Spinal bromodomain-containing protein 4 contributes to neuropathic pain induced by HIV glycoprotein 120 with morphine in rats

The symptoms of HIV-sensory neuropathy are dominated by neuropathic pain. Recent data show that repeated use of opiates enhances the chronic pain states in HIV patients. Limited attention has so far been devoted to exploring the exact pathogenesis of HIV painful disorder and opiate abuse in vivo, for which there is no effective treatment.

Bromodomain-containing protein 4 (Brd4) is a member of the bromodomain and extraterminal domain protein (BET) family and functions as a chromatin ‘reader’ that binds acetylated lysines in histones in brain neurons to mediate the transcriptional regulation underlying learning and memory. Here, we established a neuropathic pain model of interaction of intrathecal HIV envelope glycoprotein 120 (gp120) and chronic morphine in rats. The combination of gp120 and morphine (gp120/M, for 5 days) induced persistent mechanical allodynia compared with either gp120 or morphine alone. Mechanical allodynia reached the lowest values at day 10 from gp120/M application, beginning to recover from day 21. In the model, gp120/M induced overexpression of Brd4 mRNA and protein at day 10 using RT- qPCR and western blots, respectively. Immunohistochemical studies showed that Brd4 at day 10 was expressed in the neurons of spinal cord dorsal horn. BET inhibitor I-BET762 dose-dependently increased the mechanical threshold in the gp120/M pain state. The present study provides preclinical evidence for treating HIV neuropathic pain with opioids using the BET inhibitor.

Keywords: bromodomain-containing protein 4, glycoprotein 120, morphine, neuropathic pain

Introduction

Despite the effective antiretroviral therapies among HIV patients, chronic pain remains a frequently reported problem. Individuals living with HIV have an increased prevalence of conditions associated with opioid analgesic misuse [1]. HIV- 1-infected opioid abusers show more severe neuropathology than HIV-positive nondrug users [2]. Opioid use disorder is common among individuals with chronic pain conditions [3]. The exact molecular mechanisms of chronic pain experi- enced by HIV patients with opioid abuse are poorly under- stood, for which no effective treatment currently exists.

Epigenetic modifications resulting in the changes in the chromatin structure play a critical role in gene expression during the activation, proliferation, and differentiation of cells [4]. Acetylation of nucleosomal histone tails is one epigenetic mechanism that controls gene expression. The well-characterized bromodomain and extraterminal domain family proteins (BETs) bind acetyl-lysine marks on histone tails and, concurrently, can recruit other transcription fac- tors to the chromatin. The BET family comprises four bromodomains [bromodomain-containing protein (BRD)2, BRD3, BRD4, and BRDT] with two N terminal tandem bromodomains [4]. Studies have shown that BET proteins contribute toward inflammatory and immune processes [4].

Brd4 functions as a chromatin ‘reader’ that binds acetylated lysines in histones in brain neurons to mediate the tran- scriptional regulation underlying learning and memory [5]. HIV glycoprotein 120 (gp120) causes neuronal dysfunction in HIV-associated dementia [6]. However, the role of Brd4 in HIV neuropathic pain with chronic opioid use is still not clear. Here, we reported that the combination of chronic intrathecal gp120 and morphine (gp120/M) decreased the mechanical threshold using von Frey filaments. We also found that at day 10 from the intrathecal injection, gp120/ M increased Brd4 mRNA and protein using RT-qPCR and western blots, respectively. Immunohistochemical studies showed that Brd4 at day 10 was expressed in the spinal cord dorsal horn (SCDH) neurons. BET inhibitor I-BET762 dose dependently increased the mechanical threshold in the gp120/M pain state. The present studies provide preclinical evidence for treating HIV-related neu- ropathic pain using the Brd4 inhibitor.

Materials and methods

Animals

In the present studies, 90 male 7–8-week-old Sprague- Dawley rats weighing 225–250 g (Charles River Laboratories, Raleigh, North Carolina, USA) were housed one to three per cage ∼ 7 days before the initiation of all studies. All housing conditions and experimental procedures were approved by the University Animal Care and Use Committee.

Drugs

Morphine sulfate was purchased from West-Ward Pharmaceuticals (Eatontown, New Jersey, USA). Recombinant gp120 was purchased from ImmunoDX (LLC, Woburn, Massachusetts, USA) and dissolved in 0.1% rat serum albumin. I-BET762 purchased from Sigma (Sigma, St Louis, Missouri, USA) was dissolved in 5% DMSO.

Intrathecal catheter implantation

For intrathecal administration, under isoflurane anesthesia, chronic intrathecal catheters were implanted as described previously [7]. Briefly, a polyethylene-10catheter was filled with saline. The polyethylene-10 catheter, through an incision in the atlanto-occipital membrane, was advanced
8.5 cm caudally to position its tip at the level of the lumbar enlargement. The rostral tip of the catheter was passed subcutaneously, externalized on top of the skull, and sealed with a stainless-steel plug. Animals showing neurological deficits after implantation were excluded. Spinal agents
were delivered over a 60 s in a volume of 10 μl solution. Drug injection was immediately followed by a 10 μl phy- siologic saline to flush the catheter.

Model of neuropathic pain induced by gp120 with morphine

To establish neuropathic pain induced by gp120 with morphine, rats received gp120 (0.2 μg) once daily at 9 a.m., morphine (3 μg) twice daily, or a combination of gp120 once daily with morphine for 5 days. Morning morphine was injected 15–20 min following gp120. Ten milliliter saline was intrathecally injected following gp120 or morphine to ensure that drugs were injected into the central spinal fluid.

Mechanical threshold in the neuropathic pain state

We used calibrated von Frey filaments (Stoelting, Wood Dale, Illinois, USA) to determine the mechanical threshold. The filaments were applied serially to the hind-paw in ascending order of strength, and animals were placed in nontransparent plastic cubicles on a mesh floor for an acclimatization period of at least 30 min on the morning of the test day. A positive response was defined as a rapid withdrawal and/or licking of the paw immedi- ately on application of the stimulus. Whenever a positive response to a stimulus occurred, the next smaller von Frey hair was applied, and whenever a negative response occurred, the next higher force was applied. In the absence of a response at a pressure of 15.1 g, animals were assigned to this cut-off value. Mechanical threshold was determined according to the method described pre- viously with a tactile stimulus producing a 50% likelihood of withdrawal determined by using the up-and-down method [7].

Quantitative RT-PCR

The rat lumbar L4/5 SCDH tissue was collected, and total RNA was isolated using the RNeasy mini kit (cat# 74104; Qiagen, Germantown, Maryland, USA). RNA sample was treated with DNase I on column to remove genomic DNA. One µg of RNA was converted into cDNA using Superscript VILO master mix (cat# 11755-050; Invitrogen, Grand Island, New York, USA), and then real-time PCR was performed with Fast SYBR green master mix (cat# 4385612; Applied Biosystems, Grand Island, New York, USA). For real-time PCR, the following primers [8] were used: BRD4 forward 5′-ACAGCCCCAACAGAACAAAC-3′ and reverse 5′-GCT GGTTCCTTCTTGCTCAC-3′; and GAPDH forward 5′-CAGGGCTGCCTTCTCTTGTG-3′ and reverse 5′-AA CTTGCCGTGGGTAGAGTC-3′. The specificity of the PCR product was confirmed by running agarose gel electrophoresis. All reaction data were calculated using 2—DDCt methods and the gene expression levels were normalized with GAPDH.

Western blots

Under deep anesthesia with isoflurane, the L4–L5 SCDH was removed rapidly, frozen on dry ice, and stored at − 80°C. SCDH tissue was homogenized and lysed with 1 × RIPA protein lysis buffer containing protease and phosphatase inhibitor cocktail 2 and 3 (cat# P8340, P5726, P0044; Sigma). We used the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, Illinois, USA) to measure the protein concentra- tion of tissue lysates. Proteins (30 µg) were loaded onto 10% SDS/PAGE and transferred onto a PVDF membrane. The membrane was incubated with primary antibodies overnight at 4°C with rabbit anti-Brd4 (H-250) (1 : 1000, cat# sc-48772; Santa Cruz, Dallas, Texas, USA). For loading control, the blots were probed with β-actin antibody [mouse monoclonal anti-β-actin (1 : 8000, cat# A5441; Sigma)]. The membrane was incubated with secondary antibodies at room temperature and then developed in chemiluminescence solution (cat# 34076; Pierce Biotechnology). Chemiluminescence values from targeted band intensity were analyzed, quanti- fied, and normalized with β-actin using a ChemiDoc imaging system (Bio-Rad, Hercules, California, USA).

Immunohistochemistry

Ten days after gp120, animals were perfused with 4% paraformaldehyde in 0.1 M PBS. The spinal cord was postfixed and cryoprotected. Immunostaining of GFAP, OX42, NeuN, and Brd4 in the SCDH was carried out. Briefly, the cryosections of the spinal cord were probed with mouse anti-GFAP polyclonal antibody (1 : 2000, cat# G3893; Sigma), mouse anti-OX42 (1 : 200, cat# CBL1512; Chemicon International, Temecula, California, USA), mouse anti-NeuN monoclonal antibody (A60) (1 : 500, cat# MAB377; Chemicon International), and rabbit anti-Brd4 (1 : 100, cat# ARP39076; Aviva, San Diego, California, USA). These treatments were then followed by complementary secondary antibodies labeled with green-fluorescent Alexa Fluor 488, or red-fluorescent Alexa Fluor 594 (1 : 2000; Molecular Probes, Eugene, Oregon, USA), 2 h, at room tem- perature. Fluorescence images were captured by fluorescent microscopy (Fluorescent M Leica/Micro CDMI 6000B; Leica Microsystems Inc., Buffalo Grove, Illinois, USA).

Statistical analysis

For data analysis, behavioral data were analyzed by two-way repeated-measure analysis of variance (ANOVA), followed by the Bonferroni test. The area under the time–effect curves, depicting the mechanical threshold (g) over time, was calculated by the trapezoidal rule to determine the overall magnitude and duration of effect. The statistical significance of the differences of neurochemical changes was determined using a t-test or a one-way ANOVA post-hoc test following Fisher’s protected least significant difference (StatView5; SAS Institute Inc., Cary, North Carolina, USA). All data were presented as mean ± SEM, and P values less than 0.05 were considered to be statistically significant.

Results

Intrathecal gp120 and morphine induced mechanical allodynia

In the control group treated with intrathecal 0.1% rat serum albumin/saline, rats showed no significant changes com- pared with baseline in the mechanical threshold through- out the 3-week testing period after intrathecal injection. There was no significant change in the mechanical threshold in the vehicle + saline group and the vehicle + morphine group for 3 weeks. Lower mechanical threshold in the gp120 + saline group occurred, just on days 10 and 14 compared with the vehicle + saline group (P < 0.05, two- way ANOVA Bonferroni tests). HIV-1 gp120 plus mor- phine induced a persistent lowered mechanical withdrawal threshold [F(21, 175), interaction = 5.83, P < 0.0001; F(7, 175), main effect time = 14.71, P < 0.0001; F(3, 25), main effect treatment = 23.66, P < 0.0001, two-way ANOVA repeated-measures, n = 6–9 (Fig. 1a)]. In the gp120/M group, the mechanical threshold reached the lowest values at day 10. Interestingly, the mechanical threshold in the gp120 + morphine group was markedly lower than that in the vehicle + saline, vehicle + morphine, or gp120 + saline group at day 5, 6, 7, 10, 14, 17, and 21 from intrathecal injection, two-way ANOVA Bonferroni tests, n = 6–9 rats, suggesting an interaction of gp120 and morphine.

Intrathecal gp120/M upregulated the expression of Brd4 in the spinal cord dorsal horn

Behavioral time course (Fig. 1a) showed that the lowest mechanical threshold occurred at day 10 from the gp120 + morphine application, and began to recover from day 21. Thus, we compared the expression of spinal Brd4 mRNA and protein at day 10 in the vehicle + saline, vehicle + morphine, gp120 + saline, and gp120 + morphine groups. At the 10-day time point from gp120/M application, the lumbar SCDH was harvested; the expression of spinal Brd4 mRNA and protein was tested using RT-qPCR and western blots, respectively.

Brd4 mRNA in gp120 + morphine was markedly higher than that in the vehicle + saline, vehicle + morphine, or gp120 + saline group (P < 0.05), one-way ANOVA post-hoc protected least significant difference test (Fig. 2a). There was no sig- nificant difference in Brd4 protein among the vehicle + saline, vehicle + morphine, or gp120 + saline groups (Fig. 2b); how- ever, overexpression of Brd4 protein in the gp120 + M rats was found, P value of less than 0.05 versus vehicle + saline, n = 5 (Fig. 2c). To determine the location of Brd4-positive cell types, double-label immunostaining of GFAP, OX42, or NeuN with Brd4 was carried out. There was an almost complete co-localization between immunoreactivity (IR) of NeuN (red) and Brd4-IR (green) (Fig. 2d–f); however, Brd4-IR did not co-localize with GFAP-IR or OX42-IR (data not shown), which suggested that Brd4 is located in neurons, but not glia.

The antinociceptive effect of I-BED 762 on gp120/M- induced mechanical allodynia

The results above suggest that spinal Brd4 may play a role in the gp120/M-induced mechanical allodynia. To further verify the role of Brd4 in the model of gp120/M, at 10 days after gp120/M, rats received I-BET762 (5, 15, or 50 μg/20 μl) or vehicle. The mechanical threshold was measured before, and 15, 30, 60, 90, 120, and 180 min after the drug injection. I-BET762 dose dependently resulted in a sig- nificant increase in the mechanical threshold compared with vehicle [F(18, 120), interaction = 4.206, P < 0.0001, F(6, 120), main effect time = 14.19, P < 0.0001, F(3, 120), main effect drug = 12.77, P < 0.0001], two-way ANOVA repeated-measures. The I-BET762 (15 μg) group showed a significant increase in the mechanical threshold at 60, 90, and 120 min after the drug injection compared with vehicle, P value of less than 0.01 versus the vehicle group, two-way ANOVA Bonferroni post-tests, n = 6 rats. I-BET762 (50 μg) produced a further increase in the mechanical threshold at 60, 90, and 120 min, P value of less than 0.001 versus the vehicle group, two-way ANOVA Bonferroni post-tests, n = 6 rats (Fig. 3a). I-BET762 dose dependently increased the area under curves, one-way ANOVA with Bonferroni test, n = 6 rats (Fig. 3b).

Discussion

Recent studies show that concurrent pain and chronic opioid use are considered to be high-risk factors for increased pain severity and decreased pain thresholds among chronic opioid users [9]. Chronic opioid use may significantly affect individuals infected with HIV. HIV-1-infected opioid abu- sers show more severe neuropathology than HIV-1-infected nondrug users [2]. Emerging clinical data show that repeated use of opiate pain medicine might enhance the chronic pain states in HIV patients [10]. In the present studies, we established a neuropathic model induced by interaction of intrathecal gp120 and chronic morphine in rats, showing the synergistic effect of neuropathic pain. In the model, gp120/M resulted in overexpression of spinal Brd4 and BET inhibitor I-BET762 dose dependently increased the mechanical threshold.

The time course of the mechanical threshold in the neuropathic pain model induced by interactions of glycoprotein 120 (gp120) with morphine.
(a) Repeatedly intrathecal gp120 with morphine (gp120 + M) induced a persistent lowered mechanical withdrawal threshold compared with vehicle + saline (veh + sal) rats, *P < 0.05, versus vehicle + saline (veh + sal); #P < 0.05 versus vehicle + morphine (veh + M). $P < 0.05 versus gp120-saline (gp120 + sal), two-way analysis of variance (ANOVA) Bonferroni tests, n = 6–9. (b) The area under the curves in the combination of gp120 with morphine (gp120 + M) was significantly lower than that in vehicle + saline (veh + sal), vehicle + morphine (veh + M), or gp120 + saline (gp120 + sal) groups, *P < 0.05, two-way ANOVA Bonferroni tests, n = 6–9.

The expression of bromodomain-containing protein 4 (Brd4) in the combination of glycoprotein 120 (gp120) with morphine state. (a) RT-qPCR showed that the expression of Brd4 mRNA of gp120 +morphine (gp120 +M) was higher than that in vehicle +saline (veh +sal), vehicle +morphine (veh +M), or gp120 +saline (gp120 +sal),**P < 0.01, one-way analysis of variance (ANOVA) post-hoc PLSD test, n = 4. (b) Western blots showed there was no significant difference among veh +sal, veh +M, gp120 +sal. (c) Western blots showed there was a significant difference between veh +sal and gp120 +M, *P < 0.05, t-test, n = 5. (d–f) Double immunostaining showed the co-localization of Brd4-IR and NeuN, arrows showed the co-localization neurons, scale bar, 50 µm.
The antiallodynic effect of inhibitor bromodomain and extraterminal domain protein (I-BET762) on neuropathic pain induced by glycoprotein 120 + morphine (gp120 + M). (a) Intrathecal I-BET762 dose dependently increased the mechanical threshold,**P < 0.01,***P < 0.001 versus vehicle, two-way analysis of variance (ANOVA) Bonferroni post-tests, n = 6 rats. (b) I-BET762 dose dependently increased the area under curves (AUC),
*P < 0.05, ***P < 0.001 versus vehicle, one-way ANOVA with Bonferroni test, n = 6 rats.

The emerging evidence shows that BET proteins function as key factors in transcriptional activation of distinct sets of genes throughinteraction with acetylated histones and/or transcri- tional factors [19]. BET proteins have long been associated with cancer, but in recent years, important studies have shown their essential contribution toward inflammatory processes [4]. BETs act as coactivators of the NF-kB-mediated regulatory functions once these regions have been occupied by this transcription factor [4]. Blockage of BETs downregulates the expression of cytokines, TNF-α, interleukin-6, interleukin-1β, and chemokine MCP-1 by altering NF-kB activity in lipopolysaccharide induced macrophages [4]. I-BET 762 decreased c-Myc and p-ERK 1/2 protein levels and a variety of inflammatory cytokines in both immune and pancreatic cancer cells in vitro [20]. I-BET762 confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis [21]. Brd4 binds acetylated lysines in histones in brain neurons to mediate the transcriptional regulation underlying learning and memory [5]. Recent studies show that neuronal Brd4 protein expression plays an important role in autism spectrum disorder, and that inhibition of Brd4 is a viable means of alleviating epigenetic misregulation in autism [22]. In the brief report, we are the first to observe the overexpression of Brd4 in the spinal cord dorsal horn in the HIV-related pain model. I-BET762 dose-dependently increased the mechanical threshold, indicating that Brd4 plays an important role in the gp120/M neuropathic pain state. In the near future, we will determine the Brd4- related signal pathways in the neuropathic pain model.

In summary, BET inhibitors are currently emerging as promising therapeutic agents for the treatment of various pathological conditions. Several BET inhibitors are already in clinical trials for hematological malignancies, solid tumors, and cardiovascular disease [23]. The pre- sent studies provide preclinical evidence for Molibresib treating HIV neuropathic pain using the BET inhibitor.