Neuroscience Letters 709 (2019) 134370
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Role of microglia in spinal cord injury a,b,c,⁎
Antje Kroner a b c
, Jose Rosas Almanza
Department of Neurosurgery, Medical College of Wisconsin, Milwaukee, WI, United States Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI, United States Clement J. Zablocki Veterans Aﬀairs Medical Center, Milwaukee, WI, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Spinal cord injury Microglia Macrophage Astrocyte
Myeloid cells are important eﬀector cells in the injured spinal cord tissue. Microglia and monocyte-derived macrophages serve important functions in the injured spinal cord, and their distinctive roles can now be studied more eﬃciently with the help of reporter mice and cell speciﬁc markers that were described in recent years. Focusing on microglia, this review discusses the microglial response to injury, microglia speciﬁc eﬀects and the interaction between microglia and other cell types in the injured spinal cord.
1. Introduction Spinal cord injury (SCI) is a severe and life-altering condition that leads to loss or impairment of motor and sensory function below the injury level, in addition to impacting autonomic function . The SCIassociated tissue damage is characterized by cell death, axonal loss, myelin degradation, immune cell inﬁltration and activation, and disruption of the blood-spinal cord barrier [53,17,46]. The primary tissue damage caused by the mechanical trauma is further complicated and enlarged by a cascade of secondary events that can occur over a prolonged period of time possibly even months after injury. A diverse range of factors contributes to this secondary damage, including but not limited to inﬂammation, hemorrhage, ischemia, edema, production of reactive oxygen species, lipid peroxidation and excitotoxicity . In this review article, we explore the inﬂammatory response after SCI, with a focus on microglial cells. 1.1. Inﬂammation after SCI Microglia are the resident immune cells of the central nervous system (CNS), which originate from yolk sac cells populating the CNS during the embryonic phase . They should be distinguished from monocyte-derived macrophages (MDMs) that enter the CNS from the peripheral blood after injury as well as from macrophages that populate meninges or the perivascular space in healthy or pathological conditions . These myeloid cells are important responders to early CNS damage having both detrimental and beneﬁcial functions . Activated microglia and MDMs contribute to secondary damage by releasing proinﬂammatory mediators like reactive oxygen species and pro-
inﬂammatory cytokines [5,8,33], but both cell types also confer beneﬁcial eﬀects. These include protective eﬀects that are mediated by lesion containment , debris clearance  and production of antiinﬂammatory factors such as IL-10 . In the healthy CNS, microglia have a branching morphology, low immune activity and cytokine production. This homeostatic phenotype does not prevent their constant surveillance of the microenvironment by the motility of their cytoplasmic processes [7,38], which allows microglia to react rapidly to microenvironment perturbations. In the event of an injury, microglia are the ﬁrst cell type to respond. They become activated within minutes and extend cytoplasmic processes towards the lesion, as shown by two-photon live imaging after cortical injury . This reaction is protective by containing the lesion size . The early microglial response is mediated by ATP released from damaged cells, which is detected by P2Y G-protein coupled receptors on microglia . Upon activation, microglia undergo a change in morphology towards a rounded, amoeboid cell body with short, plump processes strongly resembling MDM morphology. Pro-inﬂammatory cytokines and chemokines like IL-1β, TNF, IL-6, CCL2 and CCL3 are produced within minutes after injury by neurons, astrocytes and microglia [10,41]. In parallel, damage-associated molecular patterns (DAMPs) are recognized by pattern recognition receptors (PRR). This is a typical occurrence in tissue injury when factors that are sequestered from PRRs under normal conditions, either intracellular or in the extracellular matrix, become exposed . Diﬀerent classes of PRR have been described to play important and distinct roles after SCI. To name a few, activation of Toll-like receptors (TLRs), particularly TLR2, can induce NF-κB activation, although not necessarily exerting neurotoxic eﬀects. Activation of C-type lectin
Corresponding author at: Department of Neurosurgery, Medical College of Wisconsin, Milwaukee, WI, United States. E-mail address: [email protected]
https://doi.org/10.1016/j.neulet.2019.134370 Received 7 January 2019; Received in revised form 3 July 2019; Accepted 4 July 2019 Available online 05 July 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
Neuroscience Letters 709 (2019) 134370
A. Kroner and J. Rosas Almanza
donors or recipients in bone marrow chimera experiments. As irradiation eradicates peripheral immune cells but not microglia, diﬀerentiation between microglia and MDMs can be achieved by transplanting wild type recipients with CX3CR1+/GFP bone marrow (GFP + MDMs) or vice versa for GFP + microglia. However, irradiation itself can inﬂuence the presence and function of immune cells in the CNS . To avoid this limitation in chimeric mice, CX3CR1+/GFP mice have been crossbred with CCR2+/RFP mice, which show red ﬂuorescent labeling of only MDMs [14,56]. It is important to consider that possible changes in expression levels could impact the reliability of transient markers. Lineage tracking of immune cells avoids this problem by stable labeling of target populations. A particularly useful mouse line to distinguish between MDMs and microglia is the CX3CR1CreER mouse line. These mice express Tamoxifen inducible Cre under the CX3CR1 promotor, which is widely expressed by mononuclear phagocytes. When crossbreeding CX3CR1CreER mice with mice expressing a ﬂuorescent protein like TdTomato (TdT) under the ubiquitous Rosa26 promotor (CX3CR1CreER::R26-TdT), tamoxifen treatment will initially induce labeling of all microglia and MDMs. However, while microglia have a slow turnover time and are persistently labeled, monocytes in the peripheral blood, spleen and bone marrow, have a rapid turnover, resulting in a loss of ﬂuorescent labeling in these cells [3,57]. This allows for the distinction of macrophages and microglia, independent of marker expression levels.
receptors (CLRs) such as dectin-1, in contrast, triggers macrophage activation and axonal injury . High mobility group box 1 protein (HMGB1) is another potent DAMP that is upregulated after SCI and associated with microglial activation and neurotoxic macrophage polarization . HMGB1 can bind to receptors for advanced glycation end products (RAGE), a PRR, resulting in inﬂammatory expression of IL-1β and TNF after SCI . The expression of pro-inﬂammatory molecules, such as cytokines, cyclooxygenase-2, inducible nitric oxide synthase (iNOS) and matrix metalloproteinases [26,28] accompanied with assembly and activation of inﬂammasomes , contribute to the inﬂux of immune cells from the periphery into the spinal cord lesion. Similar to other injured tissues , neutrophils are the ﬁrst cell type to inﬁltrate the injured spinal cord. They tend to peak around one day after injury, followed by a quick decline in numbers . Various reports have identiﬁed mechanisms to target neutrophils or early invading monocytes as a promising treatment approach after SCI [25,31,36]. However, other studies have reported impaired recovery and increased tissue damage when neutrophils and monocytes were depleted . MDMs start invading the injured spinal cord approximately 2–3 days after injury  and peak around day 7–10 after injury. While MDM numbers reduce over time, the resolution is incomplete . MDMs remain in the tissue for prolonged periods of time. In humans, phagocytic macrophages are detectable up to one year after injury [15,42,44]. This prolonged presence of activated MDMs and microglia in the injured tissue is a risk factor for ongoing tissue damage. Historically, the inﬁltration of myeloid cells from the circulation has made it diﬃcult to diﬀerentiate between microglia and MDMs. As previously discussed, the amoeboid appearance microglia approach during activation render morphology-based approaches ineﬀective in distinguishing activated microglia from MDMs.
1.3. Distinct roles of microglia after SCI 1.3.1. Interaction with axons Investigating the diﬀerential roles of microglia and MDMs is a recurring search for the culprit in axonal damage after SCI. A study from Jerry Silver’s lab used two-photon microscopy in a set of reporter mice with ﬂuorescently tagged axons, microglia or macrophages to assess the role of myeloid cells on axonal dieback after dorsal column crush injury . The contact between CX3CR1+ myeloid cells (microglia or MDMs) and axons was essential for the occurrence of axonal damage, deﬁned by thinning or complete severance of the axon, which occurred in about 10% of total contacts. This observation is supported by the ﬁnding that deﬁcient CX3CR1 signaling promotes recovery after SCI . Bone marrow chimeric mice were used to diﬀerentiate between MDMs and resident microglial cells revealing striking diﬀerences in the behavioral properties of both cell types: macrophages moved twice as fast as microglia and were responsible for all destructive contacts with axons. Interestingly, the total number of contacts between MDMs and axons was stable between two and eight days after injury, despite the strong increase in MDM numbers . It is worth noting that the chimeric mice used in this paper were at minimum 18 weeks old, which is twice the age of mice typically used in other studies, potentially resulting in diﬀerences in the observed immune response (Fig. 1).
1.2. Diﬀerentiation between MDMs and microglia Approaches previously used to diﬀerentiate microglia and MDMs have included determining the expression level of the pan-leukocyte marker CD45 by ﬂow cytometry. MDMs and microglia both express CD11b+ (Integrin α M) but diﬀerent levels of CD45 (MDMs: CD11b + CD45high; microglia: CD11b + CD45low). However, activated microglia can also upregulate CD45 . In recent years, a range of microglia speciﬁc or enriched markers have been described, thereby opening better possibilities to study the speciﬁc role of microglia. Markers that have been described as microglia speciﬁc or enriched in the naïve CNS include P2RY12, Tmem119, FCRLS and Sall1. Importantly, not all markers are statically expressed in the context of injury and inﬂammation. P2RY12 expression, for example, is lost early after SCI [23,3] and in active multiple sclerosis (MS) lesions . For a more detailed examination of these markers and their expression under healthy or pathological conditions, please see  for review. The use of reporter mice oﬀers an elegant option to distinguish between microglia and MDMs. Various reporter mice have been used in the context of spinal cord injury. The lysozyme M (LysM)- enhanced green ﬂuorescent protein (EGFP) reporter mouse, which expresses eGFP in MDMs and neutrophils, but not in resident microglia, has been demonstrated to be a useful model for diﬀerent pathological conditions of the CNS, including SCI . Used in combination with FCRLS and CD11b/CD45 expression levels, this model allows for a clear distinction between MDMs (EGFP+) and microglia (EGFP-) [9,23]. It is also important to note that this mouse model is diﬀerent from the LysM-Cre mouse, which can be used for gene deletion in microglial cells [21,55]. Microglia express low levels of LysM, especially during development , which is suﬃcient to induce gene deletion, but not suﬃcient to induce robust EGFP expression in microglia. In another example, in CX3CR1+/GFP mice, both microglia and MDMs express green ﬂuorescent protein (GFP), making them useful
1.3.2. Phagocytosis Using the LysM-EGFP model discussed above, the phagocytic activity of microglia and MDMs after SCI has been assessed . Phagocytosis of cell debris is a prerequisite for recovery after injury. In the ﬁrst days after injury, microglia are the main phagocytosing cell type that also come in close contact with damaged axons. When MDMs enter the injured spinal cord, they establish contact with damaged axons and take over most of the phagocytic activity by day 14. Interestingly, the phagocytosed material persists in macrophages for the entire observation period (42 days) with only transient detection in microglia. Reasons for this could be disproportional cell death in microglia or more eﬃcient processing of the phagocytosed material in microglia. Cell death after phagocytosis of myelin and cell debris was by far more frequent in MDMs compared to microglial cells both in vitro and in vivo, thereby suggesting more eﬃcient processing of phagocytosed material as a better explanation . An alternative explanation could be that 2
Neuroscience Letters 709 (2019) 134370
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Fig. 1. This summary ﬁgure describes distinct microglial functions in the injured spinal cord and interactions with other cell types.
formation. This inhibitor has also been previously shown to aﬀect primarily microglia in models of neurodegenerative diseases where it had a pronounced neuroprotective eﬀect . However, it is not entirely clear to what extent MDM proliferation is also inhibited or how this would inﬂuence the observed amelioration in recovery. In contrast, two recent studies out of the Lacroix  and the Popovich lab  administered PLX5622, a CSF1R inhibitor  that crossed the blood brain cord barrier and depleted 98% of all spinal cord microglia in uninjured mice . Based on assessments of the peripheral immune compartment as well as the use of CX3CR1CreER::R26TdT::LysM-eGFP mice, microglia but not MDMs are depleted by PLX5622 . Both studies report that microglia depletion causes impaired locomotor recovery in mice with thoracic contusion or lumbar crush injury. The timing of microglia depletion is critical, however. The strongest eﬀects were seen if PLX5622 chow was given two to three weeks prior to SCI and continuously thereafter [3,4]. Bellver-Landete et al. describe milder impairment when mice were gavage fed with PLX5622 starting at the time of the injury. No diﬀerence was detected when PLX5622 containing chow was discontinued at the time of injury or three days later, indicating that proliferating microglia are crucial in the ﬁrst week post-SCI . Brennan et al. report a slightly diﬀerent critical time window: they detected impaired functional recovery when microglia were depleted between day 8 and day 28, but not when treatment that was given prior to injury until day 7 after injury or very late (day 29- day 56) . After injury, microglia proliferate extensively, accumulate around the lesion site and form a dense interface with GFAP + astrocytes while LysM + cells, derived from the blood, predominantly populate the lesion area. CX3CR1CreER::R26-TdT::LysM-eGFP mice were used to identify diﬀerent cell types . Microglia depletion using PLX5622 results in a disruption of this astrocyte-immune interface, with reduced astrocyte proliferation and orientation and diﬀuse distribution of MDMs in the surrounding tissue [3,4], which is partly mediated by IGF-1. This phenomenon is accompanied by increased lesion sizes and loss of neurons and oligodendrocytes . While there seems to be a discrepancy between the reported improvement after use of the CSF1R inhibitor GW2580  and the deterioration and increased tissue damage in PLX5622 treated mice [3,4], the outcomes could be explained by diﬀerences in the function of the inhibitors (targeting of diﬀerent cell populations and inhibition of
microglia reverse to a more anti-inﬂammatory phenotype at later time points after injury . 1.3.3. Interaction with MDMs In deﬁning the role of microglia after SCI, it becomes increasingly important to consider cellular functions not in isolation but in the context of interactions with other cell types in the tissue environment. A recent paper from Sam David’s group highlights the importance of this interaction between microglia and MDMs after SCI . Using an in vitro co-culture system, Greenhalgh and colleagues show that MDMs and microglia can strongly inﬂuence their respective key functions. MDMs isolated from injured spinal cords actively suppress phagocytic activity of microglia, which convincingly explains earlier results highlighted above , while microglia in turn can enhance MDM phagocytosis. Furthermore, MDMs suppress LPS induced production of microglial IL-1β, TNF and IL-6, as well as other inﬂammatory genes of the NFκB pathway, via Prostaglandin E2 (PGE2) signaling. The suppressed microglial phagocytosis was not detectable in microglia from the injured cord of CCR2 null mice, which lack MDMs at the lesion site, or mpges knockout mice, which are deﬁcient in macrophage PGE2. These eﬀects of microglia-MDM interactions were conﬁrmed in SCI in vivo, where the absence of MDMs at the lesion site (CCR2 null mice) led to impaired functional recovery and upregulation of pro-inﬂammatory genes compared to control animals. In addition, MDMs do not completely impair microglial function, as they did not reduce microglial proliferation and process extension . The diﬀerential roles of MDMs and microglia are not exclusive to SCI. Some diﬀerences in inﬂammatory phenotypes between both have been previously reported in the experimental autoimmune encephalomyelitis model . 1.3.4. Microglia depletion and loss of neuroprotective tissue organization The speciﬁc role of microglia after SCI has been controversially discussed in the past. Therefore, it is of particular interest to study the eﬀect of microglia speciﬁc depletion through the use of diﬀerent Colony stimulating factor 1 receptor (CSF1R) inhibitors, which leads to elimination of microglia in the CNS . Three recent studies assessed the eﬀects of CSF1R inhibition after SCI. The ﬁrst study  used a long term treatment with an oral CSF1R inhibitor (GW2580) , which inhibits microglia/MDM proliferation in a mouse model of lateral thoracic hemisection. This treatment led to a slight improvement in locomotor recovery, reduced gliosis and reduced microcavity 3
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proliferation versus depletion) as well as the use of diﬀerent models (hemisection versus contusion or crush). It is very likely that inhibiting the proliferation of microglia and, potentially monocytes, could reduce an aggressive immune response while possibly still maintaining desirable functions. Alternatively, the complete depletion of microglia would eradicate all protective eﬀects. Furthermore, the use of injury models with diﬀerent levels of inﬂammation could also be a reason for varying eﬀects: Hemisection models have lower levels of inﬂammation as compared to contusion models , which could render a strong barrier formation and containment of macrophages less important, especially if macrophage proliferation is also inhibited.
biology of SCI will lead to better strategies in modulating cellular function in a way that preserves protective eﬀects while interfering with damaging functions to achieve better recovery after SCI. Acknowledgements The work done in the authors labs is supported by grants from Wings for Life Spinal Cord Research Foundation (WFL-US-13/16), Craig H. Neilsen Foundation (543659), Advancing a Healthier Wisconsin Endowment (5520363). References
1.3.5. Interaction with astrocytes As discussed above, microglia are essential in forming the microgliaastrocyte interface at the SCI lesion border, thereby controlling damage and functional outcome. Recent publications from related ﬁelds support the importance of microglia-astrocyte interaction in neurotrauma. In a model of traumatic brain injury, microglia derived cytokines (TNF, IL-6 and IL-1β) downregulate the P2Y1 receptor in astrocytes, resulting in scar formation and neuroprotection. Conversely, depletion or inactivation of microglia reduces the astrocytic response, leads to decreased scar formation and promotes neurodegeneration . This is in line with work from the Sofroniew group, showing that impaired proliferation of scar-forming astrocytes and STAT3 signaling astrocytes results in a loss of spontaneous regrowth of corticospinal, serotonergic and sensory axons in a severe crush SCI model . However, activated microglia can also have detrimental functions. Liddelow et al. elegantly describe how activated microglia speciﬁcally induce a neurotoxic phenotype in astrocytes , termed A1 as an analogy to the M1 pro-inﬂammatory macrophage phenotype . A1 astrocytes lose normal astrocytic functions like synapse formation and phagocytic activity and ultimately become neurotoxic. The A1 phenotype can be induced by a combination of IL-1α, TNF and C1q. Absence of each of these factors results in decreased A1 activity. In vivo, A1 astrocytes can be detected in post-mortem CNS tissue from patients with neurodegenerative and neuroinﬂammatory diseases like Alzheimer’s disease, Huntington’s disease, Parkinson’s disease as well as multiple sclerosis. A1 astrocytes also play a role in CNS trauma: in an optic nerve crush model, they were induced in the retina and associated with retinal ganglion cell (RGC) loss, which could be prevented in triple knockout mice deﬁcient in IL-1α, TNF and C1q. Strikingly, even after ablation of 95% of the microglia using the CSF1R inhibitor PLX3397, A1 astrocytes are still detectable after systemic LPS injection, as opposed to the complete absence of A1 astrocytes in CSF1R knockout mice. In line with this observation, PLX3397 did not rescue RGC survival after optic nerve injury, suggesting that a small number of microglial cells is suﬃcient to induce this damaging phenotype. Importantly, treatment of A1 astrocytes with TGFβ or ﬁbroblast growth factor (FGF) reduced reactive transcript levels and reset A1 astrocytes to a non-reactive state . This is of particular interest because microglia express high levels of TGFβ and IGF at day 7 post injury and would thus have the ability to reverse astrocyte A1 polarization towards a more neutral phenotype . It is also possible that the depletion of microglia and subsequent reduction of anti-inﬂammatory mediators help maintain the A1 phenotype. Future studies need to provide additional evidence about the beneﬁts or dangers of microglial inﬂuence on astrocytes.
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2. Concluding remarks Myeloid cells are potent contributors to the inﬂammatory response after SCI, with both beneﬁcial and detrimental eﬀects. The description and development of new reporter mice and more speciﬁc markers have led to exciting new evidence emphasizing the importance of cellular interactions after SCI to control inﬂammation, lesion development and functional outcome. Ultimately, this increased understanding of the 4
Neuroscience Letters 709 (2019) 134370
A. Kroner and J. Rosas Almanza
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