Modulators of microglial activation and polarization after intracerebral haemorrhage (original) (raw)

. Author manuscript; available in PMC: 2017 Aug 30.

Published in final edited form as: Nat Rev Neurol. 2017 May 19;13(7):420–433. doi: 10.1038/nrneurol.2017.69

Abstract

Intracerebral haemorrhage (ICH) is the most lethal subtype of stroke but currently lacks effective treatment. Microglia are among the first non-neuronal cells on the scene during the innate immune response to ICH. Microglia respond to acute brain injury by becoming activated and developing classic M1-like (proinflammatory) or alternative M2-like (anti-inflammatory) phenotypes. This polarization implies as yet unrecognized actions of microglia in ICH pathology and recovery, perhaps involving microglial production of proinflammatory or anti-inflammatory cytokines and chemokines. Furthermore, alternatively activated M2-like microglia might promote phagocytosis of red blood cells and tissue debris, a major contribution to haematoma clearance. Interactions between microglia and other cells modulate microglial activation and function, and are also important in ICH pathology. This Review summarizes key studies on modulators of microglial activation and polarization after ICH, including M1-like and M2-like microglial phenotype markers, transcription factors and key signalling pathways. Microglial phagocytosis, haematoma resolution, and the potential crosstalk between microglia and T lymphocytes, neurons, astrocytes, and oligodendrocytes in the ICH brain are described. Finally, the clinical and translational implications of microglial polarization in ICH are presented, including the evidence that therapeutic approaches aimed at modulating microglial function might mitigate ICH injury and improve brain repair.

Intracerebral haemorrhage (ICH) accounts for 10–15% of all strokes, making it the second most common type of stroke1–3. ICH is estimated to affect over 1 million people worldwide each year1, and that number is expected to rise substantially as the population ages. Most patients with ICH die or become severely disabled, and supportive care is the mainstay of current therapy4,5. Primary brain damage develops within the first few hours after ICH as a result of haematoma or oedema, formation and expansion of which induces mass effects and increased intracranial pressure that can lead to herniation and death. Secondary brain damage results from activation of microglia, which drive proinflammatory, oxidative and cytotoxic cascades causing cell death and functional impairment6.

As key innate immune cells, microglia act as guardians of the brain and are recognized to be the first non-neuronal cells to respond to various acute brain injuries7, including ICH6. Increasing evidence indicates that activated microglia are the primary source of cytokines, chemokines, prostaglandins, proteases, ferrous iron and other immunomodulatory molecules in the brain6. These molecules combine to initiate secondary brain injury and the subsequent brain repair processes. In fact, cell therapy with bone marrow-derived mononuclear cells or macrophages ameliorates injury and improves outcome after ischaemic stroke8–11. In view of these findings, research into microglial activation and function, and on cell–cell interactions, is critical to understanding ICH pathophysiology.

To develop new and effective therapies for ICH, we also need to explore the cellular and molecular mechanisms that underlie ICH-induced brain injury and repair12. As inflammation contributes to ICH-induced secondary brain injury6, modulation of the microglial phenotype might not only accelerate the absorption of haematoma and oedema, but also improve white matter integrity, brain repair and functional recovery13. The fact that clinical trials of open surgical haematoma evacuation have not shown positive results14,15 emphasizes the need to investigate endogenous mechanisms of haematoma removal. This knowledge could lead to the development of improved treatments for patients with ICH.

In this Review, we summarize key advances in our understanding of microglial function after ICH, with a particular focus on microglial activation, migration, proliferation, secretion and phagocytosis. In addition, we discuss microglial polarization, modulators and interactions with other cells. We also address the clinical and translational implications of these observations and highlight new therapeutic directions for ICH.

Microglia

Microglia are the major phagocytes of the brain. In vitro, activated microglia develop into either classically activated (M1, proinflammatory) or alternatively activated (M2, anti-inflammatory) phenotypes, a process termed polarization16,17. Alternatively activated M2 microglia are divided into three subtypes — M2a, M2b and M2c — each with different cell surface markers and distinct biological functions18: M2a microglia mainly contribute to cell regeneration, whereas M2b and M2c cells participate in phagocytosis and removal of tissue debris.

In response to acute brain injury, microglia can dynamically and temporally change their phenotype19,20. Individual classically activated and alternatively activated microglia within brain lesions can contribute to either tissue damage or repair under conditions of spinal cord injury21–23, ischaemic stroke19,24 and traumatic brain injury (TBI)20,25–27. Although the supposed dichotomy between M1 and M2 phenotypes is now recognized as an oversimplification (these two extreme activation states are only observed in vitro), this classification remains useful for understanding the function of microglia in various brain diseases28. In a mouse model of spinal cord injury, for example, although global M1 and M2 marker levels both increased in the acute phase23, flow cytometry showed that microglia and macrophages had mainly developed an M1 phenotype21. In models of TBI20,29 and ischaemic stroke30, an M2 to M1 shift was observed, whereas a mixed M1-like and M2-like response was reported in an animal model of epilepsy31. However, controversy has begun to surround the traditional concept of M1 versus M2 microglial polarization. For example, in a mouse model of experimental auto-immune encephalomyelitis32, the classic M1 marker IL-6 was induced by IL-4 and exerted an anti-inflammatory effect. Additionally, in a model of TBI, canonical markers of both polarization states were highly co-expressed by the same cell33. Hence, whether microglia actually undergo M1 or M2 polarization has been called into question34. Researchers using animal models of ICH should, therefore, pursue functional studies of microglial activities such as phagocytosis and haematoma clearance, as discussed below. The functional properties of known M1-like and M2-like markers are summarized in TABLE 1.

Table 1.

Markers of M1 and M2 microglia

Changes in M1 markers

Proinflammatory cytokines

Activation of microglia usually results in production of proinflammatory cytokines. Currently, proinflammatory cytokines are thought to be produced specifically by classically activated M1 microglia, and alterations in proinflammatory cytokine profiles and levels are thought to explain the changes in microglial function after ICH.

Microglial activation can be assessed by immunohistochemical analysis of the ICH brain, using antibodies targeting cell surface markers shared by microglia and macrophages. Suitable markers include keratan sulfate (targeted by 5D4 antibody)35,36, cell surface glycoprotein F4/80 (targeted by F4/80 antibody)35,36, CD11b37,38, Iba1 (REFS 39,40) and CD68 (REFS 41,42). However, anti-CD11b antibody cannot distinguish microglia from neutrophils and monocytes in the ICH brain: although in the intact brain CD11b is expressed only by microglia, this marker is also expressed by neutrophils and monocytes in the ICH brain39,43. Myeloperoxidase, a specific marker of neutrophils, is used to discriminate microglia from neutrophils and monocytes44. Distinguishing activated microglia from macrophages in the ICH brain by immunohistochemistry is also challenging. CD45 is helpful in this scenario because it is more highly expressed in monocytes than in microglia. Thus, CD45 expression levels can be used to separate these two cell types using flow cytometry: CD45IntCD11b+ cells are microglia and CD45HiCD11b+ cells are macrophages45–47.

Changes in M1-signature (proinflammatory) cytokines are prominent after ICH. In a clinical study of perihaematomal brain tissue, nuclear factor-κB (NF-κB) was activated and migrated into the nucleus at 13–48 h after ICH, and IL-1β and tumour necrosis factor (TNF) levels increased within 1 day after ICH48,49. These findings suggest the presence of a proinflammatory state early after ICH. In both the collagenase-induced and autologous blood-induced models of ICH (TABLE 2), IL-1β, IL-6, TNF50,51 and inducible nitric oxide synthase (iNOS)52 mRNA levels are generally elevated in the acute phase, starting to rise as early as 3 h after ICH and peaking at 3 days53–54. Changes in the corresponding protein levels follow a similar time course52,53,55. In rodents, secretion of proinflammatory cytokines IL-1β, IL-6 and TNF increased in the first 3 days56, and then returned to within the normal range on day 7 in both colla-genase-induced37 and blood-induced37,54 models of ICH. Interestingly, IL-1β, but not TNF, macrophage mannose receptor 1 (CD206) or chitinase-like protein 3 (also known as Ym1), colocalized with amoeboid microglia in rats with collagenase-induced ICH57. This colocalization pattern suggests a phagocytic and secretory phenotype that might be associated with dynamic changes in microglial morphology57. Moreover, treatment with the TNF receptor antagonist R-7050 attenuated neurovascular injury and improved functional outcomes in mice with collagenase-induced ICH58.

Table 2.

Animal models of ICH

In experimental models, IFNγ levels did not change in the first 3 days (unlike IL-1β, IL-6 and TNF levels)50,59,60, but markedly increased at later stages after ICH50,60. A reduction in the IFNγ:IL-4 ratio contributed to long-term recovery after ICH61. These results suggest that the ratio of IFNγ to M2-like cytokine levels might be a more reliable marker than IFNγ levels alone for the assessment of inflammatory status in the chronic phase after ICH. Our group has shown that M1 markers such as CD16, CD32 and iNOS are highly expressed on microglia on days 1 and 3 after ICH62, suggesting that M1-like microglial polarization occurs early in the acute phase.

Although the specific proinflammatory cytokines produced vary according to the ICH model used and time point assessed (FIG. 1), the overall direction of changes in these classic M1 markers is consistent in the acute phase after ICH. By 14 days after ICH (during the chronic phase), levels of most proinflammatory cytokines have returned to baseline60. However, whether levels of these cytokines change during the chronic phase of ICH, and whether these changes affect brain repair processes, remains unclear.

Figure 1. Dynamic changes in microglial marker levels and profiles over time after intracerebral haemorrhage.

Top panel, dashed red curve: microglial M1-like response exhibits a decreasing trend in the first 14 days. Top panel, dashed blue curve: microglial M2 like response exhibits an increasing trend in the first 14 days. Bottom panel: microglia exhibit an M1-like response as early as 6 h after intracerebral haemorrhage (ICH), as shown by upregulation of proinflammatory cytokines such as IL-1β, IL-6, tumour necrosis factor (TNF) and inducible nitric oxide synthase (iNOS). M2 markers, such as arginase-1 (Arg1), chitinase-like protein 3 (also known as Ym1), CD206, CD163 and IL-10, start to increase on day 1 after ICH. Transforming growth factor-β (TGFβ) is upregulated from day 7 until day 14 post-ICH. Although a mixed M1-like and M2-like microglial phenotype is evident during days 1 to 3, the balance of evidence supports an M1 to M2 phenotype switch in the first 7 days. IFNγ levels increase on day 7 after ICH, and levels of most proinflammatory cytokines return to baseline on day 14.

Toll-like receptors

Increased protein expression of Toll-like receptor (TLR)2 and TLR4 is associated with poor outcomes in patients with ICH63. In mice, expression of TLR2 (REFS 64,65) and TLR4 (REF. 62) proteins was upregulated (mainly in microglia) as early as 6 h after ICH and remained high throughout the first 3 days, in both collagenase-induced and blood-induced models of ICH54,62. In Tlr4−/− (REF. 54) and Tlr2−/− (REF. 65) mice after blood-induced ICH, IL-1β, IL-6 and TNF levels were substantially decreased compared with those in wild-type mice, a finding that supports a link between TLR2 and TLR4 activation and M1 microglia. Furthermore, TLR4 activates the NF-κB signalling pathway, via myeloid differentiation primary response protein MYD88 and TIR domain-containing adaptor molecule 2 (also known as TRIF-related adaptor molecule)66,67 (FIG. 2). TLR4 also upregulates M1-signature (proinflammatory) cytokines in the blood-induced model of ICH54,68–70. The same researchers demonstrated that TLR4 depletion reduced microglial autophagy, which contributes to the M1 phenotype of microglia71. In addition, treatment with a TLR4 antagonist reduced the production of proinflammatory cytokines (IL-1β, IL-6 and TNF) and inactivated microglia68. TLR2 exerts effects similar to those of TLR4 in the ICH brain, a fact that might be explained by the assembly of TLR2–TLR4 heterodimers, which trigger proinflammatory responses65.

Figure 2. Modulators of microglial polarization and phagocytosis after intracerebral haemorrhage.

Several transcription factors modulate microglial polarization towards an M1-like (left panel) or an M2-like (centre panel) phenotype. Activation of high-mobility group protein 1 (HMG1) and Toll-like receptor (TLR)2 or TLR4 promotes microglial M1-like responses. Signal transducer and activator of transcription (STAT)1 is activated by TLRs and IFNγ, promoting microglial polarization to an M1 phenotype. After intracerebral haemorrhage (ICH), TLRs modulate STAT3 phosphorylation, which increases M1-like polarization. G-protein-coupled E prostanoid (EP) receptors EP1 and EP3 are mainly expressed on microglia and drive neuronal toxicity mediated by M1 upregulation after ICH. STAT6 accumulates in response to IL-4 and is responsible for the transcription of M2-related genes. Sphingosine-1-phosphate (S1P) receptor signalling contributes to the downregulation of proinflammatory cytokines and enhances M2-like responses after ICH. Although EP2 receptors are not expressed in microglia after ICH, EP2 deletion results in an increased microglial proinflammatory response. Other modulators regulate microglial phagocytosis (right panel). Activation of the transcription factor nuclear factor erythroid 2 related factor 2 (Nrf2) is associated with microglial phagocytosis after ICH and increases CD36 and haem oxygenase-1 (HO-1) expression. The CD36 gene is a target of peroxisome proliferator-activated receptor-γ (PPARγ), which belongs to the nuclear receptor family. CD47 expression in red blood cells (RBC) can decrease their phagocytosis by microglia after ICH. Although CD36 expression is increased in the ICH brain, TLR4 activation might negatively regulate its expression and delay haematoma clearance. IFGNR, IFNγ receptor; NF-κB, nuclear factor-κB; MAL, Toll/IL-1 receptor domain-containing adaptor protein (also known as MYD88 adapter-like protein); MYD88, myeloid differentiation primary response protein MYD88; SOCS3, suppressor of cytokine signalling 3; TRAM, TIR domain-containing adaptor molecule 2 (also known as TRIF-related adaptor molecule); TRIF, TIR domain-containing adaptor molecule 1.

Complement C3a and C5a receptors

Complement C3a and C5a are thought to be involved in neuro-inflammation72, neutrophil recruitment73 and blood–brain barrier disruption45. ICH-induced microglial activation, as assessed by immunoreactivity to an anti-CD11b and anti-CD11c dual-specificity antibody (OX42), was reduced in C3-deficient mice compared with wild-type controls74. In C5a receptor knockout mice, proinflammatory cytokine levels were decreased in the acute phase after ICH75. Moreover, treatment with the C5a receptor antagonist PMX53 in combination with the thrombin antagonist argatroban resulted in decreased mRNA expression of the M1 markers TNF, IL-6 and iNOS in the mouse ICH brain76. These data indicate a prominent role for activation of complement C3a and C5a receptors in the proinflammatory response to ICH.

Changes in M2 markers

Markers of M2 microglia in ICH have been considerably less well studied than M1 markers. Nevertheless, M2 microglia do have important functions in phagocytosis and toxicity clearance in other brain diseases77, such as ischaemic stroke17,78, TBI79 and epilepsy31, suggesting that the function of M2 microglia in ICH should be explored.

Anti-inflammatory cytokines

IL-10 is an anti- inflammatory M2-signature cytokine80 secreted by M2 macrophages and microglia. Treatment with IL-10 polarizes microglia and macrophages to the M2c subtype in vitro81,82, and enhances phagocytosis by monocytes83–85. IL-10 also induces production of suppressor of cytokine signalling (SOCS)1 and SOCS3, which suppress pro-inflammatory cytokine production via signal transducer and activator of transcription (STAT)3 in macrophages86,87. Interestingly, IL-10 levels in blood88–90 and brain tissue90,91 are increased in patients with ICH, as well as in preclinical models of ICH50,92,93. IL-10 levels are increased in patients during the early acute phase of spontaneous ICH, and this rise is associated with subsequent rebleeding89, although this relationship is not necessarily causal.

IL-4 (REF. 94) and transforming growth factor (TGF) β1 (REF. 60) exert anti-inflammatory effects, promote M2-like microglial responses, and improve functional recovery after ICH. Notably, the course of changes in levels of these M2 markers apparently differs between the two principal animal models of ICH. The mRNA levels of most M2 markers — such as IL-1 receptor antagonist53, IL-10 (REF. 50), arginase-1, Ym1 and CD206 (REF. 94) — increased within 1 day after collagenase-induced ICH, whereas mRNA levels of TGFβ53 and both mRNA and protein levels of IL-4 (REF. 56) were substantially increased on day 3 post-ICH. However, in mice with blood-induced ICH, IL-10 protein levels were unchanged and IL-4 was undetectable in the perihaematomal region in the first 2 weeks60. In the latter study, TGFβ1 protein levels increased from day 1 to day 10, and remained elevated up to day 14 after ICH; this time course was delayed compared with that of the M2 marker IL-13, protein levels of which increased from day 1 up to day 3 only60. Furthermore, intracerebral TGFβ1 treatment decreased microglial Il6 gene expression and improved functional outcomes in mice with blood-induced ICH60. Interestingly, an early increase in plasma levels of TGFβ1 was associated with improved functional outcome at 90 days in patients with ICH60. The authors of this study concluded that TGFβ1 is a potential target for ICH treatment.

Peroxisome proliferator-activated receptor-γ

Peroxisome proliferator-activated receptor-γ (PPARγ) belongs to a superfamily of nuclear receptors that are important contributors to antioxidant and anti- inflammatory responses95. In rats with blood-induced ICH, binding of PPARγ to DNA in nuclear extracts was suppressed at 1 h post-ICH, and treatment with PPARγ agonists had beneficial effects on ICH owing to their ability to reduce NF-κB activation96 and decrease M1 signature cytokine levels (specifically, iNOS, TNF and IL-1)97. In mice with blood-induced ICH, treatment with a PPARγ activator promoted microglial phagocytosis of red blood cells in vitro by inducing CD36 expression97,98. This evidence supports a potential role for PPARγ activation in haematoma clearance99. The mechanisms of microglial phagocytosis and haematoma resolution after ICH are discussed in more detail below.

Mammalian target of rapamycin

Dysregulation of serine/threonine-protein kinase mTOR (mammalian target of rapamycin) activation occurs in various brain diseases, notably Alzheimer disease100, Parkinson disease101,102 and TBI103. In a rat model of subarachnoid haemorrhage104, rapamycin and another mTOR inhibitor, AZD8055, ameliorated early brain injury by inhibiting production of IL-1β and TNF. Rapamycin and AZD8055 also decreased the CD16:CD206 ratio in oxyhaemoglobin-treated primary microglia in vitro104. In rats with collagenase-induced ICH, phosphorylation of mTOR was markedly increased at 30 min. Treatment with rapamycin(50–500 μg/kg) led to a dose-dependent increase in brain levels of the M2 cytokines IL-10 and TGFβ, as well as in the ratio of IL-10 to IFNγ, after ICH105. In a separate study that used the same model106, rapamycin treatment reduced protein levels of TNF, IL-1β and IL-6. This evidence suggests a link between mTOR inhibition and microglial M2 polarization.

Other M2 markers

Upregulation of the M2 marker CD163 is observed in the brains of patients with ICH within 3 days of onset91. In mice with blood-induced ICH, global CD206 and Ym1 protein expression (detected by western blot) was increased at day 1 (REF. 107). Although our group showed that Ym1 expression was increased in microglia at 24 h and 72 h in both the thrombin-induced and collagenase-induced models of ICH62,108, the dynamics of changes in microglial M2 markers in the collagenase-induced ICH model are still unclear. Data from the limited number of available studies reviewed here60,62,107 suggest that microglial M2 markers increase by day 1 — later than M1 markers, which start to increase by 3–6 h — and remain elevated longer (that is, beyond day 7) than do M1 markers (FIG. 1).

M1 to M2 transition

Very little is known about the dynamics of changes in microglial polarization after ICH13,59. Two studies revealed changes over time in M1-like and M2-like markers in models of ICH94,107. We62 and others60 also demonstrated a similar M1 to M2 transition in both the collagenase-induced62 and blood-induced60 models of ICH. In mice with collagenase-induced ICH, we observed an M1 to M2 microglial phenotype switch from day 1 to day 3 after ICH62, whereas in mice with blood-induced ICH, the microglial phenotype switch occurred within the first week60. However, this transition could result from phenotypic transformation of a single microglial population, from M2-like microglial migration, or from infiltration of M2-like circulating blood monocytes or macrophages. These possibilities deserve further study.

In summary, by about 3 days after ICH (FIG. 1), microglia polarize toward an M1-like phenotype associated with molecules involved in short-term brain damage. Indeed, M1 microglial polarization might be the main cause of microglial activation in the acute phase after ICH, whereas M2-like microglial responses and mediators might have an important role in long-term recovery, especially in aged animals (TABLE 2). Interestingly, ageing in the nondiseased brain is associated with an overall increase in the expression of microglial genes involved in neuroprotection109. Data relating to the M2-like response also need to be expanded; in particular, the role of M2-like microglia in the subacute and chronic phase of ICH requires clarification. As some M2 surface markers and cytokines are not specific to microglia and macrophages, they should be assessed by double or triple immunofluorescence staining with microglia-specific and macrophage-specific markers. The mRNA and protein expression of these markers can be detected using fluorescence-activated cell sorting of microglia and macrophages. Unbiased genome-wide approaches such as transcriptomics and proteomics can also be applied.

Phagocytosis and haematoma clearance

Under physiological conditions, microglia act as phagocytes to scavenge plaques, neurofibrillary tangles and cell debris in the CNS. Haematoma clearance by microglia might be a major mechanism of recovery from ICH13,110–112. We previously showed that tissue plasminogen activator in microglia assists in the clearance of haematoma36.

CD36 belongs to the class B scavenger receptor family and also contributes to the phagocytic ability of microglia and macrophages113–116; cells lacking phagocytic ability acquire this capacity after being transfected with CD36 (REF. 116). In both patients and animals with ICH, haematoma absorption is increased by CD36 expression97,98,111,115. CD36 is also a pivotal target of PPARγ-mediated downstream gene transcription98. As discussed above, activation of PPARγ increases CD36 expression and thereby promotes microglial phagocytic ability, whereas CD36 expression is downregulated by TLR4 signalling in microglia and monocytes117,118. In a comparison of TLR4-deficient and wild-type mice with blood-induced ICH, perihaematomal regions in the TLR4-deficient mice showed fewer activated microglia and higher expression of CD36 and fractalkine (also known as CX3C motif chemokine 1 (CX3CL1))69. In _CD36_−/− mice with blood-induced ICH, IL-1β and TNF mRNA expression levels were higher than those in wild-type mice. The capacity of CD36−/− microglia from these mice to phagocytose red blood cells in vitro was also decreased, along with a complementary increase in IL-10 levels115. The authors concluded that CD36 deficiency led to TLR4 upregulation, suggesting that mediators downstream of the TLR4 pathway reduced microglial phagocytosis and slowed haematoma absorption by decreasing CD36 expression and IL-10 production115. In an in vitro study, Tlr4−/− microglia exposed to lysed erythrocytes exhibited reductions in autophagy and inflammation compared with similarly treated wild-type microglia71. This finding suggests that TLR4-mediated autophagy contributes to microglial polarization and function. However, whether IL-10 regulates the expression of CD36 and TLR4, thereby modulating phagocytosis of red blood cells and promoting haematoma resolution, remains unclear.

CD47 is an integrin-associated protein expressed on erythrocytes119,120 that regulates phagocytosis in microglia and macrophages121,122. In a study that compared the effects of using blood from either wild-type mice or Cd47−/− mice to induce ICH in wild-type mice, the mice receiving CD47-depleted blood showed increased expression of haem oxygenase-1 (HO-1) and accelerated haematoma clearance, in a manner that correlated with M2 microglial activation123.

Interestingly, levels of the haemoglobin scavenger receptor CD163 increased over time in parallel with haematoma resolution in piglets with blood-induced ICH110, indicating a potential role for CD163 in haematoma clearance. These observations indicate that proteins capable of modulating microglial phagocytosis (for example, IL-10, CD36, CD47 and CD163) can affect haematoma resolution (FIG. 2) and deserve further research. Identification of new targets that can regulate microglial phagocytosis and haematoma clearance might lead to the development of future therapeutic interventions for ICH.

Modulators of microglial polarization

Transcription factors

Although markers of M1 and M2 microglia and macrophage polarization are readily detectable, these proposed activation states are complex and their relevance to macrophage function in vivo is controversial124. Additionally, the signalling pathways that cause secondary damage after ICH are complex. Investigating the activation of transcription factor responses to brain injury is, therefore, important to our understanding of microglial M1 to M2 transition after ICH. However, very few studies have investigated transcription factors other than NF-κB after ICH.

NF-κB

NF-κB is a traditional transcription factor that is activated by lipopolysaccharide and regulates the expression of most M1-signature genes (those encoding proinflammatory cytokines)125. In human brain tissue, NF-κB p65 expression was detected in the nuclei of glial cells by immunochemistry126 and was associated with increases in IL-1β and TNF48. As discussed above, upregulation of TLR2 and TLR4 (which are expressed mainly in microglia) induces NF-κB activation after ICH, and increases proinflammatory cytokine levels54,65.

Signal transducer and activator of transcription proteins

The STAT family of transcription factors (STAT1–STAT6) are, like NF-κB, activated by lipopolysaccharide. The Janus activated kinase (JAK)–STAT pathway is activated by both type I and type II interferons and thereby regulates cellular proliferation, immunity, apoptosis, and inflammation127,128. However, few studies have investigated the roles of STAT proteins in ICH.

STAT family members have various roles in microglial and macrophage polarization (FIG. 2). STAT1 controls M1 macrophage polarization129, and in vitro, STAT1 levels increase in TLR3-stimulated and TLR4-stimulated microglia130. STAT1 activation also induces expression of genes encoding IL-1β, TNF and CXC motif chemokine 10 (CXCL10) in _Cryptococcus neoformans_-infected macrophages131. Conversely, loss of STAT1 in macrophages led to an increase in polarization to the M2 phenotype131. SOCS3 is a feedback inhibitor that limits excessive cytokine release resulting from persistent activation of STAT3; in vitro, SOCS3-deficient primary microglia showed increases in STAT3 activation and proinflammatory cytokine signalling, which led to their pronounced polarization to the M1 phenotype132. This polarization was associated with upregulation of iNOS, IL-1β, IL-12 p40, IL-23 p9, IL-6, CC-motif chemokine 2 (CCL2), and CXCL10 (REF. 132). In mice with collagenase-induced ICH, phosphorylated STAT3 is found mainly in microglia and macrophages, and inhibition of this molecule is associated with decreases in iNOS and cyclooxygenase-2 expression133. In this same model, levels of phosphorylated STAT3 and IL-6 decreased in Tlr4−/− and MYD88−/− mice on day 3 after induction of ICH67, suggesting that STAT3 is an important transcription factor downstream of TLR4 that mediates M1 microglial polarization in ICH. However, STAT6 became activated in microglia challenged with IL-4, which promoted microglial M2 polarization134–136. Thus, the roles of STAT proteins in ICH pathology need additional clarification.

Nuclear factor erythroid 2-related factor 2

Activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and its related signalling pathway exerts a protective effect after ICH and also contributes to haematoma clearance137,138. _Nrf2_−/− mice show more brain damage than do wild-type mice at 24 h after collagenase-induced ICH, although microglial activation status in these mice, as shown by immunostaining for allograft inflammatory factor-1 (AIF1, also known as Iba1), is not altered by the loss of Nrf2 (REF. 137). In vitro, microglial phagocytosis of red blood cells labelled with a fluorescent dye was substantially increased by Nrf2 activators such as sulforaphane139. Moreover, mRNA level of the scavenger receptor CD36 was upregulated in microglia treated with an Nrf2 activator111.

Interestingly, mRNA levels of HO-1, CD36, CD163, IL-10 and IL-1β did not change appreciably in _Nrf2_−/− mice compared with their levels in wild-type mice at 48 h after blood-induced ICH111. HO-1 deletion decreased the phagocytic ability of microglia in vitro and delayed haematoma clearance in an in vivo model of subarachnoid haemorrhage140. Additionally, HO-1 activity is elevated in the cerebrospinal fluid of patients with subarachnoid haemorrhage140, suggesting a critical role for microglial Nrf2 and HO-1 in haematoma clearance. Moreover, induction of HO-1, one of the major downstream targets of Nrf2, polarizes macrophages to the M2 phenotype141. Whether Nrf2 transcriptional activation and downstream molecules also modulate microglial polarization and function after ICH deserves further investigation.

High mobility group protein 1

Though not itself a transcription factor, high mobility group protein B1 (HMG1) is a DNA-binding protein that regulates gene transcription through its interactions with nucleosomes, transcription factors and histones142–144. Crucially, HMG1 is involved in proinflammatory cytokine gene transcription143 and is secreted by haem-stimulated microglia in vitro145. Serum levels of HMG1 are increased in patients with ICH, and correlate with levels of IL-6 and TNF, as well as with NIH Stroke Scale scores145. This evidence indicates that microglial HMG1 has a role in ICH. In addition, treatment with the nonspecific HMG1 inhibitors glycyrrhizin146 and ethyl pyruvate147 attenuates ICH injury and improves early outcomes in rodent models of ICH. Our group has shown that expression of HMG1 protein is substantially increased at 24 h after ICH39,148. HMG1 also promotes macrophage M1 polarization via increased TLR2 and TLR4 signalling, which contributes to cardiac remodelling in mice prone to accelerated senescence144. Thus, the beneficial effects of HMG1 inhibitors in rodent models of ICH might result from attenuation of microglial activation and inhibition of TLR2 and TLR4 (REF. 149).

Prostaglandin E2 receptors

Prostaglandin E2 (PGE2) is a major product of cyclo-oxygenase and prostaglandin E synthase, and is thought to be a classic proinflammatory mediator within the brain. PGE2 activates its downstream signalling pathways via the G-protein-coupled E prostanoid (EP) receptors EP1–EP4. EP1 receptors are expressed in microglia, and EP1 deletion dampens microglial activity and phagocytosis150. Furthermore, treatment with the EP1 receptor antagonist SC51089 decreases microglial activation and attenuates grey and white matter injury in mouse models of ICH39. Interestingly, in mice with collagenase-induced ICH, deletion of EP2 and EP3 produces opposite effects on ICH outcomes that correlate with microglial activation status148. Although EP2 is expressed in neurons rather than in microglia, deletion of EP2 led to an increase in M1-like microglial activation, indicating a potential EP2-mediated interaction between neurons and microglia148. By contrast, EP3 receptors are expressed in microglia108, and EP3 deletion caused decreased micro-glial activation in mice with collagenase-induced ICH151. Similarly, treatment with an EP3 antagonist inhibited microglial activation in mice with thrombin-induced ICH108, although this treatment increased the number of M2 microglia (as shown by CD11b and Ym1 double immunostaining)108. Finally, the EP2 and EP4 dual agonist misoprostol provided protection and reduced the proinflammatory response in mice with either collagenase-induced or blood-induced ICH152. Together, these results support a potential role for PGE2 and EP receptors in modulation of microglial function after ICH.

Sphingosine-1-phosphate receptors

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that exerts a wide range of effects on cells via its five known receptors (S1PR1–S1PR5) in the brain153,154. The S1PR agonist fingolimod was approved by the FDA in 2010 for the treatment of multiple sclerosis (MS). Microglia express all five S1PRs in vitro155,156, and fingolimod treatment reduced proinflammatory cytokine levels in lipopolysaccharide-induced M1-like micro-glia156,157. In vivo, fingolimod decreased the production of IL-1β and TNF in animal models of status epilepticus158 and neonatal hyperoxia159. In an animal model of MS, microglia exhibited a reduced M1-like response and an enhanced M2-like response at day 120 with fingolimod treatment160. In rodents with collagenase-induced ICH, fingolimod treatment improved functional outcomes161,162 and reduced levels of IFNγ after ICH162, and also inhibited M1-like responses by preventing T-lymphocyte infiltration into the brain162. Notably, fingolimod also accelerated haematoma clearance and improved clinical outcomes in patients with ICH163, strongly indicating the importance of S1PRs in clinical ICH. However, another group found no effects of fingolimod treatment on haematoma volume and inflammatory cells in rodents with collagenase-induced ICH164. Thus, additional studies are needed to determine the role of S1PRs and whether they mediate microglial polarization after ICH. Currently, fingolimod is being investigated in a phase II clinical trial as a treatment for ischaemic and haemorrhagic stroke165.

Microglial interactions with other cells

T lymphocytes

In patients with ICH, the frequency of both activated (CD3+CD9+) and regulatory (CD4+CD25+FOXP3+) T cells (Treg cells)166,167 was increased in the peripheral blood of patients from day 3 to day 7 after ICH90. In mice with blood-induced ICH, Treg cells45 and γδ T cells168 infiltrated the brain starting at day 4, and their levels remained elevated until day 7; furthermore, these Treg cells inhibited M1 microglia in vitro169 and in vivo170. In a mouse model of blood-induced ICH, our group found that these Treg cells promoted M2-like polarization of microglia and macrophages via the IL-10–glycogen synthase kinase-3β signalling pathway45. These data all support the premise that T-cell infiltration into the brain after ICH enhances M1-like responses of microglia and macrophages, whereas Treg cells in the brain contribute to the microglial M1 to M2 phenotype shift.

Neurons

The signalling pathway involving soluble CX3CL1, which is expressed on neurons, and its receptor CX3C chemokine receptor 1 (CX3CR1), which is expressed on microglia, mediates interactions between microglia and neurons, thereby modulating microglial activation171,172. Mice engineered to express CX3CR1 labelled with green fluorescent protein have been widely used to study microglial activation and function39,62,108,173–175. For example, after ischaemic stroke, CX3CL1 is released by neurons and participates in microglial activation via CX3CR1 (REFS 175–177), and mice deficient in CX3CL1 have a reduced susceptibility to cerebral ischaemia–reperfusion injury178. However, the roles of CX3CL1 and CX3CR1 in ICH have not been well studied.

Both CX3CL1 and CX3CR1 are expressed in the brains of patients with ICH179. In Tlr4−/− mice with blood-induced ICH, mRNA levels of both CX3CL1 and CD36 were increased compared with their levels in wild-type mice69. Moreover, mice with monocyte-specific CX3CR1 deficiency showed no defect in functional recovery after blood-induced ICH180, suggesting that neuron-secreted CX3CL1 is involved in microglial phagocytosis and functional recovery after ICH, but that monocyte recruitment after ICH is independent of signalling via CX3CR1 (REF. 180). Thus, interactions between neurons and microglia, mediated by CX3CL1 binding to CX3CR1, might be involved in the dynamic regulation of microglial phenotype after ICH.

Astrocytes

The initial observation, in 2005, that microglial processes and protrusions directly contact astrocytes, suggested an important link between these two cell types in the healthy brain181. In the CNS, astrocytes secrete proinflammatory (IL-6 and IL-1β) as well as anti-inflammatory (IL-10) cytokines182. Furthermore, astrocytes are major sources of many chemokines, such as CCL2, CXCL1, CXCL10 and CXCL12 (REFS 183,184), which might have roles in microglial differentiation. Several chemokines have been implicated in macrophage activation and polarization185. In patients with ICH, chemokines such as CCL20 (REF. 186), CXCL1 (REF. 186), CXCL2 (REF. 187), CXCL3 (REF. 187) and CCR1 (REF. 187) are upregulated in perihaematomal brain regions, indicating a potential role for these chemokines in ICH pathology. Some chemokines and their receptors have also been studied in animal models of ICH188. In rats with blood-induced ICH, gene expression microarray studies showed that, 24 h after ICH, Cxcl2 was highly upregulated in the striatum and cortex189. Moreover, the neuroprotective effect of retinoic acid receptor agonist AM80 in mice with collagenase-induced ICH is attributable to inhibition of Cxcl2 mRNA expression51.

Few studies have assessed M1 and M2 chemokine markers in ICH. However, in mice with collagenase-induced ICH, mRNA expression of the M1 marker Cxcl1 was increased at 6 h but not at 24 h after ICH, compared with its expression in control mice after sham ICH51. Astrocytes also participate in microglial M1 to M2 phenotype switching. In hyperammonaemic rats, sulforaphane promoted a switch in microglial polarization from the M1 to the M2 phenotype by decreasing membrane expression of sodium-dependent and chloride-dependent GABA transporter GAT3, mainly in activated astrocytes190.

In an in vitro study, CCL2 released from primary astrocytes contributed to M1 microglial polarization191. In mice with collagenase-induced ICH, a lack of CCL2 or CCR2 decreased the haematoma volume early after collagenase-induced ICH but resulted in delayed recovery from ICH112; these changes correlated with decreases in microglial activation and iNOS expression112. Ccr2−/− mice with blood-induced ICH also exhibited an early improvement in motor function and fewer M1 micro-glia180. A high CCL2 level at 24 h correlated with poor outcomes at day 7 in patients with ICH180.

The limited research available suggests that interactions between microglia and astrocytes can be either beneficial or harmful to neurons, but the crosstalk between microglia and astrocytes has not been fully investigated. Further study of astrocytic mediators that modulate microglial polarization, phagocytosis and other functions would improve our understanding of ICH pathology and facilitate translational research.

Oligodendrocytes

During the first 2 weeks after collagenase-induced ICH in rats, oligodendrocyte precursor cells and mature oligodendrocytes proliferate and differentiate within white matter tracts of the perihaematomal region192. In MS, M2 microglia promote oligodendrocyte proliferation and differentiation during the remyelination period, which is associated with functional recovery193. After ICH induced by injection of sonicated whole blood, haptoglobin was secreted from oligodendrocytes at 24 h and protected a mixed population of brain cells (neurons, astrocytes, and microglia) from the effects of haemoglobin toxicity194. However, our knowledge of how microglial polarization affects oligodendrocyte function is very limited.

Clinical and translational implications

Currently, several promising anti-ICH drugs are undergoing clinical trials (TABLE 3). Most exert neuroprotective effects by targeting M1-like microglia in the acute phase after ICH. Inhibition of M1-like microglial activation and enhancement of M2-like microglial anti-inflammatory responses are the major underlying mechanisms of these compounds. Additional drug candidates that target microglial polarization have demonstrated efficacy for ICH treatment in preclinical studies (TABLE 3). Although in this Review we have emphasized the importance of M2-like microglial responses (especially in the late recovery stage of ICH), treatment strategies for ICH that focus on modulating or enhancing M2-like microglia are still limited. As discussed above, rapamycin (at a low dose of 150 μg/kg) increased levels of M2-like anti-inflammatory cytokines IL-10 and TGFβ105. Sinomenine is a dextrorotatory morphinan analogue extracted from herbs used in traditional Chinese medicine195,196, and has long been used clinically for treating rheumatoid arthritis in China197. In vitro, sinomenine reduced levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF in ICH-exposed BV-2 microglia198 and increased levels of M2-like markers IL-10 and Arg1 in primary microglia exposed to erythrocyte lysate199. Sinomenine increased levels of IL10 and Arg1 mRNA in mice with blood-induced ICH199. The complicated changes in M2 phenotype microglia, as discussed above, probably make M2-like microglia a more challenging target than M1 microglia.

Table 3.

Promising anti-ICH drugs that target microglial polarization

The effects of stem cell transplantation on ICH are also now beginning to be explored in clinical trials. Preclinical studies in rats with collagenase-induced ICH showed that transplantation of mesenchymal stem cells (MSCs) obtained from umbilical cord blood improved ICH outcomes by inhibiting the M1 markers cyclooxygenase-2 and TNF200. Pneumostem, a novel agent based on human MSCs obtained from umbilical cord blood, is currently under investigation in a phase I clinical trial as a treatment for premature infants with intraventricular haemorrhage201.

Despite decades of research, effective drug treatments for ICH are still lacking. However, with the expanding investigation of microglial polarization and function in animal models of ICH, therapies that target microglial phenotype switching might soon become a clinical reality for patients affected by ICH.

Conclusions

Microglial activation has long been recognized as important in the pathology and progression of ICH6,202,203. The evidence indicates that after becoming activated, microglia undergo polarization into various phenotypes that might contribute differently to neuroinflammation in models of brain disease. Although very little is known about the dynamics of microglial polarization specifically after ICH, modulation of microglial function might be expected to mitigate ICH-related brain injury, thereby promoting tissue repair and functional recovery13. In this regard, some promising drugs strongly inhibit M1-like micro-glia, whereas others are thought to enhance microglial M2-like responses. However, both M1-like and M2-like microglia have critical roles in tissue repair at different stages after brain injury, suggesting that persistence of an M2-dominant state after ICH might impair innate immune responses and result in serious adverse effects, such as neoplasia204. Appropriate anti-ICH treatment, therefore, should aim to boost the correct microglial phenotype at the correct time, so as to maximize the natural processes of haematoma clearance and brain repair.

Considering the limitations of existing animal models of ICH and the numerous failed attempts to translate promising experimental treatments for ischaemic stroke into clinical use, studies of histopathological material from patients with ICH49 are critically needed to confirm the findings from studies in rodent models. Such a step is vitally important for future translational studies. Applications of transcriptomic, epigenomic and proteomic studies, and animal models employing conditional targeting of microglial genes, are expected to move the microglial research field forward and improve our understanding of the underlying mechanisms of ICH, potentially leading to effective treatments for affected patients.

Key points.

• Microglial polarization after intracerebral haemorrhage (ICH) modulates microglial phagocytic function and might affect haematoma clearance
• Activation of microglia to an M1-like phenotype occurs mainly in the acute phase after ICH
• M2-like microglial responses occur in the subacute and chronic phase and might contribute to phagocytosis of cell debris and haematoma clearance
• Microglial polarization can be regulated by transcription factors, chemokines, receptors and their signalling pathways, and interactions between microglia and other cells in the brain (T lymphocytes, neurons, astrocytes and oligodendrocytes)
• Data from clinical trials and preclinical studies suggest that targeting of microglial phenotype switching represents a new research direction for ICH treatment

Acknowledgments

The authors’ research work was supported by American Heart Association (AHA) Mid-Atlantic Affiliate Grant-in-Aid 13GRNT15730001, NIH grants R01NS078026 and R01AT007317, and a Stimulating and Advancing ACCM Research (StAAR) grant from the Department of Anesthesiology and Critical Care Medicine (ACCM), Johns Hopkins University (to J.W.), and an AHA Mid-Atlantic Affiliate Postdoctoral Fellowship Award 15POST25090114 (to X.L.). The authors thank Claire Levine, MS, ELS, for assistance with manuscript preparation.

Footnotes

Author contributions

X.L., X.H., and Q.L., researched the data for the article. X.L., Q.-W.Y., and J. W. provided substantial contributions to discussions of the content. X.L. and J.W. wrote and revised the manuscript before submission.

Competing interests statement

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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