Mouse monocyte cell line (J774) was obtained from Korean Cell Line Bank and cultured in RPMI 1640 medium (Hyclone, UT, USA) supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS, Hyclone), and 1% penicillin-streptomycin (Hyclone). Murine microglia cell line (BV2) was obtained from Ajou University College of Medicine, Chronic Inflammatory Disease Research Center. BV2 cells were cultured in RPMI 1640 medium (Hyclone) supplemented with 10% FBS (Hyclone) and 1% penicillin-streptomycin (Hyclone). Both cells were kept in a 37°C incubator supplemented with 5% CO₂ and sub-cultured in 90 × 20 mm cell culture dishes (2-3 days after seeding at a density of 1 × 10⁶ cells/ml) when confluence was >80%.

For the animals, healthy male C57BL/6 mice (weight, 25-30 g; age, 8-12 weeks; Central Lab Animal Inc) and CCR2:: red fluorescent protein (RFP) (weight, 25-30 g; 8-12 weeks; Jackson Lab) mice were prepared for in vivo experiments. A total of 50 mice were used in this study. All animal experiments were performed as per the guidelines of The Institutional Animal Care and Use Committee of Yonsei University Health System and according to the National Institutes of Health guidelines.

J774 or BV2 were placed in the lower compartment of a six-well chamber with poly-l-ornithine-coated cover slides attached to the base, and transwell coated with 3-µm microporous membranes was inserted in the upper compartment of the same chamber (Costar). J774 and BV2 (2 × 10⁶ cells/ml) were cross-seeded in the upper compartment with conditioned medium (CM) and CM with lipopolysaccharide (LPS; CML) from BV2 and J774, respectively. After a 48-hour reaction, cells that accumulated in the lower compartment were subjected to Hoechst staining (33342, Thermo). The remaining cells in the upper compartment were stained with crystal violet (Sigma). All slides were then viewed under an optical microscope (Eclipse Ti2; Nikon) and a confocal microscope (LSM 700; Carl Zeiss).

To screen the CM and CML of BV2, a proteome profiler assay was carried out using the Mouse Chemokine Array Panel A (R&D system) according to the manufacturer’s instructions. The blots containing captured chemokines in the membrane were oxidized with the ECL™ Western Blotting Analysis System (GE Healthcare) and visualized using the LAS 4000 mini (Fujifilm). Finally, the chemokines were quantified using HLImage++ (Western Vision software) for array analysis.

To confirm the receptor of J774, cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes and blocked with 3% bovine serum albumin for 1 hour. They were then treated with mouse anti-CCR4 (1:1000; Santa Cruz Biotechnology) and rabbit anti-CCR5 (1:1000; Santa Cruz Biotechnology) antibodies overnight at 4°C. After washing thrice with phosphate-buffered saline (PBS), the cells were treated with goat anti-mouse IgG FITC (1:1000; Abcam) and goat anti-rabbit IgG rhodamine (1:1000; Millipore). Finally, the cells were stained with 1× DAPI (Thermo) and visualized using confocal microscopy (LSM 700; Carl Zeiss). For immunoblotting, J774 cells were homogenized in a lysis buffer and centrifuged (12000 g at 4°C) for 15 minutes.

Equal amounts of protein (30 µg) from the supernatants were separated on a 10% acrylamide gel and electro-transferred onto nitrocellulose membranes. The membranes were incubated with mouse anti-CCR4 (1:2000; Santa Cruz Biotechnology), rabbit anti-CCR5 (1:2000; Santa Cruz Biotechnology), and rabbit anti-β-actin (1:2000; Santa Cruz Biotechnology) antibodies overnight at 4°C after blocking. The membranes were then incubated with goat anti-mouse and rabbit horse radish peroxidase (1:2000; Thermo). The blots were rinsed, and the protein bands were visualized and photographed using the ECL™ Western Blotting Analysis System (GE Healthcare) and the LAS 4000 mini (Fujifilm), respectively.

For the in vitro LPS treatment, BV2 and J774 cells were treated with LPS (10 ng/ml; Sigma) for 24 hours to induce inflammatory conditions. For the in vivo LPS treatment, inflammation was induced using intracerebroventricular injections for 30 minutes. Briefly, anesthetized mice were placed in a stereotaxic frame and LPS [5 ng in 5 µl PBS or sham (5 µl PBS)] was injected into the right lateral cerebral ventricle (stereotaxic coordinates: 1 mm caudal to bregma, 1.3 mm lateral to sagittal suture, and 2 mm in depth).

For antagonist treatment, J774 was treated with CAS864289-85-0 (CAS) [IC50 = 39 nM; CCR4 antagonist] and Maraviroc (Marav) [IC50 = 12.5 nM; CCR5 antagonist] for 1 hour and then seeded in the upper compartment of the transwell system. Mice were treated with CAS (5 mg) and Marav (10 mg) via an intraperitoneal injection 24 hours after LPS treatment.

Mice were injected intraperitoneally with a mixture of Zoletil (100 mg/kg) and xylazine (Rompun, 10 mg/kg) for anesthesia during imaging. Cranial window surgery and imaging were performed as described previously [15]. Body temperature was maintained at 36.5-37.5°C with a heating pad system (Live Cell Instrument). For visualization of brain vessels, mice were injected with Texas red-conjugated dextran 70-kDa (Sigma) or CF^®405M-conjugated wheat germ agglutinin (Biotium) via the retro-orbital sinus. Imaging was then performed using a two-photon microscope (LSM7MP; Carl Zeiss). Brain images were obtained with light wavelength at 820 nm or 880 nm for green, red, and blue. Imaging was performed using a resolution of 512 × 512 pixels with a 20× water-immersion objective lens. The imaging depth was 40-50 µm from the cerebral cortex surface with a z-stacking system of step sizes every 1 µm. Imaris (Bitplane) was used for 3D imaging data analysis.

After LPS treatment, brain sections (15 µm thick) were permeabilized with citrate buffer and blocked with 3% goat serum in PBS for 1 hour at room temperature. ProLong™ Diamond Antifade Mountant with DAPI (Invitrogen) was used for sample mounting and nuclear staining. A confocal microscope (LSM 700; Carl Zeiss) was used to observe the stained sections.

Cell counts and intensity of RFP expression were assessed using perfusion with 4% PFA. Each extracted whole brain was placed on a black paper to prevent autofluorescence in the background. RFP-positive cell counts were analyzed using ZEN blue desk top image analysis program (Carl Zeiss). All setting areas were analyzed by setting up ID and Area, and all groups were analyzed by “Batch” setting for constant analysis value. RFP intensity of whole brain of CCR2::RFP mice was measured using IVIS Spectrum CT (PerkinElmer).

To create transparent tissues, extracted brain was placed in 20 ml hydrogel (40% acrylamide, 2% bis-acrylamide, 10% wt VA-044 initiator, 40 ml PBS, 100 ml 16% PFA, and 210 ml dH₂O) for 2 days. To establish the bond between the acrylic amides, the thermal addition process is passed by desiccation chamber in a fume hood. Hydrogel-soaked brain sample was incubated 37°C for 3 hours on the rotator in a fume hood. Thereafter, the sample was sliced into 2-mm thick sections by vibratome and incubated in a clearing solution (123.66 g boric acid, 400 g sodium dodecyl sulphate, dH₂O, and pH 8.5) at 37°C for 7 days. The clearing solution was replaced once every 3 days. The brain was then enclosed between two cover glass-bottom petri dishes. Brain imaging was performed using confocal laser scanning microscopy (LSM 5 EXCITER; Carl Zeiss) at excitation wavelengths of 647 nm. Finally, images were analyzed using Imaris software (Bitplane).

All experiments were randomized, and all analyses were carried out by investigators blinded to experimental conditions. Statistical analyses were performed using t test (Systat Software, Inc). Values were presented as the mean ± SE. Significant differences between two groups were analyzed using an unpaired t test, with P<0.05 considered statistically significant. Multiple comparisons were performed using one-way analysis of variance followed by Bonferroni’s post hoc test.

To investigate the role of BV2 cells in J774 cell migration and vice versa, the CM and CML of each cell line were collected and cross-treated with each cell using a transwell system. The CM of BV2 and J774 did not affect migration of each cell. However, the CML of BV2 and J774 induced migration (Figure 1A). The number of Hoechst-positive J774 cells on BV2 CML was 3.5-fold higher than for Hoechst-positive BV2 cells on J774 CML (Figure 1B). Proteome profiler chemokine assay to screen for chemokine-associated migration in BV2 CML (Figure 1C) showed increased expression of several chemokines in BV2 CML. Expression levels of six chemokines, namely, CXCL10, CCL2, CCL3/4, CCL9/10, CCL5, and CCL12 were significantly higher in BV2 CML than in BV2 CM (Figure 1D). A search of major receptors related to the discovered chemokines identified CCR4 and CCR5 (Table 1).

CCR4 and CCR5 were accordingly selected as target receptors, and immunocytochemistry and immunoblotting of J774 were performed to confirm CCR4 and CCR5 expression. Immunocytochemistry showed that CCR4 and CCR5 were expressed and merged in J774 (Figure 2A). Immunoblotting also indicated positive CCR4 and CCR5 expressions and there was no statistical change in them (Figures 2B, C). Collectively, these data indicated that monocyte-derived macrophages with CCR4 and CCR5 migrate into parenchymal tissues during the neuroinflammatory response.

To determine the functional significance of CCR4 and CCR5, J774 treated with CAS and Marav cultured CML of BV2 and confirmed J774 migration using transwell system again. A high rate of Hoechst-positive J774 cells were observed in the lower compartment of CML. In addition, the Hoechst positivity rate was lower in J774 cells treated with CAS and Marav than in untreated cells. Similar findings were observed in J774 cells treated with both CAS and Marav (CAS+Marav). Meanwhile, crystal violet positivity in the upper compartment was increased in CAS, Marav, and CAS+Marav treatment groups than in the non-treatment group (Figure 3A). The number of Hoechst-positive J774 cells was lower by approximately 40-45% in the CAS and Marav groups than in the LPS group. Further, the number of Hoechst-positive cells was also lower by 38-40% in the CAS+Marav group than in the LPS group (Figure 3B). However, there was no significant difference in Hoechst-positive J774 cells among the CAS+Marav, CAS, and Marav groups.

To investigate the migration of monocyte-derived macrophage into brain parenchymal tissue under inflammatory conditions, we used CCR2::RFP mice to observe the monocyte-derived macrophage migration at the inflammation site. Two-photon intravital imaging showed CCR2::RFP cells were migrating out of dextran-stained brain blood vessels in the LPS treatment group (Figure 4A). Immunohistology indicated CAS and Marav suppressed CCR2::RFP cells in the LPS-treated parenchymal brain (Figure 4B). The number of CCR2::RFP-positive cells was lower by approximately 60-70% in the CAS, Marav, and CAS+Marav groups than in the LPS group. Among the mice treated with LPS, the CAS+Marav group showed a similar tendency in vitro (Figure 4C).

Given that immunohistology only identifies a limited area, the efficacy of CCR4 and CCR5 inhibitors on the whole brain cannot be determined. Therefore, we clarified the effect on a wider brain area and performed IVIS Spectrum and CLARITY imaging. The results of IVIS Spectrum showed stronger RFP intensity in the LPS group than in the CAS, Marav, and CAS+Marav groups (Figure 5A), with RFP intensity being approximately 50-60% lower in the CAS, Marav, and CAS+Marav groups (Figure 5B). Furthermore, CLARIITY imaging showed lower RFP positive cells in the LPS group than in the CAS, Marav, and CAS+Marav groups in parenchymal tissue of mice brain (Figure 5C). The number of CCR2::RFP-positive cells in the CAS, Marav, and CAS+Marav groups were lower by approximately 55-65% than that in the LPS group (Figure 5D). As expected, IVIS Spectrum and CLARITY imaging showed no significant difference in RFP expression between the CAS+Marav group and the CAS and Marav groups.