Blockade of IL-6 signaling prevents paclitaxel-induced neuropathy in C57Bl/6 mice

Abstract

The microtubule-stabilizing agent paclitaxel frequently leads to chemotherapy-induced peripheral neuropathy (CIN), which further increases the burden of disease and often necessitates treatment limitations. The pathophysiology of CIN appears to involve both "upstream" effects including altered intracellular calcium signaling and activation of calcium dependent proteases such as calpain as well as subsequent "downstream" neuro-inflammatory reactions with cytokine release and macrophage infiltration of dorsal root ganglia. In this study, we aimed to investigate whether these processes are linked by the pro-inflammatory cytokine interleukin-6 (IL-6). We observed that paclitaxel exposure induced IL-6 synthesis in cultured sensory neurons from postnatal Wistar rats, which could be prevented by co-treatment with a calpain inhibitor. This suggests a calcium dependent process. We demonstrate that adult C57BL/6 mice deficient in IL-6 are protected from developing functional and histological changes of paclitaxel-induced neuropathy. Furthermore, pretreatment with an IL-6-neutralizing antibody resulted in the prevention of paclitaxel-induced neuropathy in C57BL/6 mice. Electrophysiological data from our preclinical model was adequately reflected by measurements of patients undergoing paclitaxel therapy for ovarian cancer. In this cohort, measured Il-6 levels correlated with the severity of neuropathy. Our findings demonstrate that IL-6 plays a pivotal role in the pathophysiology of paclitaxel-induced neuropathy per se and that pharmacological or genetic interference with this signaling pathway prevents the development of this potentially debilitating adverse effect. These findings provide a rationale for a clinical trial with IL-6 neutralizing antibodies to prevent dose-limiting neurotoxic adverse effects of paclitaxel chemotherapy.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1. Effects of paclitaxel (PTX) treatment in wild-type and IL-6 knockout mice on motor function and mechanical allodynia.

a Schematic overview of paclitaxel-induced neuropathy: Adult male C57Bl/6J wild-type (WT) and IL-6 knockout (IL-6−/−) mice were injected intraperitoneally with 1 mg per kg bodyweight (BW) paclitaxel (PTX) or the corresponding vehicle (VEH) cremophor EL:Ethanol (1:1) four times on alternating days (cumulative dose of 4 mg per kg BW PTX). Locomotor function and mechanical withdrawal threshold were assessed with the RotaRod (RR) and von-Frey (vF) test on days 7 and 13; sensory nerve action potential (SNAP) of the caudal nerve was measured on days 7 and 14 after the initial PTX application. Tissue samples for histology were obtained in terminal anesthesia on day 14. b PTX treated mice developed a slight weight loss over the course of the treatment, which was only significant in the PTX/IL-6–/– group compared with the VEH treated IL-6–/– mice. c Locomotor function remained unaltered after PTX application in WT and IL-6–/– mice. d PTX treated WT mice developed a significant reduction of the mechanical withdrawal threshold indicative of mechanical allodynia, while IL-6–/– mice were protected from these changes. Error bars depict SEM. Statistical analysis: b–d Two-way ANOVA with Tukey post hoc analysis of n = 10 mice/group; *p < 0.05, NS not significant.

Fig. 2. Electrophysiology and histology of paclitaxel-induced neuropathy in IL-6 knockout vs. wild-type mice.

a PTX treated WT mice developed a significant reduction of the sensory nerve action potential (SNAP) amplitude, whereas IL-6–/– mice receiving PTX showed no alterations in electrophysiological measurements. b Semiautomatic quantification of semi-thin sections of the sciatic nerve revealed a decrease in fiber density after PTX treatment in WT mice, while no changes could be detected in IL-6–/– mice after PTX therapy. c PTX treatment lead to a loss of larger myelinated fibers in WT mice, while IL-6–/– mice showed no shift in fiber distribution. d We observed a moderate positive correlation between mean axon diameter and SNAP amplitude. e Measurement of IL-6 concentrations in spinal cord lysates revealed significantly higher IL-6 concentrations in paclitaxel-treated wild-type mice (LLOD: Lower limit of detection). Error bars depict SEM. Statistical analysis: a, b two-way ANOVA with Tukey post hoc analysis of n = 10 mice/group, d Pearson correlation of n = 37 mice; e one-way ANOVA with Holm-Sidak post hoc analysis. *p < 0.05, NS not significant.

Fig. 3. Paclitaxel induces IL-6 production in dorsal root ganglia neurons (DRGN).

a Exposure of DRGN to 100 nM PTX for 24 h leads to a marked decrease of cell viability. Increasing concentrations of IL-6 had no additive toxic effect on DRGN cell viability. b Cultured DRGN from postnatal rats treated with 100 nM PTX or Vehicle (VEH) for 24 h show IL-6 immunoreactivity (first and second row). Omission of the primary antibody abolished the signal (“negative control”, third row). c Elevated IL-6 concentrations were detected in the supernatant of PTX treated DRGN after 24 h exposure (100 nM). Co-incubation with the calpain inhibitor MDL28170 (CalpInh; 1 µM) as well as lithium chloride (Li+; 1 mM), but not the glycogen synthase kinase-3 inhibitor A1070722 (GSK-3-Inh; 50 nM), maintained IL-6 levels at control values after PTX exposure. d DRGN were treated for 24 h with 100 nM PTX or vehicle and subsequently underwent fractionated extraction of cytosolic (CF) and nuclear (NF) proteins. Western blots of these fractions revealed a decrease of nuclear factor-κB (NF-κB) in the cytosolic and an increase in the nuclear fraction after PTX treatment compared with VEH. e Western blot of the inhibitor of NF-κB (IκB) shows reduced immunoreactivity after PTX treatment in the cytosolic fraction. f For comparison immunoreactivity normalized to the loading control was set into relation with vehicle treatment (VEH = 100%) from three experiments. This analysis supports the observation that PTX treatment leads to a decrease of cytosolic IκB and a translocation of NF-κB from the cytosol to the nucleus. Error bars depict SEM. Statistical analysis: a, c Data obtained from n = 3 biological replicates analyzed with (a) Kruskal–Wallis with Dunn’s post hoc test and (c) one-way ANOVA with Tukey post hoc analysis; *p < 0.05, NS not significant.

Fig. 4. Effect of IL-6-neutralizing antibodies (IL-6-AB) on the development of paclitaxel-induced sensory neuropathy.

a Schematic overview of preventive treatment with IL-6-AB MAB406: Adult male C57Bl/6 mice received one intraperitoneal application of the IL-6-AB MAB406 (5 mg/kg BW) or unspecific IgG prior to PTX (4 × 1 mg/kg BW) or VEH treatment. Mice were assessed for behavioral signs of neuropathy on days 7 and 13, electrophysiological measurements were performed at baseline and on days 7 and 14 after initial PTX injection. b There were no significant weight differences between VEH and PTX treatment in any of the groups. c Locomotor function remained unchanged regardless of treatment. d PTX treated mice, which had previously received the control IgG, developed mechanical hypersensitivity while mice of the PTX/IL-6-AB group were protected from these changes. e PTX treatment led to a significant reduction of the SNAP amplitude by ~50% in mice which received the control antibody (IgG), while preventive IL-6-AB application maintained axonal integrity during PTX therapy. f Mice with the preventive IL-6-AB treatment were protected from PTX-induced neuropathy as fiber density was comparable to control values. Error bars depict SEM. Statistical analysis: b–f two-way ANOVA with Tukey post hoc of n = 6–8 mice/group; *p < 0.05, NS not significant.

Fig. 5. Paclitaxel-induced neuropathy in patients suffering from ovarian cancer.

We analyzed data from patients suffering from ovarian cancer with regards to the development of sensory neuropathy. Patients undergoing combination chemotherapy with paclitaxel (6 × 175 mg/m2 body surface area) and carboplatin (AUC 5) +/– bevacizumab developed axonal sensory neuropathy, which was marked by (a) a significant decrease of the sural nerve sensory action potential amplitude (SNAP), while (b) the nerve conduction velocity was unaffected. c The total neuropathy score (TNSr) which integrates clinical and electrophysiological parameters showed a steep increase. Severity of paclitaxel-induced neuropathy showed a positive correlation with the serum IL-6 concentration before (d) more than after (e) chemotherapy. Error bars depict SEM. Statistical analysis: a, bt-test, c Mann–Whitney-U test, d, e Spearman correlation of seven patients; *p < 0.05, NS not significant.

Fig. 6

Summary graph of molecular mechanisms involved in the pathogenesis of paclitaxel-induced neuropathy and possibilities for pharmacological modulation.