Inhibition Of Prmt6 Alleviates Postherpetic Neuralgia By Strengthening The Antiviral Innate Immunity Of Microglia

Postherpetic neuralgia(PHN)usually occurs three months after herpes zoster(HZ)infected with varicella zoster virus(VZV)and tends to occur in elderly people with weakened immunity.VZV infection can induce antiviral innate immunity and directly damage neurons and glial cells,causing the activation of neuroinflammatory responses.Neuroinflammation can disrupt the balance of facilitation and inhibition in pain transmission,resulting in synaptic plasticity remolding and central sensitization,which is essential for the progression of PHN.Refractory PHN can cause great decline in life quality of patients,and even produce depression,anxiety and other psychological disorders.PHN can last for more than one year for elderly patients with reduced immunity,some even more than ten years,causing serious physical,psychological and economic burden to patients.Therefore,exploring the pathogenesis and molecular targets of PHN is of great significance for clinical translation and drug discovery.VZV is not susceptible to animals,so the animal models of HZ and PHN were constructed by subcutaneous inoculation with herpes simplex virus type 1(HSV-1),which belongs to the α herpes subfamily the same as VZV.The body weight of HSV-1 mice decreased significantly in the acute phase of HZ,then gradually returned to normal after the HZ subsided,and decreased again in the chronic phase,which was consistent with the progression from HZ to PHN.To test whether HSV-1 retrograde infected dorsal root ganglion(DRG)and spinal dorsal horn,DNA electrophoresis was used to detect the presence of HSV-1 specific TK gene.It was found that TK gene was detected in DRG and spinal dorsal horn of HSV-1 infected mice,co-staining of HSV-1 with neuron marker Neu N also confirmed that HSV-1 infected neurons in DRG and spinal dorsal horn.Mechanical pain threshold results confirmed that allodynia and hypersensitivity in plantar,skin lesions and abdomen in the ipsilateral side of HSV-1 infected mice were significantly aggravated in the acute and chronic stage,suggesting the successful establishment of PHN model.Since PHN of mice on day 24 was most stable and significant,PHN mice on day 24 after HSV-1inoculation were mainly selected as research objects in the follow-up experiments.As is known to all,type I interferon(IFN-I)can inhibit virus replication,the levels of IFN-I(IFN-α and IFN-β)were used to evaluate the antiviral innate immunity.The production of IFN-I is mainly dependent on the expression of interferon-stimulated genes(ISGs)such as CXCL10 and ISG15.Q-PCR results showed that the levels of IFN-I,ISGs and inflammatory factors such as TNF-α,IL-1β in spinal dorsal horn and DRG of HSV-1 infected mice increased significantly,while neuroprotective factors such as brain-derived neurotrophic factor(BDNF),neurogranin(NG)and myelin-associated glycoprotein(MAG)decrease significantly.Transmission electron microscope also confirmed that the myelin lamellar layer of spinal dorsal horn axons becomes unhinged with demyelinating changes,mitochondrial edema and increased lysosome after HSV-1 infection.These results suggested that HSV-1 in DRG and spinal dorsal horn induced antiviral innate immunity,neuroinflammation and nerve injury.To further explore the effect of antiviral innate immunity on the transformation of acute pain to PHN,dexamethasone was administrated intraperitoneally on Day 14 day after HSV-1 inoculation for 3 consecutive days.Dexamethasone induced the early appearance and prolonged duration of PHN,aggravated PHN symptoms,reduced the levels of IFN-I and ISGs,resulting in increased TK gene and viral load,further indicating that dexamethasone increased viral load of HSV-1 by inhibiting antiviral innate immunity of spinal dorsal horn.To further explore the regulatory mechanism of antiviral innate immunity in PHN,transcriptomic sequencing of spinal dorsal horn was conducted on PHN and control mice.GO and KEGG suggested that differentially expressed genes(DEGs)were mainly enriched in antiviral innate immunity,immune regulatory pathway,cytokine production,inflammatory response,IFN-I synthesis,and response pathways that closely related to immunity and inflammation.The DEGs co-enriched in key pathways were screened,and found that Prmt6,IL-1α,CXCL10,ISG15,STING,and IRF5 acquired strong correlation with immune response and inflammatory stress,and their expressions were up-regulated in PHN.As a typical representative of the protein arginine methyl transferase(Prmts)family,Prmt6 acquires strong arginine methyltransferase activity,which mainly changes the biological activity of proteins by methylating the domains rich in glycine and arginine.Prmt6 mainly methylates the second arginine residue of histone H3 and the 29 th arginine residue of histone H2 A in the target protein to form asymmetric dimethylarginine(ADMA),so as to change the activity of target proteins and affect the function of downstream signaling molecules.Therefore,we focused on exploring the relationship between Prmt6 and antiviral innate immunity in PHN.By co-staining of Prmt6 with microglia marker(IBA-1),neuron marker(Neu N)and astrocyte marker(GFAP),we found that the co-expression of IBA-1 and Prmt6 increased in the spinal dorsal horn of PHN mice,while the co-expression of Neu N and Prmt6 did not change.There was almost no co-expression between GFAP and Prmt6,suggesting that Prmt6 in microglia was involved in PHN progression.Subsequently,costaining of HSV-1 and IBA-1 revealed that HSV-1 infected microglia in spinal dorsal horn.Furthermore,an increased number of activated microglial cells in spinal dorsal horn was observed after HSV-1 infection,providing further validation of microglia-mediated antiviral immune responses triggered by HSV-1.Since microglia are the main effector cells of immune response in central nervous system,primary microglia or BV-2 microglia cell lines are mainly used in subsequent cell experiments.By constructing Prmt6 knockout mice,we found that allodynia and hypersensitivity in plantar,skin lesions and abdomen of Prmt6 knockout mice were significantly relieved compared with control mice.Prmt6 knockout also increased the expressions of p-TBK1 and p-IRF3 in spinal dorsal horn,which were closely related to the production of IFN-I.Prmt6 knockout increased the levels of IFN-I and ISGs,leading to decreased viral load,alleviated neuroinflammation and cell death.The levels of neuroprotective factors were increased,demyelinating changes and mitochondrial edema were alleviated in Prmt6 knockout mice,further indicating that Prmt6 knockout relieved neuroinflammation and nerve damage to alleviate PHN by enhancing the antiviral innate immunity in spinal dorsal horn.Next,we obtained the primary microglia from Prmt6 knockout and control mice and cultured with HSV-1,and found that the expressions of p-TBK1 and p-IRF3,the levels of IFN-I and ISGs were all increased in Prmt6 knockout primary microglia compared with control microglia,leading to the decreased viral load,alleviated cell inflammation and cell death.Knocking down Prmt6 with si RNA in BV-2 cells also increased the expressions of p-TBK1 and p-IRF3,the levels of IFN-I and ISGs compared with control BV-2 cells,resulting in the decreased viral load,alleviated cell inflammation and cell death.However,overexpressing Prmt6 by plasmid in BV-2 cells decreased the expressions of p-TBK1 and p-IRF3,the levels of IFN-I and ISGs compared with control BV-2 cells,resulting in the increased viral load,aggravated cell inflammation and extensive cell death.These results all proved that Prmt6 aggravated neuroinflammation to promote PHN by negatively regulating the antiviral innate immunity of microglia.To further explore the mechanism of Prmt6 regulating the antiviral innate immunity in PHN,we performed transcriptome sequencing of spinal dorsal horn on Prmt6 knockout and control mice of PHN models,GO and KEGG suggested that DEGs were mainly enriched in immune regulation pathways,antiviral stress,pattern recognition receptor pathways,cytokine pathways,and other signaling pathways closely related to immunity and inflammation.Since the cGAS-STING pathway is a key pathway that mediates IFN-I production and antiviral innate immunity by recognizing exogenous viral DNA,which is consistent with the results of DEGs enrichment analysis,we focused on exploring whether Prmt6 regulated cGAS-STING pathway-mediated antiviral innate immunity.After knocking down Prmt6 with si RNA in BV-2 cells and stimulating with HSV-1,we found that the expressions of cGAS,p-STING and STING did not change,while co-immunoprecipitation found that Prmt6 interacted with STING,but not cGAS,TBK1,IRF3,proving that Prmt6 might affect STING activity rather than protein expressions.Subsequently,knocking down or overexpressing STING,accompanied with gradient overexpressing Prmt6 were carried out in BV-2 cells.Under STING knockdown in BV-2 cells,Prmt6 overexpression did not affect the expression of p-TBK,p-IRF3 and IFN-I.However,under STING overexpression,Prmt6 overexpression significantly reduced p-TBK,p-IRF3 and IFN-I in a dose-dependent manner,indicating that Prmt6 regulating antiviral innate immunity mainly relied on STING.Under the same concentration of Prmt6 overexpression,overexpressing STING significantly increased p-TBK 1,p-IRF3 and IFN-I levels,further indicating that Prmt6 regulating antiviral innate immunity negatively was highly dependent on STING.Prmt6 acquires strong arginine methyl transfer activity and can methylate the glycine and arginine-rich domains,so we further explore whether Prmt6 regulates the cGAS-STING pathway through its methyltransferase activity.By overexpressing STING and gradient overexpressing Prmt6 in BV-2 cells,ADMA formation was observed in total protein at 35 and 45 k D positions,and ADMA expression was dependent on Prmt6 concentration at 35 k D.Coimmunoprecipitation found that the protein precipitated by STING antibody also showed ADMA formation at 35 k D in a Prmt6 dose-dependent manner,indicating that STING was modified by Prmt6 methylation.Since the mutation of VLD at the 27 th and 29 th positions to KLA can cause the loss of methyltransferase activity of Prmt6,we constructed the mutant Prmt6 and overexpressed it in BV-2 cells,and found that overexpressing mutant Prmt6 did not change p-TBK1,p-IRF3 and IFN-I levels compared with control group,but increased pTBK1,p-IRF3 and IFN-I levels compared with overexpressing wild-type Prmt6,further indicating that Prmt6 regulating cGAS-STING negatively mainly depended on its methyltransferase activity.To validate the clinical translational value of targeting Prmt6 for the prevention and treatment of PHN,we performed intrathecal and intraperitoneal injections of small molecule inhibitor EPZ020411 of Prmt6 in PHN model for continuous three days.We found that both intrathecal and intraperitoneal administration of EPZ020411 significantly alleviated allodynia and hypersensitivity in plantar,skin lesions and abdomen in PHN mice.Moreover,EPZ020411 also increased the levels of IFN-I and ISGs,resulting in reduced viral load and inflammatory factors,and increased neuroprotective factors in spinal dorsal horn.Transmission electron microscope further confirmed that EPZ020411 intrathecally and intraperitoneally mitigated demyelination of axons and alleviated mitochondrial swelling in spinal dorsal horn of PHN mice,demonstrating that EPZ020411 enhanced antiviral capacity of microglia in spinal dorsal horn,thereby alleviating neuroinflammation and nerve damage and relieving PHN.In summary,HSV-1 infection induced an increase in Prmt6 expression in microglia of spinal dorsal horn.Increased Prmt6 inactivated STING by methylation modification,which inhibited the downstream phosphorylation,dimerization,and nuclear transposition of TBK1/IRF3,thereby leading to a decrease in IFN-I production.The reduced IFN-I weakened the ability to suppress and clear virus,resulting in a large number of viral replications,which directly induced neuroinflammation and nerve damage,and promoted the progression of PHN.The small molecule inhibitor EPZ020411 targeting Prmt6 acquired promising translational prospects for the prevention and treatment of PHN.This study proved the mechanism of Prmt6 regulating microglial antiviral innate immunity negatively to promote the pathological progression of PHN,which provided a new explanation and research target.This study can also be helpful for the development of new drugs targeting Prmt6 in dealing with PHN.