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. 2018 Jan 10;38(2):308-321.
doi: 10.1523/JNEUROSCI.2695-17.2017. Epub 2017 Nov 24.

CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia

Luiz F Ferrari  1 , Eugen V Khomula  1 , Dioneia Araldi  1 , Jon D Levine  2
Affiliations

CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia

Luiz F Ferrari et al. J Neurosci. .

Abstract

We studied, in male Sprague Dawley rats, the role of the cognate hyaluronan receptor, CD44 signaling in the antihyperalgesia induced by high molecular weight hyaluronan (HMWH). Low molecular weight hyaluronan (LMWH) acts at both peptidergic and nonpeptidergic nociceptors to induce mechanical hyperalgesia that is prevented by intrathecal oligodeoxynucleotide antisense to CD44 mRNA, which also prevents hyperalgesia induced by a CD44 receptor agonist, A6. Ongoing LMWH and A6 hyperalgesia are reversed by HMWH. HMWH also reverses the hyperalgesia induced by diverse pronociceptive mediators, prostaglandin E2, epinephrine, TNFα, and interleukin-6, and the neuropathic pain induced by the cancer chemotherapy paclitaxel. Although CD44 antisense has no effect on the hyperalgesia induced by inflammatory mediators or paclitaxel, it eliminates the antihyperalgesic effect of HMWH. HMWH also reverses the hyperalgesia induced by activation of intracellular second messengers, PKA and PKCε, indicating that HMWH-induced antihyperalgesia, although dependent on CD44, is mediated by an intracellular signaling pathway rather than as a competitive receptor antagonist. Sensitization of cultured small-diameter DRG neurons by prostaglandin E2 is also prevented and reversed by HMWH. These results demonstrate the central role of CD44 signaling in HMWH-induced antihyperalgesia, and establish it as a therapeutic target against inflammatory and neuropathic pain.SIGNIFICANCE STATEMENT We demonstrate that hyaluronan (HA) with different molecular weights produces opposing nociceptive effects. While low molecular weight HA increases sensitivity to mechanical stimulation, high molecular weight HA reduces sensitization, attenuating inflammatory and neuropathic hyperalgesia. Both pronociceptive and antinociceptive effects of HA are mediated by activation of signaling pathways downstream CD44, the cognate HA receptor, in nociceptors. These results contribute to our understanding of the role of the extracellular matrix in pain, and indicate CD44 as a potential therapeutic target to alleviate inflammatory and neuropathic pain.

Keywords: CD44; extracellular matrix; hyaluronan; hyperalgesia; nociceptor.

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Figures

Figure 1.
Figure 1.
Dose- and time-effect relationships for LMWH-induced mechanical hyperalgesia. Mechanical nociceptive threshold was evaluated before and 5, 10, 15, 20, and 30 min after intradermal injection of LMWH (0.1, 1, or 10 μg) on the dorsum of the hindpaw, in separate groups of rats. All three doses induced robust mechanical hyperalgesia (0.1 μg group: F(2.164,10.82) = 14.69, p = 0.0007; 1 μg group: F(2.366,11.83) = 38.95, p < 0.0001; 10 μg group: F(1.968,9.839) = 30.07, p < 0.0001) when the mechanical nociceptive threshold over time is compared with pre-LMWH injection baseline (repeated-measures ANOVA, followed by Bonferroni post hoc test). For the 0.1 μg dose, hyperalgesia was significant starting 15 min after injection (*p = 0.0062, when the mechanical threshold at this time point is compared with baseline). For the 1 and 10 μg doses, the hyperalgesia was already significant by 10 min (1 μg, ***p = 0.0010; 10 μg, **p = 0.0060). n = 6 paws per group.
Figure 2.
Figure 2.
Role of peptidergic (IB4−) and nonpeptidergic (IB4+) nociceptors in LMWH-induced hyperalgesia. A, Separate groups of rats received a single intrathecal injection of one of two neurotoxins, isolectin B4-saporin (IB4sap, selective for nonpeptidergic neurons, 3.2 μg, light gray bar), or [Sar9,Met(O2)11] substance P-saporin (SSPsap, selective for SP-containing fibers, 100 ng, dark gray bar), or their combination (black bar). A control group (white bar) received saline. LMWH (1 μg) was injected intradermally on the dorsum of the hindpaw 14 d later. Significant attenuation of LMWH-induced hyperalgesia was observed in all neurotoxin-treated groups (IB4sap: t(22) = 4.162, ***p = 0.0002; SSPsap: t(22) = 5.341, ****p < 0.0001; combination: t(22) = 7.762, ****p < 0.0001, compared with the control group; unpaired Student's t test). B, One week later, the control group (white bar) and the group treated with the combination of toxins (black bar), shown in A, were each further divided into 2 groups, which received an intradermal injection of either 8-bromo cAMP or ψεRACK (both 1 μg), on the dorsum of the hindpaw. At that time point, the mechanical nociceptive thresholds were not significantly different from pre-LMWH baseline (8-bromo cAMP: control group: t(5) = 0.9715, p = 0.1880; combination group: t(5) = 1.321, p = 0.1219; ψεRACK: control group: t(5) = 1.305, p = 0.1244; combination group: t(5) = 0.6697, p = 0.2664, all nonsignificant, when the mechanical nociceptive threshold before LMWH injection and immediately before 8-bromo cAMP or ψεRACK injection are compared, paired Student's t test). Robust mechanical hyperalgesia was observed in all groups, 30 min after injection (8-bromo cAMP, control: t(5) = 21.02, p < 0.0001; combination: t(5) = 4.776, p = 0.0025; ψεRACK, control: t(5) = 31.30, p < 0.0001; combination: t(5) = 6.173, p = 0.0008, when the mechanical nociceptive thresholds were compared with baseline thresholds; paired Student's t test), although in the groups pretreated with the combination of the two toxins, there was attenuation compared with the control groups (8-bromo cAMP-treated groups: t(10) = 3.907, *p = 0.0015; ψεRACK-treated groups: t(10) = 3.501, **p = 0.0029; unpaired Student's t test). A, n = 12 paws per group. B, n = 6 paws per group.
Figure 3.
Figure 3.
ODN antisense to CD44 mRNA dose-dependently attenuates LMWH- and A6-induced hyperalgesia. Groups of rats were treated daily with 1 of 3 doses (40, 80, or 120 μg, spinal intrathecal injections) of ODN, antisense (black bars) or sense (white bars) for CD44 mRNA, for 3 consecutive days. On the fourth day, LMWH (left) or A6 (right) (both 1 μg) was injected intradermally on the dorsum of the hindpaw and the mechanical nociceptive threshold evaluated 30 min later. LMWH and A6 both induced robust hyperalgesia, with no difference between the groups treated with the different doses of CD44 sense ODN (LMWH-treated groups: F(2,15) = 0.4509, p = 0.6454; A6-treated groups: F(2,15) = 0.5264, p = 0.6013, both nonsignificant, when the hyperalgesia in the three groups is compared, one-way ANOVA followed by Bonferroni post hoc test). However, the hyperalgesia induced by LMWH and A6 was dose-dependently attenuated in the groups pretreated with antisense (LMWH groups, 40 μg: t(10) = 3.981, **p = 0.0013; 80 μg: t(10) = 4.337, ###p = 0.0007; 120 μg: t(10) = 6.366, ****p < 0.0001; A6 groups, 40 μg: t(10) = 3.041, *p = 0.0062; 80 μg: t(10) = 4.600, ***p = 0.0005; 120 μg: t(10) = 5.205, ####p = 0.0002, when the respective sense and antisense groups are compared; unpaired Student's t test), with the strongest attenuation observed in the group treated with the highest dose (120 μg), for both LMWH- and A6-induced hyperalgesia. n = 6 paws per group.
Figure 4.
Figure 4.
Antihyperalgesic effect of HMWH. A, LMWH (1 μg) was injected intradermally on the dorsum of the hindpaw. Ten minutes later, HMWH (1 μg, black circles) or vehicle (open circles) was injected at the same site and the mechanical nociceptive threshold evaluated over time. Significant reversal of LMWH-induced hyperalgesia was observed in the group treated with HMWH (F(1.16) = 83.33, ****p < 0.0001, when both groups are compared, two-way repeated-measures ANOVA, followed by Bonferroni post hoc test). B, Four pronociceptive mediators, PGE2 (100 ng), epinephrine (epi, 100 ng), TNFα (100 ng), or IL-6 (10 ng), were injected intradermally on the dorsum of the hindpaw. Ten minutes after PGE2 and epinephrine, or 30 min after TNFα and IL-6, HMWH (1 μg, black bars) or vehicle (white bars) was injected at the same site. Measurement of the mechanical nociceptive threshold after an additional 30 min showed a significant attenuation of the hyperalgesia induced by all four pronociceptive mediators, in the groups treated with HMWH (PGE2 groups: t(10) = 5.676, ***p = 0.0001; epi groups: t(10) = 4.150, **p = 0.0010; TNFα groups: t(10) = 6.365, ****p < 0.0001; IL-6 groups: t(10) = 5.461, ***p = 0.0001, when the vehicle and HMWH groups are compared, unpaired Student's t test). C, Rats received four intraperitoneal injections of the neurotoxic chemotherapeutic drug paclitaxel (1 mg/kg), once every other day. Evaluation of mechanical nociceptive threshold 24 h after the last injection of paclitaxel showed robust mechanical hyperalgesia. Then HMWH (1 μg, black bar) or vehicle (white bar) was injected intradermally at the site of nociceptive testing on the dorsum of the hindpaw. Mechanical nociceptive threshold was again evaluated 30 min later. Whereas hyperalgesia was still observed in the vehicle-treated group, in the group that received HMWH, it was markedly attenuated (t(10) = 4.677, ###p = 0.0004, when control and HMWH groups are compared, unpaired Student's t test). A, Control group, n = 12 paws; HMWH group, n = 6. B, C, All groups, n = 6 paws.
Figure 5.
Figure 5.
Antihyperalgesic effect of HMWH is CD44-dependent. A, Rats were treated daily with a spinal intrathecal injection of ODN sense or antisense for CD44 mRNA (120 μg) for 3 consecutive days. On the fourth day, TNFα (100 ng, left) or IL-6 (10 ng, right) was injected intradermally on the dorsum of the hindpaw. Thirty minutes later, vehicle (white bars) or HMWH (1 μg, black bars) was injected at the same site. Evaluation of the mechanical nociceptive threshold 30 min later showed a significant reversal of the hyperalgesia induced by TNFα and IL-6 in the groups that had been treated with antisense, compared with those treated with sense (TNFα, sense groups: t(10) = 7.306, ****p < 0.0001; antisense groups: t(10) = 1.007, p = 0.1688, nonsignificant; IL-6, sense groups: t(10) = 9.077, ****p < 0.0001; antisense groups: t(10) = 0.8891, p = 0.1974, nonsignificant, when the vehicle and HMWH groups are compared, unpaired Student's t test). B, Rats received four intraperitoneal injections of the chemotherapeutic drug paclitaxel (1 mg/kg, every other day). Intrathecal injections of ODN sense or antisense were performed, once a day, from days 5–7 of paclitaxel treatment. At 24 h after the last injections of paclitaxel and ODNs, vehicle (white bars) or HMWH (1 μg, black bars) was injected intradermally on the dorsum of the hindpaw. Mechanical nociceptive threshold was evaluated before treatment with paclitaxel and 30 min after the injection of vehicle or HMWH. Significant attenuation of paclitaxel-induced hyperalgesia was observed only in the ODN sense-treated group (t(10) = 4.781, ***p = 0.0004, compared with the antisense-treated group, unpaired Student's t test). Together, these results indicate that HMWH acts at CD44 to produce antihyperalgesia. n = 6 paws, all groups.
Figure 6.
Figure 6.
HMWH attenuates mechanical hyperalgesia induced by pronociceptive second messengers. The direct activators of intracellular proalgesic signaling pathways, 8-bromo cAMP (potent PKA activator) or ψεRACK (PKCε activator), both 1 μg, were injected intradermally on the dorsum of the hindpaw; 30 min later, vehicle (white bars) or HMWH (1 μg, black bars) was injected at the same site, and the mechanical nociceptive threshold was evaluated an additional 30 min later. Significant attenuation of hyperalgesia induced by both second messengers was observed in the groups treated with HMWH (8-bromo cAMP groups: t(10) = 8.187, ****p < 0.0001; ψεRACK groups: t(10) = 5.050, ***p = 0.0002, when the vehicle and HMWH groups are compared, unpaired Student's t test), indicating that HMWH induces antihyperalgesia by triggering a signaling pathway that decreases nociceptor sensitization. n = 6 paws per group.
Figure 7.
Figure 7.
In vitro LMWH, but not HMWH, increases DRG neuron excitability. A, Control group. B, LMWH group. C, HMWH group. Left columns: Images of neurons, obtained by transmitted light (differential interference contrast, left images) or fluorescence microscopy (right images, in which the darker regions correspond to more intense IB4 binding). A, B, IB4+ neurons stained with IB4 conjugated with AlexaFluor-488 dye. C, The lack of staining determines the IB4− neuron. Arrows indicate the neurons from which the electrophysiological recordings of APs (middle and right columns) were obtained. A–C, Middle columns: The two sets of traces represent AP generation before (left) and after (right) intervention. Black traces represent AP generation in response to rheobase current injection. Gray traces represent the responses to stimulation below rheobase (no AP generation). The magnitude of current pulses is indicated above the inset boxes (gray). Right column: Traces represent the AHP development and recovery after AP induced by 1 ms current pulse before (black traces) and after (gray traces) intervention. Gray boxes represent stimulation profile in current-clamp mode. D, Pooled relative changes of AP parameters after application of perfusion solution alone (white bars), LMWH (black bars), or HMWH (gray bars). Rheobase (left) was significantly decreased in the LMWH group compared with the control and HMWH groups (F(2,28) = 11, p = 0.0003, one-way ANOVA, followed by Bonferroni post hoc test; t(22) = 3.5, **p < 0.01, when LMWH and control groups are compared, t(20) = 4.2, ###p < 0.001, when LMWH and HMWH groups are compared). In addition, there were no significant differences from baseline in the control and HMWH groups (control group: t(8) = 1.3, p = 0.2; HMWH group: t(6) = 0.0, p = 1.0, compared with 0 by one-sample Student's t test). This suggests that LMWH increases neuronal excitability by reducing rheobase, whereas HMWH does not alter rheobase. Rheobase baseline was 650 ± 130 pA (n = 32). AP threshold potential (middle) was not significantly different between control, LMWH, and HMWH groups, as well as from baseline in each group (F(2,26) = 1.5, p = 0.25, one-way ANOVA; t(6) = 1.2, p = 0.3, for the control group; t(14) = 0.2, p = 0.8, for the LMWH group; t(6) = 1.2, p = 0.3, for the HMWH group, compared with 0 by one-sample Student's t test), suggesting that neither LMWH nor HMWH alters AP threshold potential significantly, and the reduction of rheobase is not due to a shift of AP threshold potential. AP threshold potential baseline was −32 ± 3 mV (n = 29). Of note, as this value is negative, relative decrease makes it “more positive” (i.e., increases threshold, implying less excitability), and vice versa. Therefore, the vertical axis is intentionally inverted to avoid confusion. Although not significant, there is a tendency to a positive shift of AP threshold potential in HMWH group. Peak AHP (right) was significantly increased in the LMWH group, compared with the control and HMWH groups (F(2,16) = 5.0, p = 0.02, one-way ANOVA, followed by Dunnett's post hoc test: q = 2.5, *p < 0.05, when LMWH and control groups are compared; q = 2.8, #p < 0.05, when LMWH and HMWH groups are compared). In addition, there were no significant differences from baseline in the control and HMWH groups (control group: t(3) = 0.06, p = 0.96; HMWH group: t(7) = 0.4, p = 0.7, compared with 0 by one-sample Student's t test). This indicates that LMWH, but not HMWH, influences the repolarization phase of the AP, which can impact the generation of following APs. AHP baseline was 5.4 ± 0.5 mV (n = 19). In all experiments, readings were obtained 10 min after the application of perfusion solution alone (A, control; D, white bars), LMWH (B, and black bars in D) or HMWH (C, and gray bars in D) had started, when a stable effect was achieved. To avoid effects of washout, the concentration of LMWH and HMWH (0.2 mg/ml) was kept constant during recording. Of note, both IB4+ and IB4− neurons were evaluated. There was no significant difference between them in the effects of LMWH or HMWH on either parameter (data not shown). D, Left, Control group, n = 9 neurons; LMWH group, n = 15 neurons; HMWH group, n = 7 neurons. Middle, Control group, n = 7 neurons; LMWH group, n = 15 neurons; HMWH group, n = 7 neurons. Right, Control group, n = 4 neurons; LMWH group, n = 7 neurons; HMWH group, n = 8 neurons.
Figure 8.
Figure 8.
In vitro prevention and reversal of PGE2-induced nociceptor sensitization by HMWH. Neuron sensitization was assessed as relative reduction of rheobase (A, C) and latency of the first AP in ramp protocol (B, D), electrophysiological parameters characterizing electrical excitability of neurons. The effect of HMWH on PGE2-induced neuron sensitization was examined in prevention and reversal protocols. In prevention protocol (A, B), the pronociceptive mediator PGE2 (1 μm) was applied (and remained further in the bathing solution) after 30 min preincubation in the presence (black bars) or absence (white bars) of HMWH (0.2 mg/ml). In the HMWH groups, to avoid effects of washout, the HMWH concentration was kept constant in all solutions during recording, including during PGE2 application. Readings were obtained 10 min after the application of PGE2 had started, when a stable effect was achieved. As observed in the bar graphs (left side of each panel, showing the relative changes of parameters), PGE2 induced significantly smaller reduction of both rheobase (A) and latency (B) after preincubation with HMWH compared with the control groups (white bars), indicating the inhibitory effect of preincubation with HMWH on neuron sensitization induced by PGE2 (A, t(13) = 2.3, *p = 0.036; B, t(15) = 2.8, #p = 0.015; when control and HMWH groups are compared; unpaired Student's t test with Welch's correction). In reversal protocol (C, D), PGE2 was applied first (and remained further during bathing and applications). When a stable effect was achieved, the same PGE2-containing solution without (white bars) or with (black bars) HMWH was administered and remained in the experimental chamber. Reading of the parameters 10 min later revealed a tendency to attenuation of rheobase reduction (C, t(10) = 1.1, p = 0.31, not significant) and significantly smaller reduction of AP latency (D, t(7) = 2.8, ●p = 0.027, unpaired Student's t test with Welch's correction) when HMWH was administered (black bars), compared with the control (white bars), suggesting at least partial reversal of PGE2-induced neuron sensitization by HMWH. Right side of each panel: Respective original trRight side of each panelaces of membrane potential, recorded in current-clamp mode of whole-cell patch clamp, illustrating the onset of APs. Vertical calibration for all traces: 20 mV. Horizontal calibration: 20 ms for all traces, except for B (100 ms). A, C, Black traces represent AP generation in response to rheobase current injection. Gray traces represent responses to stimulation below rheobase (no AP generation). The magnitude of the current pulses is shown above the boxes illustrating the stimulation profile (gray insets). B, D, Generation of APs in response to ramp current injection. The traces corresponding to the different treatments (PGE2 or PGE2 + HMWH) are stacked vertically to emphasize changes in latency of the first AP. A, B, Right: Traces recorded in the same neuron after preincubation with HMWH, before and after PGE2 application (note the larger changes induced by PGE2 without HMWH shown in middle traces from C, D). C, Left, Middle, and Right sets of traces, D, Top, Middle, and Bottom traces: Recordings before interventions, after PGE2 and HMWH applications, correspondingly. A, Control group, n = 17 neurons; HMWH group, n = 7 neurons. B, Control group, n = 15 neurons; HMWH group, n = 6 neurons. C, Control group, n = 6 neurons; HMWH group, n = 7 neurons. D, Control group, n = 7 neurons; HMWH group, n = 6 neurons.
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