Upregulation of lysine‐specific demethylase 6B aggravates inflammatory pain through H3K27me3 demethylation‐dependent production of TNF‐α in the dorsal root ganglia and spinal dorsal horn in rats

Abstract Aims Lysine‐specific demethylase 6B (KDM6B) serves as a key mediator of gene transcription. It regulates expression of proinflammatory cytokines and chemokines in variety of diseases. Herein, the role and the underlying mechanisms of KDM6B in inflammatory pain were studied. Methods The inflammatory pain was conducted by intraplantar injection of complete Freund's adjuvant (CFA) in rats. Immunofluorescence, Western blotting, qRT‐PCR, and chromatin immunoprecipitation (ChIP)‐PCR were performed to investigate the underlying mechanisms. Results CFA injection led to upregulation of KDM6B and decrease in the level of H3K27me3 in the dorsal root ganglia (DRG) and spinal dorsal horn. The mechanical allodynia and thermal hyperalgesia following CFA were alleviated by the treatment of intrathecal injection of GSK‐J4, and by microinjection of AAV‐EGFP‐KDM6B shRNA in the sciatic nerve or in lumbar 5 dorsal horn. The increased production of tumor necrosis factor‐α (TNF‐α) following CFA in the DRGs and dorsal horn was inhibited by these treatments. ChIP‐PCR showed that CFA‐induced increased binding of nuclear factor κB with TNF‐α promoter was repressed by the treatment of microinjection of AAV‐EGFP‐KDM6B shRNA. Conclusions These results suggest that upregulated KDM6B via facilitating TNF‐α expression in the DRG and spinal dorsal horn aggravates inflammatory pain.


| INTRODUC TI ON
Chronic inflammatory pain, which lasts for 6 weeks or longer, is mainly treated by nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids in clinics, and these methods are limited due to their side effects. 1 Despite the number of studies have been performed, scientific understandings of the original mechanisms of inflammatory pain are still not fully understood. 2It has been well documented that proinflammatory mediators which released at sites of inflammation are capable of sensitizing pain-sensing primary afferent neurons, and consequently, lead to a persistent increase in pain-related synaptic transmission in the spinal dorsal horn. 3However, the regulation of these proinflammatory mediators' expression remains largely unclear.
5][6][7][8] Lysine-specific demethylase 6B (KDM6B), with another name JMJD3, is a member of the JmjC histone demethylases family that specifically demethylates the trimethylated lysine at position 27 of H3 (H3K27me3) protein to regulate correlated gene expression. 9K27me3 is associated with a condensed chromatin conformation and exerts a repressive epigenetic mark. 10The upregulation of KDM6B demethylates H3K27me3 to H3K27me2 or H3K27me1, thus leading to the removal of the methylation mark from H3K27 and actives gene transcription. 11,12Compelling evidence suggests that KDM6B is involved in the regulation of cell differentiation and inflammation by targeting distinct transcription factors which control cytokine gene expression. 10,13,146][17] Recent studies reveal that abnormal expression of KDM6B in the brain contributes to the pathogenesis of alcohol dependence, 18 cocaine reward memory, 19 and increased susceptibility to depression 20 by epigenetic regulation of proinflammatory signaling pathway.Using lumbar 5 spinal nerve ligation (SNL), we found that upregulated KDM6B in the dorsal root ganglia and spinal dorsal horn contributes to the development and maintenance of neuropathic pain via facilitating the expression of IL-6. 21Recently, Achuthan reported that granulocyte-macrophage colony-stimulating factor (GM-CSF) via enhancing JMJD3 demethylase activity mediates arthritic pain. 22But the underlying mechanisms are incompletely understood.In the current study, the inflammatory pain was conducted by intraplantar injection of complete Freund's adjuvant (CFA) in male rats.The expression and the role of KDM6B in the pathogenesis of abnormal pain were investigated.

| Animals
Male Sprague-Dawley rats weighing 180-250 g were used.The rats were housed in separate cages with free access to food and water.The room temperature was kept at 23 ± 2°C under a 12:12-h light-dark cycles.The animals were purchased from the Zhengzhou Laboratory Animal Center of Zhengzhou University in China.All animal experimental procedures were approved by the Animal Care and Use Committee of Zhengzhou University, China, and were carried out in accordance with the guidelines of the National Institutes of Health on animal care and the ethical guidelines for investigation of experimental pain in conscious animals.

| Intraplantar injection of complete Freund's adjuvant for inflammatory pain
The inflammatory pain was conducted as described previously. 23After transient anesthesia, a single hypodermic injection of 100 μL complete Freund's adjuvant (CFA) was performed in the left plantar hind paw.
For the control group, an equivalent volume of sterilized normal saline was injected in the same way in rats.

| Pain-related behavioral test
The behavioral tests were performed followed our formerly described methods. 24In brief, each animal was loosely adapted in a plastic box on a metal mesh for 3 consecutive days (15 min/day) before the baseline measurement.The paw withdrawal threshold (PWT) was identified to evaluate the mechanical sensitivity by treating the plantar surface of the hind paw with von Frey hairs, and 50% PWT was determined based on the up-down method. 25The evaluation of heat hypersensitivity was assessed by paw withdrawal latency (PWL) using a plantar analgesia tester (7370, Ugo Basile) according to the protocols published by Hargreaves et al. 26 All the evaluators were blinded to the experimental design.

| Intrathecal catheterization and drugs delivery
Drugs were delivered by intrathecal (i.t.) injection to rats.The intrathecal catheterization was performed as described previously. 27One week after the surgery, the rats were subjected to i.t.injection of GSK-J4, a specific inhibitor of KDM6B, which was performed 30 min before intraplantar injection of CFA and once daily thereafter for 3 days.The doses of GSK-J5 (5, 25, and 50 μg/10 μL, TOCRIS, 4594/10) used in the current experiment were based on a previous study. 21
Four weeks later, the CFA injection in the plantar was carried out on the AAV transfected side.

| RNA extraction and real-time quantitative polymerase chain reaction (qPCR)
qPCR was performed following the method described previously. 24,29ter the rats were sacrificed by decapitation, the L4/5 DRGs and spinal dorsal horn were harvested.Total RNA was extracted via the Trizol method.Reverse transcription was performed using oligo-dT primers and PrimeScriptII RTase (TAKARA) according to the manufacturer's protocol.Each sample was run in triplicate in a 20 μL reaction volume which contains 10 μM each of forward and reverse primers, 10 μL of SYBR Green qPCR Super Mix (Invitrogen), and 25 ng of cDNA.Reactions were performed in an Applied ABI QuantStudio 3 Real-Time PCR System.The relative expression of KDM6B and TNFα mRNA was quantified through the 2 −ΔΔCT method.The ratspecific primer sequences of KDM6B, TNFα, and GAPDH were listed in Table 1.

| Chromatin immunoprecipitation (ChIP) assays
ChIP assay was performed with a ChIP Assay Kit (Millipore, Catalog # 17-295) following our described method previously. 21,29In brief, the homogenized solution from L4-5 DRGs or spinal dorsal horn was cross-linked with 1% formaldehyde at 37°C for 10 min and terminated by the addition of 125 mM glycine.Sonication conditions to the lysed sample were tested to yield DNA fragments averaging 600-800 bp as assessed by agarose gel electrophoresis.After precleaned with protein G agarose, the samples were subjected to immunoprecipitation with 10 μg rabbit anti-NF-κB p-p65 (Invitrogen) or normal rate IgG, which served as negative control, at 4°C overnight.Ten percent of the sample was used for immunoprecipitation as the input.The precipitated protein-DNA complexes were eluted and purified and subjected to PCR for amplification of the TNFα promoter fragments, and the protein was subjected to Western blotting to examine the level of H3K27me3 at the NF-κB-binding site.
The binding sites of NF-κB in the promoter region of TNFα were predicted from the PROMO (http://alggen.lsi.upc.es) and JASPAR databases (http://jaspa r2016.genereg.net).The primers were designed to amplify NF-κB p65-bound DNA fragments (−1 to −1800 bp of the TNFα promoter region) and used to detect the TNFα promoter gene by ChIP-PCR.All the primers were listed in Table 2.

| Statistical analysis
All data are presented as the means ± SD and analyzed with GraphPad Prism 8.2.1 (GraphPad).Sample sizes per group were predicted based on a power analysis (G*Power 3.0.10)and the experience of our previous studies. 21,29All data were subjected to tests for normality by Shapiro-Wilk test.If the Data exhibit normal distribution, the TA B L E 1 Sequences (5′-3′) of primers used.

| Intrathecal injection of GSK-J4 alleviated mechanical allodynia and thermal hyperalgesia and prevented increase in the expression of TNFα in the DRG and spinal dorsal horn following CFA injection
It has been reported that GSK-J4 worked as a specific inhibitor of KDM6B in the treatment of inflammatory diseases. 32,33In the current study, the repeat intrathecal (i.t.) injections of GSK-J4 at doses study, we detected a significant increased expression of TNFα, which peaked at 6 h and lasted to day 3 in the DRG (Figure 3E).In the dorsal horn (Figure 3F), the increased production of TNFα occurred at 6 h, peaked on day 1, and persisted to day 7 following CFA injection (*p < 0.05, **p < 0.01, ***p < 0.001 vs. control group).
However, these increases in TNFα were repressed in both the DRG (Figure 3G) and dorsal horn (Figure 3H

| The cell-type of TNFα-expressing cells in the DRG and spinal dorsal horn following CFA injection
To observe the TNFα-expressing cells in the DRG and spinal dorsal horn, the double immunofluorescence was performed.The results showed that the TNFα expressed (Figure 5A-E

| The binding of NFκ B with TNFα promoter in the DRG and spinal dorsal horn depended on the demethylated activity of KDM6B following CFA injection
Previous study has shown that the LPS-induced increased expression of TNFα is inhibited by the treatment of GSK-J4. 34,35In the

| DISCUSS ION
Previous studies have implicated a critical role of KDM6B in the pathogenesis of inflammatory diseases. 13Herein, we demonstrated that KDM6B was involved in the development of inflammatory pain.
Intraplantar injection of CFA resulted in significant increase in the production of KDM6B and decrease in the level of H3K27me3 in both the DRG and spinal dorsal horn.The CFA-induced mechanical allodynia and thermal hyperalgesia were alleviated by i.t.injection of GSK-J4 or by microinjection of AAV-EGFP-KDM6B shRNA in both the sciatic nerve and L5 spinal dorsal horn.Mechanistically, the upregulated KDM6B promoted the binding of NF-κB with TNFα promoter via inducing demethylation of H3K27me3.And this combination enhanced TNFα expression in the DRG and dorsal horn following CFA injection.
These observations provided new evidence for further elucidating the mechanism of inflammatory pain.
Although the importance of epigenetically regulated gene expression in the induction and maintenance of inflammatory diseases has been well documented in the past decade, 36,37 the role of some enzymes involved in epigenetics in the pathogenesis of chronic pain still needs to be verified. 38H3K27me3 is a critical epigenetic event frequently associated with gene repression.KDM6B is a member of JmjC histone demethylases family that specifically demethylate the trimethylate lysine at position 27 of H3 protein to regulate correlated gene expression 39,40 and involves in the proinflammatory gene production. 13,17,22,35,41In our current study, we found that intraplantar injection of CFA led to increase in the expression of KDM6B and a reduction in the level of H3K27me3 in both the DRG and spinal dorsal horn.It has been demonstrated that the demethylation of H3K27me3 is one of the key steps to trigger gene expression via chromatin remodeling. 42The establishment of chronic pain, especially the peripheral sensitization in the DRG 43 and central sensitization in the spinal dorsal horn, 44 needs variety of de novo proteins to be produced.Therefore, we speculated that the upregulated KDM6B might be involved in the genesis of inflammatory pain via triggering gene expression.Our pain-related behavioral tests showed a significant alleviative mechanical allodynia and thermal hyperalgesia following Achuthan and colleagues reported that GM-CSF via enhancing JMJD3 activity mediates arthritis.Treatment with GSK-J4 reduces the production of CCL17 and alleviates arthritic pain. 22These findings agree with our present study.
Compelling evidence has highlighted the proinflammatory role of KDM6B in various inflammatory diseases by regulating the expression of several NF-κB-dependent cytokines. 13,356][47][48] In the current study, we detected an increased production of TNFα in both the DRG and spinal dorsal horn following CFA injection.But this increase was suppressed by the treatments of GSK-J4 and AAV-EGFP-KDM6B shRNA.Previous studies have shown that peripheral nerve injuryand inflammation-induced TNFα expression distributes not only in glial but also in neuronal cells in the DRG and dorsal horn. 49In the present study, we found that TNFα expressed in the same

3 | RE SULTS 3 . 1 |
Figure1K-M).Images of immunofluorescence staining showed a clear increased immunoreactivity of KDM6B protein in the ipsilateral dorsal horn than that of contralateral dorsal horn 1 day after CFA injection (Figure1N).The quantitative data showed this increase started at 6 h and persistence over 10 days after CFA injection (**P < 0.01, ***P < 0.001 vs. naive group, Figure1O-S).The Shapiro-Wilk test was used for normality, nonparametric Kruskal-Wallis test was used for difference in the

of 5 ,
25, and 50 μg were performed to examine the role of KDM6B in the CFA-induced inflammatory pain.Results of behavioral tests showed that GSK-J4 treatment prevented the reductions in PWT and PWL after CFA injection.Compared to CFA plus vehicle (Veh: i.t.normal saline) group, the i.t.injection of GSK-J4 resulted in significant increases in PWT (Figure 3A) and PWL (Figure 3B) in a dosedependent manner (*p < 0.05, **p < 0.01, and ***p < 0.001 vs. CFA or CFA + Vehicle group; ## p < 0.01, ### P < 0.001 vs. baseline).To further observe the role of KDM6B in the established inflammatory pain, the repeat i.t.injections of GSK-J4 (50 μg/10 μL) were started on day 3 after CFA treatment.The results showed that CFA-induced reductions in PWT (Figure 3C) and PWL (Figure 3D) were partially reversed by the treatment of GSK-J4 (*p < 0.05, **p < 0.01, ***p < 0.001 vs. CFA + Vehicle group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. baseline).Compelling evidence has shown the TNFα plays critical role in the development of inflammatory diseases and pain.In the current TA B L E 2 Primers to TNFα promoter region.

3 . 4 |
Figure 3J) and spinal dorsal horn (r = −0.8348;p = 0.0194, Figure 3L).It implies that the CFA-induced TNFα expression may depend on KDM6B activity.The Shapiro-Wilk test was used for normality, nonparametric Kruskal-Wallis test was used for difference in the results in Figure 3A,B,E,F, Mann-Whitney U-test was used in Figure 3C,D, and Dunnett's multiple-comparisons test was used in Figure 3G,H.
), exclusively, in neurons in the DRG on day 3 after CFA.But the satellite glial cells also expressed TNFα (Figure5F-J) in the DRG on day 7 after CFA.In the dorsal horn, the TNFα expressed in neurons, astrocytes, and microglia on day 3 after CFA (Figure5K-O), but an increased expression of TNFα in astrocytes and microglia was observed on day 7 after CFA injection (Figure5P-T).The above results showed that TNFα expressed in the same cells at almost same time point as KDM6B in both the DRG and dorsal horn.

F I G U R E 3 F I G U R E 4 F I G U R E 5 6
Figure5H,I).The CFA injection also caused a remarkable increase in the level of NF-κB p-p65 in both the DRG (Figure6J) and dorsal horn (Figure6K) (***p < 0.001 vs. control group, Figure6J,K).The Shapiro-Wilk test was used for normality, and Dunnett's multiplecomparisons test was used in Figure6C,D,F-K.
repeat i.t.injections of GSK-J4.To discriminate the different functions of upregulated KDM6B in the DRG and spinal dorsal horn during the development of inflammatory pain, the microinjections of AAV-EGFP-KDM6B shRNA in both the sciatic nerve and L5 spinal dorsal horn were performed, separately.The results showed that the CFAinduced abnormal pain was attenuated by the treatment of specific knockdown KDM6B in both the DRG and dorsal horn.These results indicate CFA-induced upregulation of KDM6B in the DRG, and dorsal horn is required in the pathogenesis of inflammatory pain.Recently, cells at almost the same time point as KDM6B in both the DRG and dorsal horn.The early increase in KDM6B and TNFα mainly comes from neurons, and the glial cells contributed to a delayed increase in KDM6B and TNFα in both the DRG and dorsal horn following CFA injection.These results indicate that CFA-induced TNFα expression depends on the regulation of KDM6B.Recently, Davis reported that the increased JMJD3 in aortic tissues reduces the repressive H3K27 trimethylation at NF-κB-binding sites on inflammatory gene promoters, and inhibition of JMJD3 via GSK-J4 reduces inflammatory cytokine expression. 41Our current results showed that CFA injection induced an increased binding of NF-κB p65 with TNFα promoter, and this increase accompanied a significant reduction in the level of H3K27me3 in the area of TNFα promoter in both the DRG and spinal dorsal horn.It indicates that the CFA-induced upregulation of KDM6B might through mediating H3K27me3 demethylation at TNFα promoter facilitates the binding of NF-κB with TNFα promoter, and subsequently promotes expression of TNFα in the dorsal horn and DRG.