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. 2015 Sep 30;35(39):13487-500.
doi: 10.1523/JNEUROSCI.1956-15.2015.

The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

Yan Li  1 , Pavel Adamek  2 , Haijun Zhang  3 , Claudio Esteves Tatsui  4 , Laurence D Rhines  4 , Petra Mrozkova  2 , Qin Li  5 , Alyssa K Kosturakis  6 , Ryan M Cassidy  7 , Daniel S Harrison  8 , Juan P Cata  5 , Kenneth Sapire  5 , Hongmei Zhang  1 , Ross M Kennamer-Chapman  7 , Abdul Basit Jawad  7 , Andre Ghetti  9 , Jiusheng Yan  5 , Jiri Palecek  2 , Patrick M Dougherty  10
Affiliations

The Cancer Chemotherapeutic Paclitaxel Increases Human and Rodent Sensory Neuron Responses to TRPV1 by Activation of TLR4

Yan Li et al. J Neurosci. .

Abstract

Peripheral neuropathy is dose limiting in paclitaxel cancer chemotherapy and can result in both acute pain during treatment and chronic persistent pain in cancer survivors. The hypothesis tested was that paclitaxel produces these adverse effects at least in part by sensitizing transient receptor potential vanilloid subtype 1 (TRPV1) through Toll-like receptor 4 (TLR4) signaling. The data show that paclitaxel-induced behavioral hypersensitivity is prevented and reversed by spinal administration of a TRPV1 antagonist. The number of TRPV1(+) neurons is increased in the dorsal root ganglia (DRG) in paclitaxel-treated rats and is colocalized with TLR4 in rat and human DRG neurons. Cotreatment of rats with lipopolysaccharide from the photosynthetic bacterium Rhodobacter sphaeroides (LPS-RS), a TLR4 inhibitor, prevents the increase in numbers of TRPV1(+) neurons by paclitaxel treatment. Perfusion of paclitaxel or the archetypal TLR4 agonist LPS activated both rat DRG and spinal neurons directly and produced acute sensitization of TRPV1 in both groups of cells via a TLR4-mediated mechanism. Paclitaxel and LPS sensitize TRPV1 in HEK293 cells stably expressing human TLR4 and transiently expressing human TRPV1. These physiological effects also are prevented by LPS-RS. Finally, paclitaxel activates and sensitizes TRPV1 responses directly in dissociated human DRG neurons. In summary, TLR4 was activated by paclitaxel and led to sensitization of TRPV1. This mechanism could contribute to paclitaxel-induced acute pain and chronic painful neuropathy. Significance statement: In this original work, it is shown for the first time that paclitaxel activates peripheral sensory and spinal neurons directly and sensitizes these cells to transient receptor potential vanilloid subtype 1 (TRPV1)-mediated capsaicin responses via Toll-like receptor 4 (TLR4) in multiple species. A direct functional interaction between TLR4 and TRPV1 is shown in rat and human dorsal root ganglion neurons, TLR4/TRPV1-coexpressing HEK293 cells, and in both rat and mouse spinal cord slices. Moreover, this is the first study to show that this interaction plays an important role in the generation of behavioral hypersensitivity in paclitaxel-related neuropathy. The key translational implications are that TLR4 and TRPV1 antagonists may be useful in the prevention and treatment of chemotherapy-induced peripheral neuropathy in humans.

Keywords: DRG; cancer; dorsal horn; neuropathy.

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Figures

Figure 1.
Figure 1.
A, B, Prevention and reversal of paclitaxel-induced hyperalgesia by intrathecal injection of a TRPV1 antagonist (AMG9810). The baseline (BL) behavioral test in A and B were collected before paclitaxel (Pac) or vehicle (Veh) treatments. In A, the gray shading indicates the time of treatment with 15 μg of AMG9810 (intrathecal) or vehicle solution. In B, paclitaxel-induced mechanical hypersensitivity was confirmed as significant from Veh–Veh-treated rats (open squares, n = 5) at 14 d after treatment (P) in two groups (open and filled circles, n = 5 each); rats were then treated with 15 μg of the TRPV1 antagonist AMG9810 intrathecally (filled circles) or vehicle solution (open circles) as indicated by the arrow. *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA followed by Bonferroni post hoc test. The representative image in C shows the baseline staining of TRPV1 (red) in the DRG in vehicle-treated rats that was not different from naive rats (data not shown); TRPV1 staining becomes elevated by day 7 after paclitaxel treatment (D). Cotreatment of rats with PBS (intrathecally) with paclitaxel did not affect the increased staining of TRPV1 (E), whereas cotreatment with LPS-RS (intrathecally) completely prevented the increase in TRPV1 (F). As indicated by the bar graphs in G, the increase in TRPV1+ neurons by paclitaxel was statistically significant (**p < 0.01), whereas in H, this was significantly less in the LPS-RS-treated rats versus the PBS-treated rats. Scale bar, 100 μm. **p < 0.01.
Figure 2.
Figure 2.
TRPV1 is colocalized with TLR4 in DRG neurons and afferent terminals in the spinal cord. TRPV1 alone is shown in red in the left column for DRG neurons in A and in spinal terminals in D. TLR4 staining in subsets of DRG neurons is shown in green in B and spinal terminals in E (center column). Colocalization of the two are shown in the merged image by yellow for DRG in the right column (C), as well as in fiber profiles in the superficial spinal dorsal horn (F). Scale bar, 100 μm. The representative recording in G shows that acute application of paclitaxel (12.5 μm) evoked spontaneous action potentials in subsets of DRG neurons from animals treated with paclitaxel. In H and I, representative action potential waveforms for the neuron in G evoked by direct current injection after 5 min of vehicle treatment and then after 5 min of acute perfusion with 12.5 μm paclitaxel are shown. The bar graphs in J show the group data for the effects of paclitaxel on several action potential properties. AP, Action potential; RMP, resting membrane potential; AHP, after-hyperpolarization; AHPxx%, interval to each percentage of maximal amplitude. *p < 0.05; **p < 0.01; ***p < 0.001 paclitaxel versus vehicle; paired t test.
Figure 3.
Figure 3.
TRPV1 sensitization by paclitaxel and LPS in DRG neurons shown using calcium imaging. Representative calcium imaging results of change in 340/380 ratio in dissociated DRG neurons after perfusions of capsaicin (CAP, 200 nm) alone and in combination with paclitaxel (12.5 μm) or paclitaxel plus LPS-RS (2 μg/ml) are shown in A–D, each colored line is a single neuron and the time of each application is indicated by the bars over the traces. The bar graphs show the grouped results for experiments testing the interactions between paclitaxel on the responses to capsaicin (E–G) on the effects of LPS on DRG neurons alone (H) and the effects of LPS on DRG neurons from vehicle- and paclitaxel-treated rats (I). **p < 0.01; ***p < 0.001, vehicle rats versus paclitaxel rats; ###p < 0.001, CAP+Pac versus CAP+Pac+LPS-RS.
Figure 4.
Figure 4.
Interactions between TLR4 and TRPV1 in HEK 293 cells studied using whole-cell patch clamp. Inward currents were recorded in HEK293 cells transfected with TLR4 only (top line), TRPV1 only (second line), and both TLR4 and TRPV1 (line 3). The bar graphs at the bottom show the summarized responses with statistical differences determined by paired t tests. Capsaicin (200 nm) did not induce inward currents in cells expressing TLR4 alone, whereas the responses to repeated capsaicin showed desensitization in cells expressing TRPV1 alone. LPS (10 ng/ml) and paclitaxel (12.5 μm) sensitized the responses to capsaicin in cells expressing both TLR4 and TRPV1. *p < 0.05, LPS+capsaicin; ***p < 0.001, paclitaxel+capsaicin; *p < 0.05 vehicle solution+capsaicin versus first capsaicin response.
Figure 5.
Figure 5.
Representative examples of whole-cell recordings for substantia gelatinosa (SG) neurons before (left) and after (right) administration of the TRPV1 antagonist AMG9810 (5 μm) in the vehicle-treated group (A, B), day 7 paclitaxel-treated group (C, D), and day 14 paclitaxel-treated group (E, F) show increased sEPSCs only in the day 7 paclitaxel-treated group that is suppressed by the AMG9810. The washout segment is not shown. Bar graphs in G and H summarize the mean (±SEM) change in amplitude (G) and frequency (H) of sEPSCs before and after 5 min of AMG9810 application. **p < 0.01, day 7 paclitaxel-treated group versus vehicle and day 14 paclitaxel-treated groups; two-way ANOVA followed by Newman–Keuls post hoc test.
Figure 6.
Figure 6.
Paclitaxel application increased mEPSC frequency in superficial dorsal horn neurons in rat spinal cord slice. A, Native recording of mEPSC activity before and after paclitaxel (50 nm) application. B, The TRPV1 antagonist SB366791 (10 μm) did not change the mEPSC frequency but prevented its increase during coapplication with paclitaxel. C, Averaged responses demonstrate that paclitaxel treatment induced a significant increase in mEPSC frequency compared with the baseline (control, 100%) value (140.7 ± 11.1%; n = 14). This increase was prevented by the TRPV1 antagonist (SB366791+paclitaxel) treatment, whereas the antagonist alone had no effect (SB366791; n = 10). D, Paclitaxel (50 nm) application did not change the frequency or amplitude of the sEPSCs or the amplitude of the dorsal root sEPSCs (E). ***p < 0.001 versus control values; ###p < 0.001 versus paclitaxel; one-way ANOVA followed by Student–Newman–Keuls test.
Figure 7.
Figure 7.
Representative traces showing that capsaicin (200 nm) increases mEPSCs in spinal neurons and that the second response was notably reduced compared with the first one in vehicle-treated mice (A). Acute application of paclitaxel (50 nm) for 10 min before the second capsaicin application prevented the decrease of the second response (B). Coapplication of paclitaxel with the TLR4 antagonist LPS-RS (2 μg/ml) prevented the effect of paclitaxel and on the second capsaicin response, with the result being desensitization, as seen in the control group (C). In D, the mean normalized responses are shown for each group (n = 8) where the responses to the second capsaicin application are expressed as a percentage of the first application. The mean increase in mEPSC frequency 5 min after the first capsaicin application was pronounced in the presence of paclitaxel (E). ***p < 0.001; *p < 0.05 versus paclitaxel-treated group; one-way ANOVA followed by Student–Newman–Keuls test.
Figure 8.
Figure 8.
Immunofluorescent double staining shows that TRPV1 (red, A) is colocalized with TLR4 (green, B) in human DRG neurons (yellow in merged image at right, C). The red arrows in the merged image indicate cells only showing TRPV1, the green arrows indicate cells only expressing TLR4, and the yellows arrow points to cells positive for both TRPV1 and TLR4. Scale bar, 200 μm. Three types of responses were observed when human DRG neurons were tested by application of capsaicin (Cap) and paclitaxel (Pac). Type 1 neurons did not respond to either capsaicin or paclitaxel (data not shown). Type 2 neurons (D) responded positively to capsaicin (left column), showed no responses to paclitaxel (center column), and then showed desensitization to a second application of capsaicin (right column). Type 3 neurons (E) showed responses to capsaicin (left column) and to paclitaxel (center column) and then showed a facilitation of response to the repeated application of capsaicin (right column). The bar graphs at the bottom show the summarized response for the type 2 and 3 neurons. The baseline response to capsaicin was not different between groups. The second response to capsaicin was significantly reduced compared with the first in the type 2 neurons. The type 3 neurons showed significantly greater responses to paclitaxel than did type 2 neurons; and type 3 neurons showed a significantly increased response to the second application of capsaicin compared with the first. *p < 0.05; Mann–Whitney U test.
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