DOK3 is involved in microglial cell activation in neuropathic pain by interacting with GPR84

Adaptor molecule downstream of kinase-3 (DOK3) is a vital regulator of innate immune responses in macrophages and B cells, and G-protein-coupled receptor 84 (GPR84) is significant in mediating the biosynthesis and maintenance of inflammatory mediators that are induced by neuropathic pain in microglia. In the present study, we determined the role of DOK3 in activating microglia-induced neuropathic pain and investigated the underlying mechanisms associated with GPR84. We found that knockdown of DOK3 in microglial cells dramatically reduced the levels of inflammatory factors, and we uncovered a physical association between DOK3 and GPR84 in the induction of inflammatory responses. We also observed that neuropathic pain and inflammatory responses induced by chronic constriction injury (CCI) of the sciatic nerve or intrathecal injection of a GPR84 agonist were compromised in DOK3-/- mice in vivo. Finally, enforced expression of DOK3 provoked inflammatory responses, and administration of pregabalin relieved neuropathic pain via inhibition of DOK3 expression. In conclusion, DOK3 induced neuropathic pain in mice by interacting with GPR84 in microglia. We hypothesize that targeting the adaptor protein DOK3 may open new avenues for pharmaceutical approaches to the alleviation of neuropathic pain in the spinal cord.

anesthetized with sodium pentobarbital (50 mg/kg body weight, i.p.), and the skin on the left mid-thigh was shaved and incised (5-8 mm). The proximal and distal parts of the biceps femoris muscle were separated to expose the common sciatic nerve, and the nerve was dissected away from surrounding tissues. Three ligatures of 6-0 silk were tied around the nerve just proximal to the trifurcation such that 1/3 to 1/2 of the nerve was held within the ligature. The desired degree of constriction retarded-but did not arrest-circulation through the superficial epineurial vasculature; and sometimes produced a small, brief twitch in the muscle surrounding the exposure. For the sham-operated controls, the mice underwent the same procedure without constriction of the nerve. The incision was closed in layers and a single dose of buprenorphine (0.05 mg/kg, s.c.) was administered. The mice were placed in clean cages and carefully monitored until fully awake [1][2][3].

Intrathecal injection and drug administration
Before intrathecal injection, the constructed cDNA plasmid encoding DOK3 protein, and non-targetingscrambled control vector were mixed with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). We mixed 1 μg of plasmid with 1 µL of reagent for in vivo experiments, and then allowed complex formation to occur for 10 min. Intrathecal injection was performed on the anesthetized mice as follows [4,5]. In brief, an injection (5 μL) into the subarachnoid space between the L5 and the L6 vertebrae was performed using a 27gauge needle; and entry of the needle was confirmed by the presence of a tail flick. Within each individual study, 1 group of 6 mice was given intrathecal injections of plasmid cDNA daily for 3 days; and simultaneously, a daily intragastric injection of pregabalin [S-(+)-3-isobutylgaba] in capsules for 10 days. Capsules were dissolved in sterile NaCl (0.9% isotonic saline) immediately before use. For the shamgavaged controls, mice underwent the same procedure with only sterile NaCl. Dosing volumes were 30 mg/kg body weight, in accordance with a previous study [6].

Behavioral testing
For behavioral analysis, animals were habituated to the testing environment daily for at least 2 days before baseline testing. They were then placed in boxes on an elevated metal mesh floor and allowed to habituate for 30 min. During testing, a probe was pressed against the lateral plantar surface of the hind paws with sufficient force to evaluate the paw-withdrawal mechanical threshold (PWMT); a positive response was noted when the paw immediately withdrew. The procedure was repeated 5 times at least 5 min apart, and the average value was used as a variable [7]. Walk-gait pattern was assessed as an index of motor function. Only animals that indicated a normal gait, without any foot deformities, were used for the following experimental procedures. Paw withdrawal latency (PWL) to heat in mice was determined with the BME-410C thermal analgesia tester (CAMS). Before testing, the mice were randomly placed in testing cubicles and acclimated for 30 minutes, and then the thermal analgesia tester was calibrated to produce a paw-withdrawal latency of 8-12 s in naive control mice. Radiant light was directed onto the center of the hind paw through the glass plate, and when the mouse lifted or licked his hind paw, PWL was measured automatically. A 25-s cutoff period was used to protect hind-paw tissue. Each hind paw was stimulated 3 times with a 5-min interval, and the mean withdrawal latency was recorded.

Microglial cell culture and treatment
BV2 microglia were maintained in MEM supplemented with 10% endotoxin-free fetal bovine serum, and incubated at 37° C in a humidified atmosphere of 5% CO2 and 95% air. We grew cells to 70%-80% confluency before being treated with different agents. DOK3 shRNA lentiviral particles and negative control shRNA were synthesized by Shanghai Genechem Co., Ltd; and shRNA lentiviral particles were used according to the manufacturer's instructions for transfection. Briefly, microglial cells were cultured to 50% confluency. The cells were infected with lentiviruses according to the recommended multiplicity of infection at approximately 10, and after 72 h, lentivirus infectionstable clones expressing the DOK3-specific shRNA were selected with puromycin (5 µg/ml).
Pregabalin powder was dissolved in phosphate-buffered saline (PBS). BV2 microglia were pre-treated with pregabalin at a concentration of 10 µg/ml for 2 hour, and then cells were incubated with LPS (1 µM) for 12 hours. The control groups were incubated with PBS without pregabalin or LPS, or with neither. The concentration of pregabalin was chosen according to a previous report [8].
After the behavioral and pain tests, mice were overdosed with sodium pentobarbital (50 mg/kg, i.p.) and perfused transcardially with 0.9% saline, followed by fixation with 4% paraformaldehyde in 0.1 M phosphate buffer. Lumbar spinal cords were dissected and assayed for DOK3, GPR84, Iba-1, p-p38, and CD11B according to previous studies [7]. Tissues were fixed in the same fixative overnight at 4° C, and then dehydrated and embedded in paraffin. A series of 4-µm paraffin sections were cut using a rotary microtome, and we heated the sections at 65° C for at least 2 h and then deparaffinized them. Antigen retrieval was accomplished with citrate buffer in a microwave oven at 92° C-98° C for 15 min. The sections were incubated separately with primary antibody (anti-Iba-1 antibody, 1:200, Abcam; anti-P-p38 antibody, 1:100, Santa Cruz; anti-CD11B antibody, 1:100, Abcam; or anti-GPR84 antibody, 1:100, Biorbyt) at 4° C overnight. The sections were then incubated using a specific secondary antibody for 2 h at room temperature, and DAB substrate solution and hematoxylin were used to develop color. As for immunofluorescence, we incubated sections with fluorescence-conjugated secondary antibody for 2 h, and nuclei were stained with 4′,6-diamidino-2-phenylindole. Labeled sections were examined under a Leica Quantimet 550 DMRXA automated research microscope (GER) and analyzed using IPP.6.