Crosstalk between macrophages and astrocytes affects proliferation, reactive phenotype and inflammatory response, suggesting a role during reactive gliosis following spinal cord injury
© Haan et al. 2015
Received: 9 January 2015
Accepted: 20 May 2015
Published: 30 May 2015
Large-scale macrophage infiltration and reactive astrogliosis are hallmarks of early spinal cord injury (SCI) pathology. The exact nature of the macrophage response and relationship between these phenomena have not been explored in detail. Here, we have investigated these responses using a combination of in vivo SCI models, organotypic and primary cultures.
In vivo macrophage response was investigated using a contusive injury mouse model. Interactions between astrocytes and macrophages were studied in primary or organotypic cultures. Proliferation was assessed though MTT assay and nucleotide incorporation and gene expression changes through qPCR.
Seven days following contusive SCI, a mixed M1/M2 macrophage response was seen in the injury site. Conditioned medium from primary M1, but not M2, macrophages are able to induce astrocyte proliferation in both organotypic spinal cord cultures and primary astrocytes. Soluble factors from M1 macrophages induce a reactive astrocyte gene expression pattern, whereas M2 factors inhibit expression of these genes. M2-stimulated astrocytes are also able to decrease both M1 and M2 macrophage proliferation and decrease TNFα production in M1 macrophages.
These results suggest a strong role of M1 macrophages in inducing reactive astrogliosis and the existence of an astrocyte-mediated negative feedback system in order to dampen the immune response. These results, combined with the poor outcomes of the current immunosuppressive steroid treatments in SCI, indicate the need for more targeted therapies, taking into account the significantly different effects of M1 and M2 macrophages, in order to optimise outcome.
Spinal cord injury (SCI) is one of the leading causes for long-term disability in the Western world. Current treatments are limited to early administration of steroids, with initial studies suggesting good outcomes , but their actual efficacy has been debated [2, 3], and significant functional improvement is still difficult to achieve.
The use of steroids illustrates the large role the immune system plays in the pathology and resolution of SCI. Following SCI, the initial phagocytotic response is from the resident microglia, however, as the blood–brain barrier is generally compromised following SCI, peripheral macrophages rapidly infiltrate the spinal cord and become the predominant phagocytosing cell type at 3 days post injury (dpi)  in mouse models. Additionally, the infiltrating peripheral macrophages are responsible for the majority of secondary neuronal cell death in the subacute phase of SCI . Indeed, complete depletion of the macrophage population following SCI led to functional improvement in one study .
However, the picture may not be that simple, as macrophages are not a monolithic cell population. Macrophages consist mainly of two subtypes, the pro-inflammatory M1 type and an anti-inflammatory/regenerative M2 type . M2 macrophages have been implicated in tissue regeneration in muscle  and skin . In an experimental demyelination model in the brain, M2 type macrophages were required for initiation of remyelination , and it has been suggested that M2 macrophages are neurosupportive in SCI as well . This indicates a potential regenerative role for these cells in the nervous system. There have been several reports about the exact nature of the macrophage response in SCI, with varying proportions of M1 and M2 having been described [11–15]. How these macrophage phenotypical differences affect the development and resolution of SCI is still unclear.
In the longer term following SCI, the formation of the glial scar by reactive astrocytes is a crucial factor in the potential long-term recovery of functionality. Although it has recently been appreciated that at least some of the glial scar may be derived from pericytes , at its heart, the glial scar is comprised of a dense network of fibrous astrocytes, filling in and surrounding the injury site. The presence of large quantities of inhibitory extracellular proteoglycans in the scar restricts the regrowth of endogenous axons into the injury area . Although traditionally viewed as detrimental to regeneration, the reactive astrocytes of the glial scar may also have beneficial effects, including aiding in the repair of the blood–brain barrier and modulation of the immune cell response [18–20].
Although the glial scar is well studied, the initiation of this astrocyte response is not well characterised. Factors known to increase glial scar formation in the spinal cord include TGFβ1  and INFγ , amongst others. It has also been suggested that Fgf2 may be used to decrease scar formation and improve axonal permissibility , suggesting growth factors are important in glial scar development.
In short, the complete picture of scar induction and formation is far from clear. It is likely that the large-scale immune cell infiltration plays in important role in this. In this study, we aim to investigate the macrophage response to SCI and the interaction between macrophages and astrocytes to elucidate the role these play in the development of the glial scar. We report that M1 macrophages, present in significant numbers following SCI, are able to induce both astrocyte proliferation and a reactive phenotype, which can be partially counteracted by M2 macrophages. Additionally, astrocytes previously stimulated by M2 macrophages are able to decrease macrophage proliferation and activity, indicating an important role of the astrocyte-macrophage axis in SCI.
Materials and methods
Spinal cord impact model
All animal work was performed according to the procedures in the Animals (Scientific Procedures) Act 1986 and local guidelines. Healthy female C57Bl/6 J mice of 18–20 g in weight were subjected to a spinal cord impact. Under isofluorane anaesthesia, the dorsal skin was opened and a laminectomy was performed at the T9 level. For sham animals, at this point, the skin and muscle layers were closed and the animals were allowed to recover. In the impact group, animals were subjected to a controlled spinal impact of 50 kDyn using the Infinite Horizon Impactor device (Precision Scientific Instrumentation, IH-0400). Following recovery of both groups, functional impairment was assessed using the BBB scale . Only animals with a BBB score of <3 were considered in the impact group and >18 in the sham group, with a minimum of four animals per group.
Tissue harvesting and preparation
Seven days following SCI surgery, after the onset of glial scar formation, animals were sacrificed using CO2 exposure and transcardially perfused with PBS followed by 4 % PFA in PBS. Spinal cords were removed and postfixed in 4 % PFA in PBS overnight. They were then transferred to 30 % sucrose for cryoprotection for 2 days, before being mounted in OCT for sectioning in 15-μm sections.
Sections were air-dried, postfixed in cold 1:1 acetone/methanol for 2 min and blocked in 2 % BSA and 1 % Triton X-100 in PBS for 1 h. Antibodies were applied overnight at 4 °C and were: anti-CD68 (Abcam, ab53444, 1:100), anti-arginase-1 (Santa Cruz Biotechnology, sc-18355, 1:50), anti-iNOS (BD Biosciences, 610329, 1:100) and anti-GFAP (Cell Signaling, 12389S, 1:250). Controls included species- and concentration-matched isotype controls, which showed no staining (data not shown). Antibodies were detected with the relevant secondary antibodies conjugated to Alexa 488 or 594 (Life Technologies, 1:500), sections were mounted in DAPI containing mounting medium and were imaged through Z-scanning on an Applied Precision DeltaVision microscope.
Spinal cord organotypic cultures
Healthy C57BL/6 J mice of 6–12 weeks of age were sacrificed through CO2 exposure and the spinal cord removed. Meninges and other remaining tissue were dissected off, and 500-μm sections of the thoracic and abdominal cord were made using a McIlwain tissue chopper. Sections were then embedded in Matrigel (Corning, #354234) and cultured in DMEM/F12 medium containing 20 % FBS, 1 % penicillin/streptomycin (P/S) and 2 mM L-glutamine at 37 °C and 5 % CO2. For immunohistochemistry and EdU detection, organotypic cultures were fixed for 30 min in 4 % PFA in PBS, cryoprotected in 30 % sucrose overnight and sectioned in the same manner as the whole cords.
Primary cell isolation and culture
Primary macrophages were cultured from bone marrow progenitors. Bone marrow was isolated from femurs by flushing the bone cavity with cold RPMI medium. Marrow was pelleted by centrifugation and erythrocytes were lysed using ammonium chloride lysing buffer (BD Pharmlyse, 55899). Following two washes in cold RPMI and straining through a 50-μm cell strainer, bone marrow progenitors were cultured in T75 flasks at 1 × 106/ml in RPMI containing 10 % FBS, 2 mM L-glutamine and 1 % P/S. For differentiation into M1 macrophages, 20 ng/ml GM-CSF was added, for M2 macrophages, 20 ng/ml of both M-CSF and Il-4 were added, all from Peprotech, products 315-03A, 315-02B and 214-14A, respectively. Following 7 days of culture, macrophages were differentiated and ready for use.
Mouse primary astrocytes were isolated from neonatal mixed glia cultures as previously described for rat .
Conditioned medium production and use
Conditioned media were prepared from M1 and M2 macrophages, as well as from astrocytes that were non-stimulated or stimulated with M1 or M2 macrophage conditioned medium. In cultures that were previously stimulated (with differentiation growth factors in the case of macrophages and conditioned medium in the case of astrocytes), cells underwent several medium changes over 24 h following stimulation to ensure none of the stimulating factors carried over into the conditioned medium. For all cell types, conditioned medium was prepared by incubating cells in their normal culture medium for 3 days. Following conditioning, medium was filtered through 0.22-μm syringe filters, aliquoted and stored at −80 °C until use.
For all conditioned medium experiments, cultures or organotypic cultures were simulated with 25 % conditioned medium for macrophage medium or 50 % for astrocyte medium, added to their usual culture medium. For astrocyte experiments, controls consisted of incubation with a matched percentage unconditioned medium of the same type as the conditioned medium. For macrophage experiments, controls consisted of conditioned medium from unstimulated astrocytes. All experiments were repeated using several batches of conditioned medium, from cells from different isolations, to avoid batch-specific effects.
Cells were seeded in 96-well plates at 5 × 104 cells/well, at 4–6 wells per condition and allowed to adhere overnight. The following day, the medium was changed to experimental conditions, and the cells were allowed to proliferate for 2, 4, or 6 days. MTT was then added to cultures to a 0.5 mg/ml working concentration in the medium from a 5-mg/ml stock solution in PBS. Following 1–3 h of incubation, medium was removed, precipitated dye was resuspended in DMSO and absorbance was measured at 570 nm. Absorbance was quantified as relative percentages compared to control conditions.
Organotypic slice cultures were incubated in experimental conditions for 72 h with an n of 4 per condition, before MTT was added at 0.5 mg/ml to the culture medium. Following 2–4 h of exposure, slice cultures were removed from the Matrigel into individual microcentrifuge tubes and dried under vacuum to obtain an accurate dry tissue weight. Tissue was then resuspended in 10 % SDS in H2O at 25 mg dry weight/ml and dissolved at 60 °C under agitation for 6 h. Following centrifugation to remove any non-dissolved tissue, absorbance was measured at 570 nm. Absorbance was quantified as relative percentages compared to control conditions.
Nucleotide incorporation assays and detection
Cells were seeded at 1 × 105 cells/well in poly-D-lysine coated 8-well chamberslides and treated identical to those used for MTT assays. Twenty-four hours prior to the end of the protocol, 10-μM EdU was added from a 100-mM 1:10 DMSO/H2O stock. For organotypic cultures, EdU was added 48 and 24 h prior to end of protocol. Cells were then fixed for 20 min in 4 % PFA in PBS, and EdU was detected as previously described . Proliferation was determined as percentage EdU incorporating cells of total number of cells as determined by DAPI nuclear staining. Organotypic cultures were sectioned prior to detection of EdU. Proliferation was quantified as the number of EdU incorporating cells per square millimetre of section, with section area being determined in ImageJ. This area measure corrected for varying sizes or organotypic cultures.
RNA was extracted from unstimulated and M1- and M2-stimulated astrocytes (n = 3) using the RNeasy kit (Qiagen), according to manufacturer’s instructions. RNA was then reverse transcribed using M-MLV (Promega, M170A). Quantitative PCR for reactive astrocyte genes GFAP, nestin, vimentin, cdk1, ccnb1, ccnd1, mki67, lcn2 and serpinA3, with gapdh as housekeeping, was performed on a Quantstudio 6 thermal cycler using PrimerDesign mastermix (PrecisionPLUS-LR-SY), using a 60 °C annealing temperature. Primers used are listed in Additional file 1: Table S1. Data was analysed using the ΔΔCt method and expressed as fold changes from control samples.
TNF production in macrophages and ELISA
M1 and M2 macrophages were stimulated with conditioned medium from astrocytes previously stimulated by M1- or M2-conditioned medium for 3 days. Following stimulation, macrophages were replated into 24-well plates at 2 × 105 cells/well, activated with 1 μg/ml of β-glucan (Sigma Aldrich, C7821), and medium was collected after 48 h. TNF levels were determined using sandwich ELISA. Plates were coated with anti-TNF antibody (eBioscience, 14-7325-85, 1:500) overnight at 4 °C, blocked with blocking buffer (eBioscience 00-4202-55), and the samples were incubated overnight at 4 °C. Following binding of biotinylated anti-TNF antibody (eBioscience, 13-7326-85, 1:500) for 2 h at room temperature, streptavidin-HRP (eBioscience, 00-4106-93, 1:250) was added for 45 min at room temperature, followed by TMB substrate (eBioscience, 00-4201-56) for 20–30 min, before being read on a spectrophotometer at 450 nm.
Groups were compared using one-way ANOVA with Tukey post hoc testing in SPSS 20. Data is expressed as average ± SEM.
SCI induces a mixed M1/M2 macrophage response
M1 macrophage soluble factors induce proliferation in spinal cord organotypic cultures
Primary astrocytes show a proliferative response to M1 macrophage soluble factors
M1 and M2 macrophages have opposite effects on astrocyte reactivity
Previously stimulated astrocytes can modulate macrophage proliferation and TNFα production
Given the current poor prognosis for functional recovery following SCI, a thorough and full understanding of the processes during that recovery period is crucial for improving treatments. Here, we have studied the interplay between infiltrating macrophages and astrocytes. In our study, we focused on a moderate contusive injury and assessed macrophage response at the crucial time point of the onset of glial scar formation. We observed a mixed M1/M2 response, with a higher proportion of M2 macrophages. Although this seems to be in contradiction with some reports suggesting the response is predominantly M1, the ratio is most likely dependant on both the severity and nature of the injury and the time point investigated.
In accordance with our hypothesis that glial proliferation in the cord following injury may be driven by macrophage infiltration, spinal cord organotypic cultures exposed to conditioned medium from pro-inflammatory M1 macrophages showed increased glial proliferation, which was further confirmed though the use of primary astrocytes. Apart from proliferation, soluble factors from M1 also induce a reactive phenotype in both isolated astrocytes and organotypic cultures, with M2 factors having the opposite effect. This shows that M1 macrophages can promote both hallmarks of reactive astrogliosis, astrocyte proliferation and induction of specific gene expression patterns. The exact mechanisms responsible for this remain to be identified.
Although this study strongly suggests that M1 macrophages play an important role in the development of astrogliosis, the efficacy of immunosuppressive therapy through steroid treatment in SCI has been under debate. This illustrates the fine balance between the pro-inflammatory and gliosis-inducing M1 macrophages and the dampening role of the M2 macrophages and drives home the need for more targeted therapies, aimed at limiting the M1 response. Achieving this will likely be a great help in SCI treatment, and, possibly in conjunction with one of the many cell transplantation strategies currently under investigation, can hopefully lead to a better functional outcome for patients.
The surgical assistance of Xiaoting Meng and Fraser Young with the SCI models and post-operative care is greatly appreciated.
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