalizing the intact and dealing with injury. Alongside their role in protect- myelin sheath [214], and presenting brain antigens to auto- ive immunity and homeostasis, they also contribute to reactive T cells [68, 129]. However, this unambiguous con- the pathology of numerous disorders. Hence, there is cept has been challenged and it is now thought that considerable interest in harnessing phagocyte function phagocytes also have beneficial properties in MS. For ex- for therapeutic benefit, either by suppressing the ac- ample, clearance of damaged myelin is essential to facilitate tivity of disease-promoting phagocytes or enhancing CNS repair [137, 168]. Moreover, phagocytes release the mobilization of phagocyte subtypes that are ad- anti-inflammatory and neurotrophic mediators in CNS le- vantageous. Such interventions require a thorough sions and can suppress the disease-promoting activity of as- understanding of the spatiotemporal phenotypes that trocytes and autoaggressive effector T cells [13, 18, 81, 167]. phagocytes display during disease progression. Of particular interest are myelin-containing foamy phago- Multiple sclerosis (MS) is an inflammatory and neurode- cytes as they make up the bulk of immune cells within ac- generative disease of the central nervous system (CNS) with tive and the rim of chronic active MS lesions (Fig. 1 and unknown etiology. While initially regarded to be a [111]). Recent evidence has shed light on the many roles lymphocyte-driven disorder, increasing evidence indicates that these cells play in promoting and suppressing MS le- that phagocytes, such as infiltrated monocyte-derived mac- sion progression, as well as the cellular mechanisms that rophages, CNS border-associated macrophages, and drive their functional properties. In this review we summarize and discuss 1) the mech- * Correspondence: Jeroen.bogie@uhasselt.be anisms involved in the uptake and cellular handling of Jerome J. A. Hendriks and Jeroen F. J. Bogie contributed equally to this myelin, 2) the spatiotemporal phenotypes that foamy work. phagocytes adopt in MS patients, and 3) the intrinsic Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium/ School of Life Sciences, Transnationale Universiteit Limburg, Diepenbeek, and extrinsic factors that impact the physiology of foamy Belgium © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 2 of 21 Fig. 1 Histopathology of inactive, chronic active, and active multiple sclerosis lesions. Inactive, chronic active, and active multiple sclerosis (MS) lesions were stained for intracellular lipid droplets (oil red o; ORO) and myelin (proteolipid protein; PLP). a and b, c and d, e and f are taken from the same lesion. Foamy phagocytes (ORO cells) are apparent in demyelinating chronic active and active MS lesions, but not in inactive lesions phagocytes. In addition, we link the physiology of foamy prior to internalization [66]. Since the discovery of phagocytes in MS to that of lipid-laden foamy macro- receptor-mediated endocytosis of myelin, researchers have phages in other disease such as atherosclerosis. Increas- attempted to identify the culprit receptors involved in the ing evidence indicates that many parallels can be drawn uptake of myelin. To date, numerous receptors such as Fc, between phagocyte subsets in various disorders. complement, and scavenger receptors are reported to To accomplish their functionally distinct roles in drive myelin internalization. In this part of review, we health and disease, tissue macrophages and monocyte- elaborate on these receptors and touch upon cell extrinsic derived macrophages can differentiate into a spectrum and intrinsic factors that influence myelin uptake by of phenotypes [208]. The ex vivo induced M1 and M2 phagocytes (Fig. 2). phenotypes represent two extremes. However, the phe- notypes found in vivo substantially differ from these Fc receptors extremes. To designate the functional properties of The discovery of immunoglobulin G (IgG) capping on phagocytes, we will utilize the term “M1-like” or “disea- the surface of phagocytes located amongst myelinated se-promoting” for phagocytes that express pro-inflam- nerve cells in active MS lesions was the first evidence matory mediators and promote MS lesion progression, for the involvement of antibody opsonization and Fc re- and “M2-like” or “disease-resolving” for those that re- ceptors in the internalization of myelin [162]. In line lease anti-inflammatory and neurotrophic mediators. with this initial discovery, a follow-up study showed that parenchymal and perivascular phagocytes in demyelinat- Myelin internalization ing MS lesions display a strong expression of Fc receptor The uptake of myelin by phagocytes is a pathological hall- I (FcRI), FcRII, and FcRIII, while microglia in the mark of MS lesions and other neurodegenerative disor- normal-appearing white matter (NAWM) barely express ders. The presence of foamy phagocytes is even used as an these receptors [192]. Subsequent in vitro studies con- index of MS lesion activity [160]. Initial evidence that firmed the contribution of Fc receptors to the internal- myelin internalization largely depends on receptor-medi- ization of myelin by showing that opsonization of myelin ated endocytosis came from the observation that myelin with anti-myelin or galactocerebroside antibodies pro- lamellae are attached to coated pits on the macrophage foundly augments the uptake of myelin by macrophages surface in an animal model for MS, experimental auto- and microglia [140, 170, 177, 179, 190]. The amount of immune encephalomyelitis (EAE) [47]. Clathrin-coated internalized myelin was further found to depend on the pits are sites where ligand-receptor complexes cluster degree of opsonization and the myelin epitope Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 3 of 21 Fig. 2 Endocytosis of myelin by phagocytes. Myelin internalization by phagocytes is dependent on the receptor repertoire as well as various intrinsic and extrinsic factors. While scavenger receptors (SR-AI/II and collectin placenta 1 (CL-P1)), Fc receptors, complement receptors (CR3), mer tyrosine kinase (MerTK), and low-density lipoprotein receptor-related protein 1 (LRP1) positively regulate the uptake of myelin, ligation of signal regulatory protein α (SIRPα) inhibits myelin uptake. Cell intrinsic and extrinsic factors, such as phagocyte polarization (M1- or M2-like), phagocyte ontogeny, (hematopoietic stem cells or yolk-sac progenitors), cellular aging, and myelin composition and modifications, can impact the capacity of phagocytes to internalize myelin recognized by the antibodies [64]. However, while activated. Collectively, these studies stress the import- anti-myelin antibodies are present in the circulation of ance of Fc receptors in the uptake of myelin but also MS patients [205], serum of MS patients does not indicate that Fc receptor-mediated uptake is fundamen- opsonize more than that of healthy controls [65]. This tally different in the central and peripheral nervous can be explained by the existence of anti-myelin anti- system. bodies in the sera of healthy controls, as their presence is not limited to MS patients [205]. To date, the opsonic Complement receptors properties of the cerebrospinal fluid (CSF) of MS pa- In addition to Fc receptors, ample evidence indicates that tients have not been determined yet. The presence of B complement receptors are involved in the uptake of mye- cell-rich meningeal follicles in the CNS of MS patients lin by phagocytes. For instance, damaged myelin in areas argues for the presence of a local, more concentrated, of active myelin breakdown and within phagocytes colo- source of myelin-directed immunoglobulins in the CSF calizes with complement components in MS lesions [2, [31]. Of interest, the microenvironment also affects Fc 20, 21]. Similar, an increased density of phagocytes ex- receptor-mediated uptake of myelin. While Ig treatment pressing complement receptors is observed in MS lesions was found to increase Fc receptor-mediated uptake of [124, 204]. In particular, early studies found that the com- myelin by macrophages in a sciatic nerve model, it did plement receptor 3 (CR3) tightly controls myelin internal- not increase myelin internalization by microglia in an ization [23, 140, 164, 165, 178]. CR3 contributes to the optic nerve model, even after addition of macrophages uptake of myelin for up to 80% in the presence of active [112]. Follow-up studies should define if the Fc receptor complement, while it was involved for 55-60% in the ab- expression profile on phagocytes differs in these models. sence of active complement [164]. Counterintuitively, In contrast to FcRI, FcRIIa, and FcRIII, FcRIIb contains myelin clearance by macrophages from CR3-KO mice an immunoreceptor tyrosine-based inhibitory motif em- is not impaired [182]. A possible explanation for this bedded in its intracellular domain [189], which might discrepancy is that CR3 can both induce and reduce negatively impact myelin internalization after being myelin phagocytosis at the same time. CR3 can reduce Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 4 of 21 uptake of myelin by phagocytes through the activation Other receptors of spleen tyrosine kinase (Syk), a non-receptor tyrosine Alongside scavenger, Fc, and complement receptors, several kinase that phagocytic receptors recruit upon activation other receptors are implicated in the endocytosis of myelin. [70]. This Syk-mediated feedback mechanism was sug- Recently, the mer tyrosine kinase (MerTK) was found to be gested to protect phagocytes from excessive intracellu- a functional regulator of myelin uptake by human lar accumulation of myelin. Collectively, these studies monocyte-derived macrophages and microglia [74]. MerTK provideevidencethatCR3-mediateduptakeofmyelin belongs to the Tyro3, Axl, and Mer (TAM) receptor family is more complex than initially regarded, being both in- and has a hand in the internalization of apoptotic cells hibitory and stimulatory. Despite the latter studies, [114, 158]. Of interest, apoptotic cell engulfment engages a anti-CR3 antibodies reduce disease severity in the EAE vicious cycle that leads to enhanced expression of MerTK model [85]. CR3 neutralization was found to reduce the [142, 145]. This vicious cycle depends on the intracellular recruitment of macrophages towards the CNS, thereby activation of the lipid-sensing liver X receptor (LXR) and ameliorating EAE disease severity. It is tempting to peroxisome proliferator-activated receptor (PPAR). Previ- speculate that a diminished phagocytic capacity may ously, we showed that myelin-containing phagocytes (mye- also underlie the reduced disease severity in EAE ani- phagocytes) also display active LXR and PPARβ signaling mals treated with anti-CR3 antibodies. [11, 15, 126]. This suggests that myelin promotes its own clearance through an LXR- and PPAR-dependent increase of MerTK. The significance of MerTK in MS pathogenesis Scavenger receptors is evidenced by the fact that polymorphisms in the MerTK Scavenger receptors are a large family of structurally di- gene are linked to MS susceptibility [87]. While the func- verse proteins, which are implicated in the binding and tional outcome of these polymorphisms remain to be clari- uptake of a wide range of molecules [26, 219]. A vast fied, they seem to depend on the genotype of individuals at amount of evidence indicates that scavenger receptors HLA-DRB1 [9], another MS risk gene [135]. In addition to mediate the uptake of myelin. By using an organ culture MerTK, the low-density lipoprotein receptor-related pro- model of peripheral nerves and a monoclonal blocking tein 1 (LRP1) is an essential receptor for myelin phagocyt- antibody, the scavenger receptors class AI/II (SR-AI/II) osis by microglia in vitro [58]. In EAE and MS lesions, the were initially found to mediate the uptake of myelin by LRP1 protein is highly expressed by phagocytes, providing rat macrophages [36]. At high antibody concentrations, evidence for involvement of LRP1 in MS pathogenesis [30, macrophage invasion of the nerves was completely abol- 58]. By using conditional knockout models, LRP1 deficiency ished, emphasizing that SR-AI/II also regulates macro- in microglia but not macrophages was found to worsen phage adhesion and migration [54, 176], similar to CR3 EAE severity [30]. Increased EAE disease severity was asso- [54, 85, 176]. Follow-up studies further defined that ciated with robust demyelination and increased infiltration SR-AI/AII blocking or knockout decreases myelin up- of immune cells. While the authors provide evidence that take by mouse macrophages and microglia [49, 164, microglia lacking LRP1 have a pro-inflammatory signature -/- 178], and that SR-A mice show reduced demyelination due to increased NF-kβ signaling, reduced microglial clear- and disease severity in the EAE model [115]. In MS le- ance of inhibitory myelin debris may also explain the ob- sions, SR-AI/II is highly expressed by foamy phagocytes served effects. Collectively, these studies stress the in the rim and by ramified microglia around chronic ac- importance of MerTK and LRP1 in the uptake of myelin by tive MS lesions [76]. This expression profile argues for phagocytes. the involvement of SR-AI/II in the uptake of myelin by phagocytes in MS lesions, and SR-AI/II being involved The inhibitory SIRPα-CD47 axis in early uptake of myelin by microglia. Aside from Aside from receptors that stimulate myelin internaliza- SR-AI/II, we recently showed that collectin placenta 1 tion, phagocytes also express receptors that inhibit the (CL-P1), a novel class A scavenger receptor [26], also uptake of particles. These receptors likely evolved to contributes to the uptake of myelin by phagocytes. In ac- limit the uptake of ‘self ’ antigens or as a feedback mech- tive demyelinating MS lesions, CL-P1 immunoreactivity anism to inhibit excessive uptake of particles. With re- colocalizes primarily with perivascular and parenchymal spect to myelin internalization, signal regulatory protein myelin-laden phagocytes. Finally, while evidence con- α (SIRPα), a membrane glycoprotein expressed primarily cerning its role in myelin clearance is still lacking, ex- by phagocytes, represents such a inhibitory receptor. pression of lectin-like oxidized low-density lipoprotein Interaction of SIRPα with the “don’t eat me” protein receptor 1 (LOX1) is elevated at sites of active demyelin- CD47 on myelin decreases the uptake of myelin by mac- ation in MS lesions [76]. Future studies should define rophages and microglia [61, 73]. Of interest, serum also whether blockage of this class E scavenger receptor im- promotes an SIRPα-dependent decrease in myelin up- pacts myelin internalization by phagocytes. take irrespective of CD47 expressed on myelin [61]. A Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 5 of 21 potential mechanism could be the transactivation of phagocytic capacity. With respect to the latter, phagocyt- SIRPα by soluble SIRPα ligands present in serum. In osis of apoptotic cells, bioparticles, and oxidized follow-up studies, SIRPα was demonstrated to inhibit low-density lipoproteins (oxLDL) is more robust in myelin internalization by remodeling of F-actin and M-CSF, IL-4/IL-10, or M-CSF/IL-10 stimulated M2-like thereby cytoskeleton function [60]. Inactivation of the phagocytes as compared to GM-CSF, IFNγ, or LPS stim- paxillin-cofilin signaling axis upon SIRPα activation un- ulated M1-like phagocytes [99, 196, 222]. The uptake of derlies the impact of SIRPα on cytoskeleton function myelin also matches the phenotype of macrophages and and myelin uptake. Of interest, the paxillin-cofilin sig- microglia. Phagocytes stimulated with the naling axis also positively regulates the uptake of myelin anti-inflammatory cytokines TGFβ, IL-4/IL13, IFNβ,or by the scavenger, complement and Fc receptors [60, 70]. IL-4/IL-13/IL-10 display a higher phagocytic capacity These findings place paxillin and cofilin centrally in the than naïve or LPS/IFNγ stimulated M1-like phagocytes process of myelin internalization. [44, 74]. These studies indicate that cytokines in the microenvironment of MS lesions, and in particular the Clearance of myelin debris presence of those cytokines that drive phagocyte Whereas internalization of the intact myelin sheath fuels polarization such as TGFβ, IFNγ, IL-10, and IL-4, regu- demyelination, ample evidence indicates that removal of late the phagocytic features of phagocytes. damaged myelin debris at the lesion site promotes CNS A number of studies further indicate that peripheral repair. Early studies already showed that myelin contains macrophages and CNS-derived microglia differ in their growth inhibitory molecules such as Nogo A, which ex- capacity to internalize myelin [44, 74, 112, 140, 178]. hibit strong inhibitory effects on neurite growth and Microglia generally show a higher capacity to internalize axonal regeneration [67]. Kotter et al. extended these myelin as compared to peripheral macrophage subsets findings by showing that myelin debris removal by [44, 74, 140]. Differences in macrophage and microglia phagocytes is a critical step for efficient remyelination ontogeny, being derived from hematopoietic stem cells [106]. Myelin debris was found to exert potent inhibitory or yolk-sac progenitors respectively, might well explain effects on the ability of oligodendrocyte progenitor cells discrepancies in their receptor expression profile and to differentiate into mature remyelinating oligodendro- phagocytic capacity [101, 173]. On that note, both the cytes [107, 159]. In concordance, by using the cupri- basal and inducible expression of MerTK and myelin zone- and lysolecithin-induced demyelination models, phagocytosis are higher in microglia as compared to reduced uptake of myelin debris by macrophages and monocyte-derived macrophages [74]. Likewise, we re- microglia resulted in inefficient axonal remyelination cently showed that myelin uptake increases the cell sur- characterized with aberrant myelin patterns in vivo [113, face expression of the phagocytic receptor CL-P1 by 147, 168]. Collectively, these studies stress that clearance mouse and human macrophages, but not by primary of myelin debris is mandatory for efficient CNS repair to mouse microglia in vitro [12]. Finally, in contrast to progress or even initiate. Interestingly, a recent study de- peripheral macrophages, immunoglobulin treatment in- fined that blood-derived macrophages and resident creases Fc receptor density on microglia [112]. Collect- microglia have functionally divergent roles in myelin in- ively, these studies suggest that differences in the density ternalization. Macrophages were found to associate with of phagocytic receptors and/or activity of signaling path- nodes of Ranvier and initiate demyelination in the EAE ways involved in driving the expression of these recep- model, whereas microglia appeared to primarily clear tors underlie discrepancies in the phagocytic properties debris [214]. To date, the mechanisms underlying this of macrophages and microglia. It is also noteworthy to difference remain elusive. Once identified they hold mention that blood-derived macrophages associate with great promise for future therapeutics aimed at improving nodes of Ranvier and initiate demyelination, whereas CNS repair in MS. microglia mainly clear myelin debris [214]. This study suggests that differences in myelin uptake might also rely on the presence of receptors that recognize cryptic Cell intrinsic and extrinsic factors influencing myelin myelin epitopes that are not exposed on intact myelin. internalization As phagocytosis experiments are generally carried out Phagocytosis is a dynamic process involving both struc- using myelin debris, differences in the recognition of tural rearrangements, complex signaling events, and a cryptic myelin epitopes by macrophages and microglia plethora of phagocytic receptors. Not surprisingly, di- remain to be determined. verse intrinsic and extrinsic factors are associated with Another factor that impacts the physiology of phago- alterations in the phagocytic capacity of macrophages cytes is aging. Several studies indicate that aged and microglia. For example, ample evidence indicates macrophages less efficiently internalize apoptotic cells that the polarization status of phagocytes drives their [102, 212], bacteria [75], latex beads, and opsonized Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 6 of 21 sheep erythrocytes [183]. By using toxin-induced focal Phenotype of myelin-containing phagocytes demyelination in the mouse spinal cord, together with Ample evidence indicates that myelin uptake changes heterochronic parabiosis, Ruckh et al. demonstrated that the functional properties of macrophages and microglia. aged blood-derived macrophages also clear myelin debris Some studies reported an M2-like phenotype of phago- less efficiently as compared to young macrophages [168]. cytes upon internalization of myelin, whereas others de- In vitro experiments using mouse macrophages and scribed no effect at all, or even an M1-like activation microglia and human monocyte-derived macrophages status. In this section, we elaborate on the phenotypes of confirmed that aging impairs myelin debris clearance by mye-phagocytes as well as the signaling pathways direct- these phagocytes [147]. The authors further show that ing these phenotypes (Fig. 3). reduced activity of the retinoid X receptor (RXR) signal- The abundant presence of foamy phagocytes in MS le- th ing pathway partially accounts for the observed differ- sions sparked interest at the end of the 20 century into ence in myelin uptake between young and old defining the phenotypes of these cells. In line with the phagocytes. Via which pathways RXR signaling decreases prevailing dogma at that time that phagocytes merely the uptake of myelin by aged phagocytes remains to be promote lesion progression, uptake of myelin was ini- clarified. While loss of RXR can directly impact the ex- tially demonstrated to promote the release of substantial pression of phagocytic receptors such as MerTK, im- amounts of TNFα and nitric oxide (NO) by macro- paired phagocytosis can also be a mere consequence of phages [194]. In agreement, myelin engulfment by adult an inability to adopt an M2-like phenotype [100]. In a human-derived microglia induced the oxidative burst follow-up study, it was demonstrated that MS-derived and the release of IL-1, TNFα, and IL-6 [210]. Further- monocytes show a reduced uptake of myelin irrespective more, exposure of M-CSF stimulated M2-like macro- of the patients’ age [148]. This finding suggests that the phages to myelin debris led to a significant decrease in disease state influences the phagocytic features of phago- the expression of M2 markers and increase in the ex- cytes in MS. It is tempting to speculate that premature pression of markers characteristic for M1-like macro- innate immunosenescence, possibly due to chronic in- phages [203]. These studies indicate that naïve as well as flammation (“inflammaging”), impacts phagocyte physi- pre-differentiated M2-like phagocytes adopt an inflam- ology in MS patients. Increasing evidence indicates that matory phenotype after uptake of myelin in vitro. Also premature aging of the immune system is apparent in in in vivo models and MS lesions, several studies defined MS patients [16]. Interestingly, in contrast to macro- the presence of M1-like mye-phagocytes. In the spinal phages, aged human microglia do not show a reduction cord injury (SCI) model, the accumulation of M1-like in myelin uptake compared to their younger counter- phagocytes closely correlates with the intracellular pres- parts [77]. This finding suggests that aging impact ence of myelin-derived lipids [110, 203]. Kroner and col- macrophages and microglia differently, and endorses the leagues extended these findings by showing that TNFα previously discussed phagocytic divergence between and iron are important determinants in inducing this in- peripheral macrophages and CNS-derived microglia. flammatory phenotype of mye-phagocytes as they pre- In addition to the polarization status, ontogeny, and vent the conversion of M1- to M2-like cells [110]. Also aging, changes in myelin itself are reported to impact its within MS lesions, numerous studies have demonstrated uptake by phagocytes. Myelin isolated from MS patients the presence of disease-promoting phagocytes in actively is more efficiently internalized by THP-1 cells, a human demyelinating lesions [14]. Interestingly, in yet another monocytic cell line, and primary human microglia as study, myelin was found to modulate microglia differen- compared to myelin isolated from healthy donors [77]. tiation with a biphasic temporal pattern. Especially dur- Enhanced uptake of myelin was not due to differences in ing the first 6h after myelin uptake, microglia display an the oxidation status of myelin. Further studies are war- inflammatory M1-like phenotype. However, prolonged ranted to define which modifications or changes in com- uptake of myelin (6-24h) quenches this initial inflamma- position underlie the increased uptake of MS-derived tory profile of mye-microglia [121]. The speed by which myelin. myelin induces the inflammatory phenotype suggests Collectively, these studies stress the complexity of that it ensues after rapid activation of receptor-mediated myelin uptake by phagocytes, being dependent on the signaling pathways, instead of relying on uptake and intra- receptor repertoire as well as various intrinsic and ex- cellular processing of myelin. In support of this hypoth- trinsic factors. Even more, while one should keep in esis, the myelin-induced release of inflammatory cytokines mind that uptake of myelin debris is advantageous for by macrophages depends on CR3 and subsequent activa- CNS repair, uptake of intact myelin causes demyelin- tion of the FAK/PI3K/Akt/NF-κB signaling pathway [182]. ation. Hence, in vitro studies using myelin debris should As scavenger and Fc receptors are also closely associated always be interpreted with caution before extrapolating with inflammatory signaling cascades [117, 219], their in- to the in vivo situation. volvement in skewing mye-phagocytes towards a more Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 7 of 21 Fig. 3 Foamy phagocyte polarization follows a triphasic pattern. Uptake of myelin initially promotes the induction of a disease-promoting phenotype of phagocytes, characterized by an increased release of inflammatory and toxic mediators, and reduced production of anti-inflammatory factors (phase I). The induction of this phenotype likely relies on the rapid activation of the FAK/PI3K/Akt/NF-κB signaling pathway following ligation of the complement receptor 3 (CR3). In time, intracellular processing of myelin will generate lipid metabolites capable of activating the anti-inflammatory liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR). Activation of these nuclear receptors will repress the inflammatory transcriptional profile in macrophages (phase II). With aging, an inability of phagocytes to process and efflux the enormous amounts of intracellular cholesterol-rich myelin debris results in the formation of cholesterol crystals that activate the NLRP3 inflammasome (phase III) inflammatory phenotype merits further investigation. In macrophages [63]. Collectively, these studies indicate that summary, these studies stress that, at least for a certain myelin uptake can direct phagocytes towards an M2-like period of time, mye-phagocytes display an M1-like phenotype. This phenotype is shared by foamy phagocytes phenotype. in other disorders, as discussed in the next sections. While early studies predominantly defined inflammatory Based on the assumption that myelin modulates features of mye-phagocytes, more recent studies indicate phagocyte differentiation with a biphasic temporal pat- that mye-phagocytes can also acquire anti-inflammatory tern [121], the delayed anti-inflammatory phenotype and wound-healing properties. Mye-phagocytes in the switch of mye-phagocytes likely relies on intracellular center of MS lesions and in in vitro cultures express a processing of myelin-derived constituents. In line with series of anti-inflammatory molecules while lacking this finding, we found that activation of the nuclear re- pro-inflammatory cytokines [18, 220], suggesting that ceptor LXR after myelin uptake and processing directs myelin uptake polarizes phagocytes towards an M2-like the less-inflammatory phenotype that mye-phagocytes phenotype. In agreement, exposure of macrophages to display [15]. LXRs are well-known to repress an inflam- sciatic or optic nerves leads to the formation of matory transcriptional profile in macrophages. More- mye-macrophages that display an unique M2-like pheno- over, LXRs are endogenously activated by cholesterol type [195]. Moreover, we and others demonstrated that metabolites, which are abundantly present in myelin or mye-phagocytes show a less-inflammatory phenotype in can be formed after engulfment and processing of response to prototypical inflammatory stimuli, suppress myelin-derived cholesterol [126]. Of interest, the deacti- autoreactive T cell proliferation, and inhibit Th1 cell vated phenotype of cholesterol-loaded macrophages in polarization [11, 13, 15, 110, 121, 198]. By using adult dor- atherosclerotic lesions also depends in part on the LXR sal root ganglia neurons, conditioned medium of signaling pathway [180]. In addition to LXRs, we also mye-macrophages even enhanced neuron survival and showed that myelin-derived phosphatidylserine activates neurite regeneration [81], suggesting that myelin uptake the fatty acid-sensing PPARβ/δ, thereby reducing the re- also increases the neurotrophic features of phagocytes. lease of inflammatory mediators such as NO [11]. Simi- While studying the phenotype of mye-phagocytes, care lar to LXRs, PPARs can repress inflammatory responses should be taken to prevent endotoxin contamination in mediated by NF-kβ in phagocytes. Active LXR and PPAR myelin isolates. In one study, endotoxin contamination signaling in lesional phagocytes further emphasizes the was found to induce insensitivity to LPS in foamy key role that these nuclear receptors play in directing Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 8 of 21 the phenotype of foamy phagocytes in MS lesions [11, located directly juxtaposed to T cells. Likewise, by using 126]. Yet another study demonstrated that the p47– ultrasound guided fine needle aspiration biopsy to ex- PHOX-mediated production of ROS after prolonged up- tract cells in vivo, macrophages containing MBP and take of myelin represses the production of inflammatory PLP were demonstrated in CLN of MS patients [51]. A mediators by microglia [121]. This study indicates that more recent study confirmed the latter two studies and ROS drives a negative-feedback-circuit aimed at limiting additionally showed that mye-phagocytes in CLNs of MS microglia inflammation. In summary, these studies patients display an M2-like phenotype and express strongly suggest that the delayed anti-inflammatory CCR7 [197]. In contrast, neuronal antigen-containing phenotype of mye-phagocytes depends on signaling phagocytes were pro-inflammatory and did not express pathways activated after myelin uptake and processing. CCR7. These findings confirm the anti-inflammatory im- Similar to the uptake of myelin, extrinsic and intrinsic pact of myelin on phagocytes. Moreover, as CCR7 is cru- factors can influence the phenotypes that mye-phagocytes cial in lymph node-directed chemotaxis [32], this study adopt. For instance, a recent study demonstrated that further suggests that myelin antigens are transported to aging skews mye-phagocytes towards an inflammatory the CNS-draining secondary lymph nodes after uptake phenotype [27]. By using the EAE and cuprizone- and by phagocytes that subsequently migrate to CLNs by lysolecithin-induced demyelination models, inflammatory chemotaxis. However, while the increase in CCR7 on foam cells harbouring large amounts of lysosomal free mye-phagocytes was functional in vitro [199], CCR7 de- cholesterol were observed in old mice. An inability of aged ficiency did not alter the number of myelin-containing phagocytes to process and efflux the high amounts of cells in CLNs of EAE mice compared to WT mice [197]. intracellular cholesterol-rich myelin debris appeared to This implies that other chemokine receptors are in- underlie the accumulation of lysosomal cholesterol. In volved or that myelin antigens are transported to time, the accumulation of free cholesterol resulted in the CNS-draining lymph nodes as soluble antigens. Of inter- formation of cholesterol crystals, which induced lysosomal est, the recently described lymphatic vasculature in the rupture and activated the NLRP3 inflammasome. This CNS, which is connected to the deep CLNs, may lend study suggests that the phenotypes that foamy phagocytes myelin or mye-phagocytes easy access to CNS-draining display in aged individuals might even be triphasic. In lymph nodes [123]. With respect to the latter, myelin anti- addition to aging, spatiotemporal-dependent differences gens and mye-phagocytes are apparent in the CSF of MS in the presence of cytokines are likely to impact the patients [105, 153], which is drained by the lymphatic ves- phenotype of mye-phagocytes differently. Future studies sels lining the dural sinuses [123]. Finally, after selective should define the precise cytokine milieu in active and killing of oligodendrocytes in an in vivo animal model, a chronic active MS lesions and determine the impact of the significant increase in intracellular lipids was found in most abundantly expressed cytokines on the functional deep CLNs, evidenced by increased Oil Red O (ORO) re- properties of mye-phagocytes. Finally, while both macro- activity [122]. ORO reactivity represented intracellular phages and microglia change their phenotype in a similar myelin, as the authors also detected increased MBP and fashion upon myelin internalization, ontogenic differences MOG levels in lumbar lymph nodes. Altogether these may impact the degree of expression of the characteristic studies indicate that CNS demyelination coincides with M1 and M2 markers. With respect to the latter, subtle the accumulation of mye-phagocytes within CNS-draining differences have been noted in the polarization of both cell lymph nodes. How myelin antigens gain excess to these types in response to LPS, IFNγ, IL-4, and IL-13 in vitro lymphoid organs, either after uptake by phagocytes that [44, 59]. In depth genomic and proteomic profiling migrate by chemotaxis or as soluble particles, remains to experiments may unravel differences in the phenotypes be clarified. that macrophages and microglia adopt upon myelin To date, the pathological impact of mye-phagocytes in internalization. CNS-draining lymph nodes remains ambiguous. Mye- phagocytes in secondary lymphoid organs may present Myelin-containing phagocytes in secondary myelin antigens to autoreactive T cells, thereby driving lymphoid organs epitope spreading and MS disease progression or even While abundantly present in MS lesions, few studies initiation [181]. Especially considering that they are lo- demonstrated the presence of mye-phagocytes in the cated directly juxtaposed to T cells and express MHC CNS-draining lymphoid organs of MS patients and EAE class II and costimulatory molecules [18, 38, 50, 198]. animals. De Vos et al. observed a redistribution of mye- Moreover, cervical lymphadenectomy reduces the level lin antigens from brain lesions to cervical lymph nodes of brain lesions in cryolesion-enhanced EAE in rats (CLNs) in primate EAE models and MS patients [38]. [157]. This argues for a key role of CLNs in the induc- Antigens were found in phagocytic cells expressing tion of EAE, possibly as a site for T cell priming. In sup- MHC class II and costimulatory molecules, which were port of an immunostimulatory role of mye-phagocytes, Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 9 of 21 human mye-macrophages were found to promote CD4 of CX3CR1 markedly delays lymphatic trafficking [94]. + hi and CD8 T cell proliferation in an allogeneic mixed These findings suggest that CX3CR1 microglia are more lymphocyte reaction and a recall response against influ- prone to home to secondary lymph nodes in MS than lo enza virus [198]. Macrophages treated with oxidized CX3CR1 monocyte subsets. However, more research is LDL and LDL did not impact lymphocyte proliferation, warranted to certify the abovementioned claims. suggesting that the immunostimulatory impact is spe- cific for myelin and not merely a hallmark of foam cells Parallels with foamy macrophages in other in general. Interestingly, the authors also show that disorders mouse mye-phagocytes reduce the release of IFNγ by Myelin-containing phagocytes are a pathological hall- Th1 cells and that MOG-pulsed mye-macrophages mark of CNS disorders such as MS. However, foamy suppress EAE severity. The latter indicates that macrophages packed with lipid bodies are also abun- mye-phagocytes in CLNs are not only aggressors in MS dantly present in many peripheral pathologies associated pathogenesis but can also dampen T cell-induced auto- with chronic inflammation, such as atherosclerosis and immunity in MS. Supportive of this notion, CLNs are re- non-alcoholic steatohepatitis (NASH), and following in- ported to be instrumental in the induction of intranasally fections with persistent pathogens like Mycobacterium induced immunological tolerance [211]. We further tuberculosis (Mtb), Chlamydia pneumoniae, and Toxo- showed that mye-macrophages inhibit TCR-triggered plasma gondii [98, 139, 161, 169, 213]. Especially in lymphocyte proliferation in an antigen-independent man- atherosclerosis, foamy macrophage physiology has been ner in vitro [13]. Inhibition of T cell proliferation thoroughly investigated. In atherosclerotic lesions, depended on direct contact between both cell types and macrophages acquire a foamy appearance through the the release of NO by mye-phagocytes. Interestingly, while uptake and degradation of native and modified lipopro- mye-phagocytes reduced proliferation of non-myelin re- teins, such as oxLDL. Generally, macrophages are well active T cells in vivo, they increased myelin-reactive T cell equipped to cope with minor intracellular increases of proliferation and worsened EAE severity. These findings LDL. However, sustained intracellular accumulation of suggest that mye-macrophages can both limit and pro- LDL-derived lipids leads to disturbances in pathways mote T cell-induced neuroinflammation, depending on that mediate the degradation, storage, and efflux of these the TCR-specificity of surrounding T cells. Of note, lymph lipids. As a consequence, macrophages become node resident CD169 macrophages activate invariant nat- engorged with lipids and obtain a disease-promoting ural killer T (iNKT) cells by presenting lipid antigens in a phenotype. In this section, we discuss and link the CD1d-dependent manner [3]. CD1d-restricted iNKT cells malfunctioning of these pathways to the development and lipid-reactive non-invariant T cells reduce neuroin- and physiology of phagocytes that internalized the flammation [39, 90]. As myelin is rich in lipids, the cap- lipid-rich myelin sheath (Fig. 4). acity of mye-phagocytes to activate these immune cells merits further investigation. Collectively, these studies Uncontrolled internalization of myelin highlight the pleiotropic impact that mye-phagocytes in In atherosclerosis, the swift removal of modified LDL CNS-draining lymph nodes may have on T cell-mediated from the intima provides protection against its cytotoxic autoimmunity in MS. and damaging effects. However, continuous uptake of To what extent extrinsic and intrinsic factors influence modified LDL by macrophages also promotes the forma- the accumulation and antigen presenting capacity of tion of inflammatory, lipid-engorged, foamy macro- mye-phagocytes in CNS lymph nodes remains to be deter- phages, which eventually may be an even more harmful mined. Interestingly, aging negatively impacts phagocyte event. Diverse studies suggest that feedback regulation migration and their antigen presenting capacity [37, 80], of receptors involved in the uptake of modified LDL and therefore might well alter the ability of mye-phago- goes awry in atherosclerosis. For example, the expression cytes to home to secondary lymph nodes and present of receptors involved in the uptake of modified LDL, myelin-derived antigens [37]. In addition, motility seems such as CD36 and SR-A, remains high throughout lesion to be differently regulated in macrophages and microglia development in atherosclerosis [138]. Similar to athero- [132], suggesting that ontogenic differences might also be sclerosis, uptake of myelin may also be a continuous involved. On that note, while both macrophages and process in neurodegenerative disorders. This is sup- microglia express CCR7 [42, 199], differences in the ex- ported by the finding that the expression of receptors in- pression of other chemokine receptors such as CX3CR1 volved in the uptake of myelin, such as FcRIII, SR-AI/II, and CCR2 are reported between microglia and specific and MerTK, is elevated in active MS lesions [76, 206]. peripheral monocyte subsets [14]. Interestingly, the trans- We further demonstrated that myelin uptake results in membrane chemokine CX3CL1 is induced in inflamed the activation of LXRs and PPARβ/δ [11, 15, 126]. Both lymphatic endothelium and dendritic cell-specific deletion nuclear receptors induce the expression of MerTK and Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 10 of 21 Fig. 4 Homeostatic and dysfunctional processing of cholesterol-containing lipid particles. During homeostasis, phagocytes are well equipped to cope with relatively minor increases of cholesterol. However, sustained intracellular accumulation of cholesterol, as observed in in many peripheral pathologies and following infections with persistent pathogens, can lead to disturbances in pathways that mediate the degradation, storage, or efflux of cholesterol. First, faulty feedback regulation of phagocytic receptors may result in an uncontrolled uptake of cholesterol-containing lipid particles. Second, lysosomal cholesterol accumulation can result in lysosomal dysfunction by reducing lysosomal acidification and causing lysosomal leakiness. In addition, sustained accumulation of cholesterol can lead to the formation of cholesterol crystals that activate the caspase-1-activating NLRP3 inflammasome. Third, persistent cholesterol trafficking to ER membranes can trigger ER stress and the unfolded protein response (UPR). Fourth, dysfunctional lipophagy machinery can hamper the capacity of foamy phagocytes to process cholesterol within lipid droplets, thereby impeding the cells’ capacity to dispose of intracellular cholesterol. Finally, quantitative and qualitative changes in lipoproteins can impact the capacity of foamy phagocytes to efflux cholesterol. Altogether, disturbances in the abovementioned pathways are well-known to promote the induction of a disease-promoting phenotype of foamy phagocytes and eventually even cause apoptosis. While ample evidence suggests that faulty regulation of these pathways also occurs in myelin-containing phagocytes, more research is warranted to define to what extent they impact their inflammatory features opsonins, such as C1qa, and C1qb [142, 145]. This in- expression of CD36 [89, 146]. Furthermore, we demon- crease in expression may augment the internalization of strated that oxidized myelin more potently increases the ex- myelin by phagocytes in demyelinating disorders. Of pression of the phagocytic scavenger receptor CL-P1 interest, continuous activation of PPARγ by modified compared to unmodified myelin [12]. More importantly, oxLDL may promote a similar vicious cycle of LDL up- while CL-P1 surface expression gradually decreases on take in oxLDL-loaded macrophages by inducing the macrophages treated with unmodified myelin, macrophages Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 11 of 21 exposedtooxidizedmyelinretain a high expression of lesions, and what the impact of lysosomal accumulation of CL-P1 over time. These findings indicate that unmodified myelin-derived cholesterol is on lysosomal integrity. and oxidized myelin impact macrophage function differ- LDL loading is reported to downregulate the expres- ently, similar to native and oxLDL. In addition, they suggest sion of Niemann Pick Disease type C1 and C2 (NPC1 an faulty feedback regulation of CL-P1 when phagocytes are NPC2) in macrophages. NPC1 and NP2 are membrane exposed to oxidized forms of myelin. While counter regula- proteins that facilitate the transfer of free cholesterol tory processes that inhibit myelin internalization such as from lysosomes to the endoplasmic reticulum (ER) for the CD47/SIRPα axis exist [61], CD47 was found to be de- further processing [91]. Hence, a reduced expression of creased at the mRNA level and expressed at low abundance NPC1 and NPC2 can augment lysosomal free cholesterol on protein level in MS lesions [73]. Even more, microRNA sequestration and lysosomal dysfunction. While no stud- profiling of MS lesions identified modulators of the regula- ies defined changes in the expression of NPC1 and tory protein CD47 [96, 187]. Reduced signaling through NPC2 in phagocytes upon myelin uptake, fingolimod this inhibitory CD47/SIRPαinhibitory pathway may further (FTY720), which is currently used for treatment of MS, boost myelin uptake and demyelination. Collectively, these increases the expression of NPC1 and NPC2 on both studies stress that faulty regulation of phagocytic and in- mRNA and protein level in NPC mutant fibroblasts hibitory receptors in MS lesions can lead to the uncon- [150]. Likewise, FTY720 increases the expression of trolled internalization of myelin by phagocytes. NPC1 in human macrophages and improves their sur- vival after sustained lipid uptake [10]. This increase in NPC expression may boost the trafficking of free choles- Lysosomal dysfunction terol to the ER in mye-phagocytes, thereby counteracting Ample evidence indicates that lysosomal dysfunction is a the accumulation of free cholesterol in lysosomes and pre- critical step in the formation of M1-like foam cells and venting lysosomal dysfunction. Thus, apart from blocking disease progression in atherosclerosis and NASH [78]. the egress of leukocytes from secondary lymph nodes [22], The sequestration of LDL-derived free cholesterol within FTY720 can suppress MS lesion progression by restoring lysosomes is regarded to underlie lysosomal dysfunction or retaining lysosomal function in mye-phagocytes. and the induction of M1-like macrophages in these dis- Whereas it is generally assumed that lysosomal dys- orders [7, 33, 46, 92, 116, 191, 218]. In MS patients, an function is a secondary event in the pathophysiology of increase in several lysosomal enzymes is apparent in pla- atherosclerosis and NASH, a genome-wide association ques, periplaque areas, NAWM, and CSF samples [35, study identified polymorphisms in the gene encoding the 45, 72], which indicates active breakdown of lipids and lysosomal enzyme galactocerebrosidase (GALC) in MS other macromolecules in the CNS. Strikingly, while ac- patients [87]. This argues for lysosomal dysfunction tive MS lesions are packed with metabolically active being a potential primary pathological event in MS. In mye-phagocytes, lysosomal function or dysfunction Krabbe disease, lack of GALC activity results in within these cells remains largely uninvestigated. Free lysosomal accumulation of galactosylcerebrosides and cholesterol is the predominant form of cholesterol in galactosphingosine in phagocytes and oligodendrocytes, myelin. Hence, continuous uptake of myelin by phago- leading to severe demyelination [103]. Haematopoietic cytes is likely to result in lysosomal accumulation of free stem cell transplantation corrects the metabolic defect cholesterol and consequently lead to lysosomal and in Krabbe disease, which indicates the importance of phagocyte dysfunction. Interestingly, an early study dysfunctional GALC in leukocytes in disease pathogen- using the EAE model demonstrated that abnormalities esis [109]. To what extent polymorphisms in the GALC in lysosomal permeability are apparent before the develop- gene impact lysosomal function and lipid accumulation ment of clinical and histological changes [56]. Similar, in phagocytes in MS upon myelin uptake remains to be cerebral lysosomes seem to be more fragile in MS white clarified. In summary, several studies suggest that lyso- matter compared to white matter of healthy controls somal dysfunction can occur in mye-phagocytes in MS [128]. Lysosomal abnormalities equally affected the lesions. However, more in-depth studies examining the plaque, periplaque, and NAWM in MS patients. These abovementioned lysosomal parameters in in vitro cul- studies suggest that lysosomes in the CNS of MS patients tured mye-phagocytes and within MS lesions are war- are more prone to become dysfunctional. A more recent ranted to certitude this claim. study showed that aged mye-phagocytes have a tendency to accumulate large amounts of lysosomal cholesterol Formation of cholesterol crystals and inflammasome [27]. Lysosomal accumulation of myelin-derived choles- activation terol led to the activation of NLRP3 inflammasome. Des- Sustained accumulation of cholesterol within foamy pite these studies, it remains unclear to what extent macrophages in atherosclerosis, NASH, and following lysosomal dysfunction occurs in foamy phagocytes in MS Mtb infections results in the formation of cholesterol Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 12 of 21 crystals [6, 25, 88]. Several studies indicate that choles- ER stress and UPR activation are known to occur in terol crystals destabilize lysosomes, thereby activating oxLDL-loaded macrophages in vitro and macrophages in the caspase-1-activating NLRP3 inflammasome and pro- human atherosclerotic lesions and apoE-knockout mice moting the release of IL-1β [43, 55, 82, 119]. Similar to [144, 217, 221]. Moreover, cholesterol trafficking to ER foamy phagocytes in these disorders, phagocytes accumu- membranes in cholesterol-loaded macrophages results in late copious amounts of cholesterol in vitro and in vivo UPR activation and promotes phagocyte apoptosis [41, following uptake of myelin [12, 126]. Moreover, several 53]. Similar to atherosclerosis, ER stress and UPR activa- studies demonstrated the presence of cholesterol tion is apparent in MS and EAE lesions. An increased crystal-like structures in mye-phagocytes [8, 27, 113]. By mRNA and protein expression of activating transcription using electron microscopy imaging, numerous mono- factor 4, CCAAT-enhancer-binding protein homologous nuclear cells containing degenerated myelin were found to protein, calreticulin, X-box-binding protein 1, and accumulate needle-shaped cholesterol structures in late immunoglobulin-heavy-chain-binding protein was found stages of Wallerian degeneration [8]. Cholesterol crystals in NAWM and demyelinating lesions of MS patients are also apparent in IBA1 mye-microglia in the corpus [34, 71, 130, 134, 143, 151]. Interestingly, calreticulin co- callosum of cuprizone-treated animals [113]. Finally, a localizes with ORO phagocytes in MS lesions, which more recent study showed that aging results in the accu- points towards ER stress and UPR activation in mulation of cholesterol crystals in mye-phagocytes, lead- mye-phagocytes [151]. Likewise, foamy phagocytes in ac- ing to NLRP3 inflammasome activation [27]. To date, it tive MS lesions show an increased expression of the remains unclear whether cholesterol crystals are also mitochondria-associated membrane protein Rab32, formed in foamy phagocytes within MS lesions, and to which is closely associated with the UPR [71]. Active what extent inflammasome activation in these cells im- UPR signaling is also observed in phagocytes, T cells, as- pacts MS lesion progression. With respect to the latter, trocytes, and oligodendrocytes during the course of EAE inflammasome activation is apparent in the CNS and per- [28, 40, 131, 151]. Importantly, inhibition of the UPR ipheral cells in several neurodegenerative disorders [79, using crocin reduces ER stress and the inflammatory 83, 136, 156]. Furthermore, mice lacking NLRP3, burden in EAE animals. The reduced EAE disease caspase-1, or IL-18 exhibit reduced neuroinflammation, severity was paralleled with preserved myelination and demyelination, and neurodegeneration [69, 79, 86, 93, 125, axonal density, and reduced immune cell infiltration and 215, 216], which underscores the pathogenic role for the phagocyte activation [40]. This study underscores the inflammasome in neurodegenerative disorders. Notably, detrimental impact of ER stress and the UPR on neuro- predominantly macrophages and microglia produce IL-1β inflammation and neurodegeneration. Remarkably, des- in EAE and MS lesions [24, 193], arguing for phagocytes pite ER stress and UPR activation, no studies have being the culprit cells involved in the abovementioned reported the presence of apoptotic and necrotic foamy knockout models. Of particular interest, the scavenger re- phagocytes in active demyelinating MS lesions yet. ceptor CD36 is closely associated with the de novo forma- Phagocyte apoptosis might be difficult to detect histolog- tion of intracellular cholesterol crystals and NLRP3 ically, owing to the fact that dying cells are rapidly inflammasome activation in oxLDL-loaded macrophages cleared by neighboring phagocytes through efferocytosis [175]. Hence, CD36 may well fulfill a similar function in [209]. Thus, while studies point towards a role for ER mye-phagocytes [49]. More in-depth studies are needed to stress and UPR activation in MS pathology, more re- define if de novo formation of cholesterol crystals under- search is warranted to define the underlying mecha- lies inflammasome activation within mye-phagocytes or if nisms, culprit cell types, and functional outcome. lysosomal destabilization due to the free cholesterol accu- mulation causes inflammasome activation. Disturbed autophagy/lipophagy Autophagy is a catabolic process essential for cellular ER stress and the unfolded protein response and tissue homeostasis. While it is crucial for the deg- The ER plays a key role in the biosynthesis, processing, radation of dysfunctional and unwanted proteins and or- and trafficking of proteins. Environmental factors or ele- ganelles, increasing evidence indicates that it also vated protein synthesis can lead to the accumulation of controls lipid degradation, a process called lipophagy misfolded or unfolded proteins in the ER, also called ER [120]. Ouimet et al. defined that lipophagy plays a key stress. ER stress triggers the unfolded protein response role in cholesterol efflux from lipid-laden macrophages (UPR), which attempts to restore ER homeostasis by [154]. During lipophagy, autophagosomes and lysosomes attenuating global protein synthesis and degrading un- fuse with lipid droplets after which esterified cholesterol folded proteins. If the UPR fails to restore ER homeosta- is hydrolyzed by specific enzymes, such as lysosomal sis, apoptotic signaling pathways are activated to remove acid lipase, into free cholesterols. Unlike esterified chol- stressed cells [202]. esterol, free cholesterol is a substrate for ABCA1 and Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 13 of 21 ABCG1-mediated efflux to apoA-I or HDL, respectively. instance, disability in MS patients is positively correlated Hence, active lipophagy represents a way to dispose to plasma LDL, apoB, and total cholesterol levels [127, intracellular cholesterol, thereby preventing their intra- 186, 207]. Furthermore, MS patients display elevated cellular accumulation. oxLDL levels in plasma and the CNS [149, 155], and an Autophagy is tightly linked to the pathogenesis of MS. increase in plasma auto-antibodies directed against However, the precise role that autophagy plays in the oxLDL [4]. In contrast to LDL, controversy exists re- pathogenesis of MS and to what extent the autophagy ma- garding HDL levels in MS patients. Whereas some stud- chinery is dysfunctional is poorly understood. To date, the ies demonstrated a decrease [133], other showed no majority of studies have focused on the impact of autoph- change or even an increase in HDL levels [4, 62, 171]. agy on lymphocyte survival and homeostasis in MS [1, 48, Of note, we recently reported that distinguishing be- 108]. However, autophagy likely also impacts foamy tween different HDL subclasses is of importance when phagocyte function in MS lesions. As autophagy regulates investigating HDL levels [95]. Irrespective of these stud- the antigen presenting capacity of dendritic cells [5], fu- ies, higher serum HDL levels correlate with reduced ture studies should define whether is it also involved in blood-brain barrier injury and a decreased infiltration of the presentation of myelin antigens by foamy phagocytes immune cells into the CSF of MS patients [52]. We re- locally in the CNS and secondary lymphoid organs. Simi- cently identified an altered lipoprotein profile in lar, the influence of autophagy/lipophagy on lipid efflux by relapsing-remitting MS (RR-MS) patients, especially in foamy phagocytes merits further investigation, in particu- low-BMI RR-MS patients, with modified and dysfunc- lar with respect to aging. Recently, aging was reported to tional HDL [95]. By using LC-MS/MS, we demonstrated hamper the efflux efficacy of mye-phagocytes in diverse that HDL is modified at its ApoA-I tyrosine and trypto- animal models for demyelination [27]. Malfunction of the phan residues. Such modifications are increasingly being lipophagy machinery may underlie the age-related dis- acknowledged to alter HDL function [104, 166, 174]. crepancy in the capacity of foamy phagocytes to dispose of Specifically, the Trp50 and Trp72 domains are respon- intracellular cholesterol. Of interest, increasing evidence sible for the initiation of lipid binding to ApoA-I [84, suggests that dysfunctional autophagy is apparent in 201]. This suggests that in in low-BMI RR-MS patients, foamy macrophages in atherosclerosis, and contributes to cholesterol efflux may be dysfunctional, potentially lead- lipid accumulation, apoptosis, and inflammasome hyper- ing to the inflammatory accumulation of myelin-derived activation in these cells [118, 163]. As autophagy regulates lipids in mye-phagocytes. In line with this hypothesis, phagocytosis by modulating the expression of phagocytic serum HDL of RR-MS patients less efficiently accepts receptors [17], defining the impact of autophagy on the cholesterol via the ABCG1 transporter compared to uptake of myelin also deserves further attention. Thus, serum HDL of healthy controls [95]. To what extent while increasingly being acknowledged to impact MS dis- tyrosine and tryptophan modifications underlie changes ease progression, more research is warranted to define the in HDL functionality in RR-MS patients remains to be role that autophagy plays in directing the functional prop- clarified. Altogether, these studies strongly suggest erties of foamy phagocytes, and elucidate whether the au- that quantitative and qualitative changes in lipopro- tophagy machinery becomes dysfunctional in phagocytes teins are apparent in MS and can impact phagocyte engorged with myelin-derived lipids. lipid load and physiology. In particular, the function of phagocytes containing abundant myelin-derived Lipoprotein alterations and modifications lipids can potentially be severely compromised by While we focused in the previous sections on intracellu- these changes in LDL and HDL. lar processes going awry in cholesterol-loaded foamy phagocytes, extracellular factors such as lipoproteins can Protective foamy macrophages also impact lipid processing, thereby directing the physi- Similar to MS, increasing evidence indicates that the ology of these foamy macrophages [172]. Generally, high phenotype of lesional macrophages in atherosclerosis is levels of LDL, and in particular modified forms of LDL, more complex than previously thought [184]. For a long drive the inflammatory activation of macrophages after time, sustained cholesterol uptake and consequent distur- sustained uptake, thereby promoting lesion formation bances in metabolic pathways were believed to promote and progression in atherosclerosis, as described in the the induction of inflammatory, pro-atherogenic macro- previous sections. In contrast, high-density lipoproteins phages, as discussed in the previous paragraphs. However, (HDL) have anti-atherogenic properties, which are at- a regulated accumulation of desmosterol following choles- tributed to their crucial role in reverse cholesterol trans- terol loading also suppresses the inflammatory activation port [166]. Ample evidence suggests that lipoprotein of foamy phagocytes in the absence of overt inflammation levels, subclasses, and function are also altered in MS [180]. Of interest, desmosterol was found to mediate its patients and associated with disease activity. For effects on the macrophage phenotype by activating LXRs. Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 14 of 21 Likewise, another study demonstrated that oxLDL loading Despite of the presence of protective foamy phago- increases the expression of the typical M2 marker arginase cytes, the majority of MS lesions evolve into chronic le- I in a PPAR-dependent manner [57]. These findings sions as disease progresses. To date, it remains elusive suggest that LDL accumulation can also suppress inflam- what causes failure of foamy phagocytes to stop lesion mation and may limit lesion progression when the inflam- progression and promote CNS repair as disease ad- matory burden in atherosclerotic lesions is low. In line vances. Aging impacts immune cell function and recent with these studies, considerable phenotypic variation of evidence indicates that aging drives foamy phagocytes macrophages is apparent in atherosclerotic lesions [19, 29, towards an inflammatory phenotype. Although MS is 97, 185]. Strikingly, similar to oxLDL-loaded macro- generally not regarded an age-related disorder, chronic phages, we found that myelin-derived lipids skew phago- inflammation might well lead to premature innate cytes towards a less inflammatory phenotype in LXR- and immunosenescence in MS patients. At the same time, PPAR-dependent manner upon uptake of myelin [11, 15]. valuable lessons can be learned from foamy macro- Also, within MS lesions considerable phenotypic variation phages in atherosclerosis and other diseases. As delin- is observed [18, 200]. Boven et al. demonstrated that eated in this review, ample evidence indicates that foamy phagocytes in the lesion center display a more checkpoints involved in lipid handling are malfunction- anti-inflammatory phenotype compared to foamy phago- ing in macrophages following massive lipid uptake, lead- cytes in the lesion rim [18]. Vogel extended these findings ing to the intracellular accumulation of inflammatory by showing the majority of foam cells within MS lesions lipids. Future studies should define whether faulty regu- have an intermediate activation status, expressing markers lation of these pathways also occurs in mye-phagocytes. that are characteristic for both inflammatory and This could lead to the identification of new targets for anti-inflammatory phagocytes [200]. Collectively, these therapeutic interventions and may open up new avenues findings indicate that foamy macrophages showing an for therapeutics currently used to treat other disorders M2-like phenotype are apparent in both MS and athero- characterized by the presence of foamy macrophages. sclerotic lesions, and that alike nuclear receptor signaling Acknowledgements pathways drive the formation of these cells. Hence, identi- This work was supported by grants of the Belgian Charcot Foundation, fying ways to specifically target these pathways in phago- Research Foundation Flanders (FWO), and the Transnational University Limburg. cytes will open therapeutic avenues for both MS and atherosclerosis. Authors’ contributions EG and JB designed and drafted the review. JH revised critically for important intellectual content. EG, JH, and JB approved the final manuscript to be Conclusions and future perspectives published. All authors read and approved the final manuscript. Our understanding of the role of foamy phagocytes in the pathophysiology of MS has increased tremendously over Competing interests the last few years. It is becoming clear that the uptake of The authors declare that they have no competing interests. myelin by phagocytes is not merely a disease-promoting process but also a prerequisite for CNS repair. By simply Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published inhibiting the uptake of myelin or reducing the number of maps and institutional affiliations. lesional phagocytes, one will suppress the clearance of in- hibitory myelin and counteract the protective phenotype Received: 24 October 2018 Accepted: 2 November 2018 that phagocytes adopt upon myelin internalization. To il- lustrate, the majority of therapeutics for MS are based on References the assumption that prevention of immune cell infiltration 1. 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The physiology of foamy phagocytes in multiple sclerosis

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Springer Journals
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Copyright © 2018 by The Author(s).
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Biomedicine; Neurosciences; Pathology; Neurology
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Abstract

Multiple sclerosis (MS) is a chronic disease of the central nervous system characterized by massive infiltration of immune cells, demyelination, and axonal loss. Active MS lesions mainly consist of macrophages and microglia containing abundant intracellular myelin remnants. Initial studies showed that these foamy phagocytes primarily promote MS disease progression by internalizing myelin debris, presenting brain-derived autoantigens, and adopting an inflammatory phenotype. However, more recent studies indicate that phagocytes can also adopt a beneficial phenotype upon myelin internalization. In this review, we summarize and discuss the current knowledge on the spatiotemporal physiology of foamy phagocytes in MS lesions, and elaborate on extrinsic and intrinsic factors regulating their behavior. In addition, we discuss and link the physiology of myelin-containing phagocytes to that of foamy macrophages in other disorders such atherosclerosis. Keywords: Macrophage, Microglia, Polarization, Neuroinflammation, Remyelination, Multiple sclerosis Introduction microglia, play an essential role in the pathogenesis of MS Macrophages are mononuclear phagocytes that reside in [14, 141]. Until recently, phagocytes were regarded to pri- every tissue of the body in which they play a crucial role marily cause lesion progression by releasing inflammatory in maintaining tissue homeostasis. They fulfill this task and toxic mediators that negatively impact neuronal and by interacting with microorganisms, remodeling tissue, oligodendrocyte integrity [152, 188], internalizing the intact and dealing with injury. Alongside their role in protect- myelin sheath [214], and presenting brain antigens to auto- ive immunity and homeostasis, they also contribute to reactive T cells [68, 129]. However, this unambiguous con- the pathology of numerous disorders. Hence, there is cept has been challenged and it is now thought that considerable interest in harnessing phagocyte function phagocytes also have beneficial properties in MS. For ex- for therapeutic benefit, either by suppressing the ac- ample, clearance of damaged myelin is essential to facilitate tivity of disease-promoting phagocytes or enhancing CNS repair [137, 168]. Moreover, phagocytes release the mobilization of phagocyte subtypes that are ad- anti-inflammatory and neurotrophic mediators in CNS le- vantageous. Such interventions require a thorough sions and can suppress the disease-promoting activity of as- understanding of the spatiotemporal phenotypes that trocytes and autoaggressive effector T cells [13, 18, 81, 167]. phagocytes display during disease progression. Of particular interest are myelin-containing foamy phago- Multiple sclerosis (MS) is an inflammatory and neurode- cytes as they make up the bulk of immune cells within ac- generative disease of the central nervous system (CNS) with tive and the rim of chronic active MS lesions (Fig. 1 and unknown etiology. While initially regarded to be a [111]). Recent evidence has shed light on the many roles lymphocyte-driven disorder, increasing evidence indicates that these cells play in promoting and suppressing MS le- that phagocytes, such as infiltrated monocyte-derived mac- sion progression, as well as the cellular mechanisms that rophages, CNS border-associated macrophages, and drive their functional properties. In this review we summarize and discuss 1) the mech- * Correspondence: Jeroen.bogie@uhasselt.be anisms involved in the uptake and cellular handling of Jerome J. A. Hendriks and Jeroen F. J. Bogie contributed equally to this myelin, 2) the spatiotemporal phenotypes that foamy work. phagocytes adopt in MS patients, and 3) the intrinsic Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium/ School of Life Sciences, Transnationale Universiteit Limburg, Diepenbeek, and extrinsic factors that impact the physiology of foamy Belgium © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 2 of 21 Fig. 1 Histopathology of inactive, chronic active, and active multiple sclerosis lesions. Inactive, chronic active, and active multiple sclerosis (MS) lesions were stained for intracellular lipid droplets (oil red o; ORO) and myelin (proteolipid protein; PLP). a and b, c and d, e and f are taken from the same lesion. Foamy phagocytes (ORO cells) are apparent in demyelinating chronic active and active MS lesions, but not in inactive lesions phagocytes. In addition, we link the physiology of foamy prior to internalization [66]. Since the discovery of phagocytes in MS to that of lipid-laden foamy macro- receptor-mediated endocytosis of myelin, researchers have phages in other disease such as atherosclerosis. Increas- attempted to identify the culprit receptors involved in the ing evidence indicates that many parallels can be drawn uptake of myelin. To date, numerous receptors such as Fc, between phagocyte subsets in various disorders. complement, and scavenger receptors are reported to To accomplish their functionally distinct roles in drive myelin internalization. In this part of review, we health and disease, tissue macrophages and monocyte- elaborate on these receptors and touch upon cell extrinsic derived macrophages can differentiate into a spectrum and intrinsic factors that influence myelin uptake by of phenotypes [208]. The ex vivo induced M1 and M2 phagocytes (Fig. 2). phenotypes represent two extremes. However, the phe- notypes found in vivo substantially differ from these Fc receptors extremes. To designate the functional properties of The discovery of immunoglobulin G (IgG) capping on phagocytes, we will utilize the term “M1-like” or “disea- the surface of phagocytes located amongst myelinated se-promoting” for phagocytes that express pro-inflam- nerve cells in active MS lesions was the first evidence matory mediators and promote MS lesion progression, for the involvement of antibody opsonization and Fc re- and “M2-like” or “disease-resolving” for those that re- ceptors in the internalization of myelin [162]. In line lease anti-inflammatory and neurotrophic mediators. with this initial discovery, a follow-up study showed that parenchymal and perivascular phagocytes in demyelinat- Myelin internalization ing MS lesions display a strong expression of Fc receptor The uptake of myelin by phagocytes is a pathological hall- I (FcRI), FcRII, and FcRIII, while microglia in the mark of MS lesions and other neurodegenerative disor- normal-appearing white matter (NAWM) barely express ders. The presence of foamy phagocytes is even used as an these receptors [192]. Subsequent in vitro studies con- index of MS lesion activity [160]. Initial evidence that firmed the contribution of Fc receptors to the internal- myelin internalization largely depends on receptor-medi- ization of myelin by showing that opsonization of myelin ated endocytosis came from the observation that myelin with anti-myelin or galactocerebroside antibodies pro- lamellae are attached to coated pits on the macrophage foundly augments the uptake of myelin by macrophages surface in an animal model for MS, experimental auto- and microglia [140, 170, 177, 179, 190]. The amount of immune encephalomyelitis (EAE) [47]. Clathrin-coated internalized myelin was further found to depend on the pits are sites where ligand-receptor complexes cluster degree of opsonization and the myelin epitope Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 3 of 21 Fig. 2 Endocytosis of myelin by phagocytes. Myelin internalization by phagocytes is dependent on the receptor repertoire as well as various intrinsic and extrinsic factors. While scavenger receptors (SR-AI/II and collectin placenta 1 (CL-P1)), Fc receptors, complement receptors (CR3), mer tyrosine kinase (MerTK), and low-density lipoprotein receptor-related protein 1 (LRP1) positively regulate the uptake of myelin, ligation of signal regulatory protein α (SIRPα) inhibits myelin uptake. Cell intrinsic and extrinsic factors, such as phagocyte polarization (M1- or M2-like), phagocyte ontogeny, (hematopoietic stem cells or yolk-sac progenitors), cellular aging, and myelin composition and modifications, can impact the capacity of phagocytes to internalize myelin recognized by the antibodies [64]. However, while activated. Collectively, these studies stress the import- anti-myelin antibodies are present in the circulation of ance of Fc receptors in the uptake of myelin but also MS patients [205], serum of MS patients does not indicate that Fc receptor-mediated uptake is fundamen- opsonize more than that of healthy controls [65]. This tally different in the central and peripheral nervous can be explained by the existence of anti-myelin anti- system. bodies in the sera of healthy controls, as their presence is not limited to MS patients [205]. To date, the opsonic Complement receptors properties of the cerebrospinal fluid (CSF) of MS pa- In addition to Fc receptors, ample evidence indicates that tients have not been determined yet. The presence of B complement receptors are involved in the uptake of mye- cell-rich meningeal follicles in the CNS of MS patients lin by phagocytes. For instance, damaged myelin in areas argues for the presence of a local, more concentrated, of active myelin breakdown and within phagocytes colo- source of myelin-directed immunoglobulins in the CSF calizes with complement components in MS lesions [2, [31]. Of interest, the microenvironment also affects Fc 20, 21]. Similar, an increased density of phagocytes ex- receptor-mediated uptake of myelin. While Ig treatment pressing complement receptors is observed in MS lesions was found to increase Fc receptor-mediated uptake of [124, 204]. In particular, early studies found that the com- myelin by macrophages in a sciatic nerve model, it did plement receptor 3 (CR3) tightly controls myelin internal- not increase myelin internalization by microglia in an ization [23, 140, 164, 165, 178]. CR3 contributes to the optic nerve model, even after addition of macrophages uptake of myelin for up to 80% in the presence of active [112]. Follow-up studies should define if the Fc receptor complement, while it was involved for 55-60% in the ab- expression profile on phagocytes differs in these models. sence of active complement [164]. Counterintuitively, In contrast to FcRI, FcRIIa, and FcRIII, FcRIIb contains myelin clearance by macrophages from CR3-KO mice an immunoreceptor tyrosine-based inhibitory motif em- is not impaired [182]. A possible explanation for this bedded in its intracellular domain [189], which might discrepancy is that CR3 can both induce and reduce negatively impact myelin internalization after being myelin phagocytosis at the same time. CR3 can reduce Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 4 of 21 uptake of myelin by phagocytes through the activation Other receptors of spleen tyrosine kinase (Syk), a non-receptor tyrosine Alongside scavenger, Fc, and complement receptors, several kinase that phagocytic receptors recruit upon activation other receptors are implicated in the endocytosis of myelin. [70]. This Syk-mediated feedback mechanism was sug- Recently, the mer tyrosine kinase (MerTK) was found to be gested to protect phagocytes from excessive intracellu- a functional regulator of myelin uptake by human lar accumulation of myelin. Collectively, these studies monocyte-derived macrophages and microglia [74]. MerTK provideevidencethatCR3-mediateduptakeofmyelin belongs to the Tyro3, Axl, and Mer (TAM) receptor family is more complex than initially regarded, being both in- and has a hand in the internalization of apoptotic cells hibitory and stimulatory. Despite the latter studies, [114, 158]. Of interest, apoptotic cell engulfment engages a anti-CR3 antibodies reduce disease severity in the EAE vicious cycle that leads to enhanced expression of MerTK model [85]. CR3 neutralization was found to reduce the [142, 145]. This vicious cycle depends on the intracellular recruitment of macrophages towards the CNS, thereby activation of the lipid-sensing liver X receptor (LXR) and ameliorating EAE disease severity. It is tempting to peroxisome proliferator-activated receptor (PPAR). Previ- speculate that a diminished phagocytic capacity may ously, we showed that myelin-containing phagocytes (mye- also underlie the reduced disease severity in EAE ani- phagocytes) also display active LXR and PPARβ signaling mals treated with anti-CR3 antibodies. [11, 15, 126]. This suggests that myelin promotes its own clearance through an LXR- and PPAR-dependent increase of MerTK. The significance of MerTK in MS pathogenesis Scavenger receptors is evidenced by the fact that polymorphisms in the MerTK Scavenger receptors are a large family of structurally di- gene are linked to MS susceptibility [87]. While the func- verse proteins, which are implicated in the binding and tional outcome of these polymorphisms remain to be clari- uptake of a wide range of molecules [26, 219]. A vast fied, they seem to depend on the genotype of individuals at amount of evidence indicates that scavenger receptors HLA-DRB1 [9], another MS risk gene [135]. In addition to mediate the uptake of myelin. By using an organ culture MerTK, the low-density lipoprotein receptor-related pro- model of peripheral nerves and a monoclonal blocking tein 1 (LRP1) is an essential receptor for myelin phagocyt- antibody, the scavenger receptors class AI/II (SR-AI/II) osis by microglia in vitro [58]. In EAE and MS lesions, the were initially found to mediate the uptake of myelin by LRP1 protein is highly expressed by phagocytes, providing rat macrophages [36]. At high antibody concentrations, evidence for involvement of LRP1 in MS pathogenesis [30, macrophage invasion of the nerves was completely abol- 58]. By using conditional knockout models, LRP1 deficiency ished, emphasizing that SR-AI/II also regulates macro- in microglia but not macrophages was found to worsen phage adhesion and migration [54, 176], similar to CR3 EAE severity [30]. Increased EAE disease severity was asso- [54, 85, 176]. Follow-up studies further defined that ciated with robust demyelination and increased infiltration SR-AI/AII blocking or knockout decreases myelin up- of immune cells. While the authors provide evidence that take by mouse macrophages and microglia [49, 164, microglia lacking LRP1 have a pro-inflammatory signature -/- 178], and that SR-A mice show reduced demyelination due to increased NF-kβ signaling, reduced microglial clear- and disease severity in the EAE model [115]. In MS le- ance of inhibitory myelin debris may also explain the ob- sions, SR-AI/II is highly expressed by foamy phagocytes served effects. Collectively, these studies stress the in the rim and by ramified microglia around chronic ac- importance of MerTK and LRP1 in the uptake of myelin by tive MS lesions [76]. This expression profile argues for phagocytes. the involvement of SR-AI/II in the uptake of myelin by phagocytes in MS lesions, and SR-AI/II being involved The inhibitory SIRPα-CD47 axis in early uptake of myelin by microglia. Aside from Aside from receptors that stimulate myelin internaliza- SR-AI/II, we recently showed that collectin placenta 1 tion, phagocytes also express receptors that inhibit the (CL-P1), a novel class A scavenger receptor [26], also uptake of particles. These receptors likely evolved to contributes to the uptake of myelin by phagocytes. In ac- limit the uptake of ‘self ’ antigens or as a feedback mech- tive demyelinating MS lesions, CL-P1 immunoreactivity anism to inhibit excessive uptake of particles. With re- colocalizes primarily with perivascular and parenchymal spect to myelin internalization, signal regulatory protein myelin-laden phagocytes. Finally, while evidence con- α (SIRPα), a membrane glycoprotein expressed primarily cerning its role in myelin clearance is still lacking, ex- by phagocytes, represents such a inhibitory receptor. pression of lectin-like oxidized low-density lipoprotein Interaction of SIRPα with the “don’t eat me” protein receptor 1 (LOX1) is elevated at sites of active demyelin- CD47 on myelin decreases the uptake of myelin by mac- ation in MS lesions [76]. Future studies should define rophages and microglia [61, 73]. Of interest, serum also whether blockage of this class E scavenger receptor im- promotes an SIRPα-dependent decrease in myelin up- pacts myelin internalization by phagocytes. take irrespective of CD47 expressed on myelin [61]. A Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 5 of 21 potential mechanism could be the transactivation of phagocytic capacity. With respect to the latter, phagocyt- SIRPα by soluble SIRPα ligands present in serum. In osis of apoptotic cells, bioparticles, and oxidized follow-up studies, SIRPα was demonstrated to inhibit low-density lipoproteins (oxLDL) is more robust in myelin internalization by remodeling of F-actin and M-CSF, IL-4/IL-10, or M-CSF/IL-10 stimulated M2-like thereby cytoskeleton function [60]. Inactivation of the phagocytes as compared to GM-CSF, IFNγ, or LPS stim- paxillin-cofilin signaling axis upon SIRPα activation un- ulated M1-like phagocytes [99, 196, 222]. The uptake of derlies the impact of SIRPα on cytoskeleton function myelin also matches the phenotype of macrophages and and myelin uptake. Of interest, the paxillin-cofilin sig- microglia. Phagocytes stimulated with the naling axis also positively regulates the uptake of myelin anti-inflammatory cytokines TGFβ, IL-4/IL13, IFNβ,or by the scavenger, complement and Fc receptors [60, 70]. IL-4/IL-13/IL-10 display a higher phagocytic capacity These findings place paxillin and cofilin centrally in the than naïve or LPS/IFNγ stimulated M1-like phagocytes process of myelin internalization. [44, 74]. These studies indicate that cytokines in the microenvironment of MS lesions, and in particular the Clearance of myelin debris presence of those cytokines that drive phagocyte Whereas internalization of the intact myelin sheath fuels polarization such as TGFβ, IFNγ, IL-10, and IL-4, regu- demyelination, ample evidence indicates that removal of late the phagocytic features of phagocytes. damaged myelin debris at the lesion site promotes CNS A number of studies further indicate that peripheral repair. Early studies already showed that myelin contains macrophages and CNS-derived microglia differ in their growth inhibitory molecules such as Nogo A, which ex- capacity to internalize myelin [44, 74, 112, 140, 178]. hibit strong inhibitory effects on neurite growth and Microglia generally show a higher capacity to internalize axonal regeneration [67]. Kotter et al. extended these myelin as compared to peripheral macrophage subsets findings by showing that myelin debris removal by [44, 74, 140]. Differences in macrophage and microglia phagocytes is a critical step for efficient remyelination ontogeny, being derived from hematopoietic stem cells [106]. Myelin debris was found to exert potent inhibitory or yolk-sac progenitors respectively, might well explain effects on the ability of oligodendrocyte progenitor cells discrepancies in their receptor expression profile and to differentiate into mature remyelinating oligodendro- phagocytic capacity [101, 173]. On that note, both the cytes [107, 159]. In concordance, by using the cupri- basal and inducible expression of MerTK and myelin zone- and lysolecithin-induced demyelination models, phagocytosis are higher in microglia as compared to reduced uptake of myelin debris by macrophages and monocyte-derived macrophages [74]. Likewise, we re- microglia resulted in inefficient axonal remyelination cently showed that myelin uptake increases the cell sur- characterized with aberrant myelin patterns in vivo [113, face expression of the phagocytic receptor CL-P1 by 147, 168]. Collectively, these studies stress that clearance mouse and human macrophages, but not by primary of myelin debris is mandatory for efficient CNS repair to mouse microglia in vitro [12]. Finally, in contrast to progress or even initiate. Interestingly, a recent study de- peripheral macrophages, immunoglobulin treatment in- fined that blood-derived macrophages and resident creases Fc receptor density on microglia [112]. Collect- microglia have functionally divergent roles in myelin in- ively, these studies suggest that differences in the density ternalization. Macrophages were found to associate with of phagocytic receptors and/or activity of signaling path- nodes of Ranvier and initiate demyelination in the EAE ways involved in driving the expression of these recep- model, whereas microglia appeared to primarily clear tors underlie discrepancies in the phagocytic properties debris [214]. To date, the mechanisms underlying this of macrophages and microglia. It is also noteworthy to difference remain elusive. Once identified they hold mention that blood-derived macrophages associate with great promise for future therapeutics aimed at improving nodes of Ranvier and initiate demyelination, whereas CNS repair in MS. microglia mainly clear myelin debris [214]. This study suggests that differences in myelin uptake might also rely on the presence of receptors that recognize cryptic Cell intrinsic and extrinsic factors influencing myelin myelin epitopes that are not exposed on intact myelin. internalization As phagocytosis experiments are generally carried out Phagocytosis is a dynamic process involving both struc- using myelin debris, differences in the recognition of tural rearrangements, complex signaling events, and a cryptic myelin epitopes by macrophages and microglia plethora of phagocytic receptors. Not surprisingly, di- remain to be determined. verse intrinsic and extrinsic factors are associated with Another factor that impacts the physiology of phago- alterations in the phagocytic capacity of macrophages cytes is aging. Several studies indicate that aged and microglia. For example, ample evidence indicates macrophages less efficiently internalize apoptotic cells that the polarization status of phagocytes drives their [102, 212], bacteria [75], latex beads, and opsonized Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 6 of 21 sheep erythrocytes [183]. By using toxin-induced focal Phenotype of myelin-containing phagocytes demyelination in the mouse spinal cord, together with Ample evidence indicates that myelin uptake changes heterochronic parabiosis, Ruckh et al. demonstrated that the functional properties of macrophages and microglia. aged blood-derived macrophages also clear myelin debris Some studies reported an M2-like phenotype of phago- less efficiently as compared to young macrophages [168]. cytes upon internalization of myelin, whereas others de- In vitro experiments using mouse macrophages and scribed no effect at all, or even an M1-like activation microglia and human monocyte-derived macrophages status. In this section, we elaborate on the phenotypes of confirmed that aging impairs myelin debris clearance by mye-phagocytes as well as the signaling pathways direct- these phagocytes [147]. The authors further show that ing these phenotypes (Fig. 3). reduced activity of the retinoid X receptor (RXR) signal- The abundant presence of foamy phagocytes in MS le- th ing pathway partially accounts for the observed differ- sions sparked interest at the end of the 20 century into ence in myelin uptake between young and old defining the phenotypes of these cells. In line with the phagocytes. Via which pathways RXR signaling decreases prevailing dogma at that time that phagocytes merely the uptake of myelin by aged phagocytes remains to be promote lesion progression, uptake of myelin was ini- clarified. While loss of RXR can directly impact the ex- tially demonstrated to promote the release of substantial pression of phagocytic receptors such as MerTK, im- amounts of TNFα and nitric oxide (NO) by macro- paired phagocytosis can also be a mere consequence of phages [194]. In agreement, myelin engulfment by adult an inability to adopt an M2-like phenotype [100]. In a human-derived microglia induced the oxidative burst follow-up study, it was demonstrated that MS-derived and the release of IL-1, TNFα, and IL-6 [210]. Further- monocytes show a reduced uptake of myelin irrespective more, exposure of M-CSF stimulated M2-like macro- of the patients’ age [148]. This finding suggests that the phages to myelin debris led to a significant decrease in disease state influences the phagocytic features of phago- the expression of M2 markers and increase in the ex- cytes in MS. It is tempting to speculate that premature pression of markers characteristic for M1-like macro- innate immunosenescence, possibly due to chronic in- phages [203]. These studies indicate that naïve as well as flammation (“inflammaging”), impacts phagocyte physi- pre-differentiated M2-like phagocytes adopt an inflam- ology in MS patients. Increasing evidence indicates that matory phenotype after uptake of myelin in vitro. Also premature aging of the immune system is apparent in in in vivo models and MS lesions, several studies defined MS patients [16]. Interestingly, in contrast to macro- the presence of M1-like mye-phagocytes. In the spinal phages, aged human microglia do not show a reduction cord injury (SCI) model, the accumulation of M1-like in myelin uptake compared to their younger counter- phagocytes closely correlates with the intracellular pres- parts [77]. This finding suggests that aging impact ence of myelin-derived lipids [110, 203]. Kroner and col- macrophages and microglia differently, and endorses the leagues extended these findings by showing that TNFα previously discussed phagocytic divergence between and iron are important determinants in inducing this in- peripheral macrophages and CNS-derived microglia. flammatory phenotype of mye-phagocytes as they pre- In addition to the polarization status, ontogeny, and vent the conversion of M1- to M2-like cells [110]. Also aging, changes in myelin itself are reported to impact its within MS lesions, numerous studies have demonstrated uptake by phagocytes. Myelin isolated from MS patients the presence of disease-promoting phagocytes in actively is more efficiently internalized by THP-1 cells, a human demyelinating lesions [14]. Interestingly, in yet another monocytic cell line, and primary human microglia as study, myelin was found to modulate microglia differen- compared to myelin isolated from healthy donors [77]. tiation with a biphasic temporal pattern. Especially dur- Enhanced uptake of myelin was not due to differences in ing the first 6h after myelin uptake, microglia display an the oxidation status of myelin. Further studies are war- inflammatory M1-like phenotype. However, prolonged ranted to define which modifications or changes in com- uptake of myelin (6-24h) quenches this initial inflamma- position underlie the increased uptake of MS-derived tory profile of mye-microglia [121]. The speed by which myelin. myelin induces the inflammatory phenotype suggests Collectively, these studies stress the complexity of that it ensues after rapid activation of receptor-mediated myelin uptake by phagocytes, being dependent on the signaling pathways, instead of relying on uptake and intra- receptor repertoire as well as various intrinsic and ex- cellular processing of myelin. In support of this hypoth- trinsic factors. Even more, while one should keep in esis, the myelin-induced release of inflammatory cytokines mind that uptake of myelin debris is advantageous for by macrophages depends on CR3 and subsequent activa- CNS repair, uptake of intact myelin causes demyelin- tion of the FAK/PI3K/Akt/NF-κB signaling pathway [182]. ation. Hence, in vitro studies using myelin debris should As scavenger and Fc receptors are also closely associated always be interpreted with caution before extrapolating with inflammatory signaling cascades [117, 219], their in- to the in vivo situation. volvement in skewing mye-phagocytes towards a more Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 7 of 21 Fig. 3 Foamy phagocyte polarization follows a triphasic pattern. Uptake of myelin initially promotes the induction of a disease-promoting phenotype of phagocytes, characterized by an increased release of inflammatory and toxic mediators, and reduced production of anti-inflammatory factors (phase I). The induction of this phenotype likely relies on the rapid activation of the FAK/PI3K/Akt/NF-κB signaling pathway following ligation of the complement receptor 3 (CR3). In time, intracellular processing of myelin will generate lipid metabolites capable of activating the anti-inflammatory liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR). Activation of these nuclear receptors will repress the inflammatory transcriptional profile in macrophages (phase II). With aging, an inability of phagocytes to process and efflux the enormous amounts of intracellular cholesterol-rich myelin debris results in the formation of cholesterol crystals that activate the NLRP3 inflammasome (phase III) inflammatory phenotype merits further investigation. In macrophages [63]. Collectively, these studies indicate that summary, these studies stress that, at least for a certain myelin uptake can direct phagocytes towards an M2-like period of time, mye-phagocytes display an M1-like phenotype. This phenotype is shared by foamy phagocytes phenotype. in other disorders, as discussed in the next sections. While early studies predominantly defined inflammatory Based on the assumption that myelin modulates features of mye-phagocytes, more recent studies indicate phagocyte differentiation with a biphasic temporal pat- that mye-phagocytes can also acquire anti-inflammatory tern [121], the delayed anti-inflammatory phenotype and wound-healing properties. Mye-phagocytes in the switch of mye-phagocytes likely relies on intracellular center of MS lesions and in in vitro cultures express a processing of myelin-derived constituents. In line with series of anti-inflammatory molecules while lacking this finding, we found that activation of the nuclear re- pro-inflammatory cytokines [18, 220], suggesting that ceptor LXR after myelin uptake and processing directs myelin uptake polarizes phagocytes towards an M2-like the less-inflammatory phenotype that mye-phagocytes phenotype. In agreement, exposure of macrophages to display [15]. LXRs are well-known to repress an inflam- sciatic or optic nerves leads to the formation of matory transcriptional profile in macrophages. More- mye-macrophages that display an unique M2-like pheno- over, LXRs are endogenously activated by cholesterol type [195]. Moreover, we and others demonstrated that metabolites, which are abundantly present in myelin or mye-phagocytes show a less-inflammatory phenotype in can be formed after engulfment and processing of response to prototypical inflammatory stimuli, suppress myelin-derived cholesterol [126]. Of interest, the deacti- autoreactive T cell proliferation, and inhibit Th1 cell vated phenotype of cholesterol-loaded macrophages in polarization [11, 13, 15, 110, 121, 198]. By using adult dor- atherosclerotic lesions also depends in part on the LXR sal root ganglia neurons, conditioned medium of signaling pathway [180]. In addition to LXRs, we also mye-macrophages even enhanced neuron survival and showed that myelin-derived phosphatidylserine activates neurite regeneration [81], suggesting that myelin uptake the fatty acid-sensing PPARβ/δ, thereby reducing the re- also increases the neurotrophic features of phagocytes. lease of inflammatory mediators such as NO [11]. Simi- While studying the phenotype of mye-phagocytes, care lar to LXRs, PPARs can repress inflammatory responses should be taken to prevent endotoxin contamination in mediated by NF-kβ in phagocytes. Active LXR and PPAR myelin isolates. In one study, endotoxin contamination signaling in lesional phagocytes further emphasizes the was found to induce insensitivity to LPS in foamy key role that these nuclear receptors play in directing Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 8 of 21 the phenotype of foamy phagocytes in MS lesions [11, located directly juxtaposed to T cells. Likewise, by using 126]. Yet another study demonstrated that the p47– ultrasound guided fine needle aspiration biopsy to ex- PHOX-mediated production of ROS after prolonged up- tract cells in vivo, macrophages containing MBP and take of myelin represses the production of inflammatory PLP were demonstrated in CLN of MS patients [51]. A mediators by microglia [121]. This study indicates that more recent study confirmed the latter two studies and ROS drives a negative-feedback-circuit aimed at limiting additionally showed that mye-phagocytes in CLNs of MS microglia inflammation. In summary, these studies patients display an M2-like phenotype and express strongly suggest that the delayed anti-inflammatory CCR7 [197]. In contrast, neuronal antigen-containing phenotype of mye-phagocytes depends on signaling phagocytes were pro-inflammatory and did not express pathways activated after myelin uptake and processing. CCR7. These findings confirm the anti-inflammatory im- Similar to the uptake of myelin, extrinsic and intrinsic pact of myelin on phagocytes. Moreover, as CCR7 is cru- factors can influence the phenotypes that mye-phagocytes cial in lymph node-directed chemotaxis [32], this study adopt. For instance, a recent study demonstrated that further suggests that myelin antigens are transported to aging skews mye-phagocytes towards an inflammatory the CNS-draining secondary lymph nodes after uptake phenotype [27]. By using the EAE and cuprizone- and by phagocytes that subsequently migrate to CLNs by lysolecithin-induced demyelination models, inflammatory chemotaxis. However, while the increase in CCR7 on foam cells harbouring large amounts of lysosomal free mye-phagocytes was functional in vitro [199], CCR7 de- cholesterol were observed in old mice. An inability of aged ficiency did not alter the number of myelin-containing phagocytes to process and efflux the high amounts of cells in CLNs of EAE mice compared to WT mice [197]. intracellular cholesterol-rich myelin debris appeared to This implies that other chemokine receptors are in- underlie the accumulation of lysosomal cholesterol. In volved or that myelin antigens are transported to time, the accumulation of free cholesterol resulted in the CNS-draining lymph nodes as soluble antigens. Of inter- formation of cholesterol crystals, which induced lysosomal est, the recently described lymphatic vasculature in the rupture and activated the NLRP3 inflammasome. This CNS, which is connected to the deep CLNs, may lend study suggests that the phenotypes that foamy phagocytes myelin or mye-phagocytes easy access to CNS-draining display in aged individuals might even be triphasic. In lymph nodes [123]. With respect to the latter, myelin anti- addition to aging, spatiotemporal-dependent differences gens and mye-phagocytes are apparent in the CSF of MS in the presence of cytokines are likely to impact the patients [105, 153], which is drained by the lymphatic ves- phenotype of mye-phagocytes differently. Future studies sels lining the dural sinuses [123]. Finally, after selective should define the precise cytokine milieu in active and killing of oligodendrocytes in an in vivo animal model, a chronic active MS lesions and determine the impact of the significant increase in intracellular lipids was found in most abundantly expressed cytokines on the functional deep CLNs, evidenced by increased Oil Red O (ORO) re- properties of mye-phagocytes. Finally, while both macro- activity [122]. ORO reactivity represented intracellular phages and microglia change their phenotype in a similar myelin, as the authors also detected increased MBP and fashion upon myelin internalization, ontogenic differences MOG levels in lumbar lymph nodes. Altogether these may impact the degree of expression of the characteristic studies indicate that CNS demyelination coincides with M1 and M2 markers. With respect to the latter, subtle the accumulation of mye-phagocytes within CNS-draining differences have been noted in the polarization of both cell lymph nodes. How myelin antigens gain excess to these types in response to LPS, IFNγ, IL-4, and IL-13 in vitro lymphoid organs, either after uptake by phagocytes that [44, 59]. In depth genomic and proteomic profiling migrate by chemotaxis or as soluble particles, remains to experiments may unravel differences in the phenotypes be clarified. that macrophages and microglia adopt upon myelin To date, the pathological impact of mye-phagocytes in internalization. CNS-draining lymph nodes remains ambiguous. Mye- phagocytes in secondary lymphoid organs may present Myelin-containing phagocytes in secondary myelin antigens to autoreactive T cells, thereby driving lymphoid organs epitope spreading and MS disease progression or even While abundantly present in MS lesions, few studies initiation [181]. Especially considering that they are lo- demonstrated the presence of mye-phagocytes in the cated directly juxtaposed to T cells and express MHC CNS-draining lymphoid organs of MS patients and EAE class II and costimulatory molecules [18, 38, 50, 198]. animals. De Vos et al. observed a redistribution of mye- Moreover, cervical lymphadenectomy reduces the level lin antigens from brain lesions to cervical lymph nodes of brain lesions in cryolesion-enhanced EAE in rats (CLNs) in primate EAE models and MS patients [38]. [157]. This argues for a key role of CLNs in the induc- Antigens were found in phagocytic cells expressing tion of EAE, possibly as a site for T cell priming. In sup- MHC class II and costimulatory molecules, which were port of an immunostimulatory role of mye-phagocytes, Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 9 of 21 human mye-macrophages were found to promote CD4 of CX3CR1 markedly delays lymphatic trafficking [94]. + hi and CD8 T cell proliferation in an allogeneic mixed These findings suggest that CX3CR1 microglia are more lymphocyte reaction and a recall response against influ- prone to home to secondary lymph nodes in MS than lo enza virus [198]. Macrophages treated with oxidized CX3CR1 monocyte subsets. However, more research is LDL and LDL did not impact lymphocyte proliferation, warranted to certify the abovementioned claims. suggesting that the immunostimulatory impact is spe- cific for myelin and not merely a hallmark of foam cells Parallels with foamy macrophages in other in general. Interestingly, the authors also show that disorders mouse mye-phagocytes reduce the release of IFNγ by Myelin-containing phagocytes are a pathological hall- Th1 cells and that MOG-pulsed mye-macrophages mark of CNS disorders such as MS. However, foamy suppress EAE severity. The latter indicates that macrophages packed with lipid bodies are also abun- mye-phagocytes in CLNs are not only aggressors in MS dantly present in many peripheral pathologies associated pathogenesis but can also dampen T cell-induced auto- with chronic inflammation, such as atherosclerosis and immunity in MS. Supportive of this notion, CLNs are re- non-alcoholic steatohepatitis (NASH), and following in- ported to be instrumental in the induction of intranasally fections with persistent pathogens like Mycobacterium induced immunological tolerance [211]. We further tuberculosis (Mtb), Chlamydia pneumoniae, and Toxo- showed that mye-macrophages inhibit TCR-triggered plasma gondii [98, 139, 161, 169, 213]. Especially in lymphocyte proliferation in an antigen-independent man- atherosclerosis, foamy macrophage physiology has been ner in vitro [13]. Inhibition of T cell proliferation thoroughly investigated. In atherosclerotic lesions, depended on direct contact between both cell types and macrophages acquire a foamy appearance through the the release of NO by mye-phagocytes. Interestingly, while uptake and degradation of native and modified lipopro- mye-phagocytes reduced proliferation of non-myelin re- teins, such as oxLDL. Generally, macrophages are well active T cells in vivo, they increased myelin-reactive T cell equipped to cope with minor intracellular increases of proliferation and worsened EAE severity. These findings LDL. However, sustained intracellular accumulation of suggest that mye-macrophages can both limit and pro- LDL-derived lipids leads to disturbances in pathways mote T cell-induced neuroinflammation, depending on that mediate the degradation, storage, and efflux of these the TCR-specificity of surrounding T cells. Of note, lymph lipids. As a consequence, macrophages become node resident CD169 macrophages activate invariant nat- engorged with lipids and obtain a disease-promoting ural killer T (iNKT) cells by presenting lipid antigens in a phenotype. In this section, we discuss and link the CD1d-dependent manner [3]. CD1d-restricted iNKT cells malfunctioning of these pathways to the development and lipid-reactive non-invariant T cells reduce neuroin- and physiology of phagocytes that internalized the flammation [39, 90]. As myelin is rich in lipids, the cap- lipid-rich myelin sheath (Fig. 4). acity of mye-phagocytes to activate these immune cells merits further investigation. Collectively, these studies Uncontrolled internalization of myelin highlight the pleiotropic impact that mye-phagocytes in In atherosclerosis, the swift removal of modified LDL CNS-draining lymph nodes may have on T cell-mediated from the intima provides protection against its cytotoxic autoimmunity in MS. and damaging effects. However, continuous uptake of To what extent extrinsic and intrinsic factors influence modified LDL by macrophages also promotes the forma- the accumulation and antigen presenting capacity of tion of inflammatory, lipid-engorged, foamy macro- mye-phagocytes in CNS lymph nodes remains to be deter- phages, which eventually may be an even more harmful mined. Interestingly, aging negatively impacts phagocyte event. Diverse studies suggest that feedback regulation migration and their antigen presenting capacity [37, 80], of receptors involved in the uptake of modified LDL and therefore might well alter the ability of mye-phago- goes awry in atherosclerosis. For example, the expression cytes to home to secondary lymph nodes and present of receptors involved in the uptake of modified LDL, myelin-derived antigens [37]. In addition, motility seems such as CD36 and SR-A, remains high throughout lesion to be differently regulated in macrophages and microglia development in atherosclerosis [138]. Similar to athero- [132], suggesting that ontogenic differences might also be sclerosis, uptake of myelin may also be a continuous involved. On that note, while both macrophages and process in neurodegenerative disorders. This is sup- microglia express CCR7 [42, 199], differences in the ex- ported by the finding that the expression of receptors in- pression of other chemokine receptors such as CX3CR1 volved in the uptake of myelin, such as FcRIII, SR-AI/II, and CCR2 are reported between microglia and specific and MerTK, is elevated in active MS lesions [76, 206]. peripheral monocyte subsets [14]. Interestingly, the trans- We further demonstrated that myelin uptake results in membrane chemokine CX3CL1 is induced in inflamed the activation of LXRs and PPARβ/δ [11, 15, 126]. Both lymphatic endothelium and dendritic cell-specific deletion nuclear receptors induce the expression of MerTK and Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 10 of 21 Fig. 4 Homeostatic and dysfunctional processing of cholesterol-containing lipid particles. During homeostasis, phagocytes are well equipped to cope with relatively minor increases of cholesterol. However, sustained intracellular accumulation of cholesterol, as observed in in many peripheral pathologies and following infections with persistent pathogens, can lead to disturbances in pathways that mediate the degradation, storage, or efflux of cholesterol. First, faulty feedback regulation of phagocytic receptors may result in an uncontrolled uptake of cholesterol-containing lipid particles. Second, lysosomal cholesterol accumulation can result in lysosomal dysfunction by reducing lysosomal acidification and causing lysosomal leakiness. In addition, sustained accumulation of cholesterol can lead to the formation of cholesterol crystals that activate the caspase-1-activating NLRP3 inflammasome. Third, persistent cholesterol trafficking to ER membranes can trigger ER stress and the unfolded protein response (UPR). Fourth, dysfunctional lipophagy machinery can hamper the capacity of foamy phagocytes to process cholesterol within lipid droplets, thereby impeding the cells’ capacity to dispose of intracellular cholesterol. Finally, quantitative and qualitative changes in lipoproteins can impact the capacity of foamy phagocytes to efflux cholesterol. Altogether, disturbances in the abovementioned pathways are well-known to promote the induction of a disease-promoting phenotype of foamy phagocytes and eventually even cause apoptosis. While ample evidence suggests that faulty regulation of these pathways also occurs in myelin-containing phagocytes, more research is warranted to define to what extent they impact their inflammatory features opsonins, such as C1qa, and C1qb [142, 145]. This in- expression of CD36 [89, 146]. Furthermore, we demon- crease in expression may augment the internalization of strated that oxidized myelin more potently increases the ex- myelin by phagocytes in demyelinating disorders. Of pression of the phagocytic scavenger receptor CL-P1 interest, continuous activation of PPARγ by modified compared to unmodified myelin [12]. More importantly, oxLDL may promote a similar vicious cycle of LDL up- while CL-P1 surface expression gradually decreases on take in oxLDL-loaded macrophages by inducing the macrophages treated with unmodified myelin, macrophages Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 11 of 21 exposedtooxidizedmyelinretain a high expression of lesions, and what the impact of lysosomal accumulation of CL-P1 over time. These findings indicate that unmodified myelin-derived cholesterol is on lysosomal integrity. and oxidized myelin impact macrophage function differ- LDL loading is reported to downregulate the expres- ently, similar to native and oxLDL. In addition, they suggest sion of Niemann Pick Disease type C1 and C2 (NPC1 an faulty feedback regulation of CL-P1 when phagocytes are NPC2) in macrophages. NPC1 and NP2 are membrane exposed to oxidized forms of myelin. While counter regula- proteins that facilitate the transfer of free cholesterol tory processes that inhibit myelin internalization such as from lysosomes to the endoplasmic reticulum (ER) for the CD47/SIRPα axis exist [61], CD47 was found to be de- further processing [91]. Hence, a reduced expression of creased at the mRNA level and expressed at low abundance NPC1 and NPC2 can augment lysosomal free cholesterol on protein level in MS lesions [73]. Even more, microRNA sequestration and lysosomal dysfunction. While no stud- profiling of MS lesions identified modulators of the regula- ies defined changes in the expression of NPC1 and tory protein CD47 [96, 187]. Reduced signaling through NPC2 in phagocytes upon myelin uptake, fingolimod this inhibitory CD47/SIRPαinhibitory pathway may further (FTY720), which is currently used for treatment of MS, boost myelin uptake and demyelination. Collectively, these increases the expression of NPC1 and NPC2 on both studies stress that faulty regulation of phagocytic and in- mRNA and protein level in NPC mutant fibroblasts hibitory receptors in MS lesions can lead to the uncon- [150]. Likewise, FTY720 increases the expression of trolled internalization of myelin by phagocytes. NPC1 in human macrophages and improves their sur- vival after sustained lipid uptake [10]. This increase in NPC expression may boost the trafficking of free choles- Lysosomal dysfunction terol to the ER in mye-phagocytes, thereby counteracting Ample evidence indicates that lysosomal dysfunction is a the accumulation of free cholesterol in lysosomes and pre- critical step in the formation of M1-like foam cells and venting lysosomal dysfunction. Thus, apart from blocking disease progression in atherosclerosis and NASH [78]. the egress of leukocytes from secondary lymph nodes [22], The sequestration of LDL-derived free cholesterol within FTY720 can suppress MS lesion progression by restoring lysosomes is regarded to underlie lysosomal dysfunction or retaining lysosomal function in mye-phagocytes. and the induction of M1-like macrophages in these dis- Whereas it is generally assumed that lysosomal dys- orders [7, 33, 46, 92, 116, 191, 218]. In MS patients, an function is a secondary event in the pathophysiology of increase in several lysosomal enzymes is apparent in pla- atherosclerosis and NASH, a genome-wide association ques, periplaque areas, NAWM, and CSF samples [35, study identified polymorphisms in the gene encoding the 45, 72], which indicates active breakdown of lipids and lysosomal enzyme galactocerebrosidase (GALC) in MS other macromolecules in the CNS. Strikingly, while ac- patients [87]. This argues for lysosomal dysfunction tive MS lesions are packed with metabolically active being a potential primary pathological event in MS. In mye-phagocytes, lysosomal function or dysfunction Krabbe disease, lack of GALC activity results in within these cells remains largely uninvestigated. Free lysosomal accumulation of galactosylcerebrosides and cholesterol is the predominant form of cholesterol in galactosphingosine in phagocytes and oligodendrocytes, myelin. Hence, continuous uptake of myelin by phago- leading to severe demyelination [103]. Haematopoietic cytes is likely to result in lysosomal accumulation of free stem cell transplantation corrects the metabolic defect cholesterol and consequently lead to lysosomal and in Krabbe disease, which indicates the importance of phagocyte dysfunction. Interestingly, an early study dysfunctional GALC in leukocytes in disease pathogen- using the EAE model demonstrated that abnormalities esis [109]. To what extent polymorphisms in the GALC in lysosomal permeability are apparent before the develop- gene impact lysosomal function and lipid accumulation ment of clinical and histological changes [56]. Similar, in phagocytes in MS upon myelin uptake remains to be cerebral lysosomes seem to be more fragile in MS white clarified. In summary, several studies suggest that lyso- matter compared to white matter of healthy controls somal dysfunction can occur in mye-phagocytes in MS [128]. Lysosomal abnormalities equally affected the lesions. However, more in-depth studies examining the plaque, periplaque, and NAWM in MS patients. These abovementioned lysosomal parameters in in vitro cul- studies suggest that lysosomes in the CNS of MS patients tured mye-phagocytes and within MS lesions are war- are more prone to become dysfunctional. A more recent ranted to certitude this claim. study showed that aged mye-phagocytes have a tendency to accumulate large amounts of lysosomal cholesterol Formation of cholesterol crystals and inflammasome [27]. Lysosomal accumulation of myelin-derived choles- activation terol led to the activation of NLRP3 inflammasome. Des- Sustained accumulation of cholesterol within foamy pite these studies, it remains unclear to what extent macrophages in atherosclerosis, NASH, and following lysosomal dysfunction occurs in foamy phagocytes in MS Mtb infections results in the formation of cholesterol Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 12 of 21 crystals [6, 25, 88]. Several studies indicate that choles- ER stress and UPR activation are known to occur in terol crystals destabilize lysosomes, thereby activating oxLDL-loaded macrophages in vitro and macrophages in the caspase-1-activating NLRP3 inflammasome and pro- human atherosclerotic lesions and apoE-knockout mice moting the release of IL-1β [43, 55, 82, 119]. Similar to [144, 217, 221]. Moreover, cholesterol trafficking to ER foamy phagocytes in these disorders, phagocytes accumu- membranes in cholesterol-loaded macrophages results in late copious amounts of cholesterol in vitro and in vivo UPR activation and promotes phagocyte apoptosis [41, following uptake of myelin [12, 126]. Moreover, several 53]. Similar to atherosclerosis, ER stress and UPR activa- studies demonstrated the presence of cholesterol tion is apparent in MS and EAE lesions. An increased crystal-like structures in mye-phagocytes [8, 27, 113]. By mRNA and protein expression of activating transcription using electron microscopy imaging, numerous mono- factor 4, CCAAT-enhancer-binding protein homologous nuclear cells containing degenerated myelin were found to protein, calreticulin, X-box-binding protein 1, and accumulate needle-shaped cholesterol structures in late immunoglobulin-heavy-chain-binding protein was found stages of Wallerian degeneration [8]. Cholesterol crystals in NAWM and demyelinating lesions of MS patients are also apparent in IBA1 mye-microglia in the corpus [34, 71, 130, 134, 143, 151]. Interestingly, calreticulin co- callosum of cuprizone-treated animals [113]. Finally, a localizes with ORO phagocytes in MS lesions, which more recent study showed that aging results in the accu- points towards ER stress and UPR activation in mulation of cholesterol crystals in mye-phagocytes, lead- mye-phagocytes [151]. Likewise, foamy phagocytes in ac- ing to NLRP3 inflammasome activation [27]. To date, it tive MS lesions show an increased expression of the remains unclear whether cholesterol crystals are also mitochondria-associated membrane protein Rab32, formed in foamy phagocytes within MS lesions, and to which is closely associated with the UPR [71]. Active what extent inflammasome activation in these cells im- UPR signaling is also observed in phagocytes, T cells, as- pacts MS lesion progression. With respect to the latter, trocytes, and oligodendrocytes during the course of EAE inflammasome activation is apparent in the CNS and per- [28, 40, 131, 151]. Importantly, inhibition of the UPR ipheral cells in several neurodegenerative disorders [79, using crocin reduces ER stress and the inflammatory 83, 136, 156]. Furthermore, mice lacking NLRP3, burden in EAE animals. The reduced EAE disease caspase-1, or IL-18 exhibit reduced neuroinflammation, severity was paralleled with preserved myelination and demyelination, and neurodegeneration [69, 79, 86, 93, 125, axonal density, and reduced immune cell infiltration and 215, 216], which underscores the pathogenic role for the phagocyte activation [40]. This study underscores the inflammasome in neurodegenerative disorders. Notably, detrimental impact of ER stress and the UPR on neuro- predominantly macrophages and microglia produce IL-1β inflammation and neurodegeneration. Remarkably, des- in EAE and MS lesions [24, 193], arguing for phagocytes pite ER stress and UPR activation, no studies have being the culprit cells involved in the abovementioned reported the presence of apoptotic and necrotic foamy knockout models. Of particular interest, the scavenger re- phagocytes in active demyelinating MS lesions yet. ceptor CD36 is closely associated with the de novo forma- Phagocyte apoptosis might be difficult to detect histolog- tion of intracellular cholesterol crystals and NLRP3 ically, owing to the fact that dying cells are rapidly inflammasome activation in oxLDL-loaded macrophages cleared by neighboring phagocytes through efferocytosis [175]. Hence, CD36 may well fulfill a similar function in [209]. Thus, while studies point towards a role for ER mye-phagocytes [49]. More in-depth studies are needed to stress and UPR activation in MS pathology, more re- define if de novo formation of cholesterol crystals under- search is warranted to define the underlying mecha- lies inflammasome activation within mye-phagocytes or if nisms, culprit cell types, and functional outcome. lysosomal destabilization due to the free cholesterol accu- mulation causes inflammasome activation. Disturbed autophagy/lipophagy Autophagy is a catabolic process essential for cellular ER stress and the unfolded protein response and tissue homeostasis. While it is crucial for the deg- The ER plays a key role in the biosynthesis, processing, radation of dysfunctional and unwanted proteins and or- and trafficking of proteins. Environmental factors or ele- ganelles, increasing evidence indicates that it also vated protein synthesis can lead to the accumulation of controls lipid degradation, a process called lipophagy misfolded or unfolded proteins in the ER, also called ER [120]. Ouimet et al. defined that lipophagy plays a key stress. ER stress triggers the unfolded protein response role in cholesterol efflux from lipid-laden macrophages (UPR), which attempts to restore ER homeostasis by [154]. During lipophagy, autophagosomes and lysosomes attenuating global protein synthesis and degrading un- fuse with lipid droplets after which esterified cholesterol folded proteins. If the UPR fails to restore ER homeosta- is hydrolyzed by specific enzymes, such as lysosomal sis, apoptotic signaling pathways are activated to remove acid lipase, into free cholesterols. Unlike esterified chol- stressed cells [202]. esterol, free cholesterol is a substrate for ABCA1 and Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 13 of 21 ABCG1-mediated efflux to apoA-I or HDL, respectively. instance, disability in MS patients is positively correlated Hence, active lipophagy represents a way to dispose to plasma LDL, apoB, and total cholesterol levels [127, intracellular cholesterol, thereby preventing their intra- 186, 207]. Furthermore, MS patients display elevated cellular accumulation. oxLDL levels in plasma and the CNS [149, 155], and an Autophagy is tightly linked to the pathogenesis of MS. increase in plasma auto-antibodies directed against However, the precise role that autophagy plays in the oxLDL [4]. In contrast to LDL, controversy exists re- pathogenesis of MS and to what extent the autophagy ma- garding HDL levels in MS patients. Whereas some stud- chinery is dysfunctional is poorly understood. To date, the ies demonstrated a decrease [133], other showed no majority of studies have focused on the impact of autoph- change or even an increase in HDL levels [4, 62, 171]. agy on lymphocyte survival and homeostasis in MS [1, 48, Of note, we recently reported that distinguishing be- 108]. However, autophagy likely also impacts foamy tween different HDL subclasses is of importance when phagocyte function in MS lesions. As autophagy regulates investigating HDL levels [95]. Irrespective of these stud- the antigen presenting capacity of dendritic cells [5], fu- ies, higher serum HDL levels correlate with reduced ture studies should define whether is it also involved in blood-brain barrier injury and a decreased infiltration of the presentation of myelin antigens by foamy phagocytes immune cells into the CSF of MS patients [52]. We re- locally in the CNS and secondary lymphoid organs. Simi- cently identified an altered lipoprotein profile in lar, the influence of autophagy/lipophagy on lipid efflux by relapsing-remitting MS (RR-MS) patients, especially in foamy phagocytes merits further investigation, in particu- low-BMI RR-MS patients, with modified and dysfunc- lar with respect to aging. Recently, aging was reported to tional HDL [95]. By using LC-MS/MS, we demonstrated hamper the efflux efficacy of mye-phagocytes in diverse that HDL is modified at its ApoA-I tyrosine and trypto- animal models for demyelination [27]. Malfunction of the phan residues. Such modifications are increasingly being lipophagy machinery may underlie the age-related dis- acknowledged to alter HDL function [104, 166, 174]. crepancy in the capacity of foamy phagocytes to dispose of Specifically, the Trp50 and Trp72 domains are respon- intracellular cholesterol. Of interest, increasing evidence sible for the initiation of lipid binding to ApoA-I [84, suggests that dysfunctional autophagy is apparent in 201]. This suggests that in in low-BMI RR-MS patients, foamy macrophages in atherosclerosis, and contributes to cholesterol efflux may be dysfunctional, potentially lead- lipid accumulation, apoptosis, and inflammasome hyper- ing to the inflammatory accumulation of myelin-derived activation in these cells [118, 163]. As autophagy regulates lipids in mye-phagocytes. In line with this hypothesis, phagocytosis by modulating the expression of phagocytic serum HDL of RR-MS patients less efficiently accepts receptors [17], defining the impact of autophagy on the cholesterol via the ABCG1 transporter compared to uptake of myelin also deserves further attention. Thus, serum HDL of healthy controls [95]. To what extent while increasingly being acknowledged to impact MS dis- tyrosine and tryptophan modifications underlie changes ease progression, more research is warranted to define the in HDL functionality in RR-MS patients remains to be role that autophagy plays in directing the functional prop- clarified. Altogether, these studies strongly suggest erties of foamy phagocytes, and elucidate whether the au- that quantitative and qualitative changes in lipopro- tophagy machinery becomes dysfunctional in phagocytes teins are apparent in MS and can impact phagocyte engorged with myelin-derived lipids. lipid load and physiology. In particular, the function of phagocytes containing abundant myelin-derived Lipoprotein alterations and modifications lipids can potentially be severely compromised by While we focused in the previous sections on intracellu- these changes in LDL and HDL. lar processes going awry in cholesterol-loaded foamy phagocytes, extracellular factors such as lipoproteins can Protective foamy macrophages also impact lipid processing, thereby directing the physi- Similar to MS, increasing evidence indicates that the ology of these foamy macrophages [172]. Generally, high phenotype of lesional macrophages in atherosclerosis is levels of LDL, and in particular modified forms of LDL, more complex than previously thought [184]. For a long drive the inflammatory activation of macrophages after time, sustained cholesterol uptake and consequent distur- sustained uptake, thereby promoting lesion formation bances in metabolic pathways were believed to promote and progression in atherosclerosis, as described in the the induction of inflammatory, pro-atherogenic macro- previous sections. In contrast, high-density lipoproteins phages, as discussed in the previous paragraphs. However, (HDL) have anti-atherogenic properties, which are at- a regulated accumulation of desmosterol following choles- tributed to their crucial role in reverse cholesterol trans- terol loading also suppresses the inflammatory activation port [166]. Ample evidence suggests that lipoprotein of foamy phagocytes in the absence of overt inflammation levels, subclasses, and function are also altered in MS [180]. Of interest, desmosterol was found to mediate its patients and associated with disease activity. For effects on the macrophage phenotype by activating LXRs. Grajchen et al. Acta Neuropathologica Communications (2018) 6:124 Page 14 of 21 Likewise, another study demonstrated that oxLDL loading Despite of the presence of protective foamy phago- increases the expression of the typical M2 marker arginase cytes, the majority of MS lesions evolve into chronic le- I in a PPAR-dependent manner [57]. These findings sions as disease progresses. To date, it remains elusive suggest that LDL accumulation can also suppress inflam- what causes failure of foamy phagocytes to stop lesion mation and may limit lesion progression when the inflam- progression and promote CNS repair as disease ad- matory burden in atherosclerotic lesions is low. In line vances. Aging impacts immune cell function and recent with these studies, considerable phenotypic variation of evidence indicates that aging drives foamy phagocytes macrophages is apparent in atherosclerotic lesions [19, 29, towards an inflammatory phenotype. Although MS is 97, 185]. Strikingly, similar to oxLDL-loaded macro- generally not regarded an age-related disorder, chronic phages, we found that myelin-derived lipids skew phago- inflammation might well lead to premature innate cytes towards a less inflammatory phenotype in LXR- and immunosenescence in MS patients. At the same time, PPAR-dependent manner upon uptake of myelin [11, 15]. valuable lessons can be learned from foamy macro- Also, within MS lesions considerable phenotypic variation phages in atherosclerosis and other diseases. As delin- is observed [18, 200]. Boven et al. demonstrated that eated in this review, ample evidence indicates that foamy phagocytes in the lesion center display a more checkpoints involved in lipid handling are malfunction- anti-inflammatory phenotype compared to foamy phago- ing in macrophages following massive lipid uptake, lead- cytes in the lesion rim [18]. Vogel extended these findings ing to the intracellular accumulation of inflammatory by showing the majority of foam cells within MS lesions lipids. Future studies should define whether faulty regu- have an intermediate activation status, expressing markers lation of these pathways also occurs in mye-phagocytes. that are characteristic for both inflammatory and This could lead to the identification of new targets for anti-inflammatory phagocytes [200]. Collectively, these therapeutic interventions and may open up new avenues findings indicate that foamy macrophages showing an for therapeutics currently used to treat other disorders M2-like phenotype are apparent in both MS and athero- characterized by the presence of foamy macrophages. sclerotic lesions, and that alike nuclear receptor signaling Acknowledgements pathways drive the formation of these cells. Hence, identi- This work was supported by grants of the Belgian Charcot Foundation, fying ways to specifically target these pathways in phago- Research Foundation Flanders (FWO), and the Transnational University Limburg. cytes will open therapeutic avenues for both MS and atherosclerosis. Authors’ contributions EG and JB designed and drafted the review. JH revised critically for important intellectual content. EG, JH, and JB approved the final manuscript to be Conclusions and future perspectives published. All authors read and approved the final manuscript. Our understanding of the role of foamy phagocytes in the pathophysiology of MS has increased tremendously over Competing interests the last few years. It is becoming clear that the uptake of The authors declare that they have no competing interests. myelin by phagocytes is not merely a disease-promoting process but also a prerequisite for CNS repair. By simply Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published inhibiting the uptake of myelin or reducing the number of maps and institutional affiliations. lesional phagocytes, one will suppress the clearance of in- hibitory myelin and counteract the protective phenotype Received: 24 October 2018 Accepted: 2 November 2018 that phagocytes adopt upon myelin internalization. To il- lustrate, the majority of therapeutics for MS are based on References the assumption that prevention of immune cell infiltration 1. 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