Gut microbiota is critical for the induction of chemotherapy-induced pain

Abstract

Chemotherapy-induced pain is a dose-limiting condition that affects 30% of patients undergoing chemotherapy. We found that gut microbiota promotes the development of chemotherapy-induced mechanical hyperalgesia. Oxaliplatin-induced mechanical hyperalgesia was reduced in germ-free mice and in mice pretreated with antibiotics. Restoring the microbiota of germ-free mice abrogated this protection. These effects appear to be mediated, in part, by TLR4 expressed on hematopoietic cells, including macrophages.

Conflict of interest statement

Competing Financial Interests Statement: The authors have no competing financial interests to report.

Figures

Fig 1. Temporary eradication of gut microbiota prevents oxaliplatin-induced mechanical hyperalgesia

a–c) Impacts of antibiotic water feeding on mice gut microbiota. Fecal samples were obtained after three weeks of antibiotic water (abx, N=10) or regular water (H2O, N=10) followed by DNA isolation from these samples. a) Antibiotics feeding reduced bacterial load as determined by semi-quantitative real-time PCR. b) Antibiotics feeding reduced the α diversity of microbiota. c) Antibiotics feeding altered bacterial community structure as shown in the phylum analysis. d) Gut microbiota eradication prevented the development of oxaliplatin-induced mechanical hyperalgesia. Mice were fed on antibiotics water (abx) or regular water (H2O) prior to oxaliplatin or saline treatment (as control). Hindpaw mechanical withdrawal threshold (HWT) was examined at indicated time points after oxaliplatin therapy. N=6 each group. * p<0.05 H2O/oxaliplatin vs. abx/oxaliplatin. **p>0.05 abx/oxaliplatin vs. abx/saline or H2O/saline. e) Germ-free (GF) status protected mice from oxaliplatin-induced mechanical hyperalgesia. GF or specific pathogen free (SPF) mice were given oxaliplatin or saline. HWT was examined at indicated time points after oxaliplatin therapy. * p<0.05 SPF, oxaliplatin vs. all other groups. N=7 each group. f) GF conventionalization abrogated the protection of mechanical hyperalgesia offered by GF status. To conventionalize GF mice, feces from SPF mice were diluted with PBS and administered daily via gastric gavage for three weeks. GF and conventionalized GF (GF conv) mice were treated with oxaliplatin or saline. Conventionalization of GF mice abrogated the protection offered by GF status. * p<0.05 GF conv, oxaliplatin vs. GF, oxaliplatin. N=8 for GF, oxaliplatin; N=7 for GF, saline; N=7 for GF conv, oxaliplatin; N=6 for GF conv, saline. g) Gut microbiota eradication did not change tissue oxaliplatin distribution (N=6 each group). Spinal cord (sc), serum and dorsal root ganglion (DRG) concentrations of platinum were determined by ICP-MS. Two-way ANOVA test suggested abx treatment did not change tissue platinum distribution (p=0.42) while types of tissues have significant influence on platinum levels (p=0.001). Post-hoc t-tests (p values in the figure). were performed to identify the impact of tissue type on platinum levels h) DRG cytokine levels for IL-6 and TNF-α were lower in mice with eradicated gut microbiota than those in mice with normal gut microbiota (N=6 each group, one-way ANOVA indicated there was significant difference in DRG samples, and subsequent post-hoc t-test was used to determine the difference between the abx and H2O groups). Sera and spinal cord levels for IL-6 and TNF-α were not significantly different among groups (N=6 each group, one-way ANOVA test for sera IL-6 samples, p=0.58; spinal cord IL-6 samples, p=0.06; sera TNF-α samples, p=0.88; spinal cord TNF-α samples, p=0.75). i) Reduced levels of reactive oxygen species (ROS) in DRG from mice with eradicated gut microbiota. At day 10 after the initiation of oxaliplatin therapy, levels of ROS were determined with IVIS using L-012 as a chemiluminescent probe. N=6 each group. One-way ANOVA test followed by post-hoc test were used for statistical analysis.

Fig 2. Gut microbiota is critical for DRG inflammatory responses

a–b) DRG flow cytometry staining for macrophages. Mice were fed on antibiotics water (abx) or regular water (H2O) prior to oxaliplatin or saline treatment. Ten days after the initiation of oxaliplatin or saline treatment, DRG samples were collected and processed for flow cytometry staining. Dot plots in a) are representative stainings for macrophages (CD11b+CD45hi cells) of each group. Percentages of macrophages in DRG cells were plotted in b). Percentages of macrophages were significantly higher in the ‘H2O, oxaliplatin’ group than in the ‘abx, oxaliplatin’ group (N=6 each group, p=0.0001 by one-way ANOVA, post-hoc t-test p=0.009). c) A permissive effect of LPS on production of IL-6 and TNF-α in macrophages. Macrophages were collected from peritoneal cavity for culture in the presence of LPS and oxaliplatin at indicated concentrations. IL-6 and TNF-α levels in culture supernatant were determined at 24 hours after oxaliplatin and LPS stimulation with ELISA. * and ** indicate post-hoc t-test performed after one-way ANOVA test suggesting significant differences exist among three groups at given LPS concentration. *p<0.05 vs oxaliplatin 0, ** p<0.05 vs oxaliplatin 1μM. Data represent quadruplicate wells, mean +/− SEM. d) LPS (endotoxin) levels in serum and DRG. Mice were fed on antibiotics water (abx) or regular water (H2O) prior to oxaliplatin treatment or saline treatment as control. Ten days after the initiation of oxaliplatin or saline treatment, serum and DRG samples were collected and processed for endotoxin assay. LPS levels in sera and DRG were higher in the ‘H2O, oxaliplatin’ group than in the ‘abx, oxaliplatin’ group. Post-hoc t-tests (p values) were performed after one-way ANOVA tests indicating significant difference present among all groups (N=6 each group).

Fig 3. TLR4 on hematopoietic cells is critical for oxaliplatin-induced mechanical hyperalgesia

a) Exogenous administration of LPS through gastric gavage reversed the effect of gut microbiota eradication. Mice were fed with antibiotic water (abx) or regular water (H2O), followed by oxaliplatin injection. LPS (3mg/Kg) or normal saline was administered on days of oxaliplatin injection and twice weekly afterwards. *p<0.05, abx,oxaliplatin; LPS gavage vs. abx, oxaliplatin; saline gavage, N=7 each group. b) Toll-like receptor 4 (TLR4) knockout mice did not develop oxaliplatin-induced mechanical hyperalgesia. TLR4 knockout (TLR4−/−) or littermate heterozygous (TLR 4+/−) mice were treated with oxaliplatin or saline and were examined at indicated time points for hindpaw mechanical withdrawal threshold (HWT) (N=6 each group). * p<0.05 WT, oxaliplatin vs. all other groups. c–d) Generation of bone marrow (BM) chimeras. c) Flow chart for BM chimeric mice generation. Recipients were irradiated 500 Rad × 2 followed by bone marrow cells injection. Donor bone marrow cells were derived from TLR4−/− or WT mice, and were injected to WT or TLR4−/− recipients, using a cross-over study design. CD 45.1 and CD45.2 congenic markers were used to distinguish between donor-derived and recipient-derived hematopoietic cells. d) Confirmation of successful generation of bone marrow chimera. Fourteen weeks after BM transplantation, DRG samples were collected and stained for CD45.1 and CD45.2 congenic markers. Contour plots were gated on CD11b+ and CD3− cells. Each panel represents 6 independent stainings. e) TLR4 on hematopoietic cells is critical for oxaliplatin-induced mechanical hyperalgesia. BM chimeric mice generated as shown in c–d) were treated with oxaliplatin (N=6 each group). In TLR4−/− to WT group, hematopoietic cells were from TLR4−/− donors. These mice were protected from oxaliplatin-induced mechanical hyperalgesia despite the presence of TLR4 on host-derived radio-resistant cells. *p<0.05 TLR4−/− to WT vs. WT to WT. In contrast, in WT to TLR4−/− group, hematopoietic cells were from WT donors, these mice developed oxaliplatin-induced mechanical hyperalgesia despite the absence of TLR4 on host-derived radio-resistant cells. ** p> 0.05 WT to TLR4−/−vs. WT to WT.