Macrophage and microglial plasticity in the injured spinal cord

Macrophage and microglial plasticity in the injured spinal cord

NSC 16551 No. of Pages 8 5 September 2015 Please cite this article in press as: David S et al. Macrophage and microglial plasticity in the injured s...

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NSC 16551

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Effects of phagocytosis of RBCs and uptake of iron by IL-4 and LPS-stimulated macrophages Conclusion Acknowledgments References

Centre for Research in Neuroscience, The Research Institute of the McGill University Health Centre, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada


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Abstract—Macrophages in the injured spinal cord arise from resident microglia and from infiltrating peripheral myeloid cells. Microglia respond within minutes after central nervous system (CNS) injury and along with other CNS cells signal the influx of their peripheral counterpart. Although some of the functions they carry out are similar, they appear to be specialized to perform particular roles after CNS injury. Microglia and macrophages are very plastic cells that can change their phenotype drastically in response to in vitro and in vivo conditions. They can change from proinflammatory, cytotoxic cells to anti-inflammatory, prorepair phenotypes. The microenvironment of the injured CNS importantly influences macrophage plasticity. This review discusses the phagocytosis and cytokine-mediated effects on macrophage plasticity in the context of spinal cord injury. Ó 2015 Published by Elsevier Ltd. on behalf of IBRO.


Key words: microglia, macrophage, spinal cord injury, macrophage activation, phagocytosis, tumor necrosis factor, inflammation. 10 11 12 13 14 15 16 17 18 19 20

Contents Introduction Early response of microglia and monocyte-derived macrophage to injury Microglia and macrophage activation Phagocytosis modulates macrophage phenotype Effects of myelin phagocytosis by LPS-stimulated macrophages Effects of phagocytosis of apoptotic neutrophils by LPS-stimulated macrophages

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*Corresponding author. Address: Centre for Research in Neuroscience, The Research Institute of the McGill University Health Centre, Livingston Hall, Room L7-210, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada. E-mail address: [email protected] (S. David). Abbreviations: BMDMs, bone marrow-derived macrophages; Ca2+, calcium; CNS, central nervous system; EAE, encephalomyelitis; FACS, fluorescence-activated cell sorting; iNOS, inducible nitric oxide synthase; MK2, protein kinase 2; MS, multiple sclerosis; RBCs, red blood cells. 0306-4522/Ó 2015 Published by Elsevier Ltd. on behalf of IBRO. 1



Macrophages play a central role in the response to central nervous system (CNS) injury and originate from two sources, one from the resident microglial population and the other of myeloid origin that infiltrate the injured CNS from the peripheral circulation. Many tissues have resident macrophages that arise from either the yolk sac, fetal liver or hematopoietic system. Microglia the resident tissue macrophages of the CNS, arise from cells originating in the yolk sac that infiltrate the brain early during embryonic development (E9.5) (Alliot et al., 1999; Ginhoux et al., 2010; Ransohoff and Cardona, 2010) and the spinal cord by E10.4 (Rigato et al., 2011). They populate the CNS and are maintained throughout life by proliferation and by their longevity (Lawson et al., 1992; Alliot et al., 1999; Ajami et al., 2007; Ginhoux et al., 2010). In extreme experimental conditions when microglia are completely depleted from the adult CNS, they appear to be replenished by CNS resident progenitors (Elmore et al., 2014). It is not clear at present whether this occurs in the normal or damaged CNS where cells can be replaced by proliferation of microglia in the vicinity. In some gray matter regions such as the cerebral cortex they show ‘‘tiling” and occupy exclusive domains but their morphology depends largely on the cytoarchitecture of the CNS region in which they reside (Lawson et al., 1990). In the normal adult CNS, microglia have a branching morphology and are relatively quiescent in terms of cytokine production and immune function. However, their branching processes show short distance motility and are constantly surveying the microenvironment for changes in homeostatic conditions. The range of motility of microglia processes is between 2 and 8 lm within 1–2 min (Davalos et al., 2005; Nimmerjahn et al., 2005). In contrast, astrocytes show process extension and retraction in the range of 8–10 lm over a 10-min period (Haber et al., 2006). Glial cells in general therefore survey and monitor their local environment, and thus respond rapidly to perturbations.

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Important functions of microglia and macrophages in response to CNS injury are to clear damaged tissue, fight infections and restore tissue homeostasis. In this review we will focus on how phagocytosis regulates macrophage phenotype to either a protective or cytotoxic state. We will discuss how what macrophages phagocytose determines their activation phenotype. In other words ‘‘what they eat” determines ‘‘what they become”. We will also discuss how TNF importantly modulates these phenotypes in vitro and in vivo in the injured spinal cord, by keeping them in a prolonged proinflammatory, cytotoxic state, that is detrimental to recovery after injury. But first we will provide a brief review of the early responses of these cells to CNS injury.

EARLY RESPONSE OF MICROGLIA AND MONOCYTE-DERIVED MACROPHAGE TO INJURY Microglia in the normal CNS have the following antigenic phenotype: CD11b+, CD45low, CX3CR1high, Ly6Clow, Gr-1low (David and Kroner, 2011). They respond rapidly to injury and as shown by 2-photon live imaging after cortical micro-lesions, they redirect and extend cytoplasmic processes toward the lesion within 5 min and by 30 min form a dense network of cytoplasmic processes surrounding the lesion (Davalos et al., 2005; Hines et al., 2009). Preventing this acute microglial response leads to an increase in the size of the lesion (Hines et al., 2009). Thus, this early microglial response is protective, as it helps contain the expansion of the lesion in the acute phase. In the mammalian cortex where microglia are densely packed, the early injury-induced response to micro lesions mainly involves redirection and extension of cytoplasmic processes (Davalos et al., 2005; Hines et al., 2009). In some cases, such as in the spinal cord (Dibaj et al., 2010), and in mouse models of Alzheimer’s disease (Fuhrmann et al., 2010) translocation of microglial cell bodies also occurs. After traumatic brain injury, microglia located close to the glia limitans acquire a honeycomb morphology aligning their processes along the astrocytic junctions and are thought to serve a protective function, or they acquire a jellyfish morphology showing cell body movement and are involved in phagocytosis of dying astrocytes (Roth et al., 2014). Migration of microglial cell bodies toward the lesion site is seen in organisms in which microglia are sparsely distributed, such as in the developing zebrafish embryo (Sieger et al., 2012). The rapid extension of microglial processes toward the lesion is mediated by purinergic receptors (P2Y12R) binding to ATP released by damaged cells or from astrocytes (Davalos et al., 2005; Haynes et al., 2006; Dibaj et al., 2010). Nitric oxide may also contribute to the ATP-mediated microglial response (Dibaj et al., 2010). In places where microglia are sparsely distributed, those located at a distance from the lesion are attracted toward the lesion by glutamate released from damaged cells that induce calcium (Ca2+) influx and the spread of Ca2+ waves in astrocytes that leads to release of ATP (Sieger et al., 2012). The ATP gradient is sensed by microglia located at a distance via the P2Y12 receptors. Microglia

and other cells in the injured CNS (astrocytes, oligodendrocytes and neurons) express chemokines and cytokines within minutes after CNS damage, including CCL2, CCL3, GM-CSF, IL-1b and TNF (Pineau and Lacroix, 2007; David et al., 2012b). Damage-associated molecular patterns (DAMPs) released from dying cells, bind to tolllike receptors and induce NFjB activation and the expression of pro-inflammatory molecules, such as cytokines, cyclooxygenase-2, inducible nitric oxide synthase (iNOS) and matrix metalloproteinases (Piccinini and Midwood, 2010; Heiman et al., 2014; Katsumoto et al., 2014). These and other factors induce the influx of monocyte-derived macrophages from the circulation into the site of CNS damage. Microglia and the other resident cells of the CNS are therefore the ‘‘first responders” to any tissue perturbation. In the normal CNS, macrophages of myeloid origin, distinct from microglia, are found in the perivascular region, choroid plexus, and the meninges (Perry et al., 1985; Ransohoff and Cardona, 2010; David and Kroner, 2011). After CNS injury, signals from microglia and other CNS cells recruit monocyte-derived macrophages from the circulation to the injury CNS in a largely CCL2-dependent manner (Lassmann et al., 1993; Mildner et al., 2007). These CX3CR1low, Ly6C+, CCR2+ monocytes are actively recruited to sites of injury or disease and are referred to as ‘inflammatory macrophages’ (Geissmann et al., 2003). Experimental evidence indicates that these cells enter the damaged CNS but do not remain in the CNS permanently. For example, in mice with experimental autoimmune encephalomyelitis (EAE), monocytederived macrophages enter the spinal cord but are cleared 3 months later when the EAE lesions have resolved (Ajami et al., 2011). These monocyte-derived macrophages therefore complement the functions of the resident microglia in responding to acute disruption of the CNS. Using LysM-eGFP knock-in mice, in which peripheral myeloid cells (macrophages and neutrophils) are tagged, we and others have shown that after SCI, macrophages enter the injury site after 2–3 days (d), reach maximum numbers at 7–10 d, and persist in the injured cord for up to 42 d (Mawhinney et al., 2012; Greenhalgh and David, 2014). We have not examined longer time points after SCI. Peripheral-derived macrophages are more susceptible to apoptotic and necrotic cell death in vitro following phagocytosis as compared to microglia (Greenhalgh and David, 2014). Furthermore, in the contusion-injured spinal cord, infiltrating peripheral macrophages show more TUNEL-positive labeling than microglia (Greenhalgh and David, 2014). Whether this cell death is accompanied by a continuing influx of monocytederived macrophages weeks or months after SCI is currently not known. The turnover rate of microglia and the mechanisms that control their numbers in the normal and injured spinal cord are still not known.




Microglia as well as macrophages that infiltrate into the injured CNS from the peripheral circulation, are highly plastic cells that can acquire very diverse activation


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phenotypes, widely referred to as polarization. In the last 15 years, two polarization phenotypes resulting from stimulation with IFN-c or IL-4, referred to as M1 and M2 macrophages with phenotypes in-between; and additional subtypes of M2 (M2a, M2b and M2c) cells resulting from stimulation with other factors have been described (Gordon, 2003; Martinez et al., 2008). A panel of experts recently re-evaluated this classification which has become imprecise with widespread use, and recommend identifying the cells in vitro based on the activation stimulus, such as M(IFN-c) for IFN-c-stimulated cells, and M(IL-4) for IL-4-stimulated cells (Murray et al., 2014). They also defined a spectrum of activation states characterized by a variety of markers (expression of various cytokines, chemokines, cell surface and intracellular molecules and transcription factors) with activation by IL-4 (Arginase-1, CD204, STAT6, SOCS2) and IFN-c (TNF, IL-12, iNOS, STAT1, SOCS1) being at opposite ends of this spectrum (Murray et al., 2014). They suggested for describing in vitro work to state the activation stimulus, the source of the macrophages, full details of the culture conditions, and the use of multiple markers (Murray et al., 2014). The in vivo application is complicated by the fact that macrophages in vivo are influenced by multiple, often antagonistic stimuli. Recent transcriptional profiling of macrophages (Martinez et al., 2006), and microglia, hematogenous macrophages, and various types of tissue macrophages have revealed their tremendous heterogeneity (Hickman et al., 2013; Butovsky et al., 2014; Wes et al., 2015). The marked differences in the transcriptional profile of different macrophage and microglial populations from different regions of the CNS are not surprising as differences in the local tissue levels of cytokines, growth factors, extracellular matrix and other factors can influence their expression profile. Microglia and macrophages are designed to respond to a large variety of environmental signals. Recent transcriptome-based network analysis indicates that although IL-4 and IFN-c stimulation result in networks existing along a virtual bipolar axis with the two conditions at opposite ends, there is heterogeneity away from this axis when macrophages are stimulated in vitro with other factors (Xue et al., 2014). Network analysis of transcriptome data obtained from macrophages stimulated with a cocktail of factors (TNF, prostaglandin E2, TLR2 ligand) led to the identification of novel signature genes in pathways that differ from the M1/M2 axis (Xue et al., 2014). This work confirms and extends the current M1/M2 polarization and accounts for the greater heterogeneity in macrophage activation states. This is particularly relevant in vivo after injury or disease when macrophages are influenced by multiple, often antagonistic stimuli that include pro- and anti-inflammatory cytokines, chemokines, growth factors, TLR ligands, bioactive lipid mediators such as prostaglandins, extracellular matrix molecules, and others (Pineau and Lacroix, 2007; David et al., 2012a,b; Bartus et al., 2014). One way to assess changes in macrophage activation in vivo after SCI would be to do a transcriptome analysis of purified macrophages and microglia for different models of SCI in different species, and at different time points after


SCI. From this analysis, relevant genes can be identified that can then be assessed by anyone to probe for effects of treatment or other experimental manipulations. It is likely that this may include some of the genes associated with M1/M2 polarization. Changes in these genes between experimental and control groups can then be compared with network analysis to defined in vitro standards, such as macrophages or microglia treated with IL-4, IL-10, TNF or TLR ligands (Xue et al., 2014). Such comparisons would allow one to assess if the cells in the experimental group move from a region of the network considered pro-inflammatory or another part that is antiinflammatory and pro-repair. This analysis can extend from quantitative real-time PCR, fluorescence-activated cell sorting (FACS) or Western blot analysis. These changes can also be correlated with functional assays carried out in vitro, with microglia and macrophages purified by sorting and probed in cell death, neurite growth or phagocytosis assays (Kigerl et al., 2009; Greenhalgh and David, 2014; Kroner et al., 2014). The concept of regional clusters within a network denoting activation states that are either detrimental or pro-repair is useful because it would allow one to define hypotheses and assess if the experimental conditions being tested drive activation toward a particular regional cluster. Until such information becomes available, another focused approach to consider would be to screen for expression of molecules functionally relevant for inflammation in SCI, such as, TNF, IL-1b, IL-10, TGFb, STAT1, STAT6, STAT4, SOCS1, SOCS3 and other pro and anti-inflammatory cytokines and transcription factors, as well as some of the M(IL-4) markers (Arg-1, CD206) and M(LPS) markers (CD16/32, CD86, IL-12) that appear to be useful markers in mouse SCI (Kigerl et al., 2009; Shechter et al., 2013; Kroner et al., 2014). This analysis can be correlated with various functional assays carried out in vitro. One can then identify if the cells shift from a pro-inflammatory, cytotoxic to an anti-inflammatory, pro-repair state or vice versa. Although this review is focused on spinal cord injury, many of the issues discussed here are likely to be of relevance for brain trauma and possibly other neurological conditions. In addition to their role in macrophage polarization, these and other cytokines and chemokines also contribute to a wide range of glial and neuronal responses after SCI (Donnelly and Popovich, 2008; David and Kroner, 2011; Bastien and Lacroix, 2014).




Microglia phagocytose damaged material within the first day after SCI (Greenhalgh and David, 2014). When peripheral macrophages from the circulation influx into the injured spinal cord 2–3 d later, they immediately begin to phagocytose damaged axons and other debris, and by day 7, when they reach maximum numbers, they far outpace microglia in phagocytosing damaged material (Greenhalgh and David, 2014). It would appear that peripheral macrophages are much more specialized to phagocytose damaged tissue in the injured spinal cord or other regions of the injured CNS, than microglia. Within


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a few days after injury after the influx of peripheral macrophages, microglia may revert back to maintaining normal tissue homeostasis and leave the business of phagocytosing damaged tissue and apoptotic cells to peripheral macrophages that enter the damaged area. Damaged tissue, apoptotic neutrophils, as well as red blood cells (RBCs) resulting from hemorrhage need to be phagocytosed after SCI to initiate tissue repair. Our in vitro work indicates that macrophages respond differently to phagocytosis of myelin, apoptotic neutrophils or RBCs (Kroner et al., 2014). In contrast, in vivo, the pro-inflammatory cytokine milieu can override some of the phagocytosisinduced changes in macrophage phenotype and promote pro-inflammatory, cytotoxic macrophages that have a detrimental effect on recovery after SCI (Kigerl et al., 2009; Kroner et al., 2014). In the following sections we will discuss the differences in the response of macrophages to phagocytosis of myelin, neutrophils, and RBCs. Effects of myelin phagocytosis by LPS-stimulated macrophages In vitro studies with human blood-derived macrophages activated with LPS showed that myelin phagocytosis reduced the mRNA expression of pro-inflammatory cytokines (IL-12 and TNF) and increased CCL18, a chemokine associated with ‘‘alternative” or M2 macrophage activation (Boven et al., 2006). We recently assessed the effects of myelin phagocytosis by LPS or LPS + IFN-c-stimulated bone marrow-derived macrophages (BMDMs) and microglia on the expression of a wider range of markers at the protein level by FACS analysis (Kroner et al., 2014). BMDMs stimulated by LPS and LPS + IFN-c, and microglia stimulated by LPS show a marked reduction in the LPS-induced increase in TNF as well as a small but significant reduction in CD16/32 (Fc c III R and Fc c II R) and CD86 both of which are increased from unstimulated levels by stimulation with LPS and LPS + IFN-c. In contrast to these markers, the scavenger receptor CD206 (MRC1) which is significantly decreased by LPS and LPS + IFN-c is markedly increased by myelin phagocytosis. Furthermore, myelin phagocytosis also reduces the stimulation- induced increase in Ly6C expression in BMDMs, and CX3CR1 expression in microglia. In addition, the increase in iNOS and reduction in arginase-1 induced in microglia by LPS is significantly reversed after myelin phagocytosis (Kroner et al., 2014). Increased expression of TNF and iNOS is associated with IFN-c stimulation of macrophages, while increased expression of CD206/MRC1 and Arg-1 is associated with IL-4 stimulation suggesting that myelin phagocytosis drives M(LPS) macrophages and microglia toward a M(IL-4) expression phenotype (according to the suggested new terminology). Myelin phagocytosis and internalization are required for changes in expression of these markers as detected using pHrodo-red-tagged myelin which fluoresces upon uptake into lysosomes (Kroner et al., 2014). More importantly, myelin phagocytosis reverses the LPS-induced cytotoxic effects of macrophages as determined in a neuronal cell death assay, and LPS-induced reduction of neurite growth from dorsal

root ganglion neurons in culture (Kroner et al., 2014). It therefore appears that phagocytosis of myelin by M (LPS) BMDMs and microglia reverses the LPS-mediated effects, and appears to push the cells toward a M(IL-4)like phenotype in vitro, which in the other terminology, would be a shift from M1 toward a M2 like phenotype. Myelin phagocytosis likely mediates its effects via multiple pathways. There is evidence, however, that myelin phagocytosis inhibits NF-jB activation (Kroner et al., 2014). There is also evidence that phagocytosis of myelin as well as liposomes containing phosphatidylserine (found in myelin) mediate reduction of nitric oxide, IL-6 in LPS-stimulated macrophages via activation of peroxisome proliferator-activated receptor b/d (PPAR b/d) (Bogie et al., 2013). Interestingly, treatment with phosphatidylserine containing liposomes is effective in reducing the severity of EAE (Bogie et al., 2013). It would appear then, that phagocytosis of myelin may be an endogenous mechanism to put the brakes on the acute inflammatory response and initiate repair processes. However, this switching-off of inflammation fails to occur in vivo in the injured spinal cord and possibly in other regions of the CNS as there is considerably stronger expression of markers associated with M(IFN-c) or M1 macrophages than M(IL-4) or M2 macrophages (Kigerl et al., 2009; Kroner et al., 2014). Our in vitro work shows that TNF contributes importantly to the failure of myelin phagocytosis to reduce expression of markers upregulated by LPS such as CD16/32 in both BMDMs and microglia (Kroner et al., 2014). Furthermore, there is an increase in the expression of the M(IL-4) markers Arg1 and CD206 in macrophages in the injured spinal cord of TNF null mice as compared to wild-type mice (Kroner et al., 2014). These changes are accompanied with improved locomotor recovery after SCI in TNF null mice. Similar changes in marker expression and functional improvement after SCI is seen in mitogenactivated protein kinase-activated protein kinase 2 (MK2) null mice (Ghasemlou et al., 2010; Kroner et al., 2014); MK2 is downstream of p38 MAPK and regulates TNF biosynthesis by targeting AU-rich elements (Winzen et al., 1999; Neininger et al., 2002). TNF expression at the protein level in the injured spinal cord is almost completely absent in MK2 null mice (Ghasemlou et al., 2010). Blocking TNF has also been shown to reduce damage and promote locomotor recovery after SCI (Novrup et al., 2014). TNF is expressed by macrophages (Pineau and Lacroix, 2007) and can thus have paracrine effects but it is also expressed by all other cell types in the injured spinal cord (Pineau and Lacroix, 2007). One of the ways in which this pro-inflammatory cytokine exerts its effects is by preventing the myelin phagocytosis-mediated change in phenotype that promotes resolution of inflammation. Cell culture work with BMDMs from wild-type and TNF null mice shows that 88% of the LPS-mediated increase in NOS2 expression and 97% of IL-12 expression is mediated via TNF. In vitro, myelin phagocytosis is able to prevent almost all of this LPS- induced increase in NOS2 and IL-12 via reducing the expression of TNF (Kroner et al., 2014). Likewise, much of the LPS-induced decrease in neurite growth from

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DRG neurons is mediated via TNF (as revealed in cultures of DRG from TNF null mice) which can be reversed by myelin phagocytosis (Kroner et al., 2014). However, as stated above, this effect of myelin phagocytosis to resolve inflammation is blocked in vivo in the injured spinal cord due to the presence of TNF produced by other cells in the injured CNS (Pineau and Lacroix, 2007), and possibly also by other cytokines. It is also important to note that in the context of EAE, soluble TNF promotes inflammation while membrane-bound TNF is neuroprotective (Taoufik et al., 2011). Earlier studies also showed that in EAE, demyelination but not inflammation is dependent on TNF receptor 1 (TNFR1) (Probert et al., 2000).

Effects of phagocytosis of apoptotic neutrophils by LPS-stimulated macrophages Earlier studies reported that phagocytosis of apoptotic cells (peripheral blood lymphocytes) by LPS-activated monocyte-derived macrophages led to increase in production of IL-10, and decrease in TNF, IL-1 and IL-12 (Voll et al., 1997). Other work has shown that LPS-stimulated macrophages that phagocytose neutrophils produce high levels of IL-10 upon re-stimulation (Filardy et al., 2013). In our hands phagocytosis of apoptotic neutrophils by LPS-stimulated BMDMs displayed noteworthy differences from similarly stimulated macrophages phagocytosing myelin (Kroner et al., 2014). While myelin phagocytosis markedly reduces the expression of TNF, IL-12, CD86 and CD16, neutrophil phagocytosis reduces expression of only TNF and CD86. In the context of CNS injury, reduction in TNF expression by these macrophages could have protective effects, however, it is not known if this change in phenotype is blocked in vivo in the CNS by cytokines. Neutrophils are the first line of defense against pathogens and microbes, and are the first cell type to enter sites of infection and injury (Mantovani et al., 2011; David et al., 2012b). They are attracted to the site of injury by chemokines, complement proteins and lipid mediators like leukotriene B4 (LTB4) (Serhan, 2010; Mantovani et al., 2011; Roth et al., 2014). Neutrophils can cause bystander damage to cells via release of proteases, reactive oxygen species, and pro-inflammatory cytokines. They enter the injured spinal cord within 24 h, reach peak numbers at 3–4 d and reduce markedly by 7–10 d (Kigerl et al., 2006; Stirling and Yong, 2008). There is some evidence that neutrophils cause tissue damage after SCI (Taoka et al., 1997; Naruo et al., 2003; Gris et al., 2004). A growing body of work suggests that the containment of neutrophil influx and phagocytosis of apoptotic neutrophils requires a class switch in the bioactive lipid mediators of inflammation from prostaglandins (PGE2, PGD2) and LTB4 produced during the acute phase to lipoxins and the synthesis of specialized proresolving lipid mediators (resolvins, protectins and maresins) arising from omega-3 fatty acids (eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) (Serhan, 2010; David et al., 2012a). This response leads to the phagocytosis and clearance of neutrophils and resolution of inflammation. How effectively this lipid mediator class


switch and the synthesis of resolvins and protectins occurs after spinal cord injury is still not known.


Effects of phagocytosis of RBCs and uptake of iron by IL-4 and LPS-stimulated macrophages


Phagocytosis of RBCs by IL-4-stimulated BMDMs increases expression of the M(IFN-c) markers TNF, IL-12 and NOS2 (as well as CD16 and CD86), and decreases M(IL-4) markers CD206 and Ym1, indicating a shift in BMDMs from a M(IL-4) phenotype toward a M (IFN-c) phenotype (Kroner et al., 2014). TNF mediates about 2/3 of this effect, indicating a TNF-dependent and a smaller TNF-independent effect of RBC phagocytosis (Kroner et al., 2014). In contrast, phagocytosis of RBCs by LPS-stimulated BMDMs induces an additional 3-fold increase in IL-12 expression and does not significantly reduce the LPS- induced increase in TNF expression (Kroner et al., 2014). On the other hand, myelin phagocytosis by IL-4-stimulated macrophages does not affect the expression of these pro-inflammatory, cytotoxic markers (unpublished data; Kroner and David). Part of the effect of RBCs is thought to be caused by iron released from hemoglobin. Interestingly, unlike RBC phagocytosis by M(IL-4) BMDMs in vitro, internalization of iron dextran by these macrophages does not have any effect on expression of M(IFN-c) or M1 markers TNF, IL-12 NOS2, CD16 and CD86, however, it reduces expression of the M(IL-4) or M2 markers CD206 and Ym1 after 3 d in vitro (Kroner et al., 2014). In contrast to these in vitro effects, transplantation into the injured spinal cord of these IL-4-stimulated BMDMs loaded with RBCs or iron dextran both induced within 3 d a large increase in expression of TNF and decrease in CD206. This illustrates that M(IL-4) anti-inflammatory macrophages appear to convert to a pro-inflammatory state after phagocytosis of RBCs and uptake of iron. Iron can be released from dying RBCs or other cells at the site of CNS injury. In addition, iron uptake by LPS- stimulated macrophages increases TNF expression in vitro (Mehta et al., 2013). Thus iron can thus have serious negative implications for preventing the timely resolution of inflammation and lead to the continuance of chronic inflammation in the injured CNS. Macrophages can either take up RBCs and recycle the iron from hemoglobin as occurs in the spleen and reticuloendothelial macrophages or they can take up and sequester iron as occurs during infections to deprive bacteria of iron and thus exert a bactericidal effect. Iron sequestration under these conditions is regulated by hepcidin-mediated internalization, ubiquitination and degradation of the iron efflux transporter ferroportin (Nemeth et al., 2004). Iron accumulation in SCI and EAE is also thought to be mediated via hepcidin-mediated loss of ferroportin (Rathore et al., 2008; Zarruk et al., 2015). Iron is required for cell proliferation and tissue repair, and in the CNS it is also required for remyelination (Schulz et al., 2012). Iron accumulation in macrophages occurs in the injured spinal cord (Rathore et al., 2008; Kroner et al., 2014), and this accumulation leads to increased TNF expression in these cells and free radical generation in vivo (Kroner et al., 2014). Similarly,


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iron accumulation in macrophages in chronic skin ulcers induces pro-inflammatory macrophages with increased TNF expression and hydroxyl radical release (Sindrilaru et al., 2011). Iron accumulation in macrophages in SCI can therefore have multiple negative consequences, such as hampering tissue repair and remyelination, and have cytotoxic effects. These iron-mediated responses are also of relevance to brain trauma. Iron accumulation also occurs in the CNS in aging and a number of neurodegenerative diseases, such as amyotrophic lateral sclerosis (Jeong et al., 2009), Parkinson’s disease and Alzheimer’s disease (Zecca et al., 2004), multiple sclerosis (MS) and EAE (Hametner et al., 2013; Schuh et al., 2014; Zarruk et al., 2015). As in spinal cord injury, microglia and macrophages in MS and EAE lesions show iron accumulation, which can influence the cytotoxic properties of these cells. A subpopulation of microglia in the aging human brain show increased expression of the iron storage protein ferritin and display a dystrophic morphology that is increased in Alzheimer’s disease (Lopes et al., 2008). Similar dystrophic iron containing microglia are also seen in MS tissue (Hametner et al., 2013). Thus excessive iron accumulation in microglia can lead to microglial degeneration that may exacerbate the underlying pathology.




Microglia and macrophages are incredibly plastic cells that can change their phenotype from pro-inflammatory, cytotoxic to anti-inflammatory, pro-repair cells. In the injured CNS, this plasticity is influenced by what they phagocytose and by the cytokines and other factors in the environment. Although much is known about the antigenic phenotype of these cells, the field is moving beyond M1/M2 polarization to identification of more specific activation states that involve changes at the network level. This will require transcriptome analysis of purified microglia and macrophages from the injured spinal cord from different SCI models in different species. Relevant genes thus identified can be screened to probe for differences in treatment or other experimental manipulations and compared in network analysis to in vitro standards. This combined with a variety of functional assays will tightly correlate the expression phenotype to function. The field is headed in this direction. Understanding this will permit one to test strategies to effectively manipulate these cells to guide them to adopt a pro-repair phenotype that promotes recovery after CNS injury or disease.

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Acknowledgments—Work done in SD’s laboratory that is presented in this review was supported by grants from the Canadian Institutes of Health Research (CIHR) (MOP-14828) and the Multiple Sclerosis Society of Canada. ADG and AK were both supported by CIHR Postdoctoral fellowships.



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(Accepted 25 August 2015) (Available online xxxx)

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