DOK3 Is Involved In Microglial Cell Activation In Neuropathic Pain By Interacting With GPR84

BackgroundNeuropathic pain—characterized by spontaneous pain and hyperalgesia to normally innocuous stimuli—is a refractory clinical problem and reflects aberrant excitability of the nervous system,with multiple anatomical and functional alterations following peripheral nerve injury.In the central nervous system(CNS),microglial cells are regarded as resident macrophages that originate from primitive macrophages of the yolk sac.These cells are widely distributed throughout the CNS and monitor the local environment to allow a rapid response to threats from a wide range of stimuli In the case of neuropathic pain,microglial molecules vary with modulations in the IFN-y/IFN-y R system;changes in toll-like receptors 2,3,or 4;upregulation of purinergic P2X4 receptors;and modifications of intracellular signaling molecules(mitogen-activated protein kinases[MAPKs]).In addition,microglia play a crucial role in the interaction with neighboring neurons in the development of pain hypersensitivity.Therefore,spinal microglia constitute a promising target in reversing disordered functioning of the nervous system,and it is thus essential to uncover the mechanisms of pain hypersensitivity to better understand microglial molecular functions and develop new approaches for neuropathic painAs molecular scaffolds,adapter proteins precisely organize positive and negative effectors into signaling complexes.A typical adaptor molecule is the DOK3(downstream of kinase-3)protein,which is preferentially expressed in immune cells such as macrophages and B cells.To the best of our knowledge,DOK3 has been principally implicated in negative-feedback regulation,and involved in facilitating or sustaining the activation of inhibitory molecules in B cells.As reported previously,overexpression of DOK3 increased the anti-inflammatory effects of vitamin B6 in macrophages,and degradation of DOK3 in TLR9 signaling increased the production of IL-6 and TNF-? in macrophages.Intriguingly,there is still controversy as to whether the primary function of some adapters is to exert positive or negative effects.With respect to synapse-associated proteins(SAP),a facilitative or inhibitory role depends upon cell type or condition.Investigators have reported that the adapter protein DOK3 plays an essential role in the evolution of B cells,and that the generation of plasma cells(PCs)was significantly attenuated in DOK3-/-mice.In addition,upon viral influenza infection,the synthesis of IFN-? was impaired in DOK3-deficient macrophages.DOK3 also plays a critical role in TLR3 signaling in macrophages.Collectively,these results are credible,although apparently contradictory in theory;and further work is thus required to provide reasonable explanations for the seemingly incongruous effects of the adapter molecule DOK3.As a membrane-bound receptor,GPR84 is markedly upregulated in macrophages and microglia in mediating the production and maintenance of inflammatory mediators in neuropathic pain,and in mice suffering from endotoxemia or multiple sclerosis,microglia express GPR84 in a strong and sustained manner.GPR84 was also a potent target in the treatment of inflammation-associated disease.Understanding mechanisms that govern the inhibition of inflammatory responses is important for treating neuroinflammation that can give rise to serious issues of neuropathic pain.Although microglia originate from macrophages,the role of DOK3 in microglia remains unclear presently,and there is a paucity of evidence regarding the association between DOK3 and GPR84 proteins.Based upon the above analysis,we speculated that DOK3 and GPR84 would interact in pain and inflammatory diseases in microglia,potentially providing novel insights into the treatment of neuropathic pain.PART I DOK3 is involved in inflammatory responses in microglial cells by interacting with GPR84Objective1.To first determine a role for DOK3 in microglial inflammatory responses.2.To research whether GPR84-increased inflammatory response in microglia was due to a direct effect of DOK3.3.To explore the relationship between DOK3 and GPR84 in microglial cells.Methods1.Microglia were infected with a DOK3-specific short hairpin RNA(shRNA)to establish stably down-regulated DOK3 expression.The efficacy of lentivirus expressing shRNA by targeting DOK3 mRNA was determined by Western blot and Real-time PCR analysis.2.Total cell lysates of DOK3-shRNA and Scr-shRNA microglia were used to determine the levels of mRNA for DOK3,TNF-?,IL-1?,IL-6,iNOS,P38,CD68,CD11B,Arg1,and CD206 by quantitative reverse transcription-polymerase chain reaction(RT-qPCR).3.Microglia were incubated with 6-n-octylaminouracil(6-OAU)for 120 min at different concentrations.DOK3 mRNA levels at different time-points were determined by RT-qPCR.DOK3,p-p38,and Iba-1 protein levels were determined using western immunoblotting analysis.4.Lentivirus-infected microglia were exposed to 6-OAU;and TNF-?,IL-1?,and IL-6 mRNA levels were determined by RT-qPCR.5.Microglia were pretreated with LPS for 4 or 8 h,and the levels of GPR84 and DOK3 protein were assayed by immunofluorescence analysis.Microglia were pretreated with 6-OAU for 1 or 2 h,and the levels of GPR84 and DOK3 protein were assayed by immunofluorescence analysis.6.Microglia were pretreated with LPS for 4 h,and the levels of GPR84 in total cell lysates were determined by co-immunoprecipitation and western blotting.Purified recombinant human GST or GST-DOK3 proteins were incubated with microglia for 2 h in reaction buffers.7.Total cell lysates or reaction products were then used in GST-pulldown assays and western blotting to determine the levels of GPR84.Results1.Identify the efficacy of lentivirus interference targeting DOK3The designs of shRNA targeting DOK3 mRNA were depicted as Figure 1A.To evaluate the lentivirus mediated shRNA against DOK3,DOK3 protein and mRNA levels of the Lv-shRNA-DOK-#3 group were remarkably decreased compared with Lv-shRNA-NC as shown in Figure 1B and C.Therefore,Lv-shRNA-DOK-#3 was chosen to silence DOK3 gene in all the following experiments.2.Knockdown of DOK3 contributes to a reduction in inflammatory factors in microgliaDOK3 protein and mRNA levels were dramatically decreased by the Lv-shR NA-DOK-#3 compared with Lv-shRNA-NC.DOK3 mRNA levels decreased by almost 50%in DOK3-shRNA cells compared with vehicle-treated microglia(Scr-shRNA),and protein levels were confirmed by western blotting analysis.Several inflammatory mediators associated with nociceptive or inflammatory signaling showed a greater down-regulation in DOK3-shRNA than in Scr-shRNA microglia,including TNF-?,IL-1?,IL-6,P38,iNOS,and CD68.Conversely,Argl and CD206 were induced to a greater degree in the DOK3-shRNA microglia.The levels of CD11B mRNA were only slightly changed,with no significant difference between the 2 groups.3.GPR84 induces inflammatory responses via DOK36-OAU in a time-dependent fashion increased DOK3 mRNA and protein at a concentration of 1 ?M and increased DOK3 in a concentration-dependent fashion after 60 min of exposure.There was an equivalent increase in both p-p38 and Iba-1 protein expression as DOK3 protein was upregulated after 6-OAU treatment,and this was commensurate with increased levels of IL-I ?,TNF-?,and IL-6After treatment with 6-OAU,the capacity to synthesize the inflammatory mediators TNF-?,IL-1?,and IL-6 was dramatically impaired under GPR84-activated conditions in DOK3-shRNA microglia compared with the Scr-shRNA group:TNF-?,IL-1?,and IL-6.Furthermore,6-OAU increased DOK3 mRNA levels,but there was no significant change in GPR84 protein expression in the 2 groups.We speculate that DOK3—as a downstream target of the GPR84-signaling pathway-is an important component in immune regulation by microglia4.Incubation of microglia with LPS or GPR84 agonist induces a physical association between DOK3 and GPR84LPS stimulated the translocation of DOK3 from the cytosol to the plasma membrane in microglia.Moreover,incubation with LPS attenuated microglial cell viability and rapidly increased TNF-?,IL-6,and IL-? mRNA levels and decreased Argl expression.the fluorescence intensity of GPR84 and DOK3 in the plasma membrane was significantly enhanced after LPS treatment compared with the control group.GPR84 and DOK3 were readily co-localized in microglia,and the merged fluorescence was progressively increased with LPS incubation.Immunofluorescence(IFC)analysis indicated that the levels of DOK3 and merged fluorescence were higher in the plasma membrane after 6-OAU treatment.Western blotting analysis showed that GPR84 was physically associated with DOK3 under either physiologic or LPS-stimulated conditions.Furthermore,Purified recombinant GST-DOK3 fusion protein was incubated with WCL in vitro.ConclusionsDOK3 is essential for up-regulating TNF-?,IL-1?,and IL-6 expression in microglia so as to promote inflammatory responses under physiologic or pathologic conditions.In its absence,the response of microglia cells to inflammatory insult was significantly impaired.DOK3 is involved in microglial inflammatory responses by a physical association with GPR84PART ? DOK3 and GPR84 are involved in neuropathic pain in spinal cord microglial cellsObjective1.To assess the role of DOK3 in CCI-induced neuropathic pain in WT and DOK3-/-mice.2.To determine the effect of DOK3 on GPR84-induced inflammatory responses in mice.3.To research whether pregabalin prevented the development of inflammatory states by inhibiting DOK3.Methods1.B129 and DOK3-/-mice underwent surgery for chronic constriction of the sciatic nerve to establish the CCI model.2.CCI-induced mechanical allodynia and GPR84 activation-induced mechanical allodynia were determined by calculating PWMT.3.The mRNA levels of DOK3,TNF-?,IL-1?,and IL-6 in mouse lumbar spinal cord were determined by RT-qPCR.Iba-1,P-p38,and GPR84 in lumbar spinal cords of mice were assayed by immunofluorescence.DOK3 protein levels were determined by western blotting.4.B129 and DOK3-/-mice were treated with 6-OAU for 0,1,2,and 4 h via intrathecal injection.5.B129 mice were treated with intrathecal injections of plasmid cDNA for 3 days and fed pregabalin simultaneously for 2 weeks.Results1.CCI-induced Neuropathic Pain and Inflammatory Responses are Alleviated in DOK3-/-MiceThe PWMT was significantly decreased from 3 to 21 days postoperatively compared with the WT controls.DOK3-/-mice exhibited normal mechanical pain thresholds equivalent to those of WT control mice;but after CCI surgery,DOK3-/-mice showed attenuated allodynia compared with the WT mice.There were similar changes in the thermal paw withdrawal latency.DOK3 mRNA levels were significantly upregulated in WT mice after CCI surgery.WT and DOK3-/-mice showed 2.2-and 1.47-fold increases in TNF-? mRNA expression,1.81-and 1.37-fold increases in IL-1? mRNA,and 1.65-and 1.64-fold increases in IL-6 mRNA expression compared with sham controls,respectively.Moreover,CCI-induced upregulation of GPR84 and CD11B mRNA levels was dramatically inhibited in DOK3-/-mice.Immunofluorescence(IFC)analysis indicated that the levels of Iba-1,p-p38,and GPR84 were increased in WT and DOK3-/-mice after CCI;but in DOK3-/-mice,they were significantly suppressed before and after surgery.2.GPR84 activation-induced mechanical allodynia and inflammatory responses are compromised in DOK3-/-miceAfter 6-OAU treatment,mechanical pain thresholds of both WT and DOK3-/-mice were reduced rapidly;however,there were significant differences between genotypes at 2 and 4 h.As expected,the levels of DOK3 mRNA were notably increased at 2 h,and this was further confirmed by western blot analysis in mouse spinal cord.In addition,after 6-OAU treatment,WT and DOK3-/-mice showed 2.8-and 1.65-fold increases in IL-1?,4.0-and 1.79-fold increases in TNF-?,and 1.9-and 1.4-fold increases in IL-6 mRNA expression in spinal cords compared with sham controls,respectively.IFC analysis demonstrated that the levels of CD11B were increased in WT and DOK3-/-mice after GPR84 activation;however,in DOK3-/-mice,they were substantially inhibited before and after treatment,consistent with mRNA levels.DOK3 was dominantly expressed in microglia in the mouse spinal cord,and the co-localization of DOK3 and GPR84 was confirmed.3.Administration of Pregabalin Attenuates Neuropathic Pain Induced by Enforced Expression of DOK3 In VivoIntrathecal injection of the DOK3 cDNA plasmid into B129 mice effectively induced neuropathic pain from days 3 to 7,which recovered to basal levels on day 14.Furthermore,the overexpression of DOK3 in spinal cord was confirmed by immunohistochemical and qPCR analyses.DOK3 overexpression activated microglial cells in the spinal cord,but no activation of astrocytes was observed.Consistent with augmented DOK3 levels,the levels of TNF-?,IL-1?,and IL-6 were markedly increased compared to vector plasmid-treated mice;as were levels of CD11B.Pregabalin significantly decreased DOK3 mRNA expression,that the inflammatory marker IL-1? was decreased by 41%,that TNF-? decreased by 33%,and that IL-6 decreased by 35%compared with plasmid cDNA-treated mice.Exposure to pregabalin caused an effective inhibition of the LPS-induced increase in DOK3 levels.However,pregabalin had no effect on basal expression of DOK3 in BV2 microglia.In summary,these data provided evidence that pregabalin relieved neuropathic pain via inhibition of DOK3 expression in mice.Thus,targeting the DOK3 gene may in the future constitute a gene-therapy approach to alleviating diseases and injury-induced neuropathic pain in the spinal cord.ConclusionsCCI-induced Neuropathic Pain and Inflammatory Responses are Alleviated in DOK3-/-Mice,GPR84 activation-induced mechanical allodynia and inflammatory responses are compromised in DOK3-/-mice,administration of Pregabalin Attenuates Neuropathic Pain Induced by Enforced Expression of DOK3 In Vivo.DOK3 plays a positive role in modulating inflammatory actions and microglial activation-induced neuropathic pain.Pregabalin relieved neuropathic pain via inhibition of DOK3 expression in mice.