IL-13 Ameliorates Neuroinflammation and Promotes Functional Recovery after Traumatic Brain Injury

Traumatic brain injury (TBI) is a common public health issue. Nearly half of the world’s population has experienced a TBI in their lifetime (1). According to the data from World Health Organization, TBI will become a major cause of death by the year 2020 (2). A large number of TBI patients are living with lifelong motor, cognitive, and/or other disabilities. In addition, TBI increases the incidence of neurodegenerative diseases, including Alzheimer disease and Parkinson disease, among survivors (3). So far, there is no effective pharmacological treatment to reduce brain injury or promote brain repair after TBI.

The severity of a TBI does not depend solely on the direct initial injury (4). The acute primary insult to the brain elicits a cascade of immune responses in the lesioned brain, which may induce secondary injuries and expand brain lesion size at the subacute stage of TBI. Indeed, prolonged immune responses could be observed in human and mouse brains years after TBI (3, 5). The severity of immune responses has been shown to associate with brain lesion size and neurologic deficits (3, 5, 6). As the first line of defense in the CNS, microglia, as well as invaded macrophages, play an essential role in immunoregulation after TBI (7, 8). In the acute phase of TBI, microglia/macrophages secrete a large number of anti-inflammatory factors, including IL-4, IL-10, and TGF-β, to limit brain damage. However, persistent microglia/macrophage activation in the subacute phase results in excessive production of proinflammatory factories such as IL-6, IL-1β, TNF-α, and IFN-γ, which can aggravate brain injury (7, 9). Shifting microglia/macrophage response to an anti-inflammatory modality that favors brain preservation and repair may therefore represent a legitimate therapy for TBI (10, 11).

IL-13 is a known anti-inflammatory cytokine released by a variety of immune cells. Emerging studies report biological significance of IL-13 in the CNS. Expression of IL-13 and its cognate functional receptor has been detected in the normal brain (12). IL-13–deficient mice exhibit a decline in working memory and reference memory, suggesting an essential role of this cytokine in cognitive performance under physiological condition (13). The functions of IL-13 in CNS diseases seem to be diverse and controversial. Some studies documented detrimental roles of IL-13 on neuronal survival in an inflammatory milieu (12, 14, 15), whereas others highlighted neuroprotective properties of this cytokine by adjusting microglia/macrophage responses or by inducing the death of inflammatory microglia/macrophages (16, 17). It is apparent that the functions of IL-13 should be explored in the context of specific CNS diseases or conditions. Elevated expression of IL-13 has been detected in the brain early after TBI (18, 19). However, the effects of IL-13 in brain lesion after TBI remain unknown.

The current study explored the effect of IL-13 treatment in a mouse model of TBI. We discovered that repeated intranasal delivery of IL-13 significantly reduced brain lesion and improved functional recovery after TBI. In vitro and in vivo studies further revealed a potent effect of IL-13 in modulating inflammatory responses and phagocytic function in microglia/macrophages. IL-13 may represent a novel immunotherapy for TBI.

Adult male C57BL/6J mice weighing 25–30 g (8 wk) were purchased from Shanghai SLAC Laboratory Animal Company, Limited (Shanghai, China). Mice were maintained under a 12–12 h light-dark cycle with food and water provided at libitum. All animal experiments were approved by the Animal Care and Use Committee of Shanghai Medical College, Fudan University (approval number 20150119-120).

TBI was induced in C57BL/6J mice by controlled cortical impact (CCI) as described previously (20). Briefly, animals were anesthetized with 3% isoflurane vaporized in 30% O₂/70% N₂ under spontaneous breathing conditions. An ∼4-mm craniotomy was performed over the right parietal-temporal cortex using a motorized drill. The CCI was centered 2 mm lateral to the midline and 0.5 mm anterior to bregma and produced with a pneumatically driven CCI device (Precision Systems and Instrumentation) using a 3-mm flat-tipped impounder (velocity, 3.5 m/s; duration, 150 ms; depth, 1.5 mm). This impact results in moderate CCI, as we described previously (21). After CCI, the scalp was sutured. Rectal temperature was maintained at 37 ± 0.5°C during surgery and for up to 30 min after TBI using a heating pad. Sham animals were subjected to all aspects of the protocol (surgery, anesthesia, craniotomy, recovery) except for CCI.

Animals were randomly assigned to receive intranasal administration of saline (vehicle) or IL-13 (60 μg/kg body weight) starting immediately after TBI. Briefly, under isoflurane anesthesia, five drops (2 μl/drop) of IL-13 were applied alternately into each nostril with a 2 min interval between drops. Control animals received an equal volume of vehicle control using the same anesthesia and delivery regimen. All animals were treated in the afternoon (between 3:00 and 5:00 pm) for five consecutive days.

Long-term sensorimotor functions were assessed during the light phase of the circadian cycle, beginning ∼4 h after the onset of the light phase. Mice were randomly coded by a researcher who was blinded to groups assignments.

The Rotarod test was performed with the Rotamex 5 apparatus (Columbus Instruments) as previously described (22). Briefly, mice were forced to run on a rotating drum with speeds starting at 4 rpm and accelerating to 40 rpm within 300 s. The latency to fall off the rotating rod was recorded. Data were expressed as the mean value from three trials.

The experimental apparatus was a steel bar (50 cm long and 2 mm diameter) resting on two vertical supports and elevated 37 cm above a flat surface. Mice were placed in the middle of the bar and were guided to climb onto the supports within 30 s. The animals were scored according to the following criteria: 0, fell off; 1, hung onto the bar with two forepaws; 2, hung onto the bar with an added attempt to climb onto the bar; 3, hung onto the bar with two forepaws and one or both hind paws; 4, hung onto the bar with all four paws and with tail wrapped around the bar; and 5, escaped to one of the supports. Mice were pretrained 3 d before TBI. After surgery, animals were tested for three trials per day up to 28 d after surgery.

The foot fault test was performed as described previously to assess sensorimotor coordination during spontaneous locomotion (23). Mice were placed on an elevated (30 cm above a flat surface) grid surface (40 cm long × 25 cm wide) with a grid opening of 2.25 cm² (1.5 × 1.5 cm square) and videotaped for 1 min from below the grid. The videotapes were analyzed by a blinded investigator to count the number of total steps and the number of foot faults made by the impaired limbs (contralateral to brain lesion side). Foot faults were determined when the mouse misplaced its impaired forepaw such that the paw fell through the grid. Data were expressed as percentages of total steps.

Mice were sacrificed and intracardial perfused with phosphate-buffered vehicle (PBS [pH 7.4], 37°C), followed by cold 4% paraformaldehyde (PFA; w/v) in PBS. Brains were postfixed in 4% PFA at 4°C for 1–2 d and cryoprotected in 30% sucrose (w/v) in PBS for at least 2 d at 4°C. Frozen serial coronal sections were sliced with a freezing microtome (Leica) at 30 μm beginning +1.98 mm to −1.98 mm from bregma. Every sixth section was selected for staining. Slices were immunohistochemically stained with the neuronal marker microtubule-associated protein 2 (MAP2; Santa Cruz Biotechnology) to measure the neuronal loss and with the nuclear marker DAPI to measure total cell loss after TBI. Tissue loss was determined using ImageJ software by an observer blinded to group assignments. The percentage of tissue loss was determined using the following equation: (area of the contralateral hemisphere ‒ nonlesioned area of the ipsilateral hemisphere)/(area of the contralateral hemisphere) ×100%.

Coronal brain sections were incubated in blocking solution containing 5% BSA, 10% donkey serum, and 0.5% Triton X-100 in PBS for 2 h at 37°C, and then incubated overnight with primary Abs (in PBS containing 3% BSA, 1% donkey serum, and 0.3% Triton X-100) at 4°C. Slices were rinsed in PBS followed by incubation with Alexa-Fluor–conjugated secondary Abs at 37°C for 2 h. Sections were then washed and mounted with Fluor-mount contained nuclear marker DAPI (Yeasen, Shanghai, China). For immunostaining using the mouse primary Ab, the M.O.M Kit (BMK-2202; Vector Laboratories; Burlingame, CA) was applied before primary Abs to block nonspecific signals, according to the manufacturer’s instructions.

Primary Abs used in this paper include the following: goat anti-CD206 (1:500, catalog no. AF2535; R&D Systems, Minneapolis, MN), rat anti-CD16/32 (1:500, catalog no. 553142; BD Biosciences, San Jose, CA), rabbit anti–myelin basic protein (MBP; 1:500, catalog no. ab40390; Abcam, Cambridge, MA), mouse antineurofilament H nonphosphorylated (SMI32, 1:500, catalog no. 801701; BioLegend, San Diego, CA), rabbit anti-Iba1 (1:2000, catalog no. 019-19741; Wako Chemicals, Richmond, VA), anti-NeuN Ab, Alexa Fluor@488 conjugated (1:500, catalog no. MAB377×; Millipore, Burlington, MA), rabbit anti-MAP2 (1:1000, catalog no. Ab5622; Millipore), and rabbit antisynaptophysin (1:500, catalog no. Ab32127; Millipore). Secondary Abs were purchased from Jackson ImmunoResearch Laboratories, and the working concentrate was 1:1000. TUNEL staining was performed with the In Situ Cell Death Detection Kit (catalog no. 12 156 792 910; Sigma-Aldrich, Louis, MO) according to the manufacturer’s protocol.

The images were acquired on an Olympus FV1000 microscope (Olympus, Tokyo, Japan) with an UPLSAPO 40× objective (N.A. = 1.25) and a Nikon Ni-E Microscope with a Plan Fluor 20× objective (N.A. = 0.75) (Nikon, Tokyo, Japan). One or two randomly selected microscopic fields in the peri-lesion areas were imaged in each section. Three sections covering the injured area were assessed for each mouse brain. The lesion area was identified as the region in which the majority of DAPI-stained nuclei were shrunken. The border of the lesion was determined by the loss of MAP2 staining and the accumulation of Iba1⁺ microglia/macrophages. We defined the peri-lesion area as the tissue that covers a radial distance of 300 μm from the border of loss of MAP2⁺ tissue. Images were coded and analyzed using Image-Pro Plus software (media cybernetics, Rockville, MD) and/or ImageJ/Fiji (National Institutes of Health, Bethesda, MD) by an investigator who was blinded of experimental groups. The three-dimensionally reconstructed images were generated by Imaris (64× version 9.2.1).

Total RNA was extracted from mouse brain tissue samples or rat culture samples using TRIzol reagent (Invitrogen), and first-stand cDNA was generated using the Moloney-Murine Leukemia Virus Reverse Transcriptase (Takara) according to the manufacturers’ protocols. The following primers were used for mouse samples: 5′-TTTGGACACCCAGATGTTTCAG-3′ (forward) and 5′-GTCTTCCTTGAGCACCTGGATC-3′ (reverse) for Cd16; 5′-CAAGGAAGGTTGGCATTTGT-3′ (forward) and 5′-CCTTTCAGTCCTTTGCAAGC-3′ (reverse) for Cd206; 5′-GACCGTTGTGTGTGTTCTGG-3′ (forward) and 5′-GATGAGCAGCATCACAAGGA-3′ (reverse) for Cd86; 5′-CCAAGACGATCTCAGCATCA-3′ (forward) and 5′-TTCTGGCTTGCTGAATCCTT-3′ (reverse) for Cd11b; 5′-AATCCTGCCGTTCCTACTGATC-3′ (forward) and 5′-GTGTCACCGTGTCTTCCTTGAG-3′ (reverse) for Cd32; 5′-GACCCTCACACTCAGATCATCTTCT-3′ (forward) and 5′-CCTCCACTTGGTGGTTTGCT-3′ (reverse) for Tnf-α; 5′-CTCCATGAGCTTTGTACAAGG-3′ (forward) and 5′-TGCTGATGTACCAGTTGGGG-3′ (reverse) for Il-1β; 5′-ACACATGTTCTCTGGGAAATC-3′ (forward) and 5′-AGTGCATCATCGTTGTTCATA-3′ (reverse) for Il-6; 5′-TGCGCTTGCAGAGATTAAAA-3′ (forward) and 5′-CGTCAAAAGACAGCCACTCA-3′ (reverse) for Tgf-β; 5′-CAGGGTAATGAGTGGGTTGG-3′ (forward) and 5′-CACGGCACCTCCTAAATTGT-3′ (reverse) for Ym1/2; and 5′-CTGCCCAGAACATCATCCCT-3′ (forward) and 5′-TGAAGTCGCAGGAGACAACC-3′ (reverse) for GAPDH. The following primers were used for rat culture samples: 5′-TCCTCCAGCAGTGGGAAACA-3′ (forward) and 5′-TTTCTAGGTTTCGGGTATCCTTGC-3′ (reverse) for Cd86; 5′-CACGCCGCGTCTTGGT-3′ (forward) and 5′-TCTAGGCTTTCAATGAGTGTGCC-3′ (reverse) for Ifn-γ; 5′-TAAGCCAACAAGTGGTATTC-3′ (forward) and 5′-AGGTATAGATTCTTCCCCTTG-3′ (reverse) for Il-1β; 5′-CAAGCTGGGAATTGGCAAAG-3′ (forward) and 5′-GGTCCAGTCCATCAACATCAAA-3′ (reverse) for Arg1; 5′-GCCCCCACAGCCAAGTCCAT-3′ (forward) and 5′-CCTCACAGCCAGCGGATCGC-3′ (reverse) for Cd11b; 5′-AAATGGGCTCCCTCTCATCAGTTC-3′ (forward) and 5′-TCTGCTTGGTGGTTTGCTACGAC-3′ (reverse) for Tnf-α; 5′-GGTTCCGGTTTGTGGAGCAG-3′ (forward) and 5′-TCCGTTTGCATTGCCCAGTA-3′ (reverse) for Cd206; and 5′-TGCTGGTGCTGAGTATGTCGTG-3′ (forward) and 5′-CGGAGATGATGACCCTTTTGG-3′ (reverse) for GAPDH.

Real-time quantitative PCR (qPCR) was performed using SYBR Green Master Mix (Yeasen) on LightCycler 480 (Eppendorf, Hamburg, Germany). All reactions were performed in triplicates, and the relative amount of mRNA was normalized to GAPDH level.

Six days after TBI or sham surgery, peripheral blood was obtained from mice by cardiac puncture. RBCs were lysed with ACK lysis buffer (Sigma-Aldrich, Burlington, MA). The ipsilateral hemisphere was collected, and single cells were extracted from the homogenates by gentle MACS Octo Dissociator with Heaters (Miltenyi Biotec, Bergisch Gladbach, Germany) and Neural Tissue Dissociation Kit(T) (catalog no. 130-093-231; Miltenyi Biotec). The single-cell suspension was prepared according to a published protocol (24, 25), using 30 and 70% Percoll gradients (GE Healthcare BioSciences, Piscataway, NJ). Cells at the interface of 30 and 70% Percoll were collected, and then gently washed with FACS buffer (1% penicillin/streptomycin antibiotic, 2 nM EDTA, 2% FBS in HBSS buffer). The cells were permeabilized and fixed with the Intracellular Staining Kit (catalog no. 88-8824-00e; Thermo Fisher Scientific, Pittsburgh, PA), and then stained with fluorophore-labeled Abs for 30 min on ice in the dark. For lymphocyte subpopulation induction, blood cells were stimulated with cell stimulation mixture (catalog no. 00-4975-03; Thermo Fisher Scientific) for 6 h, and stained with fluorophore-labeled Abs. The following Abs were used for staining: arginase 1–PE (1:50, catalog no. IC5868P; R&D Systems); CD3-allophycocyanin (1:200, catalog no. 17-0032-82; Thermo Fisher Scientific); CD3-BV510 (1:20, catalog no. 100353; BioLegend); CD4-eFluor 450 (1:100, catalog no. 48-0041-82; Thermo Fisher Scientific); CD8-BUV737 (1:200, catalog no. 564297; BD Biosciences); CD11b-allophycocyanin-cy7 (1:400, catalog no. 47-0112-82; Thermo Fisher Scientific); CD11b-BUV737 (1:400, catalog no. 564443; BD Biosciences); CD11c-Percp-cy5.5 (1:400, catalog no. 45-0114; Thermo Fisher Scientific); CD19-BUV395 (1:200, catalog no. 563557; BD Biosciences); CD19-FITC (1:200, catalog no. 561740; BD Biosciences); CD45-eFluor 450 (1:400, catalog no. 48-0451; Thermo Fisher Scientific); CD117-BV605 (1:400, catalog no. 563146; BD Biosciences); Foxp3-allophycocyanin (1:20, catalog no. 17-5773; Thermo Fisher Scientific); IFN-γ–PB (1:100, catalog no. 48-7311; Thermo Fisher Scientific); IFN-γ–PE (1:100, catalog no. 505808; BioLegend); IL-4–FITC (1:400, catalog no. 11-7042-82; Thermo Fisher Scientific); IL-10–Percp–cy5.5 (1:50, catalog no. 45-7101; Thermo Fisher Scientific); IL-17–Percp–cy5.5 (1:400, catalog no. 45-7177-82; Thermo Fisher Scientific); Ly6G-FITC (1:400, catalog no. 11-9668-82; Thermo Fisher Scientific); Ly6G-PE (1:400, catalog no. 12-9669-82; Thermo Fisher Scientific); NK1.1-allophycocyanin (0.125 μg/test, catalog no. 17-5941; Thermo Fisher Scientific); Siglec-F-PB (1:400, catalog no. 565934; BD Biosciences); and TNF-α–allophycocyanin (1:50, catalog no. 17-7321; Thermo Fisher Scientific). Appropriate isotype controls were used according to the manufacturers’ instructions (Thermo Fisher Scientific). Fluorochrome compensation was performed with single-stained OneComp eBeads (catalog no. 01-1111-42; Thermo Fisher Scientific). Flow cytometry was performed on a BD LSRII Flow Cytometer (BD Biosciences) according to the manufacturer’s instructions. Data were analyzed with FlowJo software (FlowJo).

Primary microglia–enriched cultures were prepared as described previously (20). Briefly, whole brains of 1-d-old mixed-sex Sprague Dawley rat pups were digested by Trypsin (0.01%) at 37°C for 15 min. Cells were washed with ice-cold DMEM containing 5% FBS to stop digestion, then filtered through a 70-μm filter. Cells were plated onto poly-d-lysine–coated T-flasks (one to two pups/flask) in DMEM/F12 Culture Media (Life Technologies) containing 10% heat-inactivated FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 mM nonessential amino acids, 50 U/ml penicillin, and 50 mg/ml streptomycin. After a 12- to 14-day incubation in a humidified incubator at 37°C with 5% CO₂, microglia were obtained by shaking at 180 rpm for 1 h. The enriched microglia were seeded in a PDL-coated plate for use.

Primary cortical neuronal cultures were prepared from 17-d-old Sprague Dawley rat embryos, as described previously (26). Experiments were conducted at 7 d in vitro. To induce ischemia in vitro, cultured neurons were changed in a medium containing 120 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, and 25 mM Tris HCl (pH 7.4) and were subjected to oxygen glucose deprivation (OGD) for 2 h in a sealed chamber filled with 95% N₂ and 5% CO₂. Cells were then returned to 95% air, 5% CO₂ and normal glucose medium for 24 h. Condition media were collected and used to treat cultured microglia. Dead neurons were labeled with 1 μg/ml of propidium iodide (PI) in cultured medium and incubated at 37°C for 10 min. Labeled dead neurons were used for phagocytosis assay after two washes.

Microglia were plated at the concentration of 1 × 10⁵ cells per well in a 24-well plate (Nunc; Thermo Fisher Scientific). After 3 h of treatment, microglia were incubated with Nile red fluorescent microspheres (0.03% solids, 1 × 10⁶ beads per well; Invitrogen) or PI-labeled dead neurons (5 × 10⁵ per well) for 4 h. The cells were then rinsed with PBS and fixed with 4% PFA for 15 min. Fixed cells were immunostained with Iba1 for imaging or stained with CD11b–eFluor 450 (catalog no. 48-0112-82; Thermo Fisher Scientific, eBioscience, Pittsburgh, PA) for flow cytometry.

Microglia were plated in a 96-well plate at the concentration of 1 × 10⁴ cells per well. Conditioned media were collected 24 h after treatment. Microglia viability was measured using lactate dehydrogenase assay (catalog no. L7572; Point Scientific) according to the manufacturer’s protocol.

All data were presented as mean ± SEM. GraphPad Prism software (version 8.1.0, La Jolla, CA) was used for statistical analyses. Gaussian distribution of the data were assessed by Shapiro-Wilk normality test. If the data pass the Gaussian distribution test, a parametric test was used for significance analysis, otherwise, nonparametric test was used. The two-tailed unpaired Student t test was used for comparison of two groups. The differences in means among multiple groups were analyzed using one-way ANOVA followed by Dunnett (all conditions compared with an indicated group) or Tukey (comparisons between conditions) multiple-comparisons test. Two-way ANOVA was used when two experimental groups with different treatments were analyzed, followed by post hoc Bonferroni test. In these situations, injury conditions (sham versus TBI) and treatment (vehicle versus IL-13) were between-subject factors. Differences in means across groups with repeated measurements over time were analyzed using the repeated-measures ANOVA, followed by Bonferroni multiple comparisons. In all analyses, a p value < 0.05 was considered statistically significant.

C57BL/6J mice were subjected to CCI and randomly assigned to receive IL-13 (60 μg/kg body weight) or vehicle (saline) treatment. IL-13 was intranasally delivered immediately after TBI and repeated daily for 5 d (Fig. 1A). We first evaluated the effect of IL-13 on long-term functional recovery after TBI using a battery of behavioral tests. The mice with IL-13 treatment exhibited longer duration to stay on a rotating rung in the Rotarod test (Fig. 1B), higher score in the wire hang test (Fig. 1C), and reduced percentage of forelimb fault in the foot fault test (Fig. 1D) compared with vehicle-treated mice up to 28 d after TBI, suggesting improvements in the sensorimotor performance by IL-13 treatment.

Immunohistochemical staining was used to assess the effect of IL-13 on brain tissue loss at 6 d after TBI. As shown in Fig. 1E and 1F, IL-13 treatment significantly reduced neuronal tissue loss and total cell loss, as revealed by MAP2 staining and DAPI staining, respectively. The severity of white matter damage is highly correlated with the functional deficits after TBI (27, 28). Therefore, we further evaluated the effects of IL-13 on white matter integrity. Double staining for MBP, a major myelin protein, and SMI32, a marker of demyelinated axons, was performed to assess the lesion in white matter (Fig. 1G–I). The immunofluorescence intensity of MBP staining decreased in the peri-lesion area in the striatum 6 d after TBI, indicating a significant loss of myelin protein (Fig. 1H). Meanwhile, the immunofluorescence of SMI32 increased in the same area (Fig. 1H). As a result, the SMI32/MBP ratio was significantly elevated in vehicle-treated mice (Fig. 1I). IL-13–treated mice displayed alleviated white matter damage (Fig. 1H) and a decrease in the SMI32/MBP ratio compared with vehicle-treated mice (Fig. 1I). Taken together, our result suggested a protective effect of IL-13 on both gray matter and white matter integrity after TBI, which culminate with improved long-term functional performance.

It is known that microglia/macrophages may assume different phenotypes (proinflammatory versus anti-inflammatory) after TBI, which differentially impact the development of brain injury and the execution of brain repair (29). IL-13 is a well-known anti-inflammatory factor (30). We, therefore, evaluated the effect of IL-13 on microglia/macrophage phenotypes in the peri-lesion areas. Consistent with our previous study (31), the signals of proinflammatory marker CD16 and the overall intensity of Iba1 were significantly increased 6 d after TBI (Fig. 2A, 2B, 2D). IL-13 treatment significantly reduced the expression of CD16 (Fig. 2A, 2B), which was accompanied by a significant decrease in overall Iba1 staining (Fig. 2D). The expression of anti-inflammatory marker CD206 significantly increased in IL-13–treated TBI brains but not in vehicle-treated controls (Fig. 2C). The inhibitory effect of IL-13 on CD16 and Iba1 expression lasted out for 28 d after TBI (Supplemental Fig. 1). The expression of a group of cytokines and other inflammatory factors was then quantified by qPCR at 6 h (Supplemental Fig. 2) and 6 d (Fig. 2E–M) after TBI. IL-13 treatment showed little effect on the expression of proinflammatory factors 6 h after injury (Supplemental Fig. 2A–G). At 6 d after TBI, IL-13 significantly inhibited the induction of four proinflammatory factors, including Tnf-α, Cd16, Cd86, and Cd11b (Fig. 2E–H) in the brains, but it showed no effect on other proinflammatory factors (Il-1β and Il-6, Fig. 2I, 2J). The expression of anti-inflammatory factors (Cd206, Tgf-β, and Ym1/2) remained unchanged 6 h after TBI (Supplemental Fig. 2H–J) and significantly increased 6 d after TBI (Fig. 2K–M). IL-13 treatment showed no effect on anti-inflammatory factors (Fig. 2K–M, Supplemental Fig. 2H–J). Taken together, these results indicated that IL-13 could modulate microglia/macrophage responses and suppress neuroinflammation after TBI at the subacute phase after TBI.

One of the most important roles of microglia/macrophages in an injured or diseased brain is to clear dead cells and harmful fragments (32). To study whether IL-13 affects microglia/macrophage phagocytic activity after TBI, we performed Iba1/NeuN double staining. The phagocytic clearance of neurons was determined by detecting the appearance of NeuN⁺ neuronal material within Iba1⁺ microglia/macrophages (Fig. 3A). The colocalization of NeuN and Iba1 was most frequently located at the process of microglia/macrophages in the sham brains. After TBI, the colocalization area was located in the enlarged cell body of Iba1⁺ microglia/macrophages (Fig. 3A, lower row). Line profile and three-dimensionally reconstructed images showed four types of cells: 1) Iba1⁺NeuN⁻ microglia/macrophages that were not engaged in phagocytosis; 2) Iba1⁻NeuN⁺ neurons that were not engulfed by microglia/macrophages; 3) Iba1⁺ cells partially colocalized with NeuN, which indicated microglia/macrophages engulfing neurons; and 4) Iba1⁺ cells with NeuN⁺ signal inside, which indicated microglia/macrophages with phagocytosed neurons inside (Fig. 3B, 3C). As expected, the NeuN⁺Iba1⁺ areas increased in TBI brains, indicating increased microglia/macrophage phagocytic activity. IL-13 treatment increased the NeuN⁺Iba1⁺ areas compared with vehicle treatment after TBI (Fig. 3D). The NeuN⁺Iba1⁻ area significantly decreased after TBI, consistent with the neuronal loss after TBI. IL-13 treatment rescued the neuronal loss and increased the NeuN⁺Iba1⁻ area after TBI (Fig. 3E). The phagocytosis index, which was calculated as the ratio of NeuN⁺Iba1⁺ area to Iba1⁺ area, significantly increased in TBI–IL-13 group compared with TBI-vehicle group (Fig. 3F). Iba1, NeuN, and TUNEL triple immunostaining showed that majority of engulfed neurons were TUNEL⁺ dead/dying neurons in vehicle (67.87 ± 6.89%)- and IL-13 (79.03 ± 4.74%)–treated TBI mice (Supplemental Fig. 3A, 3B). These results indicated that IL-13 enhances the clearance of dead/dying neurons by microglia/macrophages and thereby increased neuronal survival after TBI.

Microglia phagocytic ability also plays important roles in normal brain functions, such as learning-related synapse pruning. There were small and comparable NeuN⁺Iba1⁺ areas in brains collected from vehicle- or IL-13–treated sham animals (Fig. 3A, 3D), suggesting no effect of IL-13 on microglia phagocytic activity under physiological condition. Furthermore, immunostaining of synaptophysin and Iba1 was performed to assess whether IL-13 affected synaptic pruning in vehicle- or IL-13–treated brains 6 d after sham or TBI operation. The percentages of synaptophysin⁺ areas within Iba1⁺ microglia/macrophage area remained the same after IL-13 treatment in sham (Supplemental Fig. 3C, 3D) or TBI (Supplemental Fig. 3G, 3H) brains. IL-13 treatment did not change the total count (Supplemental Fig. 3E, 3I) or total intensities (Supplemental Fig. 3F, 3J) of synapse staining either. Taken together, these data suggested that IL-13 has little effect on normal microglia phagocytic activity or synaptic pruning.

To determine whether IL-13 may exert its neuroprotective roles through targeting the peripheral immune system, we evaluated the infiltration of peripheral immune cells into the brain using flow cytometry. The numbers of CD3⁺ T lymphocytes, CD19⁺ B lymphocytes, Ly6G⁺ neutrophils, CD45^highCD11b⁺CD11C⁻Ly6G⁻ macrophages, and CD11b⁺CD11c⁺ dendritic cells significantly increased 6 d after TBI, and IL-13 treatment did not alter the numbers of infiltrating immune cells after TBI (Fig. 4A, 4B). We also measured the effects of IL-13 intranasal treatment on peripheral immune cell composition in both sham animals and animals subjected to TBI. IL-13 did not change the percentages of CD19⁺ B lymphocytes, CD3⁺ total T lymphocytes, CD4⁺ T lymphocytes, or CD8⁺ T lymphocytes in blood from sham or TBI mice (Fig. 4C, 4D). Further evaluation on subpopulations of T lymphocytes revealed increases in the percentages of IFN-γ⁺ Th1 cells, IL-4⁺ Th2 cells, and IL-17⁺ Th17 cells 6 d after TBI, whereas the percentage of Foxp3⁺ regulatory T cells remained unchanged (Fig. 4D). IL-13 treatment showed no effects on the percentages of T cell subpopulations (Fig. 4D). Finally, we evaluated the effects of IL-13 on the percentages of CD11b⁺NK1.1⁻Ly6G⁻siglecF⁺ eosinophils, CD11b⁺Ly6G⁺ neutrophils, CD11b⁺NK1.1⁺ NK cells, and CD117⁺CD11b⁻ mast cells in blood (Fig. 4E, 4F). Neither TBI or IL-13 changed the number of these peripheral immune cells 6 d after TBI or sham operation. These results indicated that the protective effect of IL-13 on TBI is unlikely through modulation of the peripheral immune system.

Primary microglia were then used to further confirm the direct effect of IL-13 on microglia phenotypic alteration. IL-13 treatment at concentrations of 1–50 ng/ml did not affect cell survival with or without LPS stimulation for 24 h (Fig. 5A, 5B). LPS (100 ng/ml) induced the expression of proinflammatory marker CD16 in microglia, which was inhibited by IL-13 (20 ng/ml) treatment (Fig. 5C, 5D). The expression of anti-inflammatory factor CD206 were not affected by LPS and/or IL-13 (Fig. 5C, 5E). Real-time PCR was used to measure the expression of inflammatory factors 6 h after LPS treatment in microglia with or without IL-13 treatment (Fig. 5F–L). The quantification data showed that IL-13 partly decreased the production of proinflammatory factors, including Tnf-α and Ifn-γ (Fig. 5F, 5G), induced by LPS. These results suggested that IL-13 inhibits proinflammatory responses in microglia.

We further validated the function of IL-13 on microglia phagocytosis using primary cultures. Microglia were pretreated with LPS (100 ng/ml) and/or IL-13 (20 ng/ml) for 3 h and then incubated with Neil red fluorescent microspheres (Beads-PE) (diameter is 1 μm) for 4 h. Flow cytometry was used to measure microglia phagocytosis (Fig. 6A–C). We divided microglia into nonphagocytic, low-, and high-phagocytic groups based on the fluorescent intensity of their engulfed beads (Fig. 6A, 6B). IL-13 treatment significantly increased percentages of cells with high-phagocytic capacity and reduced the percentages of nonphagocytic and low-phagocytic cells in LPS-treated microglia (Fig. 6C).

In another set of experiments, microglia were treated with LPS and/or IL-13 followed by coincubation with PI-labeled dead neurons. After 4 h coincubation at 37°C, microglia were fixed for immunostaining. PI-labeled neuron nuclear puncta were found in microglia (Fig. 6D). Quantification data showed that IL-13 further potentiated the phagocytic ability of microglia induced by LPS (Fig. 6E). These data suggested that IL-13 enhances microglia phagocytosis in an inflammatory environment.

We then used condition medium collected from OGD challenged neurons, which contained sterile damage-associated molecular patterns, to stimulate microglia. The production of pro- and anti-inflammatory factors were measured using flow cytometry (Fig. 6F–J). We found that exposure to OGD neuron conditioned media for 6 h induced the expression of proinflammatory cytokine TNF-α (Fig. 6F, 6G) and anti-inflammatory factor arginase1 in microglia (Fig. 6F, 6I). IL-13 significantly inhibited TNF-α expression (Fig. 6G), but it had no effect on arginase 1 expression (Fig. 6I).

Recent research suggests a potential role of IL-13 in CNS homeostasis and pathology (12). As an anti-inflammatory cytokine, IL-13 has been shown to exert neuroprotection in CNS diseases and injuries (33–36). For example, lentiviral vector–mediated expression of IL-13 is able to limit lesion severity in a mouse model of multiple sclerosis (33). Local administration of IL‐13 into the brain parenchyma reduced inflammatory neuronal cell loss in regions associated with memory and prevented cognitive deficits upon LPS challenge (35). In addition, cell-based delivery of IL-13 improved functional recovery after spinal cord injury (37). In this study, we reported for the first time, to our knowledge, that repeated intranasal administration of IL-13 reduced brain lesion size and significantly promoted functional recovery after TBI. Our study not only reinforces neuroprotective roles of IL-13 but also provides a clinical feasible approach for IL-13 treatment in neurologic diseases.

Direct injection to the site of injury is the most commonly used approach for IL-13 monotherapy or IL-13 in combination with other treatments in animal models of CNS diseases (35, 38, 39). One apparent advantage for local injection is that a lower dose of IL-13 could be used to achieve immediate action on the lesion site. However, this invasive strategy has limitations for clinical application, especially for progressive neurologic disorders that require repeated dosing in long-term. The intranasal route is increasingly used for CNS drug delivery because of its capacity to deliver non–blood-brain barrier–permeable drugs to the brain parenchyma (40, 41). In this study, we delivered IL-13 intranasally at a dosage of 60 μg/kg/d (∼1.5 μg/d based on 0.025 kg average body weight). This dosage is similar to the effective intranasal dosage of IL-4 in recent studies as a treatment in models of CNS diseases (42, 43). Such low-dose intranasal cytokine treatments warrant further evaluation for clinical translation.

TBI is a progressive CNS injury. The pathological changes after TBI can be divided into three phases: acute (a few hours after initial injury), subacute (within 1 wk after injury) and chronic (1 wk after injury) phases (44). Microglia are promptly activated in acute phage after TBI and its activation can last out into the chronic phase of brain recovery. The initial activation of microglia in the acute and subacute phases intend to clear the damaged cells, resolve local inflammation and release chemokines to recruit more immune cells, including macrophages, into the battle against brain damage. During this phase, however, microglia/macrophages might be overactivated by overwhelming noxious stimulations and elicit secondary brain damage by producing plethora amounts of proinflammatory cytokines and free radicals (44). Our previous study reported the change of microglia/macrophage polarization toward proinflammatory phenotype around 7 d after TBI (31). Such a shift from anti-inflammatory to proinflammation phenotype at subacute phase of TBI provided a practical therapeutic target to reduce post-TBI injury (29, 45). Our in vitro studies confirmed the capacity of IL-13 to shift microglia/macrophages away from proinflammatory transition upon inflammatory stimulation. In a TBI model, we found that although IL-13 failed to reduce neuroinflammation 6 h after TBI, it was able to inhibit the elevation of proinflammatory cytokines and reduce the number of proinflammatory microglia/macrophages 6 d after TBI. The phenotypic adjustment by IL-13 became active when the microglia/macrophage phenotype started to steer toward detrimental phenotype after TBI. Interestingly, IL-13 seems to be less effective in increasing the number of anti-inflammatory phenotype microglia/macrophages, as demonstrated by comparable number of CD206⁺Iba1⁺ cells in the peri-lesion area and comparable number of anti-inflammatory cytokines in the lesioned brain between IL-13– and vehicle-treated TBI mice. These results suggest that IL-13 inhibits microglia/macrophages proinflammatory phenotype responses at subacute phase after TBI.

Phagocytosis is another important function of microglia/macrophages, which are essential for the clearance of damaged cells. The sequester of dead/dying cells will further reduce the release of damage-associated molecular patterns from these cells and inhibit the activation of more immune cells. Efficient microglia/macrophage phagocytosis has been shown to be beneficial for tissue preservation and functional recovery after TBI and stroke (46, 47). Microglia/macrophages in aged mice showed impairments in phagocytic activity, which was accompanied by worsened neurologic outcomes (48). In this study, we discovered that IL-13 treatment may enhance phagocytic capacity of microglia/macrophages under pathological conditions. It was reported in a zebrafish model of TBI that pharmacological or genetical removal of microglia disabled the rapid removal of cellular debris and, therefore, increased the rate of secondary cell death after TBI (47). The enhanced clearance capacity in IL-13–treated microglia/macrophages can therefore accelerate debris removal and prevent secondary tissue damage as what we observed in IL-13–treated TBI mice. It is noted that improper phagocytosis of stressed but live neurons by microglia/macrophages, which is termed as “phagoposis,” may execute neuronal death (49). Our data showed that comparable amounts of engulfed neurons were TUNEL⁺ dead/dying neurons in vehicle- or IL-13–treated TBI mice, which dispute the possible effect of IL-13 to increase phagocytosis.

IL-13 receptors are widely distributed on various cell types in addition to microglia and macrophages (30). For example, the IL-13R is known to express on T and B lymphocytes. However, we found, in this study, that repeated intranasal delivery of IL-13 had no effect on peripheral T lymphocyte subpopulations or B lymphocyte populations in the blood after sham or TBI. IL-13 treatment did not alter the brain infiltration of peripheral immune cells after TBI either. These results suggest that the protective effect of IL-13 is not due to its effects on adaptive immune responses in our TBI model. The lack of changes in systemic immune responses after intranasal cytokine application was similarly observed in recent studies (42, 43), suggesting that the intranasal route could enable CNS delivery while reducing the risk of systemic side effects.

It is noted that IL-13 is a factor that induces airway inflammation and airway hyperresponsiveness (50, 51). One important pathogenic mechanism for IL-13–induced inflammation in the lung is that IL-13 induces rapid eosinophil genesis and promotes eosinophil survival, activation, and trafficking to the site of injury. The elevation of eosinophil counts is correlated with disease severity and therefore serves as a biomarker of airway inflammation. It has been reported that intratracheal or intranasal application of IL-13 induced airway hypertension, which was accompanied by increased number of eosinophils as well as neutrophils and lymphocytes, in bronchoalveolar lavage fluid (52–54). In our study, flow cytometry analysis revealed no changes in the numbers of eosinophils, neutrophils, NK cells, mast cells, or lymphocytes in blood after IL-13 treatment, suggesting minimal effects of IL-13 on peripheral immune cells with current treatment regimen. Further study is warranted to thoroughly evaluate the off-target effects on the lung as well as other organs.

In addition to immunoregulation, IL-13 may directly target other CNS cells. It has been reported that IL-13 directly stimulated primary astrocytes to produce brain-derived neurotrophic factors, which in turn fostered cognitive functions. Astrocytes respond dynamically to brain injury with progressive changes in gene expression and proliferative function and are actively involved in neuroinflammation after TBI (55, 56). Whether IL-13 could directly regulate astrocyte activity after TBI remains unclear. IL-13 may also directly act on neurons. However, the direct effect of IL-13 on neurons seems to be destructive. The engagement of the IL-13R on dopaminergic neurons has been shown to exacerbate LPS-induced neuronal loss through increasing endoplasmic reticulum oxidative stress (14, 15, 57). Such neurotoxicity of IL-13 apparently cannot be linked to the neuroprotective effects of IL-13 in the TBI model. Nevertheless, further studies are warranted to dissect the effects of IL-13 on different CNS residential cells or infiltrated immune cells to fully elucidate the mechanisms for its neuroprotective effects in TBI brains.

We demonstrate, in this study, that intranasal IL-13 treatment reduces TBI and promotes long-term functional recovery. Specifically, IL-13 suppresses proinflammatory responses and enhances phagocytic capacity of microglia/macrophages. Therefore, IL-13 may represent a new strategy to improve functional outcomes after TBI.

The authors have no financial conflicts of interest.