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The role of microglia and myeloid immune cells in acute cerebral ischemia

The role of microglia and myeloid immune cells in acute cerebral ischemia REVIEW ARTICLE published: 14 January 2015 CELLULAR NEUROSCIENCE doi: 10.3389/fncel.2014.00461 The role of microglia and myeloid immune cells in acute cerebral ischemia Corinne Benakis, Lidia Garcia-Bonilla, Costantino Iadecola and Josef Anrather * Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, NY, USA Edited by: The immune response to acute cerebral ischemia is a major contributor to stroke Arthur Liesz, University Hospital pathobiology. The inflammatory response is characterized by the participation of brain Munich, Germany resident cells and peripheral leukocytes. Microglia in the brain and monocytes/neutrophils Reviewed by: in the periphery have a prominent role in initiating, sustaining and resolving post-ischemic Maria-Grazia De Simoni, Mario inflammation. In this review we aim to summarize recent literature concerning the origins, Negri Institute for Pharmacological research, Italy fate and role of microglia, monocytes and neutrophils in models of cerebral ischemia and Athanasios Lourbopoulos, Ludwig to discuss their relevance for human stroke. Maximilian University of Munich, Germany Keywords: cerebral ischemia, monocytes, microglia, neutrophils, myeloid cells, tissue macrophages *Correspondence: Josef Anrather, Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, 407 East 61st Street, RR-409, New York, NY 10065, USA e-mail: joa2006@med.cornell.edu INTRODUCTION microglia and infiltrating peripheral monocytes that have distinct ontogenesis (London et al., 2013; Prinz and Priller, 2014). Once in Cerebral ischemia triggers a robust activation of brain resident the injured tissue, both cell types differentiate into macrophages and peripheral immune cells, which play an active role in the and may be indistinguishable by classical histological methods acute and chronic phases of injury, as well as in subsequent reorganization and repair processes (Iadecola and Anrather, since they share similar antigens and morphologies (Prinz and Mildner, 2011). In the next sections, we will summarize recent 2011; Macrez et al., 2011; Shichita et al., 2012). Numerous experimental studies have depicted the pivotal response of findings on the origin, fate and function of myeloid immune cells and microglia particularly in the context of acute cerebral resident microglia, infiltrating monocyte-derived macrophages and neutrophils to either the development of the brain injury ischemia. or its resolution leading to conflicting interpretation of their protective or deleterious contribution in stroke (Emerich et al., ORIGIN AND FATE OF NEUTROPHILS 2002; Chiba and Umegaki, 2013), and ultimately to treat- Neutrophils are innate immune cells and are the first line of ment failure (O’Collins et al., 2006; Smith et al., 2013). Fur- defense against microbial infectious agents. They are involved in thermore, discrepancies exist towards the provenance of brain the phagocytosis, killing and degrading of microorganisms, partly macrophages, that is to say whether they are blood-borne through the generation of reactive oxygen and nitrogen species monocytes or resident microglia that further differentiate into (ROS/RNS). Neutrophils are generated in the bone marrow (BM) macrophages (London et al., 2013). A better understanding and share with monocytes the common progenitor granulocyte of the origin and function of myeloid immune cells pop- macrophage precursor (GMP; Figure 1). Only mature neutrophils ulating the ischemic brain may provide the opportunity to are normally released from the BM into circulation, where they manipulate specific subsets for therapeutic benefit. The aims exist as circulating and marginated neutrophil pools, primarily of this review are (a) to summarize the origin and develop- in the lung, which can be acutely mobilized for example by ment of leukocytes and resident microglia, (b) to delineate adrenergic agonists (Bierman et al., 1951, 1952). After termi- their contribution to ischemic injury based on recent litera- nal differentiation, under homeostatic conditions, neutrophils ture, and (c) to assess their therapeutic relevance to human remain in the BM for additional 4–6 days before being released stroke. into circulation, constituting a BM neutrophil reserve (Craddock et al., 1960). Egress is controlled by the antagonistic activities of C- ORIGIN AND DEVELOPMENT OF NEUTROPHILS, X-C chemokine receptor type 2 (CXCR2) and CXCR4 receptors. MONOCYTES AND MICROGLIA Activation of CXCR2, which binds C-X-C ligand 1 (CXCL1) Cerebral ischemia induces a time-dependent recruitment and and CXCL2 chemokines, stimulates BM egress, whereas stromal activation of leukocytes including neutrophils, monocytes and cell derived CXCL12 (SDF-1) acting on CXCR4 favors reten- lymphocytes (Iadecola and Anrather, 2011). At the site of injury, tion (Strydom and Rankin, 2013). During neutrophil maturation macrophage populations consist mainly of activated parenchymal CXCR2 expression increases, while CXCR4 is downregulated, Frontiers in Cellular Neuroscience www.frontiersin.org January 2015 | Volume 8 | Article 461 | 1 Benakis et al. Myeloid cells in ischemic stroke FIGURE 1 | Origin and trafficking of resident microglia and immune cells ischemic region or cluster into the perivascular space. During injury, CCL2 is of myeloid origin. Fetal liver and spleen/bone marrow panel: Monocytes are produced by astrocytes, macrophages/microglia and neurons. CCL2 binds its high generated from hematopoietic stem cells (HSCs) in the fetal liver and during receptor CCR2 expressed by Ly6C inflammatory monocytes, which adult life in the bone marrow (BM). The granulocyte macrophage precursors promotes their egress from the BM into the blood, and then their recruitment (GMP) give rise to immature neutrophils and the macrophage dendritic cell from the blood into the injured tissue. Here, these cells give rise to precursors (MDPs). Monocytes develop from hematopoietic stem cells (HSC) monocyte-derived DC (not shown) and monocyte-derived macrophage after myeloid lineage commitment through a series of increasingly restricted populations, which can further polarize into M1 and M2 macrophages. As the progenitors (GMP, granulocyte/monocyte progenitor; MDP, ischemic infarct develops, M1 and M2 macrophages contribute to the low monocyte/dendritic cell progenitors). The common monocyte progenitor exacerbation of the damage or wound healing, respectively. Ly6C high low (CMP) is the direct precursors of mature Ly6C and Ly6C monocytes. monocytes patrol the blood vessel lumen by associating with the vascular high low low Ly6C inflammatory monocytes might give rise to circulating Ly6C endothelium. Ly6C monocytes expressing the CX3CR1 receptor are also high monocytes directly, or via a Ly6C monocyte intermediate. Yolk sac panel: recruited to sites of inflammation and possibly contribute to wound healing by Resident brain microglia have been shown recently to have a different origin differentiating into alternatively activated M2 macrophages. Cerebral than circulating monocytes. During embryonic life, erythroblasts (not shown) ischemia/reperfusion leads to the release of damage-associated molecular and macrophage progenitors are generated in the yolk sac from the common pattern (DAMP) molecules from dying neurons. These molecules trigger the erythro-myeloid progenitor. When the blood circulation is established, activation of resident microglia and astrocytes. Activated microglia promote macrophage precursors exit the yolk sac and migrate into the developing tissue repair by producing trophic factors and by scavenging necrotic cells. brain. Embryonic microglia proliferate and are able to renew themselves Regulatory T lymphocytes (Treg) secreting IL-10 have shown a protective role during gestation and post-natal development as well as in adulthood. Blood in cerebral ischemia and might promote macrophage M2 polarization. Spleen vessel and Ischemic brain panels: Few hours after cerebral ischemia onset, panel: An interesting twist to the origin of recruited monocytes during injury mature neutrophils enter the bloodstream upon activation of the CXCR2 has been added by the recent identification of a major monocyte reservoir in receptor and infiltrate the brain in response to chemokines CKLF1 as well as the spleen of mice. Following ischemic myocardial injury, splenic monocytes CXCL1 and CXCL2 released by astrocytes. Astrocytic production of these are mobilized to the site of inflammation and participate in tissue injury chemokines is dependent upon IL-17 released from brain infiltrating gdT cells. (Swirski et al., 2009). Dashed arrows represent findings that are not clearly Neutrophils firmly adhere to the endothelium and might either invade the defined yet and need further investigations in context of cerebral ischemia. which eventually leads to neutrophils egress from the BM. Once and BM, wherein neutrophils die by apoptosis and are cleared by in the circulation, neutrophils are thought to be one of the resident macrophages. In inflammation, extravasated neutrophils most short-lived cells in the body with circulating half-life of show increased life span of several days and might die as a roughly 8 h in humans and 13 h in mice (von Vietinghoff and result of their own cytotoxic molecules by necrosis and by releas- Ley, 2008; Bugl et al., 2012). As the neutrophil age, CXCR4 ing cytotoxic neutrophil extracellular traps (NETs; Yipp et al., receptors become upregulated favoring homing to spleen, liver 2012). Frontiers in Cellular Neuroscience www.frontiersin.org January 2015 | Volume 8 | Article 461 | 2 Benakis et al. Myeloid cells in ischemic stroke MONOCYTES, THE BLOOD-BORNE PRECURSORS OF INDUCED TISSUE display proteolytic and inflammatory functions consistent with a low MACROPHAGES role in tissue damage. Ly6C monocytes are the predominant Similar to neutrophils, monocytes are generated from definitive population during the resolution phase of the inflammatory hematopoietic stem cells (HSC) in the liver and spleen during response and express repair-promoting proteins such as vascular embryonic development and primarily in the BM after birth endothelial growth factor (VEGF; Nahrendorf et al., 2007). The high low accumulation of Ly6C and Ly6C monocytes in inflamed (Auffray et al., 2009; Ginhoux and Jung, 2014; Figure 1). After lineage commitment they proceed through increasingly more tissues occurs in two phases, although the sequence of infiltra- tion by specific monocyte subsets differs between models, e.g., restricted progenitor stages to give rise to mature monocytes that are released into circulation after engagement of the C-C bacterial infection vs. sterile inflammation (Auffray et al., 2007; high low Nahrendorf et al., 2007). Thus, Ly6C and Ly6C monocytes chemokine receptor type 2 (CCR2; Tsou et al., 2007; Hettinger et al., 2013). In the circulation, they can be distinguished can have pro- or anti-inflammatory functions depending on the nature of the inflammatory stimulus, the tissue microenviron- from other leukocytes by their myeloid nature, as indicated by (a) high-level expression of CD11b/Mac-1 (a member of the ment and their molecular profile (transcription factors, gene expression, etc.). a-integrin family of proteins) and CD115 (colony simulating factor 1 receptor, CSF1R), (b) their phagocytic capacity and (c) their ability to develop into macrophages upon stimulation MICROGLIA, THE RESIDENT BRAIN MACROPHAGES with CSF-1 in vitro (Chitu and Stanley, 2006). There is sub- In contrast to monocytes, microglia originate exclusively from stantial heterogeneity in circulating monocytes and they can the yolk sac and colonize the central nervous system (CNS) be further divided into functionally distinct subsets according early during embryonic development before E9 (Ginhoux et al., to their surface expression of lymphocyte antigen 6 complex, 2010, 2013; Schulz et al., 2012; Frame et al., 2013). There, locus C1 (Ly6C; Swirski et al., 2007; Geissmann et al., 2008), yolk sac-derived macrophages proliferate and give rise to mature the CCR2 receptor (Geissmann et al., 2003; Prinz and Priller, microglia (Figure 1). It seems that microglia are long-lived under 2010) and CX3CR1—the high-affinity functional chemokine physiological conditions and are not normally replaced by BM- C high receptor for fractalkine (Imai et al., 1997). CD115 /Ly6C derived cells (Alliot et al., 1999; Ajami et al., 2007; Kierdorf monocytes that express CCR2 are termed “inflammatory” mono- et al., 2013a). Recent evidence indicates that microglia home- cytes because they are highly mobile and selectively recruited ostasis in adult mice is highly dependent on continuous pro- to inflamed tissues (Auffray et al., 2007; Swirski et al., 2010; survival signals provided by the CSF-1 receptor CD115 (Elmore C low Terry et al., 2012). In contrast, CD115 /Ly6C monocytes et al., 2014). Blockage of the receptor by selective inhibitors that express CX3CR1 and are negative for CCR2 were initially for as little as 7 days resulted in nearly complete depletion of termed “resident” monocytes because of their longer half-life brain microglia. Fast recovery of the microglia population was in circulation and their accumulation in tissues under homeo- observed after withdraw of the blocker due to proliferation of static conditions (Geissmann et al., 2003). This subset has been brain resident progenitors. These progenitors expressed the neu- later termed “patrolling” monocytes because of their crawling roectodermal marker Nestin, the hematopoietic marker CD45, behavior along the vascular endothelium, and has been shown the stem cell marker CD34 and stained positive for isolectin B4 to orchestrate the disposal of dying or infected endothelial (IB4) while being negative for myeloid markers such as ionized cells (Auffray et al., 2007; Carlin et al., 2013). Their accumu- calcium binding adapter molecule (Iba1; Elmore et al., 2014). lation in the tissue is facilitated by the expression of CX3CR1 While this is the first study to identify brain-resident microglial that mediates their adhesion and migration (Geissmann et al., progenitors, microglia expansion has been previously described 2003). Several reports have proposed that “patrolling” mono- in several disease models. In a model of amyotrophic lateral C high cytes originate from CD115 /Ly6C monocytes in the cir- sclerosis, Solomon et al. characterized the temporal and spatial culation (Sunderkötter et al., 2004; Lin et al., 2009). However, infiltration of green fluorescent protein (GFP)-BM cells trans- low recent data suggest that Ly6C monocytes are generated from planted into lethally irradiated mice. The increased number of a myeloid progenitor by the activity of the transcription factor spinal cord macrophages following disease onset was attributed NR4A1 (Nur77) in the BM (Hanna et al., 2011). Additionally, to the expansion of resident microglia rather than infiltrated BM- C high depletion of CD115 /Ly6C monocytes in the blood does derived precursors (Solomon et al., 2006). Similarly, in experi- C low not affect the numbers of circulating CD115 /Ly6C mono- mental autoimmune encephalitis (EAE), Ajami et al. found that C high cytes making it unlikely that CD115 /Ly6C cells are the only blood-borne monocytes transiently infiltrate the spinal cord but precursors of “patrolling” monocytes (Geissmann et al., 2008; do not contribute to the long-term pool of resident microglia Figure 1). (Ajami et al., 2011). Techniques such as parabiosis (Ajami et al., C low Under physiological conditions, most CD115 /Ly6C crawl- 2007; Ransohoff, 2011), whole body irradiation, head-shielded ing monocytes are found within the blood vessels and extrava- BM chimeras (Mildner et al., 2011), transgenic mice (Saederup sation rarely occurs. However, in response to tissue damage or et al., 2010) and fate mapping analysis (Ginhoux et al., 2010) C high infection, these cells extravasate faster than CD115 /Ly6C have been instrumental to better discriminate between infiltrat- monocytes and are the main TNF producers in the early phase ing myeloid immune cells and resident microglia. Furthermore, of the inflammatory response (Auffray et al., 2007). In contrast, recent data indicate that microglia express a distinctive genetic high in a model of myocardial infarction Ly6C monocytes are the signature that makes them distinguishable from neurons, astro- first population to be present in the injured myocardium and cytes, oligodendrocytes, and peripheral immune cells including Frontiers in Cellular Neuroscience www.frontiersin.org January 2015 | Volume 8 | Article 461 | 3 Benakis et al. Myeloid cells in ischemic stroke other tissue macrophages. Using transcriptomic and proteomic It must be noted that M1 and M2 are the extreme phenotypes analysis Butovsky et al. identified several genes that were specif- of the broad and heterogeneous activation states of macrophages, C low ically expressed in CD11b /CD45 microglia when compared and caution is needed in interpreting their multivalent function to blood monocytes (Butovsky et al., 2014). Among them, the under pathological conditions (Murray et al., 2014). Recently, purinergic receptor P2ry12, the proto-oncogene tyrosine kinase the M1/M2 terminology was also applied to activated microglia Mertk, and the Fc receptor-like S scavenger receptor Fcrls were (Michelucci et al., 2009; David and Kroner, 2011). Exposure of exclusively expressed in microglia in brain and spinal cord, and cultured primary microglia to LPS or IFNg induces a M1-like not in peripheral tissue macrophages, e.g., Kupfer cells, alveolar phenotype with reduced phagocytic activity and expression of macrophages, etc. P2ry12, Mertk and Fcrls are surface expressed iNOS, TNF, IL-1b and IL-6 among others, while IL-4 drives proteins enabling easy antibody-mediated detection in flow cyto- microglia towards a M2-like phenotype with increased Arg1, metric and histological assays. Given that this genetic signature Ym1 and resistin like a (RELMa, FIZZ1) expression (Michelucci is similar between mice and human microglia, a better differ- et al., 2009). Others have compared capacity of microglia and entiation of infiltrating myeloid cells and microglia in human blood- or BM-derived macrophages to assume M1 or M2 phe- stroke should also be possible. In a model of EAE, expression notypes using in vitro polarization protocols (Durafourt et al., of these genes was restricted to microglia and was not found in 2012; Girard et al., 2013). These authors assessed morphol- infiltrated monocytes (Butovsky et al., 2014). However, because ogy, surface markers, cytokine profile and phagocytic capac- the analysis was conducted at disease onset, it is not clear whether ity of both polarized blood-borne macrophages and microglia the signature is retained as the pathology progresses. It remains (Durafourt et al., 2012) or compared expression levels of M1/M2 to be determined whether expression of these marker genes is marker genes (Girard et al., 2013). Girard et al. found that both preserved during all stages of microglial activation, as observed mouse BM-derived macrophages and BV2 microglial cell line in cerebral ischemia, or whether a similar molecular signature displayed a M1 or M2 phenotypes after LPS or IL-4 stimulation, can be acquired by infiltrating monocytes that develop into respectively. In contrast, Durafourt et al. found that human tissue macrophages. For example, monocytes entering the brain microglia was able to respond to both M1 and M2-inducing parenchyma could be exposed to a brain-specific environment stimuli, but their M2 gene expression signature was restricted favoring expression of a microglia-like phenotype. Therefore, a to CD209 and the expression of CD23, CD163, and CD206 better knowledge of unique genetic signatures and reliable cellular was not increased, as observed in M2 polarized macrophages markers would greatly facilitate studies of the origin, function and (Durafourt et al., 2012). Although the functional implications fate of inflammatory cells in ischemic brain injury. remain to be established, these observations highlight important differences between human and mouse microglia. Interestingly, adult human microglia showed higher phagocytic activity and POLARIZATION OF MONOCYTES- AND MICROGLIA-DERIVED MACROPHAGES IL-10 expression than macrophages under M1 and M2 condi- tions, indicating that even in a pro-inflammatory environment Once in the damaged tissue, CD115 peripheral-blood mono- cytes have the ability to differentiate into macrophages and further microglia can retain anti-inflammatory function, which might be essential for their neuroprotective activity (Durafourt et al., polarize into several subtypes with specific functions including production of inflammatory molecules and phagocytic activity. 2012). Macrophage polarization depends on the type of injury, the CONTRIBUTION OF IMMUNE CELLS OF MYELOID ORIGIN nature of the pathogen, the organs involved and interactions AND MICROGLIA TO CEREBRAL ISCHEMIC INJURY with other immune cells. The distinct phenotypes and physio- logical activities associated with tissue macrophages were mostly Acute cerebral ischemia leads to rapid neuronal cell death and described in vitro and in non-neuronal tissue, and have only activation of resident microglia as well as the infiltration of recently been examined in the context of cerebral ischemia. blood-borne immune cells (Gelderblom et al., 2009; Dénes et al., “Classically activated macrophages” or “M1 macrophages” have 2010; Figure 2). Activated microglia become phagocytic aiming anti-microbial activity and secrete pro-inflammatory cytokines to clear the damage and promote repair (Faustino et al., 2011). and reactive oxygen species upon stimulation with interferon-g If blood flow is not rapidly restored and ischemic damage devel- (IFNg) and lipopolysaccharide (LPS; Mills et al., 2000; Gordon ops, injured neurons release damage-associated molecular pattern and Taylor, 2005; Mosser and Edwards, 2008). On the other proteins leading to the secretion of pro-inflammatory media- hand, “alternative” or M2 macrophages, induced by interleukin-4 tors and formation of ROS from parenchymal cells (del Zoppo (IL-4), IL-10 or transforming growth factor-b (TGF-b), promote et al., 2000; Shichita et al., 2012; Jackman and Iadecola, 2014). anti-inflammatory and reparative processes (Mantovani et al., Cytokines and ROS, produced both in the vascular and parenchy- 2004; Hao et al., 2012; Yang et al., 2014). Several differentially mal compartments, induce the disruption of the blood brain bar- regulated genes have been associated with each polarization state rier (BBB) facilitating the infiltration of circulating monocytes, and are used to distinguish between these two populations in neutrophils and lymphocytes, thereby promoting post-ischemic mice, including the IgG Fc receptors CD16/32 and inducible inflammation (Iadecola and Anrather, 2011; Kleinschnitz et al., nitric oxide synthase (iNOS) for the M1 phenotype, and mannose 2013; García-Bonilla et al., 2014b). However, neutrophils, as well receptor (CD206), arginase 1 (Arg1), chitinase-like 3 (Chil3 or as monocytes, some T- and B-cells and microglia may also exhibit Ym1), and IL-10 for the M2 phenotype (Table 1; Hu et al., 2012; anti-inflammatory properties that are important for limiting neu- Liu et al., 2013; Murray et al., 2014; Tang et al., 2014). ronal injury, resolving the inflammation and promoting tissue Frontiers in Cellular Neuroscience www.frontiersin.org January 2015 | Volume 8 | Article 461 | 4 Benakis et al. Myeloid cells in ischemic stroke Table 1 | Different activation states of neutrophils, circulating monocytes and tissue macrophages in mice. Neutrophils Circulating monocytes Monocyte-derived macrophages Resident microglia Subsets N1/N2 “Inflammatory” “Patrolling” M1 M2 Surveying Microglia-derived monocytes monocytes (M2a,b,c) microglia macrophages Marker iNOS iNOS Arg1, CD206, DAP12 genes Chil3 (Ym1) high high high high high int high Receptor CD45 CD45 CD45 CD45 CD45 CD45 CD45 C high low C C C C expression CD11b Ly6C Ly6C CD115 CD115 CD11b CD11b C C C C C C Ly6G CD115 CD115 CD11b CD11b CX3CR1 CD115 C C C C C CCR2 CCR2 F4/80 F4/80 Ibal F4/80 int C CX3CR1 CX3CR1 CD68 (ED-1) CD68 (ED-1) IB4 CD68 (ED-1) C C C Ly6G- Ly6G- CCR2 CX3CR1 CX3CR1 Iba1 IB4 Activated Platelets, Neutrophils, Th1, NK Th2 Neurons Dying by [cells] Endothelial cells Dying neurons neurons Activated by IL-1a CCL2 LPS, IFNg, IL- 4 and IL-13 (M2a), DAMPs, [molecules] TNF IL-10, Glucocorticoid, CX3CL1 TGFb (M2c), CCL2 (MCP1) Function Neurotoxic Recruited at sites Endothelium Cytotoxic Phagocytic Synaptic pruning Phagocytosis, (N1), of inflammation, integrity effect capacity, and remodeling Neurotoxicity Neuroprotection Precursors of Promote during (N2) peripheral neurite development, mononuclear outgrowth Homeostatic phagocytes functions Cell ROS, NO, TNF, ROS, IL-10, IL-12, IL-23, TGFb, Arg1 TGFb IL-1b, products RNS, NO, IL-1b, VEGF TNF, IL-1b, and scavenger TNF, IL-6, MMP-9, Little IL-10 IL-6, ROS receptors (M2a), Chemokines, NE IL-10 (M2c). IFNg The dichotomy in the function of each cell type shown here is an oversimplified view of their activation state. They rather undergo multiple activation phenotypes at a given time making the interpretation of their neuroprotective or/and deleterious effect in cerebral ischemia complex. Whether N1/N2 and M1/M2 phenotypes may apply to neutrophils and microglia, respectively, would need further investigation. Abbreviations: Arginase 1 (Arg1), Chemokine (C-C motif) ligand 2 (CCL2), C-C chemokine receptor type 2 (CCR2), Chitinase-like 3 (Chil3 or Ym1), CX3C chemokine receptor 1 (CX3CR1), Damage-associated molecular pattern molecules (DAMPs), DNAX activation protein of 12 kDa (DAP12), inducible nitric oxide synthase (iNOS), Interferon gamma (IFNg), Interleukin-1,4,6,10,12,23 (IL-1, IL-4, IL-6, IL-10, IL-12, IL-23), Ionized calcium binding adaptor molecule 1 (Iba1), Isolectin B4 (IB4), Lipopolysaccharide (LPS), Lymphocyte antigen 6G (Ly6G), Matrix metallopeptidase 9 (MMP-9), monocyte chemotactic protein 1 (MCP1), Natural Killer (NK), Neutrophil elastase (NE), Nitric oxide (NO), Reactive nitrogen species (RNS), Reactive oxygen species (ROS), Transforming growth factor beta (TGF- ), Tumor necrosis factor (TNF), Type 1 T helper (Th1), Type 2 T helper (Th2), Vascular Endothelial Growth Factor (VEGF). repair (Liesz et al., 2009; Yilmaz and Granger, 2010; Bodhankar NEUTROPHILS IN BRAIN ISCHEMIA et al., 2014). Characterizing the dynamics of leukocyte infiltration Neutrophils respond to sterile inflammation, such that induced after cerebral ischemia is important to understand their func- by brain ischemia, by interacting with endothelial adhesion tional role and therapeutic potential. Notably, the magnitude and molecules, mainly selectin family members and intracellular temporal profile of peripheral immune cell infiltration depends adhesion molecule-1 (ICAM-1), to slow their intravascular move- upon models of focal cerebral ischemia. For example, permanent ment and induce polarization, which results in firm adhesion ischemia leads to immune cell infiltration as early as 3 h and to the pro-inflammatory endothelium (Panés et al., 1995, 1999). a more pronounced accumulation of neutrophils compared to Rolling and adhesion of leukocytes can be observed in vivo as transient ischemia (Zhou et al., 2013; Chu et al., 2014b), wherein early as 1 h after transient middle cerebral artery (MCA) occlusion blood-borne immune cells do not appear in large numbers until and is characterized by neutrophil attachment to the endothe- 48 h after reperfusion (Stevens et al., 2002; Gelderblom et al., lium and increased platelet-neutrophil interactions which is P- 2009; García-Bonilla et al., 2014b). These differences in the timing selectin and integrin aIIbb3 dependent (Ishikawa et al., 2004). of cellular infiltration relative to the development of tissue injury Due to the proximity of platelets and neutrophils, IL-1a released hint at different roles of blood-borne immune cells in these two from activated platelets, might be an early activator of neutrophils stroke models. These considerations need to be taken into account (Thornton et al., 2010). when extrapolating findings obtained in animal models to human After initial adherence, neutrophils will follow a chemokine stroke for therapeutic purposes. and activator gradient produced by the injured tissue. Neutrophils Frontiers in Cellular Neuroscience www.frontiersin.org January 2015 | Volume 8 | Article 461 | 5 Benakis et al. Myeloid cells in ischemic stroke FIGURE 2 | Spatiotemporal profile of myeloid immune cell cellular debris and clear the damaged tissue. In the peri-infarct region activation in acute cerebral ischemia. Schematic representation of microglia have ramified processes but are negative for the phagocytic rodent brain coronal sections through the anterior commissure marker ED-1. They are present early after injury, peak between 5–7 days (bregma = 0) after transient MCA occlusion in C57BL/6 mice. The core and decrease thereafter. Neutrophils are the first myeloid immune cells of the infarct is represented in red and the peri-infarct area is shown in to invade the brain and might display a N1 or N2 phenotypes. They can light red. The evolution of the infarct is based on observations of ours be detected in the ischemic region from 1 to 5–7 days after injury. and others and depicted as follow: infarct size becomes detectable Monocytes infiltrate the ischemic parenchyma after microglia activation 1 day after transient MCA occlusion, is maximal at 3–5 days after and transform into macrophages. Between 3–5 days M2 macrophages ischemia onset and decreases afterwards together with shrinkage of are more abundant in the striatum—the core of the infarct—and decline the injured hemisphere and enlargement of the ventricles. Before injury, by 2 weeks. In contrast, pro-inflammatory M1 cells are first observed in resident microglia display ramified thin processes and are highly motile. the peri-infarct of the lesion and increase in number in the core over They scan their microenvironment to detect any disturbances. Cerebral time and outnumber the M2 macrophages later on. From 3–5 days ischemia triggers microglia to become activated and to display distinct onwards it is not clear yet whether the so called M1 and M2 shapes and expression patterns upon the course of reperfusion. Based macrophages found in the ischemic region are derived preferentially on findings depicted here, microglia become activated at the onset of from microglia or infiltrated monocytes and if they have similar cerebral ischemia—as early as 1 day after injury—and further develop functions. Preliminary data suggest that microglia-derived macrophages into macrophages. In the core of the infarct, microglia are round-shaped, can proliferate and have greater phagocytic properties than expressed ED-1 and display a M2 phenotype aimed at phagocytize the monocytes-derived macrophages. are the first blood-borne cells found in the ischemic area, and in vitro (Liu et al., 2001; Su et al., 2008). Accordingly, reach peak numbers at days 2–4 after transient ischemia CD47 deficient mice exhibit reduced brain injury and neu- and decline thereafter (Gelderblom et al., 2009; García- trophil infiltration after transient focal ischemia (Jin et al., Bonilla et al., 2014b). The CXCR2 ligands CXCL1 (KC) 2009). and CXCL2 (MIP-2a) are the main chemokines responsible Endothelial transmigration seems to be essential for the for neutrophil extravasation. In the ischemic brain CXCL1 cytotoxic activation of neutrophils (Allen et al., 2012), which is produced by activated astrocytes in an IL-17 dependent is accomplished by induction of ROS production by NADPH manner (Figure 1; Gelderblom et al., 2012). Chemokine-like oxidase and myeloperoxidase, iNOS dependent nitric oxide gen- factor 1 (CKLF1), a recently discovered chemokine, partici- eration and concomitant release of RNS, particularly peroxyni- pates in neutrophil recruitment after transient focal ischemia trite (Yilmaz and Granger, 2010). Work from our group has in rats and anti-CKLF1 antibodies diminished neuronal cell shown that iNOS expression in endothelial cells and neutrophils death in this model (Kong et al., 2014). The sequence of contributes to ischemic brain injury after transient MCA occlu- molecular events regulating neutrophil extravasation remains sion, but was dispensable for neutrophil recruitment (Iadecola incompletely understood. CD47, a immunoglobulin superfamily et al., 1997; García-Bonilla et al., 2014a). On the other hand, C=C = transmembrane glycoprotein expressed on the cell surface of adoptive transfer of iNOS neutrophils into an iNOS host neutrophils, is essential for neutrophil transmigration in vivo 24 h after MCA occlusion was sufficient to revert the protection Frontiers in Cellular Neuroscience www.frontiersin.org January 2015 | Volume 8 | Article 461 | 6 Benakis et al. Myeloid cells in ischemic stroke exerted by iNOS deletion (García-Bonilla et al., 2014a). Other MONOCYTES AND MICROGLIA IN CEREBRAL ISCHEMIA potentially neurotoxic molecules released by neutrophils are Under normal conditions, resident microglia are ramified, extracellular proteases. Among them, matrix metalloproteinase have long processes and can be morphologically discerned 9 (MMP-9), a protease released during neutrophil degranula- from macrophages (Nimmerjahn et al., 2005; Boche et al., tion, has been shown to contribute to ischemic injury (Romanic 2013). Resident microglia can be distinguished from BM- et al., 1998). Neutrophil elastase (NE), has also been impli- derived monocytes according to their level of CD45 expres- cated in brain injury after transient but not permanent focal sion: microglia are CD11b and express intermediate level of C int ischemia (Shimakura et al., 2000; Stowe et al., 2009). There CD45 (CD11b /CD45 ), whereas monocytes are also positive C high is still debate to what extent neutrophils contribute to post- for CD11b but express high level of CD45 (CD11b /CD45 ) high ischemic inflammation and neuronal cell death. While neu- (Ford et al., 1995). Monocytes can be further classified as Ly6C low trophil depletion by delivery of anti-neutrophil antibodies was pro-inflammatory monocytes or Ly6C anti-inflammatory neuroprotective in focal ischemia models (Matsuo et al., 1994), monocytes (David and Kroner, 2011). Upon injury, microglia several approaches to limit neutrophil infiltration by cytotoxic adopt ameboid morphology and become phagocytic. Activated drugs or adhesion molecule and chemokine receptor block- microglia are commonly identified by the cellular markers: IB4, ade have been unsuccessful (Soriano et al., 1999; Beray-Berthat Iba1, F4/80 or ED-1 (CD68). After cerebral ischemia, microglia et al., 2003b; Brait et al., 2011), or have suggested that the display different markers and morphology depending on their contribution of neutrophils to ischemic injury might be brain localization in the ischemic territory and the course of reper- region specific (Beray-Berthat et al., 2003a). It has also been fusion. In the ischemic core microglia have an ameboid shape, suggested that neutrophils do not penetrate the ischemic brain are Iba1 positive and ED1 positive and have increased CD11b tissue but accumulate in the perivascular space after extrava- expression, whereas in the peri-infarct area these cells have sation (Enzmann et al., 2013). However, this finding does not shorter processes than in the resting state, are Iba1 , but are exclude their participation in ischemic injury since neutrophils negative for ED1 (Ito et al., 2001; Morrison and Filosa, 2013). would be still able release soluble cytotoxic molecules such as Once microglia differentiate into macrophages they share sev- ROS, RNS, and proteases, which could induce vascular damage eral antigens and morphological features with hematogenous aggravating the ischemia or diffuse into the brain parenchyma macrophages (Patel et al., 2013), confounding the interpre- causing tissue injury. Indeed, our iNOS BM chimera and adop- tation of their origin and ultimately their function in acute tive transfer experiments (García-Bonilla et al., 2014b), collec- cerebral ischemia (Hanisch and Kettenmann, 2007; Hellwig et al., tively, support a deleterious role of neutrophils in ischemic brain 2013). injury. Several recent studies have attempted to distinguish microglia from blood-derived macrophages and to define their role in FUNCTIONAL POLARIZATION OF NEUTROPHILS IN CEREBRAL ischemic injury and repair, potentially opening novel therapeutic ISCHEMIA avenues for the treatment of stroke. To this end, several strate- In addition to their deleterious effect, neutrophils may also gies have been used (Prinz and Priller, 2014). A widely used exert neuroprotective functions as recently suggested (Cuartero approach has been to perform BM transplants with fluorescent et al., 2013). In this study it was shown that in a mouse BM (Tanaka et al., 2003). One drawback to this approach is model of permanent focal ischemia the neuroprotective effect that whole body irradiation may affect the integrity of the BBB of the PPARg agonist rosiglitazone was abolished in neutrophil and induce brain cytokine production, creating a permissive depleted animals. Rosiglitazone treatment induced an increase environment for brain engraftment of BM-derived immune cells of total number of neutrophils 24 h after ischemia and a shift normally not found in the CNS (Mildner et al., 2007). Although in the ratio of pro-inflammatory N1 neutrophils to N2 neu- head shielding during irradiation or parabiosis may avoid such trophils shown by increased expression of the M2-marker pro- disturbances, the resulting degree of chimerism (40–50%) is tein Ym1 (Chil3). N2 neutrophils were recently defined as a not as high as with whole body irradiation (>90%) (Ginhoux population of polarized cells with anti-inflammatory pheno- et al., 2013; Prinz and Priller, 2014). The reduced chimerism type, in accordance to the M1/M2 classification of macrophages, obtained with these models may not be problematic for cell and have been found in tumors where they facilitate tumor tracking and fate determination experiments, but it will con- growth by inhibiting anti-tumor T cell responses (Fridlender found the interpretation of mechanistic studies on the relative et al., 2009; Mantovani, 2009). Their role in brain ischemia, contribution of resident and blood-borne immune cells to tissue including the mechanism of neuroprotection, remains to be damage and repair. Recently, it has been shown that chemi- firmly established, but, analogous to the double-edged role cal BM ablation with busulfan leads to a better rate of blood of monocytes/macrophages, neutrophils could also have ben- chimerism with reduced inflammation and myeloid immune eficial effects, perhaps in the repair phase of the injury. cell recruitment in the mouse CNS compared to irradiation For example, neutrophils that undergo apoptosis are ingested (Kierdorf et al., 2013b). However, in our own experience, doses by microglia and macrophages, which induce these phago- of busulfan that produce a chimerism comparable to irradiation cytic cells to become anti-inflammatory or promote tissue lead to a myeloid immune cell recruitment in brain greater repair (Serhan and Savill, 2005). Therefore, manipulating the than that seen with irradiation (Sugiyama et al., 2014). There- polarization state of neutrophils could also have therapeutic fore, chemical BM ablation-based approaches require further relevance. scrutiny. Frontiers in Cellular Neuroscience www.frontiersin.org January 2015 | Volume 8 | Article 461 | 7 Benakis et al. Myeloid cells in ischemic stroke Another approach to track blood-borne myeloid cell infil- Furthermore, they demonstrated that selective deletion of tration into the ischemic area, has been to use ultrasmall CCR2 in BM-derived cells induced a delayed clinical deterio- superparamagnetic iron oxide particles (USPIO), which labels ration and hemorrhagic conversion of ischemic infarcts, sug- peripheral phagocytic cells after intravenous injection. USPIO gesting a beneficial role of CCR2 monocytes in maintaining were delivered in one case 5.5 h after permanent MCA occlusion the structure of the neurovascular unit (Gliem et al., 2012). C high (Rausch et al., 2001) and in an other study 24 h before inducing Because CCR2 /Ly6C monocytes can transform into anti- low high photothrombotic strokes (Kleinschnitz et al., 2003). Brains were inflammatory Ly6C F4/80 macrophages, it was suggested analyzed at different time points after ischemia by magnetic that lack of infiltrating CCR2 monocytes results in reduction resonance imaging (MRI) and histology. While the temporal of anti-inflammatory macrophages at later time points com- profile was slightly different between the two studies, iron par- promising repair mechanisms. However, several other studies ticles were detected late in the post-ischemic period, after most demonstrated reduced phagocytic macrophage accumulation in of the damage had occurred (Rausch et al., 2001; Kleinschnitz the brain of CCL2 or CCR2 knock-out mice together with et al., 2003). In a model of neonatal ischemia, microglia— smaller infarcts after cerebral artery occlusion (Hughes et al., C low/int identified as CD11b /CD45 by flow cytometry—were the 2002; Dimitrijevic et al., 2007; Schilling et al., 2009), suggesting predominant cell population in the infarct area compared to a deleterious role of CCR2 monocytes. These discrepancies may C high C CD11b /CD45 monocytes (Denker et al., 2007). A simi- reflect the multifunctional roles of CCR2 monocytes, which, lar pattern was described using GFP-BM chimeric mice. Thus, while contributing to early brain injury may also enable delayed Schilling et al. found that microglia were the first cells to be acti- repair processes. vated in the infarcted area, exhibited a more pronounced phago- cytic activity and out-numbered GFP-BM monocytes (Schilling CX3CR1 MONOCYTES AND MICROGLIA IN CEREBRAL ISCHEMIA et al., 2003, 2005). Collectively, these observations suggest that The chemokine CX3CL1 is found on neurons while its recep- low microglia are likely to be the first cells activated after brain tor CX3CR1 is expressed on microglia and Ly6C mono- injury, aiming at clearing the cell debris by phagocytosis and cytes (Geissmann et al., 2003; Sunnemark et al., 2005; Limatola = = contributing to the resolution of inflammation (Neumann et al., and Ransohoff, 2014). CX3CR1 or CX3CL1 mice show 2009). On the other hand, phagocytosis could also contribute reduced brain damage in transient focal ischemia models com- to neuronal cell loss after ischemia. For example, it has been pared to wild type mice (Soriano et al., 2002; Dénes et al., suggested that microglia could engulf viable neurons in the 2008; Fumagalli et al., 2013), although the effect might not be ischemic penumbra (Neher et al., 2013). Salvageable neurons sustained in time (Gliem et al., 2012). Neuroprotection found would send “eat me” signals to nearby microglia and induce in CX3CR1 mice following focal cerebral ischemia was cor- their phagocytosis that further increases brain damage (Neher related with an increase of the M2 macrophage marker genes et al., 2012). In support of this hypothesis, deficiency of the CD206 and Ym1 and a decrease of M1-iNOS gene expres- phagocytosis-related receptors Mertk and milk fat globule-EGF sion compared to wild type mice (Fumagalli et al., 2013; Tang factor 8 protein (Mfge8) on microglia promotes functional et al., 2014). These findings indicate that CX3CR1 deficient mice recovery and reduces brain atrophy in focal infarcts (Neher induce a different pattern of M1/M2 marker expression in the et al., 2013). On the other hand, deletion of triggering receptor ischemic region. The authors suggest that the lack of CX3CR1 in expressed on myeloid cells 2 (TREM2), a receptor expressed on microglia and infiltrated monocytes favors an anti-inflammatory phagocytic microglia and macrophages, did not affect infarct milieu that protects the brain from infarct development. How- size in a focal ischemia model in mice (Sieber et al., 2013), ever, intracerebroventricular delivery of CX3CL1 at the time of although TREM2 is essential for their phagocytosis capacity MCA occlusion decreased infarct volumes (Cipriani et al., 2011), (Takahashi et al., 2007). The molecular mechanisms regulating suggesting that CX3CL1 acts on microglia to reduce their acti- these microglial behaviors need to be established and may provide vation state and inhibit the release of inflammatory cytokines new insights into endogenous brain processes regulating injury (Zujovic et al., 2000; Cardona et al., 2006; Biber et al., 2007). and repair. Michaud et al. has recently shown that selective ablation of low C Ly6C /CX3CR1 monocytes, thought to preferentially develop CCR2 MONOCYTES IN CEREBRAL ISCHEMIA into M2 macrophages, does not influence stroke outcome in a Recruitment of circulating monocytes to the ischemic brain hypoxic-ischemic injury model (Michaud et al., 2014). Using = low C is orchestrated by inflammatory cytokines, de novo-expressed NR4A1 BM chimeras that lack circulating Ly6C /CX3CR1 adhesion molecules and chemokines. The monocyte chemoat- monocytes (Carlin et al., 2013), it was found that structural tractant protein (MCP-1, CCL2) and its receptor CCR2 are and functional outcome were not affected. However, brain infil- known to be involved in the inflammatory response of the trating monocytes have not been assessed in this study, and it high C injured brain after cerebral ischemia (Che et al., 2001; Chu remains an open question whether Ly6C /CCR2 monocytes low C et al., 2014a). After permanent and transient focal ischemia, in the brain parenchyma could give rise to Ly6C /CX3CR1 high Gliem et al. found an increase in Ly6C monocytes at 3 days, monocytes/macrophages, even in the absence of the NR4A1 low whereas the number of Ly6C monocytes was greatest at 6 days, transcription factor. GFP/+ paralleled by sequential peaks of CCR2 and CX3CR1 mRNA Using parabiosis of CX3CR1 mice and wild type part- as well as gene expression of the pro- and anti-inflammatory ners, Li et al. found that microglia increased gradually dur- cytokines IL-1b and TGF-b, respectively (Gliem et al., 2012). ing the first week after photothrombotic stroke (Li et al., Frontiers in Cellular Neuroscience www.frontiersin.org January 2015 | Volume 8 | Article 461 | 8 Benakis et al. Myeloid cells in ischemic stroke 2013). Hypertrophic ameboid microglia were more abundant microenvironment highlights the complexity of their response to in the periphery compared to the core of the lesion and many cerebral ischemia (Taylor and Sansing, 2013). GFP-positive microglia were labeled with the mitotic marker bromodeoxyuridine (Li et al., 2013). In the wild type para- A ROLE FOR SPLENIC MONOCYTES IN BRAIN ISCHEMIA? GFP/+ biotic partner, CX3CR1 infiltrating cells started to popu- The discovery of a splenic monocyte reservoir in adult mice, that late the brain 5 days after ischemia onset, were less numerous is mobilized following ischemic myocardial injury (Swirski et al., than activated microglia and did not proliferate, suggesting that 2009), has sparked renewed interest in the role of the spleen microglia and monocyte-derived CX3CR1 macrophages are two in brain ischemia. Previous studies have shown that splenocytes distinct populations with presumably different roles in cerebral respond to cerebral ischemia by producing large amounts of ischemia. inflammatory mediators early after injury (Offner et al., 2006b), followed later by an increase of blood monocytes and splenic M1 AND M2 PHENOTYPES IN CEREBRAL ISCHEMIA regulatory T cells, that are thought to dampen the inflammatory Recent studies have begun to investigate microglia and response (Offner et al., 2006a). In accordance with these findings, macrophage polarization in vivo. In a model of permanent chronic splenectomy (Ajmo et al., 2008) or acute spleen irradia- focal ischemia, different cell markers were used to characterize tion (Ostrowski et al., 2012) have been performed in rats to inhibit the prevalence of phagocytic cells and M2-like phenotype at systemic inflammatory responses after stroke. Although the stud- different time after ischemia (Perego et al., 2011). Soon after ies differed in the ischemic model, i.e., permanent vs. tran- injury, ED-1 positive phagocytic cells were present at the border— sient ischemia, both studies observed neuroprotection together possibly limiting the expansion of the infarct—whereas Ym1- with a decrease of immune cell counts in the ischemic region. and CD206-expressing cells were found in the ischemic core. At In addition, post-stroke treatment with the antibacterial agent 7 days, more phagocytic cells invaded the ischemic core where moxifloxacin (MFX) reduced peripheral infection and infarct they engulfed neurons. In transient focal ischemia, M2-type gene size in animal models of stroke (Meisel et al., 2004; Bao et al., expression (CD206, Arg1, CCL22, Ym1/2, IL-10, and TGF-b) was 2010). In MFX-treated mice the percentage of the splenic pro- high first apparent from 1 to 3 days after ischemia, peaked at 3–5 days, inflammatory Ly6C monocytes was reduced compared to was reduced at 7 days and returned to pre-injury levels by day vehicle animals 7 days after stroke, together with reduced expres- 14. M1-type genes (iNOS, CD11b, CD16, CD32, and CD86) were sion of CCR2 in the spleen and the brain (Bao et al., 2010). These gradually increased from day 3 and remained elevated for 14 days studies suggest that interventions aiming at reducing systemic after ischemia (Hu et al., 2012). The M1/M2 phenotype was also inflammation, specifically by targeting splenocytes, could lead to a assessed in in vitro models of ischemic injury using microglia reduction of brain immune cell infiltration and neuroprotection. and neuronal co-culture. M1-polarized microglia/macrophages However, these reports did not assess whether leukocytes, specifi- exacerbate neuronal death compared with M2 cells (Hu et al., cally monocytes, found in the brain originate from the spleen and 2012; Girard et al., 2013). Hu et al. suggested that early expression contribute directly to stroke outcome. For instance, Ajmo et al. of M2 genes may contribute to neuroprotection by enhancing the did not find alteration of immune cell counts in peripheral blood phagocytosis of dead cells, removal of tissue debris and promote following MCA occlusion in sham or splenectomized animals, recovery. At later stages, cells displaying a M1 phenotype may while spleen irradiation after MCA occlusion resulted in a drop induce pro-inflammatory mediators and exacerbate neuronal of circulating lymphocytes, but failed to lower blood monocytes death. Therefore, limiting such M2 to M1 shift and promoting M2 and neutrophils (Ajmo et al., 2008; Ostrowski et al., 2012). Thus polarization could be a potential therapeutic strategy (Hu et al., it is not clear whether peripheral immune cells are mobilized 2012). In vitro experiments have to be interpreted with caution from the spleen into the brain and contribute directly to stroke because microglia are commonly derived from neonatal tissue outcome. Recently, Kim et al. analyzed the temporal profile of high low and develop in a culture environment that cannot recapitulate brain infiltrating Ly6C and Ly6C monocytes after focal the maturation process that occurs in vivo (Hellwig et al., 2013). ischemic injury in acutely splenectomized mice (Kim et al., 2014). Importantly, the gene expression signature characteristic for adult In sham mice, they found that brain ischemia induced a reduction brain microglia is lost in primary neonatal microglia cultures of both monocytes subsets in the spleen that temporally correlated as well as in microglial cell lines including BV2 and N9 cells with their increase in the ischemic brain. In splenectomized high low (Butovsky et al., 2014). Thus, in vivo translation of findings mice, brain Ly6C and Ly6C monocytes were reduced at obtained with in vitro polarization models might not be easily 1 and 3 days after stroke compared to sham surgery. However achieved. Desestret et al. administered differentiated BM-derived no difference in infarct size was observed between groups (Kim M2 macrophages 4 days after cerebral ischemia by intravenous et al., 2014). While this study did not directly track splenic injection. M2 macrophages failed to induce protection or monocytes and the findings remain correlative, it suggests that the high low improve behavioral outcome (Desestret et al., 2013). On the spleen might contribute both Ly6C and Ly6C monocytes other hand, Girard et al. have proposed that M1 and M2 after brain ischemia. The failure to observe neuroprotection in BM-derived macrophages promote cell death in vitro, whereas this model might be related to simultaneous reduction of both high M2 microglia is neuroprotective. However, microglia does not monocyte subsets, thus balancing the loss of deleterious Ly6C low display a classic M1 and M2 phenotypes after MCA occlusion monocytes with the loss of beneficial Ly6C monocytes, which (Girard et al., 2013). The ability of microglia to differentiate into might be involved in resolution of the inflammation and tissue multiple macrophage-related phenotypes depending on their repair (Shechter et al., 2013). Frontiers in Cellular Neuroscience www.frontiersin.org January 2015 | Volume 8 | Article 461 | 9 Benakis et al. Myeloid cells in ischemic stroke MYELOID IMMUNE CELLS IN HUMAN STROKE and CD16 (Fcg receptor III). The majority of human “clas- sical” monocytes express high level of CD14 and are nega- TEMPORAL PROFILE OF MYELOID IMMUNE CELL ACTIVATION AND CC tive for CD16 (CD14 /CD16 ), whereas the minor subtypes INFILTRATION high CC C express CD16 and can be either CD14 (CD14 /CD16 ) Histological analysis of autopsy material from patients allowed low dim C or CD14 (CD14 /CD16 ), also known as intermediate to draw a temporal profile of cellular events from 1 day after and non-classical monocytes, respectively (Ziegler-Heitbrock, stroke onset to several years (Chuaqui and Tapia, 1993; Mena 1996; Ziegler-Heitbrock et al., 2010). The classical monocytes et al., 2004). Astrogliosis was observed within the first day of C high CD14 /CD16 share some features with the murine Ly6C cerebral infarct development together with neutrophils, which monocytes such as the high expression of the chemokine receptor were observed in 81% of cases between 3–37 days after stroke CCR2 (Weber et al., 2000), whereas CD16 cells express CX3CR1 onset (Mena et al., 2004). This active inflammatory phase was low similar to mouse Ly6C monocytes (Merino et al., 2011). How- accompanied by tissue necrosis and infiltration of inflammatory ever, the pro- or anti-inflammatory functions of these different cells such as mononuclear cells and macrophages, which were not monocyte subsets depend on the inflammatory context and, as observed during the first 2 days after stroke onset. Interestingly, in the mouse, a strict association of phenotype and function can inflammatory mononuclear cells and macrophages persisted up not be established (Gordon and Taylor, 2005; Ziegler-Heitbrock, to 53 years and were found in the majority of late stage specimens 2007; Merino et al., 2011). For example, blood monocyte subsets while neutrophils were absent (Mena et al., 2004). Although the from healthy donors showed different pattern of response when temporal sequence and type of cells in the infarct are similar in stimulated with LPS (Frankenberger et al., 1996; Skrzeczynska- ´ rodents and humans (Gelderblom et al., 2009), the chronology of dim C Moncznik et al., 2008). CD14 /CD16 monocytes were found events seems to be more delayed and more spread out in time in high C to produce more TNF compared to CD14 /CD16 and humans. high high C CD14 /CD16 monocytes, whereas CD14 /CD16 were the Advanced imaging techniques to visualize post-ischemic main source for IL-10, (Belge et al., 2002; Skrzeczynska-M ´ oncznik inflammation and leukocyte infiltration are increasingly being et al., 2008). employed in the clinical setting. Visualization of infiltrated Similar to the mouse (Offner et al., 2006b), stroke has a signifi- macrophages in stroke patients was performed by USPIO-MRI cant impact on the peripheral immune system in humans (Kamel (Saleh et al., 2004, 2007; Cho et al., 2007). In all patients tested, and Iadecola, 2012). Although there is significant immunosup- an increase signal of USPIO was observed by MRI in the brain pression in acute stroke (Meisel et al., 2005; Chamorro et al., parenchyma (Saleh et al., 2004). Only a minority of patients that 2006), a significant increase of circulating monocytes has also received USPIO between 24 h and 96 h after symptom onset been reported (Losy and Zaremba, 2001). The different sub- showed a positive signal in the brain (Cho et al., 2007; Saleh et al., types of blood monocytes were investigated in patients following 2007). However, iron-positive cells detected in the brain cannot stroke and were correlated with outcome severity (Urra et al., represent only monocytes since activated microglia can engulf high C 2009). The CD14 /CD16 subset was increased 48 h after iron particles as well (Oude Engberink et al., 2008; Desestret et al., dim C admission, while a parallel decrease in the CD14 /CD16 2009). Injection of iron oxide monocytes labeled ex vivo allowed high subtype was observed. An increase in classical CD14 /CD16 to follow their brain infiltration in experimental models of stroke monocytes was associated with poor outcome, whereas increased (see previous section) (Stroh et al., 2006; Oude Engberink et al., CD16 monocytes were linked to a better prognosis (Urra et al., 2008). Similarly, the use of ex vivo monocyte labeling in humans 2009). CD14 expressing cells were observed in brain tissue from may increase our understanding of the temporal pattern of blood- patients with focal infarction as early as 1 day and persisted for borne cell infiltration after stroke. Positron emission tomography months after ischemia (Beschorner et al., 2002), suggesting a (PET) has been used to label inflammatory cells in stroke patients long-term involvement in inflammatory processes at the site of using the radioligands C(R)-PK11195 for the translocator pro- injury. tein 18kDa (TSPO), commonly used as a microglial marker There is still a large knowledge gap about human microglia (Stephenson et al., 1995; Cagnin et al., 2007). Analysis in patients and immune cells of myeloid origin regarding their activation showed an increase of the marker between 3 and 150 days after state, spatiotemporal distribution in the brain and, ultimately, stroke (Gerhard et al., 2005). Positive labeling was observed early about their role in acute cerebral ischemia. Advanced brain imag- after ischemia, initially at the periphery of the lesion, then in the ing modalities in humans and new cerebral ischemic models infarct core and eventually in peri-infarct regions (Gerhard et al., more representative of human stroke would help enhance our 2005; Thiel and Heiss, 2011). Altogether, the time of magnetic understanding of the inflammatory processes occurring in acute particles or radiolabelled agents infusion might be critical to cerebral ischemia and to test therapeutic strategies directed at discriminate resident microglia from immune cells of myeloid manipulating their beneficial or deleterious effects. origin (Deddens et al., 2012). However, a limitation is that these techniques of brain macrophage visualization do not permit to TARGETING INFLAMMATION AS THERAPEUTIC STRATEGIES differentiate between their different subtypes and activation states The only successful treatment for ischemic stroke is thrombol- (Weissleder et al., 2014). ysis with tissue plasminogen activator (tPA), which, due to the MONOCYTE SUBSETS IN PATIENTS WITH CEREBRAL ISCHEMIA narrow therapeutic window (<4.5 h) (Hacke et al., 2008) and In human three distinct blood monocytes can be distinguished safety concerns, is administered to less than 5% of patients relative to the expression of the receptor CD14 (LPS co-receptor) (Fonarow et al., 2011). Different strategies aimed at preventing Frontiers in Cellular Neuroscience www.frontiersin.org January 2015 | Volume 8 | Article 461 | 10 Benakis et al. Myeloid cells in ischemic stroke the inflammatory response after cerebral ischemia have been The failure of clinical trials that targeted post-ischemic leukocyte successful in rodent models. Ischemic damage was shown to be recruitment might be in part explained by the indiscriminate attenuated, among others, by inhibition of microglial activation nature of these approaches and a lack of a nuanced understanding using the immunomodulatory antibiotic minocycline, systemic T of the impact of inflammation on damage and repair processes lymphocytes depletion using sphingosine 1-phosphate receptor in the post-ischemic brain. Our understanding of the behavior agonist (FTY720), diminishing free radical generating and pro- of myeloid immune cells and microglia in ischemic stroke is still inflammatory enzymes, such as iNOS or cyclooxygenase-2, inhi- limited. Key questions to be addressed in future research include bition of cytokines secretion, or targeting adhesion molecules (for the transcriptomic and proteomic characterization of myeloid reviews, Dirnagl et al., 1999; Iadecola and Alexander, 2001; Wang, immune cell subsets at different stages of brain ischemia, the 2005; Jordán et al., 2008). mechanism of monocytes, neutrophils and microglia polariza- Unfortunately attempts to translate anti-inflammatory thera- tion in the ischemic environment, and the neural and humoral peutic interventions into the clinics have been more disappointing signals generated by the ischemic brain to modulate periph- than promising (O’Collins et al., 2006; Moskowitz et al., 2010). eral immune cell activation and mobilization. Answering some The selective IL-1 receptor antagonist, IL1-ra, which limits the of these questions would provide the knowledge base needed pro-inflammatory action of IL-1, has been tested in random- to design more specific immunotherapies for the treatment of ized patients with acute stroke (Emsley et al., 2005). Despite a ischemic stroke. conclusive phase II study no recent publications have reported ACKNOWLEDGMENTS the recombinant human IL1-ra as a therapeutic agent for acute This work was supported by National Institutes of Health (NIH) stroke. Strategies to inhibit neutrophil infiltration have been Grants NS34179 (Costantino Iadecola) and NS081179 (Josef tested in clinical trials. Treatment with the murine mono-clonal Anrather). Corinne Benakis was supported by grant P3SMP3 anti-ICAM-1 in the Enlimomab trial (Enlimomab Acute Stroke 148367 from the Swiss National Science Foundation and the Swiss Trial Investigators, 2001) or UK-279,276 (neutrophil inhibitory Foundation for Grants in Biology and Medicine (SFGBM). factor) have failed to improve recovery in acute ischemic stroke patients (Krams et al., 2003). Likewise, NXY-059, a nitrone-based REFERENCES free radical trapping agent, has not demonstrated any benefits in Ajami, B., Bennett, J. L., Krieger, C., McNagny, K. M., and Rossi, F. M. V. (2011). stroke clinical trials (Shuaib et al., 2007). The granulocyte colony- Infiltrating monocytes trigger EAE progression, but do not contribute to the stimulating factor (G-CSF), that stimulates proliferation, survival, resident microglia pool. Nat. Neurosci. 14, 1142–1149. doi: 10.1038/nn.2887 and maturation of BM-derived cells, has shown neuroprotective Ajami, B., Bennett, J. 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Inflammatory monocytes and the pathogenesis of viral as a potential conflict of interest. encephalitis. J. Neuroinflammation 9:270. doi: 10.1186/1742-2094-9-270 Thiel, A., and Heiss, W.-D. (2011). Imaging of microglia activation in stroke. Stroke Received: 12 October 2014; accepted: 18 December 2014; published online: 14 January 42, 507–512. doi: 10.1161/STROKEAHA.110.598821 2015. Thornton, P., McColl, B. W., Greenhalgh, A., Dénes, A., Allan, S. M., and Rothwell, Citation: Benakis C, Garcia-Bonilla L, Iadecola C and Anrather J (2015) The role of N. J. (2010). Platelet interleukin-1alpha drives cerebrovascular inflammation. microglia and myeloid immune cells in acute cerebral ischemia. Front. Cell. Neurosci. Blood 115, 3632–3639. doi: 10.1182/blood-2009-11-252643 8:461. doi: 10.3389/fncel.2014.00461 Tsou, C.-L., Peters, W., Si, Y., Slaymaker, S., Aslanian, A. M., Weisberg, S. P., et al. This article was submitted to the journal Frontiers in Cellular Neuroscience. (2007). Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone Copyright © 2015 Benakis, Garcia-Bonilla, Iadecola and Anrather. This is an open- marrow and recruitment to inflammatory sites. J. Clin. Invest. 117, 902–909. access article distributed under the terms of the Creative Commons Attribution License doi: 10.1172/jci29919 (CC BY). The use, distribution and reproduction in other forums is permitted, provided Urra, X., Villamor, N., Amaro, S., Gómez-Choco, M., Obach, V., Oleaga, L., et al. the original author(s) or licensor are credited and that the original publication in this (2009). Monocyte subtypes predict clinical course and prognosis in human journal is cited, in accordance with accepted academic practice. No use, distribution or stroke. J. Cereb. Blood Flow Metab. 29, 994–1002. doi: 10.1038/jcbfm.2009.25 reproduction is permitted which does not comply with these terms. 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