Morphine and Fentanyl Repeated Administration Induces Different Levels of NLRP3‑Dependent Pyroptosis in the Dorsal Raphe Nucleus of Male Rats via Cell‑Specific Activation of TLR4 and Opioid Receptors

César J. Carranza‑Aguilar1 · Araceli Hernández‑Mendoza1 · Carlos Mejias‑Aponte2 · Kenner C. Rice3 · Marisela Morales2 · Claudia González‑Espinosa1 · Silvia L. Cruz1

Abstract

Morphine promotes neuroinflammation after NOD-like receptor protein 3 (NLRP3) oligomerization in glial cells, but the capacity of other opioids to induce neuroinflammation and its relationship to the development of analgesic tolerance is unknown. We studied the effects of morphine and fentanyl on NLRP3 inflammasome activation in glial and neuronal cells in the dorsal raphe nucleus (DRN), a region involved in pain regulation. Male Wistar rats received i.p. injections of morphine (10 mg/kg) or fentanyl (0.1 mg/kg) 3 × daily for 7 days and were tested for nociception. Two hours after the last (19th) administration, we analyzed NLRP3 oligomerization, caspase-1 activation and gasdermin D-N (GSDMD-N) expression in microglia (CD11b positive cells), astrocytes (GFAP-positive cells) and neurons (NeuN-positive cells). Tolerance developed to both opioids, but only fentanyl produced hyperalgesia. Morphine and fentanyl activated NLRP3 inflammasome in astrocytes and serotonergic (TPH-2-positive) neurons, but fentanyl effects were more pronounced. Both opioids increased GFAP and CD11b immunoreactivity, caspase-1 and GSDMD activation, indicating pyroptotic cell death. The opioid receptor antagonist (−)-naloxone, but not the TLR4 receptor antagonist (+)-naloxone, prevented microglia activation and NLRP3 oligomerization. Only (+)-naloxone prevented astrocytes’ activation. The anti-inflammatory agent minocycline and the NLRP3 inhibitor MCC950 delayed tolerance to morphine and fentanyl antinociception and prevented fentanyl-induced hyperalgesia. MCC950 also prevented opioid-induced NLRP3 oligomerization. In conclusion, morphine and fentanyl differentially induce cellspecific activation of NLRP3 inflammasome and pyroptosis in the DRN through TLR4 receptors in astrocytes and through opioid receptors in neurons, indicating that neuroinflammation is involved in opioid-induced analgesia and fentanyl-induced hyperalgesia after repeated administrations.

Keywords NLRP3 · Opioids · Glia · TLR4 · NF-κB · Serotonin

Introduction

Morphine and fentanyl are commonly used analgesics and psychoactive drugs that differ in potency and receptor selectivity. Fentanyl is 50–100 times more potent than morphine as analgesic and a selective μ-opioid receptor agonist, while morphine acts through μ- δ- and κ-opioid receptors (Schaefer et al. 2017; Suzuki and El-Haddad 2017; Vardanyan and Hruby 2014). Prolonged treatment with morphine and fentanyl can lead to tolerance to their analgesic effects (Bobeck et al. 2012; Molina-Martínez et al. 2014). Several factors contribute to the development of opioid analgesic tolerance, including membrane receptor availability, cAMP pathway adaptations, potassium and calcium channel expression, as well as changes in various neurotransmitter systems (AlHasani and Bruchas 2011; Cesselin 1995; Lueptow et al. 2018; Mayer and Mao 1999; Taylor and Fleming 2001).
Experimental data suggest that neuroinflammation also plays a role in tolerance development (Lin and Lu 2018; Lueptow et al. 2018). For example, minocycline and pentoxifylline, drugs with anti-inflammatory actions, prevent tolerance to the antinociceptive effects of morphine (Harada et al. 2013; Mika et al. 2009). Also, repeated morphine administration induces changes in morphology of astrocytes and microglia in dorsal raphe nucleus (DRN), a brain region involved in pain regulation, and periaqueductal gray (PAG), which has been identified as a critical region for the development of tolerance to opioid effects (Campion et al. 2016; Doyle et al. 2017; Harada et al. 2013; Tortorici et al. 1999). DRN is abundant in somas of serotonergic neurons (Baker et al. 1991) whose descending projections modulate pain responses at the spinal level, while their ascending projections act in sensitive neurons in the thalamus (Qing-Ping and Nakai 1994). Analgesic opioid effects occur in part through the serotonin release to spinal sites that transmit nociceptive stimuli (Bardin and Colpaert 2004; Haleem 2018).
Glial cells release pro-inflammatory mediators such as interleukin 1β (IL-1β), a cytokine that contributes to the loss of morphine-induced antinociception (Mélik Parsadaniantz et al. 2015; Shavit et al. 2005). Activation of several proteins is necessary to induce the release of IL-1β and to modulate the analgesic effect of morphine (Xu et al. 2017). Thus, repeated morphine administration increases mRNA expression of NOD-like receptor protein 3 (NLRP3) in dorsal root ganglion, which is necessary for IL-1β maturation (Ellis et al. 2016). In vitro studies have also demonstrated that morphine can activate NF-κB (nuclear factor enhancer of the kappa light chains of activated B cells), a transcription factor responsible for NLRP3 and IL-1β transcription in neurons and microglia (Chen et al. 2006; Wang et al. 2012). NF-κB activation has been documented in the spinal cord of rats with nerve damage that received morphine for 1 week (Grace et al. 2016). In addition, administration of procyanidin, an NLRP3 inhibitor, and minocycline, which effectively inhibits NF-κB, reduces tolerance development to the antinociceptive effects of morphine (Cai et al. 2016; Mika et al. 2009).
The NLRP3 inflammasome complex is formed by oligomerization of NLRP3 and pro-caspase-1, among other proteins (Kim et al. 2017). This complex has been associated to neuronal damage via IL-1β release and caspase-1 activation (Masters 2013; Zhang et al. 2016), which can lead to a type of inflammatory cell death known as pyroptosis (Guo et al. 2015; Shi et al. 2015). Pyroptotic cells are characterized by cellular swelling, membrane pore formation dependent on gasdermin D (GSDMD) cleavage in the N- and C- terminal domains, and plasma membrane rupture (Lamkanfi and Dixit 2010; Liu et al. 2018; Shi et al. 2015). There is evidence that morphine increases the expression of caspase-1, IL-1β and NLRP3 proteins in the spinal cord when it is chronically administered (Cai et al. 2016). However, to the best of our knowledge, there are no studies analyzing if pyroptosis occurs in brain areas involved in pain modulation in response to other opioid agonists.
Although the mechanisms leading to opioid-induced neuroinflammation are not fully understood, the fact that the non-specific opioid receptor antagonist (−)-naloxone prevents in vitro NLRP3 activation induced by lipopolysaccharide (LPS) indicates that opioid receptors are involved in this effect (Lin et al. 2017). On the other hand, opioids can bind to the Toll-like receptor 4 subtype (TLR4), which recognizes pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs, respectively), and pharmacological blockade of TLR4 receptors attenuates morphine-induced analgesic tolerance and hyperalgesia (Hutchinson et al. 2010). In addition, TLR4 blockade also prevents morphine-induced IL-1β release in a microglial cell line (Wang et al. 2012).
In comparison to morphine, the neuroinflammatory actions of other, even more potent opioids, are less studied. Also, the participation of neuroinflammation on the development of tolerance after repeated opioid administration is not fully understood. In this work, we tested the hypothesis that differences in the development of antinociceptive tolerance after repeated administrations of morphine and fentanyl could be related to their capacity to induce the expression of canonical markers of neuroinflammation in DRN, with differential participation of opioid and Toll-like receptors. Therefore, the aims of this study were: (a) to compare the effects of repeated morphine or fentanyl administration (enough to produce tolerance to their antinociceptive effects) on NLRP3-dependent pyroptosis in neurons, microglia and astrocytes in DRN in rats; (b) to determine the role of opioid receptors and TLR4 receptors in these effects; and (c) to study the effect of NLRP3 and inflammation blockade on the development of tolerance to morphine or fentanyl antinociceptive effects.

Materials and Methods

Animals

We used male Wistar rats (250–300 g) housed under a 12/12-h inverted light/dark cycle (light on at 10:00), with room temperature between 23–25 °C and ad libitum access to food and water. Rats were habituated to laboratory conditions by handling them for three consecutive days. Experiments to test antinociception, tolerance development and pro-inflammatory effects of repeated morphine and fentanyl administration were conducted at Cinvestav-IPN. Experiments to analyze the receptors involved in opioid-induced neuroinflammation were done at the National Institute on Drug Abuse (NIDA), Intramural Research Program. All procedures followed the Mexican Official Norm for laboratory animals (NOM-062-ZOO-1999), were approved by the Ethics Committee of Cinvestav (CICUAL, Protocols 002112 and 0074-13) and NIDA Intramural Program (Protocol # 16-INB-4), and complied with the National Institutes of Health guidelines (NIH 2011) and ARRIVE guidelines for animal research (Kilkenny et al. 2010).

Drugs

The National Institute on Drug Abuse (Research Technology Branch, Research Triangle Institute, NC, USA) and Laboratorios Psicofarma (Ciudad de México, México) kindly donated morphine sulfate and fentanyl citrate. Dr. K.C. Rice synthetized (+)-naloxone. The work of the Drug Design and Synthesis Section, Molecular Targets and Medications Discovery Branch was supported by the NIH Intramural Research Programs of NIDA and the National Institute of Alcohol Abuse and Alcoholism (NIAAA). We purchased, minocycline, MCC950, (−)-naloxone hydrochloride and LPS (Escherichia coli serotype 026:B6) from Sigma-Aldrich (Toluca, Mexico). Morphine, fentanyl, (−)-naloxone, (+)-naloxone, MCC950 and LPS were dissolved in 0.9% saline solution and injected in a final volume of 0.1 ml/100 g, i.p. Minocycline was dissolved in sterile water and administered by oral gavage in a final volume of 0.2 ml/100 g. We determined the doses for naloxone, minocycline and MCC950 to be used in this study from the literature (Chen et al. 2019; Kaneto et al. 1985; Posillico et al. 2015; Zhang et al. 2015).

Experimental Design

Independent groups of animals (n = 10 each) were randomly allocated to one of the following groups (Fig. 1): Experiment 1. Dose–response curves for antinociception produced by morphine (0.1 to 10 mg/kg) or fentanyl (0.001 to 0.1 mg/ kg). Experiment 2. Repeated administration (3x/day, 8 h apart, 19 administrations in total) of 10 mg/kg morphine or 0.1 mg/kg fentanyl. Animals were evaluated in the tail-flick test immediately after the 1st, 7th and 19th administration of each opioid to test for tolerance development. Two hours after the last saline/opioid administration and nociception testing, brains were removed and prepared for immunofluorescence studies from randomly selected animals (n = 3 of each experimental condition). Experiment 3. Same protocol as in experiment 2, but treating animals with 10 mg/ kg (−)-naloxone or 10 mg/kg (+)-naloxone 20 min prior to each opioid administration. As before, brains from 3 animals were obtained for immunofluorescence studies, 2 h after the last saline/drug administration. Experiment 4. Same drug administration schedule as in experiment 2, but in this case, we administered 5 mg/kg MCC950 or 30 mg/kg minocycline the day previous to the first opioid injection and each day, 60 min before the second daily administration of morphine or fentanyl. Although minocycline’s half-life is short (3–4 h; Colovic and Caccia 2003) we selected this administration schedule because it has been shown that pre-treatment with 30 mg/kg minocycline for three days delays morphine antinociceptive tolerance in rats with neuropathic pain (Zhang et al. 2015). The MCC950 dose and administration schedule were selected because under these conditions MCC950 effectively reduces inflammatory pain through NLRP3 inhibition (Chen et al. 2019). Control animals received only the vehicle; i.e., 0.9% saline i.p., or sterile water, p.o.

Evaluation of Antinociception

We used a standardized tail-flick apparatus (model 7360, Ugo Basile, Italy) with a variable heat source connected to an automatic timer. The heat source was adjusted to obtain a baseline latency value to tail withdrawal of 5–6 s. We tested each rat three times before the first drug injection to obtain the mean baseline latency; rats outside this range were excluded (approximately 5%). In this test, an increase or decrease in basal latency indicates antinociception or pro-nociception, respectively. To avoid tissue damage, we established a cut-off time of 15 s. The latency to tail withdrawal was determined 15, 30, 60, 90 and 120 min after drug administration. The results were expressed as percentage of the maximum possible effect: %MPE = (experimental value-baseline value)/15-baseline value × 100) and as the area under the curve (AUC) for each time course determined by trapezoidal integration.

Immunofluorescence

Two hours after the last opioid injection, animals were anesthetized with a mixture of ketamine:xylazine (90:10 mg/kg) and perfused through the ascending aorta with 4% paraformaldehyde (PFA) in 0.1 M phosphatebuffered saline (PBS), pH 7.4. Brains were removed and post-fixed during 2 h in 4% PFA, placed in 30% sucrose solution for 48 h, kept in a 30% polyethylene glycol and 30% sucrose solution and stored at − 70 °C until use. DRN coronal slices of 30 µm from bregma − 7.44 to − 7.8 (Paxinos and Watson 2009) were cut using a cryostat (Zeiss, Hyrax C25). The slices were immersed in PBS at room temperature for 1 h and then washed three times with PBS. After this, samples were blocked with 5% filtered donkey serum (Equitech, Cat. SD-0500HI) and 0.3% triton in PBS for 2 h at room temperature. Samples were then incubated overnight with primary antibodies at 4 °C and with specific secondary antibodies for 2 h at room temperature in dark conditions. Control experiments were performed using only secondary antibodies and, under these conditions, no labeling was observed. To study NLRP3 inflammasome activation, we used anti-NLRP3, anti-caspase-1 and anti-GSDMD-N (an antibody that recognizes the N-terminal GSDMD domain after cleavage). Anti-NeuN was used to stain all neurons, anti-TPH-2 (tryptophane hydroxylase-2; the rate-limiting enzyme in the synthesis of 5-HT) to identify the serotonergic neurons, anti-CD11b (cluster of differentiation molecule 11b) to stain microglia and anti-GFAP (glial fibrillary acidic protein) to detect astrocytes. All the antibodies used are listed in Table 1. DAPI (for nuclei staining) was added in a dilution 1:500 in PBS solution for 5 min, followed by three washes with PBS. The tissue samples were mounted with ProLong® Antifade Kit (Invitrogen, ref. 7481) using 10–15 μl per slice. The slides were covered with a coverslip and sealed. Samples were stored at room temperature and protected from light until further analysis.

Statistical and Data Analysis

We calculated each opioid dose to produce antinociception at 50% with their corresponding 95% confidence intervals (95% CI) using probit analysis (Probit; MedCalc version 17.8.2). For comparisons among three or more independent groups, we used a one-way analysis of variance (ANOVA) followed by Tukey test. To evaluate if hyperalgesia developed, we conducted a one-sample Wilcoxon rank-sum test at the time points at which the latencies to tail-flick test were lower than basal values. Comparisons between the proportion of TPH-2 and NLRP3 double positive cell populations were done with Fisher’s exact test. The threshold for statistical significance was p ≤ 0.05. While conducting data analysis, researchers were blinded to which samples represented treatments and controls. The software program used for statistical analysis was GraphPad Prism v.8. In order to quantify the number of positive cells to CD11b, NLRP3, caspase-1 and TPH-2, we obtained DRN images (600 × 500 µm) with an immunofluorescence microscope (Eclipse Ti-U, Nikon; 10 × objective). For more detailed images (40 × 40 µm) and complete cell analysis (10 z-stacks, 1 µm each), we used a confocal microscope (Zeiss Airyscan, LSM800; 40 × objective). To quantify the fluorescence intensity of GFAP and GSDMD-N, we converted the images to gray-scale (8-bit), subtracted the background pixels and established the threshold limit to highlight the structures of interest (cell bodies and branches); afterwards, we measured the mean fluorescence intensity of the entire image. To analyze co-localization between NLRP3/caspase-1, NLRP3/GFAP, NLRP3/ CD11b and NLRP3/NeuN, we used the Mander’s overlapping coefficient. All images were taken in triplicate from three different animals and analyzed under the same conditions with ImageJ software (version 1.52n, National Institute of Health, USA).

Results

Morphine and Fentanyl Produce Different Degrees of Tolerance to Their Antinociceptive Effects

We did dose–response curves for the acute effects of morphine or fentanyl and observed maximal antinociception with 10 mg/kg morphine or 0.1 mg/kg fentanyl (Fig. 2a), doses that were used for subsequent experiments. Values calculated from the adjusted curves were 2.96 mg/kg [95% IC: 0.52 to 5.39] for morphine and 0.03 mg/kg [95% IC: 0.01 to 0.06] for fentanyl. The time courses corresponding to the 1st, 7th and 19th administration of 10 mg/kg morphine showed that, with the first dose, peak effects were observed at 15 min and antinociception remained high for the next 90 min. After repeated administration of morphine, there was a decrease in Emax and a reduction in duration of antinociceptive effects (Fig. 2a). Emax and AUC values for each administration are compared in Fig. 2c, d. As seen, morphine’s effects significantly decreased with each injection (F(2,27) = 12.95; p = 0.0001); thus, morphine produced approximately 50% of its initial response by the 19th administration. Tolerance was more evident when the AUC values were compared because, in this case, morphine’s effects significantly decreased from the 7th administration onwards (F(2,27) = 36.3; p < 0.0001). The 1st fentanyl administration also reached maximal antinociception in the first 15 min and decreased towards basal levels afterwards (Fig. 2e).
Subsequent injections produced a slight decrease in Emax and less duration of antinociception. At the 19th administration, fentanyl still produced significant peak effects, but hyperalgesia was evident at 90 and 120 min after injection (p = 0.016; one-sample Wilcoxon rank-sum test). We did not find significant statistical differences between the 1st, 7th and 19th administration when Emax values were compared (F(2,27) = 2.26; p = 0.12; Fig. 2f), but differences were evident among AUC values (F(2,27) = 12.75; p = 0.0001; Fig. 2g).

Repeated Morphine or Fentanyl Administration Activates NLRP3, Microglia and Astrocytes in DRN

In order to evaluate the activation of microglia and astrocytes after repeated treatment with morphine or fentanyl, we analyzed NLRP3 expression in DRN 2 h after the 19th opioid administration. Figure 3 shows representative images of immunofluorescence to NLRP3, GFAP as marker of astrocytes, and CD11b, a protein expressed in microglia in control and opioid-treated groups. There was evidence of basal microglia and astrocyte activation in DRN in saline-treated rats, but only few cells were positive for NLRP3 expression (Fig. 3a). Both morphine and fentanyl significantly induced NLRP3 expression, but fentanyl levels of induction were higher than those produced by morphine (F(2,16) = 28.78; p < 0.0001; Fig. 3b). Both opioids increased the number of activated microglial cells (F(2,16) = 34.37; p < 0.0001; Fig. 3c) and GFAP fluorescence intensity (F(2,16) = 35.99; p < 0.0001; Fig. 3d) in astrocytes. We observed that GFAP-positive cells activated by morphine or fentanyl were mainly located within the medial aspects of the DRN, where serotonergic neurons are known to be concentrated (Abrams et al. 2004; Hornung 2012; Jacobs and Azmitia 1992). As a methodological control, we utilized a single injection of 1 mg/kg LPS, because it is a potent stimulator of the immune system and activates NLRP3 inflammasome (Rathinam et al. 2019). In accordance to previous reports, LPS produced an increase in NLRP3 expression as well as generalized microglia and astrocyte activation. We observed LPS-induced activation in astrocytes not only in DRN, but also within the dorsal PAG and neighboring areas (Supplementary Fig. S1).

Repeated Opioid Administration Induces NLRP3 Expression in Astrocytes and Neurons, Mainly Serotoninergic Neurons, but not in Microglia

In order to determine the cell type in which Inflammasome activation occurred, we examined NLRP3, CD11b, GFAP and NeuN markers in DRN. In saline-treated animals, some microglia, astrocytes and neurons were positive for NLRP3 expression (Fig. 4). Neither morphine nor fentanyl had an effect on microglia cells (F(2,48) = 0.41; p = 0.67; Fig. 4a, first row and Fig. 4b), but both opioids increased NLRP3 expression in neurons and astrocytes (Fig. 4a, second and third rows). The Mander’s overlapping coefficient indicated higher NLRP3 expression in astrocytes (F(2,48) = 56.61; p < 0.0001; Fig. 4c) and neurons (F(2,48) = 330.5; p < 0.0001; Fig. 4d) from animals treated with fentanyl as compared to those treated with morphine. Because 70–80% of serotonergic neuronal somas are located in DRN (Baker et al. 1991), we also did a double immunofluorescence for NLRP3 and the ratelimiting enzyme for serotonin synthesis, TPH-2. Of the total cells expressing NLRP3 in DRN, the proportion of TPH-2 positive cells was significantly higher in the group repeatedly treated with fentanyl than in the group that received morphine (579/836 cells (69.3%) vs. 176/592 cells (29.7%)); p < 0.0001; Fisher’s exact test; Fig. 4e, f).

Repeated Morphine or Fentanyl Administration Induces Caspase‑1 and GSDMD‑N Expression

To study if repeated morphine or fentanyl treatment induced NLRP3 oligomerization and functionality, we determined co-expression of NLRP3 and caspase-1 in DRN. While we observed basal expression of caspase-1 and NLRP3 in few cells (3.5 ± 1.2 cells per field) in saline-treated animals (Fig. 5), the repeated administration of either morphine or fentanyl significantly increased the number of NLRP3 and (F(2,24) = 121.2; p < 0.0001) but, as in previous experiments, fentanyl produced larger effects (Fig. 5f).

Differential Participation of Opioid and TLR4 Receptors on Opioid‑Dependent NLRP3 Expression

In order to identify the receptors involved in the observed opioid-dependent neuroinflammation, an experiment was designed using (−)-naloxone to antagonize opioid receptors and (+)-naloxone to block TLR4 receptors (Wang et al. 2016). We found that neither (−)-naloxone nor (+)-naloxone altered NLRP3, CD11b or GFAP basal expression levels (F(2,24) = 2.184; p = 0.13; white bars; Fig. 6). As we previously mentioned, both morphine and fentanyl significantly increased the number of NLRP3 positive cells. This increase was blocked by pre-treatment with (−)-naloxone, but not (+)-naloxone (Fig. 6a). Similarly, (−)-naloxone prevented the increase in CD11b positive cells produced by morphine or fentanyl, while (+)-naloxone had no effect (Fig. 6b). In contrast, the increase in fluorescence intensity to GFAP caused by morphine and fentanyl was prevented only by (+)-naloxone (Fig. 6c).

Minocycline and MCC950 Delay the Development of Tolerance Produced by Repeated Administration of Morphine or Fentanyl and Prevent Fentanyl‑Induced Hyperalgesia

Once determining the neuroinflammatory actions of morphine and fentanyl, we tested if tolerance to opioid antinociceptive effects and fentanyl-induced hyperalgesia could be prevented by the anti-inflammatory drug minocycline or by MCC950, a selective compound that averts NLRP3 inflammasome activation. To do this, we gave minocycline or MCC950 once a day (Fig. 1) and found that both inhibitors delayed tolerance development to morphine or fentanyl (Table 2). To further compare the effects of the two inhibitors tested, we analyzed the antinociception produced by the 19th administration of each opioid alone (dashed lines, Fig. 7), or with either minocycline or MCC950. Morphine reached Emax values above 70% and 85% when minocycline or MCC950 were used, in contrast with the less than 50% Emax observed with morphine alone (F(2,23) = 4.19; p = 0.028; Fig. 7a). Similar results were observed when AUC values were compared (F(2,23) = 4.69; p = 0.019). These results indicate that minocycline and MCC950 had similar effects in morphine treated groups (Fig. 7b, c). As to fentanyl, minocycline and MCC950 did not change the tolerance produced by this opioid in terms of Emax (F(2,23) = 0.7; p = 0.505; Fig. 7e), but they did in terms of AUC values (F(2,23) = 9.4; p = 0.001; Fig. 7f). Moreover, fentanyl-induced hyperalgesia did not occur in animals treated with minocycline or MCC950 (p = 0.31 and p = 0.38, respectively; one-sample Wilcoxon rank-sum test).

Oligomerization in DRN in Rats Repeatedly Injected with Morphine or Fentanyl

Once we found that MCC950 was effective in delaying opioid antinociceptive tolerance, we tested the effects of MCC950 on NLRP3 expression in DRN in rats that received 19 administrations of saline, morphine or fentanyl and were co-treated with MCC950. MCC950 had no effect on saline-treated animals, but prevented the increase in NLRP3 induced by morphine or fentanyl (F(5,48) = 24.22; p < 0.0001; Fig. 7g). The inhibitory effect of MCC950 was more evident on fentanyl-treated animals, where the number of NLRP3 positive cells diminished by 72.8% compared with those treated with morphine (64.6%). We did not find differences in NLRP3 oligomerization among the three groups treated with MCC950 (F(2,16) = 2.66; p = 0.99; Fig. 7g, h).

Discussion

Previous studies have shown that tolerance to the antinociceptive effects of morphine in rats can be attenuated by anti-inflammatory drugs, thus suggesting that neuroinflammation contributes to morphine actions (Harada et al. 2013; Mika et al. 2009), but no studies have been conducted for other opioids. Here we demonstrate that repeated administration of morphine or fentanyl produces differential expression of neuroinflammatory molecular markers, reflecting dissimilar activation of microglia and astrocytes, as well as NLRP3-dependent pyroptosis in specific cell types in the DRN by a mechanism involving opioid and TLR4 receptors. The differences between morphine and fentanyl to produce tolerance or hyperalgesia are related to their different efficacy to produce neuroinflammation (Fig. 8).
In the present study, we showed that while morphineinduced antinociception decreased in magnitude and duration with its repeated administration, fentanyl produced a transient antinociceptive effect followed by hyperalgesia. These findings further support previous results showing that fentanyl induces less tolerance than morphine (Bobeck et al. 2012; Molina-Martínez et al. 2014). While it has been reported that hyperalgesia is influenced by the experimental conditions in which chronic morphine or fentanyl are administered (Célèrier et al. 2000; Mao et al. 1995), we observed hyperalgesia induced by chronic administration of fentanyl, but not by morphine, under our tested experimental conditions. These findings from animal models are in line with clinical observation showing that repeated opioid administration induces analgesic tolerance and hyperalgesia, depending both on the opioid employed and on the administration schedule (Hayhurst and Durieux 2016).
Given the observed differences between opioids on tolerance development and hyperalgesia, we hypothesized that morphine and fentanyl also differ in their ability to induce neuroinflammation in the DRN. To test this hypothesis, we determined the effects of repeated administration of morphine or fentanyl on neuroinflammation, and compared them with those induced by LPS (Fig. 3a and Supplementary Fig. S1). By analyzing activation of microglia and astrocytes, and NLRP3 expression, we found that both morphine and fentanyl injection induced activation of astrocyte and microglia; activation that was similar in magnitude, but different in test. h Representative confocal images (600 × 500 µm) showing immunoreactivity to NLRP3 inflammasome (green) and nuclei (blue) in the DRN of rats that received 5 mg/kg MCC once a day and 19 administrations of morphine or fentanyl. Scale bar: 100 μm for complete images and 10 μm for magnification spatial distribution in comparison with the activation produced by a single LPS administration (Supplementary Fig. S1). There is evidence that morphine-induced glial activation occurs in areas related to descending pain control, such as PAG and spinal cord (Doyle et al. 2017; Harada et al. 2013; Posillico et al. 2015). Our results extend these findings to another opioid, fentanyl, and another brain region, DRN. Also, we provide evidence that neuroinflammation is drug-specific because there were regional differences in the activation of glial cells induced by LPS and by opioids.
Microglia cells are derived from early macrophage progenitors that migrate into the brain during embryonic development, while astrocytes are derived from neuronal precursor cells (Ginhoux et al. 2010, 2013; Miller and Gauthier 2007). In this work, we observed that NLRP3 inflammasome was activated after repeated opioid administration only in cells of neuronal origin; i.e., neurons and astrocytes, but not in microglia. It has been reported that LPS induces mature NLRP3 inflammasome expression in peripheral macrophages and monocytes (Awad et al. 2017), cells that arise from the same precursors as microglia (Ginhoux et al. 2010, 2013). However, other studies have found NLRP3 expression in non-immune cells under tissue damage conditions (Debye et al. 2018; von Herrmann et al. 2018). These data indicate that the role of microglia cells could be different when activated by opioids, DAMPs or PAMPS, such as LPS.
Our group and others have studied the pro- and antiinflammatory actions of opioids on other immune cells. In peritoneal mast cells of mice, acute administration of the same doses of morphine and fentanyl used in the present study inhibits the release of tumor necrosis factor (TNF) produced by LPS administration (Molina-Martínez et al. 2014). In human microglia cultures, acute morphine treatment suppresses LPS-induced chemokine release (Hu et al. 2000). Interestingly, acute fentanyl administration (0.1 μg, i.v.) induces NF-κB activation in monocytes obtained from human blood samples (Compton et al. 2015). In addition, repeated fentanyl administration (4x, 60 µg/kg, s.c., at 15-min intervals) produces pro-inflammatory cytokine release (TNF, IL-1β and IL-6) and microglia activation in rats (Chang et al. 2018). According to other authors, repeated morphine administration inhibits LPS-induced NLRP3 and cytokine expression in rats’ spleen (Mao et al. 2013), an organ with a large subpopulation of macrophages (Den Haan and Kraal 2012). Together, these data suggest that morphine and fentanyl produce different inflammatory effects depending on the cell type on which they act, and also that fentanyl produces larger pro-inflammatory effects than morphine.
Increased NLRP3 expression caused by fentanyl is associated with higher levels of caspase-1 and increased number of GSDMD-N positive cells, which strongly suggests that cell death by pyroptosis occurs mainly in fentanyl-treated rats. Because fentanyl produces hyperalgesia by the 19th administration and morphine does not, it is reasonable to suggest that cell death may be related not only to tolerance development, but also to hyperalgesia. Following this line of thought, it has been reported that increased neuroinflammation enhances nociceptive transmission (Martyn et al. 2019). Specifically, tolerance to morphine antinociception correlates with an increase in mRNA and protein levels of the pro-inflammatory cytokines IL-1β and IL-6 in spinal cord of rats (Raghavendra et al. 2002). Also, when IL-1β is intrathecally injected, it increases neuronal excitability and produces hyperalgesia in rats (Gustafson-Vickers et al. 2008; Oka et al. 1993; Reeve et al. 2000). Together, these data support the hypothesis that differences in neuroinflammation observed among opioids are positively correlated with their differences to induce analgesic tolerance and hyperalgesia. To the best of our knowledge, our study is the first to correlate opioid neuroinflammatory and antinociceptive actions.
Previous studies have proposed that morphine-induced neuroinflammation is mediated by both opioid receptors and TLR4 receptors (Hutchinson et al. 2011). Ellis and coworkers showed that morphine (10 mg/kg/day, per 2 weeks) increases pro-inflammatory cytokine release (IL-1β and TNF) in the spinal cord of rats with allodynia secondary to spinal cord injury, and proposed that this effect was mediated by TLR4 (Ellis et al. 2016). In order to determine whether opioid receptors or TLR4 receptors were involved in the observed neuroinflammation of glial and neuronal cells, we took advantage of the fact that (−)-naloxone is a non-specific opioid receptor antagonist (Lewanowitsch and Irvine 2003), while (+)-naloxone has no affinity for opioid receptors, but acts as a TLR4 receptor antagonist (Hutchinson et al. 2010; Wang et al. 2016). We found that (−)-naloxone, but not (+)-naloxone blocked morphine- and fentanyl-induced microglia activation, as well as NLRP3 expression, evidencing the role of opioid receptors in these effects. Conversely, only (+)-naloxone prevented the increase in GFAP immunoreactivity induced by morphine and fentanyl, which indicates that TLR4 receptors were involved in astrocyte activation. It is worth mentioning that both morphine and fentanyl have affinity for TLR4 receptors (Hutchinson et al. 2011; Wang et al. 2012) and that neurons and glial cells express this receptor subtype (Nicotra et al. 2012). However, we cannot discard TLR4 receptor activation by products resulting from pyroptotic cell death (Nyström et al. 2013). In this regard, it has been shown that chronic morphine can induce the release of DAMPs such as HMGB1, biglycan and heat shock protein 70 when nerve damage occurs (Grace et al. 2018; Qu et al. 2017; Yu et al. 2010). The findings in the present work showing that blockade of TLR4 receptors prevents astrogliosis, while blockade of opioid receptors prevents opioidinduced microgliosis and NLRP3 expression, indicate that opioids have differential effects on specific cells expressing these receptors. This is the first study showing that opioids produce different pro-inflammatory effects that depend on the specific cell type and receptor on which they act. More studies are needed to identify specific cellular and molecular targets that produce neuroinflammation after chronic opioid treatment.
To study if the tolerance produced by morphine or fentanyl was related to their capacity to produce neuroinflammation, we administered minocycline, which is an antibiotic with potent anti-inflammatory effects due to its inhibitory effect on NF-κB signaling pathway (Liu et al. 2017; Nikodemova et al. 2006). We did this because other authors have found that minocycline and pentoxifylline attenuate the analgesic tolerance and hyperalgesia produced by morphine in mice (Harada et al. 2013; Mika et al. 2009). Here we found that both morphine and fentanyl produce less tolerance and that fentanyl does not produce hyperalgesia in minocyclinetreated rats. This suggests that the neuroinflammatory effects mediated by the NF-κB signaling pathway contribute to both, tolerance development and hyperalgesia. Also, there is evidence that activation of opioid receptors can trigger NF-κB transcriptional activity in a neuronal environment (Ho et al. 2009). Because minocycline delays antinociceptive tolerance to morphine and fentanyl, it is reasonable to suppose that the opioid receptor-dependent pyroptosis observed in neurons, as well as the TLR4-dependent pyroptosis seen in astrocytes can be due to NF-κB activation.
To investigate a more direct correlation between neuroinflammation and antinociceptive tolerance to morphine and fentanyl, we administered MCC950, a very specific NLRP3 oligomerization inhibitor (Coll et al. 2019) that effectively crosses the blood–brain barrier when administered orally or intraperitoneally (Chen et al. 2017; Gordon et al. 2018). Systemic administration of this drug reduces neuroinflammation in animal models of brain damage (Dempsey et al. 2017; Xu et al. 2018) and attenuates inflammatory pain (Chen et al. 2019). In our study, we show that MCC950 prevents NLRP3 oligomerization in DRN induced by repeated morphine or fentanyl administration. Also, MCC950 delays antinociceptive tolerance induced by morphine or fentanyl and the hyperalgesia induced by fentanyl. These data suggest that neuroinflammation and tolerance development to morphine or fentanyl are directly correlated. Also, they provide evidence that NLRP3 inhibition could represent a novel therapeutic strategy to avoid opioid neuroinflammatory effects.
A limitation of this work is that we studied only male rats. Several studies have demonstrated that there are sex differences in opioid effects. For example, morphine is a more potent analgesic drug and develops more tolerance in males than in female rats (Craft et al. 1999). Here, we compare two opioids with similar analgesic effects and different ability to induce tolerance under the same administration schedule. Similar experiments need to be conducted in female animals using a specifically determined administration schedule due to the above-mentioned differences between males and females in opioid effects. We cannot discard the possibility that these differences in neuroimmune responses could contribute to sex differences in opioid-induced antinociception and tolerance development, but this topic must be addressed in a systematic way and for individual opioids (Doyle and Murphy 2017).

Conclusion

Repeated administration of morphine or fentanyl causes robust astrogliosis and microgliosis as well as a differential NLRP3-dependent pyroptosis in the DRN. Under identical experimental conditions, morphine produces more tolerance than fentanyl, while only fentanyl induces hyperalgesia. Opioid receptors are involved in neuronal pyroptosis and TLR4 receptors, in astrogliosis. This suggests that opioids target both receptor types in different cells to modulate specific neuroimmune responses. The finding that the NLPR3 inhibitor MCC950 and the anti-inflammatory drug minocycline delay both opioid-induced analgesia and fentanyl-induced hyperalgesia suggests that differences in neuroinflammation could determine the differences in the development of tolerance to each of these opioids and that both processes involve NF-κB and NLRP3 activation.

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