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. 2019 Aug 14;39(33):6414-6424.
doi: 10.1523/JNEUROSCI.0966-19.2019. Epub 2019 Jun 17.

Role of Nociceptor Toll-like Receptor 4 (TLR4) in Opioid-Induced Hyperalgesia and Hyperalgesic Priming

Dioneia Araldi  1   2   3 , Oliver Bogen  1   2   3 , Paul G Green  2   3   4 , Jon D Levine  5   2   3
Affiliations

Role of Nociceptor Toll-like Receptor 4 (TLR4) in Opioid-Induced Hyperalgesia and Hyperalgesic Priming

Dioneia Araldi et al. J Neurosci. .

Abstract

In addition to analgesia, opioids produce opioid-induced hyperalgesia (OIH) and neuroplasticity characterized by prolongation of inflammatory-mediator-induced hyperalgesia (hyperalgesic priming). We evaluated the hypothesis that hyperalgesia and priming induced by opioids are mediated by similar nociceptor mechanisms. In male rats, we first evaluated the role of nociceptor Toll-like receptor 4 (TLR4) in OIH and priming induced by systemic low-dose morphine (LDM, 0.03 mg/kg). Intrathecal oligodeoxynucleotide antisense to TLR4 mRNA (TLR4 AS-ODN) prevented OIH and prolongation of prostaglandin E2 hyperalgesia (priming) induced by LDM. In contrast, high-dose morphine (HDM, 3 mg/kg) increased nociceptive threshold (analgesia) and induced priming, neither of which was attenuated by TLR4 AS-ODN. Protein kinase C ε (PKCε) AS-ODN also prevented LDM-induced hyperalgesia and priming, whereas analgesia and priming induced by HDM were unaffected. Treatment with isolectin B4 (IB4)-saporin or SSP-saporin (which deplete IB4+ and peptidergic nociceptors, respectively), or their combination, prevented systemic LDM-induced hyperalgesia, but not priming. HDM-induced priming, but not analgesia, was markedly attenuated in both saporin-treated groups. In conclusion, whereas OIH and priming induced by LDM share receptor and second messenger mechanisms in common, action at TLR4 and signaling via PKCε, HDM-induced analgesia, and priming are neither TLR4 nor PKCε dependent. OIH produced by LDM is mediated by both IB4+ and peptidergic nociceptors, whereas priming is not dependent on the same population. In contrast, priming induced by HDM is mediated by both IB4+ and peptidergic nociceptors. Implications for the use of low-dose opioids combined with nonopioid analgesics and in the treatment of opioid use disorder are discussed.SIGNIFICANCE STATEMENT Opioid-induced hyperalgesia (OIH) and priming are common side effects of opioid agonists such as morphine, which acts at μ-opioid receptors. We demonstrate that OIH and priming induced by systemic low-dose morphine (LDM) share action at Toll-like receptor 4 (TLR4) and signaling via protein kinase C ε (PKCε) in common, whereas systemic high-dose morphine (HDM)-induced analgesia and priming are neither TLR4 nor PKCε dependent. OIH produced by systemic LDM is mediated by isolectin B4-positive (IB4+) and peptidergic nociceptors, whereas priming is dependent on a different class of nociceptors. Priming induced by systemic HDM is, however, mediated by both IB4+ and peptidergic nociceptors. Our findings may provide useful information for the use of low-dose opioids combined with nonopioid analgesics to treat pain and opioid use disorders.

Keywords: hyperalgesic priming; morphine; opioid-induced hyperalgesia (OIH); protein kinase epsilon (PKCε); toll-like receptor 4 (TLR4).

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Figures

Figure 1.
Figure 1.
TLR4 dependence of OIH and priming induced by systemic LDM. A. Western blot analysis of DRG extracts from rats injected with 120 μg of antisense ODN/d for 3 consecutive days revealed a significant decrease in anti-TLR4 immunoreactivity when compared with the extracts derived from mismatch treated rats (−22.07 ± 3.42%, unpaired Student's t test, *p < 0.05, n = 6). The calculated molecular weight of TLR4 is 96 kDa (according to UniProtKB database entry Q9QX05). The observed difference between the calculated and apparent molecular weight can be accounted for by the glycosylation of TLR4. β-actin, which was used as a loading control, has a calculated molecular weight of ∼42 kDa (UniProtKB database entry P60771). Groups of rats were treated intrathecally with an AS-ODN (120 μg in 20 μl/d, gray circles and bars; B,C) or MM-ODN (120 μg in 20 μl/d; black circles and bars; B,C) against TLR4 mRNA once a day for 3 consecutive days. B, On the fourth day, ∼17 h after the last intrathecal administration of ODNs, when the mechanical nociceptive threshold was not significantly different from pre-ODN baselines (t(5) = 0.8305; p = 0.4441, for the TLR4 MM-ODN-treated group and, t(5) = 1.275; p = 0.2582, for the TLR4 AS-ODN-treated group, when the mechanical nociceptive threshold is compared before and ∼17 h after the third intrathecal injection of ODNs; paired Student's t test), systemic LDM (0.03 mg/kg) was injected subcutaneously and the mechanical nociceptive threshold was evaluated 15, 30, 45, and 60 min after its injection. In the TLR4 AS-ODN-treated group, systemic LDM administration did not induce hyperalgesia measured at 30, 45, and 60 min after administration, as is observed in the TLR4 MM-ODN-treated group (F(1,10) = 236.45, ****p < 0.0001; when the hyperalgesia in the TLR4 MM-ODN-treated group is compared with TLR4 AS-ODN-treated group at 30, 45 and 60 min after systemic LDM; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). At the end of the fourth day, rats again received TLR4 AS- or MM-ODN. C, On the fifth day, ∼24 h after administration of systemic LDM, when the mechanical nociceptive threshold was not significantly different from the premorphine (mph) baselines (t(5) = 0.6143; p = 0.5659, for the TLR4 MM-ODN-treated group and, t(5) = 1.234; p = 0.2722, for the TLR4 AS-ODN-treated group, when the mechanical nociceptive threshold is compared before and 24 h after systemic LDM; paired Student's t test), PGE2 (100 ng in 5 μl) was injected intradermally and the mechanical nociceptive threshold was evaluated 30 min and 4 h later. In the group treated with TLR4 AS-ODN, which received systemic LDM, the prolongation of PGE2-induced hyperalgesia was inhibited (F(1,10) = 93.78, ****p < 0.0001; when the hyperalgesia in the TLR4 AS-ODN- and the MM-ODN-treated groups is compared at the fourth hour after intradermal PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). These findings indicate that both OIH and priming induced by systemic LDM are TLR4 dependent. (n = 6 paws per group).
Figure 2.
Figure 2.
TLR4 independence for systemic HDM-induced analgesia and priming. Rats received intrathecal injections of AS-ODN (120 μg in 20 μl/d; white circles and bars) or MM-ODN (120 μg in 20 μl/d; black circles and bars) against TLR4 mRNA daily for 3 consecutive days. A, On the fourth day, ∼17 h after the last ODN injection, systemic HDM (3 mg/kg) was injected subcutaneously and mechanical nociceptive threshold measured 15, 30, 45, and 60 min after its injection. Systemic HDM induced analgesia in both TLR4 AS- and MM-ODN-treated groups at all time points evaluated (F(1,10) = 0.137, p = 0.7184, when the analgesia in the TLR4 AS-ODN- and the MM-ODN-treated groups is compared at 15, 30, 45, and 60 min after systemic HDM; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). At the end of the fourth day, rats received another dose of TLR4 AS- or MM-ODN. B, Approximately 24 h after systemic HDM, when the mechanical nociceptive threshold was not significantly different from premorphine baseline (t(5) = 0.1334; p = 0.8991, for the MM-ODN-treated group, and t(5) = 0.2548; p = 0.8090, for the AS-ODN-treated group, when the mechanical nociceptive threshold is compared before and 24 h after systemic HDM; paired Student's t test), PGE2 (100 ng in 5 μl) was injected intradermally and the mechanical nociceptive threshold at the injection site was evaluated 30 min and 4 h after its injection. In both the TLR4 AS- and MM-ODN-treated groups, the prolongation of PGE2-induced hyperalgesia was present (ns, F(1,10) = 0.133, p = 0.7231, when the hyperalgesia in the TLR4 AS-ODN- and the MM-ODN-treated groups is compared at the fourth hour after intradermal PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that analgesia and priming induced by systemic HDM are both TLR4 independent. (n = 6 paws per group).
Figure 3.
Figure 3.
PKCε dependence of hyperalgesia and priming induced by systemically administered LDM. Rats received intrathecal injections of AS-ODN (120 μg in 20 μl/d; gray circles and bars) or MM-ODN (120 μg in 20 μl/d; black circles and bars) against PKCε mRNA daily for 3 consecutive days. A, On the fourth day, ∼17 h after the last ODN injection, when the mechanical nociceptive threshold was not significantly different from the pre-ODN baseline (t(5) = 1.904; p = 0.1152, for the MM-ODN-treated group, and t(5) = 0.7324; p = 0.4968, for the AS-ODN-treated group, when the mechanical nociceptive threshold is compared before and after systemic LDM; paired Student's t test), LDM (0.03 mg/kg) was injected subcutaneously and the mechanical nociceptive threshold was evaluated 15, 30, 45, and 60 min after its injection. Systemic LDM did not induce hyperalgesia at 30, 45 and 60 min after administration in the group treated with AS-ODN for PKCε (F(1,10) = 217.5, ****p < 0.0001, when the hyperalgesia in the PKCε AS-ODN- and the MM-ODN-treated groups is compared 30, 45 and 60 min after systemic LDM; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). At the end of the fourth day, rats again received PKCε AS- or MM-ODN. B, On the fifth day, ∼24 h after systemic LDM, when the mechanical nociceptive threshold was not significantly different from premorphine (mph) baseline (t(5) = 0.8811; p = 0.4186, for the MM-ODN-treated group, and t(5) = 0.5649; p = 0.5965, for the AS-ODN-treated group, when the mechanical nociceptive threshold is compared before and 24 h after systemic LDM; paired Student's t test), PGE2 (100 ng in 5 μl) was injected intradermally and the mechanical nociceptive threshold evaluated 30 min and 4 h later. PGE2 did not induce prolonged hyperalgesia in the AS-ODN-treated group (F(1,10) = 151.2, ****p < 0.0001, when the hyperalgesia in the PKCε AS-ODN- and the MM-ODN-treated groups is compared at the fourth hour after intradermal PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that both systemic LDM-induced hyperalgesia and priming are PKCε dependent. (n = 6 paws per group).
Figure 4.
Figure 4.
PKCε independence of analgesia and priming induced by systemic HDM. Rats received intrathecal injections of AS-ODN (120 μg in 20 μl/d; white circles and bars) or MM-ODN (120 μg in 20 μl/d; black circles and bars) against PKCε mRNA daily for 3 consecutive days. A, On the fourth day, ∼17 h after the last ODN injection, systemic HDM (3 mg/kg) was injected subcutaneously and the mechanical nociceptive threshold was evaluated 15, 30, 45, and 60 min after its injection. In both the PKCε AS- and MM-ODN-treated groups, systemic HDM induced analgesia at all time points evaluated (F(1,10) = 0.2507, p = 0.6274, when the analgesia in the AS-ODN- and the MM-ODN-treated groups is compared at 15, 30, 45, and 60 min after systemic HDM; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). At the end of the fourth day, rats received another dose of PKCε, AS- or MM-ODN. B, Approximately 24 h after systemic HDM, when the mechanical nociceptive threshold was not significantly different from the premorphine baseline (t(5) = 0.1395; p = 0.8945, for the MM-ODN-treated group, and t(5) = 1.536; p = 0.1852, for the AS-ODN-treated group, when the mechanical nociceptive threshold is compared before and after systemic HDM; paired Student's t test), PGE2 (100 ng in 5 μl) was injected intradermally and the mechanical nociceptive threshold was evaluated 30 min and 4 h after injection. The prolongation of PGE2-induced hyperalgesia was present in both the PKCε AS- and MM-ODN-treated groups (ns = not significant, F(1,10) = 1.043, p = 0.3311, when the hyperalgesia in the AS-ODN- and the MM-ODN-treated groups is compared at the fourth hour after intradermal PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). These findings support the suggestion that analgesia and priming induced by systemic HDM are both PKCε independent. (n = 6 paws per group).
Figure 5.
Figure 5.
Role of IB4+ and peptidergic nociceptors in hyperalgesia and priming induced by LDM. Rats received an intrathecal injection of vehicle (20 μl; black squares and bars), IB4-saporin (3.2 μg in 20 μl; gray circles and bars), SSP-saporin (100 ng in 20 μl; white circles and bars), or the combination of IB4-saporin (3.2 μg in 10 μl) and SSP-saporin (100 ng in 10 μl; black circles and dotted bars). A, Fourteen days later, a time at which the mechanical nociceptive threshold was not different from the pre-vehicle and pre-saporin administration baseline (t(5) = 0.107; p = 0.9190, for the vehicle-treated group, t(5) = 0.667; p = 0.5343, for the IB4-saporin-treated group, t(5) = 1.150; p = 0.1842, for the SSP-saporin-treated group, and t(5) = 0.128; p = 0.9031, for the combination of saporins-treated group, when the mechanical nociceptive threshold is compared before and 14 d after intrathecal treatments; paired Student's t test), LDM (0.03 mg/kg) was injected subcutaneously and the mechanical nociceptive threshold was evaluated 15, 30, 45, and 60 min after LDM injection. Systemic LDM-induced hyperalgesia was blocked at 30, 45, and 60 min after systemic LDM in all three groups: those previously treated with IB4-saporin, those previously treated with SSP-saporin, and those previously treated with their combination (F(3.20) = 135.3, ****p < 0.0001; when the hyperalgesia in the vehicle- and the saporin-treated groups is compared at 30, 45, and 60 min after systemic LDM; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). B, Twenty-four hours later, at which time the mechanical nociceptive threshold was not different from the premorphine baseline (t(5) = 1.772; p = 0.1366, for the vehicle-treated group, t(5) = 0.5462; p = 0.6084, for the IB4-saporin-treated group, t(5) = 1.169; p = 0.1322, for the SSP-saporin-treated group, and t(5) = 1.746; p = 0.1412, for the combination of saporin-treated group, when the mechanical nociceptive threshold is compared before and after systemic LDM; paired Student's t test), PGE2 (100 ng in 5 μl) was injected intradermally and the mechanical nociceptive threshold was evaluated 30 min and 4 h later. The prolongation of PGE2 hyperalgesia was present in all groups treated with vehicle, IB4-saporin, SSP-saporin, and the combination of saporins (ns = not significant, F(3,20) = 1.46, p = 0.2562; when the hyperalgesia in the vehicle- and saporins-treated groups is compared at the fourth hour after intradermal PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). These findings demonstrate that systemic LDM acts in both classes of nociceptors to induce hyperalgesia, but not priming, in the peripheral terminal of the nociceptor. (n = 6 paws/6 rats per group).
Figure 6.
Figure 6.
Role of IB4+ and peptidergic nociceptors in analgesia and priming induced by systemic HDM. Rats received intrathecal vehicle (20 μl; black squares and bars), IB4-saporin (3.2 μg in 20 μl; gray circles and bars), SSP-saporin (100 ng in 20 μl; white circles and bars), or the combination of IB4-saporin (3.2 μg in 10 μl) and SSP-saporin (100 ng in 10 μl; black circles and dotted bars). A, Fourteen days later, HDM (3 mg/kg) was injected subcutaneously and the mechanical nociceptive threshold was evaluated 15, 30, 45, and 60 min after HDM injection. HDM-induced analgesia was observed in all four treated-groups, without any attenuation, at any time point (F(3,20) = 1.529, p = 0.2377; when the analgesia in the vehicle-, IB4-saporin-, SSP-saporin-, and combination of saporin-treated groups is compared at 15, 30, 45, and 60 min after systemic HDM; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). B, Twenty-four hours after systemic HDM, a time at which the mechanical nociceptive threshold was not different from the premorphine baseline (t(5) = 0.5423; p = 0.6109, for the vehicle-treated group, t(5) = 1.557; p = 0.1801, for the IB4-saporin-treated group, t(5) = 0.7324; p = 0.4968, for the SSP-saporin-treated group, and t(5) = 1.536; p = 0.1852, for the combination of saporin-treated group, when the mechanical nociceptive threshold is compared before and after systemic HDM; paired Student's t test), PGE2 (100 ng in 5 μl) was injected intradermally and the mechanical nociceptive threshold was evaluated 30 min and 4 h later. In the group treated with IB4-saporin, PGE2 hyperalgesia was partially attenuated at the fourth hour (F(3,20) = 27.32, ***p = 0.0001), whereas in the group previously treated with SSP-saporin, the prolongation of PGE2 hyperalgesia was markedly attenuated (F(3,20) = 27.32, ****p < 0.0001; when the hyperalgesia in the vehicle- and in the three saporin-treated groups is compared at the fourth hour after intradermal PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that systemic HDM mainly requires peptidergic nociceptors to develop priming, but not analgesia, in the peripheral terminal of the nociceptor. Because adding IB4-saporin to SSP-saporin did not produce a further decrease, the small contribution of IB4-saporin is likely due to the overlapping population of weakly peptidergic IB4+ nociceptors (n = 6 paws/6 rats per group).
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