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. 2021 Oct;22(10):1273-1282.
doi: 10.1016/j.jpain.2021.03.152. Epub 2021 Apr 20.

Sexually Dimorphic Role of Toll-like Receptor 4 (TLR4) in High Molecular Weight Hyaluronan (HMWH)-induced Anti-hyperalgesia

Ivan J M Bonet  1 , Dionéia Araldi  1 , Paul G Green  2 , Jon D Levine  3
Affiliations

Sexually Dimorphic Role of Toll-like Receptor 4 (TLR4) in High Molecular Weight Hyaluronan (HMWH)-induced Anti-hyperalgesia

Ivan J M Bonet et al. J Pain. 2021 Oct.

Abstract

High molecular weight hyaluronan (HMWH), a prominent component of the extracellular matrix binds to and signals via multiple receptors, including cluster of differentiation 44 (CD44) and toll-like receptor 4 (TLR4). We tested the hypothesis that, in the setting of inflammation, HMWH acts at TLR4 to attenuate hyperalgesia. We found that the attenuation of prostaglandin E2 (PGE2)-induced hyperalgesia by HMWH was attenuated by a TLR4 antagonist (NBP2-26245), but only in male and ovariectomized female rats. In this study we sought to evaluated the role of the TLR4 signaling pathway in anti-hyperalgesia induced by HMWH in male rats. Decreasing expression of TLR4 in nociceptors, by intrathecal administration of an oligodeoxynucleotide (ODN) antisense to TLR4 mRNA, also attenuated HMWH-induced anti-hyperalgesia, in male and ovariectomized female rats. Estrogen replacement in ovariectomized females reconstituted the gonad-intact phenotype. The administration of an inhibitor of myeloid differentiation factor 88 (MyD88), a TLR4 second messenger, attenuated HMWH-induced anti-hyperalgesia, while an inhibitor of the MyD88-independent TLR4 signaling pathway did not. Since it has previously been shown that HMWH-induced anti-hyperalgesia is also mediated, in part by CD44 we evaluated the effect of the combination of ODN antisense to TLR4 and CD44 mRNA. This treatment completely reversed HMWH-induced anti-hyperalgesia in male rats. Our results demonstrate a sex hormone-dependent, sexually dimorphic involvement of TLR4 in HMWH-induced anti-hyperalgesia, that is MyD88 dependent. PERSPECTIVE: The role of TLR4 in anti-hyperalgesia induced by HMWH is a sexually dimorphic, TLR4 dependent inhibition of inflammatory hyperalgesia that provides a novel molecular target for the treatment of inflammatory pain.

Keywords: Hyperalgesia; anti-hyperalgesia; high molecular weight hyaluronan (HMWH); hyaluronan; prostaglandin E(2) (PGE(2)); toll-like receptor 4 (TLR4).

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Conflict of interest statement

Disclosures: All authors report no financial interests or potential conflicts of interest. This study was funded by National Institutes of Health (NIH) grant AR075334.

Figures

Figure 1.
Figure 1.. TLR4 antagonist attenuates HMWH-induced anti-hyperalgesia in male but not female rats.
Male, female or ovariectomized female rats were injected with PGE2 (100 ng/ 3 μL, i.d.) followed 5 min later by vehicle (3 μL, i.d.) or TLR4 receptor antagonist (NBP2–26245, 1 μg/ 3 μL, i.d.) and then 5 min later by HMWH (1 μg/3 μL, i.d.), all at the site of nociceptive testing on the dorsum of the hind paw. One group of rats received a single injection of TLR4 receptor antagonist (NBP2–26245, 1 μg/3 μL, i.d.) to confirm that it did not produce hyperalgesia by itself, and another group received PGE2 (100 ng/ 3 μL, i.d.) followed 5 min later by TLR4 receptor antagonist (NBP2–26245, 1 μg/ 3 μL, i.d.) to show that TLR4 receptor antagonist did not affect PGE2-induced hyperalgesia (dotted and black bars, respectively). Mechanical nociceptive threshold was measured 40 min after injection of PGE2. A. Percent reduction from baseline (top panel) in male rats: Intradermal HMWH attenuates PGE2-induced hyperalgesia (F(2,15)= 43.65, ****p<0.0001, when PGE2 was compared to PGE2 + HMWH-treated group; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). The anti-hyperalgesic effect of HMWH for PGE2-induced hyperalgesia was attenuated by the TLR4 antagonist in male rats (**p=0.0045, when HMWH-induced anti-hyperalgesia was compared between vehicle- and TLR4 antagonist-treated groups at 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). Mechanical nociceptive threshold (bottom panel) in male rats: Intradermal HMWH attenuates PGE2-induced hyperalgesia (F(2,15)= 25.42, ****p<0.0001, when PGE2- was compared to PGE2 + HMWH-treated group; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). Anti-hyperalgesic effect of HMWH was attenuated by the TLR4 antagonist (**p=0.01, when HMWH-induced anti-hyperalgesia was compared between vehicle- and TLR4 antagonist-treated groups at 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). n=6 per group. B. Percent reduction from baseline (top panel) in female rats: Intradermal PGE2-induced hyperalgesia was attenuated by HMWH, injected at the same site (F(2,15)= 43.65, ****p<0.0001, when PGE2 and PGE2 + HMWH-treated groups were compared 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). When the TLR4 antagonist was injected before HMWH, it did not affect the anti-hyperalgesic effect of HMWH for PGE2-induced hyperalgesia (ns, p>0.9, when HMWH-induced anti-hyperalgesia was compared between the vehicle- and TLR4 antagonist-treated groups at 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). Mechanical nociceptive threshold (bottom panel) in female rats: HMWH attenuated PGE2-induced hyperalgesia (F(2,15)= 46.52, ****p<0.0001, when PGE2 and PGE2 + HMWH-treated groups were compared 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). No attenuation of the anti-hyperalgesic effect of HMWH was observed in the group of female rats treated with the TLR4 antagonist (ns, p=0.67, when HMWH-induced anti-hyperalgesia was compared between vehicle- and TLR4 antagonist-treated groups at 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). n=6 per group. C. Percent reduction from baseline (top panel) in ovariectomized females: Intradermal HMWH attenuates PGE2-induced hyperalgesia in ovariectomized female rats (F(2,15)= 83.20, ****p<0.0001, when PGE2 and PGE2 + HMWH-treated groups were compared 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). An attenuation of the anti-hyperalgesic effect of HMWH, for PGE2-induced hyperalgesia, was observed in the ovariectomized female rats treated with the TLR4 antagonist (***p=0.0006, when HMWH-induced anti-hyperalgesia was compared between vehicle- and TLR4 antagonist-treated groups at 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). Nociceptive threshold (bottom panel) in ovariectomized female rats: HMWH attenuates PGE2-induced hyperalgesia (F(2,15)= 18.10, ****p<0.0001, when PGE2 and PGE2 + HMWH-treated groups were compared 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). Attenuation of the HMWH-induced anti-hyperalgesia was observed in the ovariectomized female rats treated with the TLR4 receptor antagonist (**p=0.0034, when HMWH-induced anti-hyperalgesia was compared between vehicle- and TLR4 antagonist-treated groups at 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). n=6 per group.
Figure 2.
Figure 2.. MyD88 inhibitor attenuates HMWH-induced anti-hyperalgesia in male rats.
Male rats received PGE2 (100 ng/ 3 μL, i.d.) followed 5 min later by vehicle (3 μL, i.d.) or a MyD88 inhibitor (ST2825, 1 μg/ 3 μL, i.d.), and then 5 min later by HMWH (1 μg/ 3 μL, i.d.). Mechanical nociceptive threshold was measured 40 min after intradermal PGE2. An additional group of rats received MyD88 inhibitor (1 μg/ 3 μL, i.d.) to confirm that it did not affect nociceptive threshold by itself (dotted bar), and another group of rats received PGE2 (100 ng/ 3 μL, i.d.) followed 5 min later by MyD88 inhibitor (1 μg/ 3 μL, i.d.) to show that it did not affect PGE2-induced hyperalgesia (black bar). Percent reduction from baseline (left panel): Intradermal administration of HMWH attenuate PGE2-induced hyperalgesia (F(2,15)= 35.30, ****p<0.0001, when PGE2- and PGE2 + HMWH-treated groups were compared 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). The anti-hyperalgesic effect of HMWH was attenuated by the MyD88 inhibitor (**p=0.0048, when HMWH-induced anti-hyperalgesia was compared between vehicle- and MyD88 inhibitor-treated groups at 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). Mechanical nociceptive threshold (right panel): Intradermal HMWH attenuated PGE2-induced hyperalgesia (F(2,15)= 70.50, ****p<0.0001, when PGE2- and PGE2 + HMWH-treated groups were compared 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). The anti-hyperalgesic effect of HMWH was attenuated by the MyD88 inhibitor (***p=0.0002, when HMWH-induced anti-hyperalgesia was compared between vehicle- and MyD88 inhibitor-treated groups at 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). n=6 per group.
Figure 3.
Figure 3.. IRF3 inhibitor does not attenuate HMWH induced anti-hyperalgesia in male rats.
Male rats were injected with PGE2 (100 ng/ 3 μL, i.d.) followed, 5 min later by vehicle (3 μL, i.d.) or an IRF3 inhibitor (BX-795, 1 μg/ 3 μL, i.d.), and then 5 min later by HMWH (1 μg/ 3 μL, i.d.). Mechanical nociceptive threshold was measured 40 min after intradermal PGE2. One group of rats was treated with a single injection of the IRF3 inhibitor (1 μg/ 3 μL, i.d.) to confirm that this inhibitor alone did not affect nociceptive threshold (dotted bar), and another group of rats received PGE2 (100 ng/ 3 μL, i.d.) followed 5 min later by IRF3 inhibitor (1 μg/ 3 μL, i.d.) to show that it did not affect PGE2-induced hyperalgesia (black bar). Percent reduction from baseline (left panel): Intradermal administration of HMWH attenuates PGE2-induced hyperalgesia (F(2,15)= 45.55, ****p<0.0001, when PGE2- and PGE2, + HMWH-treated groups were compared 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). The IRF3 inhibitor did not affect the anti-hyperalgesic effect of HMWH (ns, p>0.9, when HMWH-induced anti-hyperalgesia was compared between vehicle- and IRF3 inhibitor-treated groups at 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). Mechanical nociceptive threshold (right panel): PGE2-induced hyperalgesia was attenuated by intradermal HMWH (F(2,15)= 88.25, ****p<0.0001, when the PGE2 and PGE2 + HMWH-treated groups were compared 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post-hoc comparisons test). The IRF3 inhibitor did not affect the anti-hyperalgesic effect of HMWH (ns, p>0.9, when HWMH-induced anti-hyperalgesia was compared between vehicle- and IRF3 inhibitor-treated groups at 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). n=6 per group.
Figure 4.
Figure 4.. Involvement of TLR4 in HMWH-induced anti-hyperalgesia in female rats is sex hormone dependent.
A. Gonad intact, orchiectomized, and orchiectomized dihydrotestosterone treated male rats were injected with ODN antisense or mismatch (120 μg/ 20 μl, i.t.) for TLR4 mRNA, daily for 3 consecutive days. On the fourth day, approximately 17 h after the last ODN administration, PGE2 (100 ng/ 5 μL, i.d.) was injected at the site of nociceptive testing, intradermally, followed 10 min later by HMWH (1 μg/ 5 μL, i.d.) injected at the same site. Mechanical nociceptive threshold was evaluated 40 min after PGE2. Percent reduction from baseline (top panel): The anti-hyperalgesic effect of HMWH on PGE2-induced hyperalgesia was attenuated by ODN antisense to TLR4 mRNA (gonad intact groups, t(10)= 2.638, *p=0.0248; orchiectomized groups, t(10)= 4.678, ***p=0.0009; orchiectomized + dihydrotestosterone, t(10)= 4.200, ***p=0.0018, when the respective mismatch and antisense groups were compared 40 min after PGE2; unpaired Student’s t-test). Nociceptive threshold (bottom panel): Anti-hyperalgesic effect of HMWH was attenuated by ODN antisense to TLR4 mRNA (gonad intact groups, t(10)= 3.980, **p=0.0026; orchiectomized groups, t(10)= 3.343, **p=0.0075; orchiectomized + dihydrotestosterone, t(10)= 4.200, ***p=0.0001, when the respective mismatch and antisense groups were compared 40 min after PGE2; unpaired Student’s t-test). n=6 per group. B. Gonad intact, ovariectomized and 17-β estradiol replaced ovariectomized female rats were injected with ODN antisense or mismatch (120 μg/ 20 μl, i.t.) for TLR4 mRNA, daily for 3 consecutive days. On the fourth day, approximately 17 h after the last intrathecal administration of ODNs, PGE2 (100 ng/ 5 μL, i.d.) was injected intradermally, followed 10 min later by HMWH (1 μg/ 5 μL, i.d.), at the same site. Mechanical nociceptive threshold was evaluated 40 min after PGE2. Percent reduction from baseline (top panel): The anti-hyperalgesic effect of HMWH on PGE2-induced hyperalgesia was not affected by TLR4 ODN antisense in gonad-intact and 17-β estradiol-replaced ovariectomized female rats (gonad intact groups, t(10)= 0.1773, p=0.8628; ovariectomized + 17-β estradiol, t(10)= 0.6999, p=0.4999, when the respective mismatch and antisense groups are compared 40 min after PGE2; unpaired Student’s t-test). However, ovariectomized female rats show an attenuation of HWMH-induced anti-hyperalgesia by intrathecal TLR4 ODN antisense (ovariectomized groups, t(10)= 3.562, *p=0.0261; when the respective sense and antisense groups are compared 40 min after PGE2; unpaired Student’s t-test). Nociceptive threshold (bottom panel): TLR4 ODN antisense did not affect the anti-hyperalgesic effect of HMWH in gonad-intact and ovariectomized + 17-β estradiol females (gonad intact groups, t(10)= 0.5633, p=0.5856; ovariectomized + 17-β estradiol, t(10)= 0.8145, p=0.4343, when the respective mismatch and antisense groups are compared 40 min after PGE2; unpaired Student’s t-test). However, in ovariectomized female rats the intrathecal treatment with TLR4 ODN antisense attenuates HWMH-induced anti-hyperalgesia (ovariectomized group, t(10)= 2.412, *p=0.0366; when mismatch and antisense groups are compared 40 min after PGE2; unpaired Student’s t-test). n=6 per group.
Figure 5.
Figure 5.. Reversal of PGE2 hyperalgesia by HMWH is markedly attenuated by the combination of CD44 and TLR4 antisense in male rats.
Male rats were injected with the combination of ODN antisense or mismatch (120 μg/20 μl/each, i.t.) for TLR4 and CD44 mRNA, daily for 3 consecutive days. On the fourth day, approximately 17 h after the last intrathecal administration of ODN, PGE2 (100 ng/5 μL, i.d.) was injected intradermally, followed 10 min later by HMWH (1 μg/ 5 μL, i.d.) at the same site. Mechanical nociceptive threshold was evaluated 40 min after intradermal PGE2. Percent reduction from baseline (left panel): Intradermal HMWH attenuates PGE2-induced hyperalgesia (F(2,15)= 74.12, ****p<0.0001, when the PGE2- and PGE2 + HMWH-treated groups were compared 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). Intrathecal treatment with the combination of CD44 + TLR4 ODN antisense reverses the HMWH-induced anti-hyperalgesia (****p<0.0001, when HMWH-induced anti-hyperalgesia was compared between CD44 + TLR4 mismatch- and CD44 + TLR4 antisense-treated groups at 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). Nociceptive threshold (rigth panel): PGE2-induced hyperalgesia was attenuated by HMWH (F(2,15)= 108.3, ****p<0.0001, when the PGE2- and PGE2 + HMWH-treated groups were compared 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post hoc comparisons test). The combination of CD44 + TLR4 ODN antisense reverses the HMWH-induced anti-hyperalgesia (****p<0.0001, when HMWH-induced anti-hyperalgesia was compared between CD44 + TLR4 mismatch- and CD44 + TLR4 antisense-treated groups at 40 min after PGE2; one-way ANOVA followed by Bonferroni’s post-hoc comparisons test). n=6 per group.
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