In vivo immune interactions of multipotent stromal cells underlie their long-lasting pain-relieving effect

Systemic infusion of bone marrow stromal cells (BMSCs), a major type of multipotent stromal cells, produces pain relief (antihyperalgesia) that lasts for months. However, studies have shown that the majority of BMSCs are trapped in the lungs immediately after intravenous infusion and their survival time in the host is inconsistent with their lengthy antihyperalgesia. Here we show that long-lasting antihyperalgesia produced by BMSCs required their chemotactic factors such as CCL4 and CCR2, the integrations with the monocytes/macrophages population, and BMSC-induced monocyte CXCL1. The activation of central mu-opioid receptors related to CXCL1-CXCR2 signaling plays an important role in BMSC-produced antihyperalgesia. Our findings suggest that the maintenance of antihypergesia can be achieved by immune regulation without actual engraftment of BMSCs. In the capacity of therapeutic use of BMSCs other than structural repair and replacement, more attention should be directed to their role as immune modulators and subsequent alterations in the immune system.


Animal models
All surgical procedures were performed under pentobarbital sodium (50 mg/kg i.p.) anesthesia.
For the rat tendon ligation (TL) model, TL was achieved via an intraoral approach as described 5 .
On the left intraoral site, a five-mm long incision was made posterior-anteriorly lateral to the gingivobuccal margin in the buccal mucosa, beginning immediately next to the first molar. The tendon of the anterior superficial part of the rat masseter muscle was gently freed and tied with two chromic gut (4.0) ligatures, 2-mm apart. The chronic constriction injury of the infraorbital nerve (CCI-ION) mouse model was produced according to Wei et al. 54 A 5-7 mm long incision was made along the gingivobuccal margin in the buccal mucosa. The ION is freed from surrounding connective tissues. At 3-4 mm from the nerve where its branches emerge from the infraorbital fissure, the ION was loosely tied with two chromic gut (4.0) ligatures, 2-mm apart.

Behavioral testing
All behavioral tests were conducted under blind conditions. Mechanical sensitivity of the orofacial region was assessed as described elsewhere 5,45 . A series of calibrated von Frey filaments were applied to the skin above the injured tendon or the corresponding contralateral side. An active withdrawal of the head from the probing filament was defined as a response.
Each von Frey filament was applied 5 times at intervals of 5-10 seconds. The response frequencies [(number of responses/number of stimuli) X100%] to a range of von Frey filament forces were determined and a stimulus-response frequency (S-R) curve plotted. After a nonlinear regression analysis, an EF 50 value, defined as the effective von Frey filament force (g) that produces a 50% response frequency, was derived from the S-R curve (Prism, GraphPad) 5 .
A leftward shift of the S-R curve, resulting in a reduction of EF 50 , occurred after TL. This shift of the curve suggests the development of mechanical hypersensitivity, or presence of mechanical 3 hyperalgesia and allodynia since there was an increase in response to suprathreshold stimuli and a decreased response threshold for nocifensive behavior.

BMSC procedures
BMSCs were obtained from donor rats as described 5 . The rats were sacrificed with CO 2 , the both ends of the tibiae, femurs and humeri were cut off by scissors. A syringe fitted with 18gauge needle was inserted into the shaft of the bone and bone marrow was flushed out with culture medium (alpha-modified Eagle medium, Gibco, Carlsbad, CA, USA; 10% fetal bovine serum, Hyclone, Logan, UT, USA). The bone marrow was then mechanically dissociated and the suspension passed through a 100-µm cell strainer to remove debris. The cells were incubated at 37°C in 5% CO 2 in tissue-culture flasks (100 X 200 mm) (Sarstedt, Nümbrecht, Germany), and non-adherent cells removed by replacing the medium. When the cultures reached 80% confluence, the cells were washed with PBS and harvested. The cell numbers were calculated by the Hemacytometer. For intravenous administration, 1.5 X 10 6 cells (1.5 M) in 0.2 ml PBS were slowly injected into one tail vein of the anesthetized animal over a 2-minute period using a 22-gauge needle. The property of expanded cells was assessed by flow cytometry with conventional markers 5 .
Cryopreserved human primary BMSC (hBMSC) were purchased (KT-002, RoosterBio, Inc., Frederick, MD, USA). hBMSC vial from liquid nitrogen was immediately thawed in 37°C water bath. Cells were removed from water bath once only a small bit of ice was remaining. Cells were aseptically transferred into 50-ml centrifuge tube, and 4 ml of culture media were slowly added. Cells were centrifuged 200Xg for 10 min and the supernatant was carefully removed without disturbing the pellet. The cells were then resuspended in 45 ml of culture media, mixed well and seeded into three T75 vessels. After reaching 90% confluence the cells were passed into 6 10-cm culture plates. The cells were collected after reaching more than 90% confluence, and 1.5 M cells infused to the animals.
For detecting the immunoreactivity with near-infrared fluorescence using the Odyssey Infrared Imaging System (Odyssey®CLx, LI-COR, Lincoln, NE), 50 µg protein samples were denatured by boiling for 5 min and loaded onto 4-20% Bis-Tris gels (Invitrogene). After electrophoresis, proteins were transferred to nitrocellulose membranes. The membranes were blocked for 1 h with Odyssey Blocking Buffer and then incubated with primary antibodies diluted in Odyssey Blocking Buffer at 4°C overnight, followed by washing with PBS containing 0.1% Tween 20 (PBST) three times. The membranes were then incubated for 1 h with IRDye800CWconjugated goat anti-rabbit IgG and IRDye680-conjugated goat anti-mouse IgG secondary antibodies (LI-COR) diluted in Odyssey Blocking Buffer. The blots were further washed three times with PBST and rinsed with PBS. Proteins were visualized by scanning the membrane with 700-and 800-nm channels. The loading and blotting of the amount of protein was verified by reprobing the membrane with anti-b-actin and with Coomassie blue staining.

Immunocytochemistry
Rat blood monocytes (2 x 10 6 cells/well) or rat BMSC (5 x 10 5 cells/well) were cultured on Nunc™ Lab-Tek™ 8 wells Chamber Slide System (Thermo Fisher Scientific). At 1 d after the seeding of cells, the culture medium was removed, and the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde in PBS for 20 min at RT. Cells were washed 3 times with PBS, permeabilized with 0.1% Triton X-100 in PBS for 20 min at RT, and then blocked with 2% BSA in PBS for 30 min at RT. After washing 3 times with PBS, monocytes or BMSC were incubated with relevant primary antibodies overnight at 4°C. All primary antibodies were diluted with 2% BSA in PBS. After three times washing with PBS, cells were incubated with appropriate secondary antibodies IgG-Cy2 and/or IgG-Cy3 (1:500) for 2 h at RT. After washing, cultures were mounted using ProLong® Gold Antifade Reagent with DAPI (Cell Signaling Technology).

Immunohistochemistry
Rats were deeply anesthetized with pentobarbital sodium (100 mg/kg, i.p.) and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The same block of caudal brainstem tissues as that for western blot was removed, post-fixed, and transferred to 25-30% sucrose (w/v) for cryoprotection. Free-floating tissue sections (30-μm thick) were incubated with relevant antibodies with 1-3% relevant normal sera and single or double-labeling immunofluorescence was performed. Double-labeling immunofluorescence was performed with Cy2 and Cy3 (1:500, Jackson ImmunoResearch) or Alexa Fluor 488 (1:500, Invitrogen Molecular Probes) and Alexa Fluor 568 (1:500) after incubation with respective primary antibodies. Biotin-SP donkey anti-rabbit IgG (1:600, Jackson ImmunoReserch) and streptavidin Alexa Fluor 568 conjugate (1:600, Invitrogen Molecular Probes) were also used in some experiments. Control sections were processed with the same method except that the primary antibodies were omitted or adsorbed by respective antigens. The tyramide signal amplification (TSA) was used for double immunofluorescence of CXCR2 with MOR, NeuN, Iba-1 and GFAP.

ELISA
Rat CXCL1 Quantikine ELISA (R&D Systems) was performed on cerebrospinal fluid collected from medium-or PRI BMSC-treated TL rats according the manufacturer's protocol.

RT 2 PCR Array
Total RNAs from primary and 20P BMSCs as well as peripheral blood monocytes derived from BMSC or culture medium-treated rats were characterized in triplicates using The Rat Inflammatory Cytokines & Receptors RT 2 Profiler TM PCR Array (PARN-011A, Qiagen) following the manufacturer's protocol. Total RNAs were extracted using mirVana™ miRNA Isolation Kit (Applied Biosystems). Purified 1 µg of total RNA was used to prepare first-strand cDNA. The array was probed according to the manufacture's instruction in the StepOnePlus TM System (Applied Biosystems). Gene expression data were analyzed with the web-based RT 2 Profiler PCR Array Data Analysis software that performed ΔΔCt based fold-change calculations from the uploaded threshold cycle data (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php?target=upload).

Proteome Profiler TM Array
Rat cytokine antibody array (ARY008, R&D Systems) was performed on serum isolated from naïve, medium-or PRI BMSC-treated TL rats according the manufacturer's protocol. The serum (0.2 ml) was added to the array membranes coated with 29 specific cytokine antibodies.
The membranes were incubated at 4°C overnight. Array images were collected and analyzed using the LI-COR Odyssey Infrared Imaging System. The relative protein levels were obtained by subtracting the background staining and normalizing to the positive controls on the same membrane. We paid special attention to the level of CXCL1 and the results of other cytokines are not shown.

RNAi
Ccl4 shRNA (Accession and 20 ml of minimum essential medium (MEM) alpha medium (Gibco) with 10% fetal calf serum was injected i.p. at 30 minutes before isolation. Peritoneal cells were collected with a 3ml syringe, plated on culture dishes, and kept at 37°C overnight.

Monocytes isolation
Heparinized rat blood was overlaid on Ficoll solution (Ficoll-Paque Plus, GE Life Sciences) and (TreeStar, Inc., Ashland, OR). The specific staining was measured from the cross point of the isotype control with a specific antibody graph.

Brainstem microinjections 5
Rats were anesthetized with 2-3% isoflurane in a gas mixture of 30% O 2 balanced with 70% nitrogen and placed in a Kopf stereotaxic instrument (Kopf Instruments, Tujunga, CA). A midline incision was made after infiltration of lidocaine (2%) into the skin. A midline opening was made in the skull with a dental drill for inserting an injection needle into the target site. The coordinates for the rostral ventromedial medulla (RVM) were: 10.5 mm caudal to the bregma, 13 midline and 9.0 mm ventral to the surface of the cerebellum 66 . Microinjections were performed by delivering drug solutions slowly over a 10-min period using a 500 nl Hamilton syringe with a 32-gauge needle. The injection needle was left in place for at least 15 min before being slowly withdrawn. The wound was closed and animals were returned to their cages after recovering from anesthesia. For histology verification of the injection site, 30-µM coronal brainstem sections were stained with Neurotrace TM 500/525 Green fluorescent Nissal Stain (Invitrogen) (1:500 for 20 min).

Supplemental Figure Legends
Supplementary Fig. 1. a,b. Effect of a neutral opioid receptor antagonist 6-β-naltrexol on BMSC-produced attenuation of mechanical hypersensitivity. a. BMSC attenuated hyperalgesia in rats after tendon ligation injury (TL). EF 50 , the von Frey filament force (g) that produces a 50% response, was a measure of mechanical sensitivity. Primary BMSCs were infused i.v.