Azithromycin drives alternative macrophage activation and improves recovery and tissue sparing in contusion spinal cord injury

Mice were treated with AZM (160 mg/kg/day) or vehicle for 3 days prior to a moderate-severe contusion SCI. Drug administration was continued daily up to 7 days post injury (dpi). At 1, 3, and 7 dpi, n = 3–5 animals/treatment group were sacrificed and spinal cord tissue harvested for cell phenotypic analysis using fluorescent-activated cell sorting (FACS). Cells sorted from 3 and 7 dpi were further phenotypically evaluated using quantitative real-time PCR (rtPCR). Locomotor analyses (Basso Mouse Scale (BMS) and gridwalk) were conducted on a separate set of animals (n = 8–10/group) over the course of 4 weeks. At 28 dpi, these animals were sacrificed and spinal cord sections generated for histological analyses of tissue sparing and macrophage phenotype. In vitro studies were conducted using bone marrow-derived macrophages (BMDM) from adult mice. BMDMs were stimulated with AZM and/or pro-inflammatory stimuli (LPS + interferon-gamma (INFγ)). Control cells were left unstimulated. Secreted interleukin (IL)-10 and IL-12 levels were determined in the BMDM supernatant through ELISA analysis. The neurotoxicity of the BMDM supernatants was determined using the MTT assay to quantify the viability of supernatant-treated Neuro-2a cells.

Experiments were performed using 4-month-old female C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine). Animals were housed in IVC cages with ad libitum access to food and water. All procedures were performed in accordance with the guidelines and protocols of the Office of Research Integrity and with approval of the Institutional Animal Care and Use Committee at the University of Kentucky.

Animals were anesthetized via intraperitoneal (i.p.) injections of ketamine (100 mg/kg) and xylazine (10 mg/kg). Following a T9 laminectomy, a moderate-severe thoracic SCI was produced using the Infinite Horizon (IH) injury device (75-kdyn displacement; Precision Systems and Instrumentation) [22]. Any animals receiving SCI with abnormalities in the force vs. time curve generated by the IH device were excluded from analysis. These abnormalities are indicative of bone hits or instability in the spinal cord at the time of injury and occurred <10 % of the time. Mice receiving a laminectomy without injury were used as sham controls. After injury, muscle and skin incisions were closed using monofilament suture. Post surgically, animals received one subcutaneous injection of buprenorphine-SR (1 mg/kg) and antibiotic (5 mg/kg, enroloxacin 2.27 %: Norbrook Inc., Lenexa, KS) in 2 ml of saline and were housed in warming cages overnight. Animals continued to receive antibiotic subcutaneously in 1 ml saline for 5 days. Azithromycin (160 mg/kg) or vehicle (1 % methylcellulose) was delivered in 0.1-ml volume via oral gavage daily beginning 3 days prior and continuing for 7 days post injury. Food and water intake and the incision site were monitored throughout the course of the study. Bladder expression was performed on injured mice twice daily.

Following i.p. injection of ketamine (120 mg/kg) and xylazine (10 mg/kg), mice were transcardially perfused with ice cold diethyl pyrocarbonate phosphate-buffered saline (DEPC-PBS) then 1 cm of spinal cord centered on the injury site was rapidly dissected and placed in ice cold DEPC-PBS. The tissue was dissociated on ice using a size 40 mesh cell dissociation kit (Sigma, S0770) and rinsed twice with PBS. The dissociated tissue was then passed through a 70-μm screen filter (BD:352350). Cells were centrifuged at 200×g for 10 min at 4 °C, resuspended in fetal bovine serum (FBS) staining buffer (BD: 554656), and then cell numbers for each animal acquired using a hemocytometer. Cells were incubated with Fc block (BD:553142) for 15 min on ice and then were incubated with CD11b-APC, GR1-PE-Cy7, CD45-PerCP-Cy5.5, and CD206 (mannose receptor)-PE antibodies (BD Biosciences) as previously described [17]. Cell were washed twice with FBS staining buffer and resuspended in appropriate volumes of FBS staining buffer for fluorescent-activated cell sorting (FACS) analysis. Expression of these surface receptors was determined using an iCyt Synergy sorter system (Sony) in the UK Flow Cytometry Core Facility. Microglia, macrophages, and neutrophils were identified by CD11b⁺/CD45^lo/GR1^lo/neg, CD11b⁺/CD45^hi/GR1^lo/neg, and CD11b⁺/CD45^hi/GR1^hi expressions, respectively [5, 6]. CD206 expression levels were used to determine M2-polarization states. For each antibody, gating was determined based upon appropriate negative isotype-stained controls. Flow data were analyzed using FlowJo software (Tree Star). Cell numbers for each animal were estimated from cell percentages and hemocytometer counts. All investigators involved in the flow/FACS analyses have been certified for flow research methods and applications through the completion of the Annual Course in Cytometry sponsored by the Cytometry Education Association and Verity Software House.

All FACS-sorted macrophages (CD11b⁺/CD45^lo/hi/GR1^lo/neg), which consisted of both microglia- and monocyte-macrophages, were collected in FBS staining buffer (BD:554656), and 0.75 ml TRIzol LS reagent (Life Technologies) was added per 0.25 ml of suspension. Total RNA was isolated based on the manufacturer’s protocol, with an additional phase separation using BCP, precipitation with isopropanol (Sigma-Aldrich, St. Louis, MO), and wash of the isolated RNA in 70 % ethanol. Then, 1 μg RNA was reverse-transcribed using the high-capacity complementary (cDNA) reverse transcription kit (Life Technologies). Real-time PCR amplification was performed on the mixture of 100 ng cDNA sample, Taqman Universal PCR Master Mix, and Taqman Probes (Life Technologies) using the Applied Biosystems Step One Plus Real-Time PCR System. Probes included Arg1 (Mm00475988), CD206 (Mm00485148), and CD86 (Mm00444543). Expression of genes was normalized to 18S mRNA for each sample, and reported values were calculated as 2^-ΔΔCT relative to a sham reference sample.

All experimental animals were assessed using the Basso Mouse Scale (BMS) to score hindlimb function as previously described [23]. Mice were tested in an open field for 4 min before surgery and at 1, 3, 7, 14, 21, and 28 days post injury (dpi). Each hindlimb was scored separately based on movement (e.g., ankle placement and stepping), coordination, and trunk stability, and averaging both hindlimb scores generated a single score for each animal. A score of 0 indicated complete paralysis and a score of 9 indicated normal locomotion. Assessment of hindlimb function was also carried out using the gridwalk test [24]. The gridwalk utilizes a horizontal ladder with stainless steel rungs 4 mm in diameter spaced 1.2 cm apart. All experimental mice were trained before injury. On 27 dpi, only mice that could support their own body weight were tested on the apparatus. Animals were videotaped and evaluated on 30 continuous rungs on the center of the ladder. Frame-by-frame video analysis was used to track the total number of hindlimb steps/footfalls.

Mice were anesthetized and then transcardially perfused with cold PBS (0.1 M, pH 7.4), followed by perfusion with cold 4 % paraformaldehyde (PFA). Dissected spinal cords (1 cm) were post-fixed for another 2 h in 4 % PFA and subsequently rinsed and stored in cold phosphate buffer (0.2 M, pH 7.4) overnight at 4 °C. On the following day, tissues were cryoprotected in 30 % sucrose for 3 days at 4 °C, followed by rapidly freezing and blocking in optimal cutting temperature (OCT) compound (Sakura Finetek USA, Inc.) on dry ice. Tissue was systematically randomized into blocks with equal group distribution to ensure uniformity of staining across groups, and blocked tissue was stored at −80 °C before sectioning. Tissue blocks were cut in serial coronal sections (10 μm) and mounted onto Colorfrost plus slides (Fisher #12-550-17). Spinal cord sections were stained for glial fibrillary acidic protein (GFAP) or neurofilament (NF) to measure tissue sparing. Slides were incubated with chicken anti-GFAP (1:200; Aves GFAP) or chicken anti-NF (1:1500, Aves NFH 0211) primary, followed by biotinylated goat anti-chicken (1:1000; Aves B-1005) then Alexa Fluor 488 (1:1000; Invitrogen S32354) secondary antibodies. Slides were coverslipped with Immu-Mount (Thermo Scientific, Waltham, MA). GFAP or NF fluorescent images were taken using a C2+ laser scanning confocal microscope (Nikon Instruments Inc., Melville, NY). To quantify spared tissue area, the regions of dense GFAP- or NF-positive staining were outlined and measured using the MetaMorph analysis program (Molecular Devices, Sunnyvale, CA).

Bone marrow-derived macrophages (BMDMs) were extracted from the femur and tibia of female C57BL/6 mice at 8–10 weeks of age as previously reported [25, 26] and were plated at 0.8 ~ 1 × 10⁶ cells/ml in differentiation media: Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1 % penicillin/streptomycin, 1 % HEPES, 0.001 % β-mercaptoethanol, 10 % FBS, and 20 % supernatant from sL929 cells (a generous gift from Phillip Popovich, The Ohio State University). Supernatant collected from sL929 cells contains macrophage colony-stimulating factor, which helps to promote bone marrow cells’ differentiation into macrophages [27]. The BMDMs were allowed to differentiate for 7 days in culture, and cells were then replated on day 7 at a density of 1 × 10⁶ cells/ml in 12-well plates in differentiation media without L929 supernatant. On day 8, cells were stimulated to be M1 using LPS (50 ng/ml; Invivogen tlrl-eblps) plus IFNγ (20 ng/ml; eBioscience 14-8311-63) diluted in N2A growth medium. AZM (10 or 30 μM; Sigma PHR1088) or vehicle (DMSO; MP Biomedical 190186) was added at the time of stimulation. Unstimulated BMDMs were used as control. Six hours after incubation, the supernatant of the stimulated macrophages (macrophage-conditioned media (MCM)) was collected, filtered, then applied to cultured Neuro-2a cells or tested for IL-10 and IL-12p40 levels using standard ELISA kits (Thermo Scientific, Rockford, IL).

Mouse neuroblastoma cell lines (aka Neuro-2a or N2A, a gift from Chris Richards , University of Kentucky) were maintained in N2A growth medium containing 45 % DMEM, 45 % OPTI-MEM reduced-serum medium, 10 % fetal bovine serum (FBS), and 1 % penicillin/streptomycin. N2A were plated at a density of 1 × 10⁵ cells/ml in 48-well tissue culture plates and allowed to proliferate for 48 h. The neurotoxicity of MCM was evaluated as reported previously [7] using a MTT-based cell growth determination kit according to the manufacturer’s instructions (Sigma-aldrich). Briefly, on the day of testing, N2A growth media was replaced by fresh MCM, and the N2A cells were incubated in MCM for 24 h then thiazolyl blue tetrazolium bromide (MTT (5 mg/ml), 20 μl per well) was added to each well and the cells further incubated for 2 h. The tetrazolium ring of MTT can be cleaved by mitochondrial dehydrogenases of viable cells, yielding purple formazan crystals, which were then dissolved in acidified isopropanol solvent. The resulting purple solution was spectrophotometrically measured at 570 nm Epoch microplate reader (BioTek instruments, Inc., Winooski, VT) using 690 nm as a background absorbance. All measurements were done in triplicates, and at least three independent experiments were carried out.

Investigators blinded to experimental conditions performed all data acquisition and analysis. Statistical analyses were completed using GraphPad Prism 6.0 (GraphPad Software). Data were analyzed using one- or two-way ANOVA followed by Holm-Sidak’s test for multiple comparisons. F-values are reported for repeated measures. Chi square and independent sample t tests were used when appropriate. Results were considered statistically significant at p ≤ 0.05. All data are presented as mean ± SEM unless otherwise noted. Figures were prepared using Adobe Photoshop CS6 (Adobe Systems) and Prism 6.0.

To determine the effects of AZM treatment on the inflammatory cell response to SCI, mice were treated daily with AZM or vehicle beginning 3 days prior to 75-kdyn T9 contusion SCI with continued daily administration (oral gavage) up to 7 days post injury (dpi). There were no significant differences between treatment groups in spinal cord displacement at the time of SCI (p > 0.35 for any given time point between groups and p = 0.50 for overall group effect). This is an important control to ensure all animals received comparable SCI regardless of pre-injury treatment. At 1, 3, or 7 dpi, cells were isolated from the injured spinal cord for phenotypic analysis (Fig. 1). Cells were labeled with antibodies specific for neutrophils (Gr-1) and macrophages (CD11b/CD45) then the relative numbers of neutrophils (CD11b⁺/CD45^hi/GR1^hi) and monocyte- (CD11b⁺/CD45^hi/GR1^lo/neg) and microglia- (CD11b⁺/CD45^lo/GR1^lo/neg) derived macrophages were quantified. As indicated in Fig. 1a, few monocyte-derived macrophages or neutrophils were detectable 1 day after sham SCI (also see dotted line in Fig 1c). Figures 1b, c show that the number of CD11b⁺/CD45^hi/GR1^hi neutrophils in the injured spinal cord peaked at 1 dpi then decreased over time, CD11b⁺/CD45^hi/GR1^lo/neg monocyte-derived macrophage numbers remained elevated over time, and CD11b⁺/CD45^lo/GR1^lo/neg microglia increased from 1 to 7 dpi. In addition, AZM significantly attenuated the monocyte response to SCI at 3 dpi (Fig 1c, p < 0.05).

Next, we determined the phenotype of SCI macrophages using FACS to purify CD11b⁺/CD45⁺/GR-1⁻ macrophages from the spinal cord homogenates (both monocyte and microglia populations in Fig. 1b were pooled). Messenger RNA (mRNA) was isolated from the sorted, purified macrophages, and the relative gene expression of M2, anti-inflammatory (arginase, CD206), and M1, pro-inflammatory (CD86), macrophage markers was determined. Figure 1d shows that the expression of markers indicative of an anti-inflammatory phenotype, arginase and CD206, increased with SCI and AZM treatment significantly augmented expression of both arginase (F1,12 = 23.13, p = 0.0004) and CD206 (F1,12 = 11.86, p = 0.005) (Fig. 1d). Expression of the pro-inflammatory marker, CD86, increased with SCI and expression was attenuated by AZM treatment (F1,12 = 25.91, p = 0.003). There were no significant differences in macrophage phenotype between AZM- or vehicle-treated animals at 28 dpi (p > 0.8 for CD86 and arginase expression; Additional file 1: Figure S1).

Next, we examined if AZM was differentially increasing M2 marker expression on different macrophage populations. We used a CD206 antibody to determine the M2 profiles of monocyte- (CD11b⁺/CD45^hi/GR1^lo/neg) and microglia- (CD11b⁺/CD45^lo/GR1^lo/neg) derived macrophages isolated from the injured spinal cord at 3 and 7 days post-injury. Similar to what has been reported previously [6], microglia-derived macrophage expression of this M2 marker decreased over time (Fig. 1e). There was a significant time × drug interaction (F1,11 = 5.83, p = 0.03) with AZM significantly increasing the number of CD206+ microglia-derived macrophages relative to vehicle controls at 3 dpi (Fig. 1e, p = 0.01). This effect was not significant at 7 dpi (Fig. 1e, p > 0.6). In accordance with what has been previously reported for monocyte-derived macrophages [6], we observed similar levels of CD206 expression at both time points. Specifically, the numbers of CD206+ monocyte-derived macrophages were similar at 3 and 7 dpi, and there were no significant differences between AZM and vehicle at either time point (time × drug interaction (F1,11 = 0.28, p = 0.6)): 3 dpi, mean ± SEM: 1914 ± 156 (AZM) and 1988 ± 255 (veh); 7 dpi, 1945 ± 207 (AZM) and 1800 ± 155 (veh).

Collectively, these results indicate that AZM treatment alters the inflammatory response to SCI and decreases pro-inflammatory macrophage gene expression while potentiating anti-inflammatory macrophage gene expression. Further, AZM may potentiate microglia- vs. monocyte-derived M2 macrophage activation in response to SCI.

Alterations in macrophage phenotypes are associated with differences in functional recovery and tissue sparing following SCI [6]. Because AZM treatment increased indices of anti-inflammatory macrophage activation, we assessed the ability of AZM treatment to improve SCI recovery. Figure 2a–g shows that AZM treatment resulted in significantly more spared tissue at 28 dpi. Specifically, the rim of spared tissue, defined using positive GFAP immunoreactivity, was roughly twofold greater following AZM (mean ± SEM = 0.25 ± 0.04 mm²) vs. vehicle treatment (0.13 ± 0.02 mm²) (p = 0.01). The area of neurofilament-positive spared axons was also significantly improved with AZM (0.21 ± 0.05 mm²) vs. vehicle treatment (0.098 ± 0.02 mm²) (p = 0.04).

Small differences in the amount of preserved tissue at the lesion epicenter after SCI correlate with significant differences in locomotor recovery [28]. To examine functional recovery in response to AZM treatment, locomotor function was assessed using the Basso Mouse Scale (BMS) [23]. As shown in Fig. 2h, following SCI, both treatment groups had significant functional deficits that improved over time (F5,75 = 179.7, p < 0.0001). There was significant overall treatment effect (F1,15 = 9.961, p = 0.007) and significant time × treatment interaction (F5,75 = 10.75, p < 0.001) meaning that AZM treatment significantly improved both the rate of recovery and overall level of residual deficits associated with SCI (Fig. 2h). Specifically, significant improvements were detected for AZM-treated mice compared to vehicle-treated by 14 dpi. At 28 dpi, AZM-treated animals had recovered consistent stepping (BMS ~ 5) while vehicle-treated animals had some limb movement without weight-supported stepping (BMS ~ 3). These improvements were also reflected in the BMS subscore, a supplement to the BMS scale that is sensitive to treatment-specific recovery progressions that may fall outside the normal pattern of recovery [23]. Specifically, there was a main effect of treatment (F1,15 = 10.93, p = 0.005) and time × treatment interaction (F5,75 = 11.76, p < 0.0001) that manifested at 28 dpi as fore-hindlimb coordination in the AZM group (BMS subscore ~4) with no fore-hindlimb coordination in the vehicle-treated group (BMS subscore ~0) (Fig. 2i).

Coordination is an important aspect of SCI recovery; therefore, we used a variety of motor tasks to determine if AZM treatment facilitated recovery of coordinated and proprioceptive function. First, a BMS score of 5 can indicate some level of coordination between fore- and hindlimb movements [23]. Figure 2 shows that all but one animal in the AZM-treated group achieved this score while only one animal in the vehicle group achieved coordination by 28 dpi (p = 0.002) (Fig. 2j).

Next, we assessed animals on the gridwalk task at 27 dpi. The gridwalk is a horizontal ladder task that measures sensory-motor coordination [29]. Two animals from the AZM group and three animals from the vehicle group did not achieve a sufficient level of recovery for gridwalk testing. Figure 3 shows that of animals able to perform the task, those treated with AZM had ~40 % fewer footfalls compared to vehicle-treated (p = 0.05). Overall, AZM treatment improved the locomotor function and coordination of mice recovering for contusion SCI.

Because we previously observed that pro-inflammatory macrophages are neurotoxic and likely potentiate secondary injury processes while anti-inflammatory macrophages do not cause cell death and drive repair processes [3], we next sought to determine if these neuroprotective effects could be mediated through AZM altering the toxic potential of pro-inflammatory macrophages.

The effects of stimulating bone marrow-derived macrophages (BMDMs) in vitro are predictive of spinal cord macrophage responses in vivo [25, 26, 30]. Therefore, we examined the phenotype and neurotoxic potential of pro-inflammatory BMDMs treated with AZM in vitro. We used LPS + INFγ stimulation to model the pro-inflammatory, M1, macrophages that are activated in SCI [3, 6]. Figure 4a shows that AZM drives increased production of IL-10, an anti-inflammatory cytokine, while reducing pro-inflammatory IL-12 release from M1 macrophages. In the absence of the pro-inflammatory stimuli, AZM had no effect on unstimulated BMDMs (Fig. 4a). An increased ratio of IL-10:IL-12 is a defining feature of anti-inflammatory macrophages; therefore, we conclude from Fig. 4a that AZM drives pro-inflammatory macrophages toward an anti-inflammatory phenotype. As shown in Fig. 4b, this shift was associated with a significant decrease in the neurotoxicity of pro-inflammatory macrophages. Specifically, BMDMs were stimulated to be M1 in the presence or absence of AZM. The supernatants from these cells, deemed macrophage-conditioned media (MCM), were collected and used to treat neurons. The MCM from M1 cells resulted in significant reduction (~30 %, p = 0.001) in neuron viability relative to MCM from untreated cells. This toxicity was significantly attenuated with AZM treatment, as there was no significant difference in viability among neuron groups exposed to MCM from untreated vs. M1 + AZM-treated BMDMs (p > 0.15; Fig. 4b) or untreated vs. AZM-treated in the absence of M1 stimuli (p > 0.20; Fig. 4b). The results presented in Fig. 4 are representative of three independent experiments. Collectively, these results demonstrate that AZM can act directly on macrophages to alter pro-inflammatory activation and reduce macrophage-mediated neurotoxicity.